Advances in Immunology Volume 88

Contents

Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ix

CD22: A Multifunctional Receptor That Regulates B Lymphocyte Survival and Signal Transduction Thomas F. Tedder, Jonathan C. Poe, and Karen M. Haas 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CD22 Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CD22 Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CD22—A Receptor for Diverse Sialylated Ligands . . . . . . . . . . . . Effects of CD22 Engagement on B-Cell Activation and Function In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CD22 Ligand Binding Regulates B-Cell Survival and Proliferation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The CD22 Cytoplasmic Domain Regulates B-Cell Signal Transduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CD22-Directed Therapies and Clinical Trials in Oncology. . . . . . . CD22-Directed Therapies and Clinical Trials in Autoimmunity . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 2 3 4 5 7 8 15 28 30 36 37

Tetramer Analysis of Human Autoreactive CD4-Positive T Cells Gerald T. Nepom Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. pMHC Tetramers Identify Human Autoreactive T Cells . . . . . . . . 2. Autoantigen pMHC Tetramers from Mouse to Man. . . . . . . . . . . . v

51 51 59

c on t e n ts

vi

3. Tetramer-Induced Autoreactivity . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

65 67

Regulation of Phospholipase C-g2 Networks in B Lymphocytes Masaki Hikida and Tomohiro Kurosaki 1. 2. 3. 4. 5. 6. 7. 8. 9.

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . From the BCR to PLC-g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Roles for PLC-g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of IP3 Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Coupling Between Calcium Release and Calcium Influx . . . . . . . . Looking for Ca2þ Entry Channels . . . . . . . . . . . . . . . . . . . . . . . . . . NFAT and NF-kB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Modulation of Calcium Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

73 73 74 78 79 81 83 86 88 89 90

Role of Human Mast Cells and Basophils in Bronchial Asthma Gianni Marone, Massimo Triggiani, Arturo Genovese, and Amato De Paulis 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Human Mast Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Human Basophils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Experimental Evidence for Mast Cells in Bronchial Asthma . . . . . Experimental Evidence for Basophils in Bronchial Asthma . . . . . . Mast Cell and Basophil Recruitment to Asthmatic Airways . . . . . . Anatomical and Functional Evidence for Mast Cells and Basophils in Asthma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Superallergens in Bronchial Asthma . . . . . . . . . . . . . . . . . . . . . . . . Angiogenesis in Bronchial Asthma . . . . . . . . . . . . . . . . . . . . . . . . . . Tissue Remodeling in Bronchial Asthma . . . . . . . . . . . . . . . . . . . . . Pharmacologic Modulation of Human Mast Cells and Basophils in the Treatment of Bronchial Asthma . . . . . . . . . . . . . . Conclusions and Implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

97 97 101 109 116 117 118 119 121 127 128 130 136 138

c o nt e n t s

vii

A Novel Recognition System for MHC Class I Molecules Constituted by PIR Toshiyuki Takai 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PIR and LILR in Ig-like Receptor Family . . . . . . . . . . . . . . . . . . . PIR Genes and Protein Structure . . . . . . . . . . . . . . . . . . . . . . . . . . Ligand for PIR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3D Structure of PIR and LILR and Their Interaction with MHC Class I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Activation Signal via PIR-A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inhibitory Signal via PIR-B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dominant Expression of PIR-B In Vivo . . . . . . . . . . . . . . . . . . . . . Pirb–/– B Cells, Neutrophils, and Macrophages are Hyperresponsive. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Modulated Cytokine Signaling in PIR-B Deficiency . . . . . . . . . . . . PIR in Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

161 161 163 166 169 171 174 176 177 178 180 182 186 187

Dendritic Cell Biology Francesca Granucci, Maria Foti, and Paola Ricciardi-Castagnoli 1. 2. 3. 4. 5. 6.

Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DC Subtypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Deciphering DC Biology with Genomic Approaches . . . . . . . . . . . DC Interactions with the Microbial World . . . . . . . . . . . . . . . . . . . DC Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

193 193 194 196 199 209 219 219

The Murine Diabetogenic Class II Histocompatibility Molecule I-Ag7: Structural and Functional Properties and Specificity of Peptide Selection Anish Suri and Emil R. Unanue Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235

c on t e n ts

viii

2. Structural Features of I-Ag7 and DQ8 . . . . . . . . . . . . . . . . . . . . . . . 3. Biochemical Basis of Peptide Selection by I-Ag7 . . . . . . . . . . . . . . . 4. Why Is There a Difference in Results Between Binding and Peptide Selection? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. The Biological Role of I-Ag7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

236 241 254 256 259

RNAi and RNA-Based Regulation of Immune System Function Dipanjan Chowdhury and Carl D. Novina 1. 2. 3. 4. 5. 6. 7. 8.

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Short RNAs in the Mammalian Immune System . . . . . . . . . . . . . . miRNAs in Mammalian Virus Infection. . . . . . . . . . . . . . . . . . . . . . RNAi, NMD, and TCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . NMD and TCR Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . RNAi and NMD Are Genetically Linked . . . . . . . . . . . . . . . . . . . . RNAi, ADARs, and Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

267 267 271 273 274 276 278 280 284 286

Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293 Contents of Recent Volumes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305 Color Plate Section

Contributors

Numbers in parenthesis indicated the pages on which the authors’ contributions begin.

Dipanjan Chowdhury (267), Center for Blood Research and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115 Maria Foti (193), Department of Biotechnology and Bioscience, University of Milano-Bicocca, Piazza della Scienza 2, 20126 Milan, Italy Arturo Genovese (97), Division of Clinical Immunology and Allergy, Center for Basic and Clinical Immunology Research (CISI), University of Naples Federico II, School of Medicine, I-80131 Naples, Italy Francesca Granucci (193), Department of Biotechnology and Bioscience, University of Milano-Bicocca, Piazza della Scienza 2, 20126 Milan, Italy Karen M. Haas (1), Department of Immunology, Duke University Medical Center, Durham, North Carolina 27710 Masaki Hikida (73), Laboratory for Lymphocyte Differentiation, RIKEN Research Center for Allegy and Immunology, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan Tomohiro Kurosaki (73), Laboratory for Lymphocyte Differentiation, RIKEN Research Center for Allergy and Immunology, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan Gianni Marone (97), Division of Clinical Immunology and Allergy, Center for Basic and Clinical Immunology Research (CISI), University of Naples Federico II, School of Medicine, I-80131 Naples, Italy Gerald T. Nepom (51), Benaroya Research Institute at Virginia Mason, Seattle, Washington 98101 Carl D. Novina (267), Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, Massachusetts 02115; Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115 Amato De Paulis (97), Division of Clinical Immunology and Allergy, Center for Basic and Clinical Immunology Research (CISI), University of Naples Federico II, School of Medicine, I-80131 Naples, Italy ix

x

c o n tr i b u t o rs

Jonathan C. Poe (1), Department of Immunology, Duke University Medical Center, Durham, North Carolina 27710 Paola Ricciardi-Castagnoli (193), Department of Biotechnology and Bioscience, University of Milano-Bicocca, Piazza della Scienza 2, 20126 Milan, Italy Anish Suri (235), Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri 63110 Toshiyuki Takai (161), Department of Experimental Immunology and CREST Program of the Japan Science and Technology Agency, Institute of Development, Aging, and Cancer, Tohoku University, Sendai 980-8575, Japan Thomas F. Tedder (1), Department of Immunology, Duke University Medical Center, Durham, North Carolina 27710 Massimo Triggiani (97), Division of Clinical Immunology and Allergy, Center for Basic and Clinical Immunology Research (CISI), University of Naples Federico II, School of Medicine, I-80131 Naples, Italy Emil R. Unanue (235), Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri 63110

CD22: A Multifunctional Receptor That Regulates B Lymphocyte Survival and Signal Transduction Thomas F. Tedder, Jonathan C. Poe, and Karen M. Haas Department of Immunology, Duke University Medical Center, Durham, North Carolina 27710

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Abstract ........................................................................................................... Introduction ..................................................................................................... CD22 Expression............................................................................................... CD22 Structure................................................................................................. CD22—A Receptor for Diverse Sialylated Ligands .................................................. Effects of CD22 Engagement on B‐Cell Activation and Function In Vitro .............................................................................................. CD22 Ligand Binding Regulates B‐Cell Survival and Proliferation.............................. The CD22 Cytoplasmic Domain Regulates B-Cell Signal Transduction ........................ CD22‐Directed Therapies and Clinical Trials in Oncology......................................... CD22-Directed Therapies and Clinical Trials in Autoimmunity .................................. Conclusions ...................................................................................................... References .......................................................................................................

1 2 3 4 5 7 8 15 28 30 36 37

Abstract Recent advances in the study of CD22 indicate a complex role for this transmembrane glycoprotein member of the immunoglobulin superfamily in the regulation of B lymphocyte survival and proliferation. CD22 has been previously recognized as a potential lectin‐like adhesion molecule that binds a2,6‐linked sialic acid‐bearing ligands and as an important regulator of B‐cell antigen receptor (BCR) signaling. However, genetic studies in mice reveal that some CD22 functions are regulated by ligand binding, whereas other functions are ligand‐independent and may only require expression of an intact CD22 cytoplasmic domain at the B‐cell surface. Until recently, most of the functional activity of CD22 has been widely attributed to CD22’s ability to recruit potent intracellular phosphatases and limit the intensity of BCR‐generated signals. However, a more complex role for CD22 has recently emerged, including a central role in a novel regulatory loop controlling the CD19/CD21‐Src‐family protein tyrosine kinase (PTK) amplification pathway that regulates basal signaling thresholds and intensifies Src‐family kinase activation after BCR ligation. CD22 is also central to the regulation of peripheral B‐cell homeostasis and survival, the promotion of BCR‐induced cell cycle progression, and is a potent regulator of CD40 signaling. Herein we discuss our current understanding of how CD22 governs these complex and overlapping processes, how alterations in these tightly controlled regulatory activities may influence

1 advances in immunology, vol. 88 # 2005 Elsevier Inc. All rights reserved.

0065-2776/05 $35.00 DOI: 10.1016/50065-2776(05)88001-0

2

t h om a s f . t e d d er

ET AL.

autoimmune disease, and the current and future applications of CD22‐directed therapies in oncology and autoimmunity. 1. Introduction B lymphocytes are the central mediators of humoral immunity. They differentiate through highly regulated pathways before becoming mature plasma cells that secrete antigen (Ag)‐specific antibody. B cells depend on cues from their extracellular microenvironment for development, homeostasis, activation, proliferation, and effector function. These functions are regulated through cell surface molecules that generate transmembrane signals, regulate intercellular communication, and direct lymphocyte localization within tissues. These events are thought to primarily rely on signals generated by the B‐cell Ag receptor (BCR) composed of membrane immunoglobulin (Ig) noncovalently associated with disulfide‐linked CD79a/CD79b (Iga/Igb) heterodimers. Multiple other signaling molecules also provide important functional links between the cell surface and intracellular signaling (Tedder, 1998). CD22 and CD19 represent two specialized costimulatory or coreceptor cell surface molecules (Buhl and Cambier, 1997; Cyster and Goodnow, 1997; Nitschke and Tsubata, 2004; O’Rourke et al., 1997; Tedder et al., 1997a) that also function as ‘‘response regulators’’ (Tedder, 1998) to modulate the intensity, quality, and duration of homeostatic and BCR‐induced signals (Fujimoto et al., 1998; Sato et al., 1998). Response regulators carry out broader functions than costimulatory molecules because they establish intrinsic signaling thresholds that provide a context for other transmembrane and cytoplasmic signals. CD22 is also a lectin‐like member of the Ig superfamily expressed exclusively by all mature B‐lineage cells, which binds ligands in vivo to regulate BCR and CD19 signal transduction, and provide essential survival signals. In the current review, we describe a critical role for CD22 in regulating normal B‐cell function, CD19 and BCR signal transduction, BCR‐induced cell death, and the homeostatic survival of B cells in the periphery. These complex processes are differentially regulated by CD22 binding to its ligands or by intrinsic CD22 activity which functions independently of ligand engagement. Thus, CD22 is a multifunctional receptor that employs ligand‐dependent and -independent mechanisms to ultimately regulate the generation of physiologically relevant responses to foreign or self‐antigens (Ags) and govern events critical for B‐cell selection, activation, and differentiation. Because CD22 provides an important regulatory checkpoint for adjusting B‐cell function and survival, understanding CD22 function may provide mechanisms for modulating humoral immunity and treatments for malignancies or autoimmunity.

c d2 2 l i g a n d b i n d i n g r e gu l at e s b - c e ll su rv i va l

3

2. CD22 Expression CD22 expression is B‐cell‐specific and developmentally regulated in mice and humans (Fig. 1). Mouse CD22 (mCD22) was first identified as the Lyb‐8.2 Ag immunoprecipitated from mouse splenocytes as 95 and 105 kDa glycoproteins (Symington et al., 1982). Mouse CD22 is absent or expressed at low levels on the surface of pre‐B cells and newly emerging IgMþ B cells, is present at low density on immature (B220lo IgMhi) B cells, and is fully expressed by mature recirculating (B220hi IgMint IgDþ) B cells (Erickson et al., 1996). Once in the periphery, mCD22 is expressed at high levels on all B‐cell subsets. mCD22 expression increases slightly on mitogen‐stimulated B cells, but is lost during plasma cell differentiation. Human CD22 (hCD22) expression is restricted to the cytoplasm of pro‐B and pre‐B cells, and its localization shifts to the cell surface simultaneously with IgD expression (Do¨ rken et al., 1986). The vast majority of human IgMþ IgDþ B cells express cell surface CD22. In lymphoid tissues, hCD22 is expressed by follicular mantle and marginal zone (MZ) B cells, but only weakly by germinal center B cells. Upon B‐cell activation,

Figure 1 CD22 gene and protein organization, chromosome location, and protein expression during B‐cell development and activation. Ig‐like domains 1 and 2 contain the ligand‐binding region of CD22. Various effector molecules are recruited to the CD22 cytoplasmic domain when phosphorylated on one or more of six conserved tyrosine residues, several of which lie within consensus ITIM motifs in humans and mice. CD22 is expressed on the surface of mature B cells (B) within lymphoid follicle marginal zones, mantle zones, and germinal centers, but not by T cells (T), monocytes (M), neutrophils (N), red blood cells (RBC), platelets (P), or dendritic cells (DC).

4

t h om a s f . t e d d er

ET AL.

hCD22 mRNA and protein expression increase during the first 2 days of culture with mitogens, followed by a marked loss of hCD22 by 3–4 days (Do¨ rken et al., 1986; Wilson et al., 1991). Interestingly, in contrast to human adult blood B cells, neonatal cord blood B cells downregulate CD22 protein expression following BCR activation, but upregulate CD22 expression following anti‐CD40 þ IL‐4 stimulation (Viemann et al., 2000). Thus, CD22 expression may be differentially regulated depending upon the activation stimuli as well as the responding B cell. As with mCD22, hCD22 expression ceases with B‐cell differentiation into plasma cells. 3. CD22 Structure The initial isolation of cDNAs encoding CD22 revealed it to be composed of five Ig domains (Stamenkovic and Seed, 1990). Subsequently, a CD22 cDNA encoding two additional Ig domains and a full‐length 141 amino acid cytoplasmic tail was isolated (Wilson et al., 1991). The five Ig domain form of CD22 was subsequently termed CD22a, while the seven Ig domain form was termed CD22b (Stamenkovic et al., 1991). Both cDNA isoforms result from differential splicing within a single gene, with the removal of domains three and four in the CD22a form (Wilson et al., 1993). However, the seven Ig domain form of CD22 is the dominant mRNA and protein species, although a variety of rare hCD22 mRNA transcripts lacking Ig domains 3 and/or 4 can be isolated from B‐cell lines (Engel et al., 1995a). mCD22 is also composed of seven Ig domains, and is 62% identical in overall amino acid sequence with hCD22 (Torres et al., 1992). The highest level of conservation (70%) is found between the seventh Ig domains, although the 140 amino acid cytoplasmic domain and the transmembrane domains are 68% identical in sequence. CD22 in humans and mice consists of a single amino‐terminal V‐set Ig domain and six C‐2‐set Ig domains (Engel et al., 1995a; Torres et al., 1992; Wilson et al., 1991). The hCD22 and mCd22 genes have at least 15 exons (Law et al., 1993; Wilson et al., 1993). Exons 4–10 encode single Ig domains; exons 11–15 encode transmembrane and cytoplasmic domains (Fig. 1). Immunoprecipitated cell surface hCD22 generally appears as a two chain glycoprotein of 140 and 130 kDa (Boue and LeBien, 1988a,b; Do¨ rken et al., 1986; Schwarting et al., 1985; Schwartz‐Albiez et al., 1991). Although initially proposed by some to be a heterodimer, the relative intensity of the lower Mr band of hCD22 is consistently less, and varies considerably depending on the cell source examined (Engel et al., 1995a). Immunoprecipitation studies with monoclonal antibodies (mAbs) reactive with different Ig domains revealed that the 140 kDa, seven Ig domain form of hCD22 is the predominant protein species expressed on the cell surface (Engel et al., 1995a). The lower

c d2 2 l i g a n d b i n d i n g r e gu l at e s b - c e ll su rv i va l

5

Mr band variably observed in CD22 immunoprecipitations lacks domain 4, while a 120 kDa protein (<1%) that may represent CD22a is barely detectable in immunoprecipitations from some surface‐labeled B‐cell lines. Thus, the CD22 splice variants previously postulated to possess unique ligand binding activities are not present on the surface of B cells in significant amounts. Therefore, cell surface hCD22 and mCD22 are predominantly monomeric seven Ig domain molecules with identical ligand‐binding regions (Engel et al., 1995a; Law et al., 1995). CD22a and CD22b were originally proposed to bind different ligands via distinct domains within each splice variant. However, a large panel of function‐ blocking anti‐hCD22 mAbs were found to only bind epitopes localized to the first and/or second Ig‐like domains (Engel et al., 1995a). Using domain‐swap and domain‐deletion mutants of hCD22, these two amino‐terminal domains were the functional unit mediating CD22 adhesion with ligands on lymphocytes, neutrophils, monocytes, and erythrocytes. That adhesion‐blocking CD22 mAbs only bound Ig domains 1 and 2 contrasted markedly with domain mapping studies by others that reported broadly distributed CD22 ligand‐ binding sites for CD45RA and other ligands. This discrepancy led to the demonstration that soluble glycoconjugates in mouse ascites fluid are potent inhibitors of CD22 adhesion (Engel et al., 1993). Thus, the early studies of CD22 function and adhesion were misleading because soluble factors in ascites fluid were blocking CD22 function rather than the receptor‐specific mAb present in the ascites fluid. Nonetheless, it was clear that hCD22 represented a novel prototype member of the Ig superfamily that binds sialic acid– bearing ligands through its amino‐terminal Ig‐like domains. Mouse CD22 also uses its two amino‐terminal Ig domains for ligand binding, which are 55% identical in amino acid sequence with hCD22 (Law et al., 1995). 4. CD22—A Receptor for Diverse Sialylated Ligands In vitro studies have demonstrated a role for CD22 as a potent intercellular adhesion molecule in cell–cell interactions, recognizing N‐linked oligosaccharides possessing a2‐6‐linked sialic acid residues (Engel et al., 1993; Stamenkovic and Seed, 1990). Mouse CD22 is specific for ligands composed of a2,6‐linked N‐glycolylneuraminic acid (NeuGc), whereas humans only express CD22 ligands in the form of N‐acetylneuraminic acid (NeuAc) (Kelm et al., 1994; Powell and Varki, 1994; Powell et al., 1993; Sgroi et al., 1993). Thus, when expressed at high levels in ectopic cells, CD22 binds a variety of ligands that are broadly distributed on erythrocytes, T and B lymphocytes, neutrophils, and monocytes (Engel et al., 1993). Ligands have also been identified using CD22‐Ig fusion proteins, which bind to a diverse array of

6

t h om a s f . t e d d er

ET AL.

leukocytes and nonlymphoid cells. A unifying feature is that all CD22 ligands are sialic acid‐dependent, since CD22 ligand binding is eliminated by treating lymphoid and nonlymphoid target cells with neuraminidase (Engel et al., 1995a). In fact, b‐galactoside a2,6‐sialyltransferase has been identified as a golgi enzyme necessary for the generation of CD22 ligands as well as the HB‐6, CDw75, and CD76 leukocyte differentiation Ags (Bast et al., 1992; Munro et al., 1992). CD22 is now recognized as a member of the ‘‘sialoadhesin’’ or ‘‘SIgLec’’ subclass of the Ig superfamily whose members function as mammalian lectins (Engel et al., 1993; Kelm et al., 1994). The sialoadhesin family includes multiple other sialic acid‐binding receptors such as sialoadhesin, a differentiation Ag of monocytes (Crocker et al., 1994), and CD33, a differentiation Ag of myeloid progenitor cells (Simmons and Seed, 1988). CD22 ligands identified in vitro include structurally diverse sialic acid‐bearing molecules expressed by lymphocytes, neutrophils, monocytes, and erythrocytes as well as nonhematopoietic cells (Tedder et al., 1997b). Ligands identified using in vitro assays and CD22‐Ig fusion proteins, include glycoproteins, glycolipids, and gangliosides. Specific examples include CD22 itself, all isoforms of CD45, soluble IgM pentamers, haptoglobin, Ly‐6 proteins, and a variety of other diverse proteins present on leukocyte cell surfaces (Hanasaki et al., 1995; Pflugh et al., 2002; Stamenkovic et al., 1991). CD22 is also reported to bind cell surface Ig with 0.2–2% of surface Ig physically associated with immunoprecipitated cell surface CD22 (Leprince et al., 1993; Peaker and Neuberger, 1993). Whether this association is direct through the extracellular or intracellular CD22 domains or is through intermediate bridging molecules remains unknown. It is also unknown whether any of the ligands identified in vitro represent physiologic in vivo ligands, although important physiologic roles for CD22 ligand binding have been recently identified (Poe et al., 2004a). While CD22‐mediated adhesion is readily visualized during in vitro assays when CD22 is overexpressed in cell lines (Engel et al., 1993, 1995a; Stamenkovic and Seed, 1990), cis interactions between CD22 and other B‐cell surface sialoglycoproteins have been suggested to result in the occupancy or ‘‘masking’’ of CD22 on most primary mature B cells (Jin et al., 2002; Kelm et al., 2002; Razi and Varki, 1998). CD22 masking is proposed to downregulate positive signaling via cell surface receptors such as IgM and CD45, preventing B‐cell hyperactivation. However, CD22 masking is a reversible process, occurs independent of CD45 expression, and does not prevent the recruitment of CD22 to sites of cell–cell contact (Collins et al., 2004). Moreover, the presence of cell surface sialic acids does not affect the rate of CD22 internalization or interfere with CD22 interactions with CD45 or IgM (Zhang and Varki, 2004). ‘‘Unmasking’’ of some CD22 molecules also occurs following B‐cell sialidase

c d2 2 l i g a n d b i n d i n g r e gu l at e s b - c e ll su rv i va l

7

treatment or costimulation via CD40, which may relieve CD22 negative regulation of BCR signaling within germinal centers or other microenvironments (Razi and Varki, 1998). CD22 unmasking has also been proposed to regulate the migration of mature recirculating B cells to the bone marrow by promoting trans interactions with ligands expressed on bone marrow endothelium (Floyd et al., 2000; Nitschke et al., 1999), although conclusions drawn from mice expressing CD22 receptors unable to bind ligands argue against this (Poe et al., 2004a). 5. Effects of CD22 Engagement on B‐Cell Activation and Function In Vitro Various roles for CD22 adhesion have been suggested, including tissue localization, serving as a sensing mechanism for neighboring leukocytes, and as a regulator of signaling. In vitro analyses have suggested that CD22 binding to sialic acid‐bearing ligands positively influences B‐cell function. For example, the simultaneous addition of CD22 mAbs and anti‐Ig antibodies to human B‐cell cultures results in more potent B‐cell proliferation than either antibody added alone (Pezzutto et al., 1987). Treatment of B cells with a solid‐phase CD22 mAb prior to anti‐Ig stimulation also enhances proliferation and reduces the concentration of anti‐Ig antibody needed for an equivalent response without anti‐CD22 mAb pretreatment (Doody et al., 1995). Anti‐CD22 mAb binding prior to BCR engagement also enhances the increase in [Ca2þ]i observed following surface IgM cross‐linking (Pezzutto et al., 1988). In contrast, extensive CD22 cross‐linking by mAbs induces B‐cell apoptosis and potentiates anti‐IgM antibody‐induced apoptosis in malignant B cells (Chaouchi et al., 1995). Therefore, CD22 ligation generates costimulatory signals in some assay systems and apoptotic signals in others. We have also used mAbs reactive with the ligand‐binding domains of hCD22 to assess the role of CD22 in B‐cell function. Several mAbs, such as HB22‐7 and HB22‐23, stimulate potent human B‐cell proliferation directly, even in the absence of BCR ligation (Tuscano et al., 1996a). When added to costimulation assays, the HB22‐7 mAb synergizes with anti‐IgM antibodies, interleukin‐2 (IL‐2), IL‐4, or CD40 mAb to significantly augment B‐cell proliferation. Even more striking levels of B‐cell proliferation occur under culture conditions that enhance B‐cell–B‐cell interactions. Binding of the HB22‐7 mAb also augments B‐cell differentiation and triggers predominantly IgG secretion when added with IL‐2 to B‐cell cultures. By contrast, a CD22 mAb that minimally inhibits ligand binding, HB22‐5, poorly costimulates B‐ cell function. Ligation of CD22 with either the HB22‐7 or HB22‐23 mAb results in rapid CD22 tyrosine phosphorylation and in increased association of

8

t h om a s f . t e d d er

ET AL.

CD22 with Lyn, the p85 subunit of PI 3‐kinase, and Syk (Tuscano et al., 1996b). The HB22‐5 mAb has modest effects on CD22 phosphorylation. It has also been postulated that CD22‐binding to ligands on T cells regulates T‐cell function (Aruffo et al., 1992; Stamenkovic et al., 1991; Tuscano et al., 1996a). For example, inhibition of B‐cell CD22 binding to its ligand on T cells by receptor blockade with the HB22‐7 mAb significantly impairs T‐cell proliferation in costimulatory assays (Tuscano et al., 1996a). Thus, ligand binding by CD22 may provide stimulatory signals for B cells and provide costimulatory signals for T‐cell proliferation. Several studies using in vitro analyses also suggest that CD22 binding to sialic acid‐bearing ligands negatively influences B‐cell function. In one recent and provocative study, B‐cell activation by Ag‐expressing target cells that coexpressed a2‐6‐sialoglycoconjugates was suppressed only when CD22 was expressed on the B‐cell surface, with the degree of B‐cell activation determined by the upregulation or downregulation of important cell surface molecules (Lanoue et al., 2002). The authors concluded that negative regulation through CD22 dampens B‐cell reactivity to self‐Ags, preventing the activation of autoreactive B cells. In two other recent articles, the effects of CD22 ligand binding on BCR‐induced [Ca2þ]i responses and SHP1 recruitment to tyrosine‐phosphorylated CD22 were evaluated. In the first study, cDNAs encoding mutant CD22 molecules lacking ligand‐binding activity were expressed in a CD22‐ deficient B‐cell line (Jin et al., 2002). Following BCR ligation, B‐cell lines expressing mutated CD22 molecules had larger BCR‐induced [Ca2þ]i responses compared to lines expressing wild‐type CD22. Enhanced [Ca2þ]i responses correlated with decreased CD22 phosphorylation and recruitment of SHP1. In the second study, a sialic acid‐based inhibitor that selectively prevented hCD22 from binding to sialic acid‐bearing ligands expressed on other B cells also resulted in augmented [Ca2þ]i responses and decreased CD22 phosphorylation/SHP1 recruitment following BCR ligation (Kelm et al., 2002). Although these in vitro studies are useful, they depend on altered cell lines and mimetics that may not recapitulate physiologic CD22 engagement. Therefore, understanding the physiologic significance and consequences of blocking CD22 ligand binding in vivo is critical for understanding CD22 function and its regulatory role in B‐cell development. 6. CD22 Ligand Binding Regulates B‐Cell Survival and Proliferation Newly generated B cells express CD22 as they mature to acquire positive BCR signaling responses (Fig. 2). This represents a critical transition in B‐cell maturation since interactions between CD22 and its ligands in vivo are now known to be crucial for mature B‐cell survival upon entry into the periphery,

c d2 2 l i g a n d b i n d i n g r e gu l at e s b - c e ll su rv i va l

9

Figure 2 CD22 and CD40 provide essential overlapping signals for B‐cell survival and proliferation. The stages of expression of CD22 and CD40 are indicated, along with stages of expression at which signaling through these receptors regulates B‐cell homeostasis and/or activation.

in addition to regulating BCR‐induced proliferation (Poe et al., 2004a; Sato et al., 1998). 6.1. B‐Cell Survival in CD22

/

Mice

Four independent lines of CD22 / mice have been generated (Nitschke et al., 1997; O’Keefe et al., 1996; Otipoby et al., 1996; Sato et al., 1996a). Immature B‐cell numbers are normal in the bone marrow of CD22 / mice consistent with low‐level surface expression of CD22 at this stage of development (Fig. 1). However, one characteristic feature of CD22 / mice is 66% reduced numbers of circulating IgMþ B220þ B cells in blood and bone marrow (recirculating pool) relative to wild‐type littermates (Sato et al., 1996a). One of the most important feature of CD22 / B cells is their shorter lifespan, with enhanced apoptosis (Nitschke et al., 1997; Otipoby et al., 1996; Poe et al., 2004a). Despite this, B‐cell turnover is closely matched with B‐cell production in the bone marrow since only small changes in B‐cell subpopulations are observed in peripheral lymphoid tissues, with an increased frequency of peritoneal B‐1 B cells (O’Keefe et al., 1996; Sato et al., 1996a). Serum IgM levels are slightly (40%) increased in CD22 / mice, although other isotypes and humoral immune responses to T‐cell‐dependent Ags are normal (Sato et al., 1996a, 1998). Immune responses to T‐cell‐independent Ags are slightly decreased in CD22 / mice (Nitschke et al., 1997; Otipoby et al., 1996; Sato et al., 1996a). Thus, CD22 plays an important role in regulating mature B‐cell

10

t h om a s f . t e d d er

ET AL.

activation, effector function, and survival in vivo, but its loss does not have dramatic effects on early B‐cell development, maturation or differentiation. 6.2. CD22 Ligand Binding Regulates B‐Cell Survival and Proliferation Studies conducted with CD22 / mice have been unable to address the importance of CD22 ligand binding in regulating CD22 function. Therefore, to directly assess the relevance of CD22 ligand binding in B‐cell activation and survival, two lines of mice expressing mutant CD22 receptors incapable of ligand binding were generated, CD22D1‐2 and CD22AA (Poe et al., 2004a). CD22D1‐2 mice express a truncated form of CD22 lacking the two amino‐ terminal Ig domains required for ligand binding (Fig. 3). CD22AA mice express CD22 containing two point mutations (Arg130 to Ala130, Arg137 to Ala137) in the amino terminal Ig domain that results in the loss of CD22 ligand‐binding ability (van der Merwe et al., 1996). Mature naı¨ve peripheral B cells and mature recirculating bone marrow B cells in CD22 / , CD22D1‐2, and CD22AA mice have equally high in vivo turnover rates (Poe et al., 2004a). The effect of this high rate of B‐cell turnover on the peripheral B‐cell repertoire is currently unknown. CD22 ligand binding may also regulate B‐cell proliferation following Ag exposure as B cells from CD22 / , CD22D1‐2, and CD22AA mice exhibit a delayed proliferative response following in vitro stimulation with mitogenic F(ab0 )2 anti‐IgM antibodies (Poe et al., 2004a). Thus, CD22 interactions with its ligands are important for the regulation of B‐cell survival and proliferation. CD22 / B cells from mice with a C57BL/6 genetic background undergo dramatically higher rates of apoptosis than B cells from wild‐type littermates,

Figure 3 Ligand‐dependent and ligand‐independent functions of CD22 in vitro and in vivo. Protein structure of CD22 proteins produced in CD22AA and CD22D1–2 mice are illustrated.

c d2 2 l i g a n d b i n d i n g r e gu l at e s b - c e ll su rv i va l

11

while CD22 / B cells from mice with a mixed B6x129 genetic background only undergo apoptosis at slightly higher rates than wild‐type littermates. Although the genetic factors that account for this difference are currently unknown, biochemical studies have shed some light on these dramatic distinctions in the effects of CD22‐deficiency. There is a dramatic absence of lymphoblast formation in splenic C57BL/6 CD22 / B‐cell cultures after anti‐IgM treatment for 48 hr, along with a parallel increase in the percentage of apoptotic cells (Poe et al., 2004b). That CD22 / B cells predominantly respond to BCR‐induced signals by undergoing apoptosis may relate to the finding that CD22 regulates c‐Myc expression following BCR stimulation. B cells from CD22 / mice with a C57BL/6 genetic background do not upregulate c‐Myc transcriptional activity following BCR engagement (Poe et al., 2004b). As a consequence, CD22 / B cells are unable to induce Cullin 1 (Cul1), a c‐Myc‐dependent ubiquitin ligase complex scaffolding protein whose expression is required for cell cycle progression from G1 to S‐phase of the cell cycle (Dealy et al., 1999; O’Hagan et al., 2000; Zheng et al., 2002). In the absence of Cul1 expression, the proper formation of SCF/ubiquitin ligase complexes is disrupted, resulting in defective ubiquitination and degradation of cyclin‐dependent kinase inhibitors such as p27Kip1 and p21Cip1, which are critical for progression past the G1 checkpoint (O’Hagan et al., 2000; Shirane et al., 1999; Yu, Gervais, and Zhang, 1998). Follicular B cells also have high rates of apoptosis in these mice, particularly among MZ B cells. Increased numbers of apoptotic B cells is a unique property of CD22 / splenocytes because hyporesponsive B cells from CD19 / mice and hyperresponsive B cells from mice that overexpress CD19 undergo apoptosis at the same rates as wild‐type B cells (Poe et al., 2001). These data thereby indicate that CD22 / B cells in the context of a C57BL/6 background predominantly respond to BCR‐induced signals by undergoing apoptosis. Thereby, newly generated B cells unable to express CD22 in vivo may be unable to respond positively to BCR signals and mature as they enter the periphery. 6.3. CD40 Rescues B‐Cell Survival in the Absence of CD22 Engagement Like CD22, other B‐cell surface receptors also generate survival signals, including CD40 (Gordon, 1995). CD40 costimulatory signals are dominant to the death signals initiated by BCR ligation in vitro and rescue B cells from apoptosis within germinal centers (Castigli et al., 1994; Kawabe et al., 1994; Tsubata et al., 1993). Importantly, CD40 engagement rescues CD22 / B cells from BCR‐induced cell death (Poe et al., 2004b), indicating that CD22 / B cells are not preprogrammed to undergo cell death. Also, CD40 ligation alone

12

t h om a s f . t e d d er

ET AL.

induces the dramatic expansion of both wild‐type and CD22 / B cells into lymphoblasts and enhances the proliferation of BCR‐stimulated CD22 / cells. Thus, CD22 expression positively regulates mature B‐cell survival, suggesting that CD22 expression promotes the survival of the peripheral pool of naı¨ve B cells before CD40 engagement (Fig. 2). Importantly, B cells from CD22D1‐2 and CD22AA mice are also hyperproliferative to stimulation with mitogenic antibodies against CD40 (Poe et al., 2004a,b). These results suggest that CD22 may negatively regulate B‐cell stimulation or aberrant B‐cell activation during T‐cell‐dependent immune responses in which CD40 signaling is crucial. However, in the absence of both CD22 ligand binding and CD40 ligation, mature B cells may not receive the appropriate survival signals and undergo apoptosis as a consequence of BCR engagement. 6.4. CD22 Ligand Binding Regulates Cell Surface CD22 Expression CD22 undergoes constitutive internalization followed by degradation in an acidic intracellular compartment without detectable recycling of the molecule back to the cell surface (Shan and Press, 1995). Ligation of the CD22 molecule with mAbs markedly accelerates internalization and downmodulates surface Ag expression, but does not accelerate CD22 degradation (Press et al., 1989; Shan and Press, 1995; Shih et al., 1994). The kinetics of CD22 internalization (t1/2 < 1 h) in B‐cell lines and degradation (t1/2 8 h) are similar to those reported for the epidermal growth factor receptor, another terminally internalized cell surface receptor (Shan and Press, 1995). BCR ligation and intracellular kinase activation does not appear to affect the constitutive endocytosis of CD22. Interestingly, CD22 mAb binding results in rapid internalization of 80% of bound CD22 mAbs, followed by a plateau phase with apparent retention of 20% of the mAb on the cell surface (Shan and Press, 1995). Constitutive internalization of CD22 also shows biphasic internalization kinetics. This suggests the presence of a recycling pool of CD22 or the existence of two distinct pools of cell surface CD22 with different internalization and degradation kinetics. It is possible that CD22 retained on the cell surface following mAb engagement represents molecules that have engaged appropriate ligands and are thereby retained. Support for this hypothesis comes from CD22AA and CD22D1‐2 B cells (Poe et al., 2004a). Mutant CD22 expression by mature spleen and bone marrow B cells is reduced in both CD22D1‐2 and CD22AA mice. It is possible that the absence of ligand binding increases CD22 turnover since cell surface CD22 is constitutively endocytosed. Similar incremental reductions in cell surface CD22 expression by CD22D1‐2 and CD22AA B cells are unlikely to result from structural alterations in these two independently

c d2 2 l i g a n d b i n d i n g r e gu l at e s b - c e ll su rv i va l

13

targeted genes and the two mutant molecules are quite different. In addition, preliminary semi‐quantitative RT‐PCR analysis suggests that CD22 transcription is normal in CD22D1‐2 and CD22AA spleen B cells. Thus, CD22 ligand engagement may not only transduce important signals, but also retain this important regulator of signal transduction at the cell surface where it is functionally active. 6.5. CD22 Ligand Binding Regulates Cell Surface IgM Expression Peripheral B cells in CD22 / , CD22D1‐2, and CD22AA mice have decreased surface IgM expression, but increased MHC class II Ag and CD44 expression (Poe et al., 2004a; Sato et al., 1996a). Although less dramatic in these mice, this is similar to the phenotype of CD19‐overexpressing B cells (Engel et al., 1995b; Sato et al., 1997), and of SHP1‐defective B cells from moth‐eaten mice (Pani et al., 1995; Schultz et al., 1993; Sidman et al., 1978; Tsui et al., 1993). Rather than signifying different stages of differentiation, these results suggest that alterations in CD22 signaling influence the level of spontaneous BCR signaling which alters BCR turnover as in some transgenic mouse models (Bell and Goodnow, 1994). Downregulated surface IgM expression levels in mice overexpressing CD19 or in moth‐eaten mice is also thought to be a consequence of augmented transmembrane signaling through the BCR complex (Cyster and Goodnow, 1995; Engel et al., 1995b; Pani et al., 1995; Sato et al., 1997). By contrast, B cells from CD19‐deficient (CD19 / ) mice exhibit increased surface IgM expression, presumably due to hyporesponsive BCR signaling. Therefore, downregulated surface IgM levels in CD22 / mice suggests that CD22 regulates BCR signaling in the absence of Ag engagement since the majority of B cells are uniformly affected. Thus, the absence of CD22 expression may allow a higher level of spontaneous BCR signaling, which accelerates its turnover on the cell surface. CD22 interactions with its ligands may influence B‐cell signaling thresholds. Consistent with this hypothesis, the absence of CD22 ligand‐binding activity in CD22D1‐2 and CD22AA mice results in a phenotype similar to CD22 / B cells (Goodnow et al., 1988; Nitschke et al., 1997; O’Keefe et al., 1996; Otipoby et al., 1996; Poe et al., 2004a; Sato et al., 1996a). This is not simply due to decreased CD22 expression on mature B cells from CD22D1‐ 2 and CD22AA mice, because IgM and MHC class II expression are only modestly altered on B cells from CD22þ/ mice compared to the more severe defect on CD22D1‐2, CD22AA, and CD22 / B cells (Poe et al., 2004a; Sato et al., 1996a). This suggests that ligand binding by CD22 controls homeostatic BCR signaling directly, or more likely the downstream effects of homeostatic signaling that lead to B‐cell activation.

14

t h om a s f . t e d d er

ET AL.

6.6. CD22 Ligand Binding Regulates MZ B‐Cell Development MZ B cells represent a distinct nonrecirculating population localized within splenic follicles of both mouse and man. This B‐cell population demonstrates unique developmental characteristics, cell surface marker expression, and functional activity that differentiates them from mature recirculating follicular B cells (Martin and Kearney, 2002). MZ B cells may exist as a specialized B‐cell subpopulation poised to rapidly respond to blood‐borne T‐cell‐independent Ags and are thus deemed necessary for efficient humoral responses during infections (Guinamard et al., 2000; Martin and Kearney, 2000; Martin, Oliver, and Kearney, 2001). The MZ B‐cell compartment is significantly reduced in spleens of CD22 / mice (Samardzic et al., 2002; Sato et al., 1996a). The maintenance of MZ B cells also requires CD22 ligand‐binding activity, as MZ B cells are reduced equally in CD22D1‐2, CD22AA, and CD22 / mice (Poe et al., 2004a). Whether the reduction in MZ B cells results from a defect in their development, or whether CD22 ligand‐binding activity is required for MZ B‐cell localization and survival within the spleen remains to be determined. However, B cells within MZ regions of CD22 / mice appear to undergo apoptosis at higher rates than follicular B cells (Poe et al., 2004b). It is therefore possible that the overall high rate of B‐cell turnover in CD22 / , CD22D1‐2, and CD22AA mice does not permit the repopulation of MZ B cells, whereas follicular B cells are regenerated at faster rates by the bone marrow. Regardless, CD22 promotes MZ B‐cell development and/or survival through ligand‐dependent mechanisms. 6.7. CD22 Ligand Binding Does Not Regulate B‐Cell Tissue Localization It has been proposed that CD22 adhesive function regulates the migration of recirculating B cells to the bone marrow (Nitschke et al., 1999). This conclusion was based on a recombinant CD22‐Ig fusion protein binding to bone marrow sinusoidal endothelium, that treatment of mice with CD22‐Ig fusion protein or polyclonal rabbit anti‐CD22 antisera reduced the number of mature recirculating B cells in bone marrow by 50%, and that CD22 / mice have lower numbers of IgM‐secreting plasma cells in the bone marrow. Although provocative, these findings may also result from correlative changes in B‐cell function or CD22 signaling rather than CD22 serving as a specific adhesion receptor. For example, CD19 overexpressing mice also have lower numbers of circulating B cells and lower numbers of IgM‐secreting plasma cells in the bone marrow but it is unlikely that CD19 functions as an adhesion molecule (Tedder et al., 1997a). Moreover, CD22‐Ig fusion proteins bind an extremely diverse array of structurally distinct serum and cell surface sialoglycoconjugates, such

c d2 2 l i g a n d b i n d i n g r e gu l at e s b - c e ll su rv i va l

15

that it is difficult to appreciate the specificity or physiological relevance of their binding to bone marrow sinusoidal endothelium. Importantly, adoptive transfer of fluorochrome‐labeled splenic B cells from CD22D1‐2 and CD22AA mice revealed no impairment in their migration to the bone marrow or lymphoid tissues of recipient mice (Poe et al., 2004a). The importance of CD22 in B‐cell migration in vivo warrants further assessment, although there is no current evidence to suggest that CD22 functions as a ‘‘classic’’ type of adhesion molecule in vivo or that it directly regulates lymphocyte migration through mechanisms other than regulating B‐cell survival and signal transduction. 7. The CD22 Cytoplasmic Domain Regulates B-Cell Signal Transduction B cells from CD22 / , CD22D1-2, and CD22AA mice are hyperresponsive to some transmembrane stimuli while hyporesponsive to others. For example, proliferative responses induced by anti‐IgM antibodies are decreased in CD22 / B cells (Otipoby et al., 1996; Sato et al., 1996a), while responses to LPS, CD40, and CD38 plus IL‐4 are either elevated or similar to those of wild‐ type B cells. These observations indicate that CD22 functions as a complex positive and negative regulator of B lymphocyte signal transduction dependent on the context of BCR signaling (Tedder, 1998). However, significant gaps remain in our understanding of how CD22 regulates transmembrane signals and how this receptor interprets signals generated through other cell‐surface receptors. Regardless, one feature is constant; B cells from CD22 / mice have greatly increased intracellular calcium ([Ca2þ]i) responses following BCR‐cross‐linking (Fig. 4A), while B cells from CD22D1-2 and CD22AA mice generate normal [Ca2þ]i responses (Poe et al., 2004a). Thus, at least some critical features of CD22 function do not require ligand binding, but may rather depend on the membrane localization of the CD22 cytoplasmic domain. Importantly, the augmented [Ca2þ]i responses observed in CD22 / B cells are similar to [Ca2þ]i responses generated by simultaneous BCR and CD19 ligation, or responses of Lyn / B cells (Fig. 4A), suggesting that these signaling molecules are functionally linked (Fujimoto et al., 2001). 7.1. BCR Signaling Pathways After Ag is recognized by membrane Ig, immunoreceptor tyrosine‐based activation motif (ITAM; D/EX2YX2L/IX6-8YX2L) sequences of CD79a and CD79b become phosphorylated and function as signal transducing elements for nonreceptor PTKs. BCR‐mediated signaling induces the activation of

16

t h om a s f . t e d d er

ET AL.

distinct families of PTKs including Syk; Btk of the Tec family; and Lyn, Fyn, Blk, and Lck of the Src family. Lyn is the predominant Src‐family PTK member expressed in B cells. The function of Lyn in B cells is complex and includes both the initiation and subsequent termination of BCR‐generated signals (DeFranco et al., 1998). This unique role for Lyn in governing not only positive signals, but also negative feedback signals, results from Lyn phosphorylation of tyrosines within immunoreceptor tyrosine‐based inhibition motif (ITIM; V/IXYX2L) sequences of presumably negative regulatory molecules. Src‐family PTKs associate with the BCR complex in resting B cells and become activated upon BCR ligation.

Figure 4 (Continued)

c d2 2 l i g a n d b i n d i n g r e gu l at e s b - c e ll su rv i va l

17

Figure 4 CD22 regulation of [Ca2þ]i responses as described (Fujimoto et al., 2001). (A) [Ca2þ]i responses induced by simultaneous BCR (anti‐IgM F(ab0 )2 antibody fragments) and CD19 (MB19–1 mAb) ligation in B cells from wild‐type mice resembles that in B cells from CD22‐/‐ and Lyn‐/‐ mice. (B) Simultaneous BCR and CD19 engagement augments [Ca2þ]i responses and extinguishes CD22 phosphorylation and SHP1 recruitment, while extensive CD19 ligation inhibits BCR‐induced [Ca2þ]i responses. Left panel, [Ca2þ]i responses after BCR ligation with anti‐IgM or anti‐IgM plus CD19 antibody at high and low concentrations. Right panels demonstrate protein tyrosine phosphorylation (anti‐pTyr) in wild‐type B cells following BCR and/or CD19 ligation as indicated. Purified splenic B cells were incubated with medium alone, anti‐IgM F(ab0 )2 antibody fragments (40 mg/ml), CD19 antibody (40 mg/ml), or anti‐IgM (40 mg/ml) plus CD19 (10 mg/ml) antibodies for 3 min and detergent lysed. In some panels, CD19, CD22, or SHP1 were immunoprecipitated from B‐cell lysates, subjected to SDS‐PAGE analysis, and transferred onto membranes for antiphosphotyrosine (anti‐pTyr) immunoblotting. (C) Models for CD19/CD21 regulation of CD22 function. With BCR cross‐linking, CD19 augments BCR‐induced signals through its ability to amplify Src‐family PTK activation, which results in CD22 tyrosine phosphorylation. With CD19/CD21 cross‐linking, CD19 serves as a sensing mechanism for the generation of complement C3d‐Ag complexes and upregulates intrinsic levels of Lyn phosphorylation and activation, but it does not induce CD22 phosphorylation. With simultaneous BCR and CD19/ CD21 ligation, CD22 phosphorylation is inhibited, potentially due to the retention of Lyn by aggregated CD19 complexes. With simultaneous BCR and extensive CD19/CD21 ligation, CD22 and CD79 phosphorylation are both inhibited, potentially due to the sequestration of Lyn by aggregated CD19 complexes.

Following BCR ligation, activated Src‐family PTKs phosphorylate the ITAMs of CD79a and CD79b (Fig. 5A), resulting in the recruitment of Syk through its tandem Src homology 2 (SH2) domains (Saouaf et al., 1994). PTK function is additionally regulated by ‘‘cross‐talk’’ between PTKs, protein tyrosine phosphatases such as SHP1, and interactions with intracellular regulatory

18

t h om a s f . t e d d er

ET AL.

Figure 5 CD22 and CD19 regulation of [Ca2þ]i responses. (A) The CD19–CD22 regulatory loop interprets BCR‐induced signals. CD19 processive amplification of Lyn PTK activity leads to CD22 phosphorylation by Lyn. Phosphorylated CD22 binds SHP1 (in addition to the Grb2/Shc/SHIP complex), which in turn negatively regulates CD19 phosphorylation and amplification of Src family PTK activity. In addition, CD22 regulates cMyc expression through unknown pathways, which directly regulates Cul1 production and ubiquitin ligase (UL)‐dependent cell cycle progression.

c d2 2 l i g a n d b i n d i n g r e gu l at e s b - c e ll su rv i va l

19

and adapter proteins (Thomas and Brown, 1999). The net positive effects of PTK activation include tyrosine phosphorylation of important transmembrane receptors, phosphoinositide generation and turnover, and increases in [Ca2þ]i through the activation of phospholipase C‐g2 (PLC‐g2). These events in turn lead to the activation of additional PTKs, as well as serine/threonine kinases, such as mitogen‐activated protein kinases (MAPKs). Collectively these activation pathways orchestrate events critical for the function of activated cells, including restructuring of the cytoskeleton and initiation of de novo gene transcription. 7.2. CD22 Regulation of BCR Signal Transduction The approximately 140 amino acid cytoplasmic domains of hCD22 and mCD22 are highly conserved, including six tyrosine residues that are potential targets for phosphorylation following surface BCR or CD22 ligation (Torres et al., 1992; Wilson et al., 1991). The cytoplasmic domains of mCD22 and hCD22 contain two potential ITAM‐like regions (Leprince et al., 1993). In addition, the mCD22 cytoplasmic domain encodes three potential ITIMs, whereas hCD22 contains four ITIM motifs. Through these motifs, hCD22 and mCD22 provide binding sites for SHP1, PLC‐g2, phosphatidylinositol (PI) 3‐kinase, Grb2, Syk, Shc, and SHIP (Blasioli et al., 1999; Poe et al., 2000; Yohannan et al., 1999). That CD22 contains both positive and negative signaling elements may explain why both positive and negative signaling roles for CD22 are observed during in vitro experiments. Although it has become popular to categorize signaling molecules as either positive or negative regulators, this is likely to be an oversimplification because each of these molecules may influence signaling in a manner dictated by the context of the cell microenvironment, the activation or differentiation state of the cell, or by the assay utilized (Tedder, 1998). A positive signaling role for CD22 in B‐cell function is suggested by the presence of ITAM‐like regions that, when phosphorylated, serve as docking platforms for the SH2 domains of Syk kinase and Src‐family tyrosine kinases

(B) Reciprocal regulatory activities of CD19 and CD22. CD19 signal transduction and amplification of Lyn PTK activity (Fig. 4C) leads to CD22 phosphorylation and recruitment of Grb2/Shc/ SHIP. The activation and recruitment of these molecules to the plasma membrane allows Lyn to phosphorylate Btk that is recruited through its pleckstrin homology domain (PH) to bind PI(3,4,5) P3 generated from PI(4,5)P2 by PI 3‐kinase. PLC‐g2 recruitment to the membrane allows it to cleave PI(4,5)P2 and augment [Ca2þ]i responses. By contrast, SHIP bound to CD22 dephosphorylates PI(3,4,5)P3, and thereby downregulates the duration and intensity of [Ca2þ]i responses.

20

t h om a s f . t e d d er

ET AL.

such as Lyn (Law et al., 1996; Tuscano et al., 1996b). Lyn may initiate tyrosine phosphorylation of cross‐linked CD22 since low amounts of purified Lyn bind to non‐tyrosine‐phosphorylated peptides that span the distal ITAM or tyrosines 807 and 822 (Tuscano et al., 1996b). Studies with Lyn‐deficient mice have also concluded that Lyn is predominantly responsible for CD22 tyrosine phosphorylation following BCR or CD22 cross‐linking (Chan, Lowell, and DeFranco, 1998; Fujimoto et al., 2000). Consistent with a costimulatory role for CD22, positive effector molecules of B‐cell activation, PI 3‐kinase and PLC‐g2, also bind tyrosine‐phosphorylated CD22 via their SH2 domains (Law et al., 1996; Leprince et al., 1993; Tuscano et al., 1996b). Syk, Lyn, and PI 3‐kinase associate with a tyrosine‐phosphorylated peptide that spans amino acid residues 822 and 842 of hCD22 (Tuscano et al., 1996b), implicating this site as an important region in mediating CD22 signal‐transduction. Thus, CD22 binding its physiologic ligands may augment B‐cell activation through the generation of positive signals. CD22 may actually contribute significantly to the earliest signaling cascades that occur during B‐cell activation since CD22 is rapidly tyrosine phosphorylated after B‐cell activation (Schulte et al., 1992). CD22 phosphorylation by Lyn may facilitate Syk and PLC‐g2 binding. This would allow for PLC‐g2 phosphorylation by Syk, and localization of activated PLC‐g2 to the plasma membrane where its phosphoinositide substrates are present (Law et al., 1996). Thus, under different experimental conditions or in vivo, interactions between CD22, IgM, Syk, Lyn, PI 3‐kinase, and PLC‐g2 may result in proliferative signals that surpass the downregulatory effects of CD22. Consistent with a costimulatory role for CD22, in vitro CD22 cross‐linking activates the c‐Jun NH2‐terminal kinase (JNK, also known as the stress‐activated protein kinase, or SAPK) signaling cascade. Significant SAPK activation is induced after cross‐linking CD22 on primary B cells and several B‐cell lines (Tuscano et al., 1999). CD22 has the ability to generate these BCR‐independent transmembrane signals since the HS Sultan plasmacytoma cell line used as part of these studies lacks surface Ig. Co‐cross‐linking surface Ig with CD22 fails to significantly alter this effect. This is consistent with CD22 cross‐linking leading to significant SAPK activation on splenic B cells (Tooze et al., 1997). In that study, the activity of extracellular signal–regulated kinase‐2 (ERK‐2), the MAPK isoform which is preferentially activated after Ig cross‐linking, was also modulated by CD22. A modest increase in ERK‐2 activity is observed after CD22 cross‐linking alone in our studies as well (Tuscano et al., 1999). However, a modest enhancement and significant prolongation of ERK‐2 activation after CD22/BCR coligation was observed in our studies. This is consistent with CD22 generating BCR‐independent signals, which could enhance and prolong BCR‐mediated ERK‐2 activation.

c d2 2 l i g a n d b i n d i n g r e gu l at e s b - c e ll su rv i va l

21

The extensive CD22 cytoplasmic domain also serves as an interaction site for numerous negative regulatory molecules including SHP1 and SHIP, phosphatases that modify BCR‐mediated signaling, and BCR‐induced [Ca2þ]i release. All three mCD22 ITIMs provide docking sites for SH2 domains of the SHP1 protein tyrosine phosphatase (Doody et al., 1995). Following BCR cross‐linking, SHP1 associates with tyrosine‐phosphorylated CD22, inducing SHP1 phosphatase activity (Campbell and Klinman, 1995; Doody et al., 1995; Lankester et al., 1995; Law et al., 1996). In fact, CD22 and SHP1 associate in both resting and stimulated mouse splenic B cells and CD22 is the dominant phosphoprotein that associates with SHP1 (Sato et al., 1997). In addition, CD22 suppresses MAPK activation in mouse B cells when cross‐linked to membrane Ig (Tooze, et al., 1997). CD22 may also attenuate calcium signaling by potentiating plasma membrane calcium‐ATPase (PMCA) activity through a SHP1‐dependent pathway in a chicken B‐cell line (Chen et al., 2004). Interestingly, CD22 and SHP1 associate directly with PMCA4 through phosphorylation‐dependent interactions. Thus, PMCA‐mediated calcium efflux from cells may also act as a negative regulator of B‐cell signaling. These findings reinforce the predominant concept that CD22 associated with SHP1 plays a negative regulatory role in B‐cell signaling. CD22 also physically interacts with SHIP via Grb2/Shc interactions (Poe et al., 2000). SHIP independently forms complexes with the adapter proteins Grb2 and Shc (Chacko et al., 1996; Crowley et al., 1996; Harmer and DeFranco, 1998; Kavanaugh et al., 1996; Saxton et al., 1994), with Grb2 required for the efficient association of Shc and SHIP (Harmer and DeFranco, 1998). The SHIP/Grb2/Shc ternary complex has been proposed to modulate [Ca2þ]i responses and to be essential for preventing B‐cell hyperresponsiveness following BCR ligation (Harmer and DeFranco, 1998). Grb2 and Shc interact with distinct regions of CD22; Grb2 specifically interacts with a peptide containing CD22 phosphotyrosines 799/810, whereas Shc only interacts with peptides containing phosphotyrosines 755/765 and 810/825. By contrast, SHIP, Grb2, and Shc are only coprecipitated with a GST fusion protein consisting of the entire tyrosine‐phosphorylated CD22 cytoplasmic domain (GST‐CD22cyt [pTyr]), but not by any of the individual phosphopeptides. Therefore, it is likely that the interaction of SHIP and CD22 is indirect via Grb2 and Shc (Fig. 5B), and that both adapter proteins are required to form a stable Grb2/ Shc/SHIP complex (Harmer and DeFranco, 1998). That all three SHIP isoforms coprecipitate with GST‐CD22cyt further indicates that SHIP interactions with the CD22 cytoplasmic domain are indirect, and therefore likely to occur via Shc and/or Grb2 binding of SHIP phosphotyrosines. SHIP processes phosphatidylinositol 3,4,5‐trisphosphate (PI(3,4,5)P3) into the phosphatidylinositol 3,4‐bisphosphate (PI(3,4)P2) cleavage product (Fig. 5B). PI(3,4,5)P3 is

t h om a s f . t e d d er

22

ET AL.

a membrane phospholipid generated by PI 3‐kinase phosphorylation of phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P2) that is involved in the membrane recruitment of pleckstrin homology (PH) domain‐containing proteins such as Btk, a PTK that activates PLC‐g2 (Fig. 5B). PI(3,4,5)P3 is generated by PI 3‐ kinase phosphorylation of phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P2). PLC‐g2 activation results in PI(4,5)P2 hydrolysis, with the subsequent generation of inositol 1,4,5‐trisphosphate (InsP3) that mobilizes [Ca2þ]i from intracellular stores and diacylglycerol (DAG) that activates protein kinase C. By PI (3,4,5)P3 dephosphorylation, SHIP is able to reduce PI 3‐kinase‐initiated Akt phosphorylation and the membrane recruitment of Btk through its PH domain (Vanhaesebroeck et al., 2001). Thereby, the 50 ‐phosphatase activity of SHIP on PI(3,4,5)P3 negatively regulates PI 3‐kinase signaling in vivo. Thus, the CD22/SHIP/Grb2/Shc complex may facilitate SHIP recruitment to its plasma membrane substrates where it can modulate B‐cell [Ca2þ]i responses. Recently, CD22 was reported to regulate BCR signaling depending on the cell surface Ig isotype expressed (Wakabayashi et al., 2002). Cell lines expressing IgM were subject to CD22‐mediated regulation, which included inhibition of [Ca2þ]i mobilization and ERK activation following BCR cross‐linking, whereas cell lines that expressed a BCR with an IgG cytoplasmic tail were not subject to CD22‐mediated regulation. Interestingly, BCR ligation of cells expressing an IgG cytoplasmic tail did not induce CD22 tyrosine phosphorylation or subsequent SHP1 recruitment. This suggests that regulation of BCR signaling by CD22 may not be operative in class‐switched B‐cell blasts and memory B cells. Whether CD22 ligand binding alternatively continues to regulate the survival and additional effector functions of these differentiated populations remains to be determined. 7.3. Altered Signal Transduction Pathways in Primary CD22 /

/

B Cells

Biochemical studies of CD22 B cells have provided new insight into the effects of CD22 loss on BCR signaling (Sato et al., 1997). Although unexpected, overall tyrosine phosphorylation of cellular proteins in resting B cells from CD22 / mice is lower than in purified B cells from wild‐type littermates when assessed by Western blot analysis of total cell lysates or immunoprecipitated proteins with antiphosphotyrosine mAbs. Interestingly, CD19 and Vav are the only specific signaling molecules identified thus far that are more intensely tyrosine‐phosphorylated in CD22 / B cells (Sato et al., 1997). Vav, a hematopoietic cell‐specific cytoplasmic adapter protein and proto‐oncogene product, is rapidly phosphorylated following BCR ligation and is likely to be a central component of CD19 regulation of B‐cell signal transduction (Bustelo and Barbacid, 1992; Deckert et al., 1996; Fujimoto et al., 1998). Tyrosine

c d2 2 l i g a n d b i n d i n g r e gu l at e s b - c e ll su rv i va l

23

phosphorylation of Vav directly and positively modulates its guanine‐nucleotide‐exchanging activity for Rac‐1 (Crespo et al., 1997). However, basal tyrosine phosphorylation of CD79a, CD79b, Lyn, Fyn, Blk, Syk, SHP1, SHIP, and PLC‐g2 are unchanged in CD22 / B cells compared to wild‐type B cells. However, BCR‐induced tyrosine phosphorylation of CD79a, CD79b, SHIP, and PLC‐g2 are particularly lower in CD22 / B cells. Decreased CD79a and CD79b tyrosine phosphorylation does not result from reduced activation of Syk or the Src‐family PTKs (Lyn, Fyn, Blk) because their patterns of tyrosine phosphorylation and activities during in vitro kinase assays are similar in CD22 / and wild‐type B cells. MAPK activation has also been assessed in CD22 / B cells (Poe et al., 2000), demonstrating a specific reduction in JNK activity, while ERK activation is normal. Thus, eliminating CD22 expression has significant but unexpected effects on multiple signaling pathways. Although the molecular explanation for the decrease in overall protein phosphorylation in CD22 / B cells is yet to be identified, this does not readily explain their augmented [Ca2þ]i responses following BCR engagement. 7.4. A CD19/CD22 Regulatory Loop Governs [Ca2þ]i Responses Rapid CD22 tyrosine phosphorylation following BCR ligation suggests that these two receptors are functionally linked (Leprince et al., 1993; Schulte et al., 1992; Tuscano et al., 1996b). However, more recent studies indicate that CD19 and CD22 form a regulatory loop and modulate each other’s functions directly, and thereby regulate BCR signal transduction indirectly (Fujimoto et al., 1999a). In support of this, the phenotypes of CD22 / , CD19 / , and CD19‐overexpressing mice indicate that CD19 and CD22 reciprocally regulate similar, if not overlapping, signaling pathways (Tedder et al., 1997a). CD22 / mice have increased numbers of B‐1 cells and functional properties remarkably similar to mice that overexpress CD19 (O’Keefe et al., 1996; Sato et al., 1996a). In addition, B cells from CD22 / ‐ and CD19‐overexpressing mice are hyperresponsive to BCR signaling. By contrast, CD19 / B cells are hyporesponsive to mitogens and BCR ligation, and CD19 / mice have a marked loss of peritoneal B‐1 cells (Engel et al., 1995b; Rickert et al., 1995; Sato et al., 1996b; Tarakhovsky et al., 1995; Zhang et al., 1995). Biochemical studies also indicate that CD19 and CD22 reciprocally regulate BCR signal transduction. For example, CD19 phosphorylation recruits Vav into the plasma membrane‐ bound CD19 signaling complex via SH2 interactions, which facilitates the subsequent phosphorylation of Vav by activated tyrosine kinases (Weng et al., 1994). Vav tyrosine phosphorylation is modest and transient after BCR ligation in CD19 / B cells, yet uniquely augmented after BCR cross‐linking in CD22 / B cells (Sato et al., 1997). Vav is most heavily tyrosine phosphorylated

24

t h om a s f . t e d d er

ET AL.

in response to CD19 ligation, with modest phosphorylation resulting from BCR and other signaling pathways (Bustelo and Barbacid, 1992; Collins et al., 1997; Sato et al., 1997). As another example, CD19 and CD22 have counterregulatory effects on MAPK activation (Tooze et al., 1997). Biochemical studies also demonstrate that CD19 and CD22 form a regulatory loop and modulate each other’s functions directly (Fig. 5A), and regulate BCR signal transduction indirectly (Fujimoto et al., 1999a). First, CD22 tyrosine phosphorylation is greatly diminished in CD19 / B cells following BCR cross‐linking. Central to this process is the recent demonstration that CD19 regulates intrinsic and BCR‐induced Lyn kinase activity through a unique mechanism of ‘‘processive amplification’’ (Fujimoto et al., 2000). Thereby, CD19 expression and amplification of Lyn kinase activity is required for optimal CD22 phosphorylation after BCR ligation (Fujimoto et al., 1999a; Sato et al., 1997). Decreased CD22 phosphorylation in CD19 / B cells reduces the amount of associated SHP1, thereby reducing the negative regulatory effects of CD22. Thus, CD19 expression has a crucial role for initiating the CD22/SHP1/SHIP negative regulatory pathway (Fig. 5A). Surprisingly, CD19 is a major target of CD22 inhibitory signals rather than BCR‐proximal signaling pathways. For example, Syk phosphorylation and function are normal in CD22 / B cells (Fujimoto et al., 1999b). By contrast, intrinsic CD19 tyrosine phosphorylation is constitutively higher in CD22 / B cells and is increased significantly in CD22 / B cells after BCR ligation relative to wild‐ type B cells (Fujimoto et al., 1999a). CD22 presumably regulates CD19 phosphorylation directly through recruited SHP1 (Somani et al., 2001). CD19 expression upregulates tyrosine phosphorylation of CD22 and Vav following BCR cross‐linking, while CD22 most likely regulates BCR signaling indirectly by regulating CD19 phosphorylation and CD19‐mediated Lyn amplification (Sato et al., 1997). CD22 regulation of CD19 function is critically important for normal B‐cell function because a 20% increase in CD19 expression (and presumably function) confers autoimmunity in mice and potentially in humans (Sato et al., 2000; Tedder et al., 2005). Functional CD19 and CD22 interactions have also been assessed genetically through the generation of CD19/CD22 double‐deficient (CD19/CD22 / ) mice (Fujimoto et al., 1999a). The characteristics of CD19/CD22 / mice clearly support the hypothesis that CD19 expression is necessary for CD22 function after BCR ligation (Fujimoto et al., 1999a). CD19‐deficiency is dominant in CD19/CD22 / mice, rather than CD19 and CD22 deficiencies having additive effects. Indeed, CD19/CD22 / mice show a remarkably similar phenotype to CD19 / mice. For example, tyrosine phosphorylation is decreased to similar levels in both CD19 / and CD19/CD22 / B cells. Serum Ig levels are identical in CD19 / and CD19/CD22 / mice, as are

c d2 2 l i g a n d b i n d i n g r e gu l at e s b - c e ll su rv i va l

25

antibody responses to T‐dependent Ags, despite increased IgM levels and elevated IgM responses in CD22 / mice. Spleen B‐cell numbers are similarly reduced in CD19 / and CD19/CD22 / mice. As occurs in CD19 / mice, peritoneal B‐1 lineage cells are rare in CD19/CD22 / mice, despite increased B‐1 cells in CD22 / mice (O’Keefe et al., 1996; Sato et al., 1996a). BCR‐ induced tyrosine phosphorylation of total cellular proteins is equally impaired in CD19/CD22 / and CD19 / B cells. Lyn kinase activity is only modestly increased in CD19 / and CD19/CD22 / B cells after BCR ligation, while Vav tyrosine phosphorylation is decreased to a similar extent in CD19/CD22 / and CD19 / B cells. In addition, proliferative responses to mitogens are minimal in both CD19/CD22 / and CD19 / B cells. Thus, CD19 expression is necessary for optimal CD22 function. In some specific instances, CD19 and CD22 functions were additive rather than affecting identical signaling pathways in CD19/CD22 / mice (Fujimoto et al., 1999a). For example, CD22 expression influences circulating B‐cell numbers, regardless of CD19 expression. Although CD19 loss results in higher IgM expression by immature and mature B cells, CD22 loss results in lower IgM expression by more mature B cells. However, CD19/CD22 / B cells have high IgM expression early and low IgM expression later during maturation. We propose that the above events are regulated by CD22 ligand binding and may be independent of the CD19 signaling pathway, while many of the signaling events that proceed through CD19‐dependent pathways are regulated by CD22 expression independent of ligand engagement. Thus, CD19 and CD22 are likely to interact through independent pathways in addition to contributing to a common CD19/CD22 regulatory loop. 7.5. CD19/CD21 Extinction of CD22 Phosphorylation Augments [Ca2þ]i Responses A prominent feature of CD22 / mice is that their B cells exhibit dramatically increased [Ca2þ]i responses following BCR cross‐linking (Nitschke et al., 1997; O’Keefe et al., 1996; Otipoby et al., 1996; Sato et al., 1996a). Similar observations have been made in variants of the WEHI‐231 pre‐B‐cell line with diminished CD22 expression (Nadler et al., 1997). This feature appears specific to BCR signaling since significantly increased [Ca2þ]i responses are not observed following CD19 cross‐linking (Sato et al., 1996a). However, the mechanisms by which CD22 expression affects BCR‐induced [Ca2þ]i responses appear complex because multiple known and unknown signaling molecules regulate [Ca2þ]i mobilization including Syk, PI 3‐kinase, PLC‐g2, SHIP, BLNK, and Btk (Buhl et al., 1997; Hippen et al., 1997; Scharenberg and Kinet, 1996; Takata and Kurosaki, 1996).

26

t h om a s f . t e d d er

ET AL.

Because Syk is likely to regulate tyrosine phosphorylation of PLC‐g2 directly (Takata et al., 1994) and Syk phosphorylation and function are normal in CD22 / B cells (Fujimoto et al., 1999b), CD22 is unlikely to regulate [Ca2þ]i responses at the level of a BCR/Syk/PLC‐g2 pathway. Consistent with this, PLC‐g2 tyrosine phosphorylation is actually decreased in CD22 / B cells. Altered [Ca2þ]i responses could result from the failure of SHP1 to associate with CD22, given that exaggerated [Ca2þ]i responses are observed in SHP1‐defective B cells (Cyster and Goodnow, 1995). Decreased SHIP phosphorylation in CD22 / B cells may also explain augmented [Ca2þ]i responses, given that activated SHIP blocks BCR‐induced [Ca2þ]i responses (Scharenberg and Kinet, 1996). Like in CD22 / B cells, [Ca2þ]i responses are augmented in a SHIP‐deficient B‐cell line following BCR ligation (Okada et al., 1998), and B cells from SHIP / mice generate enhanced [Ca2þ]i responses following BCR‐FcgRIIB coligation (Liu et al., 1998). These findings support the notion that the normal function of the CD22/SHIP/Grb2/Shc complex is to downregulate BCR‐induced [Ca2þ]i responses (Fig. 5B) in conjunction with CD22 recruitment of SHP1 to its CD19 substrate on the plasma membrane (Fig. 5A). Studies with CD19/CD22 / B cells suggest that CD22 and CD19 actually influence [Ca2þ]i responses in an interdependent manner that can explain augmented [Ca2þ]i responses in CD22 / B cells (Fujimoto et al., 1999a). CD19 / B‐cell [Ca2þ]i responses are delayed and slightly diminished following IgM ligation compared with wild‐type B cells, while CD22 / B cells exhibit dramatically increased [Ca2þ]i responses. [Ca2þ]i responses are greatly increased in CD19/CD22 / B cells compared with wild‐type B cells, and appear to mimic the combined effects of CD19 and CD22 deficiencies. Consistent with this, simultaneous CD19 and IgM engagement generate [Ca2þ]i responses similar to those of CD22 / and Lyn / B cells (Fig. 4A). This suggests that simultaneous CD19 and IgM engagement circumvents the negative regulatory effects of CD22. Recent studies have verified this and revealed that the augmented [Ca2þ]i responses induced following simultaneous BCR and CD19 ligation result from CD19 extinction of CD22 phosphorylation (Fujimoto et al., 2001). Surprisingly, CD19 engagement with mAb at concentrations that induce optimal [Ca2þ]i responses with simultaneous BCR engagement does not augment BCR‐induced protein tyrosine phosphorylation in B cells, but actually reduces overall phosphorylation levels compared with IgM ligation alone (Fig. 4B). This treatment leads to increased CD19 phosphorylation and higher levels of Lyn bound to CD19, while CD22 phosphorylation and SHP1 binding to CD22 are significantly inhibited (Fig. 4B). This suggests a new model for CD19/CD22 regulation of [Ca2þ]i responses (Fig. 4C).

c d2 2 l i g a n d b i n d i n g r e gu l at e s b - c e ll su rv i va l

27

CD19/CD22 regulation of [Ca2þ]i may occur through Lyn retention by CD19, which prevents Lyn from phosphorylating CD22 (Fujimoto et al., 2001). CD19/CD21 complex ligation via C3d‐Ag complexes or mAb engagement generates transmembrane signals that induce Lyn recruitment and amplification by CD19 (Fig. 4C). Phosphorylated CD19 binds PI 3‐kinase (Tuscano et al., 1996b; Yohannan et al., 1999), which may also contribute to efficient [Ca2þ]i mobilization by generating PIP3 to recruit Btk to the membrane (Bolland et al., 1998; Buhl and Cambier, 1999; Li et al., 1997a,b; Scharenberg et al., 1998). However, CD19 expression is not necessary for optimal PI 3‐kinase activation (Fujimoto et al., 2000). When CD19 and cell surface IgM are engaged simultaneously, Lyn activated by BCR engagement is further amplified through CD19 ‘‘processive amplification,’’ which results in enhanced [Ca2þ]i responses. We propose that Lyn recruitment to CD19 under these conditions extinguishes the ability of Lyn to phosphorylate CD22, which also enhances [Ca2þ]i responses. By contrast, CD19 hypercross‐linking with high levels of mAb (40 mg/ml) during simultaneous BCR ligation actually inhibits [Ca2þ]i responses to the levels seen with CD19 ligation alone (Fig. 4B). This suggests that CD19 hypercross‐linking not only sequesters Lyn away from CD22, but prevents Lyn phosphorylation of Syk downstream of BCR signaling (Fig. 4C). Thus, elevated [Ca2þ]I responses in CD22 / B cells may be explained, in part, by the absence of CD22 phosphorylation, which augments CD19 phosphorylation and inhibits the recruitment and phosphorylation of SHP1 and SHIP (Sato, Jansen, and Tedder, 1997). These results further confirm complex and overlapping interactions between CD19, CD22, Lyn, and SHP1 following BCR engagement (Fig. 5). 7.6. CD22 Regulates [Ca2þ]i Responses Independent of Ligand Binding Whether CD22 ligand binding regulates [Ca2þ]i responses has been a controversial matter. Results from experiments performed using cell lines have suggested that CD22 binding to sialic acid‐bearing ligands is required for CD22 to negatively influence BCR signal transduction and dampen self reactivity. In one study, CD22 expression suppressed B‐cell activation by Ag‐expressing target cells that coexpressed a2–6‐sialoglycoconjugates (Lanoue et al., 2002). In other studies, B‐cell lines with impaired CD22 ligand binding exhibited augmented Ca2þ responses following BCR engagement, which was proposed to result from decreased CD22 tyrosine phosphorylation and decreased SHP1 recruitment to CD22 (Jin et al., 2002; Kelm et al., 2002). By contrast, analysis of ST6Gal sialyltransferase gene‐deficient (ST6Gal I / ) mice, which fail to generate CD22 ligands, suggests that CD22 exerts stronger negative regulation of B‐cell signaling in the absence of ligand engagement

28

t h om a s f . t e d d er

ET AL.

(Collins et al., 2002; Hennet et al., 1998). However, primary CD22D1‐2 and CD22AA B cells have allowed us to directly assess the role ligand binding plays in CD22‐mediated regulation of BCR‐induced Ca2þ responses. Remarkably, CD22D1‐2 and CD22AA B cells flux Ca2þ at levels identical to B cells from their wild‐type littermates following treatment with both optimal and suboptimal BCR ligation (Poe et al., 2004a). Furthermore, CD22 tyrosine phosphorylation and its subsequent association with SHP1 are not impaired in CD22D1‐2 or CD22AA B cells following BCR ligation. Thus, CD22 expression at the B‐cell surface appears to be the only requirement for it to regulate BCR‐induced [Ca2þ]i responses, with no requirement for ligand binding. This is consistent with the model for CD22 extinction following BCR and CD19 ligation since the extracellular domain of CD22 does not need to be involved in this regulatory pathway (Fig. 4C). Nonetheless, although CD22 ligand engagement is not required for BCR‐induced [Ca2þ]i responses, this does not imply that ligand binding does not influence the ability of CD22 to regulate BCR signaling; however, this remains to be demonstrated. 8. CD22‐Directed Therapies and Clinical Trials in Oncology CD22 provides an important functional link between the cell surface, B‐cell survival, and regulation of intracellular signaling. Thus, it is not surprising that CD22‐directed therapies are being developed and tested clinically for both oncology and autoimmunity applications. Moreover, therapy for B‐cell malignancies and severe autoimmune disease has primarily relied upon broadly immunosuppressive agents such as cyclophosphamide, methotrexate, cyclosporin A, and corticosteroids. Although survival rates have improved dramatically, none of these therapies offers a cure and most have significant toxicities. With the advent of mAb‐based therapies, more specific and effective therapies with lower systemic toxicity are possible. Therapies directed at specifically reducing B‐cell numbers have recently acquired a great deal of attention and enthusiasm. For example, a chimerized CD20 mAb (Rituximab) effectively reduces normal and malignant B‐cell numbers without significant toxicity (Edwards and Cambridge, 2001; Leandro et al., 2002; Protheroe et al., 1999; Saleh et al., 2000; Stasi et al., 2001, 2002; Vita et al., 2002). It is therefore being used widely in human clinical trials for patients with lymphoma or autoimmune disease. Rituximab functions by binding the membrane‐embedded CD20 surface molecule on B cells, leading to B‐cell elimination through antibody‐dependent cellular cytotoxicity mediated by monocytes (Clynes et al., 2000; Hamaguchi et al., 2005; Uchida et al., 2004). Despite the effectiveness of Rituximab and its low toxicity, new therapies evoking different

c d2 2 l i g a n d b i n d i n g r e gu l at e s b - c e ll su rv i va l

29

mechanisms of action with more selective effects on B‐cell subpopulations are needed. 8.1. CD22 Therapeutic Applications in Oncology CD22 is expressed by 60–80% of B‐cell lymphomas and leukemias, although expression density is more variable than for CD19 or CD20 (Press et al., 2001). In addition to chemotherapy, external‐beam radiation and naked mAb therapies, radioimmunotherapy is becoming a fourth mode of treatment for non‐Hodgkin’s lymphoma patients. Radiolabeled CD22 mAbs (Stein et al., 1993) have demonstrated high sensitivities in the diagnosis and the staging of B‐cell lymphomas, while therapeutic 131I‐ or 90Y‐labeled CD22 mAbs (LL2, Epratuzumab) that bind to the third extracellular Ig‐like domain of CD22 have generated partial to complete remissions (Coleman et al., 2003; Juweid et al., 1999; Siegel et al., 2003). Naked CD22 mAb (LL2) mediates antibody‐ and complement‐dependent cytotoxicity in vitro, with preclinical and early clinical studies providing a rationale for its use in immunotherapy (Carnahan et al., 2003; Leonard and Link, 2002). Because malignant B cells rapidly internalize CD22 and CD22 mAb binding leads to efficient intracellular routing of the CD22‐mAb complex to intracellular compartments (Press et al., 1994), the use of CD22 mAbs to deliver immunotoxins to non‐Hodgkin’s lymphoma and chronic lymphocytic leukemia cells has received considerable attention (Press et al., 1989; Sieber et al., 2003). This includes CD22 immunotoxins containing ricin A‐chain (Ghetie et al., 1988; 1992; 1994; Shen et al., 1988; Van Horssen et al., 1996), single‐chain ribosome‐inactivating proteins (Bolognesi et al., 1998; Bonardi et al., 1993; French et al., 1995b), Pseudomonas exotoxin‐A fragment (Decker et al., 2004; Kreitman et al., 1999; Mansfield et al., 1997a,b), and chemotherapeutic agents such as calicheamicin (DiJoseph et al., 2004, 2005). CD22 mAb‐immunotoxins containing ricin A‐chain (Amlot et al., 1993; Sausville et al., 1995; Vitetta et al., 1991) and ribosome‐inactivating proteins (Bonardi et al., 1992; French et al., 1995a, 1996) have been evaluated in phase I–II clinical trials, with partial or transient clinical remissions, potentially due to large tumor burdens in treated patients. Despite these clinical responses, severe and in certain cases fatal vascular leak syndrome was dose limiting. However, a more recent clinical trial using a recombinant CD22‐Pseudomonas exotoxin‐based immunotoxin induced complete remissions in 68% of chemorefractory hairy‐cell leukemia patients (Kreitman et al., 2001). Thus, multiple studies have validated CD22 as a rational target for treating B‐cell malignancies.

30

t h om a s f . t e d d er

ET AL.

8.2. Is Blocking CD22 Ligand Binding Therapeutic in Oncology? The importance of CD22 ligand binding for normal B‐cell survival suggests that blocking this interaction may influence malignant B‐cell survival. In addition, CD22 mAbs that block CD22‐ligand interactions have distinct functional properties during in vitro assays with B‐cell lymphoma cell lines. For example, binding of CD22 with these mAbs results in a 3‐ to 5‐fold induction of SAPK activity, and efficient and effective induction of apoptosis (Tuscano et al., 1999). That ligand‐blocking mAbs would have potent in vivo effects on lymphoma growth was validated using a Raji lymphoma xenograft model (Tuscano et al., 2003) and the HB22‐7 mAb (Engel et al., 1993). These Raji xenograft studies also assessed whether blocking CD22 ligand binding would be additive or synergistic when combined with radioimmunotherapy to enhance apoptosis induced by low dose–rate radiation. Surprisingly, mice treated with naked HB22‐7 mAb alone had impressive tumor volume reductions 14 days after treatment, with superior cure and survival rates when compared to other treatment groups. The combination of HB22‐7 mAb with radioimmunotherapy also significantly enhanced the magnitude of tumor volume responses if HB22‐7 mAb was given simultaneously or 24 hours after radioimmunotherapy, with superior response rates and survival. Based on these preclinical studies, the National Cancer Institute has humanized the HB22‐7 mAb, with lymphoma clinical trials scheduled to start in 2006. In addition, we have generated mouse anti‐mouse CD22 mAbs that block ligand binding (unpublished). In early preclinical studies, mAbs that block CD22 ligand binding also appear effective in inhibiting syngeneic tumor growth in mice. These findings complement the findings obtained with normal B cells and further suggest that both normal and malignant B cells require CD22 ligand binding for survival. 9. CD22-Directed Therapies and Clinical Trials in Autoimmunity Autoimmune diseases represent complex disorders characterized by adaptive immune responses that are inappropriately directed against self‐Ags. Despite this complexity, B cells are important contributors in the pathogenesis of autoimmunity, beyond their ability to produce autoantibodies (Chan et al., 1999; Chan et al., 1999; Madaio, 1998; Tuscano et al., 2003). This has provided new incentives to identify the multiple checkpoints that control both positive and negative selection of B cells, both centrally in the bone marrow and in peripheral lymphoid tissues. This complex series of checks and balances is necessary to allow the production of a large and diverse population of B cells capable of generating high affinity effector antibodies that have been purged of pathological autoreactivity. As a result, perturbations in select regulatory

c d2 2 l i g a n d b i n d i n g r e gu l at e s b - c e ll su rv i va l

31

pathways that affect B‐cell function or selection may lead to autoimmune disease. Two broad categories of defects that lead to autoimmunity have been identified: those that alter signal transduction thresholds for cellular activation and those that alter B‐cell longevity. Given that CD22 regulates both signal transduction thresholds and B‐cell longevity, it is not surprising that CD22 has been implicated in the development of autoimmunity, at least in mouse models of disease. 9.1. Altered B‐Cell Activation and Survival Can Lead to Autoimmunity Because the generation and maintenance of self‐reactive B cells is regulated in part by signaling through the BCR complex, defective activation pathways that regulate BCR signaling can predispose to autoimmunity (Tedder, 1998). Consequently, peripheral tolerance is disrupted in mice that overexpress CD19 (Engel et al., 1995b), which results in hyperactive B cells and the spontaneous production of IgG subclass autoantibodies (Inaoki et al., 1997; Sato et al., 1996b). In fact, mice that have only a 15–30% increase in CD19 expression have a profoundly different phenotype from normal controls, developing autoimmune disease‐like manifestations in normally nonautoimmune strains of mice (Sato et al., 2000). Surprisingly, CD19 expression levels are also 20% higher on B cells from systemic sclerosis (SSc) patients compared with healthy individuals (Sato et al., 2000). Autoantibodies are detected in more than 90% of SSc patients and are considered to play a critical role in SSc pathogenesis (Okano, 1996). Like mice that overexpress CD19, the tight‐skin mouse, a genetic model for human SSc, also contains spontaneously activated B cells and autoantibodies against SSc‐specific auto‐Ags (Saito et al., 2002). CD19‐deficiency in tight‐skin mice results in quiescent B cells, with significantly reduced autoantibody production and skin fibrosis (Saito et al., 2002). Thus, modest alterations in CD19 or CD22 function could contribute to autoantibody development in humans. B cells from mice expressing a hypomorphic CD21 molecule or CD21 / mice express 30–50% increased densities of cell surface CD19 (Haas et al., 2002; Hasegawa et al., 2001). Multiple studies have suggested that altered CD21 function correlates with autoimmunity in mouse models (Boackle et al., 2001; Prodeus et al., 1998). Moreover, subtle alterations in the expression or function of other regulatory molecules involved in the CD19 signal transduction pathway may also predetermine autoimmunity susceptibility. Lyn‐deficient mice exhibit glomerulonephritis due to the presence of autoantibody immune complexes (Hibbs et al., 1995; Nishizumi et al., 1995). Moth‐eaten viable mice with catalytically compromised SHP1 (Kozlowski et al., 1993; Schultz et al., 1993) also demonstrate elevated levels of spontaneous autoantibodies,

32

t h om a s f . t e d d er

ET AL.

hypergammaglobulinemia, and tissue deposition of immune complexes (Tsui et al., 1993). Thus, mice with altered CD22, CD19, CD21, Lyn, or SHP1 expression are predisposed to produce autoantibodies and develop autoimmunity to varying degrees. Although speculative, it is possible that graded alterations in expression or function in these or other ‘‘response‐regulators’’ may result in the spectra of autoantibody specificities that characterize different autoimmune diseases. These findings suggest that antagonizing B‐cell signal transduction is a valid therapeutic strategy for patients with autoimmune disease. Altered expression of factors that regulate B‐cell survival can also lead to autoimmunity. One of the first examples of dysregulated apoptotic regulatory genes leading to autoimmunity was transgenic expression of dysregulated Bcl‐2 in mice, which allowed the inappropriate survival of autoreactive B‐cell clones that normally are lost through negative selection and apoptosis (Strasser et al., 1991). Bcl‐2 transgenic mice develop anti‐nuclear antibodies and have glomerulonephritis due to immune complex deposition. MRL mice provide a second example where homozygous mutations in the Fas gene (MRLlpr/lpr), encoding a death‐inducing receptor required for normal regulation of lymphocyte life spans, leads to a spectrum of autoreactivity resembling that found in human systemic lupus erythematosus (SLE) and other autoimmune diseases (Theofilopoulos, 1992). B‐cell depletion in autoimmune MRL mice blocks the manifestations of nephritis such as glomerulonephritis due to immune complex deposition, and also blocks cellular infiltrates into the kidney and vasculitis (Shlomchik et al., 1994). Intrinsic defects in external signals can also allow autoreactive B cells to escape deletion. One such signal comes from cytokines that are particularly important in B‐cell growth, differentiation, and survival. BAFF (B‐cell Activating Factor; also known as BlyS, TALL‐1, THANK, and zTNF4) is a member of the TNF family of cytokines that is produced by neutrophils, dendritic cells, and macrophages (Mackay et al., 2005). It induces immature B‐cell survival and growth of mature B cells within peripheral lymphoid tissues. BAFF binds three receptors; BCMA (B‐cell maturation Ag), TACI (transmembrane activator and calcium‐modulator and cyclophilin ligand interacter), and BAFF‐ receptor. Through these receptors, BAFF acts as a potent costimulator for B‐cell survival when coupled with BCR ligation. BAFF ligation correlates with increased Bcl‐2 expression and increased activation of NF‐kB, both of which increase B‐cell survival. Mice transgenic for either BCMA or BAFF have SLE‐ like disease, with anti‐DNA antibodies, elevated serum IgM, vasculitis, and glomerulonephritis (Mackay et al., 2005). Moreover, BAFF expression is elevated in MRLlpr/lpr mice and (NZB X NZW) F1 hybrid mice and correlates with disease progression in both models of autoimmunity (Gross et al., 2000).

c d2 2 l i g a n d b i n d i n g r e gu l at e s b - c e ll su rv i va l

33

Elevated BAFF levels are also found in a significant number of SLE patients. These findings suggest that antagonizing B‐cell survival is a valid therapeutic strategy for patients with autoimmune disease. 9.2. Potential Role for CD22 in Autoimmunity In addition to regulating B‐cell apoptosis and normal BCR and CD19 signal transduction, defects in CD22 or its related signaling pathways contribute to the pathogenesis of abnormal B‐cell function. That the extracellular ligand‐ binding domains of CD22 are divergent in autoimmune strains of mice suggests that CD22 may directly influence autoantibody production or may contribute to the severity of autoimmune disorders (Mary et al., 2000). First, at least three alleles of the mCd22 gene have been identified that encode proteins that are 3% different in amino acid sequence (Law et al., 1993). One of these, the Cd22a allele, is uniquely found in some autoimmune‐prone strains of mice (DBA/1, DBA/2J, NZB, NZW, NZC, PL/J, and AKR/J) (Lajaunias et al., 1999; Nadler et al., 1997). The Lyb‐8.2 allele found in BALB/c and most other mouse strains is named Cd22b/Lyb‐8b and the DBA mouse allele is named Cd22a/Lyb‐8a (Law et al., 1993). Dramatic differences occur in the two amino‐terminal Ig domains encoded by these alleles; 16 amino acid substitutions are found as well as a 6 amino acid insertion in the first Ig domain of the Lyb‐8.2 allele. These changes explain the presence of the Lyb‐8.2 allele defined by the CY34 mAb that binds an epitope dependent on residues in both domains 1 and 2, while the NIM‐R6 mAb binds more membrane proximal Ig domains (Law et al., 1995; van der Merwe et al., 1996). One Thr‐to‐Met amino acid substitution occurs in the cytoplasmic domain, but none of the six Tyr residues conserved between hCD22 and mCD22 are affected. A third Cd22c/Lyb‐8c allele has been identified in BXSB mice which are lupus‐prone (Lajaunias et al., 1999). Furthermore, there are six gene sequence differences between BALB/c and C57BL/6 mice (Lajaunias et al., 1999; van der Merwe et al., 1996). CD22 in MRL mice is identical to CD22 of BALB/c mice with the exception of single amino acid changes in Ig domains 2 and 7, and the transmembrane domain, which are id entical to those found in C57BL/6 mice (Lajaunias et al., 1999). Nonetheless, the Cd22a allele present in multiple autoimmune‐prone strains of mice is likely to have been disseminated by a precursor strain that contributed to all of these lines. Although not proven, multiple investigators postulate that CD22 allelic differences contribute to autoimmunity. Consistent with this, the proximal region of chromosome 7 containing the Cd22 locus is implicated in the development of SLE‐like disease in NZM and NZW mice (Kono et al., 1994; Morel et al., 1994; Santiago et al., 1998). In addition, NZW

34

t h om a s f . t e d d er

ET AL.

mice synthesize aberrant CD22 mRNAs, some of which interfere with upregulated cell surface CD22 expression on NZW B cells following LPS exposure (Mary et al., 2000). Thus, CD22 genetic polymorphisms may contribute to autoimmunity. A second line of evidence that CD22 influences the development of autoimmunity is that one CD22 / mouse line has increased autoantibody production with age, including antibodies against double‐stranded (ds) DNA and dsDNA‐histone complexes (O’Keefe et al., 1996, 1999). Heterozygous CD22þ/ C57BL/6 mice carrying the autoimmune acceleration gene, Yaa, also have markedly increased production of IgG anti‐DNA autoantibodies (Mary et al., 2000). The hyperactivated status of CD22 / B cells is consistent with susceptibility to autoimmunity such that CD22 may augment autoimmune disease once induced or when combined with increasing age, other disease susceptibility loci, or pathogens. Alternatively, it has been proposed that CD22 ligand engagement prevents the development of autoimmunity (Lanoue et al., 2002). By contrast, our line of CD22 / mice develops hypergammaglobulinemia with age, with autoantibody levels merely paralleling increased serum Ig levels (unpublished observations). Thus, in this line of mice, CD22‐deficiency appears to result in overall B‐cell hyperactivity rather than preferentially supporting the expansion of autoreactive clones. Furthermore, estrogen administration, which accelerates and exacerbates autoimmunity in mice (Grimaldi et al., 2001) specifically upregulates CD22 and SHP1 levels in B cells (Grimaldi et al., 2002). The authors suggest that CD22 overexpression may reduce the occurrence of BCR‐induced apoptosis in autoreactive cells and promote their survival. Thus, there is evidence for CD22 deficiency as well as CD22 overexpression in influencing autoimmunity. A third line of evidence is that CD22 is the primary phosphoprotein associated with SHP1 in B cells (Sato et al., 1997). CD22 also regulates CD19 function. We have shown that augmented CD19 function induces autoimmunity in mice independent of its effects on the B‐1 population of B cells (Fujimoto et al., 1999a; Sato et al., 1996b). In fact, only a 20% increase in CD19 expression induces autoimmunity in mice with the onset and severity of autoimmunity linearly correlated with CD19 expression levels (Sato et al., 1996b, 2000) that correlates with a breakdown in B‐cell tolerance (Inaoki et al., 1997). Moreover, B cells from SHP1‐defective, CD22 / , and CD19‐ overexpressing mice are all hyperresponsive to activation signals (Pani et al., 1995) and have an IgMlo MHC class IIhi phenotype (Cyster and Goodnow, 1995). That autoimmunity and immune responses are regulated or ‘‘fine‐ tuned’’ by CD19‐dependent signal transduction pathways explains why subtle increases in CD19 expression or decreased CD22 function affects the onset

c d2 2 l i g a n d b i n d i n g r e gu l at e s b - c e ll su rv i va l

35

or development of autoimmunity (Sato et al., 1996b, 2000; Tedder et al., 1997a, 2000). Whether these principles apply to humans remains unknown because combinations of subtle genetic alterations are likely to dysregulate normal B‐ cell function and thereby confer susceptibility to autoimmune disease. Regardless, these studies provide sufficient justification to assess this possibility and to determine whether alterations in CD22‐regulated signaling pathways and the resulting defects in B‐cell survival contribute to or inhibit autoimmunity. 9.3. Potential Role for MZ B Cells in Autoimmunity MZ B cells represent a B‐cell population that is highly susceptible to alterations in CD22 expression and function (Poe et al., 2004a; Samardzic et al., 2002; Sato et al., 1996a). This is important because MZ B cells display a diverse repertoire of BCR specificities that bind T‐cell‐independent and T‐cell‐dependent Ags, as well as self‐Ags (Martin and Kearney, 2002). Indeed, self‐reactive MZ B‐cell clones are found in both normal (Chen et al., 1997) and autoimmune strains of mice (Wellmann et al., 2001). The MZ B‐cell population is expanded in (NZB X NZW) F1 mice prior to disease development (Wither et al., 2000) and transgenic mice overexpressing BAFF exhibit MZ B‐cell expansion (Batten et al., 2000; Mackay et al., 1999). Similarly, pristane administration causes lupus‐like symptoms in mice and also leads to MZ B‐cell expansion (Yang et al., 2003). MZ B cells can produce autoreactive antibodies either spontaneously or under activating conditions. CD1dhi MZ B cells from (NZB X NZW) F1 mice spontaneously produce high amounts of IgM anti‐ DNA Abs (Zeng et al., 2000). Furthermore, estrogen treatment results in the expansion and activation of autoreactive MZ B‐cell clones in anti‐DNA heavy chain transgenic BALB/c mice (Grimaldi et al., 2001). The expansion and activation of autoreactive MZ B cells may be an important observation for certain disease pathologies as salivary glands become infiltrated with B cells expressing a MZ B‐cell phenotype in autoimmune mice overexpressing BAFF (Groom et al., 2002). B cells with a MZ B‐cell phenotype are also found to infiltrate the thyroid in patients suffering from Graves’ disease (Segundo et al., 2001). Thus, there is considerable evidence that MZ B cells participate in the development and exacerbation of autoimmunity. Because altered CD22 expression or blocking CD22 ligand binding can dramatically attenuate MZ B‐cell numbers, CD22 may be an important therapeutic target for eliminating MZ B cells. The development of BAFF antagonists encompasses a similar rationale. Thus, CD22‐directed therapeutics may allow for the manipulation of this unique population such that their involvement in autoimmunity can be controlled.

36

t h om a s f . t e d d er

ET AL.

9.4. CD22 Therapeutic Applications in Autoimmunity The importance of B‐cell survival in vivo is well appreciated, leading to the pursuit of therapies that influence this potent biological outcome. By interfering with B‐cell survival and enhancing B‐cell turnover, it is envisioned that B cells or B‐cell subsets can be specifically targeted without the risk of eliminating bulk B‐cell populations as with CD20‐directed therapies. Moreover, it may be possible to effect therapeutic benefit by attenuating B‐ cell responses to transmembrane signals without eliminating B cells that give rise to humoral immunity. Although CD40–CD40 ligand (CD40L) interactions are critical for normal B‐ and T‐cell interactions and mAbs against these therapeutic targets have demonstrated efficacy in mice, human clinical trials targeting this pathway have been disappointing. In fact, two trials assessing anti‐CD40L mAbs have been abandoned due to either lack of efficacy or unexpected toxicity (Davis et al., 2001; Huang et al., 2002). Other potential targets for treating B‐cell‐mediated human autoimmune diseases include BAFF antagonists and BMCA‐Ig and TACI‐Ig fusion proteins as decoy BAFF receptors (Gross et al., 2001). Administration of BMCA‐Ig or TACI‐Ig to (NZB X NZW) F1 mice leads to increased survival, decreased proteinuria, and slowed disease progression. Recent trials of CTLA‐4‐Ig fusion proteins that disrupt T‐cell–B‐cell interactions and T‐cell activation have been promising in the treatment of rheumatoid arthritis, showing moderate efficacy with no evidence of significant toxicity (Kremer et al., 2003). Accelerated B‐cell apoptosis and turnover by therapeutic mAbs that block CD22 ligand engagement may also have considerable benefit for the treatment of autoimmunity (Tuscano, Harris, and Tedder, 2003). Thus, interfering with B‐cell survival represents a valid and exciting approach for the treatment of autoimmunity. 10. Conclusions CD22 acts as a multifunctional receptor by participating in ligand‐dependent and ligand‐independent pathways, which may function independently or converge to ultimately regulate the generation of physiologically relevant responses to foreign or self‐Ags. Although a considerable amount of new information is available regarding CD22 function, further knowledge is essential for understanding how CD22 governs events critical for B‐cell selection, activation, and differentiation during immune responses. Better understanding of CD22 function and regulation may provide mechanisms by which humoral immunity can be modulated, leading to the development of new strategies to augment antimicrobial defense and acquired immune responses, and the development of novel approaches for modulation of chronic infections and

c d2 2 l i g a n d b i n d i n g r e gu l at e s b - c e ll su rv i va l

37

inflammatory disorders. Because B cells contribute substantially to many human autoimmune diseases and the prevalence of hematologic malignancies, it is anticipated that new drugs targeting CD22 directly and B‐cell function in general will be developed. References Amlot, P. L., Stone, M. J., Cunningham, D., Fay, J., Newman, J., Collins, R., May, R., McCarthy, M., Richardson, J., and Ghetie, V. (1993). A phase I study of anti‐CD22‐deglycosylated ricin A chain immunotoxin in the treatment of B‐cell lymphomas resistant to conventional therapy. Blood 82, 2624–2633. Aruffo, A., Kanner, S. B., Sgroi, D., Ledbetter, J. A., and Stamenkovic, I. (1992). CD22‐mediated stimulation of T cells regulates T‐cell receptor/CD3‐induced signaling. Proc. Natl. Acad. Sci. USA 89, 10242–10246. Bast, B. J., Zhou, L.‐J., Freedman, G. J., Colley, K. J., Ernst, T. J., Munro, J. M., and Tedder, T. F. (1992). The HB‐6, CDw75, and CD76 differentiation antigens are unique cell‐surface carbohydrate determinants generated by the b‐galactoside a2,6‐sialyltransferase. J. Cell Biol. 116, 423–435. Batten, M., Groom, J., Cachero, T. G., Qian, F., Schneider, P., Tschopp, J., Browning, J. L., and Mackay, F. (2000). BAFF mediates survival of peripheral immature B lymphocytes. J. Exp. Med. 192, 1453–1466. Bell, S. E., and Goodnow, C. C. (1994). A selective defect in IgM antigen receptor synthesis and transport causes loss of cell surface IgM expression on tolerant B lymphocytes. EMBO J. 13, 816–826. Blasioli, J., Paust, S., and Thomas, M. L. (1999). Definition of the sites of interaction between the protein tyrosine phosphatase SHP‐1 and CD22. J. Biol. Chem. 274, 2303–2307. Boackle, S. A., Holers, V. M., Chen, X., Szakonyi, G., Karp, D. R., Wakeland, E. K., and Morel, L. (2001). Cr2, a candidate gene in the murine Sle1c lupus susceptibility locus, encodes a dysfunctional protein. Immunity 15, 775–785. Bolland, S., Pearse, R. N., Kurosaki, T., and Ravetch, J. V. (1998). SHIP modulates immune receptor responses by regulating membrane association of Btk. Immunity 8, 509–516. Bolognesi, A., Tazzari, P. L., Oliveri, F., Polito, L., Lemoli, R., Rerenzi, A., Pasqualucci, L., Falini, B., and Stirpe, F. (1998). Evaluation of immunotoxins containing single‐chain ribosome‐ inactivating proteins and an anti‐CD22 monoclonal antibody (OM124): In vitro and in vivo studies. Brit. J. Hematol. 101, 179–188. Bonardi, M. A., Bell, A., French, R. R., Gromo, G., Hamblin, T., Modena, D., Tutt, A. L., and Glennie, M. J. (1992). Initial experience in treating human lymphoma with a combination of bispecific antibody and saporin. Intl. J. Cancer—Supplement 7, 73–77. Bonardi, M. A., French, R. R., Amlot, P., Gromo, G., Modena, D., and Glennie, M. J. (1993). Delivery of saporin to human B‐cell lymphoma using bispecific antibody: Targeting via CD22 but not CD19, CD37, or immunoglobulin results in efficient killing. Cancer Res. 53, 3015–3021. Boue, D. R., and LeBien, T. W. (1988). Expression and structure of CD22 in acute leukemia. Blood 71, 1480–1486. Boue, D. R., and LeBien, T. W. (1988). Structural characterization of the human B lymphocyte‐ restricted differentiation antigen CD22. Comparison with CD21 (complement receptor type 2/Epstein Barr virus receptor). J. Immunol. 140, 192–199. Buhl, A. M., and Cambier, J. C. (1997). Co‐receptor and accessory regulation of B‐cell antigen receptor signal transduction. Immunol. Rev. 160, 127–138.

38

t h om a s f . t e d d er

ET AL.

Buhl, A. M., and Cambier, J. C. (1999). Phosphorylation of CD19 Y484 and Y515, and linked activation of phosphatidylinositol 3‐kinase, are required for B‐cell antigen receptor‐mediated activation of Bruton’s tyrosine kinase. J. Immunol. 162, 4438–4446. Buhl, A. M., Pleiman, C. M., Rickert, R. C., and Cambier, J. C. (1997). Qualitative regulation of B‐ cell antigen receptor signaling by CD19: Selective requirement for PI3‐kinase activation, inositol‐1,4,5‐trisphosphate production and Ca2þ mobilization. J. Exp. Med. 186, 1897–1910. Bustelo, X. R., and Barbacid, M. (1992). Tyrosine phosphorylation of the Vav proto‐oncogene product in activated B cells. Science 256, 1196–1199. Campbell, M. A., and Klinman, N. R. (1995). Phosphotyrosine‐dependent association between CD22 and protein tyrosine phosphatase 1C. Eur. J. Immunol. 25, 1573–1579. Carnahan, J., Wang, P., Kendall, R., Chen, C., Hu, S., Boone, T., Juan, T., Talvenheimo, J., Montestruque, S., Sun, J., Elliott, G., Thomas, J., Ferbas, J., Kern, B., Briddell, R., Leonard, J. P., and Cesano, A. (2003). Epratuzumab, a humanized monoclonal antibody targeting CD22: Characterization of in vitro properties. Clin. Cancer Res. 9, 3982S–3990S. Castigli, E., Alt, F. W., Davidson, L., Bottaro, A., Mizoguchi, E., Bhan, A. K., and Geha, R. S. (1994). CD40‐deficient mice generated by recombination‐activating gene‐2‐deficient blastocyst complementation. Proc. Natl. Acad. Sci. USA 91, 12135–12139. Chacko, G. W., Tridandapani, S., Damen, J. E., Liu, L., Krystal, G., and Coggeshall, K. M. (1996). Negative signaling in B lymphocytes induces tyrosine phosphorylation of the 145‐kDa inositol polyphosphate 5‐phosphatase, SHIP. J. Immunol. 157, 2234–2238. Chan, O. T., Hannum, L. G., Haberman, A. M., Madaio, M. P., and Shlomchik, M. J. (1999). A novel mouse with B cells but lacking serum antibody reveals an antibody‐independent role for B cells in murine lupus. J. Exp. Med. 189, 1639–1648. Chan, O. T., Madaio, M. P., and Shlomchik, M. J. (1999). The central and multiple roles of B cells in lupus pathogenesis. Immunol. Rev. 169, 107–121. Chan, V. W. F., Lowell, C. A., and DeFranco, A. L. (1998). Defective negative regulation of antigen receptor signaling in Lyn‐deficient B lymphocytes. Curr. Biol. 8, 545–553. Chaouchi, N., Vazquez, A., Galanaud, P., and Leprince, C. (1995). B‐cell antigen receptor‐ mediated apoptosis. Importance of accessory molecules CD19 and CD22, and of surface IgM cross‐linking. J. Immunol. 154, 3096–3104. Chen, J., McLean, P. A., Neel, B. G., Okunade, G., Shull, G. E., and Wortis, H. H. (2004). CD22 attenuates calcium signaling by potentiating plasma membrane calcium‐ATPase activity. Nat. Immunol. 5, 651–657. Chen, X., Martin, F., Forbush, K. A., Perlmutter, R. M., and Kearney, J. F. (1997). Evidence for selection of a population of multi‐reactive B cells into the splenic marginal zone. Int. Immunol. 9, 27–41. Clynes, R. A., Towers, T. L., Presta, L. G., and Ravetch, J. V. (2000). Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets. Nat. Med. 6, 443–446. Coleman, M., Goldenberg, D. M., Siegel, A. B., Ketas, J. C., Ashe, M., Fiore, J. M., and Leonard, J. P. (2003). Epratuzumab: Targeting B‐cell malignancies through CD22. Clin. Cancer Res. 9, 3991S–3994S. Collins, B. E., Blixt, O., Bovin, N. V., Danzer, C.‐P., Chui, D., Marth, J. D., Nitschke, L., and Paulson, J. C. (2002). Constitutively unmasked CD22 on B cells of ST6Gal I knockout mice: Novel sialoside probe for murine CD22. Glycobiology 12, 563–571. Collins, B. E., Blixt, O., De Sieno, A. R., Bovin, N., Marth, J. D., and Paulson, J. C. (2004). Masking of CD22 by cis ligands does not prevent redistribution of CD22 to sites of cell contact. Proc. Natl. Acad. Sci. USA 101, 6104–6109. Collins, T. L., Deckert, M., and Altman, A. (1997). Views on Vav. Immunol. Today 18, 221–225.

c d2 2 l i g a n d b i n d i n g r e gu l at e s b - c e ll su rv i va l

39

Crespo, P., Schuebel, K. E., Ostrom, A. A., Gutkind, J. S., and Bustelo, X. R. (1997). Phosphotyrosine‐dependent activation of Rac‐1 GDP/GTP exchange by the Vav proto‐oncogene product. Nature 385, 169–172. Crocker, P. R., Mucklow, S., Bouckson, V., McWilliam, A., Willis, A. C., Gordon, S., Milon, G., Kelm, S., and Bradfield, P. (1994). Sialoadhesin, a macrophage sialic acid‐binding receptor for haemopoietic cells with 17 immunoglobulin‐like domains. EMBO J. 13, 4490–4503. Crowley, M. T., Harmer, S. L., and DeFranco, A. L. (1996). Activation‐induced association of a 145‐kDa tyrosine‐phosphorylated protein with Shc and Syk in B lymphocytes and macrophages. J. Biol. Chem. 271, 1145–1152. Cyster, J. G., and Goodnow, C. C. (1995). Protein tyrosine phosphatase 1C negatively regulates antigen receptor signaling in B lymphocytes and determines thresholds for negative selection. Immunity 2, 13–24. Cyster, J. G., and Goodnow, C. C. (1997). Tuning antigen receptor signaling by CD22: Integrating cues from antigens and the microenvironment. Immunity 6, 509–517. Davis, J. C., Jr., Totoritis, M. C., Rosenberg, J., Sklenar, T. A., and Wofsy, D. (2001). Phase I clinical trial of a monoclonal antibody against CD40‐ligand (IDEC‐131) in patients with systemic lupus erythematosus. J. Rheumatol. 28, 95–101. Dealy, M. J., Nguyen, K. V., Lo, J., Gstaiger, M., Krek, W., Elson, D., Arbeit, J., Kipreos, E. T., and Johnson, R. S. (1999). Loss of Cul1 results in early embryonic lethality and dysregulation of cyclin E. Nat. Genet. 23, 245–248. Decker, T., Oelsner, M., Kreitman, R. J., Salvatore, G., Wang, Q. C., Pastan, I., Peschel, C., and Licht, T. (2004). Induction of caspase‐dependent programmed cell death in B‐cell chronic lymphocytic leukemia by anti‐CD22 immunotoxins. Blood 103, 2718–2726. Deckert, M., Tertare‐Deckert, S., Couture, C., Mustelin, T., and Altman, A. (1996). Functional and physical interactions of Syk family kinases with the Vav proto‐oncogene product. Immunity 5, 591–604. DeFranco, A. L., Chan, V. W. F., and Lowell, C. A. (1998). Positive and negative roles of the tyrosine kinase Lyn in B‐cell function. Semin. Immunol. 10, 299–308. DiJoseph, J. F., Armellino, D. C., Boghaert, E. R., Khandke, K., Dougher, M. M., Sridharan, L., Kunz, A., Hamann, P. R., Gorovits, B., Udata, C., Moran, J. K., Popplewell, A. G., Stephens, S., Frost, P., and Damle, N. K. (2004). Antibody‐targeted chemotherapy with CMC‐544: A CD22‐ targeted immunoconjugate of calicheamicin for the treatment of B‐lymphoid malignancies. Blood 103, 1807–1814. DiJoseph, J. F., Popplewell, A., Tickle, S., Ladyman, H., Lawson, A., Kunz, A., Khandke, K., Armellino, D. C., Boghaert, E. R., Hamann, P., Zinkewich‐Peotti, K., Stephens, S., Weir, N., and Damle, N. K. (2005). Antibody‐targeted chemotherapy of B‐cell lymphoma using calicheamicin conjugated to murine or humanized antibody against CD22. Cancer Immunol. Immunother. 54, 11–24. Doody, G. M., Justement, L. B., Delibrias, C. C., Mathews, R. J., Lin, J., Thomas, M. L., and Fearon, D. T. (1995). A role in B‐cell activation for CD22 and the protein tyrosine phosphatase SHP. Science 269, 242–244. Do¨ rken, B., Moldenhauer, G., Pezzutto, A., Schwartz, R., Feller, A., Kiesel, S., and Nadler, L. M. (1986). HD39 (B3), a B lineage‐restricted antigen whose cell surface expression is limited to resting and activated human B lymphocytes. J. Immunol. 136, 4470–4479. Edwards, J. C. W., and Cambridge, G. (2001). Sustained improvement in rheumatoid arthritis following a protocol designed to deplete B lymphocytes. Rheumatology 40, 1–7. Engel, P., Nojima, Y., Rothstein, D., Zhou, L.‐J., Wilson, G. L., Kehrl, J. H., and Tedder, T. F. (1993). The same epitope on CD22 of B lymphocytes mediates the adhesion of erythrocytes, T and B lymphocytes, neutrophils and monocytes. J. Immunol. 150, 4719–4732.

40

t h om a s f . t e d d er

ET AL.

Engel, P., Wagner, N., Miller, A., and Tedder, T. F. (1995). Identification of the ligand binding domains of CD22, a member of the immunoglobulin superfamily that uniquely binds a sialic acid‐dependent ligand. J. Exp. Med. 181, 1581–1586. Engel, P., Zhou, L.‐J., Ord, D. C., Sato, S., Koller, B., and Tedder, T. F. (1995). Abnormal B lymphocyte development, activation and differentiation in mice that lack or overexpress the CD19 signal transduction molecule. Immunity 3, 39–50. Erickson, L. D., Tygrett, L. T., Bhatia, S. K., Grabstein, K. H., and Waldschmidt, T. J. (1996). Differential expression of CD22 (Lyb8) on murine B cells. Int. Immunol. 8, 1121–1129. Floyd, H., Nitschke, L., and Crocker, P. R. (2000). A novel subset of murine B cells that expreses unmasked forms of CD22 is enriched in the bone marrow: implications for B‐cell homing to the bone marrow. Immunology 101, 342–347. French, R. R., Bell, A. J., Hamblin, T. J., Tutt, A. L., and Glennie, M. J. (1996). Response of B‐cell lymphoma to a combination of bispecific antibodies and saporin. Leuk. Res. 20, 607–617. French, R. R., Hamblin, T. J., Bell, A. J., Tutt, A. L., and Glennie, M. J. (1995). Treatment of B‐cell lymphomas with combination of bispecific antibodies and saporin. Lancet 346, 223–224. French, R. R., Penney, C. A., Browning, A. C., Stirpe, F., George, A. J., and Glennie, M. J. (1995). Delivery of the ribosome‐inactivating protein, gelonin, to lymphoma cells via CD22 and CD38 using bispecific antibodies. Brit. J. Cancer 71, 986–994. Fujimoto, M., Bradney, A. P., Poe, J. C., Steeber, D. A., and Tedder, T. F. (1999). Modulation of B lymphocyte antigen receptor signal transduction by a CD19/CD22 regulatory loop. Immunity 11, 191–200. Fujimoto, M., Fujimoto, Y., Poe, J. C., Jansen, P. J., Lowell, C. A., DeFranco, A. L., and Tedder, T. F. (2000). CD19 regulates Src‐family protein tyrosine kinase activation in B lymphocytes through processive amplification. Immunity 13, 47–57. Fujimoto, M., Poe, J. C., Hasegawa, M., and Tedder, T. F. (2001). CD19 amplification of B lymphocyte Ca2þ responses: A role for Lyn sequestration in extinguishing negative regulation. J. Biol. Chem. 276, 44820–44827. Fujimoto, M., Poe, J. C., Inaoki, M., and Tedder, T. F. (1998). CD19 regulates B lymphocyte responses to transmembrane signals. Semin. Immunol. 10, 267–277. Fujimoto, M., Poe, J. C., Jansen, P. J., Sato, S., and Tedder, T. F. (1999). CD19 amplifies B lymphocyte signal transduction by regulating Src‐family protein tyrosine kinase activation. J. Immunol. 162, 7088–7094. Ghetie, M.‐A., May, R. D., Till, M., Uhr, J. W., Ghetie, V., Knowles, P. P., Relf, M., Brown, A., Wallace, P. M., Janossy, G., Amlot, P., and Vitetta, E. S. (1988). Evaluation of ricin A chain‐ containing immunotoxins directed against CD19 and CD22 antigens on normal and malignant human B‐cells as potential reagents for in vivo therapy. Cancer Res. 48, 2610–2617. Ghetie, M.‐A., Picker, L. J., Richardson, J. A., Tucker, K., Uhr, J. W., and Vitetta, E. S. (1994). Anti‐CD19 inhibits the growth of human B‐cell tumor lines in vitro and of Daudi cells in SCID mice by inducing cell cycle arrest. Blood 83, 1329–1336. Ghetie, M.‐A., Tucker, K., Richardson, J., Uhr, J. W., and Vitetta, E. S. (1992). The antitumor activity of an anti‐CD22 immunotoxin in SCID mice with disseminated Daudi lymphoma is enhanced by either an anti‐CD19 antibody or an anti‐CD19 immunotoxin. Blood 80, 2315–2320. Goodnow, C. C., Crosbie, J., Adelstein, S., Lavoie, T. B., Smith‐Gill, S. J., Brink, R. A., Pritchard‐ Briscoe, H., Wotherspoon, J. S., Loblay, R. H., Raphael, K., Trent, R. J., and Basten, A. (1988). Altered immunoglobulin expression and functional silencing of self‐reactive B lymphocytes in transgenic mice. Nature 334, 676–682. Gordon, J. (1995). CD40 and its ligand: Central players in B lymphocyte survival, growth, and differentiation. Blood Rev. 9, 53–56.

c d2 2 l i g a n d b i n d i n g r e gu l at e s b - c e ll su rv i va l

41

Grimaldi, C. M., Cleary, J., Dagtas, A. S., Moussai, D., and Diamond, B. (2002). Estrogen alters thresholds for B‐cell apoptosis and activation. J. Clin. Invest. 109, 1625–1633. Grimaldi, C. M., Michael, D. J., and Diamond, B. (2001). Cutting Edge: Expansion and activation of a population of autoreactive marginal zone B cells in a model of estrogen‐induced lupus. J. Immunol. 167, 1886–1890. Groom, J., Kalled, S. L., Cutler, A. H., Olson, C., Woodcock, S. A., Schneider, P., Tschopp, J., Cachero, T. G., Batten, M., Wheway, J., Mauri, D., Cavill, D., Gordon, T. P., Mackay, C. R., and Mackay, F. (2002). Association of BAFF/BLyS overexpression and altered B‐cell differentiation with Sjo¨ gren’s syndrome. J. Clin. Invest. 109, 59–68. Gross, J. A., Dillon, S. R., Mudri, S., Johnston, J., Littau, A., Roque, R., Rixon, M., Schou, O., Foley, K. P., Haugen, H., McMillen, S., Waggie, K., Schreckhise, R. W., Shoemaker, K., Vu, T., Moore, M., Grossman, A., and Clegg, C. H. (2001). TACI‐Ig neutralizes molecules critical for B‐ cell development and autoimmune disease. Impaired B‐cell maturation in mice lacking BLyS. Immunity 15, 289–302. Gross, J. A., Johnston, J., Mudri, S., Enselman, R., Dillon, S. R., Madden, K., Xu, W., Parrish‐ Novak, J., Foster, D., Lofton‐Day, C., Moore, M., Littau, A., Grossman, A., Haugen, H., Foley, K., Blumberg, H., Harrison, K., Kindsvogel, W., and Clegg, C. H. (2000). TACI and BCMA are receptors for a TNF homologue implicated in B‐cell autoimmune disease. Nature 404, 995–999. Guinamard, R., Okigaki, M., Schlessinger, J., and Ravetch, J. V. (2000). Absence of marginal zone B cells in Pyk‐2‐deficient mice defines their role in the humoral responses. Nat. Immunol. 1, 31–36. Haas, K. M., Hasegawa, M., Steeber, D. A., Poe, J. C., Zabel, M. D., Bock, C. B., Karp, D. R., Briles, D. E., Weis, J. H., and Tedder, T. F. (2002). Complement receptors CD21/35 link innate and protective immunity during Streptococcus pneumoniae infection by regulating IgG3 antibody responses. Immunity 17, 713–723. Hamaguchi, Y., Uchida, J., Cain, D. W., Venturi, G. M., Poe, J. C., Haas, K. M., and Tedder, T. F. (2005). The peritoneal cavity provides a protective niche for B1 and conventional B lymphocytes during anti‐CD20 immunotherapy in mice. J. Immunol. 7, 4389–4399. Hanasaki, K., Powell, L. D., and Varki, A. (1995). Binding of human plasma sialoglycoproteins by the B‐cell‐specific lectin CD22: Selective recognition of immunoglobulin M and haptoglobin. J. Biol. Chem. 270, 7543–7550. Harmer, S. L., and DeFranco, A. L. (1998). The Src homology domain 2‐containing inositol phosphatase SHIP forms a ternary complex with Shc and Grb2 in antigen receptor‐stimulated B lymphocytes. J. Biol. Chem. 274, 12183–12191. Hasegawa, M., Fujimoto, M., Poe, J. C., Steeber, D. A., and Tedder, T. F. (2001). CD19 can regulate B lymphocyte signal transduction independent of complement activation. J. Immunol. 167, 3190–3200. Hennet, T., Chui, D., Paulson, J. C., and Marth, J. D. (1998). Immune regulation by the ST6Gal sialyltransferase. Proc. Natl. Acad. Sci. USA 95, 4504–4509. Hibbs, M. L., Tarlinton, D. M., Armes, J., Grail, D., Hodgson, G., Maglitto, R., Stacker, S. A., and Dunn, A. R. (1995). Multiple defects in the immune system of Lyn‐deficient mice, culminating in autoimmune disease. Cell 83, 301–311. Hippen, K. L., Buhl, A. M., D’ Ambrosio, D., Nakamura, K., Persin, C., and Cambier, J. C. (1997). FcgRIIB1 inhibition of BCR‐mediated phosphoinositide hydrolysis and Ca2þ mobilization is integrated by CD19 dephosphorylation. Immunity 7, 49–58. Huang, W., Sinha, J., Newman, J., Reddy, B., Budhai, L., Furie, R., Vaishnaw, A., and Davidson, A. (2002). The effect of anti‐CD40 ligand antibody on B cells in human systemic lupus erythematosus. Arthritis Rheum. 46, 1554–1562.

42

t h om a s f . t e d d er

ET AL.

Inaoki, M., Sato, S., Weintraub, B. C., Goodnow, C. C., and Tedder, T. F. (1997). CD19‐regulated signaling thresholds control peripheral tolerance and autoantibody production in B lymphocytes. J. Exp. Med. 186, 1923–1931. Jin, L., McLean, P. A., Neel, B. G., and Wortis, H. H. (2002). Sialic acid binding domains of CD22 are required for negative regulation of B‐cell receptor signaling. J. Exp. Med. 195, 1199–1205. Juweid, M. E., Stadtmauer, E., Hajjar, G., Sharkey, R. M., Suleiman, S., Luger, S., Swayne, L. C., Alavi, A., and Goldenberg, D. M. (1999). Pharmacokinetics, dosimetry, and initial therapeutic results with 131I‐ and (111)In‐/90Y‐labeled humanized LL2 anti‐CD22 monoclonal antibody in patients with relapsed, refractory non‐Hodgkin’s lymphoma. Clin. Cancer Res. 5, 3292–3302. Kavanaugh, W. M., Pot, D. A., Chin, S. M., Deuter‐Reinhard, M., Jefferson, A. B., Norris, F. A., Masiarz, F. R., Cousens, L. S., Majerus, P. W., and Williams, L. T. (1996). Multiple forms of an inositol polyphosphate 5‐phosphatase form signaling complexes with Shc and Grb2. Curr. Biol. 6, 438–445. Kawabe, T., Naka, T., Yoshida, K., Tanaka, T., Fujiwara, H., Suematsu, N., Yoshida, N., Kishimoto, T., and Kikutani, H. (1994). The immune responses in the CD40‐deficient mice: Impaired immunoglobulin class switching and germinal center formation. Immunity 1, 167–178. Kelm, S., Gerlach, J., Brossmer, R., Danzer, C.‐P., and Nitschke, L. (2002). The ligand‐binding domain of CD22 is needed for inhibition of the B‐cell receptor signal, as demonstrated by a novel human CD22‐specific inhibitor compound. J. Exp. Med. 195, 1207–1213. Kelm, S., Pelz, A., Schauer, R., Filbin, M. T., Tang, S., de Bellard, M.‐E., Schnaar, R. L., Mahoney, J. A., Hartnell, A., Bradfield, P., and Crocker, P. R. (1994). Sialoadhesin, myelin‐associated glycoprotein and CD22 define a new family of sialic acid‐dependent adhesion molecules of the immunoglobulin superfamily. Curr. Biol. 4, 965–972. Kono, D. H., Burlingame, R. W., Owens, D. G., Kuramochi, A., Balderas, R. S., Balomenos, D., and Theofilopoulos, A. N. (1994). Lupus susceptibility loci in New Zealand mice. Proc. Natl. Acad. Sci. USA 91, 10168–10172. Kozlowski, M., Mlinaric‐Rascan, I., Feng, G. S., Shen, R., Pawson, T., and Siminovitch, K. A. (1993). Expression and catalytic activity of the tyrosine phosphatase PTP1C is severely impaired in motheaten and viable motheaten mice. J. Exp. Med. 178, 2157–2163. Kreitman, R. J., Wang, Q. C., Fitz Gerald, D. J., and Pastan, I. (1999). Complete regression of human B‐cell lymphoma xenografts in mice treated with recombinant anti‐CD22 immunotoxin RFB4(dsFv)‐PE38 at doses tolerated by cynomolgus monkeys. Intl. J. Cancer 81, 148–155. Kreitman, R. J., Wilson, W. H., Bergeron, K., Raggio, M., Stetler‐Stevenson, M., Fitz Gerald, D. J., and Pastan, I. (2001). Efficacy of the anti‐CD22 recombinant immunotoxin BL22 in chemotherapy‐resistant hairy‐cell leukemia. N. Engl. J. Med. 345, 241–247. Kremer, J. M., Westhovens, R., Leon, M., Di Giorgio, E., Alten, R., Steinfeld, S., Russell, A., Dougados, M., Emery, P., Nuamah, I. F., Williams, G. R., Becker, J. C., Hagerty, D. T., and Moreland, L. W. (2003). Treatment of rheumatoid arthritis by selective inhibition of T‐cell activation with fusion protein CTLA4Ig. N. Engl. J. Med. 349, 1907–1915. Lajaunias, F., Ibnou‐Zekri, N., Jimack, L. F., Chichepertiche, Y., Parkhouse, R. M. E., Reininger, C. M. L., Brighouse, G., and Izui, S. (1999). Polymorphisms in the Cd22 gene of inbred mouse strains. Immunogenetics 49, 991–995. Lankester, A. C., van Schijndel, G. M., and van Lier, R. A. (1995). Hematopoietic cell phosphatase is recruited to CD22 following B‐cell antigen receptor ligation. J. Biol. Chem. 270, 20305–20308. Lanoue, A., Batista, F. D., Stewart, M., and Neuberger, M. S. (2002). Interaction of CD22 with a2,6‐linked sialoglycoconjugates: Innate recognition of self to dampen B‐cell autoreactivity? Eur. J. Immunol. 32, 348–355.

c d2 2 l i g a n d b i n d i n g r e gu l at e s b - c e ll su rv i va l

43

Law, C.‐L., Aruffo, A., Chandran, K. A., Doty, R. T., and Clark, E. A. (1995). Ig domains 1 and 2 of murine CD22 constitute the ligand‐binding domain and bind multiple sialylated ligands expressed on B and T cells. J. Immunol. 155, 3368–3376. Law, C.‐L., Sidorenko, S. P., Chandran, K. A., Zhao, Z., Shen, S.‐H., Fischer, E. H., and Clark, E. A. (1996). CD22 associates with protein tyrosine phosphatase 1C, Syk, and phospholipase C‐g1 upon B‐cell activation. J. Exp. Med. 183, 547–560. Law, C.‐L., Torres, R. M., Sundberg, H. A., Parkhouse, R. M. E., Brannan, C. I., Copeland, N. G., Jenkins, N. A., and Clark, E. A. (1993). Organization of the murine Cd22 locus. Mapping to chromosome 7 and characterization of two alleles. J. Immunol. 151, 17–87. Leandro, M. J., Edwards, J. C., and Cambridge, G. (2002). Clinical outcome in 22 patients with rheumatoid arthritis treated with B lymphocyte depletion. Ann. Rheum. Dis. 61, 883–888. Leandro, M. J., Edwards, J. C., Cambridge, G., Ehrenstein, M. R., and Isenberg, D. A. (2002). An open study of B lymphocyte depletion in systemic lupus erythematosis. Arthritis Rheum. 46, 2673–2677. Leonard, J. P., and Link, B. K. (2002). Immunotherapy of non‐Hodgkin’s lymphoma with hLL2 (epratuzumab, an anti‐CD22 monoclonal antibody) and Hu1D10 (apolizumab). Semin. Oncol. 29, 81–86. Leprince, C., Draves, K. E., Geahlen, R. L., Ledbetter, J. A., and Clark, E. A. (1993). CD22 associates with the human surface IgM‐B‐cell antigen receptor complex. Proc. Natl. Acad. Sci. USA 90, 3236–3240. Li, T. J., Rawlings, D. J., Park, H., Kato, R. M., Witte, O. N., and Satterthwaite, A. B. (1997a). Constitutive membrane association potentiates activation of Bruton’s tyrosine kinase. Oncogene 15, 1375–1383. Li, Z. M., Wahl, M. I., Eguinoa, A., Stephens, L. R., Hawkins, P. T., and Witte, O. N. (1997b). Phosphatidylinositol 3‐kinase‐g activates Bruton’s tyrosine kinase in concert with Src family kinases. Proc. Natl. Acad. Sci. USA 94, 13820–13825. Liu, Q., Oliveira‐Dos Santos, A. J., Mariathasan, S., Bouchard, D., Jones, J., Sarao, R., Kozieradzki, I., Ohashi, P. S., Pennigner, J. M., and Dumont, D. J. (1998). The inositol polyphosphate 5‐phosphatase Ship is a crucial negative regulator of B‐cell antigen receptor signaling. J. Exp. Med. 188, 1333–1342. Mackay, F., Sierro, F., Grey, S. T., and Gordon, T. P. (2005). The BAFF/APRIL system: An important player in systemic rheumatic diseases. Curr. Dir. Autoimmunity 8, 243–265. Mackay, F., Woodcock, S. A., Lawton, P., Ambrose, C., Baetscher, M., Schneider, P., Tschopp, J., and Browning, J. L. (1999). Mice transgenic for BAFF develop lymphocytic disorders along with autoimmune manifestations. J. Exp. Med. 190, 1697–1710. Madaio, M. P. (1998). B cells and autoantibodies in the pathogenesis of lupus nephritis. Immunol. Res. 17, 123–132. Mansfield, E., Amlot, P., Pastan, I., and Fitz Gerald, D. J. (1997a). Recombinant RFB4 immunotoxins exhibit potent cytotoxic activity for CD22‐bearing cells and tumors. Blood 90, 2020–2026. Mansfield, E., Chiron, M. F., Amlot, P., Pastan, I., and Fitz Gerald, D. J. (1997b). Recombinant RFB4 single‐chain immunotoxin that is cytotoxic towards CD22‐positive cells. Biochem. Soc. Trans. 25, 709–714. Martin, F., and Kearney, J. F. (2000). B‐cell subsets and the mature preimmune repertoire. Marginal zone and B1 B cells as part of a ‘‘natural immune memory’’. Immunol. Rev. 175, 70–79. Martin, F., and Kearney, J. F. (2002). Marginal‐zone B cells. Nat. Rev. Immunol. 2, 323–335. Martin, F., Oliver, A. M., and Kearney, J. F. (2001). Marginal zone and B1 B cells unite in the early response against T‐independent blood‐borne particulate antigens. Immunity 14, 617–629. Mary, C., Laporte, C., Parzy, D., Santiago, M.‐L., Stefani, F., Lajaunias, F., Parkhouse, R. M. E., O’Keefe, T. L., Neuberger, M. S., Izui, S., and Reininger, L. (2000). Dysregulated expression

44

t h om a s f . t e d d er

ET AL.

of the Cd22 gene as a result of a short interspersed nucleotide element insertion in Cd22a lupus‐ prone mice. J. Immunol. 165, 2987–2996. Morel, L., Rudofsky, U. H., Longmate, J. A., Schiffenbauer, J., and Wakeland, E. K. (1994). Polygenic control of susceptibility to murine systemic lupus erythematosus. Immunity 1, 219–229. Munro, S., Bast, B. J. E. G., Colley, K. J., and Tedder, T. F. (1992). B lymphocyte surface antigen CD75 is not a‐2,6‐sialyltransferase but is a carbohydrate antigen modified by the enzyme. Cell 68, 1003–1004. Nadler, M. J. S., McLean, P. A., Neel, B. G., and Wortis, H. H. (1997). B‐cell antigen receptor‐ evoked calcium influx is enhanced in CD22‐deficient B‐cell lines. J. Immunol. 159, 4233–4243. Nishizumi, H., Horikawa, K., Mlinaric‐Rascan, I., and Yamamoto, T. (1998). A double‐edged kinase Lyn: A positive and negative regulator for antigen receptor‐mediated signals. J. Exp. Med. 187, 1343–1348. Nishizumi, H., Taniuchi, I., Yamanashi, Y., Kitamura, D., Ilic, D., Mori, S., Watanabe, T., and Yamamoto, T. (1995). Impaired proliferation of peripheral B cells and indication of autoimmune disease in lyn‐deficient mice. Immunity 3, 549–560. Nitschke, L., Carsetti, R., Ocker, B., Kohler, G., and Lamers, M. C. (1997). CD22 is a negative regulator of B‐cell receptor signaling. Curr. Biol. 7, 133–143. Nitschke, L., Floyd, H., Ferguson, D. J. P., and Crocker, P. R. (1999). Identification of CD22 ligands on bone marrow sinusoidal endothelium implicated in CD22‐dependent homing of recirculating B cells. J. Exp. Med. 189, 1513–1518. Nitschke, L., and Tsubata, T. (2004). Molecular interactions regulate BCR signal inhibition by CD22 and CD72. Trends Immunol. 25, 543–550. O’Hagan, R. C., Ohh, M., David, G., de Alboran, I. M., Alt, F. W., Kaelin, J., W. G., and De Pinho, R. A. (2000). Myc‐enhanced expression of Cul1 promotes ubiquitin‐dependent proteolysis and cell cycle progression. Genes & Dev. 14, 2185–2191. O’Keefe, T. L., Williams, G. T., Batista, F. D., and Neuberger, M. S. (1999). Deficiency in CD22, a B‐cell‐specific inhibitory receptor, is sufficient to predispose to development of high affinity autoantibodies. J. Exp. Med. 189, 1307–1313. O’Keefe, T. L., Williams, G. T., Davies, S. L., and Neuberger, M. S. (1996). Hyperresponsive B cells in CD22‐deficient mice. Science 274, 798–801. O’Rourke, L., Tooze, R., and Fearon, D. T. (1997). Co‐receptors of B lymphocytes. Curr. Opin. Immunol. 9, 324–329. Okada, H., Bolland, S., Hashimoto, A., Kurosaki, M., Kabuyama, Y., Iino, M., Ravetch, J. V., and Kurosaki, T. (1998). Role of the inositol phosphatase SHIP in B‐cell receptor‐induced Ca2þ oscillatory response. J. Immunol. 161, 5129–5132. Okano, Y. (1996). Antinuclear antibody in systemic sclerosis (scleroderma). Rheum. Dis. Clin. North Am. 22, 709–735. Otipoby, K. L., Andersson, K. B., Draves, K. E., Klaus, S. J., Farr, A. G., Kerner, J. D., Perlmutter, R. M., Law, C.‐L., and Clark, E. A. (1996). CD22 regulates thymus‐independent responses and the lifespan of B cells. Nature 384, 634–637. Pani, G., Kozlowski, M., Cambier, J. C., Mills, G. B., and Siminovitch, K. A. (1995). Identification of the tyrosine phosphatase PTP1C as a B‐cell antigen receptor‐associated protein involved in the regulation of B‐cell signaling. J. Exp. Med. 181, 2077–2084. Peaker, C. J. G., and Neuberger, M. S. (1993). Association of CD22 with the B‐cell antigen receptor. Eur. J. Immunol. 23, 1358–1363. Pezzutto, A., Do¨ rken, B., Moldenhauer, G., and Clark, E. A. (1987). Amplification of human B‐cell activation by a monoclonal antibody to the B‐cell‐specific antigen CD22, Bp130/140. J. Immunol. 138, 98–103.

c d2 2 l i g a n d b i n d i n g r e gu l at e s b - c e ll su rv i va l

45

Pezzutto, A., Rabinovitch, P. S., Do¨ rken, B., Moldenhauer, G., and Clark, E. A. (1988). Role of the CD22 human B‐cell antigen in B‐cell triggering by anti‐immunoglobulin. J. Immunol. 140, 1791–1795. Pflugh, D. L., Maher, S. E., and Bothwell, A. L. M. (2002). Ly‐6 superfamily members Ly‐6A/E, Ly‐6C, and Ly‐6I recognize two potential ligands expressed by B lymphocytes. J. Immunol. 169, 5130–5136. Poe, J. C., Fujimoto, M., Jansen, P. J., Miller, A. S., and Tedder, T. F. (2000). CD22 forms a quaternary complex with SHIP, Grb2 and Shc. A pathway for regulation of B lymphocyte antigen receptor‐induced calcium flux. J. Biol. Chem. 275, 17420–17427. Poe, J. C., Fujimoto, Y., Hasegawa, M., Haas, K. M., Miller, A. S., Sanford, I. G., Bock, C. B., Fujimoto, M., and Tedder, T. F. (2004a). CD22 regulates B lymphocyte function in vivo through both ligand‐dependent and ligand‐independent mechanisms. Nat. Immunol. 5, 1078–1087. Poe, J. C., Haas, K. M., Uchida, J., Lee, Y., Fujimoto, M., and Tedder, T. F. (2004b). Severely‐ impaired B lymphocyte proliferation, survival and induction of the c‐Myc:Cullin 1 ubiquitin ligase pathway resulting from CD22 deficiency on the C57BL/6 genetic background. J. Immunol. 172, 2100–2110. Poe, J. C., Hasegawa, M., and Tedder, T. F. (2001). CD19, CD21 and CD22: Multifaceted response regulators of B lymphocyte signal transduction. Int. Rev. Immunol. 20, 739–762. Powell, L. D., Sgroi, D., Sjoberg, E. R., Stamenkovic, I., and Varki, A. (1993). Natural ligands of the B‐cell adhesion molecule CD22b carry N‐linked oligosaccharides with a‐2,6‐linked sialic acids that are required for recognition. J. Biol. Chem. 268, 7019–7027. Powell, L. D., and Varki, A. (1994). The oligosaccharide binding specificities of CD22‐b, a sialic acid‐specific lectin of B‐cells. J. Biol. Chem. 269, 10628–10636. Press, O. W., Farr, A. G., Borroz, K. I., Anderson, S. K., and Martin, P. J. (1989). Endocytosis and degradation of monoclonal antibodies targeting human B‐cell malignancies. Cancer Res. 49, 4906–4912. Press, O. W., Howell‐Clark, J., Anderson, S., and Bernstein, I. (1994). Retention of B‐cell‐specific monoclonal antibodies by human lymphoma cells. Blood 83, 1390–1397. Press, O. W., Leonard, J. P., Coiffier, B., Levy, R., and Timmerman, J. (2001). Immunotherapy of Non‐Hodgkin’s lymphomas. Hematology (Am. Soc. Hematol. Educ. Program), 221–240. Prodeus, A. P., Goerg, S., Shen, L. M., Pozdnyakova, O. O., Chu, L., Alicot, E. M., Goodnow, C. C., and Carroll, M. C. (1998). A critical role for complement in maintenance of self‐tolerance. Immunity 9, 721–731. Protheroe, A., Edwards, J. C., Simmons, A., Maclennan, K., and Selby, P. (1999). Remission of inflammatory arthropathy in association with anti‐CD20 therapy for non‐Hodgkin’s lymphoma. Rheumatology 38, 1150–1152. Razi, N., and Varki, A. (1998). Masking and unmasking of the sialic acid‐binding lectin activity of CD22 (Siglec‐2) on B lymphocytes. Proc. Natl. Acad. Sci. USA 95, 7469–7474. Rickert, R. C., Rajewsky, K., and Roes, J. (1995). Impairment of T‐cell‐dependent B‐cell responses and B‐1 cell development in CD19‐deficient mice. Nature 376, 352–355. Saito, E., Fujimoto, M., Hasegawa, M., Komura, K., Hamaguchi, Y., Kaburagi, Y., Nagaoka, T., Takehara, K., Tedder, T. F., and Sato, S. (2002). CD19‐dependent B lymphocyte signaling thresholds influence skin fibrosis and autoimmunity in the tight‐skin mouse. J. Clin. Invest 109, 1453–1462. Saleh, M. N., Gutheil, J., Moore, M., Bunch, P. W., Butler, J., Kunkel, L., Grillo‐Lopez, A. J., and Lo Buglio, A. F. (2000). A pilot study of the anti‐CD20 monoclonal antibody rituximab in patients with refractory immune thrombocytopenia. Semin. Oncol. 27, 99–103. Samardzic, T., Marinkovic, D., Danzer, C.‐P., Gerlach, J., Nitschke, L., and Wirth, T. (2002). Reduction of marginal zone B cells in CD22‐deficient mice. Eur. J. Immunol. 32, 561–567.

46

t h om a s f . t e d d er

ET AL.

Santiago, M. L., Mary, C., Parzy, D., Jacquet, C., Montagutelli, X., Parkhouse, R. M. E., Lemoine, R., Izui, S., and Reininger, L. (1998). Linkage of a major quantitative trait locus to Yaa gene‐induced lupus‐like nephritis in (NZW x C57BL/6)F1 mice. Eur. J. Immunol. 28, 4257–4267. Saouaf, S. J., Mahajan, S., Rowley, R. B., Kut, S. A., Fargnoli, J., Burkhardt, A. L., Tsukada, S., Witte, O. N., and Bolen, J. B. (1994). Temporal differences in the activation of three classes of non‐transmembrane protein tyrosine kinases following B‐cell antigen receptor surface engagement. Proc. Natl. Acad. Sci. USA 91, 9524–9528. Sato, S., Hasegawa, M., Fujimoto, M., Tedder, T. F., and Takehara, K. (2000). Quantitative genetic variation in CD19 expression correlates with autoimmunity in mice and humans. J. Immunol. 165, 6635–6643. Sato, S., Jansen, P. J., and Tedder, T. F. (1997). CD19 and CD22 reciprocally regulate Vav tyrosine phosphorylation during B lymphocyte signaling. Proc. Natl. Acad. Sci. USA 94, 13158–13162. Sato, S., Miller, A. S., Inaoki, M., Bock, C. B., Jansen, P. J., Tang, M. L. K., and Tedder, T. F. (1996). CD22 is both a positive and negative regulator of B lymphocyte antigen receptor signal transduction: Altered signaling in CD22‐deficient mice. Immunity 5, 551–562. Sato, S., Ono, N., Steeber, D. A., Pisetsky, D. S., and Tedder, T. F. (1996). CD19 regulates B lymphocyte signaling thresholds critical for the development of B‐1 lineage cells and autoimmunity. J. Immunol. 157, 4371–4378. Sato, S., Steeber, D. A., Jansen, P. J., and Tedder, T. F. (1997). CD19 expression levels regulate B lymphocyte development: Human CD19 restores normal function in mice lacking endogenous CD19. J. Immunol. 158, 4662–4669. Sato, S., Tuscano, J. M., Inaoki, M., and Tedder, T. F. (1998). CD22 negatively and positively regulates signal transduction through the B lymphocyte antigen receptor. Semin. Immunol. 10, 287–297. Sausville, E. A., Headlee, D., Stetler‐Stevenson, M., Jaffe, E. S., Solomon, D., Figg, W. D., Herdt, J., Kopp, W. C., Rager, H., and Steinberg, S. M. (1995). Continuous infusion of the anti‐CD22 immunotoxin IgG‐RFB4‐SMPT‐dgA in patients with B‐cell lymphoma: a phase I study. Blood 85, 3457–3465. Saxton, T. M., van Oostveen, I., Bowtell, D., Aebersold, R., and Gold, M. R. (1994). B‐cell antigen receptor cross‐linking induces phosphorylation of the p21ras oncoprotein activators SHC and mSOS1 as well as assembly of complexes containing SHC, GRB‐2, mSOS1, and a 145‐kDa tyrosine‐phosphorylated protein. J. Immunol. 153. Scharenberg, A. M., El‐Hillal, O., Fruman, D. A., Beitz, L. O., Li, Z., Lin, S., Gout, I., Cantaley, L. C., Rawlings, D. J., and Kinet, J. P. (1998). Phosphatidylinositol‐3,4,5‐triphosphate (PtdIns‐ P3)/Tec kinase‐dependent calcium signaling pathway: A target for SHIP‐mediated inhibitory signals. EMBO J. 17, 1961–1972. Scharenberg, A. M., and Kinet, J.‐P. (1996). The emerging field of receptor‐mediated inhibitory signaling: SHP or SHIP? Cell 87, 961–964. Schulte, R. J., Campbell, M. A., Fischer, W. H., and Sefton, B. M. (1992). Tyrosine phosphorylation of CD22 during B‐cell activation. Science 258, 1001–1004. Schultz, L. D., Schweitzer, P. A., Ranjan, T. V., Yi, T., Ihle, J. N., Matthews, R. J., Thomas, M. L., and Beier, D. R. (1993). Mutations at the murine motheaten locus are within the hematopoietic cell protein‐tyrosine phosphatase (Hcph) gene. Cell 73, 1445–1454. Schwarting, R., Stein, H., and Wang, C. Y. (1985). The monoclonal antibodies aS‐HCL 1 (aLeu‐14) and aS‐HCL 3 (aLeu‐M5) allow the diagnosis of hairy cell leukemia. Blood 65, 974–983.

c d2 2 l i g a n d b i n d i n g r e gu l at e s b - c e ll su rv i va l

47

Schwartz‐Albiez, R., Do¨ rken, B., Monner, D. A., and Moldenhauer, G. (1991). CD22 antigen: Biosynthesis, glycosylation, and surface expression of a B lymphocyte protein involved in B‐cell activation and adhesion. Int. Immunol. 3, 623–633. Segundo, C., Rodriguez, C., Garcia‐Poley, A., Aguilar, M., Gavilan, I., Bellas, C., and Brieva, J. A. (2001). Thyroid‐infiltrating B lymphocytes in Graves’ disease are related to marginal zone and memory B‐cell compartments. Thyroid 11, 525–530. Sgroi, D., Varki, A., Braesch‐Andersen, S., and Stamenkovic, I. (1993). CD22, a B‐cell‐specific immunoglobulin superfamily member, is a sialic acid‐binding lectin. J. Biol. Chem. 268, 7011–7018. Shan, D., and Press, O. W. (1995). Constitutive endocytosis and degradation of CD22 by human B cells. J. Immunol. 154, 4466–4475. Shen, G.‐L., Li, J.‐A., Ghetie, M.‐A., Ghetie, V., May, R. D., Till, M., Brown, A. N., Relf, M., Knowles, P., Uhr, J. W., Janossy, G., Amlot, P., Vitetta, E. S., and Thorpe, P. E. (1988). Evaluation of four CD22 antibodies as ricin A‐chain‐containing immunotoxins for the in vivo therapy of human B‐cell leukemias and lymphomas. Intl. J. Cancer 42, 792–797. Shih, L. B., Lu, H. H. Z., Xuan, H., and Goldenberg, D. M. (1994). Internalization and intracellular processing of an anti‐B‐cell lymphoma monoclonal antibody, LL2. Int. J. Cancer 56, 538–545. Shirane, M., Harumiya, Y., Ishida, N., Hirai, A., Miyamoto, C., Hatakeyama, S., Nakayama, K., and Kitagawa, M. (1999). Down‐regulation of p27Kip1 by two mechanisms, ubiquitin‐mediated degradation and proteolytic processing. J. Biol. Chem. 274, 13886–13893. Shlomchik, M. J., Madaio, M. P., Ni, D., Trounstine, M., and Huszar, D. (1994). The role of B cells in lpr/lpr‐induced autoimmunity. J. Exp. Med. 180, 1295–1306. Sidman, C. L., Shultz, L. D., and Unanue, E. R. (1978). The mouse mutant ‘‘Motheaten.’’ Functional studies of the immune system. J. Immunol. 121, 2399–2404. Sieber, T., Schoeler, D., Ringel, F., Pascu, M., and Schreiver, F. (2003). Selective internalization of monoclonal antibodies by B‐cell chronic lymphocytic leukaemia cells. Brit. J. Hematol. 121, 458–461. Siegel, A. B., Goldenberg, D. M., Cesano, A., Coleman, M., and Leonard, J. P. (2003). CD22‐ directed monoclonal antibody therapy for lymphoma. Semin. Oncol. 30, 457–464. Simmons, D., and Seed, B. (1988). Isolation of a cDNA encoding CD33, a differentiation antigen of myeloid progenitor cells. J. Immunol. 141, 2797–2800. Somani, A., Yuen, K., Xu, F., Zhang, J., Branch, D. R., and Siminovitch, K. A. (2001). The SH2 domain containing tyrosine phosphatase‐1 down‐regulates activation of Lyn and Lyn‐induced tyrosine phosphorylation of the CD19 receptor in B cells. J. Biol. Chem. 276, 1938–1944. Stamenkovic, I., and Seed, B. (1990). The B‐cell antigen CD22 mediates monocyte and erythrocyte adhesion. Nature 344, 74–77. Stamenkovic, I., Sgroi, D., Aruffo, A., Sy, M. S., and Anderson, T. (1991). The B lymphocyte adhesion molecule CD22 interacts with leukocyte common antigen CD45RO on T cells and a2,6 sialyltransferase, CD75, on B cells. Cell 66, 1133–1144. Stasi, R., Pagano, A., Stipa, E., and Amadori, S. (2001). Rituximab chimeric anti‐CD20 monoclonal antibody treatment for adults with chronic idiopathic thrombocytopenic purpura. Blood 98, 952–957. Stasi, R., Stipa, E., Forte, V., Meo, P., and Amadori, S. (2002). Variable patterns of response to Rituximab threatment in adults with chronic idiopathic thrombocytopenic purpura. Blood 99, 3872–3873. Stein, R., Belisle, E., Hansen, H. J., and Goldenberg, D. M. (1993). Epitope specificity of the anti‐ (B‐cell lymphoma) monoclonal antibody, LL2. Cancer Immunol. Immunother. 37, 293–298.

48

t h om a s f . t e d d er

ET AL.

Strasser, A., Whittingham, S., Vaux, D. L., Bath, M. L., Adams, J. M., Cory, S., and Harris, A. W. (1991). Enforced BCL2 expression in B‐lymphoid cells prolongs antibody responses and elicits autoimmune disease. Proc. Natl. Acad. Sci. USA 88, 8661–8665. Symington, F. W., Subbarao, B., Mosier, D. E., and Sprent, J. (1982). Lyb‐8.2: A new B‐cell antigen defined and characterized with a monoclonal antibody. Immunogenetics 16, 381–391. Takata, M., and Kurosaki, T. (1996). A role for Bruton’s tyrosine kinase in B‐cell antigen receptor‐ mediated activation of phospholipase C‐g2. J. Exp. Med. 184, 31–40. Takata, M., Sabe, H., Hata, A., Inazu, T., Homma, Y., Nukada, T., Yamamura, H., and Kurosaki, T. (1994). Tyrosine kinases Lyn and Syk regulates B‐cell receptor‐coupled Ca2þ mobilization through distinct pathways. EMBO J. 13, 1341–1349. Tarakhovsky, A., Turner, M., Shaal, S., Mee, P. J., Duddy, L. P., Rajewsky, K., and Tybulewicz, V. (1995). Defective antigen receptor‐mediated proliferation of B and T cells in the absence of Vav. Nature 374, 467–470. Tedder, T. F. (1998). Response‐regulators of B lymphocyte signaling thresholds provide a context for antigen receptor signal transduction. Semin. Immunol. 10, 259–265. Tedder, T. F., Inaoki, M., and Sato, S. (1997a). The CD19/21 complex regulates signal transduction thresholds governing humoral immunity and autoimmunity. Immunity 6, 107–118. Tedder, T. F., Poe, J. C., Fujimoto, M., Haas, K. M., and Sato, S. (2005). The CD19‐CD21 signal transduction complex of B lymphocytes regulates the balance between health and autoimmune disease: systemic sclerosis as a model system. Curr. Dir. Autoimmunity 8, 55–90. Tedder, T. F., Sato, S., Poe, J. C., and Fujimoto, M. (2000). CD19 and CD22 regulate a B lymphocyte signal transduction pathway that contributes to autoimmunity. Keio J. Med. 49, 1–13. Tedder, T. F., Tuscano, J., Sato, S., and Kehrl, J. H. (1997b). CD22, a B lymphocyte‐specific adhesion molecule that regulates antigen receptor signaling. Ann. Rev. Immunol. 15, 481–504. Theofilopoulos, A. N. (1992). In ‘‘Systemic Lupus Erythematosus’’ (R. G. Lahita, Ed.), pp. 121–195. Churchill Livingston, Edinburgh. Thomas, M. L., and Brown, E. J. (1999). Positive and negative regulation of Src‐family membrane kinases by CD45. Immunol. Today. 20, 406–411. Tooze, R. M., Doody, G. M., and Fearon, D. T. (1997). Counterregulation by the coreceptors CD19 and CD22 of MAP kinase activation by membrane immunoglobulin. Immunity 7, 59–67. Torres, R. M., Law, C.‐L., Santos‐Argumedo, L., Kirkham, P. A., Grabstein, K., Parkhouse, R. M. E., and Clark, E. A. (1992). Identification and characterization of the murine homologue of CD22, a B lymphocyte‐restricted adhesion molecule. J. Immunol. 149, 2641–2649. Tsubata, T., Wu, J., and Honjo, T. (1993). B‐cell apoptosis induced by antigen receptor crosslinking is blocked by a T‐cell signal through CD40. Nature 12, 645–648. Tsui, H. W., Siminovitch, K. A., de Souza, L., and Tsui, F. W. L. (1993). Motheaten and viable motheaten mice have mutations in the hemopoietic cell phosphatase gene. Nat. Genet. 4, 124–129. Tuscano, J., Engel, P., Tedder, T. F., and Kehrl, J. H. (1996). Engagement of the adhesion receptor CD22 triggers a potent stimulatory signal for B cells and blocking CD22/CD22L interactions impairs T‐cell proliferation. Blood 87, 4723–4730. Tuscano, J. M., Engel, P., Tedder, T. F., Agarwal, A., and Kehrl, J. H. (1996). Involvement of p72syk kinase, p53/56lyn kinase and phosphatidyl inositol‐3 kinase in signal transduction via the human B lymphocyte antigen CD22. Eur. J. Immunol. 26, 1246–1252. Tuscano, J. M., Harris, G., and Tedder, T. F. (2003). B lymphocytes contribute to autoimmune disease pathogenesis: Current trends and clinical implications. Autoimmunity Rev. 2, 101–108. Tuscano, J. M., O’Donnell, R. T., Miers, L. A., Kroger, L. A., Kukis, D. L., Lamborn, K. R., Tedder, T. F., and De Nardo, G. L. (2003). The anti‐CD22 ligand blocking antibody, HB22.7,

c d2 2 l i g a n d b i n d i n g r e gu l at e s b - c e ll su rv i va l

49

has independent lymphomacidal properties, and augments the efficacy of 90Y‐DOTA‐peptide‐ Lym‐1 in lymphoma xenografts. Blood 101, 3641–3647. Tuscano, J. M., Riva, A., Toscano, S. N., Tedder, T. F., and Kehrl, J. H. (1999). CD22 cross‐linking generates B‐cell antigen receptor‐independent signals that activate the JNK/SAPK signaling cascade. Blood 94, 1382–1392. Uchida, J., Hamaguchi, Y., Oliver, J. A., Ravetch, J. V., Poe, J. C., Haas, K. M., and Tedder, T. F. (2004). The innate mononuclear phagocyte network depletes B lymphocytes through Fc receptor‐dependent mechanisms during anti‐CD20 antibody immunotherapy. J. Exp. Med. 199, 1659–1669. van der Merwe, P. A., Crocker, P. R., Vinson, M., Barclay, A. N., Schauer, R., and Kelm, S. (1996). Localization of the putative sialic acid‐binding site on the immunoglobulin superfamily cell‐ surface molecule CD22. J. Biol. Chem. 271, 9273–9280. Van Horssen, P. J., Preijers, F. W. M. B., Van Oosterhout, Y. V. J. M., and De Witte, T. (1996). Highly potent CD22‐recombinant ricin A results in complete cure of disseminated malignant B‐ cell xenografts in SCID mice but fails to cure solid xenografts in nude mice. Intl. J. Cancer 68, 378–383. Vanhaesebroeck, B., Leevers, S. J., Ahmadi, K., Timms, J., Katso, R., Driscoll, P. C., Woscholski, R., Parker, P. J., and Waterfield, M. D. (2001). Synthesis and function of 3‐phosphorylated inositol lipids. Ann. Rev. Biochem. 70, 535–602. Viemann, D., Schlenke, P., Hammers, H. J., Kirchner, H., and Kruse, A. (2000). Differential expression of the B‐cell‐restricted molecule CD22 on neonatal B lymphocytes depending upon antigen stimulation. Eur. J. Immunol. 30, 550–559. Vita, S. D., Zaja, F., Sacco, S., Candia, A. D., Fanin, R., and Ferraccioli, G. (2002). Efficacy of selective B‐cell blockade in the treatment of rheumatoid arthritis: evidence for a pathogenetic role of B cells. Arthritis Rheum. 46, 2029–2033. Vitetta, E. S., Stone, M., Amlot, P., Fay, J., May, R., Till, M., Newman, J., Clark, P., Collins, R., and Cunningham, D. (1991). Phase I immunotoxin trial in patients with B‐cell lymphoma. Cancer Res. 51, 4052–4058. Wakabayashi, C., Adachi, T., Wienands, J., and Tsubata, T. (2002). A distinct signaling pathway used by the IgG‐containing B‐cell antigen receptor. Science 298, 2392–2395. Wellmann, U., Werner, A., and Winkler, T. H. (2001). Altered selection processes of B lymphocytes in autoimmune NZB/W mice, despite intact central tolerance against DNA. Eur. J. Immunol. 31, 2800–2810. Weng, W. K., Jarvis, L., and LeBien, T. W. (1994). Signaling through CD19 activates vav/mitogen‐ activated protein kinase pathway and induces formation of a CD19/vav/phosphatidylinositol 3‐ kinase complex in human B‐cell precursors. J. Biol. Chem. 269, 32514–32521. Wilson, G. L., Fox, C. H., Fauci, A. S., and Kehrl, J. H. (1991). cDNA cloning of the B‐cell membrane protein CD22: A mediator of B–B‐cell interactions. J. Exp. Med. 173, 137–146. Wilson, G. L., Najfeld, V., Kozlow, E., Menniger, J., Ward, D., and Kehrl, J. H. (1993). Genomic structure and chromosomal mapping of the human CD22 gene. J. Immunol. 150, 5013–5024. Wither, J. E., Roy, V., and Brennan, L. A. (2000). Activated B cells express increased levels of costimulatory molecules in young autoimmune NZB and (NZB x NZW)F(1) mice. Clin. Immunol. 94, 51–63. Yang, J. Q., Singh, A. K., Wilson, M. T., Satoh, M., Stanic, A. K., Park, J. J., Hong, S., Gadola, S. D., Mizutani, A., Kakumanu, S. R., Reeves, W. H., Cerundolo, V., Joyce, S., Van Kaer, L., and Singh, R. R. (2003). Immunoregulatory role of CD1d in the hydrocarbon oil‐induced model of lupus nephritis. J. Immunol. 171, 2142–2153.

50

t h om a s f . t e d d er

ET AL.

Yohannan, J., Wienands, J., Coggeshall, K. M., and Justement, L. B. (1999). Analysis of tyrosine phosphorylation‐dependent interactions between stimulatory effector proteins and the B‐cell co‐receptor CD22. J. Biol. Chem. 274, 18769–18776. Yu, Z. K., Gervais, J. L., and Zhang, H. (1998). Human CUL‐1 associates with the SKP1/SKP2 complex and regulates p21(CIP1/WAF1) and cyclin D proteins. Proc. Natl. Acad. Sci. USA 95, 11324–11329. Zeng, D., Lee, M. K., Tung, J., Brendolan, A., and Strober, S. (2000). Cutting edge: A role for CD1 in the pathogenesis of lupus in NZB/NZW mice. J. Immunol. 164, 5000–5004. Zhang, M., and Varki, A. (2004). Cell surface sialic acids do not affect primary CD22 interactions with CD45 and surface IgM nor the rate of constitutive CD22 endocytosis. Glycobiology 14, 939–949. Zhang, R., Alt, F. W., Davidson, L., Orkin, S. H., and Swat, W. (1995). Defective signaling through the T‐ and B‐cell antigen receptors in lymphoid cells lacking the Vav proto‐oncogene. Nature 374, 470–473. Zheng, N., Schulman, B. A., Song, L., Miller, J. J., Jeffrey, P. D., Wang, P., Chu, C., Koepp, D. M., Elledge, S. J., Pagano, M., Conaway, R. C., Conaway, J. W., Harper, J. W., and Pavletich, N. P. (2002). Structure of the Cul1‐Rbx1‐Skp1‐F boxSkp2 SCF ubiquitin ligase complex. Nature 416, 703–709.

Tetramer Analysis of Human Autoreactive CD4‐Positive T Cells Gerald T. Nepom Benaroya Research Institute at Virginia Mason, Seattle, Washington 98101 Abstract............................................................................................................. 1. pMHC Tetramers Identify Human Autoreactive T Cells ............................................. 2. Autoantigen pMHC Tetramers from Mouse to Man................................................... 3. Tetramer‐Induced Autoreactivity ............................................................................ References .........................................................................................................

51 51 59 65 67

Abstract Self‐reactivity is an intrinsic property of the human immune system. Autoreactive T cells derive directly from the developmental requirement for TCR engagement by self‐antigens during lymphocyte maturation. The fundamental questions implicating these autoreactive cells in human autoimmunity then, are not ‘‘Where do they come from?’’, but rather ‘‘Why do they persist?’’, ‘‘How do they become activated?’’, and ‘‘How are they regulated or deleted?’’. New technologies, in which peptide‐MHC (pMHC) ligands used for T‐cell recognition are utilized as soluble fluorescent multimers, now permit the direct visualization of antigen‐specific autoreactive T‐lymphocytes. By using multimer technology to study self‐reactive cells present in autoimmune patients and control individuals, a very broad range of autoreactive potential has been identified. 1. pMHC Tetramers Identify Human Autoreactive T Cells Structural studies demonstrate that the variable chain segments of the T‐cell receptor bind to the pMHC complex through a combination of conserved structural determinants and unique contact interactions with highly variable residues. A single T‐cell receptor can bind and interact with a single pMHC complex; however, T‐cell activation requires at least a dimer or pseudo‐dimer interaction (Krogsgaard et al., 2005), and higher order multimers lead to more avid binding properties (Cochran, Cameron, and Stern, 2000). This property is exploited in the construction of soluble fluorescent pMHC ligands, which are generally used in the form of tetramers, in which four biotinylated pMHC monomers are joined via binding to streptavidin (Novak et al., 1999; Altman et al., 1996). These tetramers are surrogate ligands for recognition by the antigen‐specific T‐cell receptor, and bind to T cells based on the antigen specificity and MHC restriction properties of an individual T cell.

51 advances in immunology, vol. 88 # 2005 Elsevier Inc. All rights reserved.

0065-2776/05 $35.00 DOI: 10.1016/S0065-2776(05)88002-2

52

g e ra l d t. n e po m

Figure 1 illustrates the identification of antigen‐specific T cells from peripheral blood, in which HLA‐DR4 MHC molecules and peptides from viral proteins of herpes simplex virus 2 or influenza virus, or from the self‐antigen glutamic acid decarboxylase, were utilized to make different pMHC tetramers. In the example shown, a large number of influenza‐specific T cells were visualized by flow cytometry, but no HSV‐specific T cells were present. T cells specific for the autoantigen GAD65 were also readily observed. Indeed, detection of autoreactive T cells using tetramers is not rare, and has been reported in a number of interesting clinical situations. For example, autoantigen tetramer‐binding CD8þ T cells were detected in peripheral blood from patients who had received allogeneic heart transplants (Barber et al., 2004). In this study, six patients who had antivimentin antibodies after cardiac transplantation were tested, and two reacted with specific HLA A*0201 Class I tetramers containing vimentin peptides. There are many reports of similar pMHC tetramer reactivity for peptide‐ HLA A2 tetramers representing cancer‐associated antigens; for example, recognizing the P53 (264–272) peptide (Hoffmann et al., 2002) or a Her2/Neu E75 peptide in peripheral blood of breast and prostate cancer patients receiving Her2/Neu vaccine immunotherapy (Woll et al., 2004). In other examples, the pMHC epitopes recognized are not associated with known oncogenes, but are associated with self‐proteins expressed or overexpressed in growing tumors; for example, HLA‐A2 tetramers with an a‐actinin peptide interacting with CTL clones derived from PBL of a patient with lung cancer (Mami‐ Chouaib et al., 2002) and HLA‐A24 tetramers containing an Syt‐SSX peptide which identified CTL in the blood of patients with synovial carcinomas (Sato et al., 2002). The most detailed studies of self‐antigen tetramers for detecting CD8 T cells directed to tumors come from the study of malignant melanoma, in which tetramers to MELAN‐A/MART‐1, MAGE‐4, NY‐ESO‐1, and GP100 have all been used successfully to detect antigen‐specific CD8þ T cells in patients before or after immunotherapy (Cohen et al., 2003; Hu et al., 2004; Jager et al., 2002; Kobayashi et al., 2003; Reynolds et al., 1998; Speiser et al., 2002; Valmori et al., 2003; van Rhee et al., 2005). With respect to the CD4þ T‐cell response to autoantigens, identification of tetramer‐binding T cells may simply be a matter of looking in the right way. That is, it is possible that the peripheral T‐cell repertoire is capable of recognizing self‐specificities for most dominant peptide‐epitopes, likely reflecting the developmental history of T cells that encountered similar pMHC targets during selection. This concept was directly tested (Danke et al., 2004) by generating three diverse pMHC tetramers to self‐specificities, one associated with the melanocyte differentiation antigen tyrosinase, one for the cancer/testes tumor antigen NY‐ESO‐1, and one for the diabetes-associated

53 Figure 1 Flow cytometry of peripheral blood lymphocytes (PBL) utilizing pMHC tetramers. Two‐color fluorescence detection of cells that bind anti‐ CD4 mAb and pMHC tetramers are shown for HLA‐DR4 MHC molecules containing peptides from epitopes of influenza (HA), HSV‐2 (control), and the autoantigen glutamic acid decarboxylase (GAD65). PBL from an HLA DR4‐positive subject were cultured in vitro with the corresponding antigen for 10 days to expand the responding population prior to cytometry analysis. Large numbers of HA‐specific and GAD‐specific CD4þ T cells were present, illustrating the utility of this methodology for detection of T cells responding to foreign and self‐antigens, respectively.

54

g e ra l d t. n e po m

auto‐antigen glutamic acid decarboxylase. CD4þ T cells specific for each of these tetramers were readily identified in peripheral blood of normal, healthy human individuals; notably, however, detection was possible only after removal of regulatory T‐cell populations, using specific antibody depletion of CD4 þ CD25 þ T cells (Treg). The most straightforward interpretation of these results is that autoreactive cells to many self‐antigen pMHC targets may well be present in most or all individuals, with the caveat that detection using pMHC tetramers requires matching the MHC, choosing a reasonably dominant peptide epitope, and importantly, unmasking the autoreactive specificities by depletion of regulatory elements which suppress the antigen‐induced expansion of the autoreactive populations. 1.1. Stages of Autoimmune T‐Cell Expansion and Activation The concept that pMHC tetramer‐positive autoreactive cells circulate in the periphery of normal individuals is consistent with models of autoimmunity in which the MHC genetic predisposition to specific autoimmune diseases is reflected in the developmental T‐cell selection of potentially autoreactive T cells. Figure 2 illustrates such a model, previously developed to track progression of autoimmunity in people at risk for type‐1 diabetes (T1D) (Nepom and Kwok, 1998). In this model, the first key step is the selection of a repertoire of thymocyte specificities based on pMHC interactions during T‐cell maturation in the thymus. In this stage, the HLA allelic polymorphism of each individual determines the spectrum of dominant autoreactive epitopes used as a basis for T‐cell selection. These T cells mature into peripheral populations which are potentially autoreactive, restricted by the MHC used for selection, and can be directly observed by expansion from peripheral blood using either the original self‐antigen or a peptide mimetic. Data indicating that expansion of these cells is unmasked by depletion of regulatory T‐cell populations in normal individuals suggest that the potential for autoreactivity is actually a normal, unavoidable, and readily documented result of this first step of the autoimmunity pathway model (Danke et al., 2004). Tetramer analysis of CD4þ T cells in the murine model of diabetes in the NOD mouse also supports this paradigm. In one study (Stratmann et al., 2003), pMHC tetramers with the NOD MHC IAg7 were constructed using peptide mimetics for a specificity known to be represented in the T‐cell populations infiltrating islets in diabetic mice. Tetramer-positive T cells were identified in these studies in the pancreatic lymph nodes associated with diabetic and prediabetic animals. Notably, however, they were also identified in the nondiabetic strains of mice that shared the same IAg7 MHC. In other

t et r a m e r a na ly s i s o f h u m a n au to r e act i v e t c el l s

55

Figure 2 The ‘‘hopscotch’’ pathway toward autoimmune disease. Selection, expansion, and activation of autoreactive T cells occur in an ordered progression of steps punctuated by functional stages of disease. In the first stage, genetic traits, particularly MHC susceptibility genes, are permissive for selection of autoreactive specificities during thymocyte development. The specific pMHC interactions with TCR at this stage determine thresholds for T‐cell activation and survival, which create an avidity window for future events. Individuals with disease‐associated MHC genes jump to this first stage expressing potentially autoreactive peripheral T‐cell repertoires. In the second stage, these autoreactive cells encounter stimuli that lead to proliferation and expansion. These peripheral stimuli are likely to be very diverse and variable between different individuals, and include various viral exposures, transient tissue damage, and even nonspecific inflammatory events; The increase in the circulating number of autoreactive T cells at this step overcomes a stochastic barrier such that specific autoreactive cells may now encounter their cognate autoantigen, resulting in activation and triggering a complex cascade of immunologic effector functions. In the third stage, autoreactive lymphocytes must jump regulatory barriers, which include a set of protective mechanisms such as activation‐induced cell death and transacting suppressor cells. These regulatory barriers arrest progressive autoimmunity in most cases, but when overcome or bypassed, are manifest as the final stage of overt autoimmune diseases.

words, the primary determinant of the presence of tetramer-positive autoreactive cells was not the disease phenotype, but was the MHC of the host. This is consistent with the preceding model, namely that the first stage in the autoimmune pathway, the selection of potentially autoreactive T cells, appears to be a normal physiologic consequence of T‐cell selection, and can be directly observed both in humans and mice, using pMHC tetramers with dominant self‐epitopes. 1.2. Properties Associated with Self‐pMHC Recognition Although studies using tetramers of self‐pMHC complexes readily identify autoreactive T cells, there may nevertheless be key differences between pMHC tetramers containing self‐antigens, compared to similar tetramers with foreign nonself antigens. The frequency of specific pMHC tetramer-binding cells and the avidity of the TCR interaction with the pMHC tetramer both

g e ra l d t. n e po m

56

appear to be much lower for self‐pMHC recognition. Table 1 summarizes data from several studies in which pMHC tetramers specific for the hemagglutinin of influenza and pMHC tetramers specific for the GAD65 epitope associated with autoimmune diabetes were tested in the same individuals. In general, three properties distinguish the self and the nonself recognition profiles in these studies. First, the frequency of T cells autoreactive to GAD65 is lower than the frequency of cells reactive to influenza HA. While this undoubtedly reflects the fact that most people have either been infected or vaccinated with influenza, it also may indicate that there are biological constraints on the in vivo expansion of the autoreactive specificities. A second difference is the overall TCR‐pMHC avidity, as determined by tetramer binding. The vast majority of influenza HA reactive CD4þ T cells derived from peripheral blood will bind MHC tetramers containing the relevant specificity (Novak et al., 2001). In contrast, only one third to one half of the GAD65-reactive T cells derived from peripheral blood are highly avid binders to pMHC tetramers containing the relevant GAD epitopes (Reijonen et al., 2004). In the latter study, the GAD-responsive T cells represented a broad spectrum of receptor specificities and pMHC binding avidities, and measures of T‐cell activation, such as cytokine release and proliferation, were more sensitive for detection of the antigen‐specific populations than pMHC tetramer binding, which was limited to the high‐avidity cells. In these examples, the self‐ and non‐self‐peptides are both immunodominant in the context of the same HLA class II molecules, and both have similar binding affinities for the peptide‐MHC interaction, so that differences in pMHC tetramer binding to T cells largely reflects the receptor avidity properties of the T‐cell specificity. These differences not only reflect inherent differences between self and nonself recognition, but also provide important clues regarding the Table 1 Characteristics of pMHC Tetramer Interactions Property of CD4þ T cell 1. Frequency in peripheral blood 2. Proportion of antigen‐responsive T‐cell population which binds pMHC Tetramers 3. Avidity for pMHC 4. Lineage

HA (nonself) recognition

GAD (self‐antigen)

High (1/6,000–1/30,000)

Low (1/20,000–1/100,000)

High

Low

High TH1 > TH0

Low TH0 > TH1

Differences which have been observed between CD4þ T cells that bind self‐pMHC tetramers compared to non‐self‐antigens. For the most part, these properties are characteristic of the average response within a broad distribution, so that while high‐avidity cells do exist within the self‐pMHC binding population, this is much reduced compared to a foreign antigen response.

t et r a m e r a na ly s i s o f h u m a n au to r e act i v e t c el l s

57

amplification and the activation steps in the cascade of autoimmune progression presented in Fig. 2. Overall, the spectrum of avidity for dominant self‐antigens may be quite a bit lower than the spectrum for traditional foreign antigen responses, a result which was previously suggested by studies of HLA transgenic mice immunized with either HA or GAD65 (Gebe et al., 2003). Whether this difference is due to the lack of survival of high‐avidity autoreactive T cells through a process of thymic negative selection, or whether it is due to activation‐induced cell death and selective deletion of high‐avidity cells in the peripheral T‐cell population, is not known. In either case, however, it may reflect a fundamental fail‐safe mechanism that the immune system has utilized to attempt to prevent deleterious autoimmunity. In the pathway model described in Fig. 2, after initial selection of the autoreactive T cell, some peripheral expansion is necessary to raise the frequency of these cells in the periphery, to the point where they achieve a threshold poised for subsequent activation. Definitive precursor frequency calculations using self‐antigen pMHC tetramers have not yet been reported for human CD4þ autoreactive T cells. Using tetramer binding as a rough correlate of T‐cell frequency, however, suggests that there is at least a 5‐ to 10‐ fold expansion of autoreactive T cells distinguishing patients with clinical autoimmunity from at‐risk or normal HLA‐matched individuals (Reijonen et al., 2002). Extrapolation of these results to data derived from studies of influenza HA pMHC tetramers, in which tetramer binding correlated directly with measured precursor frequencies (Danke and Kwok, 2003), suggests that this amplification checkpoint may represent a transition from an autoreactive T‐cell frequency of less than 1 in 100,000 to a frequency as high as 1 in 20,000. Subsequent activation of these autoreactive T‐cells, termed ‘‘autoimmune triggering’’ in Fig. 2, very likely requires specific antigen presentation and local microenvironments associated with proinflammatory immune responses. Indeed, given the presence of autoreactive cells as a normal physiologic state, this step, which probably involves dendritic cell presentation of tissue‐specific antigens occurring in complex lymphoid environments, is the key triggering step in autoimmunity and plays a major role in directing the subsequent immune response toward specific outcomes. During this triggering event, establishing initial T‐cell phenotypes becomes one of the key elements that control the distinction between pathological expansion and a benign, subclinical persistence of autoreactive cells. In our studies of T‐cell phenotypes expressed by tetramer‐binding cells in human peripheral blood, the majority of pMHC‐positive cells derived to foreign antigens like influenza HA express a functional phenotype, which is partially polarized toward TH1 characteristics. Thus, these cells are commonly producers of g‐interferon, and are often low in the production of TH2‐like cytokines

58

g e ra l d t. n e po m

such as IL10 and IL4. In contrast, the pMHC tetramer‐positive cells specific for the GAD65 autoantigen have been less TH1 polarized. In most cases, they produce a multitude of heterogeneous cytokines, including IL13, IL5, and lower levels of g‐interferon, a phenotype sometimes referred to as TH0. This phenotypic generalization partially correlates with the avidity differences which correspond to self‐ and non‐self‐recognition profiles, but is not absolute, and numerous exceptions exist (Holzer et al., 2003). 1.3. Regulatory Control of pMHC Reactivity The last stage in Fig. 2 represents the regulatory immune response, which is the final arbiter of clinical autoimmunity in the face of activated autoreactive cells. Various mechanisms for immune regulation coexist in multiple compartments and involve many different cells. The studies discussed previously (Danke and Kwok, 2003; Danke et al, 2004), in which normal individuals show GAD65 pMHC tetramer‐positive cells after depletion of regulatory lymphocytes, contrast with the similar observation of GAD65 pMHC tetramer-positive cells in the blood of T1D subjects, which indicates that overt autoimmunity is detectable in these subjects without removal of regulatory cells (Reijonen et al., 2002). This observation suggests the hypothesis that autoimmunity has progressed to disease in T1D patients in part due to a spontaneous loss of regulatory competence. Either the autoreactive T cells have become refractory to regulation, or the regulatory components of the immune response failed to function properly. This regulatory step, the final stage in the proposed pathway model, has only recently been investigated using pMHC tetramers. Characteristics of human Treg were analyzed in terms of lineage and antigen specificity. In their initial report (Walker et al., 2003), a system was described for generating human CD4þ CD25þ T cells in vitro which function to suppress other T‐cell responses. This suppressor function was dependent upon activation via the T‐cell receptor. Once activated, these Treg were able to suppress bystander T‐cell responses, consistent with their activity in animal models of autoimmunity in which a broad therapeutic benefit was achieved with transfer of similar antigen‐specific Treg (Tang et al., 2004; Tarbell et al., 2004). These studies were then extended by utilizing pMHC tetramers to identify and generate antigen‐specific Treg (Walker et al., 2005). In this study, a population of Treg was generated through the antigen‐specific expansion of previously nonregulatory CD4þ CD25‐ responder T cells, followed by isolation of pMHC tetramer-binding CD4þ CD25þ cells utilizing flow cytometry. These tetramer‐positive cells were then secondarily expanded in the presence of antigen to yield a population of Treg that functions in an antigen‐specific manner. That

t et r a m e r a na ly s i s o f h u m a n au to r e act i v e t c el l s

59

is, triggering of regulatory function in these cells required contact with specific peptide MHC ligands. As with other Treg populations, the effector function of the antigen‐activated regulatory cells provided bystander suppression for other T‐cell responses as well. These findings suggest several opportunities for utilizing specific pMHC recognition to expand and elicit regulatory function in an antigen‐specific manner. In the context of human autoimmunity, identification of dominant pMHC epitopes is a necessary prerequisite for testing this novel therapeutic pathway, and is a logical, practical application for utilizing tetramers with autoreactive specificity. 2. Autoantigen pMHC Tetramers from Mouse to Man The essential requirements for using pMHC tetramers to study autoimmune disease are: knowledge of the relevant MHC, information regarding dominant T‐cell epitopes, and suitable access to lymphoid cells for analysis. All three of these requirements pose major challenges in human clinical applications, with useful insights coming from mouse models. Tetramers have been used to study autoreactive T cells in spontaneously occurring T1D models in the NOD mouse, and in the antigen‐elicited models of experimental autoimmune encephalomyelitis (EAE) and collagen‐induced arthritis (CIA). In the NOD diabetes model, both CD4 and CD8þ lymphocytes are known to be important for disease pathogenesis. However, in neither case are dominant pMHC targets known. To circumvent this problem, pMHC tetramers have been devised that utilize peptide‐mimetic sequences, selected by library screening approaches, which act as surrogates for the autoantigen pMHC recognition. In these studies (Jang, Seth, and Wucherpfennig, 2003; Stratmann et al., 2003), MHC class II molecules from the IAg7 haplotype were used for tetramers coupled to a peptide mimotope which acted as a surrogate ligand for islet‐specific T cells. This mimotope was originally isolated from a chemically synthesized random peptide library, based on its ability to stimulate a pathogenic T‐cell clone known as BDC‐2.5 (Yoshida et al., 2002). The BDC‐ 2.5 T cell had originally been cloned from diabetic NOD mice, and was shown to be able to transfer diabetes, therefore qualifying as a pathogenic T‐cell specificity, even though the native target is not known. It was demonstrated that tetramers containing IAg7 with the peptide mimotope reacted with T cells in the pancreatic and iliac lymph nodes, with frequencies from 0.05–0.15%, and were found as early as 2 weeks of age, well in advance of clinical disease (Stratmann et al., 2003). In histologic sections of pancreas, pMHC tetramerbinding T cells were also directly identified by immunocytochemistry. Tetramer‐ positive cells increased in number as the mice aged, and recovery of some

60

g e ra l d t. n e po m

of the tetramer‐positive cells yielded T‐cell clones with a wide range of TCR usage, antigen avidity, and cytokine production. A notable finding in this study was that congenic strains of mice that carry the same MHC haplotype as the NOD mouse but differed in other loci, were also studied. These strains are fully resistant to diabetes, yet the tetramer‐ positive cells were easily detectable in lymphoid organs, with distribution and numbers comparable to the NOD mouse. This is an important observation, which clarifies the concept that the autoreactive‐pMHC specificity is a necessary, but not a sufficient pathogenic element required for autoimmune disease. In the T‐cell progression model outlined in Fig. 2, this pMHC specificity drives the first stage, in which autoreactive T-cell potential is selected, but does not determine T‐cell or disease outcome. An additional notable feature of their work was the use of a high‐avidity peptide mimotope, in order to successfully design pMHC tetramers which detected autoreactive peripheral T cells (Stratmann et al., 2003). This strategy, which has the potential to bypass the constraints of low‐avidity self‐antigen reactivity, was also utilized in prior studies of class I restricted CD8þ T‐cell autoreactivity in NOD mice. In another study, class I pMHC tetramers were designed using H2‐Kd, containing a peptide known as NRP‐V7 (Trudeau et al., 2003). This peptide is a high‐avidity mimetic for a CD8þ T‐cell epitope associated with diabetes in the NOD mouse, and tetramers using this pMHC mimetic detected autoreactive T cells infiltrating the pancreatic islets of diabetic NOD mice. Indeed, up to 10% of CD8þ-infiltrating T cells in the islets were bound by the high‐avidity NRP‐V7 pMHC tetramer. Notably, a tetramer made with the lower‐avidity native sequence NRP peptide identified few or none of the cells. Also, relative to the islet infiltrates, the frequency of tetramer‐positive cells in peripheral blood was very low, in all cases less than 1% of CD8þ cells. On the other hand, in mice who progressed to diabetes, most showed tetramer‐positive cells in the blood on at least one prior occasion. This study, similar to that of Stratmann, illustrates the advantages of using high‐avidity pMHC tetramers to detect what appear to be low‐avidity responses to islet-associated self‐antigens. The other notable finding in this study was that detection in peripheral blood is challenged by the low frequency of circulating autoreactive cells, relative to a much higher frequency in target organs. Two studies (Chen et al., 2003; Liu et al., 2000) generated pMHC tetramers using IAg7 complexed with epitopes from GAD65, as previous studies have implicated autoimmunity in GAD65 in pathogenesis of diabetes in the NOD model (Jun et al., 2002; Sercarz, 2003). However, no NOD T cells were identified which bound these tetramers, unless the animals were deliberately immunized to GAD65. Interestingly, in the latter case, tetramer‐ binding T cells were subsequently identified which expressed regulatory

t et r a m e r a na ly s i s o f h u m a n au to r e act i v e t c el l s

61

cytokines and inhibited diabetes development. This finding is consistent with other observations in transgenic and knockout genetic models which suggest that immunity to GAD65 in the NOD mouse may be predominantly biased toward disease suppression (Jaeckel et al., 2003; Kim et al., 2004). Recently three pMHC class I tetramers were described for additional CD8þ T‐cell epitopes associated with spontaneous autoimmune diabetes in NOD mice (Lieberman et al., 2004). In this study, in which b‐cell-reactive T cells were cultured from the islets of individual NOD mice, each individual mouse exhibited a distinct profile of pMHC tetramer‐positive T cells. This high degree of variation, even within a single inbred strain of diabetic mouse, highlights the challenge for identifying immunodominant epitopes, which can be well represented in the outbred and highly diverse human population. Class II MHC tetramers have also been used in two other murine models of autoimmunity which, in contrast to the NOD, do not get spontaneous disease but which respond to self‐antigen immunization with progressive autoimmune symptoms resembling clinical pathology. In the case of murine experimental autoimmune encephalomyelitis (EAE), disease in H‐2u animals is elicited using an immunodominant epitope of myelin basic protein, MBP1–9. In H2S animals, disease is induced with proteolipid protein 139–151. In studies in these models, pMHC tetramers containing the immunizing peptide were used to detect CD4þ autoreactive cells following immunization (Bischof et al., 2004; Radu et al., 2000; Reddy et al., 2003). Self‐antigen‐specific CD4þ T cells accumulated in the lymph nodes draining the immunization site, similar to standard primary immune responses, and subsequently entered the central nervous system. Also, tetramer‐positive cells which entered the CNS displayed activation markers that distinguish them from the larger pool of tetramer‐ positive cells that remain in the periphery. Tetramer studies in human subjects with autoimmunity are confounded by the same issues observed in the animal models, namely low‐frequency, low‐ avidity, and the constraints of utilizing peripheral blood for analysis, rather than lymph nodes or target tissue. The most extensive studies in humans have been in subjects with T1D, utilizing MHC class II tetramers (Reijonen et al., 2003; 2004; Reijonen, Kwok, and Nepom, 2003). In these studies, pMHC tetramers using HLA DR4 molecules complexed with an immunodominant peptide from the islet autoantigen glutamic acid decarboxylase (GAD65) have been used to evaluate CD4þ antigen‐specific T‐cell frequencies in the blood of T1D subjects and controls. Because the majority of patients with T1D carry at least one HLA DR4 haplotype, patient populations can be surveyed using a reasonably small number of pMHC combinations. Due to the very low frequency of autoantigen‐specific CD4þ T cells in peripheral blood, detection of tetramer interactions with specific T cells in these studies requires the in vitro

62

g e ra l d t. n e po m

expansion of the antigen‐responsive population. In the studies, this is accomplished by incubation of peripheral lymphocytes with antigen for 7–14 days prior to flow cytometry analysis using fluorescent pMHC tetramers (Reijonen et al., 2003, 2004). The majority of HLA DR4 T1D subjects tested have pMHC‐staining T cells in this system. Single cell sorting experiments were used to confirm the antigen specificity of the tetramer population which in addition to being specific for GAD65, tended to make a heterogenous set of cytokines following antigen activation, with some cells expressing TH1 properties and the majority expressing TH0 phenotypes. Analysis of the tetramer‐ positive T‐cell clones revealed a broad range of pMHC avidity, with some very high‐avidity responses and some very low. Thus, within a single individual, both high‐ and low‐avidity T cells coexist to the same autoantigen. Nondiabetic individuals are classified as ‘‘at risk’’ or ‘‘prediabetic’’ if they carry T1D susceptibility genes and if autoantibodies to islet antigens are present in serum. Interestingly, some but not all of these at‐risk HLA DR4 subjects were found to have peripheral CD4þ T cells binding the same pMHC tetramers. Whether this type of analysis will be a useful prognostic tool for disease progression and individual assessment of disease risk will require more extensive longitudinal studies. Class II pMHC tetramers for two other human autoimmune diseases have also been described, an HLA DQ2 tetramer containing peptides derived from gliadin for the study of celiac disease, and HLA DR4 tetramers containing collagen peptides for the study of rheumatic disease. A study used the gliadin pMHC tetramers to stimulate and bind T‐cell clones and lines which were expanded in vitro after isolation from intestinal biopsy tissue (Quarsten et al., 2001). Specificities associated with at least three different peptides from gliadins were identified, and it remains to be seen whether there are any immunodominant epitopes suitable for direct detection studies in blood or in tissue. In the case of the collagen pMHC tetramers, two studies (Buckner et al., 2002; Kotzin et al., 2000) described the use of HLA DR4 complexed with type II collagen epitopes, but failed to directly detect CD4þ antigen‐specific T cells in patients with rheumatoid arthritis or relapsing polychondritis, respectively. In the latter study, however, peripheral blood‐ derived T cells from patients with relapsing polychondritis were expanded in vitro, and subsequently shown to bind the collagen pMHC tetramer, which was also capable of activating the specific T cells (Kwok et al., 2002). Similar tetramers were used in a humanized animal model of arthritis, in which HLA DR4 transgenic mice were immunized with collagen and developed acutely arthritic joints (Svendsen et al., 2004). In these studies, tetramer‐positive cells that were present in blood after immunization accumulated in the lymph nodes, while levels declined in synovial fluid and blood. The mechanism for this shift was not determined, but as in the NOD model studies described

t et r a m e r a na ly s i s o f h u m a n au to r e act i v e t c el l s

63

previously, suggest the importance of evaluating different tissue compartments to fully evaluate the distribution of tetramer‐positive responses. These studies of tetramer‐binding properties reinforce the general features previously discussed, in that autoreactive T cells are generally of low‐frequency, low‐avidity, and variable phenotype when studied in the periphery of the autoimmune patient. However, they also shed new light on the biology of the autoreactive CD4þ T cell, in several respects. For example, the frequency of pMHC tetramer‐positive T cells waxes and wanes, as documented in the murine NOD (Trudeau et al., 2003) and CIA (Svendsen et al., 2004) studies, and both find that the peripheral blood compartment is quite different from tissue and lymph node compartments proximal to the autoimmune target. This is not particularly surprising, but does highlight the fact that human studies of autoimmune patients, using peripheral blood as the source of autoreactive T cells, need to be interpreted with the recognition that the temporal and spatial picture is incomplete. Even more problematic is the issue of TCR avidity for the pMHC complex controlling T‐cell fate and, potentially, immunologic outcome. A class I pMHC tetramer analysis of patients with vitiligo suggests that both high‐frequency and high‐avidity responses correlate with clinical autoimmunity. In this study, T cells interacting with pMHC tetramers for MelanA/MART‐1, Tyrosinase, and GP100 were found in all vitiligo patients studied, and include some high‐ avidity populations (Palermo et al., 2001). This contrasts with observations in the studies of relapsing polychondritis and in T1D (Buckner et al., 2002; Reijonen et al., 2002), in which autoreactive cells were of low frequency and low avidity. Many more dominant epitopes need to be examined to understand the possible immunologic basis for such differences. There is an additional confounding issue with respect to class II pMHC tetramers: namely, that single epitopes may be displayed on antigen‐presenting cells in a variety of peptides of different length, and this may lead to heterogenous T‐cell recognition profiles. Peptide epitopes for CD4þ T‐cell recognition in the context of MHC class II restriction are known to involve peptides of variable length. For example, in our studies of the naturally processed autoantigen GAD65, peptides eluted from HLA‐DR4 molecules encompassed a set of sequences surrounding a single immunodominant epitope, with a peptide length of 13 amino acids in the core sequence of GAD65 from residues 555–567 (Nepom et al., 2001). This epitope occurs within a nested set of naturally processed peptides, including two longer variants, representing residues 552–572, and residues 554–570. Figure 3 presents a comparison of T‐cell activation derived from stimulation of a panel of T‐cell clones, using all three of these naturally processed peptides. The T‐cell clones were derived by cell sorting of pMHC tetramer-binding CD4þ cells from peripheral blood of

64

g e ra l d t. n e po m

Figure 3 Epitope length matters. An immunodominant epitope of the autoantigen GAD65 encompasses residues 555–567, which are contained within two larger naturally processed peptides 554–570 and 552–572 (Nepom et al., 2001). Four CD4þ T‐cell lines are shown which recognize this epitope, resulting in proliferation (SI ¼ stimulation index, the ratio of T‐cell response to specific antigen divided by response in the absence of antigen, determined by thymidine uptake) and in cytokine release (gIFN, measured in ng/ml in supernatants from antigen‐responsive cultured cells). Also shown for each T‐cell line responding to each peptide are the flow cytometry profiles with pMHC tetramer binding on the vertical axis. In each case, the specific tetramer contained HLA DR4 complexed with peptides of different length, as shown. Some T cells (e.g., #123) recognize and respond to the longer 552–572 peptide better than to the shorter variants, whereas other T cells (e.g., #307, #325) show the best binding to the short 555–567 epitope tetramer. T‐cell #228 is an example of an antigen‐responsive cell that nonetheless fails to show detectable levels of pMHC tetramer binding. These highly variable responses coexist within the heterogeneous population of peripheral autoreactive GAD65-specific T cells.

T1D subjects. Variable levels of response, and indeed differences in cytokine release profiles, were seen for different length variants. Also shown in Fig. 3 are the tetramer‐binding profiles of these T‐cell clones for pMHC tetramers containing the three different length variants. Again, differences are seen that reflect different avidity of each T‐cell receptor for the three length variants. The implication of this complex relationship between TCR, peptide, and MHC is that in the natural processed environment a multitude of signals of different strength are offered, and the specific T‐cell outcome may well depend upon complex variables, including processing by the APC which determines antigen

t et r a m e r a na ly s i s o f h u m a n au to r e act i v e t c el l s

65

length, which then guides the quality and quantity of the subsequent immune response. 3. Tetramer‐Induced Autoreactivity In addition to visualizing autoreactive T cells, pMHC tetramers can be used to probe T‐cell responses. Just as anti‐CD3 antibodies trigger TCR activation cascades, albeit in an antigen nonspecific manner, pMHC tetramers act as ligands for analyzing antigen‐specific receptor‐initiated events, similarly triggering activation initiated by the TCR. One of the key determinants of differential outcomes for T‐cell responses is the TCR avidity for the pMHC complex. Studies (Mallone et al., 2004) compared T‐cell responses following activation through the TCR utilizing autoreactive pMHC tetramers, containing the GAD65 epitope. T cells in these studies were derived from patients with T1D, and were selected for either high or low avidity for antigen based on tetramer‐binding properties. Both high‐ and low‐avidity T cells showed dose‐dependent activation profiles to pMHC stimulation, with the high‐avidity T cells responding to much lower concentrations of stimuli. One notable difference was the sensitivity to activation‐induced cell death (AICD). Tetramer binding triggered a Fas‐dependent apoptotic mechanism in the high‐avidity T cells, but not in the low‐avidity cells. This very likely represents only one of a set of differential signals that are avidity‐dependent, reflected in different duration of TCR signaling, or perhaps, differential recruitment of accessory molecules. The potential for activation through pMHC interactions has attractive therapeutic possibilities. In animal studies of autoimmunity, administration of various pMHC complexes can have dramatic outcomes, including immune deviation, TH2 cytokine release, and deletional tolerance through activation‐induced cell death (Casares et al., 1999; 2002; Masteller et al., 2003; Sharma et al., 1991; Spack et al., 1995). Further studies with human T‐cell clones responding to myelin basic protein or to GAD65 similarly indicate the potential for immune deviation or anergy induction (Appel et al., 2001; Mallone et al., in press). The wide spectrum of differential avidity to self‐pMHC complexes in subjects with autoimmunity, and the differential activation program of these T cells when stimulated through the antigen‐specific TCR, raises important questions about disease pathogenesis: Are autoreactive pathogenic T cells the high‐avidity population? And if so, how did they escape negative selection during thymic development? Alternatively, are the pathogenic cells low‐avidity autoreactive T cells that escaped negative selection but became aberrantly activated in the periphery due to exposure to high‐avidity mimetics, high‐ antigen density, or costimulation, which lowered the threshold for activation?

66

g e ra l d t. n e po m

Although these are fundamental questions in understanding autoimmunity, answers are not known, and tetramer studies to date are consistent with all possibilities. As noted above, high‐ and low‐avidity autoreactive cells coexist in the peripheral blood of patients with autoimmune disease, suggesting that there is no strong bias toward expansion and deletion in vivo of either population. While the high‐avidity cell population may be sensitive to activationinduced cell death in the presence of autoantigens, this stimuli may, under some circumstances, preferentially induce anergy and allow for the persistence of these cells (Mallone et al., in press). A study (Ober et al., 2000) directly compared the avidity of TCR binding determined by pMHC tetramers with the thymic selection thresholds observed in vivo. High‐affinity binding of the HY self‐antigen indeed correlated with negative selection, whereas low‐affinity binding correlated with positive selection of the CD8þ T cells in this class I pMHC system. Nevertheless, the presence of high‐avidity self‐reactive T cells in the periphery of subjects with autoimmunity, and in the blood of normal human subjects (Danke et al., 2004) suggests that this negative selection mechanism is often incomplete. Similar to the observations in T1D subjects, studies using class I pMHC tetramers to the melanoma‐associated MAGE antigens have also documented a wide spectrum of tetramer staining intensity by peripheral T cells, correlating with high‐ and low‐avidity recognition of antigen. In this CD8þ T‐cell system, similar to the CD4þ T cells in T1D subjects, a wide range of functional programs were represented in the diverse T‐cell population (Dutoit et al., 2001). The avidity of T‐cell recognition is dependent on three interactions: The T‐cell receptor binding to pMHC, the peptide–MHC affinity, and the clustering of these complexes in the cell membrane. The interplay between these factors was evaluated using pMHC tetramers with class I molecules complexed with cartilage-associated aggrecan peptides (Appel et al., 2004). In this study, the low‐avidity peptide–MHC interaction was manipulated by mutagenesis of a cystine in the binding pocket of the HLA B27 class I molecule. When this interaction was of low avidity, tetramer stability and binding to TCR was impaired; when this interaction was of higher avidity due to the presence of the native cystine, the pMHC stability was restored, and the tetramer binding increased correspondingly. This observation illustrates how disease‐associated MHC molecules interacting with self‐peptides may directly contribute to skewing of the T‐cell avidity profile in the autoreactive population. Coupled with the use of altered peptide ligands which function as high‐avidity mimetics, as described previously, this has the potential to be useful in defining the avidity maturation of an ongoing autoimmune response and also lead to the improved design of therapeutic mimetics designed to elicit AICD.

t et r a m e r a na ly s i s o f h u m a n au to r e act i v e t c el l s

67

3.1. Tetramer‐Assisted Perspectives on Autoimmunity Tetramers are valuable additions to the immunology toolkit. They provide a molecular tag for one of the most important elements of the adaptive immune response, namely the specificity of recognition by T cells. Their use in studies of autoimmunity has already provided several new insights, which have implications for both basic and clinical immunology: First, tetramer studies directly detect high‐avidity autoreactive cells present in the peripheral circulation of human subjects. This indicates that selection mechanisms that delete autoreactive high‐avidity cells are incomplete most of the time, and directs new attention to studies that are focused on discovering what these high‐ avidity cells are destined to become. Second, tetramer studies have demonstrated that normal individuals have autoreactive circulating cells that expand when regulatory controls are released. This fundamentally illustrates the underlying autoreactive potential which is inherent in the immune system and which presents both a major therapeutic challenge and an exciting opportunity through manipulation of the regulatory pathway. Third, soluble tetramers provide very specific ways to engage the TCR on antigen‐specific lymphocytes, which can be used to initiate activation pathways ranging from expansion to cell death. Subtle modifications in the tetramer structure reveal a spectrum of TCR avidity within a polyclonal T‐cell response, which can be manipulated to direct a variety of different T‐cell outcomes. And finally, the clinical use of tetramers to evaluate progressive autoimmunity and response to immunotherapy, although in its infancy, promises to expand the types of individualized patient management strategies that may become commonplace in the era of personalized molecular medicine. Acknowledgments These studies were supported by grants from the National Institutes of Health and the Juvenile Diabetes Research Foundation. The work of many colleagues and members of the Nepom laboratory contributed to the studies cited, and their contributions are gratefully acknowledged.

References Altman, J. D., Moss, P. A. H., Goulder, P. J. R., Barouch, D. H., McHeyzer‐Williams, M. G., Bell, J. I., McMichael, A. J., and Davis, M. M. (1996). Phenotypic analysis of antigen‐specific T lymphocytes. Science 274, 94–96. Appel, H., Kuon, W., Kuhne, M., Hulsmeyer, M., Kollnberger, S., Kuhlmann, S., Weiss, E., Zeitz, M., Wucherpfennig, K., Bowness, P., and Sieper, J. (2004). The solvent‐inaccessible Cys67 residue of HLA‐B27 contributes to T cell recognition of HLA‐B27/peptide complexes. J. Immunol. 173, 6564–6573.

68

g e ra l d t. n e po m

Appel, H., Seth, N. P., Gauthier, L., and Wucherpfennig, K. W. (2001). Anergy induction by dimeric TCR ligands. J. Immunol. 166, 5279–5285. Barber, L. D., Whitelegg, A., Madrigal, J. A., Banner, N. R., and Rose, M. L. (2004). Detection of vimentin‐specific autoreactive CD8þ T cells in cardiac transplant patients. Transplantation 77, 1604–1609. Bischof, F., Hofmann, M., Schumacher, T. N., Vyth‐Dreese, F. A., Weissert, R., Schild, H., Kruisbeek, A. M., and Melms, A. (2004). Analysis of autoreactive CD4 T cells in experimental autoimmune encephalomyelitis after primary and secondary challenge using MHC class II tetramers. J. Immunol. 172, 2878–2884. Buckner, J. H., Van, L. M., Kwok, W. W., and Tsarknaridis, L. (2002). Identification of type II collagen peptide 261–273‐specific T cell clones in a patient with relapsing polychondritis. Arthritis. Rheum. 46, 238–244. Casares, S., Hurtado, A., McEvoy, R., Sarukhan, A., von Boehmer, H., and Brumeanu, T. D. (2002). Down‐regulation of diabetogenic CD4þ T cells by a soluble dimeric peptide‐MHC class II chimera. Nat. Immunol. 3, 383–391. Casares, S., Zong, C. S., Radu, D. L., Miller, A., Bona, C. A., and Brumeanu, T. D. (1999). Antigen‐ specific signaling by a soluble, dimeric peptide/major histocompatibility complex class II/Fc chimera leading to T helper cell type 2 differentiation. J. Exp. Med. 190, 543–553. Chen, C., Lee, W. H., Yun, P., Snow, P., and Liu, C. P. (2003). Induction of autoantigen‐specific Th2 and Tr1 regulatory T cells and modulation of autoimmune diabetes. J. Immunol. 171, 733–744. Cochran, J. R., Cameron, T. O., and Stern, L. J. (2000). The relationship of MHC‐peptide binding and T cell activation probed using chemically defined MHC class II oligomers. Immunity 12, 241–250. Cohen, C. J., Denkberg, G., Schiffenbauer, Y. S., Segal, D., Trubniykov, E., Berke, G., and Reiter, Y. (2003). Simultaneous monitoring of binding to and activation of tumor‐specific T lymphocytes by peptide‐MHC. J. Immunol. Methods 277, 39–52. Danke, N. A., Koelle, D. M., Yee, C., Beheray, S., and Kwok, W. W. (2004). Autoreactive T cells in healthy individuals. J. Immunol. 172, 5967–5972. Danke, N. A., and Kwok, W. W. (2003). HLA class II‐restricted CD4þ T cell responses directed against influenza viral antigens postinfluenza vaccination. J. Immunol. 171, 3163–3169. Dutoit, V., Rubio‐Godoy, V., Dietrich, P. Y., Quiqueres, A. L., Schnuriger, V., Rimoldi, D., Lienard, D., Speiser, D., Guillaume, P., Batard, P., Cerottini, J. C., Romero, P., and Valmori, D. (2001). Heterogeneous T‐cell response to MAGE‐A10(254–262): High avidity‐specific cytolytic T lymphocytes show superior antitumor activity. Cancer Res. 61, 5850–5856. Gebe, J. A., Falk, B. A., Rock, K. A., Kochik, S. A., Heninger, A. K., Reijonen, H., Kwok, W. W., and Nepom, G. T. (2003). Low avidity recognition by CD4þ T cells directed to self‐antigens. Eur. J. Immunol. 33, 1409–1417. Hoffmann, T. K., Donnenberg, A. D., Finkelstein, S. D., Donnenberg, V. S., Friebe‐Hoffmann, U., Myers, E. N., Appella, E., De Leo, A. B., and Whiteside, T. L. (2002). Frequencies of tetramerþ T cells specific for the wild‐type sequence p53(264–272) peptide in the circulation of patients with head and neck cancer. Cancer Res. 62, 3521–3529. Holzer, U., Kwok, W. W., Nepom, G. T., and Buckner, J. H. (2003). Differential antigen sensitivity and costimulatory requirements in human Th1 and Th2 antigen‐specific CD4þ cells with similar TCR avidity. J. Immunol. 170, 1218–1223. Hu, H. M., Dols, A., Meijer, S. L., Floyd, K., Walker, E., Urba, W. J., and Fox, B. A. (2004). Immunological monitoring of patients with melanoma after peptide vaccination using soluble peptide/HLA‐A2 dimer complexes. J. Immunother. 27, 48–59.

t et r a m e r a na ly s i s o f h u m a n au to r e act i v e t c el l s

69

Jaeckel, E., Klein, L., Martin‐Orozco, N., and von Boehmer, H. (2003). Normal incidence of diabetes in NOD mice tolerant to glutamic acid decarboxylase. J. Exp. Med. 197, 1635–1644. Jager, E., Hohn, H., Necker, A., Forster, R., Karbach, J., Freitag, K., Neukirch, C., Castelli, C., Salter, R. D., Knuth, A., and Maeurer, M. J. (2002). Peptide‐specific CD8þ T‐cell evolution in vivo: Response to peptide vaccination with Melan‐A/MART‐1. Int. J. Cancer 98, 376–388. Jang, M. H., Seth, N. P., and Wucherpfennig, K. W. (2003). Ex vivo analysis of thymic CD4 T cells in nonobese diabetic mice with tetramers generated from I‐A(g7)/class II‐associated invariant chain peptide precursors. J. Immunol. 171, 4175–4186. Jun, H. S., Chung, Y. H., Han, J., Kim, A., Yoo, S. S., Sherwin, R. S., and Yoon, J. W. (2002). Prevention of autoimmune diabetes by immunogene therapy using recombinant vaccinia virus expressing glutamic acid decarboxylase. Diabetologia 45, 668–676. Kim, S. K., Tarbell, K. V., Sanna, M., Vadeboncoeur, M., Warganich, T., Lee, M., Davis, M., and McDevitt, H. O. (2004). Prevention of type I diabetes transfer by glutamic acid decarboxylase 65 peptide 206–220‐specific T cells. Proc. Natl. Acad. Sci. USA 101, 14204–14209. Kobayashi, T., Lonchay, C., Colau, D., Demotte, N., Boon, T., and van der, B. P. (2003). New MAGE‐4 antigenic peptide recognized by cytolytic T lymphocytes on HLA‐A1 tumor cells. Tissue Antigens 62, 426–432. Kotzin, B. L., Falta, M. T., Crawford, F., Rosloniec, E. F., Bill, J., Marrack, P., and Kappler, J. (2000). Use of soluble peptide‐DR4 tetramers to detect synovial T cells specific for cartilage antigens in patients with rheumatoid arthritis. Proc. Natl. Acad. Sci. USA 97, 291–296. Krogsgaard, M., Li, Q. J., Sumen, C., Huppa, J. B., Huse, M., and Davis, M. M. (2005). Agonist/ endogenous peptide‐MHC heterodimers drive T cell activation and sensitivity. Nature 434, 238–243. Kwok, W. W., Ptacek, N. A., Liu, A. W., and Buckner, J. H. (2002). Use of class II tetramers for identification of CD4þ T cells. J. Immunol. Methods 268, 71–81. Lieberman, S. M., Takaki, T., Han, B., Santamaria, P., Serreze, D. V., and Di Lorenzo, T. P. (2004). Individual nonobese diabetic mice exhibit unique patterns of CD8þ T cell reactivity to three islet antigens, including the newly identified widely expressed dystrophia myotonica kinase. J. Immunol. 173, 6727–6734. Liu, C.‐P., Jiang, K., Wu, C.‐H., Lee, W.‐H., and Lin, W.‐J. (2000). Detection of glutamic acid decarboxylase‐activated T cells with I‐Ag7 tetramers. Proc. Natl. Acad. Sci. USA 97, 14596–14601. Mallone, R., Kochik, S. A., Laughlin, E., Gersuk, V., Reijonen, H., Kwok, W. W., and Nepom, G. T. (2004). Differential recognition and activation thresholds in human autoreactive GAD‐specific T‐cells. Diabetes 53, 971–977. Mallone, R., Kochik, S. A., Reijonen, H., Carson, B. D., Ziegler, S. F., Kwok, W. W., and Nepom, G. T. (2005). T‐Cell Receptor Avidity Determines Different Apoptotic and Regulatory Outcomes in Human GAD‐specific CD4þ T‐cells. Blood, in press. Mami‐Chouaib, F., Echchakir, H., Dorothee, G., Vergnon, I., and Chouaib, S. (2002). Antitumor cytotoxic T‐lymphocyte response in human lung carcinoma: Identification of a tumor‐associated antigen. Immunol. Rev. 188, 114–121. Masteller, E. L., Warner, M. R., Ferlin, W., Judkowski, V., Wilson, D., Glaichenhaus, N., and Bluestone, J. A. (2003). Peptide‐MHC class II dimers as therapeutics to modulate antigen‐ specific T cell responses in autoimmune diabetes. J. Immunol. 171, 5587–5595. Nepom, G. T., and Kwok, W. W. (1998). Molecular basis for HLA‐DQ associations with IDDM. Diabetes 47, 1177–1184. Nepom, G. T., Lippolis, J. D., White, F. M., Masewicz, S., Marto, J. A., Herman, A., Falk, B., Shabanowitz, J., Hunt, D. F., Engelhard, V. H., and Nepom, B. S. (2001). Identification and modulation of a naturally processed T cell epitope from the diabetes‐associated autoantigen human glutamic acid decarboxylase 65 (hGAD65). Proc. Natl. Acad. Sci. USA 98, 1763–1768.

70

g e ra l d t. n e po m

Novak, E. J., Liu, A. W., Nepom, G. T., and Kwok, W. W. (1999). MHC class II tetramers identify peptide‐specific human CD4(þ) T cells proliferating in response to influenza A antigen. J. Clin. Invest. 104, R63–R67. Novak, E. J., Masewicz, S. A., Liu, A. W., Lernmark, A˚ ., Kwok, W. W., and Nepom, G. T. (2001). Activated human epitope‐specific T cells identified by class II tetramers reside within a CD4high, proliferating subset. Int. Immunol. 13, 799–806. Ober, B. T., Hu, Q., Opferman, J. T., Hagevik, S., Chiu, N., Wang, C. R., and Ashton‐Rickardt, P. G. (2000). Affinity of thymic self‐peptides for the TCR determines the selection of CD8(þ) T lymphocytes in the thymus. Int. Immunol. 12, 1353–1363. Palermo, B., Campanelli, R., Garbelli, S., Mantovani, S., Lantelme, E., Brazzelli, V., Ardigo, M., Borroni, G., Martinetti, M., Badulli, C., Necker, A., and Giachino, C. (2001). Specific cytotoxic T lymphocyte responses against Melan‐A/MART1, tyrosinase and gp100 in vitiligo by the use of major histocompatibility complex/peptide tetramers: The role of cellular immunity in the etiopathogenesis of vitiligo. J. Invest Dermatol. 117, 326–332. Quarsten, H., McAdam, S. N., Jensen, T., Arentz‐Hansen, H., Molberg, O., Lundin, K. E., and Sollid, L. M. (2001). Staining of celiac disease‐relevant T cells by peptide‐DQ2 multimers. J. Immunol. 167, 4861–4868. Radu, C. G., Anderton, S. M., Firan, M., Wraith, D. C., and Ward, E. S. (2000). Detection of autoreactive T cells in H‐2u mice using peptide‐MHC multimers. Int. Immunol. 12, 1553–1560. Reddy, J., Bettelli, E., Nicholson, L., Waldner, H., Jang, M. H., Wucherpfennig, K. W., and Kuchroo, V. K. (2003). Detection of autoreactive myelin proteolipid protein 139–151‐specific T cells by using MHC II (IAs) tetramers. J. Immunol. 170, 870–877. Reijonen, H., Kwok, W. W., and Nepom, G. T. (2003). Detection of CD4þ autoreactive T cells in T1D using HLA class II tetramers. Ann. N. Y. Acad. Sci. 1005, 82–87. Reijonen, H., Mallone, R., Heninger, A., Laughlin, E., Kochik, S., Falk, B., Kwok, W. W., Greenbaum, C. J., and Nepom, G. T. (2004). GAD65 Specific CD4þ T‐cells with High Antigen Avidity are Prevalent in Peripheral Blood of Type 1 Diabetes Patients. Diabetes 53, 1987–1994. Reijonen, H., Novak, E. J., Kochik, S., Heninger, A., Liu, A., Kwok, W., and Nepom, G. (2002). Detection of GAD65 specific T‐cells by major histocompatibility complex class II tetramers in type 1 diabetes patients and at‐risk subjects. Diabetes 51, 1375–1382. Reynolds, S. R., Celis, E., Sette, A., Oratz, R., Shapiro, R. L., Johnston, D., Fotino, M., and Bystryn, J. C. (1998). HLA‐independent heterogeneity of CD8þ T cell responses to MAGE‐3, Melan‐A/MART‐1, gp100, tyrosinase, MC1R, and TRP‐2 in vaccine‐treated melanoma patients. J. Immunol. 161, 6970–6976. Sato, Y., Nabeta, Y., Tsukahara, T., Hirohashi, Y., Syunsui, R., Maeda, A., Sahara, H., Ikeda, H., Torigoe, T., Ichimiya, S., Wada, T., Yamashita, T., Hiraga, H., Kawai, A., Ishii, T., Araki, N., Myoui, A., Matsumoto, S., Umeda, T., Ishii, S., Kawaguchi, S., and Sato, N. (2002). Detection and induction of CTLs specific for SYT‐SSX‐derived peptides in HLA‐A24(þ) patients with synovial sarcoma. J. Immunol. 169, 1611–1618. Sercarz, E. E. (2003). Large antigens such as GAD‐65 become involved in pathogenic self‐ reactivity, but fortunately only rarely. J. Autoimmun. 20, 203–206. Sharma, S. D., Nag, B., Su, X.‐M., Green, D., Spack, E., Clark, B. R., and Sriram, S. (1991). Antigen‐specific therapy of experimental allergic encephalomyelitis by soluble class II major histocompatibility complex‐peptide complexes. Proc. Natl. Acad. Sci. USA 88, 11465–11469. Spack, E. G., McCutcheon, M., Corbelletta, N., Nag, B., Passmore, D., and Sharma, S. D. (1995). Induction of tolerance in experimental autoimmune myasthenia gravis with solubilized MHC class II: Acetylcholine receptor peptide complexes. J. Autoimmun. 8, 787–807. Speiser, D. E., Lienard, D., Pittet, M. J., Batard, P., Rimoldi, D., Guillaume, P., Cerottini, J. C., and Romero, P. (2002). In vivo activation of melanoma‐specific CD8(þ) T cells by endogenous tumor

t et r a m e r a na ly s i s o f h u m a n au to r e act i v e t c el l s

71

antigen and peptide vaccines. A comparison to virus‐specific T cells. Eur. J. Immunol. 32, 731–741. Stratmann, T., Martin‐Orozco, N., Mallet‐Designe, V., Poirot, L., McGavern, D., Losyev, G., Dobbs, C. M., Oldstone, M. B., Yoshida, K., Kikutani, H., Mathis, D., Benoist, C., Haskins, K., and Teyton, L. (2003). Susceptible MHC alleles, not background genes, select an autoimmune T cell reactivity. J. Clin. Invest 112, 902–914. Svendsen, P., Andersen, C. B., Willcox, N., Coyle, A. J., Holmdahl, R., Kamradt, T., and Fugger, L. (2004). Tracking of proinflammatory collagen‐specific T cells in early and late collagen‐induced arthritis in humanized mice. J. Immunol. 173, 7037–7045. Tang, Q., Henriksen, K. J., Bi, M., Finger, E. B., Szot, G., Ye, J., Masteller, E. L., McDevitt, H., Bonyhadi, M., and Bluestone, J. A. (2004). In vitro‐expanded antigen‐specific regulatory T cells suppress autoimmune diabetes. J. Exp. Med. 199, 1455–1465. Tarbell, K. V., Yamazaki, S., Olson, K., Toy, P., and Steinman, R. M. (2004). CD25þ CD4þ T cells, expanded with dendritic cells presenting a single autoantigenic peptide, suppress autoimmune diabetes. J. Exp. Med. 199, 1467–1477. Trudeau, J. D., Kelly‐Smith, C., Verchere, C. B., Elliott, J. F., Dutz, J. P., Finegood, D. T., Santamaria, P., and Tan, R. (2003). Prediction of spontaneous autoimmune diabetes in NOD mice by quantification of autoreactive T cells in peripheral blood. J. Clin. Invest 111, 217–223. Valmori, D., Dutoit, V., Ayyoub, M., Rimoldi, D., Guillaume, P., Lienard, D., Lejeune, F., Cerottini, J. C., Romero, P., and Speiser, D. E. (2003). Simultaneous CD8þ T cell responses to multiple tumor antigen epitopes in a multipeptide melanoma vaccine. Cancer Immun. 3, 15. van Rhee, F., Szmania, S. M., Zhan, F., Gupta, S. K., Pomtree, M., Lin, P., Batchu, R. B., Moreno, A., Spagnoli, G., Shaughnessy, J., and Tricot, G. J. (2005). NY‐ESO‐1 is highly expressed in poor prognosis multiple myeloma and induces spontaneous humoral and cellular immune responses. Blood 105, 3939–3944. Walker, M. R., Carson, B. D., Nepom, G. T., Ziegler, S. F., and Buckner, J. H. (2005). De novo generation of antigen‐specific CD4þCD25þ regulatory T cells from human CD4þ. Proc. Natl. Acad. Sci. USA 102, 4103–4108. Walker, M. R., Kasprowicz, D. J., Gersuk, V. H., Benard, A., Van Landeghen, M., Buckner, J. H., and Ziegler, S. F. (2003). Induction of FoxP3 and acquisition of T regulatory activity by stimulated human CD4þCD25‐ T cells. J. Clin. Invest. 112, 1437–1443. Woll, M. M., Fisher, C. M., Ryan, G. B., Gurney, J. M., Storrer, C. E., Ioannides, C. G., Shriver, C. D., Moul, J. W., McLeod, D. G., Ponniah, S., and Peoples, G. E. (2004). Direct measurement of peptide‐specific CD8þ T cells using HLA‐A2:Ig dimer for monitoring the in vivo immune response to a HER2/neu vaccine in breast and prostate cancer patients. J. Clin. Immunol. 24, 449–461. Yoshida, K., Martin, T., Yamamoto, K., Dobbs, C., Munz, C., Kamikawaji, N., Nakano, N., Rammensee, H. G., Sasazuki, T., Haskins, K., and Kikutani, H. (2002). Evidence for shared recognition of a peptide ligand by a diverse panel of nonobese diabetic mice‐derived, islet‐ specific, diabetogenic T cell clones. Int. Immunol. 14, 1439–1447.

Regulation of Phospholipase C‐g2 Networks in B Lymphocytes Masaki Hikida and Tomohiro Kurosaki Laboratory for Lymphocyte Differentiation, RIKEN Research Center for Allergy and Immunology, Tsurumi‐ku, Yokohama, Kanagawa 230‐0045, Japan

1. 2. 3. 4. 5. 6. 7. 8. 9.

Abstract............................................................................................................. Introduction ....................................................................................................... From the BCR to PLC‐g ...................................................................................... Roles for PLC‐g .................................................................................................. Regulation of IP3 Receptors .................................................................................. Coupling Between Calcium Release and Calcium Influx ............................................. Looking for Ca2þ Entry Channels .......................................................................... NFAT and NF‐kB ............................................................................................... Modulation of Calcium Signaling............................................................................ Concluding Remarks............................................................................................ References .........................................................................................................

73 73 74 78 79 81 83 86 88 89 90

Abstract The modulation of inositol‐1,4,5‐trisphosphate (IP3), a product of phospholipase C (PLC) activity, is one of a common signaling mechanism used in many biological systems. B lymphocytes also rely on IP3 and subsequent calcium signaling to ensure appropriate developmental outcomes, as well as antigen‐ specific responses. In establishing the optimal intensity and duration of the PLC‐g activity, an important role has emerged for adaptor molecules, which direct the appropriate subcellular localization of PLC‐g and induce its conformational changes. Generated IP3 binds to IP3 receptors located on the endoplasmic reticulum (ER), which in turn is essential for triggering calcium release from the ER and subsequent entry of extracellular calcium by so‐called Ca2þ entry channels. Recent data has begun to shed new light on the connection between the calcium release and the influx of extracellular calcium, and the molecular identity of the Ca2þ entry channels. 1. Introduction The tremendous diversity and plasticity of the immune responses are well exemplified by the B‐cell limb of the immune system. To accomplish this, B‐cell signaling through its antigen receptor (BCR) leads to a wide range of biological outputs, depending upon developmental stages and properties of the antigen. A simple ‘‘on‐and‐off’’ mode of signal transduction would not allow for such a diversity of responses. Rather, BCR signals must be precisely

73 advances in immunology, vol. 88 # 2005 Elsevier Inc. All rights reserved.

0065-2776/05 $35.00 DOI: 10.1016/S0065-2776(05)88003-4

74

m as a ki h i ki da a n d t o m oh i ro k u ro s a k i

regulated in terms of their magnitude and duration. This is typically shown in the case of the PLC‐g‐calcium pathway. A large transient calcium rise activates NF‐kB, whereas NFAT is activated by a slow but sustained calcium plateau (Dolmetsch et al., 1997). Given the importance of the transcription factors (NF‐kB and NFAT) in B‐cell differentiation and survival (Choi et al., 1994; Henderson and Calame, 1998; Li and Verma, 2002; Peng et al., 2001; Venkataraman et al., 1994), these findings have spurred interest in uncovering when and how such differential Ca2þ signals are generated, and how these differential signals are translated into biological outcomes. Thus, the purpose of this review is to summarize the major advances in the PLC‐g‐calcium pathway in B cells, with specific emphasis on the significance of positive‐ or negative‐feedback loops in this pathway. In these regards, the first section addresses the roles of BCR‐proximal signaling molecules in the regulation of PLC‐g. Secondly, we address how IP3 and diacylglycerol (DAG), both of which are products of the PLC‐g action, trigger the Ca2þ release channels on the ER, and subsequently the Ca2þ entry channels in the plasma membrane. Finally, we discuss the role of PLC‐g‐calcium‐regulated effectors in B‐cell activation. 2. From the BCR to PLC‐g T cells mainly express the PLC‐g1 isoform, whereas PLC‐g2 is predominantly expressed in B cells (Hashimoto et al., 2000; Irvin et al., 2000; Park et al., 1991; Rhee and Bae, 1997; Secrist, et al.,1991; Wang et al., 2000). PLC‐g2 is activated by, at least, tyrosine phosphorylation by a complex of proteins connected to the BCR and the coordinated actions of recruitment to the inner face of the plasma membrane, where its substrate, PIP2, is located. Among three distinct families of nonreceptor tyrosine kinases (Lyn, Syk, and Btk), Syk and Btk are obligatory kinases in PLC‐g2 activation that follows BCR stimulation (Fig. 1) (Kurosaki, 1999). Indeed, the reduced levels of BCR‐induced IP3 production and calcium mobilization, which reflect PLC‐g activation, are seen in B cells from X‐linked agammaglobulinemia (XLA) patients and X‐linked immunodeficiency (Xid) mice (Fluckiger et al., 1998; Rigley et al., 1989), both of who have defective Btk genes. This defective PLC‐g2‐calcium signal in XLA patients and Xid mice is likely to cause the impaired maturation of B cells and compromised T‐cell‐independent immune responses (Rawlings and Witte, 1994; Smith et al., 1994). In the case of disruption of Syk, compared with that of Btk, a more severe phenotype in PLC‐g activation is seen, simply suggesting that Syk lies upstream of Btk in terms of BCR‐mediated PLC‐g2 activation (Takata et al., 1994; Takata and Kurosaki, 1996).

r eg u l at i o n of pl c -g2 n e t w o rk s

75

Figure 1 Activation mechanisms of PLC‐g2. Upon BCR stimulation, Iga and Igb are doubly phosphorylated by Src‐PTKs, which leads Syk recruitment. BLNK, after being phosphorylated by activated Syk, binds Btk and PLC‐g2 in a SH2‐phosphotyrosine‐dependent manner. This association brings Btk and PLC‐g2 close to each other and also supports recruitment of these molecules to the plasma membrane. Thus, PLC‐g2 is phosphorylated by Btk and gains access to its substrate PIP2. After hydrolysis of PIP2 by PLC‐g2, generated IP3 binds to the IP3 receptor and stimulates the subsequent Ca2þ release from the intracellular pool.

For full activation of Syk, it requires not only an open conformation by binding of its two tandem SH2 domains to the phosphorylated Iga and Igb, but also the autophosphorylation of Syk at the regulatory loop of the kinase domain (Kurosaki et al., 1995; Rowley et al., 1995). In addition, Syk also undergoes phosphorylation on several other tyrosine residues that are located in the linker region connecting the two tandem SH2 domains (interdomain A), or the SH2 domains to the kinase domain (interdomain B) including Tyr317 (Keshvara et al., 1998). Although phosphorylation of these sites occurs as a consequence of Syk activation, this phosphorylation, in turn, could function as an initiator to attenuate the activation of Syk, thereby contributing to formation of a negative‐feedback regulation loop for Syk activation. For instance, Tyr317 in Syk is phosphorylated after BCR cross‐linking, thereby providing a binding site for Cbl and Cbl‐b (Deckert et al., 1998). Considering that the ring finger domains of the Cbl family act as E3 ubiquitin ligases that downregulate the activities of signaling molecules through protein ubiquitination/degradation (Joazeiro et al., 1999; Levkowitz et al., 1999; Yokouchi et al., 1999), a promising model has emerged that proposes that Cbl and/or Cbl‐b, after their

76

m as a ki h i ki da a n d t o m oh i ro k u ro s a k i

Figure 2 Models of negative‐feedback regulatory loops for Syk and Btk, and positive‐forward regulatory loops for PLC‐g2. (A) Activated Syk autophosphorylates Tyr317, in addition to its substrates such as BLNK. This Tyr317 phosphorylation provides a binding site for Cbl, which acts as E3 ubiquitin ligase. Ubiquitin‐mediated degradation of Syk results in the attenuation of BCR signaling. (B) In this model, PLC‐g2 is activated by virtue of Btk action, leading to generation of DAG. Then, the generated DAG recruits PKCb to the plasma membrane, wherein PKCb is activated. Phosphorylation of Btk by PKCb downregulates Btk kinase activity and decreases its membrane localization, both of which in turn inhibit PLC‐g2 activity. (C) Calcium influx can be

r eg u l at i o n of pl c -g2 n e t w o rk s

77

binding to the phosphorylated Tyr317 in Syk, dampen the Syk activity (Fig. 2A) (Thien and Langdon, 2001). In support of this model, B cells from Cbl‐b‐ deficient mice demonstrate reduced ubiquitination and subsequent prolonged phosphorylation of Syk during BCR stimulation (Sohn et al., 2003). The importance of kinase activity of Btk is clear; after being activated by its recruitment to the plasma membrane, Btk phosphorylates Tyr753 and Tyr759 on PLC‐g2, thereby augmenting the enzymatic activity of PLC‐g2 (Fig. 1) (Humphries et al., 2004; Kim et al., 2004; Rodriguez et al., 2001; Watanabe et al., 2001). But, new data suggest that Btk plays an additional role in a kinase‐ independent manner as well (Schwartzberg, 2003). The Btk PH/TH domain associates with phosphatidylinositol‐4‐phosphate 5 kinase (PIP5K) and participates in recruitment of PIP5K to the plasma membrane (Saito et al., 2003). Because PIP2, a substrate for both PI3K and PLC‐g2, is synthesized from PIP by PIP5K, this PIP5K recruitment results in generation of more PIP2. Thus, Btk plays two roles in PLC‐g2 activation. In addition to phosphorylating Tyr753 and Tyr759 on PLC‐g2, the recruitment of the Btk‐PIP5K complex to the membrane ensures that the activated PLC‐g2 does not run out of its substrate, thereby contributing to efficient production of IP3 and DAG. The existence of a negative‐feedback regulatory loop for Btk activation has been suggested by recent results (Kang et al., 2001; Yao et al., 1994). A first clue to this negative loop came from the observation that BCR‐mediated tyrosine phosphorylation of Btk is increased and prolonged in PKCb‐deficient B cells, which indicates PKCb functions as a potent inhibitor of Btk (Kang et al., 2001). As Btk has a positive role in the PLC‐g2‐calcium‐PKC pathway, a model is that PKCb, after being activated by a Btk‐dependent mechanism, phosphorylates Btk, which in turn, inactivates Btk kinase activity and subsequent PLC‐g2 activity (Fig. 2B). Indeed, Ser180 in the TH domain of Btk has been identified as a PKCb phosphorylation site, and the Ser180Ala Btk mutant is hyperactive, presumably owing to enhanced membrane localization as well as enhanced enzymatic activity. Furthermore, inhibition of PKCs leads to augmented PLC‐g2 activation (Kim et al., 2004). So, this negative‐feedback mechanism could contribute to controlling the duration of Btk residency in the plasma membrane and subsequent PLC‐g2 activation. Adaptor molecules serve as a substrate of BCR‐induced tyrosine kinases and phosphorylation of these molecules is essential for subsequent PLC‐g2 activation (Kurosaki, 2002). BLNK (also known as SLP‐65, BASH, and BCA), induced by PLC‐g2 and subsequent calcium release from ER. This calcium influx mediated by SOCs has been shown to recruit PLC‐g2 to the plasma membrane in the C2 domain‐dependent manner, thereby amplifying hydrolysis of PIP2 in the plasma membrane.

78

m as a ki h i ki da a n d t o m oh i ro k u ro s a k i

after being phosphorylated by Syk, binds to Btk, Vav, and PLC‐g2 in a SH2‐phosphotyrosine‐dependent manner (Chiu et al., 2002; Fu et al., 1998; Hashimoto et al., 1999; Ishiai et al., 1999; Johmura et al., 2003). As BLNK is translocated to the plasma membrane after BCR stimulation, these binding causes two consequences; recruiting Btk, Vav, and PLC‐g2 to the plasma membrane, and bringing them into close proximity with each other. Thus, the former mechanism makes PLC‐g2 gain access to its substrate PIP2, located in the plasma membrane, while the latter one facilitates phosphorylation of Tyr753 and Tyr759 on PLC‐g2 by Btk (Humphries et al., 2004; Kim et al., 2004; Watanabe et al., 2001) (Fig. 1). In addition to Tyr753 and Tyr759, PLC‐g 2 undergoes phosphorylation at Tyr1217 in a Btk‐independent manner (Kim et al., 2004). Although the importance of this phosphorylation in the BCR signaling context is clear, this is probably not caused by direct activation of the PLC‐g2 lipase activity. Rather, phosphorylation on Tyr1217 seems to induce association with as‐yet unidentified SH2‐containing molecules, thereby stabilizing the PLC‐g2 residency in the plasma membrane and/or enhancing accessibility of PLC‐g2 to its substrate. PI3K activation is thought to contribute to PLC‐g2 activation through stabilizing the association of Btk and PLC‐g2 to the plasma membrane, given that both Btk and PLC‐g2 possess PH domains that bind selectively to the PI3K product PIP3 (Fig. 1). In fact, the importance of PIP3 in PLC‐g2 activation is further evidenced by deletion of SHIP (SH2 domain‐containing 5’ inositol phosphatase), a counteracting enzyme toward the action of PI3K (Brauweiler et al., 2000; Okada et al., 1998). A deficiency in this lipid phosphatase leads to increased PIP3, thereby enhancing PLC‐g2 phosphorylation and its subsequent activation (Brauweiler et al., 2000). 3. Roles for PLC‐g A central role for PLC‐g2 in calcium mobilization in B lymphocytes has been demonstrated using gene‐targeting experiments both in the cell line and in mice (Hashimoto et al., 2000; Takata et al., 1995; Wang et al., 2000). PLC‐g2‐ deficient mice exhibit a partial block at the developmental transition after pre‐BCR signaling and a more complete block at the transition from immature to mature B cells (Bell et al., 2004; Hashimoto et al., 2000; Hikida et al., 2003; Wang et al., 2000). As the defect in the pre‐BCR checking point is similar to, but less complete than, the block in Btk/Tec double knockout mice, low‐level expression of PLC‐g1 in pre‐B cells has been thought to compensate for PLC‐g2. Indeed, this idea is recently substantiated by the observation that PLC‐g1 haploid insufficiency in a PLC‐g2 null background resulted in a more complete block from pro‐ to pre‐B‐cell stage. More interestingly, allelic

r eg u l at i o n of pl c -g2 n e t w o rk s

79

exclusion of the immunoglobulin heavy chain was perturbed in these mice (Wen et al., 2004). Hence, assuming that PLC‐g1 mediates a qualitatively identical signal with PLC‐g2, a certain level of PLC‐g activity is required for progression through the pre‐B‐cell stage as well as inducing allelic exclusion, and higher PLC‐g threshold appears to be set for further making up the mature B‐cell pools. 4. Regulation of IP3 Receptors After PLC‐g2 activation, the generated IP3 binds to IP3 receptors located in the ER, thereby stimulating the release of calcium from internal stores. It is thought that, once these calcium stores are emptied, the entry of external calcium is triggered through so‐called store‐operated calcium channels (SOCs) in the plasma membrane (Parekh and Penner, 1997). Three distinct IP3 receptor genes are identified with differential expression patterns, depending upon distinct cell types (Dent et al., 1996; Miyakawa et al., 1999; Newton et al., 1994; Wojcikiewicz et al., 1994). Triple knockout of all three IP3 receptor isoforms in DT40 B cells abolishes the BCR‐induced calcium mobilization both from internal stores and from extracellular stores, whereas this calcium mobilization still occurs by a single knockout of these three receptors. Thus, overall, three IP3 receptors are essential and functional redundant mediators for BCR‐mediated calcium mobilization (Miyakawa et al., 1999). And these data appear to support the store‐operated coupling model mentioned previously. Although expression of only one isoform does not abolish calcium mobilization, detailed calcium‐signaling patterns differ significantly among these three IP3 receptor isoforms. For instance, DT40 B cells expressing only the type 2 IP3 receptor showed regular and robust calcium oscillations upon BCR ligation, whereas monophasic calcium transient or rapidly dampened calcium oscillations were observed in mutant B cells expressing type 3 or type 1 alone, respectively (Miyakawa et al., 1999). Hence, differential and combinatorial expression of these three IP3 receptors is one of the critical determinants for creating transient or oscillatory calcium signals, which in turn could regulate the selectivity of transcriptional factors in B cells. For instance, NF‐kB is activated by a large transient calcium rise, whereas NFAT requires a slow, but sustained calcium plateau (Dolmetsch et al., 1997). Then, the question arises about the mechanisms underlying such differential calcium oscillation patterns. Although being not entirely clear, a clue came from mutational analysis of the type 1 IP3 receptor (Miyakawa et al., 2001). Binding of IP3 to the IP3 receptors is essential, but not sufficient to open the calcium‐release channel embedded in these receptors, and calcium by itself is

80

m as a ki h i ki da a n d t o m oh i ro k u ro s a k i

Figure 3 Models of SOCs activation. (A) Left, the direct activation model by the diffusible messenger. In this model, it is postulated that depletion of calcium from intracellular store induces production of calcium influx factor (CIF). CIF diffuses to the plasma membrane and activates SOCs directly. Right, the indirect activation model by the diffusible messenger. CIF produced by depletion of the internal calcium pool diffuses and activates iPLA2 by displacement of inhibitory calmodulin (CaM). Once iPLA2 gets activated, it generates lysophospholipids, which in turn activate SOCs. (B) Left, the direct conformational‐coupling model. In this model, it is postulated

r eg u l at i o n of pl c -g2 n e t w o rk s

81

thought to be required as a coagonist for the IP3 receptors. Asp2100 is responsible for such a calcium sensor, because its replacement with Glu causes a 10‐fold decrease in calcium sensitivity, but bears normal IP3 sensitivity. This Asp2100Glu mutant exhibits a dramatic decrease in calcium oscillation activity. Furthermore, consistent with the ability of the type 2 IP3 receptor to create robust calcium oscillations, this receptor, compared with type 1 and type 3, possesses higher calcium sensitivity (Miyakawa et al., 1999). Therefore, these data provide compelling evidence that calcium initially released by IP3 receptors feeds back to augment further calcium release in a positively cooperative fashion, thereby contributing to generation of calcium oscillations. 5. Coupling Between Calcium Release and Calcium Influx The mechanism by which SOCs are activated by intracellular calcium release still remains a mystery. SOCs are named functionally, particularly in terms of electrophysiological criteria, but not molecularly. Hence, for solving this important question, two interrelated issues should be addressed: the molecular identity of SOCs, and the nature of the signal that activates them. In the first issue, much recent interest has been directed toward the TRP‐family of ion channels and they have been considered likely to encode components of SOCs, as discussed in greater detail in the next section. Because of a limited amount of information about these two issues in lymphocytes, it is important to incorporate more from other cell types in this section. There are currently three models (Fig. 3), for activation of SOCs, but they are not mutually exclusive. Recent studies have suggested that these three models could coexist in the same cells and cooperate even in a single receptor system. The diffusible messenger model is the oldest one (Randriamampita and Tsien, 1993). According to this model, calcium influx factor (CIF) is produced by depleted internal stores and it triggers activation of SOCs (Fig. 3A, left panel). After initial excitement, the CIF model was strongly criticized because of a continuous uncertainty about the molecular nature, but a few

that conformational change of the IP3 receptor takes place by its opening and this change is transmitted to SOCs by direct interaction and activation of SOCs. Right, the indirect conformational‐coupling model. Instead of the IP3 receptor by itself, other ER‐resident molecules sense the conformational change of the IP3 receptor and directly interact to the SOC, thereby activating them. (C) The secretion model. This model suggests a mechanism by which depletion of calcium from intracellular store initiates the vesicular translocation and insertion of calcium channels to the plasma membrane. Then, delivery of these channels to the membrane might be a trigger for cation influx.

82

m as a ki h i ki da a n d t o m oh i ro k u ro s a k i

groups have continued their struggle, attempting to identify native CIF and determine the CIF‐mediated pathway (Randriamampita and Tsien, 1995; Rzigalinski et al., 1999; Takemura and Ohshika, 1999; Thomas and Hanley, 1995; Trepakova et al., 2000). Although the original CIF model hypothesized that CIF directly binds and activates SOCs, an indirect activation model has been proposed recently (Smani et al., 2003; 2004). In this model, CIF‐induced displacement of inhibitory calmodulin from iPLA2 is a key event, leading to iPLA2 activation. Once iPLA2 is activated by CIF, lysophospholipids are generated, which in turn activate SOCs. Hence, according to this variant model, a more direct activator for SOCs is considered to be lysophospholipids rather than CIF (Fig. 3A, right panel). The conformational coupling model postulates that a direct interaction between the ER‐resident proteins such as IP3 receptors and SOCs is required for opening SOCs (Fig. 3B, left panel). This mechanism has been particularly appealing, because structural and functional coupling of transient receptor protein C3 (TRPC3) channel, possibly one of SOCs, and IP3 receptor was demonstrated in an in vitro system (Kiselyov et al., 2000). However, in contradiction with this model, triple knockout of all three IP3 receptor isoforms in DT40 B cells still exhibits proper calcium influx after pharmacological emptying of the ER calcium stores such as thapsigargin or ionomycin treatment (Sugawara et al., 1997). One interpretation that reconciles these data is that opening mechanisms for SOCs might differ between BCR signaling and pharmacological contexts, although some overlapping exists. In support of this explanation, in a triple knockout background, BCR‐mediated calcium influx can be restored by type 1 IP3 receptor mutants that have IP3 binding, but not channel activity (van Rossum et al., 2004). Conversely, the IP3 binding mutants cannot restore, simply suggesting that IP3‐mediated conformational change in the IP3 receptor is required for opening SOCs in the BCR signaling context. As an obvious extension of this model, this conformational change in the IP3 receptor can be transmitted to SOCs through other ER‐resident and/or intermediate molecules (Fig. 3B, right panel) (Yuan et al., 2003). In these regards, upon binding of IP3 to its receptors, conformationally coupled junctions might be newly formed between ER and plasma membrane, thereby contributing to activated SOCs. This idea appears to be consistent with studies that show the involvement of a GTP‐regulatory step in the activation of SOCs (Bird and Putney, 1993; Fasolato et al., 1993; Fernando et al., 1997; Yao et al., 1999), because these molecules are well‐known to participate in intracellular trafficking events. Assuming that SOCs comprise TRP‐family members, a series of recent studies suggest that recruitment of TRPs to the plasma membrane is the third mechanism by which SOCs are activated (Fig. 3C) (Clapham, 2003).

r eg u l at i o n of pl c -g2 n e t w o rk s

83

Such a concept has considerable appeal as many TRP channels expressed in vitro are constitutively active. If this is also true in vivo, then BCR‐induced delivery of TRPs to the plasma membrane could be one trigger for SOCs‐ mediated cation influx. The first report corroborating an exocytic mechanism for a TRP channel was the demonstration that TRPV2 translocates to the plasma membrane after stimulation of the insulin growth factor using an overexpression system (Kanzaki et al., 1999). Since then, several TRP family members, TRPC3 (Xu and Sternberg, 2003), TRPC5 (Bezzerides et al., 2004), TRPV5, and TRPV6 (van de Graaf et al., 2003), have been shown to move to the plasma membrane after receptor stimulation (Montell, 2004). In the case of the TRPC5 in hippocampal neurons, incorporation of this channel into the plasma membrane is initiated by growth factors such as NGF that stimulate receptor tyrosine kinases (RTKs), and this incorporation appears to be dependent on PI3K, Rac, and PIP5K in the NGF signaling context (Bezzerides et al., 2004). Because these enzymes and GTP‐binding proteins are also activated in the BCR‐signaling context, the previous findings that PI3K is required for BCR‐mediated calcium influx (Clayton et al., 2002; Jou et al., 2002) might be accounted for by its involvement in translocation of SOCs to the plasma membrane. 6. Looking for Ca2þ Entry Channels Proteins of the TRP family are presently the best candidates for the pore‐ forming subunit of SOCs. The TRP family can be classified into six subfamilies (TRPC, TRPV, TRPM, TRPA, TRPP, and TRPML), and the total number of these genes has now turned out to be 28 in mice (see Fig. 4 for their structures) (Clapham, 2003). Among them, TRPV5 and TRPV6 seem to be stronger candidates, but their physiological properties, when overexpressed each alone in heterologous cells, such as HEK293 cells, do not fully resemble those of endogenous SOCs (Hoenderop et al., 2001; Vennekens et al., 2000; Voets et al., 2001). Thus, the best speculation at present is that SOCs in B lymphocytes are heteromultimeric complexes of TRP components, and additional regulatory or adaptor subunits are needed to promote channel assembly and subsequent coupling to store depletion previously mentioned. Functionally, the TRPV6 channel exhibited several key features of SOCs when expressed in CHO cells, including activation of TRPV6 through store depletion using either IP3 or thapsigargin, and a high degree of calcium selectivity in the presence of divalent cations (Yue et al., 2001). In contrast to these initial observations, a subsequent study reported that no activation of the TRPV6 channels was observed in HEK293 cells after ionomycin‐induced store release (Voets et al., 2001). One of the explanations for this disparity, among

84 Figure 4 Schematic structure of TRP‐family ion channels. All the members of the family consist of six transmembrane domain‐containing ion channels flanked with two cytoplasmic tails that are characteristic to each member. The TRP box is EWKFAR in TRPC, but is less conserved in TRPV and TRPM. CC indicates coiled‐coil domain. Ankyrin repeats (AnkR) range from 0 to 14 in number. CIRB stands for putative calmodulin‐ and IP3 receptor‐binding domain. EF hand, canonical helix‐loop‐helix Ca2þ‐binding domain; PDZ, amino acids‐binding PDZ domains; PLIK, phospholipase‐ C‐interacting kinase; Nudix, NUDT9 hydrolase protein homologue‐binding ADP ribose.

r eg u l at i o n of pl c -g2 n e t w o rk s

85

many, is that both the function and pharmacological properties of TRPV6 are highly expression‐dependent. Thus, at low‐expression levels (in the case of CHO cells), the channel is store‐dependent, similar to native SOCs, whereas at higher expression levels (in the case of HEK293 cells), the TRPV6 is constitutively active. Similarly, expression‐dependent alterations of the channel properties were also observed in the case of TRPC3 (Vazquez et al., 2003). TRPC3, when expressed in HEK293 cell (corresponding to higher levels of expression), exhibited a constitutive activity that was substantially enhanced by agonist stimulation such as carbachol, but not by store depletion. However, in the relatively lower expression of TRPC3 in DT40 B cells, this channel behaved as a store‐operated channel. Then the question arises about the underlying mechanisms. At least two possible explanations seem to be possible. First, under low or endogenous levels of expression, store‐depletion may cause recruitment of TRPs in intracellular compartments to the plasma membrane. If so, overexpression of TRPs skews their expression from intracellular compartments to the plasma membrane even before stimulation, wherein TRPs exhibit no more store‐dependency. Second, under low‐expression conditions, heteromeric complexes are formed between the expressed TRP and regulatory factors, thereby leading to store‐dependency of these channels. On the other hand, overexpression of the individual TRP most likely generates predominantly homomultimers, losing the store‐dependency. Hence, to complement the overexpression experiments, knockout approach is undoubtedly required. Apart from the store‐operated mechanism, recent analyses of each TRP channel have made a novel paradigm emerge that many TRP channels respond to multiple inputs including lipid metabolites. For instance, opening of TRPM7 is positively controlled by PIP2; hence activation of PLC‐g‐coupled receptors such as BCR is able to cause channel inactivation in the case of TRPM7 (Runnels et al., 2002). Conversely, TRPC3, TRPC6, and TRPC7 are directly activated by DAG, a PLC‐g product (Hofmann et al., 1999; Okada et al., 1999; Venkatachalam et al., 2001). Thus, once PLC‐g is activated, not only IP3 but also DAG appears to be involved in modulation of TRP channels. After calcium influx occurs, it is proposed that PLC‐g2 is activated again, thereby forming a positive‐forward loop between PLC‐g2 and calcium, based upon the existence of the C2 calcium‐binding domain in PLC‐g2 (Nishida et al., 2003). Supporting this model, the PLC‐g2 mutant devoid of this C2 domain exhibited no more such amplification. Given the physical association between PLC‐g2 and TRP channels such as TRPC3 (Nishida et al., 2003), this model also suggests that the PLC‐g2 C2 domain could quickly sense the alteration of the calcium concentration just induced by TRPC3, possibly

86

m as a ki h i ki da a n d t o m oh i ro k u ro s a k i

making a positive loop in a very‐restricted region inside B lymphocytes (Fig. 2C). In contrast to significant progress regarding how calcium signals are generated, how calcium signals are terminated is largely unknown. Several mechanisms, including inhibition of SOCs, SERCA‐mediated uptake of Ca2þ into intracellular Ca2þ stores, and the action of Ca2þ pumps (PMCAs) that pump Ca2þ out the cells, are proposed (Berridge et al., 2003). Indeed, the importance of PMCAs was recently shown in B lymphocytes as well as T lymphocytes (Bautista et al., 2002; Chen et al., 2004). 7. NFAT and NF‐kB Increased intracellular calcium promotes nuclear translocation of two important sets of transcription factors, namely the NFAT and the NF‐kB family (Fig. 5) (Dolmetsch et al., 1997). Activation of the NF‐kB transcription factors NF‐kB1, NF‐kB2, c‐Rel, RelA, and RelB is essential for B‐cell development as well as immune responses (Henderson and Calame, 1998; Li and Verma, 2002). Extraordinary progress has been made defining molecular events leading to NF‐kB activation (Ghosh and Karin, 2002). NF‐kB is released from inhibitors of the IkB family by phosphorylation and ubiquitination‐mediated proteolysis. And this IkB phosphorylation is triggered by a multimolecular complex comprising two serine threonine kinases, IKKa and IKKb, and a regulatory subunit IKKg. Thus, activation of the IKK complex is essential for subsequent NF‐kB activation. Deficiency in PKCb has a dramatic impact on the phosphorylation of IKKa and, to a lesser extent, IKKb, thereby reducing the activation status of the IKK complex (Saijo et al., 2002; Su et al., 2002). The proteins CARMA1, Bcl10, and MALT1 are involved in NF‐kB activation downstream of PKCb (Guo et al., 2004; Thome, 2004). Probably CARMA1, Bcl10, and MALT1 are physically associated with each other, and PKCb could mediate phosphorylation on Bcl10, which in turn facilitates and/or stabilizes these associations. Then, the question arises about connection between the CARMA1/Bcl10/MALT1 and the IKK complex. A new study shows the intriguing possibility that Bcl10 induces IKKg polyubiquitination in a MALT1‐, Ubc13‐, TRAF2‐, and TRAF6‐dependent manner, which in turn is important for activation of the IKK complex (Zhou et al., 2004). Because PKCb is more critically regulated by DAG, rather than calcium, the preceding mechanism suggests that NF‐kB activation in the BCR signaling context could be accounted for mainly by the DAG axis rather than calcium. However, although the experiments were not performed in lymphocytes, new data suggest that calcium pathway might be involved in NF‐kB activation, independently of PKCb activation. Mitochondrial inhibitors such as CCCP

r eg u l at i o n of pl c -g2 n e t w o rk s

87

Figure 5 Regulation of NF‐kB and NFAT by PLC‐g2. BCR‐mediated PLC‐g2 activation causes elevation of cytoplasmic concentration of Ca2þ, which leads to the activation of calcineurin. NFAT is dephosphorylated by calcineurin and translocates to the nucleus. DAG, generated by PIP2 hydrolysis, activates PKCb, which then activates IKK complex presumably by promoting membrane recruitment and aggregation of the CARMA1/bcl10/MALT1 complex. This complex activates IKKs, resulting in the phosphorylation and ubiquitin‐mediated degradation of IkB. Released rel/p50 complex translocates to the nucleus. It has been reported that inhibition of calcineurin blocks the activation of NF‐kB suggesting that calcineurin is involved in NF‐kB activation pathway (Biswas et al., 2003).

increase intracellular calcium levels and activate NF‐kB in several cell types. More importantly, this NF‐kB activation is blocked by FK506 or cyclosporin A (Biswas et al., 2003; Venkatesha et al., 2004), suggesting involvement of calcineurin, a calcium‐sensitive serine threonine phosphatase (Fig. 5). CCCP appears to promote degradation of IkBb‐ (but not conventional Ikba‐) containing NF‐kB complexes (Biswas et al., 2003). Hence, if this mechanism takes place in B lymphocytes, calcineurin could participate in not only NFAT, as discussed in the following, but also NF‐kB activation. There are four calcium‐regulated members of the NFAT family (Feske et al., 2003; Rao et al., 1997). The activity of these proteins is determined by their phosphorylation status, which is tightly regulated by the interplay between calcineurin and opposing kinases. When calcineurin is activated through an increase in calcium levels, NFAT is dephosphorylated at a large number of phosphorylated serine residues and rapidly enters the nucleus (Okamura et al., 2000; Shibasaki et al., 1996). Conversely in stimulated cells in which NFAT is

88

m as a ki h i ki da a n d t o m oh i ro k u ro s a k i

already localized to the nucleus, termination of calcium signaling results in rephosphorylation of NFAT, exposure of a nuclear export signal that is bound by the nuclear export receptor Crm1, and transport of NFAT back to the cytoplasm (Kehlenbach et al., 1998; Klemm et al., 1997). Hence, an implication of these findings is that in order to maintain NFAT in a dephosphorylated state for subsequent gene regulation, calcineurin should be present in the nucleus of stimulated cells. Furthermore, calcineurin‐mediated dephosphorylation must be capable of being reversed by NFAT kinases (Hogan et al., 2003; Kiani et al., 2000). More importantly, the property of reversible activation and deactivation by calcineurin and NFAT kinase, respectively, confers on NFAT a remarkable activity to sense dynamic changes in [Ca2þ]i in lymphocytes (Dolmetsch et al., 1997; Dolmetsch et al., 1998; Li et al., 1998; Tomida et al., 2003). Thus, the differential responsiveness of NFAT versus NF‐kB activation in calcium changes, as observed in activation of NF‐kB and NFAT by a large transient calcium rise and a slow, but sustained calcium plateau, respectively, appears to reflect the fact that NF‐kB activation requires irreversible degradation of IkB during activation processes, whereas NFAT is reversibly activated and deactivated through the calcium‐sensing enzyme calcineurin and opposing kinases. Although degradation of IkB is irreversible in a course of one hour range after stimulation, IkB is again transcribed and synthesized after a lag time, because IkB is well known to be a target gene of NF‐kB pathway (Li and Verma, 2002). Hence, in a longer time length such as 5 hours after stimulation, NF‐kB components are reversibly back into a calcium‐sensitive state again. 8. Modulation of Calcium Signaling Transgenic mice expressing a well‐defined HEL‐specific BCR on B cells in combination with mice harboring soluble HEL as a self‐antigen provide a well‐ controlled model to analyze anergic B cells. In this model, a distinct pattern of signaling by BCRs, activating NFAT or ERK, but not NF‐kB or JNK, characterizes anergic B cells (Glynne et al., 2000; Healy et al., 1997). These results indicate that chronic, suboptimal stimulation induces an inhibitory feedback to selectively uncouple the receptor from activation‐signaling pathways. In anergic B cells, basal calcium level is high and antigen‐mediated calcium oscillations are dampened (Healyet al., 1997). This differential calcium signaling, given the evidence that NF‐kB, but not NFAT, requires a large transient calcium rise, could explain why NF‐kB pathway is selectively uncoupled in anergic cells. Therefore, the preceding findings raise the question of how PLC‐g2‐calcium signaling properties are modulated by the inhibitory feedback operating in anergic B cells. Several data suggest ubiquitin E3 ligases (E3s) as an important

r eg u l at i o n of pl c -g2 n e t w o rk s

89

possible mediator for this feedback (Davis and Ben‐Neriah, 2004; Liu, 2004). In the case of T cells, T cells are induced to the anergy state through anergy‐ inducing stimuli such as TCR stimulation without costimulation and treatment of the calcium ionophore ionomycin (Heissmeyer et al., 2004; Macian et al., 2002). Indeed, after ionomycin stimulation, at least three E3s: Itch, Cbl‐b, and GRAIL are upregulated at the mRNA and protein levels (Heissmeyer et al., 2004). When anergic T cells are restimulated, Itch and closely related HECT‐ domain E3 Nedd4 becomes localized to the membrane, whereby they target key signaling molecules, including PLC‐g1 and PKCy for degradation, thereby diminishing calcium mobilization. Hence, if a similar mechanism also operates in B cells, signaling molecules involved in PLC‐g‐calcium pathway, such as Syk, BLNK, PLC‐g2, IP3R, or TRPs, could be a target for ubiquitin‐mediated downregulation, thereby dampening calcium signaling observed in anergic B cells. As previously discussed, Syk was indeed shown to be a potential target for Cbl and/or Cbl‐b, thereby undergoing poly‐ubiquitination‐induced degradation (Sohn et al., 2003). In addition to poly‐ubiquitin‐mediated degradation, studies in yeast and in mammalian cells have shown that mono‐ubiquitin tags are necessary and sufficient to trigger endocytosis (Dupre et al., 2004). For instance, binding of epidermal growth factor (EGF) to its receptor (EGFR) stimulates receptor internalization, and EGF‐dependent mono‐ubiquitination of the EGFR by the E3 ligase Cbl triggers a sorting event on the early endosome (Levkowitz et al., 1998). Sorting involves recognition of the mono‐ubiquitinated receptors by proteins, such as Hrs and Tsg101, that contain ubiquitin‐binding domains (Chin et al., 2001; Lu et al., 2003). Hence, if Cbl and/or Cbl‐b are upregulated in anergic B cells, like ionomycin‐induced T‐cell anergy, enhanced cycles of endocytic sequestration and recycling would be expected. This enhanced endocytosis is likely to terminate BCR‐mediated calcium signaling prematurely by sequestering the early signaling complexes from the plasma membrane supply of PIP2 and PIP3 needed for Btk and PLC‐g2 activity. Supporting this possibility, endocytosis of BCRs to a large pool of recycling intracellular receptors is enhanced in anergic B cells compared with that of their naı¨ve counterparts (Heltemes‐Harris et al., 2004; Morris et al., 2000). Thus, E3s could modulate calcium signaling by at least two ways, degradation of signaling proteins and enhancing endocytosis. 9. Concluding Remarks Significant progress has been made defining the role of PLC‐g in B lymphocyte development and activation and deciphering the means by which BCR regulates PLC‐g. However, the molecular identification of calcium entry channels

90

m as a ki h i ki da a n d t o m oh i ro k u ro s a k i

and elucidation of biochemical sequence of events leading to turn on and off these channels are still poorly understood. Given that many channels, pumps, and adaptors may functionally connect to each other, which in turn contributes to spatiotemporal regulation of effective calcium influx, an answer to this question will require deletion or activation of wild‐type and mutated genes under regulated conditions, as well as the use of multiple fluorescent proteins whose location and expression can be followed simultaneously inside living cells. References Bautista, D. M., Hoth, M., and Lewis, R. S. (2002). Enhancement of calcium signaling dynamics and stability by delayed modulation of the plasma‐membrane calcium‐ATPase in human T cells. J. Physiol. 541, 877–894. Bell, S. E., Vigorito, E., McAdam, S., Reynolds, H. M., Caraux, A., Colucci, F., and Turner, M. (2004). PLCg‐2 regulates Bcl‐2 levels and is required for survival rather than differentiation of marginal zone and follicular B cells. Eur. J. Immunol. 34, 2237–2247. Berridge, M. J., Bootman, M. D., and Roderick, H. L. (2003). Calcium signalling: dynamics, homeostasis and remodeling. Nat. Rev. Mol. Cell. Biol. 4, 517–529. Bezzerides, V. J., Ramsey, I. S., Kotecha, S., Greka, A., and Clapham, D. E. (2004). Rapid vesicular translocation and insertion of TRP channels. Nat. Cell. Biol. 6, 709–720. Bird, G. S., and Putney, J. W., Jr. (1993). Inhibition of thapsigargin‐induced calcium entry by microinjected guanine nucleotide analogues. Evidence for the involvement of a small G‐protein in capacitative calcium entry. J. Biol. Chem. 268, 21486–21488. Biswas, G., Anandatheerthavarada, H. K., Zaidi, M., and Avadhani, N. G. (2003). Mitochondria to nucleus stress signaling: a distinctive mechanism of NFkB/Rel activation through calcineurin‐ mediated inactivation of IkBb. J. Cell. Biol. 161, 507–519. Brauweiler, A., Tamir, I., Dal Porto, J., Benschop, R. J., Helgason, C. D., Humphries, R. K., Freed, J. H., and Cambier, J. C. (2000). Differential regulation of B cell development, activation, and death by the src homology 2 domain‐containing 5’ inositol phosphatase (SHIP). J. Exp. Med. 191, 1545–1554. Chen, J., McLean, P. A., Neel, B. G., Okunade, G., Shull, G. E., and Wortis, H. H. (2004). CD22 attenuates calcium signaling by potentiating plasma membrane calcium‐ATPase activity. Nat. Immunol. 5, 651–657. Chin, L. S., Raynor, M. C., Wei, X., Chen, H. Q., and Li, L. (2001). Hrs interacts with sorting nexin 1 and regulates degradation of epidermal growth factor receptor. J. Biol. Chem. 276, 7069–7078. Chiu, C. W., Dalton, M., Ishiai, M., Kurosaki, T., and Chan, A. C. (2002). BLNK: molecular scaffolding through ‘cis’‐mediated organization of signaling proteins. EMBO J. 21, 6461–6472. Choi, M. S., Brines, R. D., Holman, M. J., and Klaus, G. G. (1994). Induction of NF‐AT in normal B lymphocytes by anti‐immunoglobulin or CD40 ligand in conjunction with IL‐4. Immunity 1, 179–187. Clapham, D. E. (2003). TRP channels as cellular sensors. Nature 426, 517–524. Clayton, E., Bardi, G., Bell, S. E., Chantry, D., Downes, C. P., Gray, A., Humphries, L. A., Rawlings, D., Reynolds, H., Vigorito, E., and Turner, M. (2002). A crucial role for the p110d subunit of phosphatidylinositol 3‐kinase in B cell development and activation. J. Exp. Med. 196, 753–763.

r eg u l at i o n of pl c -g2 n e t w o rk s

91

Davis, M., and Ben‐Neriah, Y. (2004). Behind the scenes of anergy: a tale of three E3s. Nat. Immunol. 5, 238–240. Deckert, M., Elly, C., Altman, A., and Liu, Y. C. (1998). Coordinated regulation of the tyrosine phosphorylation of Cbl by Fyn and Syk tyrosine kinases. J. Biol. Chem. 273, 8867–8874. Dent, M. A., Raisman, G., and Lai, F. A. (1996). Expression of type 1 inositol 1,4,5‐trisphosphate receptor during axogenesis and synaptic contact in the central and peripheral nervous system of developing rat. Development 122, 1029–1039. Dolmetsch, R. E., Lewis, R. S., Goodnow, C. C., and Healy, J. I. (1997). Differential activation of transcription factors induced by Ca2þ response amplitude and duration. Nature 386, 855–858. Dolmetsch, R. E., Xu, K., and Lewis, R. S. (1998). Calcium oscillations increase the efficiency and specificity of gene expression. Nature 392, 933–936. Dupre, S., Urban‐Grimal, D., and Haguenauer‐Tsapis, R. (2004). Ubiquitin and endocytic internalization in yeast and animal cells. Biochim. Biophys. Acta 1695, 89–111. Fasolato, C., Hoth, M., and Penner, R. (1993). A GTP‐dependent step in the activation mechanism of capacitative calcium influx. J. Biol. Chem. 268, 20737–20740. Fernando, K. C., Gregory, R. B., Katsis, F., Kemp, B. E., and Barritt, G. J. (1997). Evidence that a low‐molecular‐mass GTP‐binding protein is required for store‐activated Ca2þ inflow in hepatocytes. Biochem. J. 328(Pt 2), 463–471. Feske, S., Okamura, H., Hogan, P. G., and Rao, A. (2003). Ca2þ/calcineurin signalling in cells of the immune system. Biochem. Biophys. Res. Commun. 311, 1117–1132. Fluckiger, A. C., Li, Z., Kato, R. M., Wahl, M. I., Ochs, H. D., Longnecker, R., Kinet, J. P., Witte, O. N., Scharenberg, A. M., and Rawlings, D. J. (1998). Btk/Tec kinases regulate sustained increases in intracellular Ca2þ following B‐cell receptor activation. EMBO J 17, 1973–1985. Fu, C., Turck, C. W., Kurosaki, T., and Chan, A. C. (1998). BLNK: A central linker protein in B cell activation. Immunity 9, 93–103. Ghosh, S., and Karin, M. (2002). Missing pieces in the NF‐kB puzzle. Cell 109(Suppl.), S81–S96. Glynne, R., Akkaraju, S., Healy, J. I., Rayner, J., Goodnow, C. C., and Mack, D. H. (2000). How self‐tolerance and the immunosuppressive drug FK506 prevent B‐cell mitogenesis. Nature 403, 672–676. Guo, B., Su, T. T., and Rawlings, D. J. (2004). Protein kinase C family functions in B‐cell activation. Curr. Opin. Immunol. 16, 367–373. Hashimoto, S., Iwamatsu, A., Ishiai, M., Okawa, K., Yamadori, T., Matsushita, M., Baba, Y., Kishimoto, T., Kurosaki, T., and Tsukada, S. (1999). Identification of the SH2 domain binding protein of Bruton’s tyrosine kinase as BLNK—functional significance of Btk‐SH2 domain in B‐cell antigen receptor‐coupled calcium signaling. Blood 94, 2357–2364. Hashimoto, A., Takeda, K., Inaba, M., Sekimata, M., Kaisho, T., Ikehara, S., Homma, Y., Akira, S., and Kurosaki, T. (2000). Cutting edge: essential role of phospholipase C‐g2 in B cell development and function. J. Immunol. 165, 1738–1742. Healy, J. I., Dolmetsch, R. E., Timmerman, L. A., Cyster, J. G., Thomas, M. L., Crabtree, G. R., Lewis, R. S., and Goodnow, C. C. (1997). Different nuclear signals are activated by the B cell receptor during positive versus negative signaling. Immunity 6, 419–428. Heissmeyer, V., Macian, F., Im, S. H., Varma, R., Feske, S., Venuprasad, K., Gu, H., Liu, Y. C., Dustin, M. L., and Rao, A. (2004). Calcineurin imposes T cell unresponsiveness through targeted proteolysis of signaling proteins. Nat. Immunol. 5, 255–265. Heltemes‐Harris, L., Liu, X., and Manser, T. (2004). Progressive surface B cell antigen receptor down‐regulation accompanies efficient development of antinuclear antigen B cells to mature, follicular phenotype. J. Immunol. 172, 823–833.

92

m as a ki h i ki da a n d t o m oh i ro k u ro s a k i

Henderson, A., and Calame, K. (1998). Transcriptional regulation during B cell development. Annu. Rev. Immunol. 16, 163–200. Hikida, M., Johmura, S., Hashimoto, A., Takezaki, M., and Kurosaki, T. (2003). Coupling between B cell receptor and phospholipase C‐g2 is essential for mature B cell development. J. Exp. Med. 198, 581–589. Hoenderop, J. G., Vennekens, R., Muller, D., Prenen, J., Droogmans, G., Bindels, R. J., and Nilius, B. (2001). Function and expression of the epithelial Ca2þ channel family: comparison of mammalian ECaC1 and 2. J. Physiol. 537, 747–761. Hofmann, T., Obukhov, A. G., Schaefer, M., Harteneck, C., Gudermann, T., and Schultz, G. (1999). Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol. Nature 397, 259–263. Hogan, P. G., Chen, L., Nardone, J., and Rao, A. (2003). Transcriptional regulation by calcium, calcineurin, and NFAT. Genes. Dev. 17, 2205–2232. Humphries, L. A., Dangelmaier, C., Sommer, K., Kipp, K., Kato, R. M., Griffith, N., Bakman, I., Turk, C. W., Daniel, J. L., and Rawlings, D. J. (2004). Tec kinases mediate sustained calcium influx via site‐specific tyrosine phosphorylation of the phospholipase Cg Src homology 2‐Src homology 3 linker. J. Biol. Chem. 279, 37651–37661. Irvin, B. J., Williams, B. L., Nilson, A. E., Maynor, H. O., and Abraham, R. T. (2000). Pleiotropic contributions of phospholipase C‐g1 (PLC‐g1) to T‐cell antigen receptor‐mediated signaling: reconstitution studies of a PLC‐g1‐deficient Jurkat T‐cell line. Mol. Cell. Biol. 20, 9149–9161. Ishiai, M., Kurosaki, M., Pappu, R., Okawa, K., Ronko, I., Fu, C., Shibata, M., Iwamatsu, A., Chan, A. C., and Kurosaki, T. (1999). BLNK required for coupling Syk to PLCg2 and Rac1‐JNK in B cells. Immunity 10, 117–125. Joazeiro, C. A., Wing, S. S., Huang, H., Leverson, J. D., Hunter, T., and Liu, Y. C. (1999). The tyrosine kinase negative regulator c‐Cbl as a RING‐type, E2‐dependent ubiquitin‐protein ligase. Science 286, 309–312. Johmura, S., Oh‐hora, M., Inabe, K., Nishikawa, Y., Hayashi, K., Vigorito, E., Kitamura, D., Turner, M., Shingu, K., Hikida, M., and Kurosaki, T. (2003). Regulation of Vav localization in membrane rafts by adaptor molecules Grb2 and BLNK. Immunity 18, 777–787. Jou, S. T., Carpino, N., Takahashi, Y., Piekorz, R., Chao, J. R., Carpino, N., Wang, D., and Ihle, J. N. (2002). Essential, nonredundant role for the phosphoinositide 3‐kinase p110d in signaling by the B‐cell receptor complex. Mol. Cell. Biol. 22, 8580–8591. Kang, S. W., Wahl, M. I., Chu, J., Kitaura, J., Kawakami, Y., Kato, R. M., Tabuchi, R., Tarakhovsky, A., Kawakami, T., Turck, C. W., Witte, O. N., and Rawlings, D. J. (2001). PKCb modulates antigen receptor signaling via regulation of Btk membrane localization. EMBO J 20, 5692–5702. Kanzaki, M., Zhang, Y. Q., Mashima, H., Li, L., Shibata, H., and Kojima, I. (1999). Translocation of a calcium‐permeable cation channel induced by insulin‐like growth factor‐I. Nat. Cell. Biol. 1, 165–170. Kehlenbach, R. H., Dickmanns, A., and Gerace, L. (1998). Nucleocytoplasmic shuttling factors including Ran and CRM1 mediate nuclear export of NFAT In vitro. J. Cell. Biol. 141, 863–874. Keshvara, L. M., Isaacson, C. C., Yankee, T. M., Sarac, R., Harrison, M. L., and Geahlen, R. L. (1998). Syk‐ and Lyn‐dependent phosphorylation of Syk on multiple tyrosines following B cell activation includes a site that negatively regulates signaling. J. Immunol. 161, 5276–5283. Kiani, A., Rao, A., and Aramburu, J. (2000). Manipulating immune responses with immunosuppressive agents that target NFAT. Immunity 12, 359–372. Kim, Y. J., Sekiya, F., Poulin, B., Bae, Y. S., and Rhee, S. G. (2004). Mechanism of B‐cell receptor‐ induced phosphorylation and activation of phospholipase C‐g2. Mol. Cell. Biol. 24, 9986–9999.

r eg u l at i o n of pl c -g2 n e t w o rk s

93

Kiselyov, K. I., Shin, D. M., Wang, Y., Pessah, I. N., Allen, P. D., and Muallem, S. (2000). Gating of store‐operated channels by conformational coupling to ryanodine receptors. Mol. Cell 6, 421–431. Klemm, J. D., Beals, C. R., and Crabtree, G. R. (1997). Rapid targeting of nuclear proteins to the cytoplasm. Curr. Biol. 7, 638–644. Kurosaki, T. (1999). Genetic analysis of B cell antigen receptor signaling. Annu. Rev. Immunol. 17, 555–592. Kurosaki, T. (2002). Regulation of B‐cell signal transduction by adaptor proteins. Nat. Rev. Immunol. 2, 354–363. Kurosaki, T., Johnson, S. A., Pao, L., Sada, K., Yamamura, H., and Cambier, J. C. (1995). Role of the Syk autophosphorylation site and SH2 domains in B cell antigen receptor signaling. J. Exp. Med. 182, 1815–1823. Levkowitz, G., Waterman, H., Ettenberg, S. A., Katz, M., Tsygankov, A. Y., Alroy, I., Lavi, S., Iwai, K., Reiss, Y., Ciechanover, A., Lipkowitz, S., and Yarden, Y. (1999). Ubiquitin ligase activity and tyrosine phosphorylation underlie suppression of growth factor signaling by c‐Cbl/Sli‐1. Mol. Cell 4, 1029–1040. Levkowitz, G., Waterman, H., Zamir, E., Kam, Z., Oved, S., Langdon, W. Y., Beguinot, L., Geiger, B., and Yarden, Y. (1998). c‐Cbl/Sli‐1 regulates endocytic sorting and ubiquitination of the epidermal growth factor receptor. Genes. Dev. 12, 3663–3674. Li, Q., and Verma, I. M. (2002). NF‐kB regulation in the immune system. Nat. Rev. Immunol. 2, 725–734. Li, W., Llopis, J., Whitney, M., Zlokarnik, G., and Tsien, R. Y. (1998). Cell‐permeant caged InsP3 ester shows that Ca2þ spike frequency can optimize gene expression. Nature 392, 936–941. Liu, Y. C. (2004). Ubiquitin ligases and the immune response. Annu. Rev. Immunol. 22, 81–127. Lu, Q., Hope, L. W., Brasch, M., Reinhard, C., and Cohen, S. N. (2003). TSG101 interaction with HRS mediates endosomal trafficking and receptor down‐regulation. Proc. Natl. Acad. Sci. USA 100, 7626–7631. Macian, F., Garcia‐Cozar, F., Im, S. H., Horton, H. F., Byrne, M. C., and Rao, A. (2002). Transcriptional mechanisms underlying lymphocyte tolerance. Cell 109, 719–731. Miyakawa, T., Maeda, A., Yamazawa, T., Hirose, K., Kurosaki, T., and Iino, M. (1999). Encoding of Ca2þ signals by differential expression of IP3 receptor subtypes. EMBO J. 18, 1303–1308. Miyakawa, T., Mizushima, A., Hirose, K., Yamazawa, T., Bezprozvanny, I., Kurosaki, T., and Iino, M. (2001). Ca2þ‐sensor region of IP3 receptor controls intracellular Ca2þ signaling. EMBO J. 20, 1674–1680. Montell, C. (2004). Exciting trips for TRPs. Nat. Cell. Biol. 6, 690–692. Morris, S. C., Moroldo, M., Giannini, E. H., Orekhova, T., and Finkelman, F. D. (2000). In vivo survival of autoreactive B cells: characterization of long‐lived B cells. J. Immunol. 164, 3035–3046. Newton, C. L., Mignery, G. A., and Sudhof, T. C. (1994). Co‐expression in vertebrate tissues and cell lines of multiple inositol 1,4,5‐trisphosphate (InsP3) receptors with distinct affinities for InsP3. J. Biol. Chem. 269, 28613–28619. Nishida, M., Sugimoto, K., Hara, Y., Mori, E., Morii, T., Kurosaki, T., and Mori, Y. (2003). Amplification of receptor signalling by Ca2þ entry‐mediated translocation and activation of PLCg2 in B lymphocytes. EMBO J. 22, 4677–4688. Okada, H., Bolland, S., Hashimoto, A., Kurosaki, M., Kabuyama, Y., Iino, M., Ravetch, J. V., and Kurosaki, T. (1998). Role of the inositol phosphatase SHIP in B cell receptor‐induced Ca2þ oscillatory response. J. Immunol. 161, 5129–5132. Okada, T., Inoue, R., Yamazaki, K., Maeda, A., Kurosaki, T., Yamakuni, T., Tanaka, I., Shimizu, S., Ikenaka, K., Imoto, K., and Mori, Y. (1999). Molecular and functional characterization of a novel

94

m as a ki h i ki da a n d t o m oh i ro k u ro s a k i

mouse transient receptor potential protein homologue TRP7. Ca2þ‐permeable cation channel that is constitutively activated and enhanced by stimulation of G protein‐coupled receptor. J. Biol. Chem. 274, 27359–27370. Okamura, H., Aramburu, J., Garcia‐Rodriguez, C., Viola, J. P., Raghavan, A., Tahiliani, M., Zhang, X., Qin, J., Hogan, P. G., and Rao, A. (2000). Concerted dephosphorylation of the transcription factor NFAT1 induces a conformational switch that regulates transcriptional activity. Mol. Cell 6, 539–550. Parekh, A. B., and Penner, R. (1997). Store depletion and calcium influx. Physiol. Rev. 77, 901–930. Park, D. J., Rho, H. W., and Rhee, S. G. (1991). CD3 stimulation causes phosphorylation of phospholipase C‐g1 on serine and tyrosine residues in a human T‐cell line. Proc. Natl. Acad. Sci. USA 88, 5453–5456. Peng, S. L., Gerth, A. J., Ranger, A. M., and Glimcher, L. H. (2001). NFATc1 and NFATc2 together control both T and B cell activation and differentiation. Immunity 14, 13–20. Randriamampita, C., and Tsien, R. Y. (1993). Emptying of intracellular Ca2þ stores releases a novel small messenger that stimulates Ca2þ influx. Nature 364, 809–814. Randriamampita, C., and Tsien, R. Y. (1995). Degradation of a calcium influx factor (CIF) can be blocked by phosphatase inhibitors or chelation of Ca2þ. J. Biol. Chem. 270, 29–32. Rao, A., Luo, C., and Hogan, P. G. (1997). Transcription factors of the NFAT family: regulation and function. Annu. Rev. Immunol. 15, 707–747. Rawlings, D. J., and Witte, O. N. (1994). Bruton’s tyrosine kinase is a key regulator in B‐cell development. Immunol. Rev. 138, 105–119. Rhee, S. G., and Bae, Y. S. (1997). Regulation of phosphoinositide‐specific phospholipase C isozymes. J. Biol. Chem. 272, 15045–15048. Rigley, K. P., Harnett, M. M., Phillips, R. J., and Klaus, G. G. (1989). Analysis of signaling via surface immunoglobulin receptors on B cells from CBA/N mice. Eur. J. Immunol. 19, 2081–2086. Rodriguez, R., Matsuda, M., Perisic, O., Bravo, J., Paul, A., Jones, N. P., Light, Y., Swann, K., Williams, R. L., and Katan, M. (2001). Tyrosine residues in phospholipase Cg2 essential for the enzyme function in B‐cell signaling. J. Biol. Chem. 276, 47982–47992. Rowley, R. B., Burkhardt, A. L., Chao, H. G., Matsueda, G. R., and Bolen, J. B. (1995). Syk protein‐tyrosine kinase is regulated by tyrosine‐phosphorylated Iga/Igb immunoreceptor tyrosine activation motif binding and autophosphorylation. J. Biol. Chem. 270, 11590–11594. Runnels, L. W., Yue, L., and Clapham, D. E. (2002). The TRPM7 channel is inactivated by PIP2 hydrolysis. Nat. Cell. Biol. 4, 329–336. Rzigalinski, B. A., Willoughby, K. A., Hoffman, S. W., Falck, J. R., and Ellis, E. F. (1999). Calcium influx factor, further evidence it is 5, 6‐epoxyeicosatrienoic acid. J. Biol. Chem. 274, 175–182. Saijo, K., Mecklenbrauker, I., Santana, A., Leitger, M., Schmedt, C., and Tarakhovsky, A. (2002). Protein kinase C b controls nuclear factor kB activation in B cells through selective regulation of the IkB kinase a. J. Exp. Med. 195, 1647–1652. Saito, K., Tolias, K. F., Saci, A., Koon, H. B., Humphries, L. A., Scharenberg, A., Rawlings, D. J., Kinet, J. P., and Carpenter, C. L. (2003). BTK regulates PtdIns‐4,5‐P2 synthesis: importance for calcium signaling and PI3K activity. Immunity 19, 669–678. Schwartzberg, P. L. (2003). Amplifying Btk’s signal. Immunity 19, 634–636. Secrist, J. P., Karnitz, L., and Abraham, R. T. (1991). T‐cell antigen receptor ligation induces tyrosine phosphorylation of phospholipase C‐g1. J. Biol. Chem. 266, 12135–12139. Shibasaki, F., Price, E. R., Milan, D., and McKeon, F. (1996). Role of kinases and the phosphatase calcineurin in the nuclear shuttling of transcription factor NF‐AT4. Nature 382, 370–373. Smani, T., Zakharov, S. I., Csutora, P., Leno, E., Trepakova, E. S., and Bolotina, V. M. (2004). A novel mechanism for the store‐operated calcium influx pathway. Nat. Cell. Biol. 6, 113–120.

r eg u l at i o n of pl c -g2 n e t w o rk s

95

Smani, T., Zakharov, S. I., Leno, E., Csutora, P., Trepakova, E. S., and Bolotina, V. M. (2003). Ca2þ‐ independent phospholipase A2 is a novel determinant of store‐operated Ca2þ entry. J. Biol. Chem. 278, 11909–11915. Smith, C. I., Islam, K. B., Vorechovsky, I., Olerup, O., Wallin, E., Rabbani, H., Baskin, B., and Hammarstrom, L. (1994). X‐linked agammaglobulinemia and other immunoglobulin deficiencies. Immunol. Rev. 138, 159–183. Sohn, H. W., Gu, H., and Pierce, S. K. (2003). Cbl‐b negatively regulates B cell antigen receptor signaling in mature B cells through ubiquitination of the tyrosine kinase Syk. J. Exp. Med. 197, 1511–1524. Su, T. T., Guo, B., Kawakami, Y., Sommer, K., Chae, K., Humphries, L. A., Kato, R. M., Kang, S., Patrone, L., Wall, R., Teitell, M., Leitges, M., Kawakami, T., and Rawlings, D. J. (2002). PKC‐b controls IkB kinase lipid raft recruitment and activation in response to BCR signaling. Nat. Immunol. 3, 780–786. Sugawara, H., Kurosaki, M., Takata, M., and Kurosaki, T. (1997). Genetic evidence for involvement of type 1, type 2 and type 3 inositol 1,4,5‐trisphosphate receptors in signal transduction through the B‐cell antigen receptor. EMBO J. 16, 3078–3088. Takata, M., Homma, Y., and Kurosaki, T. (1995). Requirement of phospholipase C‐g2 activation in surface immunoglobulin M‐induced B cell apoptosis. J. Exp. Med. 182, 907–914. Takata, M., and Kurosaki, T. (1996). A role for Bruton’s tyrosine kinase in B cell antigen receptor‐ mediated activation of phospholipase C‐g2. J. Exp. Med. 184, 31–40. Takata, M., Sabe, H., Hata, A., Inazu, T., Homma, Y., Nukada, T., Yamamura, H., and Kurosaki, T. (1994). Tyrosine kinases Lyn and Syk regulate B cell receptor‐coupled Ca2þ mobilization through distinct pathways. EMBO J. 13, 1341–1349. Takemura, H., and Ohshika, H. (1999). Capacitative Ca2þ entry involves Ca2þ influx factor in rat glioma C6 cells. Life. Sci. 64, 1493–1500. Thien, C. B., and Langdon, W. Y. (2001). Cbl: many adaptations to regulate protein tyrosine kinases. Nat. Rev. Mol. Cell. Biol. 2, 294–307. Thomas, D., and Hanley, M. R. (1995). Evaluation of calcium influx factors from stimulated Jurkat T‐lymphocytes by microinjection into Xenopus oocytes. J. Biol. Chem. 270, 6429–6432. Thome, M. (2004). CARMA1, BCL‐10, and MALT1 in lymphocyte development and activation. Nat. Rev. Immunol. 4, 348–359. Tomida, T., Hirose, K., Takizawa, A., Shibasaki, F., and Iino, M. (2003). NFAT functions as a working memory of Ca2þ signals in decoding Ca2þ oscillation. EMBO J. 22, 3825–3832. Trepakova, E. S., Csutora, P., Hunton, D. L., Marchase, R. B., Cohen, R. A., and Bolotina, V. M. (2000). Calcium influx factor directly activates store‐operated cation channels in vascular smooth muscle cells. J. Biol. Chem. 275, 26158–26163. van de Graaf, S. F., Hoenderop, J. G., Gkika, D., Lamers, D., Prenen, J., Rescher, U., Gerke, V., Staub, O., Nilius, B., and Bindels, R. J. (2003). Functional expression of the epithelial Ca2þ channels (TRPV5 and TRPV6) requires association of the S100A10‐annexin 2 complex. EMBO J. 22, 1478–1487. van Rossum, D. B., Patterson, R. L., Kiselyov, K., Boehning, D., Barrow, R. K., Gill, D. L., and Snyder, S. H. (2004). Agonist‐induced Ca2þ entry determined by inositol 1,4,5‐trisphosphate recognition. Proc. Natl. Acad. Sci. USA 101, 2323–2327. Vazquez, G., Wedel, B. J., Trebak, M., St John Bird, G., and Putney, J. W., Jr. (2003). Expression level of the canonical transient receptor potential 3 (TRPC3) channel determines its mechanism of activation. J. Biol. Chem. 278, 21649–21654. Venkatachalam, K., Ma, H. T., Ford, D. L., and Gill, D. L. (2001). Expression of functional receptor‐coupled TRPC3 channels in DT40 triple receptor InsP3 knockout cells. J. Biol. Chem. 276, 33980–33985.

96

m as a ki h i ki da a n d t o m oh i ro k u ro s a k i

Venkataraman, L., Francis, D. A., Wang, Z., Liu, J., Rothstein, T. L., and Sen, R. (1994). Cyclosporin‐A sensitive induction of NF‐AT in murine B cells. Immunity 1, 189–196. Venkatesha, R. T., Ahamed, J., Nuesch, C., Zaidi, A. K., and Ali, H. (2004). Platelet‐activating factor‐induced chemokine gene expression requires NF‐kB activation and Ca2þ/calcineurin signaling pathways. Inhibition by receptor phosphorylation and b‐arrestin recruitment. J. Biol. Chem. 279, 44606–44612. Vennekens, R., Hoenderop, J. G., Prenen, J., Stuiver, M., Willems, P. H., Droogmans, G., Nilius, B., and Bindels, R. J. (2000). Permeation and gating properties of the novel epithelial Ca2þ channel. J. Biol. Chem. 275, 3963–3969. Voets, T., Prenen, J., Fleig, A., Vennekens, R., Watanabe, H., Hoenderop, J. G., Bindels, R. J., Droogmans, G., Penner, R., and Nilius, B. (2001). CaT1 and the calcium release‐activated calcium channel manifest distinct pore properties. J. Biol. Chem. 276, 47767–47770. Wang, D., Feng, J., Wen, R., Marine, J. C., Sangster, M. Y., Parganas, E., Hoffmeyer, A., Jackson, C. W., Cleveland, J. L., Murray, P. J., and Ihle, J. N. (2000). Phospholipase Cg2 is essential in the functions of B cell and several Fc receptors. Immunity 13, 25–35. Watanabe, D., Hashimoto, S., Ishiai, M., Matsushita, M., Baba, Y., Kishimoto, T., Kurosaki, T., and Tsukada, S. (2001). Four tyrosine residues in phospholipase C‐g2, identified as Btk‐dependent phosphorylation sites, are required for B cell antigen receptor‐coupled calcium signaling. J. Biol. Chem. 276, 38595–38601. Wen, R., Chen, Y., Schuman, J., Fu, G., Yang, S., Zhang, W., Newman, D. K., and Wang, D. (2004). An important role of phospholipase Cg1 in pre‐B‐cell development and allelic exclusion. EMBO J. 23, 4007–4017. Wojcikiewicz, R. J., Furuichi, T., Nakade, S., Mikoshiba, K., and Nahorski, S. R. (1994). Muscarinic receptor activation down‐regulates the type I inositol 1,4,5‐trisphosphate receptor by accelerating its degradation. J. Biol. Chem. 269, 7963–7969. Xu, X. Z., and Sternberg, P. W. (2003). A C. elegans sperm TRP protein required for sperm‐egg interactions during fertilization. Cell 114, 285–297. Yao, L., Kawakami, Y., and Kawakami, T. (1994). The pleckstrin homology domain of Bruton tyrosine kinase interacts with protein kinase C. Proc. Natl. Acad. Sci. USA 91, 9175–9179. Yao, Y., Ferrer‐Montiel, A. V., Montal, M., and Tsien, R. Y. (1999). Activation of store‐operated Ca2þ current in Xenopus oocytes requires SNAP‐25 but not a diffusible messenger. Cell 98, 475–485. Yokouchi, M., Kondo, T., Houghton, A., Bartkiewicz, M., Horne, W. C., Zhang, H., Yoshimura, A., and Baron, R. (1999). Ligand‐induced ubiquitination of the epidermal growth factor receptor involves the interaction of the c‐Cbl RING finger and UbcH7. J. Biol. Chem. 274, 31707–31712. Yuan, J. P., Kiselyov, K., Shin, D. M., Chen, J., Shcheynikov, N., Kang, S. H., Dehoff, M. H., Schwarz, M. K., Seeburg, P. H., Muallem, S., and Worley, P. F. (2003). Homer binds TRPC family channels and is required for gating of TRPC1 by IP3 receptors. Cell 114, 777–789. Yue, L., Peng, J. B., Hediger, M. A., and Clapham, D. E. (2001). CaT1 manifests the pore properties of the calcium‐release‐activated calcium channel. Nature 410, 705–709. Zhou, H., Wertz, I., O’ Rourke, K., Ultsch, M., Seshagiri, S., Eby, M., Xiao, W., and Dixit, V. M. (2004). Bcl10 activates the NF‐kB pathway through ubiquitination of NEMO. Nature 427, 167–171.

Role of Human Mast Cells and Basophils in Bronchial Asthma Gianni Marone, Massimo Triggiani, Arturo Genovese, and Amato De Paulis Division of Clinical Immunology and Allergy, Center for Basic and Clinical Immunology Research (CISI), University of Naples Federico II, School of Medicine, I‐80131 Naples, Italy Abstract ........................................................................................................... Introduction ..................................................................................................... Human Mast Cells ............................................................................................. Human Basophils .............................................................................................. Experimental Evidence for Mast Cells in Bronchial Asthma ...................................... Experimental Evidence for Basophils in Bronchial Asthma ........................................ Mast Cell and Basophil Recruitment to Asthmatic Airways ........................................ Anatomical and Functional Evidence for Mast Cells and Basophils in Asthma............... Superallergens in Bronchial Asthma ...................................................................... Angiogenesis in Bronchial Asthma ........................................................................ Tissue Remodeling in Bronchial Asthma ................................................................ Pharmacologic Modulation of Human Mast Cells and Basophils in the Treatment of Bronchial Asthma ................................................................... 12. Conclusions and Implications............................................................................... References .......................................................................................................

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

97 97 101 109 116 117 118 119 121 127 128 130 136 138

Abstract Mast cells and basophils are the only cells expressing the tetrameric (abg2) structure of the high affinity receptor for IgE (FcERI) and synthesizing histamine in humans. Human FcERIþ cells are conventionally considered primary effector cells of bronchial asthma. There is now compelling evidence that these cells differ immunologically, biochemically, and pharmacologically, which suggests that they might play distinct roles in the appearance and fluctuation of the asthma phenotype. Recent data have revealed the complexity of the involvement of human mast cells and basophils in asthma and have shed light on the control of recruitment and activation of these cells in different lung compartments. Preliminary evidence suggests that these cells might not always be detrimental in asthma but, under some circumstances, they might exert a protective effect by modulating certain aspects of innate and acquired immunity and allergic inflammation. 1. Introduction Bronchial asthma is a genetically complex disease clinically recognized since ancient times. The term ‘‘asthma’’ was coined by Hippocrates (460–377 BC) in the Corpus Hippocraticum to describe the attacks of breathlessness and

97 advances in immunology, vol. 88 # 2005 Elsevier Inc. All rights reserved.

0065-2776/05 $35.00 DOI: 10.1016/S0065-2776(05)88004-6

98

g i a nn i m a r o ne

ET AL.

wheezing experienced by sufferers. Early contributors to our understanding of asthma include Galen and Moses Maimonides. In 1819, John Bostock first described ‘‘catarrus aestivus,’’ or hay fever, and in 1873 Charles Blackley recognized that pollen grains were the causative agents of hay fever (Cohen and Samter, 1992). In 1923, Coca and Cooke coined the term ‘‘atopy’’ to describe the ‘‘familiar occurrence of asthma, allergic rhinitis, and atopic dermatitis associated with positive skin tests to environmental antigens’’ (Coca and Cooke, 1923). In 1966, Kimishige and Teruko Ishizaka purified a new class of immunoglobulin, denominated ‘‘E’’ (¼ erythema), from the serum of a highly allergic patient (Ishizaka and Ishizaka, 1966). The identification of an IgE myeloma protein by Gunnar Johansson, Leif Wide, and Hans Bennich made reagents available for measurement of total and specific IgE (Wide et al., 1967). Bronchial asthma is a chronic inflammatory disease characterized by episodes of airflow obstruction, which reflects bronchial smooth muscle contraction, bronchial wall edema, and mucus plugging (Marone et al., 2004a). This syndrome is the most common cause of significant respiratory morbidity in adults and children in westernized countries. In the 1800s, ‘‘allergy’’ was described as a rare disorder restricted to the privileged class (Cohen and Samter, 1992). This is clearly no longer the case. Atopic disorders now affect up to 40% of populations in western civilizations (Bach, 2002; Holford‐Strevens et al., 1984). Epidemiological data show a steady rise in the incidence of allergic diseases (asthma, rhinitis, and atopic dermatitis) in developed countries over the last four decades (ISAAC Steering Committee, 1998; Upton et al., 2000; Williams, 1992). It is also important to note that the incidence of autoimmune diseases (multiple sclerosis, insulin‐dependent diabetes [type 1 diabetes], and Crohn’s disease) is increasing (Bach, 2002). Asthma is not one disease but a group of diseases resulting from the interplay of environmental factors and the expression of several genes on different chromosomes (Holgate et al., 2004). It is estimated that at least a dozen polymorphic genes regulate asthma, by controlling the inflammatory response, IgE synthesis, cytokine and chemokine production, as well as airway function and airway remodeling (Cookson, 1998; Holgate et al., 2004; Wills‐ Karp and Ewart, 2004). Although the genetic composition of populations in industrialized countries has not changed significantly in the last 20 years, it is likely that the environment in these countries has changed dramatically. However, the specific conditions in industrialized societies that affect asthma pathogenesis are not yet clear. The time factor adds to the complexity of the genes–environment interaction. Compelling evidence now exists of the importance of the appearance and disappearance of the allergic phenotype during an individual’s lifespan. In fact,

h u m a n m a s t ce l l s an d bas o p h i l s i n a s t h m a

99

certain genetic and environmental factors (e.g., diet, microbial exposure, antibiotics, vaccinations) might be relevant to the development of allergies only during specific ‘‘windows of opportunity.’’ Similarly, it is possible that certain immune cells might play different (pathogenetic or even protective) roles in the appearance and fluctuation of the asthma phenotype. Despite the phenotypic and genetic complexity of asthma, the different clinical forms of this disorder share various pathological features: epithelial cell disruption, goblet cell metaplasia, infiltration of the submucosa and epithelium by mast cells, basophils, T cells and eosinophils, deposition of tenascin and collagen beneath the subepithelial basal lamina, and hyperplasia of airway smooth muscle (ASM) and vasculature (Flood‐Page et al., 2003; Holgate et al., 2000; Ordonez et al., 2001). Th2 cells and their cytokines (interleukin‐4 [IL‐4] and IL‐13) are implicated in atopic, nonatopic, and occupational asthma (Larche´ et al., 2003; Romagnani, 2004). Mast cells and basophils, described by Paul Ehrlich in 1879, are unique in being the only cells to express the tetrameric (abg2) structure of the high affinity receptor for IgE (Fc
RI) and to synthesize histamine (Ehrlich, 1879). However, they differ in various aspects. Mast cells are traditionally considered tissue‐resident cells, whereas basophils are normally found only in peripheral blood. Both cell types are highly mobile and can readily infiltrate tissues at sites of inflammation (de Paulis et al., 2004b; Ying et al., 1999). The role of mast cells in the pathogenesis of asthma has been extensively investigated, whereas the role of basophils and their mediators is less well defined, mainly because of limitations in the experimental models available at present. Mast cells and basophils have different strategic microlocalizations in the human lung compartments and synthesize different sets of proinflammatory mediators, cytokines, and chemokines. For instance, mast cells, but not basophils, are specifically scattered throughout the ASM bundles (Brightling et al., 2002). Basophils, not found in normal lung tissue, infiltrate the sites of allergic airway inflammation (KleinJan et al., 2000; Ying et al., 1999). A wide range of newly identified chemotactic receptors (e.g., CC and CXC chemokine receptors, c‐kit, CRTH2, FPR, FPRL1, FPRL2, Flt‐1, Flk‐1, uPAR, C3aR, C5aR) are selectively displayed on basophils and mast cells and are responsible for their recruitment to distinct compartments of asthmatic airways. There are four canonical mechanisms whereby cross‐linking IgE‐high affinity receptor for the IgE (Fc
RI) network can induce the release of mediators from human mast cells and basophils in vitro and, presumably, in vivo (Fig. 1). In the classical model a multivalent antigen cross‐links at least two specific IgE bound to Fc
RIþ cells. Alternatively, antibodies antihuman IgE possess two binding sites for the Fc
region of IgE and can be an effective stimulus for histamine and cytokine release from these cells. Similarly, antibodies directed

100

g i a nn i m a r o ne

ET AL.

Figure 1 Schematic representation of the four canonical mechanisms by which cross‐linking of the IgE‐Fc
RI network can induce the release of mediators from human basophils and mast cells. (A) A multivalent antigen can cross‐link at least two specific IgE molecules bound to Fc
RIþ cells to release mediators. (B) Anti‐human IgE (anti‐IgE) antibodies possess two binding sites for the Fc
region of human IgE and activate mediator secretion from these cells. (C) Antibodies directed against an epitope of the a chain of Fc
RI (anti‐Fc
RIa) can also trigger the release of mediators. (D) Immune complexes containing IgG anti‐IgE and anti‐IgG can activate human basophils in vitro. Modified, with permission, from Marone et al., 2004b.

against an epitope of the a chain of human Fc
RI can trigger the release of mediators from Fc
RIþ cells. Finally, immune complexes containing IgG against human IgE can activate human basophils (Marone et al., 2004b). A fifth mechanism was identified with the observation that IgE‐mediated activation of human mast cells and basophils can also be induced by endogenous, bacterial, and viral superallergens (Marone et al., 2004b). The binding of certain types of monomeric IgE to Fc
RI in the absence of any apparent IgE cross‐linking (e.g., with specific antigen or anti‐IgE) can influence not only receptor expression but also murine mast cell survival, apoptosis (Asai et al., 2001), and cytokine production (Kalesnikoff et al., 2001; Lam et al., 2003; Pandey et al., 2004). Exposure of mouse bone marrow‐derived mast cells (BMMC) to monomeric IgE, but not to IgG induces

h u m a n m a s t ce l l s an d bas o p h i l s i n a s t h m a

101

histidine decarboxylase (HDC) expression and, consequently, affects levels of stored histamine (Tanaka et al., 2002). Clarification of the underlying molecular mechanism(s) and assessment of their relevance in vivo and in human in vitro models will shed further light on these enigmatic observations. Interestingly, research (MacGlashan, Jr., 2003) found that three purified monoclonal IgE do not induce signaling or mediator release from human basophils. The purpose of this review is to summarize the experimental and clinical evidence that human mast cells and basophils play key roles in the pathogenesis of asthma. 2. Human Mast Cells 2.1. Origin of Human Mast Cells The human mast cell is a tissue‐based immune cell of bone marrow origin. Mast cell precursors derived from human peripheral blood, bone marrow, and fetal liver can be grown in long‐term culture in the presence of Stem Cell Factor (SCF), the ligand for c‐kit, and other cytokines in liquid culture. Early studies demonstrated the presence of mast cell precursors in human bone marrow (Horton and O’Brien, 1983) and peripheral blood (Denburg et al., 1983). It was later shown that SCF induces in vitro differentiation of human mast cells from their CD34þ bone marrow and peripheral blood progenitor cells in long‐ term culture (Kirshenbaum et al., 1991; Valent et al., 1992). Human fetal liver cells are also a rich source of mast cell progenitors that differentiate and mature in response to SCF in liquid culture (Nilsson et al., 1993). It is now well established that mast cells cultured from peripheral blood of normal donors and patients with mastocytosis originate from a CD34þ/Fc
RI– cell population (Rottem et al., 1994). Mature mast cells develop from their committed progenitors thanks to input from signals that direct their trafficking from the bone marrow, circulation, and maturation. A crucial signal is the interaction between the membrane receptor c‐kit (Geissler et al., 1988; Kitamura et al., 1978), which is highly expressed by mast cells, and its ligand, SCF (Huang et al., 1990; Kitamura and Go, 1979). The latter is expressed constitutively by endothelial cells, fibroblasts, and other stromal cells, among them bronchial smooth muscle (Page et al., 2001). Mast cells in tissue frequently juxtapose with the SCF‐producing stromal cells. SCF is chemotactic for mast cells (Nilsson et al., 1994), triggers their adhesion (Lorentz et al., 2002), and sustains their survival, differentiation, and maturation (Irani et al., 1992; Mitsui et al., 1993). Therefore, the widespread constitutive expression of SCF and the presence of the c‐kit receptor at all stages of mast cell maturation ensure that mast cells are present in tissues under normal conditions. An increase in local

102

g i a nn i m a r o ne

ET AL.

concentrations of SCF may underlie mast cell hyperplasia—a possible example being hyperplasia of mast cells in asthmatic smooth muscle (Brown et al., 2002; Page et al., 2001). 2.2. Morphological and Ultrastructural Characteristics of Human Mast Cells The morphological and ultrastructural features of human basophils and mast cells have been extensively reviewed (Dvorak, 1995, 2000). Human basophils and mast cells differ morphologically, ultrastructurally, and in their granule architecture. Mast cells are generally ovoid or irregularly elongated in tissue (Patella et al., 1995). Mature human mast cells are large, mononuclear cells filled with membrane‐bound secretory granules. The nucleus has a partially condensed chromatin pattern; nucleoli are usually absent. The secretory granules exhibit metachromasia when stained with toluidine blue and have variable ultrastructural patterns. Other cytoplasmic features include small Golgi structures and different amounts of spherical lipid bodies (Fig. 2). Human mast cells extrude membrane‐free granules either into newly formed degranulation channels in the cytoplasm or individually through

Figure 2 Isolated human lung mast cell. Note the narrow surface folds, a single‐lobed nucleus, the large number of scroll‐packed secretory granules, and six lipid bodies that are larger than granules, osmiophilic, and do not contain scrolls. 14,000. Photo kindly provided by Ann M. Dvorak.

h u m a n m a s t ce l l s an d bas o p h i l s i n a s t h m a

103

pores in the plasma membrane to the exterior environment, when appropriately stimulated. Ultrastructural studies of human biopsies including lung parenchyma show that piecemeal degranulation is the most prevalent expression of secretion in human mast cells in vivo. This secretory process characteristically empties granules in place, leaving their containers intact. 2.3. Mediators of Human Mast Cells Mast cells produce a wide array of mediators and cell–cell signaling molecules, and this variety may account for the unique features of the mast cell in the immune system (Fig. 3). Human mast cells synthesize histamine, which is stored in secretory granules as a preformed mediator (3 pg/cell). These granules also contain a variety of proteolytic enzymes (a and b tryptase, chymase, carboxypeptidase A, and cathepsin G) (Goldstein et al., 1987;

Figure 3 Immunologically activated human mast cells release preformed and lipid mediators, cytokines, and chemokines. Human mast cells synthesize histamine, which is stored in secretory granules as a preformed mediator (3 pg/cell). Human mast cells from different anatomic sites contain in their secretory granules various concentrations of proteolytic enzymes (b and
a tryptase, chymase, carboxypeptidase A, and cathepsin G) and proteoglycans (heparin and chondroitin sulphate E). Human mast cells are a major source of a wide spectrum of cytokines, chemokines, and VEGF‐A. Interestingly, stem cell factor (SCF), the principal growth, differentiating, and chemotactic factor for human mast cells, is present in and released by lung mast cells. Immunological activation of all human mast cells leads to the synthesis of approximately 80 ng PGD2/106 cells. By contrast, the synthesis of LTC4 varies significantly among mast cells isolated from different anatomic sites. Immunologically activated lung mast cells produce PAF and a PAF analog 1‐acyl‐2‐acetyl‐GPC (AAGPC).

104

g i a nn i m a r o ne

ET AL.

Schechter et al., 1990, 2000) and proteoglycans (heparin and chondroitin sulphate E). The concentration of these enzymes varies significantly among mast cells isolated from different anatomical sites and among mast cells within the same localization. For instance, lung mast cells contain an average of 10 pg/ cell tryptase, whereas human skin mast cells contain a mean of 35 pg/cell (Patella et al., 1995). Human mast cells are a major source of arachidonic acid‐derived lipid mediators that can be secreted after their immunologic activation. Human lung mast cells contain 8 nmol of arachidonic acid/106 cells esterified into cellular lipids (Triggiani et al., 1995b). Upon IgE‐mediated activation of lung mast cells, only a small portion (<5%) of cellular arachidonic acid is released and metabolized through the cyclooxygenase (COX) and the 5‐lipoxygenase (5‐LO) pathways. The bulk of arachidonic acid for lipid mediator synthesis in mast cells is provided by a cytosolic group IV phospholipase A2. However, recent evidence suggests that another class of low‐molecular‐weight secretory phospholipase A2 (group IIA and X) may contribute to arachidonate generation in human mast cells (Triggiani et al., unpublished observations). All human mast cells synthesize prostaglandin D2 (PGD2) (80 ng/106 cells) subsequent to Fc
RI cross‐linkage. By contrast, there is striking tissue‐to‐tissue heterogeneity in the quantity of cysteinyl leukotriene generation. For example, mast cells isolated from human lung parenchyma generate an average of 60 ng LTC4/106 cells when stimulated by IgE‐receptor cross‐linking (Patella et al., 1995). In contrast, mast cells isolated from human heart generate 20 ng LTC4/106 cells, and skin mast cells generate little or no LTC4 (Patella et al., 1995). These wide variations in CysLT production among different human tissue mast cells suggest that this biochemical property is modulated by tissue‐ specific factors. However, the apparent consistency in PGD2 production suggests that this is an innate characteristic of the human mast cell lineage. There is increasing evidence that a number of lipid molecules other than the eicosanoids may play an important role in allergic inflammation. Triggiani et al. have shown that immunologically activated human lung mast cells synthesize Platelet Activating Factor (PAF). However, they produce larger amounts of a PAF analog containing a 1‐acyl group at the sn‐1 position of the molecule (1‐acyl‐2‐acetyl‐GPC:AAGPC) (Triggiani et al., 1990). IgE‐mediated activation of human lung mast cells induces the synthesis of 6 pmol of AAGPC/106 cells and 2 pmol of PAF/106 cells. The complete biological activities of the 2‐acetylated phospholipid analogs of PAF remain to be determined. Immunologic stimulation of human mast cells activates a specific program of gene expression leading to de novo synthesis of a wide spectrum of cytokines (IL‐3, IL‐5, IL‐6, IL‐13, IL‐16, IL‐18, IL‐25, TGF‐b, SCF, GM‐CSF, TNF‐a) (Bressler et al., 1997; Burd et al., 1995; Gauchat et al., 1993; Ohkawara et al.,

h u m a n m a s t ce l l s an d bas o p h i l s i n a s t h m a

105

1992; Okayama et al., 1998; Patella et al., 1998b; Pawankar et al., 1997; Rumsaeng et al., 1997) and chemokines (IL‐8/CXCL8, MIP‐1aCCL3, MCP‐ 1/CCL2, I‐309/CCL1) (Baghestanian et al., 1997; Gilchrest et al., 2003; Mo¨ ller et al., 1993; Yano et al., 1997). Interestingly, SCF, the principal growth, differentiating, and chemotactic factor for mast cells (de Paulis et al., 1999; Tsai et al., 1991), is synthesized (Zhang et al., 1998) and released by lung mast cells (Patella et al., 1998b). Thus, SCF, locally released by mast cells, might represent an autocrine factor that sustains mast cell hyperplasia in asthma. These results emphasize the finely tuned interactions between mast cells and their mediators. 2.4. Heterogeneity of Eicosanoid Metabolism in Human Mast Cells A body of biochemical, physiological, and pharmacological data implicate cysteinyl leukotrienes (CysLTs) in the pathogenesis of asthma. Mast cells are a heterogeneous source of CysLTs. Changes in the mechanisms that govern CysLT generation by mast cells could affect the development and severity of asthma and other allergic diseases. 5‐LO and the LTC4 synthase (LTC4S) are involved in the regulation of CysLT production. Genetic variants of the 5‐LO and LTC4S genes have been identified in humans. Variants of the 5‐LO core promoter sequence appear to be related to reduced sensitivity to 5‐LO inhibitors in asthmatic subjects (Drazen et al., 1999). Asthmatic patients with acetylsalicylic acid (ASA) sensitivity, and enhanced CysLT production, have an increased prevalence of heterozygosity for the LTC4S polymorphism in some (Kawagishi et al., 2002; Sanak et al., 2000), but not all (Van Sambeek et al., 2000) ethnic populations. Thus, there are polymorphisms at two potential control points (5‐LO and LTC4S), which could lead to intrasubject variability in CysLT generation by mast cells and other inflammatory cells. Locally produced cytokines greatly affect 5‐LO and LTC4S expression and activity. Human mast cells obtained from cord blood generate little CysLTs when activated by Fc
RI cross‐linking. When these cells were primed with IL‐4, there was a marked increase in CysLT production (Hsieh et al., 2001) caused by a rapid induction of LTC4S. Based on these studies, it appears that interactions between genetic factors and locally produced cytokines regulate, in a complex fashion, CysLT generation by mast cells in asthma. 2.5. Surface Markers of Human Mast Cells Human mast cells and basophils express the tetrameric (abg2) high affinity receptor for IgE (Fc
RI). However, the two cells selectively display a variety of membrane receptors (Fig. 4). After IFN‐g exposure, human mast cells express

106

g i a nn i m a r o ne

ET AL.

Figure 4 Selective display of membrane receptors on human mast cells. Mast cells and basophils express the tetrameric high affinity receptor for IgE (Fc
RI). IFN‐g‐primed mast cells express FcgRI, whose cross‐linking can induce the release of mediators. Mast cells, but not basophils, express the c‐kit receptor, which is activated by stem cell factor (SCF). These cells also express Toll‐like receptor 2 (TLR2), TLR3, TLR4, TLR5, TLR6, TLR7, and TLR9. Activators of TLR2, TLR3, and TLR4 selectively induce the release of various cytokines. Mast cells also express at least two receptors for CysLTs (CysLTR1 and CysLTR2). Mast cells express at least four chemokine receptors: CCR3, CXCR1, CXCR3, and CXCR4. Human skin and heart mast cells, but apparently not lung mast cells, express the receptor for C5a. Cultured mast cells express low levels of IL‐4Ra, IL‐5Ra, and IFN‐gRa. These cells also express a4b1, b7, a1b2, and aMb2 integrins and P‐selectin ligand, and bind to E‐selectin, P‐selectin, and VCAM‐1. Lung mast cells express the receptor for VEGF and PlGF, and the urokinase plasminogen activator (uPA) receptor (uPAR). Human mast cells express histamine H4 receptor, whose activation induces chemotaxis.

FcgRI, the cross‐linking of which can induce the release of mediators (Tkaczyk et al., 2004). Mast cells, but not basophils, typically express high levels of the c‐kit receptor, which is activated by SCF (Galli et al., 1993). Cultured mast cells express low levels of IL‐4Ra, IL‐5Ra, and IFN‐gRa (Kulka and Metcalfe, 2005). Bacterial microbes can trigger mast cells through Toll‐like receptors (TLRs), endowing them with the broad ‘‘pattern recognition’’ capability of the TLR system, which is probably an important element of their antibacterial

h u m a n m a s t ce l l s an d bas o p h i l s i n a s t h m a

107

responses (Supajatura et al., 2001). Human mast cells express TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, and TLR9 (Kulka et al., 2004). Activation of TLR2, TLR3, and TLR4 selectively induces the release of various cytokines (McCurdy et al., 2003). Mast cells also express at least two receptors for CysLTs (CysLTR1, CysLTR2) (Kanaoka and Boyce, 2004). The activation of various receptors for chemokines (CCR3, CXCR1, CXCR3, and CXCR4) on mast cells induces chemotaxis and/or mediator release (de Paulis et al., 2000; 2001; Ochi et al., 1999; Romagnani et al., 1999; 2000). Adhesion molecules play a role in recruitment of mast cell precursors to the selective localization in different compartments of the airways. Cultured human mast cells express a4b1, b7, a1b2, and aMb2 integrins and P‐selectin ligand, and bind to E‐selectin, P‐selectin, and vascular cell adhesion molecule‐1 (VCAM‐1) (Boyce et al., 2002). Human mast cells express the histamine H4 receptor, whose activation induces chemotaxis (Hofstra et al., 2003). Human skin and heart mast cells, but apparently not lung mast cells, express the receptor for anaphylatoxin C5a (Patella et al., 1995). Both lung mast cells and basophils express the high affinity urokinase plasminogen activator receptor (uPAR) (de Paulis et al., 2004a; Sillaber et al., 1997). 2.6. Histamine and Eicosanoid Receptors on Human Mast Cells Histamine was conventionally considered a mediator that causes the acute symptoms of allergic inflammation. However, increasing evidence suggests that histamine also participates in the long‐term regulation of immune cell function. For example, histamine induces lysosomal enzyme release and IL‐6 and TNF‐a production from human lung macrophages by activating the H1 receptor (Triggiani et al., 2001). In addition, histamine might induce mast‐cell chemotaxis by interacting with the H4 receptor (Hofstra et al., 2003). Interestingly, histamine inhibits the release of mediators from human basophils through the engagement of the H2 receptor (Lichtenstein and Gillespie, 1973). Immunological activation of lung mast cells induces the release of IL‐16, which is a chemoattractant for CD4þ T cells (Rumsaeng et al., 1997). IL‐16 has also been detected in bronchoalveolar lavage fluid from asthmatics (Mashikian et al., 1998). Histamine induces IL‐16 release by CD4þ T cells and bronchial epithelial cells (Mekori and Metcalfe, 1999). The release of histamine from Fc
RIþ cells might thus enhance IL‐16 secretion from other sources in the lung microenvironment. This complex interaction provides an explanation for the persistence of CD4þ T‐cell infiltration at allergic sites. CysLTs produced by immunologically activated Fc
RIþ cells exert a variety of responses by activating at least two receptors, CysLTR1 and CysLTR2.

108

g i a nn i m a r o ne

ET AL.

These receptors are expressed on bronchial smooth muscle (Heise et al., 2000; Lynch et al., 1999), human lung macrophages (Heise et al., 2000; Lynch et al., 1999), mast cells (Mellor et al., 2001), and peripheral blood leukocytes (Figueroa et al., 2001). Coincubation of human mast cells with IL‐4 upregulates the expression of both CysLTR1 (Mellor et al., 2002) and LTC4 synthase (Hsieh et al., 2001). This might be an additional mechanism by which basophils, a major source of IL‐4, in addition to directing the IgE switch in B cells, are able to influence the expression of the effector phenotype of human mast cells. Activation of CysLTRs not only elicits direct responses but also primes target cells to other stimuli. For example, CysLTs potentiate exocytosis and cytokine production in histamine‐activated lung macrophages. Macrophages are the predominant (80%) immune cells in both bronchial alveolar lavage (BAL) and lung parenchyma. Two subpopulations of human lung macrophages have been identified: low‐density macrophages (LDM) and high‐density macrophages (HDM) (Triggiani et al., 2004b). HDM account for about two thirds of macrophages retrieved from lung tissue, whereas LDM constitute the majority of cells in BAL. Interestingly, histamine induces IL‐6 release only from HDM and it has no effect on LDM (Triggiani et al., 2004b). CysLTs exert powerful vasoactive and bronchoactive effects in humans. They are remarkably powerful bronchoconstrictors (Davidson et al., 1987; Griffin et al., 1983), and cause bronchial eosinophilia (Laitinen et al., 1993), edema (Soter et al., 1983), and mucous hypersecretion (Marom et al., 1982) in vivo and/or in vitro. In a chronic allergen inhalation mouse model of asthma, CysLTs are also involved in the development of bronchial smooth muscle hyperplasia and submucosal collagen deposition (Henderson et al., 2002). Thus, CysLTs have both acute and chronic effects on airway functions. CysLTs act through at least two G‐protein‐coupled receptors (GPCRs), that is, the CysLT1 and CysLT2 receptors (Heise et al., 2000; Lynch et al., 1999). Both the 5‐LO inhibitor zileuton (Israel et al., 1996), which affects CysLT synthesis, and CysLT1 receptor antagonists (Altman et al., 1998) improve lung function in asthmatic subjects, and CysLT1 receptor antagonists attenuate the early and late‐phase pulmonary responses to inhaled allergen in asthmatics (Hamilton et al., 1998). Thus, CysLTs are involved in asthma and in airway dysfunction consequent to mast cell activation in vivo. PGD2 acts through a specific 7 transmembrane‐spanning GPCR, the DP receptor (Noguchi et al., 2002), to elicit bronchoconstriction in asthmatics (Hardy et al., 1984). Also the PGD2 receptor CRTH2 is chemotactic for eosinophils, basophils, and Th2 cells (Hirai et al., 2001). Experimental and clinical studies have confirmed the importance of DP receptors in asthma. The prostanoid DP receptor is required for the development of sensitization in a

h u m a n m a s t ce l l s an d bas o p h i l s i n a s t h m a

109

mouse model of asthma. The asthma phenotype does not develop in mice with a nonfunctional DP receptor (Matsuoka et al., 2000). Oguma et al. (2004) were the first to observe an association between DP gene (PTGDR) polymorphisms and asthma in case‐control studies of white and black Americans. They demonstrated that the implicated polymorphisms affect the binding of regulatory factors to the PTGDR gene promoter and that these polymorphisms alter the level of transcription of the gene. The variation in expression of the receptor influencing susceptibility to asthma suggests that the prostanoid DP receptor is a therapeutic target in allergic disorders. 3. Human Basophils 3.1. Origin of Human Basophils Basophils are granulocytes that circulate in human peripheral blood where they represent less than 1% of leukocytes. They are believed to represent a separate lineage from mast cells. Peripheral blood also contains basophil precursors (Denburg et al., 1983). IL‐3 is the principal cytokine responsible for human basophil growth and differentiation (Valent et al., 1989, 1990) from CD34þ pluripotent progenitor cells (Kirshenbaum et al., 1992). Other cytokines (GM‐CSF, IL‐4, IL‐5, SCF, and NGF) are also important for basophil growth, differentiation, and mediator production (Denburg, 1995). 3.2. Morphological and Ultrastructural Characteristics of Human Basophils Human basophils display polylobed nuclei that have condensed chromatin and inapparent nucleoli (Fig. 5). They are commonly identified by their metachromatic staining with basic dyes, such as toluidine blue. The surface profile of human basophils has irregular, broad processes. The cytoplasm contains membrane‐bound secretory granules, mitochondria, and small Golgi structures. The secretory granules of basophils are of two types. The most numerous granules are large, round, and filled with electron‐dense particles and/or finely granular material. An infrequent, small, paranuclear granule with homogenous content has also been observed (Dvorak, 1995). Human basophils extrude membrane‐free granules to the external microenvironment when stimulated with any of a variety of triggering agents. Exposure of basophils to multivalent stimuli bridging the IgE‐Fc
RI network in vitro initiates a series of biochemical and ultrastructural alterations, termed ‘‘anaphylactic degranulation,’’ which culminate in fusion of cytoplasmic granule membranes with the plasma membrane (Dvorak, 2000). Using electron

110

g i a nn i m a r o ne

ET AL.

Figure 5 Human peripheral blood basophil. Note the irregular blunt surface processes, polylobed nucleus, and large oval secretory granules filled with particles. Clusters of electron‐dense glycogen particles are present in the cytoplasm. 21,500. Photo kindly provided by Ann M. Dvorak.

microscopy, it was found that basophils that infiltrated certain immunologic responses in man underwent a progressive loss of cytoplasmic granule contents without evidence of anaphylactic degranulation (Dvorak, 2000). These basophils contain numerous small cytoplasmic vesicles, some of which fused to granules or plasma membranes. In this case, the loss of granule contents occurs by means of exocytotic vesicles that bud from the granule membrane, carrying with them some of the granule material. Upon fusion with the plasma membrane, these vesicles discharge their contents into the extracellular space. This vesicle‐mediated release of granule content is called ‘‘piecemeal degranulation.’’ Until a few years ago, technical limitations hampered the identification of basophils infiltrating the sites of inflammation. In fact, partially degranulated basophils are difficult to identify with conventional morphological techniques. The availability of monoclonal antibodies (BB1 and 2D7) that identify specific basophil epitopes (e.g., basogranulin) (Irani et al., 1998; McEuen et al., 2001) represents a technological breakthrough in these studies.

h u m a n m a s t ce l l s an d bas o p h i l s i n a s t h m a

111

3.3. Mediators of Human Basophils Human basophils contain 1 pg/cell of histamine (Fig. 6). Basogranulin, a basophil‐specific granule protein recognized by mAb BB1, used to identify basophils in tissues (de Paulis et al., 2004b; McEuen et al., 2001), is secreted together with histamine in response to IgE‐dependent activation (Mochizuki et al., 2003). Basophils express mature and enzymatically active tryptase at levels of less than 1% of those of mast cells unrelated to the a/b‐tryptase genotype (Foster et al., 2002; Jogie‐Brahim et al., 2004). Human basophils immunologically activated produce only LTC4 (30 ng/106 cells). Arachidonic acid to be converted to LTC4 in basophils is almost exclusively provided by the cytosolic group IV PLA2 (Triggiani et al., 2004a), even though these cells contain one or more secretory PLA2 isoforms (Hundley et al., 1998). No COX metabolite has been conclusively demonstrated in these cells. This may be due, among other possibilities, to the lack of COX activity in human basophils or to the distribution of arachidonic acid into phospholipid pools

Figure 6 Human peripheral blood basophils synthesize histamine, which is stored in secretory granules as a preformed mediator (1 pg/cell). Basophils contain in their secretory granules basogranulin, a specific granule protein recognized by the MoAb BB1, and mature and enzymatically active tryptase at levels of less than 1% of those of mast cells. Immunologic activation of basophils leads to the production of a restricted profile of TH2‐like cytokines (IL‐4 and IL‐13). Basophils also express the chemokines IL‐8/CXCL8 and MIP‐1a/CCL3 and several isoforms of VEGF‐A. Immunologically activated basophils produce only LTC4 (30 ng/106 cells); no COX metabolite has been conclusively demonstrated in these cells. Activated basophils also synthesize PAF and AAGPC in a ratio of 1:2.

112

g i a nn i m a r o ne

ET AL.

not accessible to COX. However, PGD2, unlike other prostaglandins, enhances histamine release from immunologically activated basophils. In addition, PGD2 also reverses the inhibition of release produced by drugs and hormones that activate adenylate cyclase to increase cellular cyclic AMP (cAMP) (Peters et al., 1984). Immunologically activated basophils produce both AAGPC and PAF in a ratio of 2:1 (Triggiani et al., 1991, 1995a). Interestingly, these two acetylated phospholipids exert divergent effects on basophils primed with cytokines (Brunner et al., 1991; Columbo et al., 1993b). Several laboratories have demonstrated that immunologic activation of human basophils leads to an increase in IL‐4 mRNA transcription and IL‐4 secretion (Genovese et al., 2003; MacGlashan, Jr., et al., 1994; Ochensberger et al., 1996; Redrup et al., 1998). Basophils secrete an average of 30 pg/106 cells of IL‐4 with a range of 10–80 pg/106 basophils; IL‐4 secretion starts 1 hour after stimulation and peaks 6 hours poststimulation. IL‐13 production is characterized by a different kinetic; in this case, there is a lag period of 4 hours and a plateau occurs 18–24 hours after anti‐IgE stimulation. Under these conditions in which purified basophils stimulated with various immunologic stimuli expressed mRNA for IL‐4 and IL‐13, there was no evidence of IFN‐g mRNA. Therefore, it appears that human basophils are unique because they express large amounts of IL‐4 and IL‐13 without any Th1‐like cytokine (e.g., IFN‐g) and without most proinflammatory cytokines (TNF‐a, IL‐1, IL‐6) (Dahinden, 2000). This indicates that basophils are not simply inflammation effector cells, but they also play a key immunoregulatory role by skewing immune responses toward the Th2 type. These results also demonstrate that basophils, differently from mast cells, express a restricted cytokine profile. The different profile of cytokines synthesized and released by basophils and mast cells provides indirect evidence that these cells have distinct roles in the appearance and fluctuation (exacerbations and remissions) of the asthma phenotype. Basophils also express the chemokines IL‐8/CXCL8 and MIP‐1a/CCL3 upon IgE receptor cross‐linking (Li et al., 1996). However, the biological meaning of this observation is unclear, because the quantities of these chemokines produced by basophils are rather small compared to those produced by other immune cells. Recently, VEGF‐A has also been identified in the supernatants of activated basophils (Marone et al., 2005a). 3.4. Surface Markers of Human Basophils Basophils express an impressively broad array of cell–cell signaling molecules and this might explain why these cells can be attracted and/or activated by a very wide variety of inflammatory and immune stimuli (Fig. 7). In addition to

h u m a n m a s t ce l l s an d bas o p h i l s i n a s t h m a

113

Figure 7 Selective display of membrane receptors on human basophils. Basophils express the tetrameric high‐affinity receptor for IgE (Fc
RI). These cells also express the FcgRIIb, whose cross‐linking inhibits the release of mediators. Basophils express Toll‐like receptor 2 (TLR2), TLR3, TLR4, TLR5, TLR6, TLR7, and TLR9. Basophils express various receptors for chemokines (CCR1, CCR2, CCR3, CXCR1, and CXCR4), and cytokines (IL‐3Ra, IL‐5Ra, GM‐CSFRa, and TRAK‐A), whose activation induces chemotaxis and/or mediator release. Basophils express the seven‐transmembrane receptor CRTH2, whose activation by prostaglandin D2 (PGD2) induces chemotaxis, and a receptor for PAF. They also express at least two receptors for CysLTs (CysLTR1 and CysLTR2) and three formyl peptide receptors (FPR, FPRL1, and FPRL2). Basophils express receptors for anaphylatoxins (C3a and C5a), for VEGF and PlGF (Flt‐1 and Flk‐1) and for IGF (IGF‐1R and IGF‐2R). Basophils express the urokinase plasminogen (uPA) receptor (uPAR). Human basophils express the histamine H2 receptor, whose activation inhibits the release of mediators. Basophils express low levels of LIR3 and LIR7; cross‐linking of LIR7 induces the secretion of mediators, whereas coligation of LIR3 and Fc"RI inhibits the release of mediators.

Fc
RI, human basophils express the inhibitory receptor FcgRIIb (Kepley et al., 2000). Coligation of FcgRIIb and Fc
RI inhibits the release of mediators from basophils. Of particular importance for the basophil response to agonists is the priming effect of certain cytokines that enhance effector functions, such as mediator release and cytokine expression. For example, IL‐3, IL‐5, GM‐ CSF, and NGF prime basophils with identical efficacies, but different potencies (IL‐3 > NGF  IL‐5  GM‐CSF) (Bischoff and Dahinden, 1992; Bischoff

114

g i a nn i m a r o ne

ET AL.

et al., 1990; Brunner et al., 1993). These effects are mediated by specific surface receptors. In particular, the priming effect of IL‐3, IL‐5, and GM‐ CSF is mediated by the expression of the common b‐chain in their receptors (Ochensberger et al., 1999). The potent priming and modulatory effect of NGF is mediated by the expression of the high affinity receptor TRK‐A (Burgi et al., 1996). The effects of nerve growth factor (NGF) on human basophils highlight the link between the nervous system and allergic inflammation in bronchial asthma. Basophils express various receptors for chemokines (CCR1, CCR2, CCR3, CXCR1, and CXCR4), whose activation induces chemotaxis and/or mediator release. The eotaxin receptor, CCR3, is expressed on the vast majority (80%) of human basophils (de Paulis et al., 2000, 2001; Uguccioni et al., 1997). This receptor is also expressed on human eosinophils, lung mast cells (20%) (de Paulis et al., 2000; Romagnani et al., 1999, 2000), dendritic cells (Beaulieu et al., 2002), and a small percentage of Th2 lymphocytes (Romagnani et al., 1999; Sallusto et al., 1997). CCR3 is activated by eotaxin/CCL11, eotaxin‐2/ CCL24, eotaxin‐3/CCL26, RANTES/CCL5, and mediates mostly chemotaxis to sites of allergic inflammation. Basophils express also high levels of CCR2, which can be activated by MCP‐1/CCL2, MCP‐2/CCL8, MCP‐3/CCL7, or MCP‐4/CCL13. Activation of CCR2 seems to mediate predominantly mediator release from basophils with only weak migratory responses (Bischoff et al., 1992; Uguccioni et al., 1997). As mentioned previously, an important function of basophils is their ability to migrate from peripheral blood to sites of allergic inflammation. This occurs thanks to a complex interplay of different chemotactic factors of various origins that act on a wide spectrum of surface receptors. PGD2, a major mast‐cell mediator released during the allergic response (Genovese et al., 2000; Schleimer et al., 1985), is chemotactic for basophils, eosinophils, mast cells, and Th2 cells through activation of the CRTH2 receptor (Hirai et al., 2001). The anaphylatoxins C3a and C5a are potent chemoattractants for basophils and induce mediator release particularly in basophils primed with certain cytokines (IL‐3, IL‐5, GM‐CSF) (Bischoff et al., 1990; Ochensberger et al., 1995, 1996). The effects of anaphylatoxins are mediated by specific seven transmembrane (STM) G‐protein‐coupled receptors C3aR and C5aR. Among this family of STM, de Paulis and collaborators have recently identified in human basophils at least three receptors that bind several natural N‐formyl peptides, including the prototype N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP) (de Paulis et al., 2004a,b). Basophils express the high affinity receptor FPR and its homologues FPR‐like‐1 (FPRL1) and FPR‐like‐2 (FPRL2). The latter two receptors serve as chemotactic receptors for endogenous or viral products. For example, two HIV‐1 gp41 peptides act as chemoattractants for

h u m a n m a s t ce l l s an d bas o p h i l s i n a s t h m a

115

basophils by interacting with FPRL1 (de Paulis et al., 2002). It was also found that urokinase induces basophil chemotaxis through a urokinase receptor epitope that is an endogenous ligand for FPRL1 and FPRL2 (de Paulis et al., 2004a). These results provide the first evidence that the uPA/uPAR system is involved in allergic disorders. Activated Fc
RIþ cells express the ligand for CD40 (CD40L), which can provide the cell contact signal required for IgE synthesis by human B cells (Gauchat et al., 1993). Mast cells and basophils may therefore play a key role in allergic disorders, not only by producing inflammatory and fibrogenic mediators, but also by directly (CD40L) and indirectly (IL‐4 and IL‐13) regulating IgE synthesis independently of T cells. Recent evidence demonstrates that human basophils can contribute to various aspects of angiogenesis. Basophils synthesize and release such potent proangiogenic factors as vascular endothelial growth factor (VEGF). Furthermore, VEGF and placental growth factor (PlGF) are chemotactic for basophils through the activation of VEGF receptors (Marone et al., 2005a). The leukocyte immunoglobulin‐like receptors (LIRs) comprise a family of immunoregulatory cell surface receptors that include both activating and inhibitory receptors (Arm, 2004). Human basophils express low levels of LIR3 and LIR7 (Sloane et al., 2004). Cross‐linking of LIR7 induces the secretion of proinflammatory mediators and IL‐4, whereas coligation of LIR3 and Fc
RI inhibits the release of mediators. CD203c (ecto‐nucleotide pyrophosphatase/phosphodiesterase 3), a transmembrane protein has been described as being selectively expressed on basophils, mast cells, and their CD34þ progenitors. As CD203c is rapidly upregulated after allergen challenge in sensitized patients, it has been proposed as a new tool for allergy diagnosis (Boumiza et al., 2003). 3.5. Autocrine and Paracrine Effects of Mediators on Mast Cells and Basophils Infiltrating basophils and resident mast cells probably continuously release mediators (histamine, LTC4, PGD2, tryptase, and chymase), as well as cytokines and chemokines, in the airways of asthmatics. The local concentration of these mediators might be important not only in sustaining chronic inflammation, but also by influencing the homing and functions of immune cells in the lung, such as Fc
RIþ cells, T cells, macrophages, ASM, and epithelial cells. For example, histamine inhibits the release of mediators from human basophils through the engagement of the histamine H2 receptor (Lichtenstein and Gillespie, 1973). Interestingly, mast cells produce IL‐25 (Ikeda et al., 2003),

116

g i a nn i m a r o ne

ET AL.

a cytokine that induces IL‐4 and IL‐13 gene expression, and is thus capable of amplifying allergic inflammation. A striking example of the paracrine effects exerted by cytokines on the activation of Fc
RIþ cells is the observation that IL‐3, IL‐5, and GM‐CSF produced by mast cells enhance histamine secretion and IL‐4 synthesis in basophils. Similarly, NGF and IL‐18 enhance histamine and cytokine release by basophils (Sin et al., 2001; Yoshimoto et al., 1999), whereas IL‐4 from basophils enhances the production of proinflammatory mediators (PGD2 and LTC4) (Hsieh et al., 2001) and various cytokines (Ochi et al., 2000) by mast cells. Thus, depending on the setting, Th2 (IL‐4, IL‐5, IL‐3) or Th1 (IL‐18) cytokines can induce changes in the biosynthetic pathways of basophils and mast cells at the loci of allergic inflammation. SCF is the principal growth, differentiating, and chemotactic factor for human mast cells (Galli et al., 1993). Interestingly, SCF is present in and immunologically released by lung mast cells (de Paulis et al., 1999) and represents an autocrine factor that sustains mast‐cell hyperplasia in asthma. 4. Experimental Evidence for Mast Cells in Bronchial Asthma In murine models of asthma‐like disease, systemic sensitization is obtained using adjuvant‐conjugated antigens such as ovalbumin (Corry et al., 1996; Foster et al., 1996), and those derived from cockroach (Lukacs et al., 2002), dust mite (Sadakane et al., 2002), mold (Schuh et al., 2002), or parasites (Mochizuki et al., 2001); this is followed by repeated inhalation challenge with the same antigens. Animals so sensitized produce allergen‐specific IgE and Th2 cells and develop pulmonary eosinophilia, cytokine production, and airway hyperresponsiveness (AHR) in response to methacholine. Evidence that mast cells are primary effector cells in asthma derives from these and various experimental models of atopic and nonatopic asthma (Kobayashi et al., 2000; Kraneveld et al., 2002; Mayr et al., 2002; Yu and Chen, 2003). However, the results obtained in experimental studies in rodents are conflicting regarding the relative importance of individual cytokines and the relevance of mast cells in the pathogenesis of lung inflammation (Takeda et al., 1997). Mast cells appear to be required for the expression of bronchial eosinophilia and AHR in some (Kobayashi et al., 2000; Williams and Galli, 2000), but not all systems (Ogawa et al., 2002). Consequently, depending on the asthma model investigated, mast cells can play a crucial role, or not, in multiple features of allergic airway responses. It is important to note that rodent mast cells differ immunologically, phenotypically, biochemically, and pharmacologically from human mast cells (Marone et al., 2000). Even among human mast cells there is a significant

h u m a n m a s t ce l l s an d bas o p h i l s i n a s t h m a

117

degree of heterogeneity between cells isolated from different tissues presumably because of the influence of the microenvironment. This should be kept in mind when conducting studies with human mast cells cultured in vitro from progenitors in cord or in peripheral blood. It is likely that the different experimental conditions (e.g., different cytokines) may not fully reproduce the biochemical and phenotypical characteristics of the lung mast cells. Therefore, pathogenetic and pharmacological studies relevant to asthma should be always performed/confirmed with mast cells obtained from different compartments (lung parenchyma, BAL) of the human lung. 5. Experimental Evidence for Basophils in Bronchial Asthma Based on their apparent similarity to mast cells, basophils have often been considered (and neglected) as minor and possible redundant ‘‘circulating mast cells.’’ It is now evident that basophils derive from their own specific progenitor and their main growth and differentiating factor is IL‐3 (Kirshenbaum et al., 1992; Saito et al., 1988). In addition, human basophils differ from mast cells morphologically and ultrastructurally; they produce a different set of preformed and de novo synthesized mediators. More importantly, basophils express a restricted profile of Th2‐like cytokines (IL‐4 and IL‐13), whereas mast cells synthesize a wide spectrum of proinflammatory and immunoregulatory cytokines. These marked differences suggest that the two cell types could have evolved to play different roles in the pathogenesis of allergic disorders. Basophils are normally confined to the circulation and are not found in normal tissues. The involvement of basophils in asthma derives from indirect evidence. It is well documented that basophils infiltrate the sites of allergic inflammation (Irani et al., 1998; KleinJan et al., 2000; Ying et al., 1999). In addition, basophils have been found in the airways of asthmatics (Gauvreau et al., 2000; Liu et al., 1991; Macfarlane et al., 2000), in postmortem cases of fatal asthma (Kepley et al., 2001; Koshino et al., 1993) and after antigen challenge of airway mucosa (KleinJan et al., 2000; Nouri‐Aria et al., 2001). From a functional viewpoint, IgE‐mediated basophil releasability (i.e., the ability of a basophil/mast cell to release a given percentage of histamine in response to a given immunological stimulus) is increased in asthma (Casolaro et al., 1990). More importantly, allergen‐induced asthmatic responses are accompanied by infiltration of basophils that express IL‐4 mRNA (Nouri‐Aria et al., 2001). Thus, basophils might represent an important source of Th2‐like cytokines (IL‐4 and IL‐13) in the lung microenvironment. Past studies of the role of basophils in health and disease were curtailed by the lack of suitable experimental tools. The recent availability of techniques for the isolation and purification of basophils from peripheral blood and of

118

g i a nn i m a r o ne

ET AL.

monoclonal antibodies (2D7 and BB1) that recognize specific epitopes in these cells (Irani et al., 1998; McEuen et al., 2001) should encourage further studies of basophil functions in allergic diseases (Falcone et al., 2000; Marone et al., 2005b). 6. Mast Cell and Basophil Recruitment to Asthmatic Airways Studies of chemokines and their receptors have provided the basis for better understanding of the processes leading to Fc
RIþ cell recruitment in asthmatic airways. Human lung mast cells (Fig. 4) and basophils (Fig. 7) are endowed with a wide set of chemotactic receptors. Basophils constitutively express several chemokine receptors (CCR1, CCR2, CCR3, CXCR1, CXCR3, and CXCR4). Approximately 80% of basophils express CCR3, which can be activated by eotaxin/CCL11, eotaxin‐2/CCL24, eotaxin‐3/CCL26, RANTES/CCL5, MCP‐3/CCL7, and MCP‐4/CCL13 (Romagnani et al., 1999; Uguccioni et al., 1997). CCR3 is also expressed by 20% of lung mast cells (Romagnani et al., 1999). Eotaxin does not induce histamine release from mast cells (Romagnani et al., 1999) but increases Fc
RI‐dependent IL‐4 and IL‐13 generation by basophils (Devouassoux et al., 1999) and mast cells (Price et al., 2003), respectively. It is likely that RANTES/CCL5, eotaxin/CCL11, and eotaxin‐2/CCL24 production by human airway epithelial cells (Stellato et al., 1997) and smooth muscle (Hirst et al., 2002) accounts in part for the recruitment of basophils and mast cells to the lung during allergic inflammation. Two cytokines, IL‐4 and tumor necrosis factor‐a (TNF‐a), produced by basophils and mast cells, respectively, increase eotaxin mRNA stability in airway epithelial cells, thereby further amplifying the recruitment of inflammatory cells (Atasoy et al., 2003). Asthmatic ASM specifically synthesizes IP‐10/CXCL10, which activates the lung mast cells that express CXCR3. This could be a major pathway that facilitates the migration of mast cells into the ASM bundles in asthmatics. ASM (Page et al., 2001) and mast cells generate SCF (de Paulis et al., 1999), which is chemotactic for human mast cells through activation of the c‐kit receptor (Galli et al., 1993). Adhesion molecules also play an important role in the recruitment of mast‐ cell precursors to the airways, and possibly in selective localization of these cells within the tissue. In fact, cultured human mast cells express various integrins and VCAM‐1 (Boyce et al., 2002). Interestingly, binding of mast cells to IL‐4‐activated human umbilical vein endothelial cells (HUVEC) is completely blocked by antibodies to a4b1, although binding to TNF‐a‐ activated HUVEC is not (Boyce et al., 2002). Thus, a4b1 integrin may play a

h u m a n m a s t ce l l s an d bas o p h i l s i n a s t h m a

119

role in mast‐cell progenitor recruitment to the asthmatic airways, where IL‐4 and IL‐13 upregulate VCAM‐1 expression. Recently, insulin‐like growth factor‐1 (IGF‐1) and IGF‐2 have been identified as selective basophil chemoattractants in human nasal polyps (Hartnell et al., 2004). These factors induce chemotaxis of basophils, but not eosinophils or neutrophils. Therefore, IGF‐1 and IGF‐2 may account for the preferential infiltration of basophils observed in certain allergic disorders, although in vivo studies are required to confirm this hypothesis. These observations demonstrate that chemotactic factors produced by resident structural cells, such as ASM and airway epithelial cells, have a pivotal role in mast cell and basophil microlocalization in distinct compartments of asthmatic airways. In addition, mast cells also produce chemotactic mediators (SCF, histamine, and PGD2), thereby sustaining an autocrine loop that might further increase Fc
RIþ cell recruitment. 7. Anatomical and Functional Evidence for Mast Cells and Basophils in Asthma Mast cells are unique among immune cells in that they are distributed in all vascularized tissues and are particularly abundant at the interfaces with the external environment (skin, respiratory, gastrointestinal, and genitourinary tracts) where they are primary effector cells in the immediate responses that can occur when sensitized individuals come in contact with allergens. Normal human bronchi contain mast cells in submucosal connective tissue, but generally not in the smooth muscle or in the epithelial layers. In contrast, mast cells are present in different compartments of the lung of asthmatic individuals (i.e., the bronchoalveolar space [Casolaro et al., 1989], beneath the basement membrane, adjacent to blood vessels, surrounding the submucosal glands, and scattered throughout the ASM bundles [Brightling et al., 2002]). Microlocalization throughout the ASM bundles is a specific feature of the asthmatic phenotype and mast‐cell density in the bronchial smooth muscle from asthmatics is correlated with the degree of airway hyperresponsiveness to methacholine (Brightling et al., 2002). The specificity of this observation is supported by the finding that mast cells were not found in ASM bundles in eosinophilic bronchitis, a condition similar to asthma and characterized by similar inflammatory infiltrates in the submucosa of the lower airways. More recently, IL‐4þ and IL‐13þ mast cells have been found within the ASM bundles of asthmatics suggesting that these cytokines have an important role in mast cell–ASM interactions in asthma (Brightling et al., 2003). As mentioned previously, the location of mast cells within the hyperplastic bronchial smooth muscle layer of asthmatics could also reflect interactions

120

g i a nn i m a r o ne

ET AL.

between ASM and mast cells. Asthmatic bronchial smooth muscle cells generate SCF, which is the mast‐cell growth factor. Mast cells, through the mitogenic effects of their tryptases, could in turn promote smooth‐muscle hyperplasia (Brown et al., 2002). Mast‐cell density is increased in bronchial biopsy specimens of both atopic and nonatopic asthmatics (Amin et al., 2000; Chetta et al., 2003). The positive correlation between the thickness of the tenascin and laminin layers and mast‐ cell density in asthmatics suggests that mast cells are involved in tissue remodeling (Amin et al., 2000). In addition, the positive correlation between mast‐cell density and the number of blood vessels suggests that these cells play a role in angiogenesis in asthma (Chetta et al., 2003). An increase in circulating mast‐cell progenitors might contribute to the recruitment of these cells to sites of asthmatic mucosal inflammation (Mwamtemi et al., 2001). A characteristic of asthma is mast‐cell activation in vivo. Autopsy specimens from subjects who died of asthma, and biopsy specimens from asthmatic volunteers, show evidence of mast‐cell degranulation (Carroll et al., 2002; Pesci et al., 1993). The notion that altered or amplified mast‐cell function is a feature of asthma is supported by the finding that CysLT release consequent to endobronchial allergen challenge was 10‐ to 20‐fold greater in asthmatics than in controls (Heard et al., 1990). Inhalation challenge with specific antigen of asthmatic adults rapidly (15–30 min) causes bronchial smooth muscle constriction and bronchial wall edema (early phase of asthmatic response) and hence airflow obstruction (Inman et al., 1995). Mast cell/basophil‐derived mediators (histamine, CysLTs, PGD2, and tryptase) occur in bronchial lavage fluid during the early phase response (Casale et al., 1987; Liu et al., 1991), confirming Fc
RIþ cell activation. In about half of subjects, the early phase response is followed hours later by a longer period of airflow obstruction, accompanied by cellular infiltration (eosinophils and basophils) of the bronchial mucosa and enhanced nonspecific AHR (late phase response) (Gauvreau et al., 2000; Liu et al., 1991). Studies with selective pharmacologic antagonists indicate that mast cell‐derived mediators are involved in both the early and the late phase of the allergic pulmonary response. For example, the early‐phase response is almost completely prevented by preadministration of a combination of H1‐receptor antagonists and CysLT1‐receptor antagonists. The same combination greatly but not totally attenuates the late‐phase response (Hamilton et al., 1998; Roquet et al., 1997). Also preadministration of a nonanaphylactogenic anti‐IgE monoclonal antibody affects both phases (Fahy et al., 1997). With the caveat that experimental allergen challenge does not completely reproduce the pathophysiology of asthma, the aforementioned studies show that Fc
RIþ cell activation may alter airway function.

h u m a n m a s t ce l l s an d bas o p h i l s i n a s t h m a

121

8. Superallergens in Bronchial Asthma Mast cells occur in all tissues. They are particularly abundant in tissues that have contact with the external environment such as the lung, where they exert homeostatic and protective functions. Studies conducted during recent decades have demonstrated a direct role for human mast cells and basophils in host defense against bacterial and viral pathogens (Abraham and Malaviya, 2000; Marshall and Jawdat, 2004; Patella et al., 2000a). Unlike normal congenic control mice, mast cell‐deficient mice have poor survival when subjected experimentally to septic peritonitis or gram‐negative pneumonia (Echtenacher et al., 1996; Malaviya et al., 1996; Prodeus et al., 1997). The latter protective effects of mast cells are mainly due to their ability to enhance the recruitment of leukocytes through the release of cytokines (Echtenacher et al., 1996; Malaviya et al., 1996; Prodeus et al., 1997). However, other aspects of Fc
RIþ cell activation probably contribute to the appropriate local response to bacterial and viral infections. The production of antimicrobial peptides by mast cells is another potentially important aspect of their function in innate host defense. Cathelicidins and defensins are major families of antimicrobial peptides in mammals. Their function is to disrupt the integrity of the microbial membrane. The human cathelicidin (LL‐37) and the cathelicidin‐related peptide (CRAMP) are both expressed by mast cells (Di Nardo et al., 2003). Cathelicidin LL‐37 is also chemotactic (Niyonsaba et al., 2002) and induces the release of mediators from mast cells (Niyonsaba et al., 2001). Complement fixation is a feature of the innate response to different infections and tissue damage. Human mast cells and basophils express multiple receptors for complement components (C3a and C5a) (Fu¨ reder et al., 1995; Patella et al., 1995). Compared with normal congenic control mice, complement‐deficient mice exhibited reductions in mast‐cell degranulation, production of TNF‐a, neutrophil infiltration, and clearance of bacteria (Prodeus et al., 1997). There are a variety of other receptor systems implicated in the Fc
RIþ cell’s innate response to pathogens. A recent and intriguing example is the ability of the FMLP receptors FPRL1 and FPRL2 to mediate the activation and chemotaxis of basophils in response to viral (gp41 of HIV‐1) and bacterial [Hp (2–20)] peptides (de Paulis et al., 2002, 2004b). IL‐4‐primed mast cells express Toll‐like receptor 4 (TLR4), whose activation by lipopolysaccharide (LPS) induces the release of Th2 cytokines (Okumura et al., 2003). Activators of TLR2 and TLR3 selectively induce the release of leukotrienes, GM‐CSF, and IL‐1b (Kulka et al., 2004; McCurdy et al., 2003). Human cultured mast cells also express TLR1, TLR2, TLR4, TLR5, TLR6, TLR7, and TLR9 (Kulka et al., 2004).

122

g i a nn i m a r o ne

ET AL.

Consequent to these observations, it became evident that human mast cells and basophils express several receptors that play a role in the innate pattern recognition of microbes (TLRs, FPR, FPRL1, FPRL2, CD48, and complement receptors) (Gommerman et al., 2000; Malaviya et al., 1999; Supajatura et al., 2001, 2002). These receptors can directly activate Fc
RIþ cells in response to specific microbial constituents or complement fragments, irrespective of antibody and T‐cell education—the so‐called ‘‘innate’’ immunity. Thus the concept emerged that mast cells are specialized to receive danger signals in infection and injury, and they release mediators that initiate a protective leukocyte response, which facilitate the subsequent repair and healing. This function is teleologically consistent with the strategic localization of mast cells in mucosal (respiratory, gastrointestinal, and genitourinary tracts) and skin surfaces, which regularly contact microbes. Amplification of these processes could lead to persistent inflammation and excessive collagen deposition, angiogenesis, and smooth muscle hyperplasia that are characteristic of bronchial asthma. Human mast‐cell lines can be infected in vitro with rhinovirus, the major agent involved in 80% of asthma exacerbations (Johnston et al., 1995), through intracellular adhesion molecule (ICAM)‐1, which lowers their threshold for Fc
RI‐dependent exocytosis and cytokine generation (Hosoda et al., 2002). This lowered threshold could be associated with the observation that experimental rhinovirus infection enhances allergen‐induced late‐phase responses and histamine release in subjects suffering from allergic rhinitis (Calhoun et al., 1991). A novel mechanism by which human Fc
RIþ cells can be activated by bacterial and viral proteins, acting as immunoglobulin superantigens, has recently been identified (Marone et al., 2004b). This mechanism might physiologically play a protective role in certain bacterial and viral infections. Amplification of this mechanism could lead to superantigen activation of Fc
RIþ cells, thus providing the means by which certain viruses and bacteria can cause asthma exacerbations. Superantigens are characterized by their ability to induce changes in the composition of a lymphocyte repertoire. A conventional antigen can usually stimulate less than 0.001% of the naı¨ve lymphocyte pool, whereas a superantigen can stimulate greater than 5% of the naı¨ve lymphocytes (Silverman, 1998). This immunologic property derives from the unique ability of superantigens to interact with most lymphocytes that express antigen receptors from a particular variable (V) region gene family (Silverman, 1997). Classical superantigens are T‐cell superantigens (Staphylococcal enterotoxins and toxic shock syndrome toxin‐1). Some naturally occurring proteins are superantigens for B‐lymphocytes. B‐cell superantigens are endowed with unconventional immunoglobulin‐binding

h u m a n m a s t ce l l s an d bas o p h i l s i n a s t h m a

123

capacities that parallel the properties of T‐cell superantigens to activate lymphocytes. The best characterized of these immunoglobulin superantigens is Staphylococcus aureus protein A, which is the B‐cell superantigen prototype (Graille et al., 2000; Ingana¨ s et al., 1980). Other B‐cell superantigens are the gp120 envelope glycoprotein of HIV‐1 (Townsley‐Fuchs et al., 1997), a human gut‐associated sialoprotein termed ‘‘protein Fv’’ (Guihard et al., 1997), and protein L from Peptostreptococcus magnus (Bjo¨ rck, 1988). The concept of immunoglobulin superantigens applied to the pathophysiology of allergic disorders could be translated as ‘‘superallergens’’ to indicate proteins of various origins able to activate Fc
RIþ cells through interaction with membrane‐bound IgE. It is generally thought that four canonical mechanisms of IgE‐mediated activation of human Fc
RIþ cells are responsible for the pathophysiologic involvement of these cells in the majority of allergic disorders (Marone, 1998a). However, evidence exists that a significant percentage of allergic diseases (e.g., certain cases of asthma) cannot be explained by the four classical mechanisms of Fc
RIþ cell activation. Therefore we have investigated the hypothesis that immunoglobulin superantigens of various origins (endogenous, bacterial, and viral) can activate Fc
RIþ cells to release proinflammatory mediators and cytokines. 8.1. Protein Fv: An Endogenous Immunoglobulin Superallergen Skin rashes and urticarial reactions occur in about 5% of patients with acute and chronic viral hepatitis (Popp et al., 1981; Segool et al., 1975; Vaida et al., 1983). Protein Fv is a sialoprotein produced in the human liver and released in biological fluids during viral hepatitis A, B, C, and E (Bouvet et al., 1990). Protein Fv binds to the variable domain of the heavy (H) chains of immunoglobulin (Ig), irrespective of Ig class, subclass, and light (L) chain type (Bouvet et al., 1991b). A single protein Fv molecule can bind six F(ab’)2 fragments (Bouvet et al., 1991a) of human IgM, IgG, and IgE (Bouvet et al., 1990, 1991a, b). Binding of protein Fv to the VH3 region of immunoglobulins occurs in a domain external to the conventional antigen‐binding pocket (Bouvet et al., 1991a,b). We found that protein Fv is the most potent IgE‐mediated stimulus for the activation of human basophils and lung mast cells (Patella et al., 1998a). Protein Fv is approximately 100 times more potent than anti‐IgE and therefore acts as a complete secretagogue on Fc
RIþ cells by interacting with IgE VH3þ. The VH3 is the largest immunoglobulin family within the human repertoire (50%) (Karray and Zouali, 1997; Karray et al., 1998; Silverman, 1997). Therefore, protein Fv can function as an endogenous immunoglobulin superantigen that

124

g i a nn i m a r o ne

ET AL.

interacts with a high frequency with IgE VH3þ bound to Fc
RIþ cells (Patella et al., 1993, 1998a). We also demonstrated that low concentrations of protein Fv can induce IL‐4 secretion from basophils through interaction with IgE VH3þ (Patella et al., 1998a). This was the first evidence that an endogenous superallergen, protein Fv, released during viral infections, can induce the synthesis and secretion of such an important cytokine as IL‐4 from basophils. IL‐4 is a critical cytokine in the regulation of IgE synthesis by B lymphocytes (Del Prete et al., 1988) and it is intriguing that some patients with viral hepatitis have high serum IgE levels (Van Epps et al., 1976). The in vitro potency of protein Fv raises the possibility that it exerts important effects in vivo. It is conceivable that protein Fv released in the blood of patients with acute and chronic HCV, HAV, or HBV infection contributes, through the release of mediators from Fc
RIþ, to the allergic manifestations observed in some of these patients (Miadonna et al., 1982; Segool et al., 1975; Vaida et al., 1983). The mechanism of Fc
RIþ cell activation by protein Fv represents a new pathogenetic cascade consisting of viral infection, endogenous immunoglobulin superantigen production, activation of Fc
RIþ cells, and tissue injury (Fig. 8). More generally, this sequence of events raises the possibility that additional endogenous immunoglobulin superantigens induced by viruses can cause tissue injury in allergic inflammation through this mechanism that involves Fc
RIþ cell activation. 8.2. HIV‐1 gp120: A Viral Superallergen Serum IgE levels are increased in HIV‐1‐infected children (Koutsonikolis et al., 1996; Onorato et al., 1999; Vigano` et al., 1995) and adults (Paganelli et al., 1995; Rancinan et al., 1998; Wright et al., 1990). Interestingly, HIV‐1‐ infected patients have an increased prevalence and/or severity of allergic reactions and adverse reactions to drugs (Coopman et al., 1993; Kaplan et al., 1987). Elevated IgE levels have been associated with the progression of HIV‐l disease (Israe¨ l‐Biet et al., 1992; Koutsonikolis et al., 1996; Onorato et al., 1999; Rancinan et al., 1998; Vigano` et al., 1995). HIV‐1 gp120 is a member of the immunoglobulin superantigen family (Karray and Zouali, 1997) and immunoglobulins VH3þ are a ligand for gp120 (Berberian et al., 1993). We found that four recombinant gp120 derived from divergent HIV‐1 isolates from different viral clades of various geographical origins stimulated the release of IL‐4 and IL‐13 parallel to the secretion of histamine from basophils (Florio et al., 2000; Patella et al., 2000b). By contrast, IFN‐g mRNA was not detected in any of the gp120‐stimulated basophil

h u m a n m a s t ce l l s an d bas o p h i l s i n a s t h m a

125

Figure 8 A new pathogenic link between endogenous, viral, and bacterial superallergens and tissue injury. Protein Fv and gp120 are two immunoglobulin superantigens that enable viruses to activate Fc
RIþ cells. Protein Fv, synthesized in low amounts in normal liver, is released in biological fluids of patients affected by acute and chronic viral hepatitis. Protein Fv has six binding sites for the VH3 region of human immunoglobulin (Ig) and is a potent stimulator of histamine and cytokine release from Fc
RIþ cells through the interaction with VH3þ IgE. HIV‐1 gp120 from divergent HIV‐1 isolates from different viral clades of various geographical regions activate human Fc
RIþ cells through interaction with membrane‐bound VH3þ IgE. Protein A, a cell‐wall protein of Staphylococcus aureus, has a classical binding site for Fcg and an alternative site that binds the Fab portion of 15–50% of human polyclonal IgM, IgA, IgG, and IgE. Protein A induces mediator release from human basophils and mast cells through interaction with the VH3 domain of IgE. Peptostreptococcus magnus is a bacterium expressing a cell‐wall protein L that binds human Ig regardless of the heavy‐chain class, through high‐affinity interaction with k light chains, and is thus an Ig superantigen. Protein L is a potent stimulator of the activation of human Fc
RIþ cells through interaction with the k light chain of membrane‐bound IgE. This illustrates a novel mechanism by which endogenous (protein Fv), bacterial (protein A and protein L), and viral proteins (gp120) activate human Fc
RIþ cells, thereby acting as superallergens. Modified with permission, from Marone et al., 2004b.

preparations. This indicated that gp120‐mediated stimulation of Fc
RIþ cells induced only cytokines of the TH2 profile (Marone et al., 2001). Preincubation of gp120 with three preparations of human monoclonal IgM VH3þ inhibited the effect of gp120 on IL‐4 and IL‐13 secretion from basophils. Therefore, the viral superantigen gp120 can rapidly activate Fc
RIþ cells through interaction with IgE VH3þ (Fig. 8). These results represent a new pathogenetic cascade consisting of viral infection, viral immunoglobulin superallergen, activation of Fc
RIþ cells, and allergic disorders. This raises the possibility that other viral immunoglobulin superantigens can cause allergic diseases through this novel mechanism.

126

g i a nn i m a r o ne

ET AL.

8.3. Protein A: A Bacterial Immunoglobulin Superallergen Most clinical isolates of Staphylococcus aureus synthesize protein A, a cell‐wall protein that has unique immunoglobulin‐binding properties. Protein A has a classical site that binds the Fcg of IgG, and an alternative site that binds the Fab portion of 15–50% of human polyclonal IgG, IgM, IgA, and IgE (Ingana¨ s, 1981). S. aureus Cowan 1, which synthesize protein A, and soluble protein A induce gradual increases in histamine release from basophils (Marone et al., 1982) and lung mast cells (Genovese et al., 2000). S. aureus Wood 46 which does not contain protein A, does not activate these cells (Marone et al., 1987). Protein A mediates the Staphylococcus‐induced activation of Fc
RIþ cells through the interaction of the alternative binding site with IgE VH3þ. The mechanism of Fc
RIþ cell activation by protein A represents a novel pathogenetic cascade consisting of S. aureus infection, bacterial superallergen production, activation of Fc
RIþ cells, and tissue injury (Fig. 8). This raises the possibility that exacerbations of atopic dermatitis (Leung and Bloom, 2003) and certain forms of asthma (Suh et al., 2004) associated with S. aureus infection can be caused through this mechanism. 8.4. Protein L: A Bacterial Immunoglobulin Superallergen Protein L, a cell‐wall protein synthesized by the bacterium Peptostreptococcus magnus (Bjo¨ rck, 1988), consists of up to five repeated Ig‐binding domains (B1–B5) (Kastern et al., 1992) and appears to be a virulence determinant (Ricci et al., 2001). Each homologous domain binds with high affinity to the variable domain of the VKI, VKIII, and VKIV subgroups, but does not bind to Ig H chains, l chains, the CL domains of k L chains, or the variable domain of the VKII subgroup (Nilson et al., 1992). Thus, protein L binds human Ig regardless of the H chain class, is mitogenic for B cells (Axcrona et al., 1995), and is an immunoglobulin superantigen (Silverman, 1997). We demonstrated that protein L induces the release of proinflammatory mediators and cytokines (IL‐4 and IL‐13) from basophils and lung mast cells by interacting with the IgE bound to Fc
RI (Genovese et al., 2000, 2003; Patella et al., 1990). This is the first demonstration that a bacterial protein (protein L) activates human Fc
RIþ cells through the interaction with the k light chains of IgE. The association between certain viral infections and the induction and/or exacerbation of allergic reactions is well established (Gern and Busse, 2000; Holtzman et al., 2002; Johnston, 1997). Moreover, certain bacterial infections (e.g., Staphylococcus aureus) can also induce exacerbation of atopic dermatitis (Leung et al., 1998) and certain forms of asthma (Suh et al., 2004). Our results

h u m a n m a s t ce l l s an d bas o p h i l s i n a s t h m a

127

provide a novel mechanism by which viral and bacterial infections can be involved in the induction and/or exacerbation of certain allergic reactions. Figure 8 schematically depicts a new pathogenetic link between viral and bacterial infections, the production of superallergens, and tissue injury in allergic inflammation. The in vivo implications of IgE‐mediated activation of human Fc
RIþ cells by these immunoglobulin superallergens are yet to be defined. 9. Angiogenesis in Bronchial Asthma The formation of new blood vessels (angiogenesis) is crucial for numerous inflammatory and immune disorders including asthma (Carmeliet, 2003). Angiogenesis is a multistep process, requiring the timely expression of various growth factors and receptors on endothelial cell progenitors (Levi‐Schaffer and Pe’er, 2001). Angiogenesis is a prerequisite for airway remodeling. Several growth factors could play important roles in allergic inflammation and angiogenesis. VEGF and placental growth factor (PlGF) are among the most potent proangiogenetic factors (De Falco et al., 2002; Nagy et al., 2003). Asthmatic airways have more vessels and a greater vascular area than controls (Chetta et al., 2003; Hoshino et al., 2001; Li and Wilson, 1997; Salvato, 2001). Asthmatic subjects exhibited higher VEGF and basic fibroblast growth factor (bFGF) in the submucosa than did controls (Hoshino et al., 2001). In addition, concentrations of VEGF in sputum obtained from acute asthmatics are increased in comparison with controls (Lee and Lee, 2001). These results are compatible with the hypothesis that increased vascularity of the bronchial mucosa in asthmatics is related to the expression of angiogenic factors that might contribute to the pathogenesis of asthma. Fc
RIþ cells, which are closely associated with blood vessels and are increased at angiogenic sites, can contribute to various aspects of angiogenesis (Hiromatsu and Toda, 2003; Ribatti et al., 2002). Mast cells synthesize and release various proangiogenic factors (histamine, tryptase, TGF‐b, IL‐8, VEGF, and I‐309/CCL1) (Boesiger et al., 1998; Gilchrest et al., 2003; Gru¨ tzkau et al., 1998). Production of VEGF by mast cells is increased by PGE2 and other cAMP‐elevating agents (Abdel‐Majid and Marshall, 2004). In patients with asthma, the increase in bronchial mast‐cell density is correlated with the increase in the number of vessels, suggesting that Fc
RIþ cells could have a role in asthmatic angiogenesis (Chetta et al., 2003). Expression of VEGF and its receptors and their functional interactions in human basophils has been recently explored (Marone et al., 2005a). Basophils constitutively express several isoforms of VEGF‐A (VEGF‐A121, VEGF‐A165,

128

g i a nn i m a r o ne

ET AL.

and VEGF‐A189) and their immunologic activation induces the release of VEGF‐A. Interestingly, VEGF‐A is chemotactic for basophils presumably through interaction with VEGFR‐1/Flt‐1 and VEGFR‐2/KDR, which are phenotypically expressed on the vast majority of these cells. It is intriguing that basophils constitutively express also the soluble VEGFR‐1 (sVEGFR‐1), which has been recently found to be biologically active in blocking endogenously expressed VEGF activity (Eubank et al., 2004). Murine mast cell‐derived angiopoietin‐1 plays a critical role in promoting the growth of plasma cell tumors, acting in conjunction with VEGF‐A (Nakayama et al., 2004). Moreover, angiopoietin‐1 induces eosinophil chemotaxis through interaction with Tie‐2 receptor (Feistritzer et al., 2004). The expression and functions of angiopoietins and their receptors in human Fc
RIþ cells remain to be elucidated. The complexity of the roles of Fc
RIþ cells in asthmatic angiogenesis is illustrated by the observation that two angiostatic chemokines, IP‐10/CXCL10 and I‐TAC/CXCL11, synthesized by ASM, inhibit endothelial cell proliferation through activation of CXCR3‐B (Romagnani et al., 2004). Interestingly, these chemokines, synthesized by asthmatic ASM, are chemotactic for lung mast cells. The urokinase plasminogen activator (uPA) and its high‐affinity receptor (uPAR) are involved in tissue remodeling and vessel sprouting (Carmeliet, 2003). The uPA receptor (uPAR) is expressed by human basophils (de Paulis et al., 2004a) and by mast cells (Sillaber et al., 1997). uPA is a potent chemoattractant for basophils through exposure of a chemotactic uPAR epitope, which is a ligand for FPRL1 and FPRL2 (de Paulis et al., 2004a). These findings illustrate the complex roles played by Fc
RIþ cells in the fine regulation of the homeostatic control of angiogenesis that occurs during chronic allergic inflammation. Various aspects of asthmatic angiogenesis might be pharmacologically modulated by targeting Fc
RIþ cells. 10. Tissue Remodeling in Bronchial Asthma Asthma is a chronic inflammatory disease accompanied by histologic features of airway remodeling, that is, bronchial subepithelial fibrosis, hypertrophy/ hyperplasia of ASM, increase in mucous glands, and changes in airway blood vessels (Boulet et al., 1997; Brewster et al., 1990). Thickening of the reticular basement membrane results from the deposition, probably by activated fibroblasts, of collagens I, III, and V and fibronectin (Brewster et al., 1990; Roche et al., 1989). It is likely that cytokines and other mediators released in the asthmatic inflammatory microenvironment activate the repair process that leads to fibroblast proliferation and collagen synthesis. In fact, cellular

h u m a n m a s t ce l l s an d bas o p h i l s i n a s t h m a

129

response to cytokines are modified in asthmatic bronchial fibroblasts (Dube´ et al., 1998). A number of clinical and experimental observations have implicated mast cells and their mediators in the pathogenesis of airway fibrosis in bronchial asthma. Mast cell density is increased in different compartments of human lung in various fibrotic lung disorders (Chanez et al., 1993; Kawanami et al., 1979). Activated mast cells enhance fibroblast proliferation and collagen production (Levi‐Schaffer and Rubinchik, 1995). In addition, coculture of human lung mast cells with fibroblasts can prolong mast‐cell survival (Levi‐Schaffer et al., 1987). These pioneering studies highlighted the bidirectional interactions between mast cells and fibroblasts. More recently, it has been demonstrated that human mast cells stimulate fibroblast proliferation depending on the heterotypic cell–cell contact (Trautmann et al., 1998) or through the release of several mediators (Artuc et al., 2002; Garbuzenko et al., 2002). The mast‐cell mediators responsible for fibroblast proliferation and collagen synthesis have, at least in part, been identified. Histamine (Garbuzenko et al., 2002; Russel et al., 1977), cysteinyl leukotrienes (Baud et al., 1987), tryptase (Cairns and Walls, 1997; Garbuzenko et al., 2002; Ruoss et al., 1991), IL‐8, IL‐13 (Jinnin et al., 2004), and TGF‐b1 (Levi‐Schaffer et al., 1999) stimulate the synthesis of collagen in human fibroblasts. In particular, TGF‐b, which is released by mast cells, is a potent stimulus for fibroblast proliferation and collagen synthesis (Levi‐Schaffer et al., 1999). Interestingly, TGF‐b1 also promotes growth and differentiation of human basophils (Sillaber et al., 1992). Histamine also participates in the chronic evolution of asthma by inducing the release of lysosomal enzymes and cytokines (IL‐6 and TNF‐a) from lung macrophages through activation of the H1 receptor (Triggiani et al., 2001). IL‐13 is now thought to be especially critical in animal models of allergic asthma. In fact, blockade of IL‐13 markedly inhibits allergen‐induced AHR, mucus production, and eosinophilia (Grunig et al., 1998; Wills‐Karp et al., 1998; Zhu et al., 1999). It has recently been demonstrated that IL‐13 is a potent stimulus for the transcription of human type I collagen gene in human fibroblasts (Jinnin et al., 2004). Previous studies showed that tenascin‐C and collagen IV genes are induced by IL‐13 in human bronchial epithelial cells (Lee et al., 2001; Yuyama et al., 2002). In addition, IL‐13 induces mucus hypersecretion and goblet cell metaplasia (Grunig et al., 1998; Kuperman et al., 2002; Wills‐Karp et al., 1998; Zhu et al., 1999). These studies suggest that IL‐13, produced by human Fc
RIþ cells, is a potent fibrogenic cytokine. This set of findings emphasizes the multiple interactions between fibroblasts, macrophages, mast cells, and basophils. However, it is important to note that fibrosis is a complex dynamic effect characterized by the synthesis

130

g i a nn i m a r o ne

ET AL.

and degradation of collagen. Moreover, a single cell alone could not be responsible for such a complex biological event. Therefore, it is conceivable that mast cells, basophils, and their mediators contribute to airway fibrosis in bronchial asthma together with many other inflammatory and mesenchymal cells. 11. Pharmacologic Modulation of Human Mast Cells and Basophils in the Treatment of Bronchial Asthma Mast cells, basophils, and their mediators play a pivotal role in most of the manifestations of asthma in humans. This finding has prompted a race within the pharmaceutical industry to achieve optimal therapeutic targeting of Fc
RIþ cells in asthma. The difficulty of this goal was underestimated in the 1970s because the complexity of the human Fc
RIþ‐IgE system was unknown. It is now evident that this ambitious therapeutic target is more difficult than originally thought. First, it has been demonstrated that mast cells differ immunologically, biochemically, and pharmacologically from human basophils. Second, mast cells and basophils have different strategic microlocalizations in the human lung compartments and synthesize different sets of proinflammatory mediators, cytokines, and chemokines. Moreover, different subpopulations of mast cells or mast cells at different stages of maturation might have different or even, in some cases, protective roles in the appearance and disappearance of the asthmatic phenotype. In addition, Fc
RIþ cells, under some circumstances, play important homeostatic roles. Finally, human Fc
RIþ cells can be activated by a variety of immunologic and nonimmunologic stimuli, in addition to IgE cross‐linking. 11.1. Blockade of Activating Receptors There are four main areas of therapeutic targeting of Fc
RIþ cells in asthma. The first is by interfering with activating receptors (Fc
RI, C3a, C5a, cytokine and chemokine receptors, etc.) on these cells. This strategy using a monoclonal antibody anti‐IgE has been partially successful in certain forms of allergic asthma (Milgrom et al., 1999). The partial efficacy of the latter approach could be because other receptors, in addition to Fc
RI, trigger the release of mediators from human Fc
RIþ cells. Surface receptors (e.g., chemokine and angiogenic receptors) on Fc
RIþ cells also can be blocked by small‐molecular‐ weight compounds (Lukacs, 2001; Wood et al., 2000) or peptide antagonists (Holash et al., 2002; Lukacs, 2001). Chemokine receptor antagonists represent another area of great pharmacologic potential in the prevention/treatment of allergic disorders. Several

h u m a n m a s t ce l l s an d bas o p h i l s i n a s t h m a

131

chemokine receptors are selectively displayed on human basophils and mast cells (Dahinden, 2000; Romagnani et al., 2000). Their antagonism with high‐ affinity small‐molecular‐weight compounds can interfere with chemotaxis and/ or secretion of Fc
RIþ cells (Lukacs, 2001). The redundancy of chemokines and their receptors is a relevant problem in achieving specific inhibitory effects. VEGF and its receptors are expressed in human Fc
RIþ cells (Marone et al., 2005a). Increased bronchial vessels in asthmatics (Chetta et al., 2003; Hoshino et al., 2001; Li and Wilson, 1997; Salvato, 2001) might be related to the expression of angiogenic factors and their receptors in Fc
RIþ cells. Small‐ molecular‐weight antagonists of VEGF receptors are candidates for the prevention/treatment of certain aspects of tissue remodeling in asthma (Wood et al., 2000). Alternative modulation of VEGF‐induced angiogenesis include the use of humanized monoclonal antibodies against VEGF (Presta et al., 1997) or its receptor Flk‐1/KDR (Prewett et al., 1999), or a decoy form of this receptor (Wood et al., 2000). 11.2. Inhibition of Signal Transduction A second therapeutical strategy for asthma is to inhibit Fc
RIþ cell activation by interfering with one or more biochemical events essential for signal transduction. Studies of mice with targeted gene mutations have demonstrated that mast‐cell degranulation and/or cytokine production are deficient in Syk‐/‐, Btk‐/‐, SLP‐76‐/‐, LAT‐/‐, and Vav‐/‐ mice (Siraganian, 2003). Several excellent reviews have focused on the molecular consequences of mast cell (Bastan et al., 2001; Blank and Rivera, 2004; MacGlashan, Jr., et al., 2000a; Siraganian, 2003; Wymann et al., 2003) and basophil (Luskova´ and Dra´ ber, 2004; MacGlashan, Jr., et al., 2000b) activation and their pharmacological modulation. Diverse steps in the signal transduction process appear promising targets for the modulation of the release of mediators, cell growth, proliferation, and survival of Fc
RIþ cells. In particular, tyrosine kinases (Syk, Btk, Lyn, Fyn, etc.), protein kinase C, phosphoinositide‐3‐kinase (PI3‐K), and adaptor molecules (Gab1, Gab2, Cbl, LAT, Grb2, Vav, etc.) might qualify as drug targets for the treatment of allergic disorders. The pharmacologic effects of most of these compounds have been so far evaluated in in vitro models using rodent and/or mast‐cell lines. Their pharmacologic properties must be confirmed using human mast cells and basophils before they can be considered for clinical trials. 11.2.1. Adenylate Cyclase Activators and Phosphodiesterase Inhibitors Catecholamines inhibit IgE‐mediated histamine release from basophils by binding to b2‐adrenergic receptors (Lichtenstein and Margolis, 1968). Fenoterol rapidly inhibits antigen‐induced histamine secretion from human lung

132

g i a nn i m a r o ne

ET AL.

mast cells and basophils (Marone et al., 1984) and the dose‐response inhibition curve is paralleled by a fenoterol‐induced increase in cyclic AMP (cAMP) levels. Salmeterol and formoterol, two long‐acting b2‐adrenergic agonists also modulate the release of mediators from lung mast cells (Nials et al., 1994). Phosphodiesterases (PDEs) are responsible for cAMP hydrolysis. At least eleven different classes of cAMP PDE isoenzymes have been identified. Both cAMP PDE3 and PDE4 have been found in basophils and mast cells (Essayan, 2001). PDE4 has attracted particular attention and four variants have been identified (PDE4A, PDE4B, PDE4C, and PDE4D) (Giembycz, 2000). The four gene products of PDE4 are characterized by selective, high‐affinity hydrolysis of cAMP and sensitivity to inhibition by rolipram. Rolipram inhibits PAF‐ and anti‐IgE‐induced mediator release and increases cAMP levels in basophils (Columbo et al., 1993a; Peachell et al., 1992). Inhibitors of PDE4 also regulate mast cell functions (Torphy et al., 1992). In contrast, mediator release is not inhibited by PDE3 and PDE5 inhibitors. It thus appears that PDE4 is the isoenzyme mainly responsible for modulating Fc
RIþ cells. 11.2.2. Glucocorticoids Glucocorticoids are one of the therapeutic mainstays in bronchial asthma. These drugs exert multiple biological and pharmacological effects (Leung and Bloom, 2003). Intravenously injected glucocorticoids in humans cause rapid basopenia, whereas skin tissue histamine remains unchanged (Dunsky et al., 1979). In contrast, prolonged treatment with topical glucocorticoids reduces skin mast‐cell density and inhibits the allergen‐induced wheal‐and‐flare response (Lavker and Schechter, 1985). High doses of inhaled glucocorticoids in mild‐to‐moderate asthmatics reduce mast‐cell density and the number of bronchial vessels (Chetta et al., 2003). Prolonged (12–24 hr) in vitro incubation with glucocorticoids inhibits IgE‐ mediated histamine release from basophils (Schleimer et al., 1981). In contrast, incubation for up to 24 hr of mast cells isolated from lung parenchyma with glucocorticoids does not alter their immunologic release of histamine, PGD2, or LTC4 (Schleimer et al., 1983). By contrast, dexamethasone inhibits Fc
RI‐mediated activation of PI3‐K and downstream responses in RBL‐2H3 (Andrade et al., 2004). The latter examples emphasize the pharmacological heterogeneity of human Fc
RIþ cells and the differences between human and rodent mast cells. Short preincubation (1 hr) of human basophils with glucocorticoids inhibits the release of IL‐4 (Schroeder et al., 1997), without exerting any inhibitory effect on the release of histamine. These results suggest that the mechanisms controlling the release of cytokines differ from those controlling

h u m a n m a s t ce l l s an d bas o p h i l s i n a s t h m a

133

the release of histamine from basophils. The inhibitory effect of glucocorticoids on cytokine production is probably mediated by blockade of NF‐kB, a family of DNA‐binding proteins that play a critical role in experimental asthma (Desmet et al., 2004). Evidence exists that glucocorticoids can also modulate SCF‐induced migration of mast cells (Jeong et al., 2003) and maturation, and Fc
RI expression of human mast cells (Smith et al., 2002). 11.2.3. Immunophilin Ligands Researchers identified cyclophilin (CyP), a protein with high affinity for cyclosporin A (CsA) (Hait et al., 1986). CyP belongs to a family of intracellular proteins, the immunophilins, which include the FK‐binding proteins (FKBPs) (Standaert et al., 1990). Two natural macrolides, FK‐506 (tacrolimus) and rapamycin (RAP) bind with high affinity to FKBPs (Standaert et al., 1990). CsA‐CyP complexes, as well as FK‐506‐FKBP complexes, bind to a protein identified as calcineurin (Cn) (Stellato et al., 1992). Calcineurin has a catalytic A subunit (CnA) and a regulatory B subunit (CnB). The CnA subunit has a binding site for calmodulin (CaM) and for the CnB subunit. Complexes of CsA or tacrolimus and their respective intracellular binding proteins inhibit the CaM‐dependent protein phosphatase 2B, which is essential in the signal transduction pathway for basophils (Cirillo et al., 1990; de Paulis et al., 1991) and mast cells (Cirillo et al., 1990; de Paulis et al., 1992; Stellato et al., 1992). RAP binds to FKBP, but the molecular mechanism of its immunosuppressive action is different from that of tacrolimus. Low concentrations of CsA inhibit histamine and LTC4 release from basophils challenged with IgE‐mediated stimuli (Cirillo et al., 1990; de Paulis et al., 1992). CsH, which has an extremely low affinity for CyP, does not affect the IgE‐mediated activation of basophils, but is a competitive antagonist of FPR on these cells (de Paulis et al., 1996, 2004a,b). CsA also inhibits the release of preformed and de novo synthesized mediators from human lung and skin mast cells, by interacting with CyP (Stellato et al., 1992). A single oral dose of CsA in normal volunteers caused rapid inhibition of histamine release from basophils obtained ex vivo, which provides a rare example of how an in‐vivo‐administered drug can modulate basophil releasability ex vivo (Casolaro et al., 1993). Tacrolimus also inhibits histamine and LTC4 release from mast cells and basophils challenged with antigen and anti‐IgE, whereas RAP has little or no effect. RAP acts as a competitive antagonist of tacrolimus, presumably at the level of FKBP (de Paulis et al., 1991, 1992). Thus, binding to FKBP is necessary, but not sufficient in itself to deliver the inhibitory signal for the release of mediators from basophils and mast cells.

134

g i a nn i m a r o ne

ET AL.

CsA and tacrolimus are also potent inhibitors of the de novo synthesis of IL‐4 and IL‐13 from immunologically activated basophils (Florio et al., 2000; Genovese et al., 2003; Patella et al., 1998a). Tacrolimus ointment is rapidly effective in the treatment of patients with atopic dermatitis (Ruzicka et al., 1997). Skin mast cells and infiltrating basophils play a prominent role in atopic dermatitis (Mitchell et al., 1982). Tacrolimus exerts potent antiinflammatory effects by inhibiting the release of histamine and eicosanoids from skin mast cells (de Paulis et al., 1991, 1992). These findings, together with the rapid clinical efficacy of tacrolimus in atopic dermatitis, suggest that the antiinflammatory properties of this compound are largely responsible for its beneficial effects in vivo (Marone, 1998b). Pimecrolimus, another immunophilin ligand, has also been shown to prevent the release of mediators from mast cells (Gupta and Chow, 2003). It is likely that the antiinflammatory properties of immunophilin‐binding drugs could be beneficial in the treatment of allergic disorders of the respiratory tract. 11.3. Receptor Antagonists of Fc
RIþ Cell‐Derived Mediators A third strategic approach to asthma treatment is to antagonize receptors activated by mediators synthesized by human Fc
RIþ cells. These classes of compounds are widely used in the treatment of allergic disorders. The main property of H1‐antihistamines is to antagonize the effects of this mediator at the H1 receptor level in different organs (Marone, 1997). In addition, high concentrations of first‐generation antihistamines inhibit in vitro antigen‐induced histamine release from human basophils (Lichtenstein and Gillespie, 1975). Certain antihistamines prevent the release not only of histamine, but also of other proinflammatory mediators such as LTC4, PAF, and PGD2 (Church and Gradidge, 1980; Genovese et al., 1997; Marone et al., 1999; Patella et al., 1996). It is important to emphasize that not all H1 antagonists exert antiinflammatory activity in vitro. For instance, mizolastine inhibits the de novo synthesis of LTC4 from basophils, but it potentiates the secretion of histamine (Triggiani et al., 2004a). Loratadine and desloratadine inhibit histamine release from human basophils induced by both IgE‐dependent and IgE‐independent mechanisms (Genovese et al., 1997). These results were extended to human lung mast cells in which desloratadine inhibits preformed (histamine and tryptase) and de novo synthesized mediators (LTC4 and PGD2) (Genovese et al., 1997). Desloratadine also inhibits IL‐4 and IL‐13 synthesis induced by anti‐IgE in human basophils (Schroeder et al., 2001). These results indicate that the inhibitory effect of certain H1 receptor antagonists on mediator release from human Fc
RIþ cells is not a class‐specific effect and it is not a general property of all antihistamines.

h u m a n m a s t ce l l s an d bas o p h i l s i n a s t h m a

135

The identification of histamine H3 and H4 receptors opens the possibility that specific inhibitors of these receptors might modulate certain functions of effector cells of allergic inflammation. Activation of H4 receptors mediates mast cell chemotaxis (Hofstra et al., 2003) and their antagonism might be beneficial in certain clinical conditions. Selective CysLT1 receptor antagonists, such as montelukast, have been successfully employed in the treatment of asthma (Barnes et al., 2005). CysLT1 and CysLT2 receptors are also expressed on lung macrophages (Heise et al., 2000; Lynch et al., 1999), mast cells (Mellor et al., 2001), and leukocytes (Figueroa et al., 2001). It is possible that CysLT antagonists modulate certain aspects of the activation of these cells. For instance, high concentrations of montelukast directly inhibit 5‐LO activity in human mast cells (Ramires et al., 2004). PGD2 acts through the activation of two important receptors: the DP receptor, whose activation elicits bronchoconstriction in asthmatics (Hardy et al., 1984) and the CRTH2 receptor present on human basophils and mast cells (Hirai et al., 2001). Specific antagonists of these receptors are being evaluated for the treatment of allergic disorders. b tryptase and chymase, stored and released by human mast cells, exert several biochemical and proinflammatory effects (Caughey, 2004; Stevens et al., 2004). Specific small‐molecular‐weight inhibitors of b tryptase and chymase are undergoing investigation for the treatment of allergic disorders. Several VEGF‐A isoforms have been identified in human mast cells (Boesiger et al., 1998; Gru¨ tzkau et al., 1998) and basophils (Marone et al., 2005a). In addition, immunologic activation of human Fc
RIþ cells induces the release of VEGF‐A (Boesiger et al., 1998; Gru¨ tzkau et al., 1998), which is chemotactic for human mast cells and basophils through the activation of Flt‐1/KDR (Gru¨ tzkau et al., 1998). Therefore, the antagonism of VEGFs and their receptors by different strategic approaches may be effective in interrupting an autocrine loop relevant in certain aspects of tissue remodeling and angiogenesis in asthma. 11.4. Inhibitors of Proliferation and Inducers of Apoptosis A fourth therapeutic strategy for asthma is inhibition of the in vivo proliferation and/or induction of apoptosis of basophils and mast cells. Some evidence indicates that glucocorticoids might interfere with mast‐cell survival. It has been shown that dexamethasone inhibits development of human cord blood‐ and fetal liver‐derived mast cells (Irani et al., 1995; Smith et al., 2002). It is likely that glucocorticoids induce these effects through the expression of either antiapoptotic or proapoptotic molecules in mast‐cell progenitors (Smith et al., 2002). Tamoxifen inhibits human lung mast‐cell proliferation, possibly through

136

g i a nn i m a r o ne

ET AL.

ion channel modulation (Duffy et al., 2003). Retinoic acid inhibits the in vitro development of human mast cells (Hjertson et al., 2003). Another approach is to block the specific tyrosine kinases activated by the engagement of c‐kit by SCF. STI571 (Imatinib) inhibits SCF‐dependent mast cell growth from cord blood‐derived mast cell progenitors through the inhibition of tyrosine phosphorylation of c‐kit (Akin et al., 2003; Takeuchi et al., 2003). 17‐allylamino‐17‐demethoxygeldanamycin (17‐AAG), a derivative of the antibiotic geldanamycin, is effective in downregulating mutated, constitutively activated c‐kit in human mast cells (Fumo et al., 2004). The development of safe compounds that modulate the in vivo proliferation and/or survival of human mast cells and basophils is a promising avenue for the treatment and prevention of allergic diseases. 12. Conclusions and Implications The unique microlocalization of mast cells in specific lung tissue compartments, the ability of basophils to migrate to sites of allergic inflammation, the powerful effector repertoire of both cell types, the recognition of their different microbial‐related activating ligands, and their plasticity in response to various signals suggest that Fc
RIþ cells have a central role in most of the variants of bronchial asthma (Fig. 9). It is now evident that mast cells differ from basophils immunologically, biochemically, and pharmacologically and, therefore, these cells have distinct roles in the orchestration of inflammation in the airways. Moreover, it is likely that different subpopulations of mast cells or mast cells at different stages of maturation might have different or even, in some cases, protective roles in the appearance of the asthmatic phenotype. The extent to which each effector cell contributes toward each phase of allergic inflammation and tissue repair remains to be fully elucidated. Optimal therapeutic targeting of mast cells and/or basophils in asthma must therefore take into account that the pharmacological modulation of these cells could inhibit or even exacerbate certain aspects of the inflammatory response. Where should researchers go now? First, defining the cytokine and chemokine and their counterpart receptors acting on or released by human basophils and mast cells from different lung compartments should continue. Second, elucidating the complex biochemical steps induced in human mast cells and basophils activated by different immunological and nonimmunological stimuli must occur. Regarding the genetics of asthma, it remains to be determined whether susceptibility genes might influence Fc
RIþ cell releasability (e.g., aspirin‐induced asthma) and/or their localization in different lung

h u m a n m a s t ce l l s an d bas o p h i l s i n a s t h m a

137

Figure 9 Hypothetical scheme of the central role of lung mast cells and basophils in bronchial asthma. Resident mast cells and infiltrating basophils, attracted by CC (RANTES, eotaxin) and CXC chemokines [IP‐10/CXCL10, Mig/CXCL9, and I‐TAC/CXCL11], stem cell factor (SCF), prostaglandin D2 (PGD2), and histamine are a major source of cytokines that polarize T cells to preferentially differentiate into the Th2 subset. Activation of mast cells induces the synthesis of IL‐16, which is a chemoattractant for CD4þ T cells. IL‐4 and IL‐13 and cognate interaction between CD40L and CD40 favor IgE synthesis by B cells. Certain T cells also produce IL‐3 and transforming growth factor‐b (TGF‐b), which augment and inhibit mast‐cell proliferation, respectively. CysLTs produced by immunologically activated mast cells and basophils exert a variety of responses by activating the CysLTR1 receptor expressed on lung macrophages, mast cells, and basophils. CysLTs potentiate exocytosis and cytokine production in histamine‐activated lung macrophages. Histamine also participates in the chronic evolution of asthma by inducing the release of lysosomal enzymes and cytokines (IL‐6 and TNF‐a) from lung macrophages through activation of the H1 receptor. Interestingly, histamine induces chemotaxis of mast cells by interacting with H4 receptor and inhibits the release of mediators from human basophils through the engagement of the H2 receptor. Modified with permission, from Marone et al., 2005b.

compartments. The fourth area to explore is paracrine and autocrine interactions of Fc
RIþ cells on other immune cells (e.g., macrophages, Th2 cells, eosinophils, B cells) that play a critical role in the pathogenesis of asthma. Finally, it remains to be elucidated whether mast cells and basophils might have a role(s), even in some cases protective, in angiogenesis and tissue remodeling. In summary, better knowledge of human Fc
RIþ cell biology is necessary to be able to modify the ‘‘bad’’ behavior of these cells without compromising their

138

g i a nn i m a r o ne

ET AL.

homeostatic and protective roles in innate and acquired immunity. Studies are also warranted to unravel the basophil–mast cell dilemma: are mast cells and basophils friends as well as foes in asthma? Perhaps lessons learned from studies of Fc
RIþ cells in asthma will also pay dividends for patients with other chronic inflammatory diseases (including rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, etc.) in which these cells play a pathogenetic role. Acknowledgments This work was supported by grants from the Ministero dell’Istruzione, Universita` e Ricerca, the Istituto Superiore di Sanita` (AIDS Project 40D.57), and Ministero della Salute ‘‘Alzheimer Project’’ (Rome, Italy). We thank Jean Gilder and Giorgio Giannattasio for critical reading of the manuscript and Francesco Granata for the artwork. We also wish to acknowledge the important contribution of colleagues whose work has not been included due to space constraints. Gianni Marone is the recipient of the Esculapio Award 2003 (Accademia Tiberina, Rome, Italy).

References Abdel‐Majid, R. M., and Marshall, J. S. (2004). Prostaglandin E2 induces degranulation‐independent production of vascular endothelial growth factor by human mast cells. J. Immunol. 172, 1227–1236. Abraham, S. N., and Malaviya, R. (2000). Mast cell‐enterobacteria interactions during infection. In ‘‘Mast Cells and Basophils’’ (G. Marone, L. M. Lichtenstein, and S. J. Galli, Eds.), pp. 381–396. Academic Press, San Diego. Akin, C., Brockow, K., D’ Ambrosio, C., Kirshenbaum, A. S., Ma, Y., Longley, B. J., and Metcalfe, D. D. (2003). Effects of tyrosine kinase inhibitor STI571 on human mast cells bearing wild‐type or mutated c‐kit. Exp. Hematol. 31, 686–692. Altman, L. C., Munk, Z., Seltzer, J., Noonan, N., Shingo, S., Zhang, J., and Reiss, T. F. (1998). A placebo‐controlled, dose‐ranging study of montelukast, a cysteinyl leukotriene‐receptor antagonist. Montelukast Asthma Study Group. J. Allergy Clin. Immunol. 102, 50–56. Amin, K., Ludviksdottir, D., Janson, C., Nettelbladt, O., Bjornsson, E., Roomans, G. M., Boman, G., Seveus, L., and Venge, P. (2000). Inflammation and structural changes in the airways of patients with atopic and nonatopic asthma. BHR Group. Am. J. Respir. Crit. Care Med. 162, 2295–2301. Andrade, M. V., Hiragun, T., and Beaven, M. A. (2004). Dexamethasone suppresses antigen‐ induced activation of phosphatidylinositol 3‐kinase and downstream responses in mast cells. J. Immunol. 172, 7254–7262. Arm, J. P. (2004). Generation of cytokines through the leukocyte immunoglobulin‐like receptors. In ‘‘Asthma and Allergic Diseases. Physiology, Immunopharmacology and Treatment’’ (G. Marone, K. F. Austen, S. T. Holgate, A. B. Kay, and L. M. Lichtenstein, Eds.), pp. 162–166. Blackwell, London. Artuc, M., Steckelings, U. M., and Henz, B. M. (2002). Mast cell‐fibroblast interactions: human mast cells as source and inducers of fibroblast and epithelial growth factors. J. Invest. Dermatol. 118, 391–395.

h u m a n m a s t ce l l s an d bas o p h i l s i n a s t h m a

139

Asai, K., Kitaura, J., Kawakami, Y., Yamagata, N., Tsai, M., Carbone, D. P., Liu, F. T., Galli, S. J., and Kawakami, T. (2001). Regulation of mast cell survival by IgE. Immunity 14, 791–800. Atasoy, U., Curry, S. L., Lopez de Silanes, I., Shyu, A. B., Casolaro, V., Gorospe, M., and Stellato, C. (2003). Regulation of eotaxin gene expression by TNF‐a and IL‐4 through mRNA stabilization: involvement of the RNA‐binding protein HuR. J. Immunol. 171, 4369–4378. Axcrona, K., Bjorck, L., and Leanderson, T. (1995). Multiple ligand interactions for bacterial immunoglobulin‐binding proteins on human and murine cells of the hematopoetic lineage. Scand. J. Immunol. 42, 359–367. Bach, J. F. (2002). The effect of infections on susceptibility to autoimmune and allergic diseases. N. Engl. J. Med. 347, 911–920. Baghestanian, M., Hofbauer, R., Kiener, H. P., Bankl, H. C., Wimazal, F., Willheim, M., Scheiner, O., Fureder, W., Muller, M. R., Bevec, D., Lechner, K., and Valent, P. (1997). The c‐kit ligand stem cell factor and anti‐IgE promote expression of monocyte chemoattractant protein‐1 in human lung mast cells. Blood 90, 4438–4449. Barnes, N., Thomas, M., Price, D., and Tate, H. (2005). The national montelukast survey. J. Allergy Clin Immunol 115, 47–54. Bastan, R., Peirce, M. J., and Peachell, P. T. (2001). Regulation of immunoglobulin E‐mediated secretion by protein phosphatases in human basophils and mast cells of skin and lung. Eur. J. Pharmacol. 430, 135–141. Baud, L., Perez, J., Denis, M., and Ardaillou, R. (1987). Modulation of fibroblast proliferation by sulfidopeptide leukotrienes: effect of indomethacin. J. Immunol. 138, 1190–1195. Beaulieu, S., Robbiani, D. F., Du, X., Rodrigues, E., Ignatius, R., Wei, Y., Ponath, P., Young, J. W., Pope, M., Steinman, R. M., and Mojsov, S. (2002). Expression of a functional eotaxin (CC chemokine ligand 11) receptor CCR3 by human dendritic cells. J. Immunol. 169, 2925–2936. Berberian, L., Goodglick, L., Kipps, T. J., and Braun, J. (1993). Immunoglobulin VH3 gene products: natural ligands for HIV gp120. Science 261, 1588–1591. Bischoff, S. C., and Dahinden, C. A. (1992). Effect of nerve growth factor on the release of inflammatory mediators by mature human basophils. Blood 79, 2662–2669. Bischoff, S. C., de Weck, A. L., and Dahinden, C. A. (1990). Interleukin 3 and granulocyte/ macrophage‐colony‐stimulating factor render human basophils responsive to low concentrations of complement component C3a. Proc. Natl. Acad. Sci. USA 87, 6813–6817. Bischoff, S. C., Krieger, M., Brunner, T., and Dahinden, C. A. (1992). Monocyte chemotactic protein 1 is a potent activator of human basophils. J. Exp. Med. 175, 1271–1275. Bjo¨ rck, L. (1988). Protein L. A novel bacterial cell wall protein with affinity for Ig L chains. J. Immunol. 140, 1194–1197. Blank, U., and Rivera, J. (2004). The ins and outs of IgE‐dependent mast‐cell exocytosis. Trends Immunol. 25, 266–273. Boesiger, J., Tsai, M., Maurer, M., Yamaguchi, M., Brown, L. F., Claffey, K. P., Dvorak, H. F., and Galli, S. J. (1998). Mast cells can secrete vascular permeability factor/vascular endothelial cell growth factor and exhibit enhanced release after immunoglobulin E‐dependent upregulation of Fc
receptor I expression. J. Exp. Med. 188, 1135–1145. Boulet, L. P., Laviolette, M., Turcotte, H., Cartier, A., Dugas, M., Malo, J. L., and Boutet, M. (1997). Bronchial subepithelial fibrosis correlates with airway responsiveness to methacholine. Chest 112, 45–52. Boumiza, R., Monneret, G., Forissier, M. F., Savoye, J., Gutowski, M. C., Powell, W. S., and Bienvenu, J. (2003). Marked improvement of the basophil activation test by detecting CD203c instead of CD63. Clin. Exp. Allergy 33, 259–265.

140

g i a nn i m a r o ne

ET AL.

Bouvet, J. P., Pires, R., Lunel‐Fabiani, F., Crescenzo‐Chaigne, B., Maillard, P., Valla, D., Opolon, P., and Pillot, J. (1990). Protein F. A novel F(ab)‐binding factor, present in normal liver, and largely released in the digestive tract during hepatitis. J. Immunol. 145, 1176–1180. Bouvet, J. P., Pires, R., Charlemagne, J., Pillot, J., and Iscaki, S. (1991a). Non‐immune binding of human protein Fv to immunoglobulins from various mammalian and non‐mammalian species. Scand. J. Immunol. 34, 491–496. Bouvet, J. P., Pires, R., Quan, C., Iscaki, S., and Pillot, J. (1991b). Non‐immune VH‐binding specificity of human protein Fv. Scand. J. Immunol. 33, 381–386. Boyce, J. A., Mellor, E. A., Perkins, B., Lim, Y. C., and Luscinskas, F. W. (2002). Human mast cell progenitors use a4‐integrin, VCAM‐1, and PSGL‐1 E‐selectin for adhesive interactions with human vascular endothelium under flow conditions. Blood 99, 2890–2896. Bressler, R. B., Lesko, J., Jones, M. L., Wasserman, M., Dickason, R. R., Huston, M. M., Cook, S. W., and Huston, D. P. (1997). Production of IL‐5 and granulocyte‐macrophage colony‐ stimulating factor by naive human mast cells activated by high‐affinity IgE receptor ligation. J. Allergy Clin. Immunol. 99, 508–514. Brewster, C. E., Howarth, P. H., Djukanovic, R., Wilson, J., Holgate, S. T., and Roche, W. R. (1990). Myofibroblasts and subepithelial fibrosis in bronchial asthma. Am. J. Respir. Cell Mol. Biol. 3, 507–511. Brightling, C. E., Bradding, P., Symon, F. A., Holgate, S. T., Wardlaw, A. J., and Pavord, I. D. (2002). Mast‐cell infiltration of airway smooth muscle in asthma. N. Engl. J. Med. 346, 1699–1705. Brightling, C. E., Symon, F. A., Holgate, S. T., Wardlaw, A. J., Pavord, I. D., and Bradding, P. (2003). Interleukin‐4 and ‐13 expression is co‐localized to mast cells within the airway smooth muscle in asthma. Clin. Exp. Allergy 33, 1711–1716. Brown, J. K., Jones, C. A., Rooney, L. A., Caughey, G. H., and Hall, I. P. (2002). Tryptase’s potent mitogenic effects in human airway smooth muscle cells are via nonproteolytic actions. Am. J. Physiol. Lung Cell. Mol. Physiol. 282, L197–206. Brunner, T., de Weck, A. L., and Dahinden, C. A. (1991). Platelet‐activating factor induces mediator release by human basophils primed with IL‐3, granulocyte‐macrophage colony‐stimulating factor, or IL‐5. J. Immunol. 147, 237–242. Brunner, T., Heusser, C. H., and Dahinden, C. A. (1993). Human peripheral blood basophils primed by interleukin 3 (IL‐3) produce IL‐4 in response to immunoglobulin E receptor stimulation. J. Exp. Med. 177, 605–611. Burd, P. R., Thompson, W. C., Max, E. E., and Mills, F. C. (1995). Activated mast cells produce interleukin 13. J. Exp. Med. 181, 1373–1380. Burgi, B., Otten, U. H., Ochensberger, B., Rihs, S., Heese, K., Ehrhard, P. B., Ibanez, C. F., and Dahinden, C. A. (1996). Basophil priming by neurotrophic factors. Activation through the trk receptor. J. Immunol. 157, 5582–5588. Cairns, J. A., and Walls, A. F. (1997). Mast cell tryptase stimulates the synthesis of type I collagen in human lung fibroblasts. J. Clin. Invest. 99, 1313–1321. Calhoun, W. J., Swenson, C. A., Dick, E. C., Schwartz, L. B., Lemanske, R. F., Jr., and Busse, W. W. (1991). Experimental rhinovirus 16 infection potentiates histamine release after antigen bronchoprovocation in allergic subjects. Am. Rev. Respir. Dis. 144, 1267–1273. Carmeliet, P. (2003). Angiogenesis in health and disease. Nat. Med. 9, 653–660. Carroll, N. G., Mutavdzic, S., and James, A. L. (2002). Distribution and degranulation of airway mast cells in normal and asthmatic subjects. Eur. Respir. J. 19, 879–885. Casale, T. B., Wood, D., Richerson, H. B., Zehr, B., Zavala, D., and Hunninghake, G. W. (1987). Direct evidence of a role for mast cells in the pathogenesis of antigen‐induced bronchoconstriction. J. Clin. Invest. 80, 1507–1511.

h u m a n m a s t ce l l s an d bas o p h i l s i n a s t h m a

141

Casolaro, V., Galeone, D., Giacummo, A., Sanduzzi, A., Melillo, G., and Marone, G. (1989). Human basophil/mast cell releasability. V. Functional comparisons of cells obtained from peripheral blood, lung parenchyma, and bronchoalveolar lavage in asthmatics. Am. Rev. Respir. Dis. 139, 1375–1382. Casolaro, V., Spadaro, G., and Marone, G. (1990). Human basophil releasability. VI. Changes in basophil releasability in patients with allergic rhinitis or bronchial asthma. Am. Rev. Respir. Dis. 142, 1108–1111. Casolaro, V., Spadaro, G., Patella, V., and Marone, G. (1993). In vivo characterization of the anti‐ inflammatory effect of cyclosporin A on human basophils. J. Immunol. 151, 5563–5573. Caughey, G. H. (2004). Genetic insights into mast cell chymase and tryptase function. In ‘‘Asthma and Allergic Diseases. Physiology, Immunopharmacology and Treatment’’ (G. Marone, K. F. Austen, S. T. Holgate, A. B. Kay, and L. M. Lichtenstein, Eds.), pp. 96–101. Blackwell, London. Chanez, P., Lacoste, J. Y., Guillot, B., Giron, J., Barne´ on, G., Enander, I., Godard, P., Michel, F. B., and Bousquet, J. (1993). Mast cells’ contribution to the fibrosing alveolitis of the scleroderma lung. Am. Rev. Respir. Dis. 147, 1497–1502. Chetta, A., Zanini, A., Foresi, A., Del Donno, M., Castagnaro, A., D’ Ippolito, R., Baraldo, S., Testi, R., Saetta, M., and Olivieri, D. (2003). Vascular component of airway remodeling in asthma is reduced by high dose of fluticasone. Am. J. Respir. Crit. Care Med. 167, 751–757. Church, M. K., and Gradidge, C. F. (1980). Inhibition of histamine release from human lung in vitro by antihistamines and related drugs. Br. J. Pharmacol. 69, 663–667. Cirillo, R., Triggiani, M., Siri, L., Ciccarelli, A., Pettit, G. R., Condorelli, M., and Marone, G. (1990). Cyclosporin A rapidly inhibits mediator release from human basophils presumably by interacting with cyclophilin. J. Immunol. 144, 3891–3897. Coca, A. F., and Cooke, R. A. (1923). On the classification of the phenomena of hypersensitization. J. Immunol. 8, 163–182. Cohen, S. G., and Samter, M. (1992). ‘‘Classics in allergy.’’ NIH Symposia Foundation, Carlsbad (CA). Columbo, M., Horowitz, E. M., McKenzie‐White, J., Kagey‐Sobotka, A., and Lichtenstein, L. M. (1993a). Pharmacologic control of histamine release from human basophils induced by platelet‐ activating factor. Int. Arch. Allergy Immunol. 102, 383–390. Columbo, M., Horowitz, E. M., Patella, V., Kagey‐Sobotka, A., Chilton, F. H., and Lichtenstein, L. M. (1993b). A comparative study of the effects of 1‐acyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine and platelet activating factor on histamine and leukotriene C4 release from human leukocytes. J. Allergy Clin. Immunol. 92, 325–333. Cookson, W. (1998). Genetics and asthma. In ‘‘Asthma and Allergic Diseases. Physiology, Immunopharmacology and Treatment’’ (G. Marone, K. F. Austen, S. T. Holgate, A. B. Kay, and L. M. Lichtenstein, Eds.), pp. 13–23. Academic Press, San Diego. Coopman, S. A., Johnson, R. A., Platt, R., and Stern, R. S. (1993). Cutaneous disease and drug reactions in HIV infection. N. Engl. J. Med. 328, 1670–1674. Corry, D. B., Folkesson, H. G., Warnock, M. L., Erle, D. J., Matthay, M. A., Wiener‐Kronish, J. P., and Locksley, R. M. (1996). Interleukin 4, but not interleukin 5 or eosinophils, is required in a murine model of acute airway hyperreactivity. J. Exp. Med. 183, 109–117. Dahinden, C. A. (2000). Differential regulation of basophils functions by chemokines. In ‘‘Mast Cells and Basophils’’ (G. Marone, L. M. Lichtenstein, and S. J. Galli, Eds.), pp. 567–577. Academic Press, San Diego. Davidson, A. B., Lee, T. H., Scanlon, P. D., Solway, J., McFadden, E. R., Jr., Ingram, R. H., Jr., Corey, E. J., Austen, K. F., and Drazen, J. M. (1987). Bronchoconstrictor effects of leukotriene E4 in normal and asthmatic subjects. Am. Rev. Respir. Dis. 135, 333–337.

142

g i a nn i m a r o ne

ET AL.

De Falco, S., Gigante, B., and Persico, M. G. (2002). Structure and function of placental growth factor. Trends Cardiovasc. Med. 12, 241–246. de Paulis, A., Cirillo, R., Ciccarelli, A., Condorelli, M., and Marone, G. (1991). FK‐506, a potent novel inhibitor of the release of proinflammatory mediators from human Fc
RIþ cells. J. Immunol. 146, 2374–2381. de Paulis, A., Stellato, C., Cirillo, R., Ciccarelli, A., Oriente, A., and Marone, G. (1992). Anti‐ inflammatory effect of FK‐506 on human skin mast cells. J. Invest. Dermatol. 99, 723–728. de Paulis, A., Ciccarelli, A., de Crescenzo, G., Cirillo, R., Patella, V., and Marone, G. (1996). Cyclosporin H is a potent and selective competitive antagonist of human basophil activation by N‐formyl‐methionyl‐leucyl‐phenylalanine. J. Allergy Clin. Immunol. 98, 152–164. de Paulis, A., Minopoli, G., Arbustini, E., de Crescenzo, G., Dal Piaz, F., Pucci, P., Russo, T., and Marone, G. (1999). Stem cell factor is localized in, released from, and cleaved by human mast cells. J. Immunol. 163, 2799–2808. de Paulis, A., De Palma, R., Di Gioia, L., Carfora, M., Prevete, N., Tosi, G., Accolla, R. S., and Marone, G. (2000). Tat protein is an HIV‐1‐encoded b‐chemokine homolog that promotes migration and up‐regulates CCR3 expression on human Fc
RIþ cells. J. Immunol. 165, 7171–7179. de Paulis, A., Annunziato, F., Di Gioia, L., Romagnani, S., Carfora, M., Beltrame, C., Marone, G., and Romagnani, P. (2001). Expression of the chemokine receptor CCR3 on human mast cells. Int. Arch. Allergy Immunol. 124, 146–150. de Paulis, A., Florio, G., Prevete, N., Triggiani, M., Fiorentino, I., Genovese, A., and Marone, G. (2002). HIV‐1 envelope gp41 peptides promote migration of human Fc
RIþ cells and inhibit IL‐13 synthesis through interaction with formyl peptide receptors. J. Immunol. 169, 4559–4567. de Paulis, A., Montuori, N., Prevete, N., Fiorentino, I., Rossi, F. W., Visconte, V., Rossi, G., Marone, G., and Ragno, P. (2004a). Urokinase induces basophil chemotaxis through a urokinase receptor epitope that is an endogenous ligand for formyl peptide receptor‐like 1 and ‐like 2. J. Immunol. 173, 5739–5748. de Paulis, A., Prevete, N., Fiorentino, I., Walls, A. F., Curto, M., Petraroli, A., Castaldo, V., Ceppa, P., Fiocca, R., and Marone, G. (2004b). Basophils infiltrate human gastric mucosa at sites of Helicobacter pylori infection, and exhibit chemotaxis in response to H. pylori‐derived peptide Hp(2–20). J. Immunol. 172, 7734–7743. Del Prete, G., Maggi, E., Parronchi, P., Chretien, I., Tiri, A., Macchia, D., Ricci, M., Banchereau, J., De Vries, J., and Romagnani, S. (1988). IL‐4 is an essential factor for the IgE synthesis induced in vitro by human T cell clones and their supernatants. J. Immunol. 140, 4193–4198. Denburg, J. A. (1995). Differentiation of human basophils and mast cells. In ‘‘Human Basophils and Mast Cells: Biological Aspects’’ (G. Marone, Ed.), pp. 49–71. Karger, Basel. Denburg, J. A., Richardson, M., Telizyn, S., and Bienenstock, J. (1983). Basophil/mast cell precursors in human peripheral blood. Blood 61, 775–780. Desmet, C., Gosset, P., Pajak, B., Cataldo, D., Bentires‐Alj, M., Lekeux, P., and Bureau, F. (2004). Selective blockade of NF‐kB activity in airway immune cells inhibits the effector phase of experimental asthma. J. Immunol. 173, 5766–5775. Devouassoux, G., Metcalfe, D. D., and Prussin, C. (1999). Eotaxin potentiates antigen‐dependent basophil IL‐4 production. J. Immunol. 163, 2877–2882. Di Nardo, A., Vitiello, A., and Gallo, R. L. (2003). Cutting edge: mast cell antimicrobial activity is mediated by expression of cathelicidin antimicrobial peptide. J. Immunol. 170, 2274–2278. Drazen, J. M., Yandava, C. N., Dube, L., Szczerback, N., Hippensteel, R., Pillari, A., Israel, E., Schork, N., Silverman, E. S., Katz, D. A., and Drajesk, J. (1999). Pharmacogenetic association between ALOX5 promoter genotype and the response to anti‐asthma treatment. Nat. Genet. 22, 168–170.

h u m a n m a s t ce l l s an d bas o p h i l s i n a s t h m a

143

Dube´ , J., Chakir, J., Laviolette, M., Saint Martin, S., Boutet, M., Desrochers, C., Auger, F., and Boulet, L. P. (1998). In vitro procollagen synthesis and proliferative phenotype of bronchial fibroblasts from normal and asthmatic subjects. Lab. Invest. 78, 297–307. Duffy, S. M., Lawley, W. J., Kaur, D., Yang, W., and Bradding, P. (2003). Inhibition of human mast cell proliferation and survival by tamoxifen in association with ion channel modulation. J. Allergy Clin. Immunol. 112, 965–972. Dunsky, E. H., Zweiman, B., Fischler, E., and Levy, D. A. (1979). Early effects of corticosteroids on basophils, leukocyte histamine, and tissue histamine. J. Allergy Clin. Immunol. 63, 426–432. Dvorak, A. M. (1995). Ultrastructural analysis of human mast cells and basophils. In ‘‘Human Basophils and Mast Cells: Biological Aspects’’ (G. Marone, Ed.), pp. 1–33. Karger, Basel. Dvorak, A. M. (2000). Ultrastructural features of human basophil and mast cell secretory function. In ‘‘Mast Cells and Basophils’’ (G. Marone, L. M. Lichtenstein, and S. J. Galli, Eds.), pp. 63–88. Academic Press, San Diego. Echtenacher, B., Mannel, D. N., and Hultner, L. (1996). Critical protective role of mast cells in a model of acute septic peritonitis. Nature 381, 75–77. Ehrlich, P. (1879). Beitra¨ ge zur kenntniss der granulirten bindegewebszellen und der eosinophilen leukocythen. Arch. Anat. Physiol. (Leipzig) 3, 166–169. Essayan, D. M. (2001). Cyclic nucleotide phosphodiesterases. J. Allergy Clin. Immunol. 108, 671–680. Eubank, T. D., Roberts, R., Galloway, M., Wang, Y., Cohn, D. E., and Marsh, C. B. (2004). GM‐CSF induces expression of soluble VEGF receptor‐1 from human monocytes and inhibits angiogenesis in mice. Immunity 21, 831–842. Fahy, J. V., Fleming, H. E., Wong, H. H., Liu, J. T., Su, J. Q., Reimann, J., Fick, R. B., Jr., and Boushey, H. A. (1997). The effect of an anti‐IgE monoclonal antibody on the early‐ and late‐ phase responses to allergen inhalation in asthmatic subjects. Am. J. Respir. Crit. Care Med. 155, 1828–1834. Falcone, F. H., Haas, H., and Gibbs, B. F. (2000). The human basophil: A new appreciation of its role in immune responses. Blood 96, 4028–4038. Feistritzer, C., Mosheimer, B. A., Sturn, D. H., Bijuklic, K., Patsch, J. R., and Wiedermann, C. J. (2004). Expression and function of the angiopoietin receptor Tie‐2 in human eosinophils. J. Allergy Clin. Immunol. 114, 1077–1084. Figueroa, D. J., Breyer, R. M., Defoe, S. K., Kargman, S., Daugherty, B. L., Waldburger, K., Liu, Q., Clements, M., Zeng, Z., O’Neill, G. P., Jones, T. R., Lynch, K. R., Austin, C. P., and Evans, J. F. (2001). Expression of the cysteinyl leukotriene 1 receptor in normal human lung and peripheral blood leukocytes. Am. J. Respir. Crit. Care Med. 163, 226–233. Flood‐Page, P., Menzies‐Gow, A., Phipps, S., Ying, S., Wangoo, A., Ludwig, M. S., Barnes, N., Robinson, D., and Kay, A. B. (2003). Anti–IL‐5 treatment reduces deposition of ECM proteins in the bronchial subepithelial basement membrane of mild atopic asthmatics. J. Clin. Invest. 112, 1029–1036. Florio, G., Petraroli, A., Patella, V., Triggiani, M., and Marone, G. (2000). The immunoglobulin superantigen‐binding site of HIV‐1 gp120 activates human basophils. AIDS 14, 931–938. Foster, B., Schwartz, L. B., Devouassoux, G., Metcalfe, D. D., and Prussin, C. (2002). Characterization of mast‐cell tryptase‐expressing peripheral blood cells as basophils. J. Allergy Clin. Immunol. 109, 287–293. Foster, P. S., Hogan, S. P., Ramsay, A. J., Matthaei, K. I., and Young, I. G. (1996). Interleukin 5 deficiency abolishes eosinophilia, airways hyperreactivity, and lung damage in a mouse asthma model. J. Exp. Med. 183, 195–201.

144

g i a nn i m a r o ne

ET AL.

Fumo, G., Akin, C., Metcalfe, D. D., and Neckers, L. (2004). 17‐Allylamino‐17‐demethoxygeldanamycin (17‐AAG) is effective in down‐regulating mutated, constitutively activated KIT protein in human mast cells. Blood 103, 1078–1084. Fu¨ reder, W., Agis, H., Willheim, M., Bankl, H. C., Maier, U., Kishi, K., Muller, M. R., Czerwenka, K., Radaszkiewicz, T., Butterfield, J. H., Klappacher, G. W., Sperr, W. R., Oppermann, M., Lechner, K., and Valent, P. (1995). Differential expression of complement receptors on human basophils and mast cells. Evidence for mast cell heterogeneity and CD88/C5aR expression on skin mast cells. J. Immunol. 155, 3152–3160. Galli, S. J., Tsai, M., and Wershil, B. K. (1993). The c‐kit receptor, stem cell factor, and mast cells. What each is teaching us about the others. Am. J. Pathol. 142, 965–974. Garbuzenko, E., Nagler, A., Pickholtz, D., Gillery, P., Reich, R., Maquart, F. X., and Levi‐Schaffer, F. (2002). Human mast cells stimulate fibroblast proliferation, collagen synthesis and lattice contraction: A direct role for mast cells in skin fibrosis. Clin. Exp. Allergy 32, 237–246. Gauchat, J. F., Henchoz, S., Mazzei, G., Aubry, J. P., Brunner, T., Blasey, H., Life, P., Talabot, D., Flores‐Romo, L., Thompson, J., Kishi, K., Butterfield, J., Dahinden, C. A., and Bonnefoi, J. Y. (1993). Induction of human IgE synthesis in B cells by mast cells and basophils. Nature 365, 340–343. Gauvreau, G. M., Lee, J. M., Watson, R. M., Irani, A. M., Schwartz, L. B., and O’Byrne, P. M. (2000). Increased numbers of both airway basophils and mast cells in sputum after allergen inhalation challenge of atopic asthmatics. Am. J. Respir. Crit. Care Med. 161, 1473–1478. Geissler, E. N., Ryan, M. A., and Housman, D. E. (1988). The dominant‐white spotting (W) locus of the mouse encodes the c‐kit proto‐oncogene. Cell 55, 185–192. Genovese, A., Patella, V., De Crescenzo, G., de Paulis, A., Spadaro, G., and Marone, G. (1997). Loratadine and desethoxylcarbonyl‐loratadine inhibit the immunological release of mediators from human Fc
RIþ cells. Clin. Exp. Allergy 27, 559–567. Genovese, A., Bouvet, J. P., Florio, G., Lamparter‐Schummert, B., Bjo¨ rck, L., and Marone, G. (2000). Bacterial immunoglobulin superantigen proteins A and L activate human heart mast cells by interacting with immunoglobulin E. Infect. Immun. 68, 5517–5524. Genovese, A., Borgia, G., Bjo¨ rck, L., Petraroli, A., de Paulis, A., Piazza, M., and Marone, G. (2003). Immunoglobulin superantigen protein L induces IL‐4 and IL‐13 secretion from human Fc
RIþ cells through interaction with the k light chains of IgE. J. Immunol. 170, 1854–1861. Gern, J. E., and Busse, W. W. (2000). The role of viral infections in the natural history of asthma. J. Allergy Clin. Immunol. 106, 201–212. Giembycz, M. A. (2000). Phosphodiesterase 4 inhibitors and the treatment of asthma: where are we now and where do we go from here? Drugs 59, 193–212. Gilchrest, H., Cheewatrakoolpong, B., Billah, M., Egan, R. W., Anthes, J. C., and Greenfeder, S. (2003). Human cord blood‐derived mast cells synthesize and release I‐309 in response to IgE. Life Sci. 73, 2571–2581. Goldstein, S. M., Kaempfer, C. E., Proud, D., Schwartz, L. B., Irani, A. M., and Wintroub, B. U. (1987). Detection and partial characterization of a human mast cell carboxypeptidase. J. Immunol. 139, 2724–2729. Gommerman, J. L., Oh, D. Y., Zhou, X., Tedder, T. F., Maurer, M., Galli, S. J., and Carroll, M. C. (2000). A role for CD21/CD35 and CD19 in responses to acute septic peritonitis: A potential mechanism for mast cell activation. J. Immunol. 165, 6915–6921. Graille, M., Stura, E. A., Corper, A. L., Sutton, B. J., Taussig, M. J., Charbonnier, J. B., and Silverman, G. J. (2000). Crystal structure of a Staphylococcus aureus protein A domain complexed with the Fab fragment of a human IgM antibody: Structural basis for recognition of B‐cell receptors and superantigen activity. Proc. Natl. Acad. Sci. USA 97, 5399–5404.

h u m a n m a s t ce l l s an d bas o p h i l s i n a s t h m a

145

Griffin, M., Weiss, J. W., Leitch, A. G., McFadden, E. R., Jr., Corey, E. J., Austen, K. F., and Drazen, J. M. (1983). Effects of leukotriene D on the airways in asthma. N. Engl. J. Med. 308, 436–439. Grunig, G., Warnock, M., Wakil, A. E., Venkayya, R., Brombacher, F., Rennick, D. M., Sheppard, D., Mohrs, M., Donaldson, D. D., Locksley, R. M., and Corry, D. B. (1998). Requirement for IL‐13 independently of IL‐4 in experimental asthma. Science 282, 2261–2263. Gru¨ tzkau, A., Kruger‐Krasagakes, S., Baumeister, H., Schwarz, C., Kogel, H., Welker, P., Lippert, U., Henz, B. M., and Mo¨ ller, A. (1998). Synthesis, storage, and release of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) by human mast cells: Implications for the biological significance of VEGF206. Mol. Biol. Cell 9, 875–884. Guihard, A. I., Pires, R., and Bouvet, J. P. (1997). An endogenous superantigen in the rat gut lumen as a model to study the role of human protein‐Fv. Scand. J. Immunol. 45, 36–42. Gupta, A. K., and Chow, M. (2003). Pimecrolimus: A review. J. Eur. Acad. Dermatol. Venereol. 17, 493–503. Hait, W. N., Harding, M. W., and Handschumacher, R. E. (1986). Calmodulin, cyclophilin, and cyclosporin A. Science 233, 987–989. Hamilton, A., Faiferman, I., Stober, P., Watson, R. M., and O’Byrne, P. M. (1998). Pranlukast, a cysteinyl leukotriene receptor antagonist, attenuates allergen‐induced early‐ and late‐phase bronchoconstriction and airway hyperresponsiveness in asthmatic subjects. J. Allergy Clin. Immunol. 102, 177–183. Hardy, C. C., Robinson, C., Tattersfield, A. E., and Holgate, S. T. (1984). The bronchoconstrictor effect of inhaled prostaglandin D2 in normal and asthmatic men. N. Engl. J. Med. 311, 209–213. Hartnell, A., Heinemann, A., Conroy, D. M., Wait, R., Sturm, G. J., Caversaccio, M., Jose, P. J., and Williams, T. J. (2004). Identification of selective basophil chemoattractants in human nasal polyps as insulin‐like growth factor‐1 and insulin‐like growth factor‐2. J. Immunol. 173, 6448–6457. Heard, B. E., Dewar, A., Nunn, A. J., and Kay, A. B. (1990). Heterogeneous ultrastructure of human bronchial mast cells: Morphometric subdivision of cell types and evidence for a degranulation gradient. Am. J. Respir. Cell Mol. Biol. 3, 71–78. Heise, C. E., O’Dowd, B. F., Figueroa, D. J., Sawyer, N., Nguyen, T., Im, D. S., Stocco, R., Bellefeuille, J. N., Abramovitz, M., Cheng, R., Williams, D. L., Jr., Zeng, Z., Liu, Q., Ma, L., Clements, M. K., Coulombe, N., Liu, Y., Austin, C. P., George, S. R., O’Neill, G. P., Metters, K. M., Lynch, K. R., and Evans, J. F. (2000). Characterization of the human cysteinyl leukotriene 2 receptor. J. Biol. Chem. 275, 30531–30536. Henderson, W. R., Jr., Tang, L. O., Chu, S. J., Tsao, S. M., Chiang, G. K., Jones, F., Jonas, M., Pae, C., Wang, H., and Chi, E. Y. (2002). A role for cysteinyl leukotrienes in airway remodeling in a mouse asthma model. Am. J. Respir. Crit. Care Med. 165, 108–116. Hirai, H., Tanaka, K., Yoshie, O., Ogawa, K., Kenmotsu, K., Takamori, Y., Ichimasa, M., Sugamura, K., Nakamura, M., Takano, S., and Nagata, K. (2001). Prostaglandin D2 selectively induces chemotaxis in T helper type 2 cells, eosinophils, and basophils via seven‐transmembrane receptor CRTH2. J. Exp. Med. 193, 255–261. Hiromatsu, Y., and Toda, S. (2003). Mast cells and angiogenesis. Microsc. Res. Tech. 60, 64–69. Hirst, S. J., Hallsworth, M. P., Peng, Q., and Lee, T. H. (2002). Selective induction of eotaxin release by IL‐13 or IL‐4 in human airway smooth muscle cells is synergistic with IL‐1b and is mediated by the IL‐4 receptor a‐chain. Am. J. Respir. Crit. Care Med. 165, 1161–1171. Hjertson, M., Kivinen, P. K., Dimberg, L., Nilsson, K., Harvima, I. T., and Nilsson, G. (2003). Retinoic acid inhibits in vitro development of mast cells but has no marked effect on mature human skin tryptase‐ and chymase‐positive mast cells. J. Invest. Dermatol. 120, 239–245.

146

g i a nn i m a r o ne

ET AL.

Hofstra, C. L., Desai, P. J., Thurmond, R. L., and Fung‐Leung, W. P. (2003). Histamine H4 receptor mediates chemotaxis and calcium mobilization of mast cells. J. Pharmacol. Exp. Ther. 305, 1212–1221. Holash, J., Davis, S., Papadopoulos, N., Croll, S. D., Ho, L., Russell, M., Boland, P., Leidich, R., Hylton, D., Burova, E., Ioffe, E., Huang, T., Radziejewski, C., Bailey, K., Fandl, J. P., Daly, T., Wiegand, S. J., Yancopoulos, G. D., and Rudge, J. S. (2002). VEGF‐Trap: A VEGF blocker with potent antitumor effects. Proc. Natl. Acad. Sci. USA 99, 11393–11398. Holford‐Strevens, V., Warren, P., Wong, C., and Manfreda, J. (1984). Serum total immunoglobulin E levels in Canadian adults. J. Allergy Clin. Immunol. 73, 516–522. Holgate, S. T., Lackie, P., Wilson, S., Roche, W., and Davies, D. (2000). Bronchial epithelium as a key regulator of airway allergen sensitization and remodeling in asthma. Am. J. Respir. Crit. Care Med. 162, S113–117. Holgate, S. T., Davies, D. E., Rorke, S., Cakebread, J., Murphy, G., Powell, R. M., and Holloway, J. W. (2004). Identification and possible functions of ADAM 33 as an asthma susceptibility gene. In ‘‘Asthma and Allergic Diseases. Physiology, Immunopharmacology and Treatment’’ (G. Marone, K. F. Austen, S. T. Holgate, A. B. Kay, and L. M. Lichtenstein, Eds.), pp. 49–55. Blackwell, London. Holtzman, M. J., Morton, J. D., Shornick, L. P., Tyner, J. W., O’Sullivan, M. P., Antao, A., Lo, M., Castro, M., and Walter, M. J. (2002). Immunity, inflammation, and remodeling in the airway epithelial barrier: epithelial‐viral‐allergic paradigm. Physiol. Rev. 82, 19–46. Horton, M. A., and O’ Brien, H. A. (1983). Characterization of human mast cells in long‐term culture. Blood 62, 1251–1260. Hoshino, M., Nakamura, Y., and Hamid, Q. A. (2001). Gene expression of vascular endothelial growth factor and its receptors and angiogenesis in bronchial asthma. J. Allergy Clin. Immunol. 107, 1034–1038. Hosoda, M., Yamaya, M., Suzuki, T., Yamada, N., Kamanaka, M., Sekizawa, K., Butterfield, J. H., Watanabe, T., Nishimura, H., and Sasaki, H. (2002). Effects of rhinovirus infection on histamine and cytokine production by cell lines from human mast cells and basophils. J. Immunol. 169, 1482–1491. Hsieh, F. H., Lam, B. K., Penrose, J. F., Austen, K. F., and Boyce, J. A. (2001). T helper cell type 2 cytokines coordinately regulate IgE‐dependent cysteinyl leukotriene production by human cord blood‐derived mast cells: Profound induction of leukotriene C4 synthase expression by IL‐4. J. Exp. Med. 193, 123–133. Huang, E., Nocka, K., Beier, D. R., Chu, T. Y., Buck, J., Lahm, H. W., Wellner, D., Leder, P., and Besmer, P. (1990). The hematopoietic growth factor KL is encoded by the Sl locus and is the ligand of the c‐kit receptor, the gene product of the W locus. Cell 63, 225–233. Hundley, T. R., Marshall, L. A., Hubbard, W. C., and MacGlashan, D. W., Jr. (1998). Characteristics of arachidonic acid generation in human basophils: relationship between the effects of inhibitors of secretory phospholipase A2 activity and leukotriene C4 release. J. Pharmacol. Exp. Ther. 284, 847–857. Ikeda, K., Nakajima, H., Suzuki, K., Kagami, S., Hirose, K., Suto, A., Saito, Y., and Iwamoto, I. (2003). Mast cells produce interleukin‐25 upon Fc
RI‐mediated activation. Blood 101, 3594–3596. Ingana¨ s, M. (1981). Comparison of mechanisms of interaction between protein A from Staphylococcus aureus and human monoclonal IgG, IgA and IgM in relation to the classical Fcg and the alternative F(ab’)2
protein A interactions. Scand. J. Immunol. 13, 343–352. Ingana¨ s, M., Johansson, S. G., and Bennich, H. H. (1980). Interaction of human polyclonal IgE and IgG from different species with protein A from Staphylococcus aureus: demonstration of protein‐A‐reactive sites located in the Fab’2 fragment of human IgG. Scand. J. Immunol. 12, 23–31.

h u m a n m a s t ce l l s an d bas o p h i l s i n a s t h m a

147

Inman, M. D., Watson, R., Cockcroft, D. W., Wong, B. J., Hargreave, F. E., and O’Byrne, P. M. (1995). Reproducibility of allergen‐induced early and late asthmatic responses. J. Allergy Clin. Immunol. 95, 1191–1195. Irani, A. M., Nilsson, G., Miettinen, U., Craig, S. S., Ashman, L. K., Ishizaka, T., Zsebo, K. M., and Schwartz, L. B. (1992). Recombinant human stem cell factor stimulates differentiation of mast cells from dispersed human fetal liver cells. Blood 80, 3009–3021. Irani, A. A., Nilsson, G., Ashman, L. K., and Schwartz, L. B. (1995). Dexamethasone inhibits the development of mast cells from dispersed human fetal liver cells cultured in the presence of recombinant human stem cell factor. Immunology 84, 72–78. Irani, A. M., Huang, C., Xia, H. Z., Kepley, C., Nafie, A., Fouda, E. D., Craig, S., Zweiman, B., and Schwartz, L. B. (1998). Immunohistochemical detection of human basophils in late‐phase skin reactions. J. Allergy Clin. Immunol. 101, 354–362. ISAAC Steering, Committee (1998). Worldwide variation in prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and atopic eczema: ISAAC. Lancet 351, 1225–1232. Ishizaka, K., and Ishizaka, T. (1966). Physicochemical properties of human reaginic antibody. I. Association of reaginic activity with an immunoglobulin other than A or G globulin. J. Allergy 37, 169. Israel, E., Cohn, J., Dube, L., and Drazen, J. M. (1996). Effect of treatment with zileuton, a 5‐lipoxygenase inhibitor, in patients with asthma. A randomized controlled trial. Zileuton Clinical Trial Group. JAMA 275, 931–936. Israe¨ l‐Biet, D., Labrousse, F., Tourani, J. M., Sors, H., Andrieu, J. M., and Even, P. (1992). Elevation of IgE in HIV‐infected subjects: A marker of poor prognosis. J. Allergy Clin. Immunol. 89, 68–75. Jeong, H. J., Na, H. J., Hong, S. H., and Kim, H. M. (2003). Inhibition of the stem cell factor‐ induced migration of mast cells by dexamethasone. Endocrinology 144, 4080–4086. Jinnin, M., Ihn, H., Yamane, K., and Tamaki, K. (2004). Interleukin‐13 stimulates the transcription of the human a2(I) collagen gene in human dermal fibroblasts. J. Biol. Chem. 279, 41783–41791. Jogie‐Brahim, S., Min, H. K., Fukuoka, Y., Xia, H. Z., and Schwartz, L. B. (2004). Expression of a‐tryptase and b‐tryptase by human basophils. J. Allergy Clin. Immunol. 113, 1086–1092. Johnston, S. L. (1997). Bronchial hyperresponsiveness and cytokines in virus‐induced asthma exacerbations. Clin. Exp. Allergy 27, 7–9. Johnston, S. L., Pattemore, P. K., Sanderson, G., Smith, S., Lampe, F., Josephs, L., Symington, P., O’Toole, S., Myint, S. H., Tyrrell, D. A., and Holgate, S. T. (1995). Community study of role of viral infections in exacerbations of asthma in 9–11 year old children. BMJ 310, 1225–1229. Kalesnikoff, J., Huber, M., Lam, V., Damen, J. E., Zhang, J., Siraganian, R. P., and Krystal, G. (2001). Monomeric IgE stimulates signaling pathways in mast cells that lead to cytokine production and cell survival. Immunity 14, 801–811. Kanaoka, Y., and Boyce, J. A. (2004). Cysteinyl leukotrienes and their receptors: Cellular distribution and function in immune and inflammatory responses. J. Immunol. 173, 1503–1510. Kaplan, M. H., Sadick, N., McNutt, N. S., Meltzer, M., Sarngadharan, M. G., and Pahwa, S. (1987). Dermatologic findings and manifestations of acquired immunodeficiency syndrome (AIDS). J. Am. Acad. Dermatol. 16, 485–506. Karray, S., and Zouali, M. (1997). Identification of the B cell superantigen‐binding site of HIV‐1 gp120. Proc. Natl. Acad. Sci. USA 94, 1356–1360. Karray, S., Juompan, L., Maroun, R. C., Isenberg, D., Silverman, G. J., and Zouali, M. (1998). Structural basis of the gp120 superantigen‐binding site on human immunoglobulins. J. Immunol. 161, 6681–6688. Kastern, W., Sjobring, U., and Bjorck, L. (1992). Structure of peptostreptococcal protein L and identification of a repeated immunoglobulin light chain‐binding domain. J. Biol. Chem. 267, 12820–12825.

148

g i a nn i m a r o ne

ET AL.

Kawagishi, Y., Mita, H., Taniguchi, M., Maruyama, M., Oosaki, R., Higashi, N., Kashii, T., Kobayashi, M., and Akiyama, K. (2002). Leukotriene C4 synthase promoter polymorphism in Japanese patients with aspirin‐induced asthma. J. Allergy Clin. Immunol. 109, 936–942. Kawanami, O., Ferrans, V. J., Fulmer, J. D., and Crystal, R. G. (1979). Ultrastructure of pulmonary mast cells in patients with fibrotic lung disorders. Lab. Invest. 40, 717–734. Kepley, C. L., Cambier, J. C., Morel, P. A., Lujan, D., Ortega, E., Wilson, B. S., and Oliver, J. M. (2000). Negative regulation of Fc
RI signaling by FcgRII costimulation in human blood basophils. J. Allergy Clin. Immunol. 106, 337–348. Kepley, C. L., McFeeley, P. J., Oliver, J. M., and Lipscomb, M. F. (2001). Immunohistochemical detection of human basophils in postmortem cases of fatal asthma. Am. J. Respir. Crit. Care Med. 164, 1053–1058. Kirshenbaum, A. S., Kessler, S. W., Goff, J. P., and Metcalfe, D. D. (1991). Demonstration of the origin of human mast cells from CD34þ bone marrow progenitor cells. J. Immunol. 146, 1410–1415. Kirshenbaum, A. S., Goff, J. P., Kessler, S. W., Mican, J. M., Zsebo, K. M., and Metcalfe, D. D. (1992). Effect of IL‐3 and stem cell factor on the appearance of human basophils and mast cells from CD34þ pluripotent progenitor cells. J. Immunol. 148, 772–777. Kitamura, Y., and Go, S. (1979). Decreased production of mast cells in S1/S1d anemic mice. Blood 53, 492–497. Kitamura, Y., Go, S., and Hatanaka, K. (1978). Decrease of mast cells in W/Wv mice and their increase by bone marrow transplantation. Blood 52, 447–452. KleinJan, A., McEuen, A. R., Dijkstra, M. D., Buckley, M. G., Walls, A. F., and Fokkens, W. J. (2000). Basophil and eosinophil accumulation and mast cell degranulation in the nasal mucosa of patients with hay fever after local allergen provocation. J. Allergy Clin. Immunol. 106, 677–686. Kobayashi, T., Miura, T., Haba, T., Sato, M., Serizawa, I., Nagai, H., and Ishizaka, K. (2000). An essential role of mast cells in the development of airway hyperresponsiveness in a murine asthma model. J. Immunol. 164, 3855–3861. Koshino, T., Teshima, S., Fukushima, N., Takaishi, T., Hirai, K., Miyamoto, Y., Arai, Y., Sano, Y., Ito, K., and Morita, Y. (1993). Identification of basophils by immunohistochemistry in the airways of post‐mortem cases of fatal asthma. Clin. Exp. Allergy 23, 919–925. Koutsonikolis, A., Nelson, R. P., Jr., Fernandez‐Caldas, E., Brigino, E. N., Seleznick, M., Good, R. A., and Lockey, R. F. (1996). Serum total and specific IgE levels in children infected with human immunodeficiency virus. J. Allergy Clin. Immunol. 97, 692–697. Kraneveld, A. D., van der Kleij, H. P., Kool, M., van Houwelingen, A. H., Weitenberg, A. C., Redegeld, F. A., and Nijkamp, F. P. (2002). Key role for mast cells in nonatopic asthma. J. Immunol. 169, 2044–2053. Kulka, M., and Metcalfe, D. D. (2005). High‐resolution tracking of cell division demonstrates differential effects of TH1 and TH2 cytokines on SCF‐dependent human mast cell production in vitro: Correlation with apoptosis and Kit expression. Blood 105, 592–599. Kulka, M., Alexopoulou, L., Flavell, R. A., and Metcalfe, D. D. (2004). Activation of mast cells by double‐stranded RNA: Evidence for activation through Toll‐like receptor 3. J. Allergy Clin. Immunol. 114, 174–182. Kuperman, D. A., Huang, X., Koth, L. L., Chang, G. H., Dolganov, G. M., Zhu, Z., Elias, J. A., Sheppard, D., and Erle, D. J. (2002). Direct effects of IL‐13 on epithelial cells cause airway hyperreactivity and mucus overproduction in asthma. Nat. Med. 8, 885–889. Laitinen, L. A., Laitinen, A., Haahtela, T., Vilkka, V., Spur, B. W., and Lee, T. H. (1993). Leukotriene E4 and granulocytic infiltration into asthmatic airways. Lancet 341, 989–990.

h u m a n m a s t ce l l s an d bas o p h i l s i n a s t h m a

149

Lam, V., Kalesnikoff, J., Lee, C. W., Hernandez‐Hansen, V., Wilson, B. S., Oliver, J. M., and Krystal, G. (2003). IgE alone stimulates mast cell adhesion to fibronectin via pathways similar to those used by IgE þ antigen but distinct from those used by Steel factor. Blood 102, 1405–1413. Larche´ , M., Robinson, D. S., and Kay, A. B. (2003). The role of T lymphocytes in the pathogenesis of asthma. J. Allergy Clin. Immunol. 111, 450–463. Lavker, R. M., and Schechter, N. M. (1985). Cutaneous mast cell depletion results from topical corticosteroid usage. J. Immunol. 135, 2368–2373. Lee, J. H., Kaminski, N., Dolganov, G., Grunig, G., Koth, L., Solomon, C., Erle, D. J., and Sheppard, D. (2001). Interleukin‐13 induces dramatically different transcriptional programs in three human airway cell types. Am. J. Respir. Cell Mol. Biol. 25, 474–485. Lee, Y. C., and Lee, H. K. (2001). Vascular endothelial growth factor in patients with acute asthma. J. Allergy Clin. Immunol. 107, 1106. Leung, D. Y., and Bloom, J. W. (2003). Update on glucocorticoid action and resistance. J. Allergy Clin. Immunol. 111, 3–22. Leung, D. Y., Hauk, P., Strickland, I., Travers, J. B., and Norris, D. A. (1998). The role of superantigens in human diseases: Therapeutic implications for the treatment of skin diseases. Br. J. Dermatol. 139, 17–29. Levi‐Schaffer, F., and Pe’er, J. (2001). Mast cells and angiogenesis. Clin. Exp. Allergy 31, 521–524. Levi‐Schaffer, F., and Rubinchik, E. (1995). Activated mast cells are fibrogenic for 3T3 fibroblasts. J. Invest. Dermatol. 104, 999–1003. Levi‐Schaffer, F., Austen, K. F., Caulfield, J. P., Hein, A., Gravallese, P. M., and Stevens, R. L. (1987). Co‐culture of human lung‐derived mast cells with mouse 3T3 fibroblasts: Morphology and IgE‐mediated release of histamine, prostaglandin D2, and leukotrienes. J. Immunol. 139, 494–500. Levi‐Schaffer, F., Garbuzenko, E., Rubin, A., Reich, R., Pickholz, D., Gillery, P., Emonard, H., Nagler, A., and Maquart, F. A. (1999). Human eosinophils regulate human lung‐ and skin‐ derived fibroblast properties in vitro: A role for transforming growth factor b (TGF‐b). Proc. Natl. Acad. Sci. USA 96, 9660–9665. Li, H., Sim, T. C., Grant, J. A., and Alam, R. (1996). The production of macrophage inflammatory protein‐1a by human basophils. J. Immunol. 157, 1207–1212. Li, X., and Wilson, J. W. (1997). Increased vascularity of the bronchial mucosa in mild asthma. Am. J. Respir. Crit. Care Med. 156, 229–233. Lichtenstein, L. M., and Gillespie, E. (1973). Inhibition of histamine release by histamine controlled by H2 receptor. Nature 244, 287–288. Lichtenstein, L. M., and Gillespie, E. (1975). The effects of the H1 and H2 antihistamines on ‘‘allergic’’ histamine release and its inhibition by histamine. J. Pharmacol. Exp. Ther. 192, 441–450. Lichtenstein, L. M., and Margolis, S. (1968). Histamine release in vitro: Inhibition by catecholamines and methylxanthines. Science 161, 902–903. Liu, M. C., Hubbard, W. C., Proud, D., Stealey, B. A., Galli, S. J., Kagey‐Sobotka, A., Bleecker, E. R., and Lichtenstein, L. M. (1991). Immediate and late inflammatory responses to ragweed antigen challenge of the peripheral airways in allergic asthmatics. Cellular, mediator, and permeability changes. Am. Rev. Respir. Dis. 144, 51–58. Lorentz, A., Schuppan, D., Gebert, A., Manns, M. P., and Bischoff, S. C. (2002). Regulatory effects of stem cell factor and interleukin‐4 on adhesion of human mast cells to extracellular matrix proteins. Blood 99, 966–972. Lukacs, N. W. (2001). Role of chemokines in the pathogenesis of asthma. Nat. Rev. Immunol. 1, 108–116.

150

g i a nn i m a r o ne

ET AL.

Lukacs, N. W., John, A., Berlin, A., Bullard, D. C., Knibbs, R., and Stoolman, L. M. (2002). E‐ and P‐selectins are essential for the development of cockroach allergen‐induced airway responses. J. Immunol. 169, 2120–2125. Luskova´ , P., and Dra´ ber, P. (2004). Modulation of the Fc
RI signaling by tyrosine kinase inhibitors: Search for therapeutic targets of inflammatory and allergy diseases. Curr. Pharm. Des. 10, 1727–1737. Lynch, K. R., O’Neill, G. P., Liu, Q., Im, D. S., Sawyer, N., Metters, K. M., Coulombe, N., Abramovitz, M., Figueroa, D. J., Zeng, Z., Connolly, B. M., Bai, C., Austin, C. P., Chateauneuf, A., Stocco, R., Greig, G. M., Kargman, S., Hooks, S. B., Hosfield, E., Williams, D. L., Jr., Ford‐Hutchinson, A. W., Caskey, C. T., and Evans, J. F. (1999). Characterization of the human cysteinyl leukotriene CysLT1 receptor. Nature 399, 789–793. Macfarlane, A. J., Kon, O. M., Smith, S. J., Zeibecoglou, K., Khan, L. N., Barata, L. T., McEuen, A. R., Buckley, M. G., Walls, A. F., Meng, Q., Humbert, M., Barnes, N. C., Robinson, D. S., Ying, S., and Kay, A. B. (2000). Basophils, eosinophils, and mast cells in atopic and nonatopic asthma and in late‐phase allergic reactions in the lung and skin. J. Allergy Clin. Immunol. 105, 99–107. MacGlashan, D., Jr. (2003). Two regions of down‐regulation in the IgE‐mediated signaling pathway in human basophils. J. Immunol. 170, 4914–4925. MacGlashan, D. W., Jr., Lavens‐Phillips, S., and Miura, K. (2000a). Perspectives on the regulation of secretion from human basophils and mast cells. In ‘‘Mast Cells and Basophils’’ (G. Marone, L. M. Lichtenstein, and S. J. Galli, Eds.), pp. 195–207. Academic Press, San Diego. MacGlashan, D. W., Jr., Miura, K., and Lavens‐Phillips, S. (2000b). Pharmacology of IgE‐mediated desensitization of human basophils: Effects of protein kinase C and src‐family kinase inhibitors. Biochem. Pharmacol. 60, 1717–1727. MacGlashan, D. W., Jr., White, J. M., Huang, S. K., Ono, S. J., Schroeder, J. T., and Lichtenstein, L. M. (1994). Secretion of IL‐4 from human basophils. The relationship between IL‐4 mRNA and protein in resting and stimulated basophils. J. Immunol. 152, 3006–3016. Malaviya, R., Ikeda, T., Ross, E., and Abraham, S. N. (1996). Mast cell modulation of neutrophil influx and bacterial clearance at sites of infection through TNF‐a. Nature 381, 77–80. Malaviya, R., Gao, Z., Thankavel, K., van der Merwe, P. A., and Abraham, S. N. (1999). The mast cell tumor necrosis factor alpha response to FimH‐expressing Escherichia coli is mediated by the glycosylphosphatidylinositol‐anchored molecule CD48. Proc. Natl. Acad. Sci. USA 96, 8110–8115. Marom, Z., Shelhamer, J. H., Bach, M. K., Morton, D. R., and Kaliner, M. (1982). Slow‐reacting substances, leukotrienes C4 and D4, increase the release of mucus from human airways in vitro. Am. Rev. Respir. Dis. 126, 449–451. Marone, G. (1997). Milestones in the biology and pharmacology of H1‐receptor antagonists. Allergy 52, 7–13. Marone, G. (1998a). Asthma: Recent advances. Immunol. Today 19, 5–9. Marone, G. (1998b). Tacrolimus ointment for atopic dermatitis. N. Engl. J. Med. 339, 1788. Marone, G., Poto, S., Petracca, R., Triggiani, M., de Lutio di Castelguidone, E., and Condorelli, M. (1982). Activation of human basophils by staphylococcal protein A. I. The role of cyclic AMP, arachidonic acid metabolites, microtubules and microfilaments. Clin. Exp. Immunol. 50, 661–668. Marone, G., Ambrosio, G., Bonaduce, D., Genovese, A., Triggiani, M., and Condorelli, M. (1984). Inhibition of IgE‐mediated histamine release from human basophils and mast cells by fenoterol. Int. Arch. Allergy Appl. Immunol. 74, 356–361. Marone, G., Tamburini, M., Giudizi, M. G., Biagiotti, R., Almerigogna, F., and Romagnani, S. (1987). Mechanism of activation of human basophils by Staphylococcus aureus Cowan 1. Infect. Immun. 55, 803–809.

h u m a n m a s t ce l l s an d bas o p h i l s i n a s t h m a

151

Marone, G., Granata, F., Spadaro, G., Onorati, A. M., and Triggiani, M. (1999). Antiinflammatory effects of oxatomide. J. Invest. Allergol. Clin. Immunol. 9, 207–214. Marone, G., Lichtenstein, L. M., and Galli, S. J. (2000). ‘‘Mast Cells and Basophils.’’ Academic Press, San Diego. Marone, G., Florio, G., Petraroli, A., Triggiani, M., and de Paulis, A. (2001). Role of human Fc
RIþ cells in HIV‐1 infection. Immunol. Rev. 179, 128–138. Marone, G., Austen, K. F., Holgate, S. T., Kay, A. B., and Lichtenstein, L. M. (2004a). ‘‘Asthma and Allergic Diseases. Physiology, Immunopharmacology and Treatment.’’ Blackwell, London. Marone, G., Rossi, F. W., Bova, M., Detoraki, B., Liccardo, B., and Petraroli, A. (2004b). Superallergens: a novel mechanism of IgE‐mediated activation of human basophils and mast cells. In ‘‘Asthma and Allergic Diseases. Physiology, Immunopharmacology and Treatment’’ (G. Marone, K. F. Austen, S. T. Holgate, A. B. Kay, and L. M. Lichtenstein, Eds.), pp. 64–75. Blackwell, London. Marone, G., Prevete, N., De Rienzo, G., Fiorentino, I., Rossi, F. W., Liccardo, B., and de Paulis, A. (2005a). Role of mast cells and basophils in angiogenesis in respiratory allergies. XVIII IFOS World Congress, Rome. Marone, G., Triggiani, M., and de Paulis, A. (2005b). Mast cells and basophils: Friends as well as foes in bronchial asthma? Trends Immunol. 26, 25–31. Marshall, J. S., and Jawdat, D. M. (2004). Mast cells in innate immunity. J. Allergy Clin. Immunol. 114, 21–27. Mashikian, M. V., Tarpy, R. E., Saukkonen, J. J., Lim, K. G., Fine, G. D., Cruikshank, W. W., and Center, D. M. (1998). Identification of IL‐16 as the lymphocyte chemotactic activity in the bronchoalveolar lavage fluid of histamine‐challenged asthmatic patients. J. Allergy Clin. Immunol. 101, 786–792. Matsuoka, T., Hirata, M., Tanaka, H., Takahashi, Y., Murata, T., Kabashima, K., Sugimoto, Y., Kobayashi, T., Ushikubi, F., Aze, Y., Eguchi, N., Urade, Y., Yoshida, N., Kimura, K., Mizoguchi, A., Honda, Y., Nagai, H., and Narumiya, S. (2000). Prostaglandin D2 as a mediator of allergic asthma. Science 287, 2013–2017. Mayr, S. I., Zuberi, R. I., Zhang, M., de Sousa‐Hitzler, J., Ngo, K., Kuwabara, Y., Yu, L., Fung‐ Leung, W. P., and Liu, F. T. (2002). IgE‐dependent mast cell activation potentiates airway responses in murine asthma models. J. Immunol. 169, 2061–2068. McCurdy, J. D., Olynych, T. J., Maher, L. H., and Marshall, J. S. (2003). Cutting edge: Distinct Toll‐like receptor 2 activators selectively induce different classes of mediator production from human mast cells. J. Immunol. 170, 1625–1629. McEuen, A. R., Calafat, J., Compton, S. J., Easom, N. J., Buckley, M. G., Knol, E. F., and Walls, A. F. (2001). Mass, charge, and subcellular localization of a unique secretory product identified by the basophil‐specific antibody BB1. J. Allergy Clin. Immunol. 107, 842–848. Mekori, Y. A., and Metcalfe, D. D. (1999). Mast cell–T cell interactions. J. Allergy Clin. Immunol. 104, 517–523. Mellor, E. A., Maekawa, A., Austen, K. F., and Boyce, J. A. (2001). Cysteinyl leukotriene receptor 1 is also a pyrimidinergic receptor and is expressed by human mast cells. Proc. Natl. Acad. Sci. USA 98, 7964–7969. Mellor, E. A., Austen, K. F., and Boyce, J. A. (2002). Cysteinyl leukotrienes and uridine diphosphate induce cytokine generation by human mast cells through an IL‐4‐regulated pathway that is inhibited by leukotriene receptor antagonists. J. Exp. Med. 195, 583–592. Miadonna, A., Tedeschi, A., Leggieri, E., Invernizzi, F., and Zanussi, C. (1982). Basophil activation in idiopathic mixed cryoglobulinemia. J. Clin. Immunol. 2, 309–313. Milgrom, H., Fick, R. B., Jr., Su, J. Q., Reimann, J. D., Bush, R. K., Watrous, M. L., and Metzger, W. J. (1999). Treatment of allergic asthma with monoclonal anti‐IgE antibody. rhuMAb‐E25 Study Group. N. Engl. J. Med. 341, 1966–1973.

152

g i a nn i m a r o ne

ET AL.

Mitchell, E. B., Crow, J., Chapman, M. D., Jouhal, S. S., Pope, F. M., and Platts‐Mills, T. A. (1982). Basophils in allergen‐induced patch test sites in atopic dermatitis. Lancet 1, 127–130. Mitsui, H., Furitsu, T., Dvorak, A. M., Irani, A. M., Schwartz, L. B., Inagaki, N., Takei, M., Ishizaka, K., Zsebo, K. M., Gillis, S., and Ishizaka, T. (1993). Development of human mast cells from umbilical cord blood cells by recombinant human and murine c‐kit ligand. Proc. Natl. Acad. Sci. USA 90, 735–739. Mochizuki, A., Tamura, N., Yatabe, Y., Onodera, S., Hiruma, T., Inaba, N., Kusunoki, J., and Tomioka, H. (2001). Suppressive effects of F‐1322 on the antigen‐induced late asthmatic response and pulmonary eosinophilia in guinea pigs. Eur. J. Pharmacol. 430, 123–133. Mochizuki, A., McEuen, A. R., Buckley, M. G., and Walls, A. F. (2003). The release of basogranulin in response to IgE‐dependent and IgE‐independent stimuli: Validity of basogranulin measurement as an indicator of basophil activation. J. Allergy Clin. Immunol. 112, 102–108. Mo¨ ller, A., Lippert, U., Lessmann, D., Kolde, G., Hamann, K., Welker, P., Schadendorf, D., Rosenbach, T., Luger, T., and Czarnetzki, B. M. (1993). Human mast cells produce IL‐8. J. Immunol. 151, 3261–3266. Mwamtemi, H. H., Koike, K., Kinoshita, T., Ito, S., Ishida, S., Nakazawa, Y., Kurokawa, Y., Shinozaki, K., Sakashita, K., Takeuchi, K., Shiohara, M., Kamijo, T., Yasui, Y., Ishiguro, A., Kawano, Y., Kitano, K., Miyazaki, H., Kato, T., Sakuma, S., and Komiyama, A. (2001). An increase in circulating mast cell colony‐forming cells in asthma. J. Immunol. 166, 4672–4677. Nagy, J. A., Dvorak, A. M., and Dvorak, H. F. (2003). VEGF‐A(164/165) and PlGF: Roles in angiogenesis and arteriogenesis. Trends Cardiovasc. Med. 13, 169–175. Nakayama, T., Yao, L., and Tosato, G. (2004). Mast cell‐derived angiopoietin‐1 plays a critical role in the growth of plasma cell tumors. J. Clin. Invest. 114, 1317–1325. Nials, A. T., Ball, D. I., Butchers, P. R., Coleman, R. A., Humbles, A. A., Johnson, M., and Vardey, C. J. (1994). Formoterol on airway smooth muscle and human lung mast cells: A comparison with salbutamol and salmeterol. Eur. J. Pharmacol. 251, 127–135. Nilson, B. H., Solomon, A., Bjo¨ rck, L., and Akerstrom, B. (1992). Protein L from Peptostreptococcus magnus binds to the k light chain variable domain. J. Biol. Chem. 267, 2234–2239. Nilsson, G., Forsberg, K., Bodger, M. P., Ashman, L. K., Zsebo, K. M., Ishizaka, T., Irani, A. M., and Schwartz, L. B. (1993). Phenotypic characterization of stem cell factor‐dependent human foetal liver‐derived mast cells. Immunology 79, 325–330. Nilsson, G., Butterfield, J. H., Nilsson, K., and Siegbahn, A. (1994). Stem cell factor is a chemotactic factor for human mast cells. J. Immunol. 153, 3717–3723. Niyonsaba, F., Someya, A., Hirata, M., Ogawa, H., and Nagaoka, I. (2001). Evaluation of the effects of peptide antibiotics human b‐defensins‐1/‐2 and LL‐37 on histamine release and prostaglandin D2 production from mast cells. Eur. J. Immunol. 31, 1066–1075. Niyonsaba, F., Iwabuchi, K., Someya, A., Hirata, M., Matsuda, H., Ogawa, H., and Nagaoka, I. (2002). A cathelicidin family of human antibacterial peptide LL‐37 induces mast cell chemotaxis. Immunology 106, 20–26. Noguchi, E., Shibasaki, M., Kamioka, M., Yokouchi, Y., Yamakawa‐Kobayashi, K., Hamaguchi, H., Matsui, A., and Arinami, T. (2002). New polymorphisms of haematopoietic prostaglandin D synthase and human prostanoid DP receptor genes. Clin. Exp. Allergy 32, 93–96. Nouri‐Aria, K. T., Irani, A. M., Jacobson, M. R., O’Brien, F., Varga, E. M., Till, S. J., Durham, S. R., and Schwartz, L. B. (2001). Basophil recruitment and IL‐4 production during human allergen‐ induced late asthma. J. Allergy Clin. Immunol. 108, 205–211. Ochensberger, B., Rihs, S., Brunner, T., and Dahinden, C. A. (1995). IgE‐independent interleukin‐4 expression and induction of a late phase of leukotriene C4 formation in human blood basophils. Blood 86, 4039–4049.

h u m a n m a s t ce l l s an d bas o p h i l s i n a s t h m a

153

Ochensberger, B., Daepp, G. C., Rihs, S., and Dahinden, C. A. (1996). Human blood basophils produce interleukin‐13 in response to IgE‐receptor‐dependent and ‐independent activation. Blood 88, 3028–3037. Ochensberger, B., Tassera, L., Bifrare, D., Rihs, S., and Dahinden, C. A. (1999). Regulation of cytokine expression and leukotriene formation in human basophils by growth factors, chemokines and chemotactic agonists. Eur. J. Immunol. 29, 11–22. Ochi, H., Hirani, W. M., Yuan, Q., Friend, D. S., Austen, K. F., and Boyce, J. A. (1999). T helper cell type 2 cytokine‐mediated comitogenic responses and CCR3 expression during differentiation of human mast cells in vitro. J. Exp. Med. 190, 267–280. Ochi, H., De Jesus, N. H., Hsieh, F. H., Austen, K. F., and Boyce, J. A. (2000). IL‐4 and ‐5 prime human mast cells for different profiles of IgE‐dependent cytokine production. Proc. Natl. Acad. Sci. USA 97, 10509–10513. Ogawa, K., Kaminuma, O., Kikkawa, H., Nakata, A., Asahina, M., Egan, R. W., Akiyama, K., and Mori, A. (2002). Transient contribution of mast cells to pulmonary eosinophilia but not to hyper‐ responsiveness. Clin. Exp. Allergy 32, 140–148. Oguma, T., Palmer, L. J., Birben, E., Sonna, L. A., Asano, K., and Lilly, C. M. (2004). Role of prostanoid DP receptor variants in susceptibility to asthma. N. Engl. J. Med. 351, 1752–1763. Ohkawara, Y., Yamauchi, K., Tanno, Y., Tamura, G., Ohtani, H., Nagura, H., Ohkuda, K., and Takishima, T. (1992). Human lung mast cells and pulmonary macrophages produce tumor necrosis factor‐a in sensitized lung tissue after IgE receptor triggering. Am. J. Respir. Cell Mol. Biol. 7, 385–392. Okayama, Y., Kobayashi, H., Ashman, L. K., Dobashi, K., Nakazawa, T., Holgate, S. T., Church, M. K., and Mori, M. (1998). Human lung mast cells are enriched in the capacity to produce granulocyte‐macrophage colony‐stimulating factor in response to IgE‐dependent stimulation. Eur. J. Immunol. 28, 708–715. Okumura, S., Kashiwakura, J., Tomita, H., Matsumoto, K., Nakajima, T., Saito, H., and Okayama, Y. (2003). Identification of specific gene expression profiles in human mast cells mediated by Toll‐like receptor 4 and Fc
RI. Blood 102, 2547–2554. Onorato, J., Esposito, S., Scovena, E., Morandi, B., Morelli, M., Pizzi, M., Zisa, G., Marchisio, P., and Principi, N. (1999). Eosinophil involvement and serum IgE level in HIV‐1‐infected children. J. Allergy Clin. Immunol. 104, 245–247. Ordonez, C. L., Khashayar, R., Wong, H. H., Ferrando, R., Wu, R., Hyde, D. M., Hotchkiss, J. A., Zhang, Y., Novikov, A., Dolganov, G., and Fahy, J. V. (2001). Mild and moderate asthma is associated with airway goblet cell hyperplasia and abnormalities in mucin gene expression. Am. J. Respir. Crit. Care Med. 163, 517–523. Paganelli, R., Scala, E., Mezzaroma, I., Pinter, E., D’Offizi, G., Fanales‐Belasio, E., Rosso, R. M., Ansotegui, I. J., Pandolfi, F., and Aiuti, F. (1995). Immunologic aspects of hyperimmunoglobulinemia E‐like syndrome in patients with AIDS. J. Allergy Clin. Immunol. 95, 995–1003. Page, S., Ammit, A. J., Black, J. L., and Armour, C. L. (2001). Human mast cell and airway smooth muscle cell interactions: Implications for asthma. Am. J. Physiol. Lung Cell. Mol. Physiol. 281, 1313–1323. Pandey, V., Mihara, S., Fensome‐Green, A., Bolsover, S., and Cockcroft, S. (2004). Monomeric IgE stimulates NFAT translocation into the nucleus, a rise in cytosol Ca2þ, degranulation, and membrane ruffling in the cultured rat basophilic leukemia‐2H3 mast cell line. J. Immunol. 172, 4048–4058. Patella, V., Casolaro, V., Bjo¨ rck, L., and Marone, G. (1990). Protein L. A bacterial Ig‐binding protein that activates human basophils and mast cells. J. Immunol. 145, 3054–3061.

154

g i a nn i m a r o ne

ET AL.

Patella, V., Bouvet, J. P., and Marone, G. (1993). Protein Fv produced during viral hepatitis is a novel activator of human basophils and mast cells. J. Immunol. 151, 5685–5698. Patella, V., Marino`, I., Lamparter, B., Arbustini, E., Adt, M., and Marone, G. (1995). Human heart mast cells. Isolation, purification, ultrastructure, and immunologic characterization. J. Immunol. 154, 2855–2865. Patella, V., de Crescenzo, G., Marino`, O., Spadaro, G., Genovese, A., and Marone, G. (1996). Oxatomide inhibits the release of proinflammatory mediators from human basophils and mast cells. Int. Arch. Allergy Immunol. 111, 23–29. Patella, V., Giuliano, A., Bouvet, J. P., and Marone, G. (1998a). Endogenous superallergen protein Fv induces IL‐4 secretion from human Fc
RIþ cells through interaction with the VH3 region of IgE. J. Immunol. 161, 5647–5655. Patella, V., Marino`, I., Arbustini, E., Lamparter‐Schummert, B., Verga, L., Adt, M., and Marone, G. (1998b). Stem cell factor in mast cells and increased mast cell density in idiopathic and ischemic cardiomyopathy. Circulation 97, 971–978. Patella, V., Florio, G., Oriente, A., Spadaro, G., Forte, V., Genovese, A., and Marone, G. (2000a). Human mast cells and basophils in immune responses to infectious agents. In ‘‘Mast Cells and Basophils’’ (G. Marone, L. M. Lichtenstein, and S. J. Galli, Eds.), pp. 397–418. Academic Press, San Diego. Patella, V., Florio, G., Petraroli, A., and Marone, G. (2000b). HIV‐1 gp120 induces IL‐4 and IL‐13 release from human Fc
RIþ cells through interaction with the VH3 region of IgE. J. Immunol. 164, 589–595. Pawankar, R., Okuda, M., Yssel, H., Okumura, K., and Ra, C. (1997). Nasal mast cells in perennial allergic rhinitics exhibit increased expression of the Fc
RI, CD40L, IL‐4, and IL‐13, and can induce IgE synthesis in B cells. J. Clin. Invest. 99, 1492–1499. Peachell, P. T., Undem, B. J., Schleimer, R. P., Mac Glashan, D. W., Jr., Lichtenstein, L. M., Cieslinski, L. B., and Torphy, T. J. (1992). Preliminary identification and role of phosphodiesterase isozymes in human basophils. J. Immunol. 148, 2503–2510. Pesci, A., Foresi, A., Bertorelli, G., Chetta, A., and Olivieri, D. (1993). Histochemical characteristics and degranulation of mast cells in epithelium and lamina propria of bronchial biopsies from asthmatic and normal subjects. Am. Rev. Respir. Dis. 147, 684–689. Peters, S. P., Kagey‐Sobotka, A., Mac Glashan, D. W., Jr., and Lichtenstein, L. M. (1984). Effect of prostaglandin D2 in modulating histamine release from human basophils. J. Pharmacol. Exp. Ther. 228, 400–406. Popp, J. W., Jr., Harrist, T. J., Dienstag, J. L., Bhan, A. K., Wands, J. R., La Mont, J. T., and Mihm, M. C., Jr. (1981). Cutaneous vasculitis associated with acute and chronic hepatitis. Arch. Intern. Med. 141, 623–629. Presta, L. G., Chen, H., O’Connor, S. J., Chisholm, V., Meng, Y. G., Krummen, L., Winkler, M., and Ferrara, N. (1997). Humanization of an anti‐vascular endothelial growth factor monoclonal antibody for the therapy of solid tumors and other disorders. Cancer Res. 57, 4593–4599. Prewett, M., Huber, J., Li, Y., Santiago, A., O’Connor, W., King, K., Overholser, J., Hooper, A., Pytowski, B., Witte, L., Bohlen, P., and Hicklin, D. J. (1999). Antivascular endothelial growth factor receptor (fetal liver kinase 1) monoclonal antibody inhibits tumor angiogenesis and growth of several mouse and human tumors. Cancer Res. 59, 5209–5218. Price, K. S., Friend, D. S., Mellor, E. A., De Jesus, N., Watts, G. F., and Boyce, J. A. (2003). CC chemokine receptor 3 mobilizes to the surface of human mast cells and potentiates immunoglobulin E‐dependent generation of interleukin 13. Am. J. Respir. Cell Mol. Biol. 28, 420–427. Prodeus, A. P., Zhou, X., Maurer, M., Galli, S. J., and Carroll, M. C. (1997). Impaired mast cell‐ dependent natural immunity in complement C3‐deficient mice. Nature 390, 172–175.

h u m a n m a s t ce l l s an d bas o p h i l s i n a s t h m a

155

Ramires, R., Caiaffa, M. F., Tursi, A., Haeggstrom, J. Z., and Macchia, L. (2004). Novel inhibitory effect on 5‐lipoxygenase activity by the anti‐asthma drug montelukast. Biochem. Biophys. Res. Commun. 324, 815–821. Rancinan, C., Morlat, P., Chene, G., Guez, S., Baquey, A., Beylot, J., and Salamon, R. (1998). IgE serum level: A prognostic marker for AIDS in HIV‐infected adults? J. Allergy Clin. Immunol. 102, 329–330. Redrup, A. C., Howard, B. P., Mac Glashan, D. W., Jr., Kagey‐Sobotka, A., Lichtenstein, L. M., and Schroeder, J. T. (1998). Differential regulation of IL‐4 and IL‐13 secretion by human basophils: their relationship to histamine release in mixed leukocyte cultures. J. Immunol. 160, 1957–1964. Ribatti, D., Polimeno, G., Vacca, A., Marzullo, A., Crivellato, E., Nico, B., Lucarelli, G., and Dammacco, F. (2002). Correlation of bone marrow angiogenesis and mast cells with tryptase activity in myelodysplastic syndromes. Leukemia 16, 1680–1684. Ricci, S., Medaglini, D., Marcotte, H., Olsen, A., Pozzi, G., and Bjorck, L. (2001). Immunoglobulin‐binding domains of peptostreptococcal protein L enhance vaginal colonization of mice by Streptococcus gordonii. Microb. Pathog. 30, 229–235. Roche, W. R., Beasley, R., Williams, J. H., and Holgate, S. T. (1989). Subepithelial fibrosis in the bronchi of asthmatics. Lancet 1, 520–524. Romagnani, P., de Paulis, A., Beltrame, C., Annunziato, F., Dente, V., Maggi, E., Romagnani, S., and Marone, G. (1999). Tryptase‐chymase double‐positive human mast cells express the eotaxin receptor CCR3 and are attracted by CCR3‐binding chemokines. Am. J. Pathol. 155, 1195–1204. Romagnani, P., de Paulis, A., Beltrame, C., Marone, G., and Romagnani, S. (2000). Chemokine receptors on human mast cells. In ‘‘Mast Cells and Basophils’’ (G. Marone, L. M. Lichtenstein, and S. J. Galli, Eds.), pp. 579–596. Academic Press, San Diego. Romagnani, P., Lasagni, L., Annunziato, F., Serio, M., and Romagnani, S. (2004). CXC chemokines: the regulatory link between inflammation and angiogenesis. Trends Immunol. 25, 201–209. Romagnani, S. (2004). The increased prevalence of allergy and the hygiene hypothesis: Missing immune deviation, reduced immune suppression, or both? Immunology 112, 352–363. Roquet, A., Dahlen, B., Kumlin, M., Ihre, E., Anstren, G., Binks, S., and Dahlen, S. E. (1997). Combined antagonism of leukotrienes and histamine produces predominant inhibition of allergen‐induced early and late phase airway obstruction in asthmatics. Am. J. Respir. Crit. Care Med. 155, 1856–1863. Rottem, M., Okada, T., Goff, J. P., and Metcalfe, D. D. (1994). Mast cells cultured from the peripheral blood of normal donors and patients with mastocytosis originate from a CD34þ/Fc
RI‐ cell population. Blood 84, 2489–2496. Rumsaeng, V., Cruikshank, W. W., Foster, B., Prussin, C., Kirshenbaum, A. S., Davis, T. A., Kornfeld, H., Center, D. M., and Metcalfe, D. D. (1997). Human mast cells produce the CD4þ T lymphocyte chemoattractant factor, IL‐16. J. Immunol. 159, 2904–2910. Ruoss, S. J., Hartmann, T., and Caughey, G. H. (1991). Mast cell tryptase is a mitogen for cultured fibroblasts. J. Clin. Invest. 88, 493–499. Russel, J. D., Russell, S. B., and Trupin, K. M. (1977). The effect of histamine on the growth of cultured fibroblasts isolated from normal and keloid tissue. J. Cell. Physiol. 93, 389–393. Ruzicka, T., Bieber, T., Schopf, E., Rubins, A., Dobozy, A., Bos, J. D., Jablonska, S., Ahmed, I., Thestrup‐Pedersen, K., Daniel, F., Finzi, A., and Reitamo, S. (1997). A short‐term trial of tacrolimus ointment for atopic dermatitis. European Tacrolimus Multicenter Atopic Dermatitis Study Group. N. Engl. J. Med. 337, 816–821. Sadakane, K., Ichinose, T., Takano, H., Yanagisawa, R., Sagai, M., Yoshikawa, T., and Shibamoto, T. (2002). Murine strain differences in airway inflammation induced by diesel exhaust particles and house dust mite allergen. Int. Arch. Allergy Immunol. 128, 220–228.

156

g i a nn i m a r o ne

ET AL.

Saito, H., Hatake, K., Dvorak, A. M., Leiferman, K. M., Donnenberg, A. D., Arai, N., Ishizaka, K., and Ishizaka, T. (1988). Selective differentiation and proliferation of hematopoietic cells induced by recombinant human interleukins. Proc. Natl. Acad. Sci. USA 85, 2288–2292. Sallusto, F., Mackay, C. R., and Lanzavecchia, A. (1997). Selective expression of the eotaxin receptor CCR3 by human T helper 2 cells. Science 277, 2005–2007. Salvato, G. (2001). Quantitative and morphological analysis of the vascular bed in bronchial biopsy specimens from asthmatic and non‐asthmatic subjects. Thorax 56, 902–906. Sanak, M., Pierzchalska, M., Bazan‐Socha, S., and Szczeklik, A. (2000). Enhanced expression of the leukotriene C4 synthase due to overactive transcription of an allelic variant associated with aspirin‐intolerant asthma. Am. J. Respir. Cell Mol. Biol. 23, 290–296. Schechter, N. M., Irani, A. M., Sprows, J. L., Abernethy, J., Wintroub, B., and Schwartz, L. B. (1990). Identification of a cathepsin G‐like proteinase in the MCTC type of human mast cell. J. Immunol. 145, 2652–2661. Schechter, N. M., Barbosa Pereira, P. J., and Strobl, S. (2000). Structure and function of human chymase. In ‘‘Mast Cells and Basophils’’ (G. Marone, L. M. Lichtenstein, and S. J. Galli, Eds.), pp. 275–290. Academic Press, San Diego. Schleimer, R. P., Lichtenstein, L. M., and Gillespie, E. (1981). Inhibition of basophil histamine release by anti‐inflammatory steroids. Nature 292, 454–455. Schleimer, R. P., Schulman, E. S., Mac Glashan, D. W., Jr., Peters, S. P., Hayes, E. C., Adams, G. K., 3rd, Lichtenstein, L. M., and Adkinson, N. F., Jr. (1983). Effects of dexamethasone on mediator release from human lung fragments and purified human lung mast cells. J. Clin. Invest. 71, 1830–1835. Schleimer, R. P., Fox, C. C., Naclerio, R. M., Plaut, M., Creticos, P. S., Togias, A. G., Warner, J. A., Kagey‐Sobotka, A., and Lichtenstein, L. M. (1985). Role of human basophils and mast cells in the pathogenesis of allergic diseases. J. Allergy Clin. Immunol. 76, 369–374. Schroeder, J. T., Mac Glashan, D. W., Jr., Mac Donald, S. M., Kagey‐Sobotka, A., and Lichtenstein, L. M. (1997). Regulation of IgE‐dependent IL‐4 generation by human basophils treated with glucocorticoids. J. Immunol. 158, 5448–5454. Schroeder, J. T., Schleimer, R. P., Lichtenstein, L. M., and Kreutner, W. (2001). Inhibition of cytokine generation and mediator release by human basophils treated with desloratadine. Clin. Exp. Allergy 31, 1369–1377. Schuh, J. M., Power, C. A., Proudfoot, A. E., Kunkel, S. L., Lukacs, N. W., and Hogaboam, C. M. (2002). Airway hyperresponsiveness, but not airway remodeling, is attenuated during chronic pulmonary allergic responses to Aspergillus in CCR4‐/‐ mice. FASEB J. 16, 1313–1315. Segool, R. A., Lejtenyi, C., and Taussig, L. M. (1975). Articular and cutaneous prodromal manifestations of viral hepatitis. J. Pediatr. 87, 709–712. Sillaber, C., Baghestanian, M., Hofbauer, R., Virgolini, I., Bankl, H. C., Fureder, W., Agis, H., Willheim, M., Leimer, M., Scheiner, O., Binder, B. R., Kiener, H. P., Bevec, D., Fritsch, G., Majdic, O., Kress, H. G., Gadner, H., Lechner, K., and Valent, P. (1997). Molecular and functional characterization of the urokinase receptor on human mast cells. J. Biol. Chem. 272, 7824–7832. Sillaber, C., Geissler, K., Scherrer, R., Kaltenbrunner, R., Bettelheim, P., Lechner, K., and Valent, P. (1992). Type b transforming growth factors promote interleukin‐3 (IL‐3)‐dependent differentiation of human basophils but inhibit IL‐3‐dependent differentiation of human eosinophils. Blood 80, 634–641. Silverman, G. J. (1997). B‐cell superantigens. Immunol. Today 18, 379–386. Silverman, G. J. (1998). B‐cell superantigens: Possible roles in immunodeficiency and autoimmunity. Semin. Immunol. 10, 43–55.

h u m a n m a s t ce l l s an d bas o p h i l s i n a s t h m a

157

Sin, A. Z., Roche, E. M., Togias, A., Lichtenstein, L. M., and Schroeder, J. T. (2001). Nerve growth factor or IL‐3 induces more IL‐13 production from basophils of allergic subjects than from basophils of nonallergic subjects. J. Allergy Clin. Immunol. 108, 387–393. Siraganian, R. P. (2003). Mast cell signal transduction from the high‐affinity IgE receptor. Curr. Opin. Immunol. 15, 639–646. Sloane, D. E., Tedla, N., Awoniyi, M., Mac Glashan, D. W., Jr., Borges, L., Austen, K. F., and Arm, J. P. (2004). Leukocyte immunoglobulin‐like receptors: Novel innate receptors for human basophil activation and inhibition. Blood 104, 2832–2839. Smith, S. J., Piliponsky, A. M., Rosenhead, F., Elchalal, U., Nagler, A., and Levi‐Schaffer, F. (2002). Dexamethasone inhibits maturation, cytokine production and Fc
RI expression of human cord blood‐derived mast cells. Clin. Exp. Allergy 32, 906–913. Soter, N. A., Lewis, R. A., Corey, E. J., and Austen, K. F. (1983). Local effects of synthetic leukotrienes (LTC4, LTD4, LTE4, and LTB4) in human skin. J. Invest. Dermatol. 80, 115–119. Standaert, R. F., Galat, A., Verdine, G. L., and Schreiber, S. L. (1990). Molecular cloning and overexpression of the human FK506‐binding protein FKBP. Nature 346, 671–674. Stellato, C., de Paulis, A., Ciccarelli, A., Cirillo, R., Patella, V., Casolaro, V., and Marone, G. (1992). Anti‐inflammatory effect of cyclosporin A on human skin mast cells. J. Invest. Dermatol. 98, 800–804. Stellato, C., Collins, P., Ponath, P. D., Soler, D., Newman, W., La Rosa, G., Li, H., White, J., Schwiebert, L. M., Bickel, C., Liu, M., Bochner, B. S., Williams, T., and Schleimer, R. P. (1997). Production of the novel C‐C chemokine MCP‐4 by airway cells and comparison of its biological activity to other C‐C chemokines. J. Clin. Invest. 99, 926–936. Stevens, R. L., Yang, Y., and Li, L. (2004). RasGRP4 is a lineage‐restricted, guanine nucleotide exchange factor/phorbol ester receptor that controls mast cell development. In ‘‘Asthma and Allergic Diseases. Physiology, Immunopharmacology and Treatment’’ (G. Marone, K. F. Austen, S. T. Holgate, A. B. Kay, and L. M. Lichtenstein, Eds.), pp. 102–110. Blackwell, London. Suh, Y. J., Yoon, S. H., Sampson, A. P., Kim, H. J., Kim, S. H., Nahm, D. H., Suh, C. H., and Park, H. S. (2004). Specific immunoglobulin E for staphylococcal enterotoxins in nasal polyps from patients with aspirin‐intolerant asthma. Clin. Exp. Allergy 34, 1270–1275. Supajatura, V., Ushio, H., Nakao, A., Okumura, K., Ra, C., and Ogawa, H. (2001). Protective roles of mast cells against enterobacterial infection are mediated by Toll‐like receptor 4. J. Immunol. 167, 2250–2256. Supajatura, V., Ushio, H., Nakao, A., Akira, S., Okumura, K., Ra, C., and Ogawa, H. (2002). Differential responses of mast cell Toll‐like receptors 2 and 4 in allergy and innate immunity. J. Clin. Invest. 109, 1351–1359. Takeda, K., Hamelmann, E., Joetham, A., Shultz, L. D., Larsen, G. L., Irvin, C. G., and Gelfand, E. W. (1997). Development of eosinophilic airway inflammation and airway hyperresponsiveness in mast cell‐deficient mice. J. Exp. Med. 186, 449–454. Takeuchi, K., Koike, K., Kamijo, T., Ishida, S., Nakazawa, Y., Kurokawa, Y., Sakashita, K., Kinoshita, T., Matsuzawa, S., Shiohara, M., Yamashita, T., Nakajima, M., and Komiyama, A. (2003). STI571 inhibits growth and adhesion of human mast cells in culture. J. Leukoc. Biol. 74, 1026–1034. Tanaka, S., Takasu, Y., Mikura, S., Satoh, N., and Ichikawa, A. (2002). Antigen‐independent induction of histamine synthesis by immunoglobulin E in mouse bone marrow‐derived mast cells. J. Exp. Med. 196, 229–235. Tkaczyk, C., Okayama, Y., Metcalfe, D. D., and Gilfillan, A. M. (2004). Fcg receptors on mast cells: Activatory and inhibitory regulation of mediator release. Int. Arch. Allergy Immunol. 133, 305–315.

158

g i a nn i m a r o ne

ET AL.

Torphy, T. J., Livi, G. P., Balcarek, J. M., White, J. R., Chilton, F. H., and Undem, B. J. (1992). Therapeutic potential of isozyme‐selective phosphodiesterase inhibitors in the treatment of asthma. Adv. Second Messenger Phosphoprotein Res. 25, 289–305. Townsley‐Fuchs, J., Neshat, M. S., Margolin, D. H., Braun, J., and Goodglick, L. (1997). HIV‐1 gp120: A novel viral B cell superantigen. Int. Rev. Immunol. 14, 325–338. Trautmann, A., Krohne, G., Brocker, E. B., and Klein, C. E. (1998). Human mast cells augment fibroblast proliferation by heterotypic cell–cell adhesion and action of IL‐4. J. Immunol. 160, 5053–5057. Triggiani, M., Hubbard, W. C., and Chilton, F. H. (1990). Synthesis of 1‐acyl‐2‐acetyl‐sn‐glycero‐3‐ phosphocholine by an enriched preparation of the human lung mast cell. J. Immunol. 144, 4773–4780. Triggiani, M., Schleimer, R. P., Warner, J. A., and Chilton, F. H. (1991). Differential synthesis of 1‐acyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine and platelet‐activating factor by human inflammatory cells. J. Immunol. 147, 660–666. Triggiani, M., Oriente, A., de Crescenzo, G., and Marone, G. (1995a). Metabolism of lipid mediators in human basophils and mast cells. Chem. Immunol. 61, 135–147. Triggiani, M., Oriente, A., de Crescenzo, G., Rossi, G., and Marone, G. (1995b). Biochemical functions of a pool of arachidonic acid associated with triglycerides in human inflammatory cells. Int. Arch. Allergy Immunol. 107, 261–263. Triggiani, M., Gentile, M., Secondo, A., Granata, F., Oriente, A., Taglialatela, M., Annunziato, L., and Marone, G. (2001). Histamine induces exocytosis and IL‐6 production from human lung macrophages through interaction with H1 receptors. J. Immunol. 166, 4083–4091. Triggiani, M., Giannattasio, G., Balestrieri, B., Granata, F., Gelb, M. H., de Paulis, A., and Marone, G. (2004a). Differential modulation of mediator release from human basophils and mast cells by mizolastine. Clin. Exp. Allergy 34, 241–249. Triggiani, M., Giannattasio, G., Balestrieri, B., Loffredo, S., Forte, V., and Granata, F. (2004b). Phenotypical and functional heterogeneity of human lung macrophages. In ‘‘Asthma and Allergic Diseases. Physiology, Immunopharmacology and Treatment’’ (G. Marone, K. F. Austen, S. T. Holgate, A. B. Kay, and L. M. Lichtenstein, Eds.), pp. 129–134. Blackwell, London. Tsai, M., Shih, L. S., Newlands, G. F., Takeishi, T., Langley, K. E., Zsebo, K. M., Miller, H. R., Geissler, E. N., and Galli, S. J. (1991). The rat c‐kit ligand, stem cell factor, induces the development of connective tissue‐type and mucosal mast cells in vivo. Analysis by anatomical distribution, histochemistry, and protease phenotype. J. Exp. Med. 174, 125–131. Uguccioni, M., Mackay, C. R., Ochensberger, B., Loetscher, P., Rhis, S., La Rosa, G. J., Rao, P., Ponath, P. D., Baggiolini, M., and Dahinden, C. A. (1997). High expression of the chemokine receptor CCR3 in human blood basophils. Role in activation by eotaxin, MCP‐4, and other chemokines. J. Clin. Invest. 100, 1137–1143. Upton, M. N., McConnachie, A., McSharry, C., Hart, C. L., Smith, G. D., Gillis, C. R., and Watt, G. C. (2000). Intergenerational 20 year trends in the prevalence of asthma and hay fever in adults: The Midspan family study surveys of parents and offspring. BMJ 321, 88–92. Vaida, G. A., Goldman, M. A., and Bloch, K. J. (1983). Testing for hepatitis B virus in patients with chronic urticaria and angioedema. J. Allergy Clin. Immunol. 72, 193–198. Valent, P., Schmidt, G., Besemer, J., Mayer, P., Zenke, G., Liehl, E., Hinterberger, W., Lechner, K., Maurer, D., and Bettelheim, P. (1989). Interleukin‐3 is a differentiation factor for human basophils. Blood 73, 1763–1769. Valent, P., Besemer, J., Kishi, K., Kaltenbrunner, R., Kuhn, B., Maurer, D., Lechner, K., and Bettelheim, P. (1990). IL‐3 promotes basophilic differentiation of KU812 cells through high affinity binding sites. J. Immunol. 145, 1885–1889.

h u m a n m a s t ce l l s an d bas o p h i l s i n a s t h m a

159

Valent, P., Spanblo¨ chl, E., Sperr, W. R., Sillaber, C., Zsebo, K. M., Agis, H., Strobl, H., Geissler, K., Bettelheim, P., and Lechner, K. (1992). Induction of differentiation of human mast cells from bone marrow and peripheral blood mononuclear cells by recombinant human stem cell factor/ kit‐ligand in long‐term culture. Blood 80, 2237–2245. Van Epps, E., Husby, G., Williams, R. C., Jr., and Strickland, R. G. (1976). Liver disease—a prominent cause of serum IgE elevation. Clin. Exp. Immunol. 23, 444–450. Van Sambeek, R., Stevenson, D. D., Baldasaro, M., Lam, B. K., Zhao, J., Yoshida, S., Yandora, C., Drazen, J. M., and Penrose, J. F. (2000). 50 flanking region polymorphism of the gene encoding leukotriene C4 synthase does not correlate with the aspirin‐intolerant asthma phenotype in the United States. J. Allergy Clin. Immunol. 106, 72–76. Vigano`, A., Principi, N., Crupi, L., Onorato, J., Vincenzo, Z. G., and Salvaggio, A. (1995). Elevation of IgE in HIV‐infected children and its correlation with the progression of disease. J. Allergy Clin. Immunol. 95, 627–632. Wide, L., Bennich, H., and Johansson, S. G. (1967). Diagnosis of allergy by an in vitro test for allergen antibodies. Lancet 2, 1105–1107. Williams, C. M., and Galli, S. J. (2000). Mast cells can amplify airway reactivity and features of chronic inflammation in an asthma model in mice. J. Exp. Med. 192, 455–462. Williams, H. C. (1992). Is the prevalence of atopic dermatitis increasing? Clin. Exp. Dermatol. 17, 385–391. Wills‐Karp, M., and Ewart, S. L. (2004). Time to draw breath: Asthma‐susceptibility genes are identified. Nat. Rev. Genet. 5, 376–387. Wills‐Karp, M., Luyimbazi, J., Xu, X., Schofield, B., Neben, T. Y., Karp, C. L., and Donaldson, D. D. (1998). Interleukin‐13: Central mediator of allergic asthma. Science 282, 2258–2261. Wood, J. M., Bold, G., Buchdunger, E., Cozens, R., Ferrari, S., Frei, J., Hofmann, F., Mestan, J., Mett, H., O’Reilly, T., Persohn, E., Rosel, J., Schnell, C., Stover, D., Theuer, A., Towbin, H., Wenger, F., Woods‐Cook, K., Menrad, A., Siemeister, G., Schirner, M., Thierauch, K. H., Schneider, M. R., Drevs, J., Martiny‐Baron, G., and Totzke, F. (2000). PTK787/ZK 222584, a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor‐induced responses and tumor growth after oral administration. Cancer Res. 60, 2178–2189. Wright, D. N., Nelson, R. P., Jr., Ledford, D. K., Fernandez‐Caldas, E., Trudeau, W. L., and Lockey, R. F. (1990). Serum IgE and human immunodeficiency virus (HIV) infection. J. Allergy Clin. Immunol. 85, 445–452. Wymann, M. P., Zvelebil, M., and Laffargue, M. (2003). Phosphoinositide 3‐kinase signaling— which way to target? Trends Pharmacol. Sci. 24, 366–376. Yano, K., Yamaguchi, M., de Mora, F., Lantz, C. S., Butterfield, J. H., Costa, J. J., and Galli, S. J. (1997). Production of macrophage inflammatory protein‐1a by human mast cells: Increased anti‐IgE‐dependent secretion after IgE‐dependent enhancement of mast cell IgE‐binding ability. Lab. Invest. 77, 185–193. Ying, S., Robinson, D. S., Meng, Q., Barata, L. T., McEuen, A. R., Buckley, M. G., Walls, A. F., Askenase, P. W., and Kay, A. B. (1999). C‐C chemokines in allergen‐induced late‐phase cutaneous responses in atopic subjects: Association of eotaxin with early 6‐hour eosinophils, and of eotaxin‐2 and monocyte chemoattractant protein‐4 with the later 24‐hour tissue eosinophilia, and relationship to basophils and other C‐C chemokines (monocyte chemoattractant protein‐3 and RANTES). J. Immunol. 163, 3976–3984. Yoshimoto, T., Tsutsui, H., Tominaga, K., Hoshino, K., Okamura, H., Akira, S., Paul, W. E., and Nakanishi, K. (1999). IL‐18, although antiallergic when administered with IL‐12, stimulates IL‐4 and histamine release by basophils. Proc. Natl. Acad. Sci. USA 96, 13962–13966.

160

g i a nn i m a r o ne

ET AL.

Yu, C. K., and Chen, C. L. (2003). Activation of mast cells is essential for development of house dust mite Dermatophagoides farinae‐induced allergic airway inflammation in mice. J. Immunol. 171, 3808–3815. Yuyama, N., Davies, D. E., Akaiwa, M., Matsui, K., Hamasaki, Y., Suminami, Y., Yoshida, N. L., Maeda, M., Pandit, A., Lordan, J. L., Kamogawa, Y., Arima, K., Nagumo, F., Sugimachi, M., Berger, A., Richards, I., Roberds, S. L., Yamashita, T., Kishi, F., Kato, H., Arai, K., Ohshima, K., Tadano, J., Hamasaki, N., Miyatake, S., Sugita, Y., Holgate, S. T., and Izuhara, K. (2002). Analysis of novel disease‐related genes in bronchial asthma. Cytokine 19, 287–296. Zhang, S., Anderson, D. F., Bradding, P., Coward, W. R., Baddeley, S. M., Mac Leod, J. D., McGill, J. I., Church, M. K., Holgate, S. T., and Roche, W. R. (1998). Human mast cells express stem cell factor. J. Pathol. 186, 59–66. Zhu, Z., Homer, R. J., Wang, Z., Chen, Q., Geba, G. P., Wang, J., Zhang, Y., and Elias, J. A. (1999). Pulmonary expression of IL‐13 causes inflammation, mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and eotaxin production. J. Clin. Invest. 103, 779–788.

A Novel Recognition System for MHC Class I Molecules Constituted by PIR Toshiyuki Takai Department of Experimental Immunology and CREST Program of the Japan Science and Technology Agency, Institute of Development, Aging, and Cancer, Tohoku University, Sendai 980‐8575, Japan

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Abstract ........................................................................................................... Introduction ..................................................................................................... PIR and LILR in Ig‐like Receptor Family .............................................................. PIR Genes and Protein Structure ......................................................................... Ligand for PIR.................................................................................................. 3D Structure of PIR and LILR and Their Interaction with MHC Class I..................... Activation Signal via PIR‐A.................................................................................. Inhibitory Signal via PIR‐B.................................................................................. Dominant Expression of PIR‐B In Vivo ................................................................. Pirb–/– B Cells, Neutrophils, and Macrophages are Hyperresponsive ........................... Modulated Cytokine Signaling in PIR‐B Deficiency ................................................. PIR in Transplantation........................................................................................ Conclusion ....................................................................................................... References .......................................................................................................

161 161 163 166 169 171 174 176 177 178 180 182 186 187

Abstract The paired immunoglobulin (Ig)‐like receptors (PIRs) represent a typical receptor pair of the Ig‐like receptor family in which various combinations of ligand‐ receptor interaction provide a positive and negative regulation of immune cells, thus enabling those cells to respond properly to extrinsic stimuli. Activating PIR‐A and inhibitory PIR‐B are expressed in a wide range of cells in the murine immune system, such as B cells, mast cells, macrophages, and dendritic cells, mostly in a pair‐wise fashion. PIRs bind to MHC class I molecules expressed ubiquitously on hematopoietic as well as nonhematopoietic cells. The unbalanced binding of PIR‐A and PIR‐B to MHC class I molecules may lead to the perturbation of cell development, regulation, and function as observed in PIR‐ B‐deficient mice. Thus, PIR‐A and PIR‐B are indispensable for the regulation of cellular signaling and important for homeostasis of the immune system. 1. Introduction The immune system possesses two types of major histocompatibility complex (MHC)‐restricted self‐recognition molecules, namely the T‐cell receptor (TCR) of T cells and killer‐cell receptor of NK cells (Fig. 1). These receptors

161 advances in immunology, vol. 88 # 2005 Elsevier Inc. All rights reserved.

0065-2776/05 $35.00 DOI: 10.1016/S0065-2776(05)88005-8

162 Figure 1 Schematic structures of MHC class I molecules and the related proteins expressed on human and mouse T cells and NK cells. Structures of classical and nonclassical MHC class I molecules and class I‐like molecules (top panel) and the receptors for them (bottom panel) are shown. These TCR and killer‐cell receptors constitute unique recognition system for normal or ‘‘self’’ or ‘‘self with stress’’ such as a viral infection. Classical MHC class I molecules with peptides are ubiquitously distributed on various hematopoietic and nonhematopoietic cells, whereas the expression and the associating peptide of nonclassical MHC class I molecules are restricted. Expression of MHC class I‐related proteins is induced with various stress such as a viral infection. ULBP, UL16‐binding protein; MIC, MHC class I‐related chain.

r ec o g n i t i o n sys t em o f m h c cl a s s i m o l e cu l e s

163

allow a positive and negative selection for T cells in the adaptive immunity and control the activation against nonself cells for NK cells in the innate immunity as well as in feto–maternal immunity, respectively. In the TCR‐mediated self‐ recognition, MHC class I and class II molecules presenting self‐peptides on thymic stromal cells are utilized for the positive selection of T cells, with TCR possessing a weak reactivity to self, while the negative selection process eliminates most autoreactive T cells with TCR having a strong reactivity to self. T cells with no reactivity to MHC class I or class II molecules are thought to die from neglect. In the periphery, the engagement of TCRs on cytotoxic T cells (CTLs) with MHC class I with a nonself peptide, or an allogeneic MHC class I expressed by target cells triggers the elimination of these cells by CTLs. Conversely, in killer cell receptor‐mediated self‐recognition, inhibitory killer receptors expressed on NK cells, such as human killer immunoglobulin (Ig)‐ like receptor (KIR)2DL and murine Ly49A (Fig. 1), recognize MHC class I molecules expressed by self‐cells, thereby avoiding damage to self tissues (Cerwenka and Lanier, 2001; Ka¨rre, 2002; Lanier and Bakker, 2000; Ravetch and Lanier, 2000). Recently, it has become increasingly evident that mammalian B cells and myeloid cells possess a third self‐recognizing system, which may constitutively regulate these cells. In this review, the nature of this novel MHC class I‐recognition system composed of murine paired Ig‐like receptors (PIRs) expressed on B cells and myeloid cells is discussed regarding their physiological and pathological significance, and is compared with those of their close relatives or orthologs in humans, the leukocyte Ig‐like receptors (LILRs). 2. PIR and LILR in Ig‐like Receptor Family PIRs were first identified as those homologous to the human Fc receptor (FcR) for IgA, FcaRI. In 1997, a study reported the isolation of several cDNA clones coding for a novel molecule from a B10.A mouse macrophage cDNA library during the course of the experiments to obtain a hypothetical murine counterpart of FcaRI. The gene product was initially designated as p91 due to the calculated molecular weight of the mature polypeptide backbone (Hayami et al., 1997). A similar approach (Kubagawa et al., 1997) yielded a novel gene family, with the members constituting a set of PIR‐A and PIR‐B genes in the BALB/c splenic library. As p91 and PIR‐B were the most plausibly identical molecules based on their 98% sequence identity, the nomenclature has been standardized as PIR to avoid confusion (Yamashita et al., 1998b) (Fig. 2). PIR‐B contains immunoreceptor tyrosine‐based inhibitory motifs (ITIMs) in its cytoplasmic portion and can inhibit receptor‐mediated activation signaling in vitro upon cellular engagement with other activating‐type receptors, such as the B cell antigen receptor (BCR) (see following) (Ble´ry et al., 1998; Maeda et al., 1998b; Yamashita et al., 1998b).

164

t o s h i y u k i tak a i

r ec o g n i t i o n sys t em o f m h c cl a s s i m o l e cu l e s

165

Initial studies by the groups of Takai and Cooper could not detect PIR binding to IgA or other immunoglobulins (Hayami et al., 1997; Kubagawa et al., 1997), suggesting that they are not receptors for immunoglobulins. Instead, PIR‐A and PIR‐B are now proposed as close relatives or orthologs of human LILR, also termed Ig‐like transcripts/leukocyte Ig‐like receptors/ monocyte/macrophage Ig‐related receptors (ILT/LIR/MIR), based on their similarities in structure, expression profiles, and genomic localization (Hayami et al., 1997; Kubagawa et al., 1997; Takai and Ono, 2001; Wende et al., 1999; Yamashita et al., 1998a). The analogy of PIR‐B to the inhibitory isoform of LILR, namely LILRB, and the findings that constitutive phosphorylation of PIR‐B in splenocytes was reduced in b2‐microglobulin (b2M)‐deficient (B2m–/–) mice (Ho et al., 1999), led to the notion that PIR‐B may recognize classical or nonclassical MHC class I molecule(s) (Ho et al., 1999; Takai and Ono, 2001). The observation that human HLA‐G, a nonclassical MHC class I molecule expressed on fetal trophoblast cells (Fig. 1) binds to murine PIR‐B (Liang et al., 2002) supports this notion. Research has shown that, in fact, the extracellular portion of PIR can bind to murine MHC class I molecules both in vitro and in vivo (Nakamura et al., 2004). LILRs expressed on various human lymphoid and myeloid cells have been shown to bind to MHC class I or related molecules (Borges and Cosman, 2000; Colonna et al., 1999b; Long, 1999) (Fig. 2). LILRs comprise at least five inhibitory (LILRB‐1–5) and three activating (LILRA1–3) receptors characterized by either two or four extracellular C2‐type Ig‐like domains (Colonna and Samaridis, 1995). LILRAs deliver activating signals through the associating FcR common g subunit (FcRg) that harbors an immunoreceptor tyrosine‐based activation motif (ITAM) (Nakajima et al., 1999). The engagement of LILRB1 or LILRB2 and the inhibitory LILRs harboring ITIMs in their cytoplasmic tails with MHC class I delivers a negative signal that downregulates CD40 ligand‐mediated secretion of various cytokines such as IL‐1a, TNF‐a, and IL‐6 by myelomonocytic cells (Colonna et al., 1997; 1998; 1999a; 1999b). More

Figure 2 Schematic structures of PIR‐A and PIR‐B and their relatives or related molecules, their ligands, and distribution. Activating Ig‐like receptors with one or more Ig‐like domains (orange sphere) have a positively charged amino acid, Arg, Lys, or His, (not shown in the figure) in their transmembrane domains, which is involved in their interaction with negatively charged Asp residues (not shown) of homodimeric FcRg, DAP12, or CD3z having ITAMs (red barrel). In contrast, inhibitory Ig‐like receptors have one or more ITIM or ITIM‐like sequences (green barrel) in their cytoplasmic portions important for SHP‐1 or SHIP‐1 recruitment upon tyrosine phosphorylation. GenBank accession numbers for PIR: U83172, AF040946–040953 and AF041035–041036 (as p91), and U96682–96693. Repertoires of LILR are more complicated than those illustrated here (see http://www.gene.ucl.ac.uk/nomenclature/genefamily/lilr.html).

166

t o s h i y u k i tak a i

recently, LILRB2 and LILRB4 have been suggested to be involved in graft survival in allogeneic heart transplants (Chang et al., 2002; Manavalan et al., 2003). However, many aspects of the physiological and pathological relevance of human LILRs remain to be verified, mainly due to the absence of a suitable animal model. Many receptors similar to PIRs and LILRs have been identified, with many comprising the activating and inhibitory paired‐receptors, whose recognition of each ligand is supposed or verified to control intracellular signaling of hematopoietic cells (Fig. 2). These include human and murine FcRs for IgG (FcgRs) (Takai, 2002), myeloid‐associated Ig‐like receptor (MAIR)‐I and MAIR‐II (CMRF orthologs) (Yotsumoto et al., 2003), signal regulatory protein (SIRP) a and SIRPb1 (Brooke et al., 2004), leukocyte‐associated Ig‐like receptor (LAIR) (Lebbink et al., 2004), triggering receptors expressed in myeloid cells (TREM) (Colonna, 2003), KIR2DS and KIR2DL (Hoelsbrekken et al., 2003), murine gp49A and gp49B1 (Castells et al., 2001; Lee et al., 2000), human CMRF‐35A and CMRF‐35H (Clark et al., 2000), NKp46 (Mandelboim et al., 2001), NKp44 (Moretta and Moretta, 2004), and paired Ig‐like type 2 receptor (PILR) a and PILRb (Fournier et al., 2000; Mousseau et al., 2000; Shiratori et al., 2004) (Fig. 2). Some of the signaling and regulatory aspects of these receptors have been reviewed in detail elsewhere (Colonna, 2003; Colonna et al., 1999a,b; Daheshia et al., 2001; Long, 1999; Mandelboim and Porgador, 2001; Moretta and Moretta, 2004; Oshima et al., 2002; Ravetch and Lanier, 2000; Takai, 2002; Takai and Ono, 2001; van der Berg et al., 2004). 3. PIR Genes and Protein Structure By employing molecular cloning, one study (Yamashita et al., 1998a) attempted to isolate human homologs of PIR by screening a human splenocyte cDNA library with a mouse PIR as a probe. They characterized several positive clones for the hybridization using a probe corresponding to a common extracellular portion of PIR. Homology search indicated that these sequences were nearly identical to those of human LILRs, again supporting the notion that the murine PIR and human LILR gene families are close relatives or orthologs. The PIR genes are located at the proximal end of the mouse chromosome 7 (Kubagawa et al., 1997; Yamashita et al., 1998a) (Fig. 3A). By searching the mouse genome database and molecular cloning of genes, at least six PIR‐A genes (Pira) and a single PIR‐B gene (Pirb) have been identified in this region (Lebbink et al., 2004; Tun et al., 2003), which is a syntenic position of human chromosome 19q13.3–13.4 harboring the leukocyte receptor complex (LRC). In LRC, genes for LILRs, LAIRs, and KIRs have been mapped (Colonna et al., 1997; Wende et al., 1999; Wilson et al., 2000). Sequencing of several

r ec o g n i t i o n sys t em o f m h c cl a s s i m o l e cu l e s

167

Figure 3 Pira/Pirb locus, Schematic structures of genomic DNA and diagram of the predicted protein domains of PIRs. (A) Initial studies identified the Pir locus on centromeric region of mouse chromosome 7 by FISH analysis as well as by the analysis of segregation patterns of the PIR genes with microsatellite DNA markers in the backcross mice (Kubagawa et al., 1997; Yamashita et al., 1998a). Recombination distances between loci are shown in centiMorgan (cM). Mouse genome data analysis roughly identified the number of PIR genes (open arrowhead), the direction of transcription, and the relative positions to other genes (closed arrowhead) (modified from Lebbink et al., 2004; Tun et al., 2003). The maps are not to scale. RPS9, ribosomal protein S9; Ttyh1, Tweety homologue 1; LENG, LRC‐encoded novel gene; BORG, binder of Rho GTPase; Ly94, lymphocyte antigen 94 (or natural cytotoxicity triggering receptor 1 or NCR‐1); GP‐VI, platelet glycoprotein VI. (B) Schematic representation of the exon/intron structures of PIR‐B and PIR‐A genes (Pirb and Pira, respectively) and the corresponding protein domains are shown. The protein‐coding

168

t o s h i y u k i tak a i

different genes for PIRs from some strains of mice (Chen et al., 2000; Kubagawa et al., 1997; Tun et al., 2003; Yamashita et al., 1998a) yielded the notion that the Pirb gene from 129/Sv and BALB/c mice is composed of 15 exons coding for a leader peptide, extracellular Ig‐like domains, pretransmembrane and transmembrane domains, and a cytoplasmic portion (Fig. 3B). On the other hand, Pira genes from 129/Sv and 129/Ola harbor nine or 10 exons with similar compositions of the Pirb gene, with the last exon coding for the cytoplasmic portion being significantly short (Fig. 3B). Considering the gene evolution of PIR, it is necessary to examine the PIR from other species. Structures of rat PIR‐A and PIR‐B homologs (Dennis et al., 1999) (Fig. 2) were quite similar to those of the respective mouse PIR, indicating that the structural hallmarks of the PIR gene family are conserved in rats and mice. The rat PIR genes are localized to the distal end of q21.1—the proximal end of q21.3 on chromosome 1, where conserved linkage homology to mouse chromosome 7 has been identified (Matsuda et al., 1992). Similarly, genes for chicken PIR homologs (termed CHIR) have been identified (Dennis et al., 2000, 2001) (Fig. 2). Although the putative activating‐type CHIR‐A and inhibitory CHIR‐B have only two Ig‐like domains in their extracellular portion, other structural characteristics were similar to mouse and rat PIR‐A and PIR‐ B. A basic histidine residue was located in the CHIR‐A transmembrane region and two tyrosine residues embedded in the ITIM consensus sequences were present in the CHIR‐B cytoplasmic portion. The coordinate expression of CHIR‐A and CHIR‐B mRNA was observed in B‐ and T‐cell lines. Amino acid sequences of PIR‐A and PIR‐B ectodomains are highly homologous (over 92% identity) (Hayami et al., 1997; Kubagawa et al., 1997). The deduced structure of PIR‐B is a type I transmembrane glycoprotein with six extracellular Ig‐like domains, a hydrophobic transmembrane segment, and an intracellular polypeptide with four ITIM or ITIM‐like sequences (consensus: (I/L/V/S)xYxx(L/V); Fig. 2, Fig. 3B). The PIR‐B is highly homologous to several human and mouse Ig‐like receptors, including murine gp49B1 (31% homology at the amino acid level) (Castells et al., 1994), human KIR2DL (34%) (Colonna and Samaridis, 1995; Litwin et al., 1994; Phillips et al., 1995; Wagtmann et al., 1995), human FcaRI (29%) (Maliszewski et al., 1990), bovine Fcg2R (32%) (Zhang et al., 1995), and less homologous to human and mouse FcgRIIB (17%) sequences are denoted as closed boxes, and the noncoding sequences are open boxes. The relative positions of the isolated genomic clones were shown below each restriction map. The protein structure is subdivided into the signal peptide (S), extracellular Ig‐like domains (EC1 to EC6), pretransmembrane (pre‐TM), transmembrane (TM), and cytoplasmic domain (CP). The positions of 12 cysteines forming potential disulfide bonds (C–C) in the extracellular domain are shown. * indicates the position of the ITIM or ITIM‐like sequences.

r ec o g n i t i o n sys t em o f m h c cl a s s i m o l e cu l e s

169

(Hayami et al., 1997; Ravetch et al., 1986). Similarly, PIR‐A molecules have six Ig‐like extracellular domains, but in contrast to PIR‐B, they contain unique pretransmembrane, transmembrane, and short cytoplasmic sequences harboring no ITIM‐like motifs (Fig. 2, Fig. 3B). In addition, their transmembrane domains contain a positively charged residue, arginine, which presumably is crucial for the association of FcRg, which harbors an ITAM and is a common subunit for several activating receptors, and is critical for the expression of PIR‐A on the cell surface and for the delivery of the activation signal (Kubagawa et al., 1999; Ono et al., 1999; Yamashita et al., 1998b). Although the comparison of the available sequences of PIR extracellular portions from 129/Sv, B10.A and BALB/c mice indicate a fairly high sequence similarity, multiple substitutions of amino acid residues were observed, especially in the first four extracellular domains (Kubagawa et al., 1997; Yamashita et al., 1998a) (Fig. 4). The polymorphic nature of PIR supports the notion that PIR can bind to polymorphic MHC class I molecules, similar to the situation for LILR and KIR, which have many polymorphic substitutions in their extracellular domains (Colonna et al., 1997; Wagtmann et al., 1995), some of which recognize MHC class I molecules. PIR‐A and PIR‐B are expressed on various hematopoietic cell lineages, including B cells, mast cells, macrophages, granulocytes, and DCs, mostly in a pair‐wise fashion, but are not expressed on T and NK cells (Hayami et al., 1997; Kubagawa et al., 1997, 1999) (Fig. 2), suggesting that PIRs play a regulatory role mainly in B cells and myeloid cells. Monoclonal and polyclonal antibodies to PIR have identified cell‐surface glycoproteins of approximately 85 and 120 kDa on B cells, granulocytes, and macrophages (Kubagawa et al., 1999). Using a fibroblast transfection experiment as well as FcRg‐deficient (Fcrg–/–) mice, it was shown that approximately 120 kDa PIR‐B is normally expressed on the cell surface without any other subunits, whereas approximately 85 kDa PIR‐A requires an association with a homodimeric FcRg for its cell surface expression (Kubagawa et al., 1999; Maeda et al., 1998a; Ono et al., 1999; Yamashita et al., 1998b). Interestingly, the cell surface levels of PIR molecules on myeloid and B‐lineage cells increased with cellular differentiation and activation (Kubagawa et al., 1999). Surface PIR levels are highest on marginal zone B cells and the B1 B cells express higher PIR levels than the B2 B cells (Kubagawa et al., 1999). 4. Ligand for PIR PIR‐B tyrosine phosphorylation was examined in either B2m–/–, TAP1‐deficient, or MHC class II‐deficient mice (Ho et al., 1999), and the level of PIR‐B tyrosine phosphorylation was reduced by approximately 50% in B2m–/– mice,

170 Figure 4 Polymorphisms in the amino acid sequences of PIR‐B. Alignment of amino acid sequences of PIR‐B proteins from three different strains of mice are shown, subdividing into six extracellular Ig‐like domains (EC1 to EC6) (Yamashita et al., 1998a). For comparison between PIR‐B and PIR‐A sequences, two PIR‐A sequences from 129/Sv mice are shown together. Arbitrary numbers are assigned to these PIR‐A and shown in parentheses. Dashes indicate the identical residues. Bold Cs identify the conserved cysteine residues involved in potential disulfide bonds. The position numbers of the residues are given in the right side of each line. Similar analysis is also found in elsewhere (Chen et al., 1999).

r ec o g n i t i o n sys t em o f m h c cl a s s i m o l e cu l e s

171

but was not significantly altered in TAP1‐ or MHC class II‐deficient mice. Nonclassical MHC class I‐ or class I‐like molecules were speculated to be more likely candidates for native PIR ligands than classical MHC class I molecules (Ho et al., 1999). To obtain direct evidence for the PIR binding to MHC class I molecules, researchers took advantage of surface plasmon resonance analysis of the possible interaction between recombinant PIR‐B ectodomain and various murine MHC class I (H‐2) molecules (Nakamura et al., 2004). They found that the recombinant PIR‐B ectodomain was bound to the monomeric H‐2 molecules, H‐2Ld, H‐2Dd, H‐2Kb, H‐2Kk, and H‐2Kd, at affinities of the submicromolar range Kd, the values comparable, for example, to those between IgG and low‐affinity FcgRs, FcgRIIB, and FcgRIII (Hogarth, 2002; Takai, 2002). The low‐affinity FcgRs can bind multimeric IgG or IgG‐immune complexes very efficiently in vitro and in vivo (Hogarth, 2002; Takai, 2002). In fact, the recombinant PIR‐B ectodomain bound tetrameric H‐2 at higher affinities of nanomolar Kd, indicating the physiological significance of the low but substantial affinities of PIR to monomeric MHC class I molecules. Similar in the way that human LILRB1 binds to b2M of MHC class I (Shiroishi et al., 2003), recombinant PIR‐B binds to mouse b2M at a relatively high affinity (Nakamura et al., 2004), suggesting that at least one of the contact sites between recombinant PIR‐B and H‐2 is located on b2M, which may account for the apparently broad binding specificities of recombinant PIR‐B to H‐2 monomers. H‐2 binding to native PIR molecules expressed on a cell surface has also been verified (Nakamura et al., 2004). When splenic B cells were analyzed by confocal microscopy after labeling with either FITC‐conjugated H‐2Ld tetramer and phycoerythrin‐tagged monoclonal antibody to PIR‐A/B, 6C1, the fluorescence of 6C1 was substantially colocalized with that of H‐2Ld tetramer (Fig. 5). Likewise, native PIR‐A expressed on macrophages was shown to bind the H‐2 tetramer. These observations strongly suggest that native PIR‐B, and substantially native PIR‐A as well, efficiently bind tetrameric H‐2 molecules added extrinsically (Nakamura et al., 2004), in addition to their binding to a human classical MHC class I molecule, HLA‐B27 (Kollnberger et al., 2004), and nonclassical MHC class I molecule, HLA‐G (Liang et al., 2002). 5. 3D Structure of PIR and LILR and Their Interaction with MHC Class I Considering the 3D structure of PIR and its ligand interaction, it is noteworthy to look at those of human LILR and their interaction to MHC class I molecules. Another study (Willcox et al., 2003) described the 3.4‐A˚ crystal structure of a complex between the LILRB1 domains 1 and 2 (D1D2) and the MHC class I HLA‐A2, and found that LILRB1 D1D2 contacts b2M and a‐3 domains

172

t o s h i y u k i tak a i

Figure 5 H‐2 tetramer binds to PIR‐B on splenic B cells. Confocal microscopy analysis revealed the binding reaction between H‐2Ld tetramer and PIR‐B on splenic B cells. Upper panels, Confocal immunofluorescent images are shown (original magnification;
60) after double‐staining of splenic B cells from wild‐type C57BL/6 (B6) mice with FITC‐conjugated H‐2Ld tetramer and phycoerythrin‐conjugated anti‐PIR‐A/B, 6C1 (anti‐PIR). Merged image demonstrated colocalization of H2‐Ld and PIR‐B on the cell surface of B6 mice. Lower panels, As a negative control, H‐2Ld and anti‐PIR showed no significant binding in B cells from Pirb–/– mice.

of HLA‐A2 (Fig. 6). The D1D2 inter‐domain hinge region and a patch at the D1 tip contained the residues for the binding. The interaction is therefore consistent with the recognition by LILRB1 of a broad range of MHC class I molecules in a peptide‐independent way. A similar mode of MHC class I recognition seemed to be utilized by other LILR family members as well as by PIR because of the similarities between human LILR and murine PIR. The crystal structure of LILR shows a 1:1 LILRB1:HLA‐A2 stoichiometry, as predicted by analytical ultracentrifugation studies (Chapman et al., 1999), with no evidence of LILRB1 binding to HLA‐A2 dimers or oligomers. The binding structure is most consistent with a trans interaction involving the recognition of an MHC class I molecule on a target cell by a LILRB1 protein on an opposing effector cell. The 1:1 binding stoichiometry and specificity for a broad range of MHC class I molecules distinguishes LILRB1 and LILRB2 from KIR ligand recognition, and indicates that the strength of LILRB1/ 2 signals may reflect the overall expression of MHC class I molecules on the target cell. Researchers (Shiroishi et al., 2003) used surface plasmon resonance to analyze the interaction of soluble forms of LILRB1 and LILRB2 with several MHC class I molecules. LILRB1 and LILRB2 bound to a nonclassical HLA‐G

r ec o g n i t i o n sys t em o f m h c cl a s s i m o l e cu l e s

173

Figure 6 Schematic three‐dimensional (3D) structure of the N‐terminal two Ig‐like domains of LILRB binding to an MHC class I molecule. Domains 1 and 2 (D1D2) of LILRB are shown in red, a1–3 domains of HLA‐A2 are in purple, and b2‐microglobulin (b2M) is in green. A peptide accommodating the groove of a1/a2 domains is also shown in a red curve. Putative amino acid residues contributing to the binding between MHC class I and LILRB molecules are shown in spheres.

molecule (Fig. 1) with a threefold to fourfold higher affinity than that of classical MHC class I molecules, suggesting that LILR/HLA‐G recognition may play a dominant role in the regulation of natural killer‐, T‐, and myelomonocytic cell activation. Interestingly, these LILRBs effectively compete with CD8 for MHC class I binding, raising the possibility that LILRB1, expressed also in the subsets of NK and T cells other than myeloid cells, can modulate CD8þ T‐cell activation by blocking the CD8 binding to MHC class I on target cells. Considering the ubiquitous expression of MHC class I molecules on hematopoietic as well as nonhematopoietic cells, the question arises as to whether PIR can recognize MHC class I molecules on the same cell (in cis) or those on different cells (in trans) or both. A recent report on the cis/trans binding of Ly49A on NK cells to its ligand H‐2Dd may provide a clue. Others showed that the inhibitory Ly49A not only binds to its H‐2Dd ligand expressed on the potential target cells (in trans) but also is constitutively associated with H‐2Dd in cis (on the same cell) (Doucey et al., 2004). Cis association and trans interaction occur through the same binding site. Consequently, cis association restricts the number of Ly49A receptors available for the binding of H‐2Dd on target cells, reducing NK‐cell inhibition through Ly49A by lowering the

174

t o s h i y u k i tak a i

threshold at which NK‐cell activation exceeds NK‐cell inhibition. Cis interaction allows for optimal discrimination of normal and abnormal host cells. The structural resolution of the Ly49A–H‐2Dd cocrystal shows two possible interaction sites: one uses N‐terminal residues of the a 1 helix and C‐terminal residues of the a 2 helix of H‐2Dd (Site 1), and a second site is located beneath the floor of the peptide‐binding groove, making contact with residues of the a2 and a3 domains and with b2M (Site 2). In this study, it was shown that Ly49A and H‐2Dd are constitutively associated on the surface of cells. This interaction in cis and the functional binding mediating NK‐cell inhibition (trans) are mediated by Site 2. This binding mode excludes simultaneous cis and trans interaction through Site 2 by a single, homodimeric Ly49A receptor. Therefore, cis interaction restricts the availability of Ly49A molecules to interact in trans. Although it is unclear whether PIR and MHC class I can interact in cis and/or trans configurations, this possible two‐way mode of interaction may provide a different regulation of effector cells expressing PIR. 6. Activation Signal via PIR‐A Engagement with antibodies of transfected PIR‐A expressed on the rat mast cell line RBL‐2H3 elicited a calcium mobilization and degranulation response, indicating that PIR‐A plays a role in the activation of mast cells (Yamashita et al., 1998b). The molecule associated with the PIR‐A transmembrane region was FcRg (Kubagawa et al., 1999; Maeda et al., 1998a; Ono et al., 1999; Taylor and McVicar, 1999) and the Fc
RIb chain in RBL‐2H3 (Ono et al., 1999), in which two positively and negatively charged residues of PIR‐A, Arg626 and Glu643, respectively, were pivotal in the activating function of PIR‐A and its association with the FcR subunits (Ono et al., 1999). Events in the downstream signal of FcRg have been studied extensively in relation to Fc
RI on mast cells and activating type FcRs, FcgRI, and FcgRIII on macrophages (Takai, 2002). However, the exact physiological nature of PIR‐A/FcRg signaling has not been studied until recently because of a lack of information regarding the ligand for PIR‐A. Verification of the tetrameric H‐2 binding to cellular PIR‐A and PIR‐B suggests that H‐2 molecules expressed on neighboring cells (trans) or on the same cell (cis) may induce the constitutive interaction to PIR, which could lead to a continuous delivery of signals that will induce cell activation or silencing due to the continuous phosphorylation of ITAMs of FcRg in the PIR‐A receptor complex or ITIMs of the PIR‐B cytoplasmic domain (Fig. 7). It was examined whether the binding of tetrameric H‐2 molecules to PIR‐A on macrophages or PIR‐B on B cells could induce upregulated tyrosine phosphorylation of the receptor system. The phosphotyrosylation of FcRg was

r ec o g n i t i o n sys t em o f m h c cl a s s i m o l e cu l e s

175

Figure 7 Schematic positive and negative signaling via PIR. (Left) PIR‐A on myeloid cells such as macrophages and DCs are expressed as a complex with homodimeric FcRg, which have ITAM. Interaction of PIR‐A and PIR‐B with MHC class I molecules on themselves (cis) or neighboring cells (trans) will induce tyrosine phosphorylation of ITAMs of FcRg and ITIMs of PIR‐B by Src family protein kinases. The constitutive SHP‐1 association to the phosphorylated ITIM of PIR‐B will block inadequate activation signal via PIR‐A/FcRg, and modulate cytokine, chemokine, and integrin signaling. (Right) schematic illustration of negative signaling via PIR‐B on B cells upon constitutive interaction with MHC class I molecules in cis or trans. SHP‐1 constitutively associate with the tyrosine‐phosphorylated ITIM of PIR‐B in B cells, suggesting that it is not necessary to co‐cross‐link between activating receptors, such as BCR. In Pirb–/– B cells, BCR is hyperresponsive to anti‐BCR stimulation (Ujike et al., 2002).

substantially upregulated in peritoneal macrophages from wild‐type mice upon tetrameric H‐2 stimulation (Nakamura et al., 2004). It should be noted however, that the constitutive phosphotyrosylation of FcRg occurred in peritoneal macrophages without any specific stimuli, suggesting the constitutive stimulation of PIR‐A by surrounding or neighboring native H‐2 molecules expressed on cells. The phosphorylation was further increased in PIR‐B deficiency (Nakamura et al., 2004). These observations indicate that PIR‐A can deliver an activation signal intracellularly via tyrosine phosphorylation of FcRg upon

176

t o s h i y u k i tak a i

interaction of PIR‐A with H‐2 molecules, with the signal being continuously inhibited by PIR‐B coexpressed on the same cells (Fig. 7). 7. Inhibitory Signal via PIR‐B Taking advantage of B cells that express PIR‐B but not PIR‐A (Fig. 2), the signaling nature via PIR‐B upon interaction with H‐2 has been investigated (Nakamura et al., 2004). Various H‐2 tetramers were added to splenic B cells, and then phosphotyrosylation of PIR‐B was monitored by immunoprecipitation and immunoblot analysis. The enhanced phosphotyrosylation of PIR‐B after stimulation with every H‐2 tetramer was observed, although the enhancement was rather small, about 1.8‐fold higher than the nonstimulation control at most. Importantly, the constitutive PIR‐B tyrosine phosphorylation in splenic B cells was observed without any specific stimuli. These notions are consistent with the hyperactivated state of B cells from PIR‐B‐deficient (Pirb–/–) mice in the absence of any specific stimulation of BCR in vitro and in vivo (Ujike et al., 2002) (Fig. 7). Most of the inhibitory isoforms of Ig‐like receptors exert their negative regulation of cells by recruiting Src homology 2 domain‐containing protein tyrosine phosphatases SHP‐1 and/or SHP‐2 to their phosphorylated ITIM (Long, 1999; Ravetch and Lanier, 2000; Takai, 2002). Exceptionally, a unique inhibitory FcgR, namely FcgRIIB, recruits SH2‐domain‐containing inositol 5‐phosphatase SHIP‐1 to the phosphorylated ITIM (Long, 1999; Ravetch and Lanier, 2000; Takai, 2002). PIR‐B functions as the former type receptor. In vitro mutation analysis of cytoplasmic tyrosine residues in the ITIM of PIR‐B indicated that the tyrosine in the third ITIM plays the most crucial role in mediating the inhibitory signal for BCR‐mediated cell activation as assessed by calcium mobilization and NF‐AT activation (Maeda et al., 1998b). Also in vitro, synthetic phosphotyrosyl peptides corresponding to the third and fourth ITIMs of PIR‐B can bind SHP‐1, SHP‐2, and SHIP‐1 in cell extracts from macrophages, mast cells, as well as in a B‐cell line (Maeda et al., 1998b; Uehara et al., 2001; Yamashita et al., 1998a). However, the PIR‐B‐mediated inhibition was markedly reduced in SHP‐1‐ and SHP‐2‐double‐deficient DT40 chicken B cells, whereas this inhibition was unaffected in SHIP‐deficient cells, suggesting that PIR‐B can negatively regulate BCR activation through the redundant functions of SHP‐1 and SHP‐2, but not by SHIP (Maeda et al., 1998b). In addition, in the mast cell line RBL‐2H3, coaggregation of transfected PIR‐B with Fc
RI induced the PIR‐B to recruit SHP‐1 but not SHP‐2 nor SHIP (Ble´ ry et al., 1998). Even in the absence of the coaggregation, a weak constitutive association of the PIR‐B cytoplasmic domain with SHP‐1 was observed (Ble´ ry et al., 1998). The third and fourth ITIM tyrosine residues

r ec o g n i t i o n sys t em o f m h c cl a s s i m o l e cu l e s

177

were crucial for inhibition of RBL‐2H3 cell degranulation and calcium mobilization (Yamashita et al., 1998a). A similar approach with bone marrow‐ derived cultured mast cells (Uehara et al., 2001) reached the conclusion that PIR‐B expressed on bone marrow mast cells was constitutively phosphorylated and associated with SHP‐1. Coligation of PIR‐B and Fc
RI by antibodies inhibited IgE‐mediated mast cell activation. Researchers noted that the inhibitory activity of PIR‐B was unimpaired in SHP‐1‐deficient mast cells (Uehara et al., 2001). They proposed a third functional tyrosine‐based inhibitory motif, one that fails to bind the SHP‐1, SHP‐2, and SHIP phosphatases, suggesting a potential of inhibition mediated via a SHP‐1‐independent pathway. A reverse approach yielded an intriguing observation about the preferential association of SHP‐1 to PIR‐B. The SHP‐1 was shown to associate with a 130‐kDa tyrosyl‐phosphorylated species, termed P130, in murine macrophages, suggesting that the P130 may be an SHP‐1 regulator and/or substrate (Timms et al., 1998). Interestingly, P130 consisted of two transmembrane glycoproteins, namely PIR‐B and SIRPa (also termed BIT, SHPS‐1, P84) (Fig. 2). Furthermore, PIR‐B was hyperphosphorylated in macrophages from SHP‐1‐inactive motheaten viable mice, whereas it was hypophosphorylated in SHP‐1‐deficient motheaten macrophages, suggesting a model in which SHP‐1 dephosphorylates specific sites on PIR‐B while protecting other sites from dephosphorylation via its SH2 domains. The PIR‐B also associated with two tyrosyl phosphoproteins and a tyrosine kinase activity (Timms et al., 1998). 8. Dominant Expression of PIR‐B In Vivo Because the physiologic ligand for PIR was identified as MHC class I molecules, PIR may modulate inflammatory and immune responses by constitutive engagement with self H‐2 molecules. Recent research on Pirb–/– mice has provided us with an insight into the physiological significance of the H‐2 recognition by PIRs in the immune response, especially in antigen presentation, humoral immunity, and transplantation as follows (Nakamura et al., 2004; Ujike et al., 2002). Because an available monoclonal antibody to PIR, 6C1, recognizes both PIR‐A and PIR‐B, it was not known which receptor is dominantly expressed on various cell surfaces. Given that the deletion of one receptor does not influence the expression of the other, PIR‐B deficiency will reveal the surface expression of PIR‐A by flow cytometry with 6C1, whereas cells devoid of FcRg will enable us to estimate PIR‐B expression. Comparison of flow cytometric data on cells from either Pirb–/– (Ujike et al., 2002) or Fcrg–/– mice (Takai et al., 1994) revealed the more dominant expression of PIR‐B than

178

t o s h i y u k i tak a i

PIR‐A on splenic macrophages, splenic DC, and bone marrow‐derived cultured mast cells and exclusive expression of PIR‐B on splenic B cells. Protein blot analysis also demonstrated the dominant expression of the inhibitory isoform in mast cells (Uehara et al., 2001). The consistent observations from either PIR‐B or FcRg deletion suggested that the PIR‐B deletion did not largely alter the PIR‐A expression in these cells and vice versa. Thus, the PIR‐ A and PIR‐B surface expression is characteristic for each cell type, at least in its resting state, and the suppression of these cells by dominantly expressed PIR‐B may have a physiological importance, such as in maintaining their resting state upon a continuous interaction with H‐2. Physiological upregulation or downmodulation of the expression level of PIR‐A or PIR‐B on the cell surface would lead to an increase or decrease in the intensity of H‐2‐induced, PIR‐mediated signal transduction. We now know that PIR‐B expression level increases during the development of B cells (Kubagawa et al., 1999), and that PIR‐A and PIR‐B expression levels on DCs significantly increase during graft‐ versus‐host disease (GVHD) (see later), although the mechanism for these changes in gene expression is not well understood. In relation to this issue, researchers observed that IL‐4 reduced the expression of PIR‐B on B cells together with the decrease in other inhibitory receptors CD22 and FcgRIIB (Rudge et al., 2002). PIR‐B and these inhibitory receptors on B cells would contribute to the maintenance of B‐cell tolerance in the absence of T‐cell help. 9. Pirb–/– B Cells, Neutrophils, and Macrophages are Hyperresponsive The PIR‐B molecules in macrophages and B cells are constitutively phosphorylated (Ho et al., 1999), and this is presumably induced by the constitutive interaction with H‐2 molecules. Pirb–/– splenic B cells showed a significantly enhanced proliferation upon anti‐BCR F(ab0 )2 stimulation. When stimulated with anti‐BCR whole IgG antibodies after blocking with anti‐FcgRIIB monoclonal antibody, the enhanced Pirb–/– B‐cell proliferation was more pronounced due to the masking of an inhibitory effect by FcgRIIB. This indicates that the inhibitory effects by PIR‐B and FcgRIIB are additive, possibly because of the fact that PIR‐B uses the SHP‐1 cascade for inhibition, whereas FcgRIIB uses SHIP (Ujike et al., 2002). These results indicate that Pirb–/– B cells are hypersensitive to stimulation via BCR ligation. Consistent with this observation, Pirb–/– mice showed a higher IgM response against T‐ independent antigens TNP‐Ficoll and TNP‐LPS. The enhanced response upon TNP‐LPS challenge suggested that the PIR‐B inhibitory effect could also be exerted independently of BCR ligation. Enhanced tyrosine phosphorylation of cellular proteins in Pirb–/– B cells, even in the resting state, indicated the constitutive activation of Pirb–/– B cells (Ujike et al., 2002). Thus, PIR‐B

r ec o g n i t i o n sys t em o f m h c cl a s s i m o l e cu l e s

179

may downregulate BCR signaling by interacting with H‐2 molecules (Fig. 7). A PIR‐B deficiency may generally render B cells active and hyperresponsive to stimulation via BCR. However, serum IgM levels were not increased in naı¨ve Pirb–/– mice. In addition, anti‐double‐stranded DNA antibodies were not detected in adult Pirb–/– mice of C57BL/6 background, in contrast with CD22‐deficient mice, in which hyper‐IgM and autoantibody production is evident (O’Keefe et al., 1996; Otipoby et al., 1996). It was pointed out that PIR‐B is directly involved in integrin signaling in neutrophils and macrophages. Researchers observed that Lyn‐deficient neutrophils showed an enhanced respiratory burst, granule release, and a hyperadhesive phenotype upon engagement of surface integrins (Pereira and Lowell, 2003). Lyn‐deficient macrophages also showed a hyperadhesive phenotype. It was shown that the Lyn plays an essential role in the adhesion‐ dependent phosphorylation of the ITIM of the inhibitory receptors SIRPa and PIR‐B, which in turn recruits the phosphatase SHP‐1. The reduced mobilization of SHP‐1 to the membrane in Lyn‐deficient neutrophils results in a hyperadhesive and hyperactive phenotype. Thus, Lyn kinase functions in these cases as a negative regulator in integrin signaling via PIR‐B and SIRPa. In line with this observation, Pirb–/– neutrophils displayed an enhanced respiratory burst, secondary granule release, and a hyperadhesive phenotype upon integrin engagement (Pereira and Lowell, 2003). Bone marrow‐derived macrophages from Pirb–/– mice were also hyperadhesive and spread more rapidly than wild‐type cells upon crosslinking of the cellular b2 integrins. Phosphorylation and activation of proteins involved in integrin signaling were observed in such Pirb–/– macrophages. Thus, PIR‐B is critical in the regulation of neutrophil and macrophage integrin signaling, suggesting a significant role of PIR in inflammatory responses (Fig. 7). They extended their observations to the detailed kinase signaling in neutrophils and DCs. Another study (Zhang et al., 2005) found that neutrophils and DCs from Pirb–/– mice were hyperresponsive to chemokine stimulation. Similarly, neutrophils derived from hck–/–fgr–/– mice and DCs from fgr–/– mice manifested significantly higher intracellular signaling in terms of calcium influx, MAP kinase activation, and actin polymerization, and functional responses such as chemotaxis in vitro and migration in vivo to a number of different chemokines. Thus, these kinases might mediate their effect through PIR‐B (Fig. 7). In wild‐type cells, dephosphorylation of PIR‐B was associated with maximal chemokine signaling, whereas in hck–/–fgr–/– cells PIR‐B was unphosphorylated. These results support a model in which the Src family kinases Hck and Fgr function as negative regulators of myeloid cell chemokine signaling by maintaining the constitutive phosphorylation of PIR‐B (Zhang et al., 2005). In classical descriptions of leukocyte chemokine signaling, Src

180

t o s h i y u k i tak a i

family kinases were thought to function in a positive fashion by coupling receptor‐associated Ga subunits to downstream MAP kinase activation. However, their results strongly suggest that these kinases mediate their ‘‘positive’’ effect through the ‘‘inhibitory’’ receptor PIR‐B (Fig. 7). 10. Modulated Cytokine Signaling in PIR‐B Deficiency Th2‐type humoral responses were augmented in Pirb–/– mice upon immunization with TNP‐KLH or OVA with alum adjuvant, in terms of both IL‐4‐rich and IFN‐g‐poor cytokine profiles and enhanced IgG1 and IgE production (Ujike et al., 2002). At least one of the mechanisms for the Th2‐skewed responses in Pirb–/– mice was suggested to be the immature phenotype of DCs. Flow cytometric analysis of surface markers, such as MHC class II, CD80, and CD86, on bone marrow‐derived cultured DCs (BMDCs) before and after antigen loading revealed that the BMDCs from Pirb–/– mice were immature. The production of IL‐12, a Th1‐polarizing cytokine (Magram et al., 1996), was diminished upon the antigen loading of BMDCs from Pirb–/– mice. The successful adoptive transfer of a Th2‐prone response by Pirb–/– BMDCs into wild‐type mice strongly suggested that the impaired maturation of DCs would be responsible for the skewing. To test the possibility that intracellular signaling may differ between Pirb–/– BMDCs and wild‐type cells, BMDCs were stimulated with GM‐CSF, an inducer cytokine for DC development, and their protein tyrosine phosphorylation profile were examined (Ujike et al., 2002). PIR‐B was tyrosine phosphorylated in resting wild‐type DCs similar to that found in resting B cells and mast cells (Ho et al., 1999; Uehara et al., 2001). The tyrosine phosphorylation of PIR‐B was augmented upon GM‐CSF stimulation, indicating that PIR‐B is involved in the cytokine signaling. Upon GM‐CSF stimulation, altered phosphorylation profiles of total cellular phosphotyrosyl proteins as well as cytokine receptor common b chain were observed in Pirb–/– DC when compared to those of the wild‐type cells (Ujike et al., 2002). It is conceivable that the PIR‐B deficiency leads to altered phosphorylation profiles of GM‐CSF signaling in the absence of an inhibitory signal initiated upon H‐2 binding of PIR‐B, which yields immature DCs. Thus, the PIR‐B and H‐2 interaction is critical for DC maturation and for regulating humoral responses (Fig. 7). The IL‐3 receptor‐mediated signal effects progenitor, myeloid, and mast cells to induce cell proliferation, survival, and facilitate differentiation. Upon IL‐3 binding to the receptor‐specific a chain and cytokine receptor common b chain, the activation of members of Jak and Src family kinases takes place followed by rapid and reversible tyrosine phosphorylation of a number of cellular proteins. The association of SHP1 and SHP2 with IL‐3 receptor

r ec o g n i t i o n sys t em o f m h c cl a s s i m o l e cu l e s

181

common b chain (Bone et al., 1997) suggests that SHP1 plays a negative regulatory role in IL‐3 signaling. In response to IL‐3, SHP1 recruitment to PIR‐B was shown to increase, suggesting a functional link between inhibitory PIR‐B signaling and IL‐3 receptor signaling (Wheadon et al., 2002). These observations suggest that PIR‐B is significantly involved in IL‐3 and GM‐ CSF signal cascades, which may effect mast cell and DC development and function, respectively (Fig. 7). The detailed mode of involvement remains to be investigated. Recent observation in bone formation of mutant mice devoid of the DAP12 and/or FcRg gene has revealed an unexpected involvement of multiple Ig‐like receptors including PIR in osteoclastogenesis. The development and homeostasis of the skeletal system depends on the balanced bone formation and resorption by osteoblasts and osteoclasts, respectively (Karsenty and Wagner, 2002; Rodan and Martin, 2000). Osteoclasts are derived from monocyte/ macrophage‐lineage cells in bone marrow. Receptor activator of NF‐kB ligand (RANKL) expressed on osteoblasts as well as stromal cells critically regulates the differentiation of the bone‐resorbing osteoclasts in the presence of macrophage‐colony stimulating factor (M‐CSF) (Boyle et al., 2003; Teitelbaum and Ross, 2003). It remains unclear how RANKL activates the calcium signal leading to the induction of nuclear factor of activated T cells c1 (NFATc1), a key transcription factor for osteoclastogenesis (Takayanagi et al., 2002). It was shown (Koga et al., 2004) that the combined deficiency of ITAM‐harboring adaptors, FcRg, and DNAX‐activating protein (DAP)12 resulted in severe osteopetrosis due to impaired osteoclast differentiation. In osteoclast precursor cells, FcRg and DAP12 were associated with multiple Ig‐like receptors (Dietrich et al., 2000; Hayami et al., 1997; Kim et al., 2002; Kubagawa et al., 1997; Tomasello et al., 2000). Putative FcRg‐ and DAP12‐associating receptors were detected on the surface of the osteoclast lineage using cell‐surface labeling with biotin and immunoprecipitation experiments. They confirmed the pairing of PIR‐A, FcgRIII, and osteoclast‐associated receptor (OSCAR) with FcRg and that of TREM‐2 and SIRPb1 with DAP12 in the osteoclast lineage. To test whether these receptor‐mediated signals promote osteoclast differentiation by associating with FcRg or DAP12, they stimulated bone marrow monocyte/macrophage lineage cells with plate‐bound monoclonal antibodies to OSCAR, PIR, TREM‐2, and SIRPb1. Triggering of either receptor accelerated RANKL‐induced osteoclast differentiation, indicating the activating role of these receptors in osteoclastogenesis. In the absence of RANKL, the stimulation of these receptors alone could not induce the osteoclast differentiation, suggesting that these receptor‐mediated signals act cooperatively with RANKL but cannot substitute the signal. The ITAM‐mediated signal activated calcium oscillation through PLCg. Thus, ITAM‐dependent

182

t o s h i y u k i tak a i

stimulatory signals activated by multiple Ig‐like receptors including PIR are indispensable for the maintenance of bone homeostasis, indicating that the RANK and M‐CSF receptor are not sufficient to activate signals required for osteoclastogenesis. 11. PIR in Transplantation Feto‐maternal tolerance is important in the physiological system for maternal unresponsiveness to the fetus, a type of transplant for mothers. The nonclassical MHC class I molecule HLA‐G (Fig. 1) is selectively expressed on fetal trophoblast tissue at the feto‐maternal interface in pregnancy (Clements et al., 2005). It has long been suggested that HLA‐G may inhibit maternal NK cells through its interaction with particular inhibitory KIRs. However, this feto‐maternal tolerance may be substantially supported by inhibitory Ig‐like receptors expressed on maternal DCs and monocytes. On transfectants in fact, it is known that HLA‐G tetramers can bind to inhibitory LILRB1 and LILRB2. The HLA‐ G tetramer that binds to blood monocytes was largely due to binding to LILRB2 (Allan et al., 1999). Interestingly, the human HLA‐G can also bind to murine PIR‐B and thus modifies the function of murine DCs (Liang et al., 2002). This observation is not surprising because PIR binds to b2M (Nakamura et al., 2004), which exhibits a high amino acid‐sequence homology (about 70%) between mice and humans. HLA‐G tetrameric complexes inhibited maturation of murine BMDCs in vitro, similar to what occurred in HLA‐G‐transgenic mice. PIR‐ B was highly phosphorylated on BMDCs from HLA‐G‐transgenic mice. It is important to dissect these observations in the murine system, in which mouse nonclassical MHC class I molecules, such as Qa‐1 (Jensen et al., 2004) (Fig. 1), might modulate the PIR‐B inhibitory function in murine DCs. The ‘‘self‐recognition system’’ provided by TCR of T cells is critical in tissue or hematopoietic cell transplantation. Engagement of TCRs on CTLs with allogeneic MHC class I expressed by target cells triggers the elimination of transplanted or host cells. Therefore, matching MHC class I molecules are critical for successful tissue or bone marrow transplantation between allogeneic individuals. Conversely, bone marrow transplantation into immunocompromised individuals causes GVHD, in which alloreactive CD4 T cells of donor origin respond to allogeneic MHC class II molecules on the activated recipient’s antigen‐presenting cells (APCs) such as DCs. Recipient DCs are required for the induction of GVHD (Shlomchik et al., 1999) suggesting that donor T cell–host APC interaction is essential for triggering graft‐versus‐host reaction. The alloreactive CD4 T cells then become effector CD4 T cells and activate, in combination with recipient DCs bearing allogeneic MHC class I molecules, alloreactive donor CD8 T cells to become effector CD8 CTLs

r ec o g n i t i o n sys t em o f m h c cl a s s i m o l e cu l e s

183

(Fig. 8A). These activated CTLs can induce deleterious damage in host tissues (Shlomchik et al., 1999). The development of allogeneic responses is mainly due to the presence of a relatively high percentage of alloreactive T cells (1– 10%) in the periphery of normal individuals as compared to the much smaller percentage of specific antigen‐reactive T cells (around 0.001%) (Heeger, 2003). Alloreactive T cells are believed not to be generated aiming at transplant rejection but are the consequence of an unexpected strong recognition of allogeneic MHC class I‐presenting peptides to their TCRs, which are weakly reactive to self‐peptide MHC class I molecules. To test the possible interaction between allogeneic MHC class I molecules and PIR in transplantation, a lethal GVHD model was employed, in which sublethally irradiated Pirb–/– or wild‐type host mice received allogeneic

Figure 8 Proposed role of PIRs on DCs during the interaction with allogeneic MHC class I molecules in GVHD. PIR‐B and PIR‐A on DCs recognize self‐MHC class I molecules on neighboring cells or on themselves and balance the cellular activation of DCs. PIR‐B negatively regulates the cellular signaling induced by activating‐type receptors including PIR‐A. (A) In the development of GVHD, alloreactive CD4þ T cells of donor origin respond to allogenic MHC class II molecules on activated, recipient DCs, and become effector CD4þ T cells, which then activate alloreactive donor CD8þ T cells to become activated CD8þ CTLs. The activated donor CD8þ T cells target recipient tissues through their effector molecules such as IFN‐g, leading to GVHD. (B) When PIR‐B was deficient, recipient DCs were more activated with increased PIR‐A expression, and then robustly activate alloreactive effector CD4þ and CD8þ T cells, resulting in more severe GVHD.

184

t o s h i y u k i tak a i

splenocytes intravenously. Pirb–/– mice showed accelerated, lethal GVHD (Nakamura et al., 2004) (Fig. 9). When splenocytes in the recipients were analyzed by flow cytometry 6 days after transplantation, the numbers of both IFN‐gþ CD4 T cells and IFN‐gþ CD8 T cells were significantly higher in Pirb–/– B6 recipients than wild‐type B6 recipients. An increased mortality of mice and increased numbers of IFN‐gþ CD4 or CD8 T cells in Pirb–/– recipients were also noticed for the opposite donor and recipient combination, in which Pirb–/– or wild‐type BALB/c received B6 splenocytes. It was confirmed that T cells found in recipient mouse spleens 6 days posttransplant were exclusively derived from donor splenocytes. In GVHD, activation of donor alloreactive CTLs requires prior activation of donor CD4þ T cells and recognition of host allogeneic MHC class I molecules with peptides on the host APCs (Shlomchik et al., 1999). Consistent with this notion, day 4 splenic DCs from host wild‐type or Pirb–/– BALB/c mice who received B6 splenocytes were similarly activated as compared to those from the control mice who received no transplant. These host splenic DCs had an augmented expression of MHC class I and costimulatory molecules, including CD40, CD80, and CD86. Concomitant with this DC activation, the expression of PIR‐A and PIR‐B or PIR‐A was significantly elevated on DCs from wild‐ type or Pirb–/– recipient BALB/c mice, respectively, compared to DCs from those mice that received no transplant. These results suggest the critical role of PIR‐A and PIR‐B in the modulation of alloreactive CD4 and CD8 T cells (Fig. 8). DCs from Pirb–/– recipients were shown to be significantly more

Figure 9 Exacerbated GVHD in Pirb–/– mice. Survival curves of GVHD in Pirb–/– B6 (n ¼ 16, closed square) or B6 mice (n ¼ 16, closed circle) are shown (Nakamura et al., 2004). 4
107 BALB/c splenocytes were injected into 6 Gy‐irradiated Pirb–/– B6 or B6 recipients. Mortality was assessed every 24 h for 21 d. Statistical analyses were performed using Fisher’s exact test. *P < 0.05 between Pirb–/– B6 and B6 mice.

r ec o g n i t i o n sys t em o f m h c cl a s s i m o l e cu l e s

185

positive for IFN‐g than those from the wild‐type recipient mice, indicating that the PIR‐B‐deficient DCs of Pirb–/– recipient mice were more activated than DCs of wild‐type recipients (Fig. 8B). The interaction between allogeneic H‐2 molecules on donor T cells and PIR‐A, which increased on the recipient’s DCs might further enhance DC activation, resulting in the augmented production of one of the key effector cytokines, IFN‐g. Blocking Programmed Death‐1 (PD‐1), one of the inhibitory Ig‐like receptors (Fig. 2), accelerates GVHD lethality through an IFN‐g‐dependent mechanism (Blazar et al., 2003), supports the critical role of IFN‐g in lethal GVHD. Thus, PIR‐B could render APCs such as DCs capable of regulating CTLs through the suppression of at least IFN‐g production (Fig. 8).

Figure 10 Receptors and costimulatory ligands at a T cell and an APC interface. Interaction between TCR/CD3 complex on a donor T cell and an MHC class I or class II molecule on a recipient APC leads to the donor T‐cell activation in conjunction with stimulating interactions between costimulatory molecules and ligands such as CD28/CD80/86 and ICOS/ICOSL. These stimulatory signals are inhibited by CTLA‐4/CD80/86, PD‐1/PD‐L1/L2, and B‐ and T‐lymphocyte attenuator (BTLA)/herpes virus entry mediator (HVEM) interactions. At this interface of T cell/ APC, PIRs on APC can recognize MHC class I molecules on donor T cells and will control the APC activity, which leads to regulated production of IFN‐g, a key cytokine for GVHD induction.

186

t o s h i y u k i tak a i

In a rat model of heart transplantation, the importance of CD8 T‐suppressor cells was reported (Liu et al., 2004). CD8 T‐suppressor cells from tolerant rats could transfer the tolerance to naı¨ve hosts and induce the upregulation of rat PIR‐B in allogeneic DCs and heart endothelial cells. The heart allograft with PIR‐B‐positive endothelial cells were retransplanted to a secondary allogeneic recipient, and it was found that they did not elicit rejection. In the human system, it was reported that human T‐suppressor cells rendered professional and nonprofessional APCs such as DC and endothelial cells tolerogenic, and they also induced the downregulation of costimulatory molecules and the upregulation of the inhibitory Ig‐like receptors of human PIR‐B orthologs, LILRB2 and LILRB4 on APCs (Manavalan et al., 2003). T cell and APC interaction would be a crucial step toward the priming of transplant rejection and GVHD. In this interface of a T cell and an APC, multiple costimulatory or coinhibitory receptor/ligand interactions exist (Fig. 10). Exacerbated GVHD in Pirb–/– mice shed light on the novel and significant interaction between MHC class I molecules on T cells and PIR‐A and PIR‐B on APCs, in addition to those interactions made by CD28/CTLA‐4 and CD80/CD86, ICOS and ICOSL, PD‐1 and PD‐L1/L2, B7‐H3/H4, and BTLA and HVEM (Gonzalez et al., 2005; Greenwald et al., 2005; Sedy et al., 2005; Wang et al., 2005). 12. Conclusion Various immune cells may have adopted the recognition of ubiquitously expressed, MHC class I molecules as a common strategy to inhibit cellular activation. Thus, PIR‐A and PIR‐B provide a novel system for MHC class I recognition with physiological and pathological significance, where PIR‐A and PIR‐B regulate the threshold for the activation signaling of cells, such as B cells, mast cell, neutrophils, macrophages, DCs, and osteoclasts, and prevent the undesired reaction to autologous tissues in a constitutive fashion. They become critical for successful tissue and hematopoietic stem cell transplantation. Further analysis of the detailed structural features of interaction between PIR and classical and nonclassical MHC class I molecules, as well as the characteristics of Pirb–/– mice, will provide us with further information about the physiological role of PIR and pathophysiological aspects of its involvement in infection, hypersensitivity, and autoimmune diseases. It is also important to identify the pathophysiological aspects of LILR polymorphism, and the regulation of their expression on hematopoietic cells to apply the knowledge of PIR to various immune disorders in the clinical setting.

r ec o g n i t i o n sys t em o f m h c cl a s s i m o l e cu l e s

187

Acknowledgments The author’s work cited in the present review is supported by the CREST Program of Japan Science and Technology Agency and by research grants from the Ministry of Education, Science, Sports and Culture of Japan.

References Allan, D. S., Colonna, M., Lanier, L. L., Churakova, T. D., Abrams, J. S., Ellis, S. A., McMichael, A. J., and Braud, V. M. (1999). Tetrameric complexes of human histocompatibility leukocyte antigen (HLA)‐G bind to peripheral blood myelomonocytic cells. J. Exp. Med. 189, 1149–1156. Blazar, B. R., Carreno, B. M., Panoskaltsis‐Mortari, A., Carter, L., Iwai, Y., Yagita, H., Nishimura, H., and Taylor, P. A. (2003). Blockade of programmed death‐1 engagement accelerates graft‐versus‐host disease lethality by an IFN‐g‐dependent mechanism. J. Immunol. 171, 1272–1277. Ble´ ry, M., Kubagawa, H., Chen, C.‐C., Ve´ ly, F., and Cooper, M. D. (1998). The paired Ig‐like receptor PIR‐B is an inhibitory receptor that recruits the protein‐tyrosine phosphatase SHP‐1. Proc. Natl Acad. Sci. USA 95, 2446–2451. Bone, H., Dechert, U., Jirik, F., Schrader, J. W., and Welham, M. J. (1997). SHP1 and SHP2 protein‐tyrosine phosphatases associate with betac after interleukin‐3‐induced receptor tyrosine phosphorylation. Identification of potential binding sites and substrates. J. Biol. Chem. 272, 14470–14476. Brooke, G., Holbrook, J. D., Brown, M. H., and Barclay, A. N. (2004). Human lymphocytes interact directly with CD47 through a novel member of the signal regulatory protein (SIRP) family. J. Immunol. 173, 2562–2570. Borges, L., and Cosman, D. (2000). LIRs/ILTs/MIRs, inhibitory and stimulatory Ig‐superfamily receptors expressed in myeloid and lymphoid cells. Cytokine Growth Factor Rev. 11, 209–217. Boyle, W. J., Simonet, W. S., and Lacey, D. L. (2003). Osteoclast differentiation and activation. Nature 423, 337–342. Castells, M. C., Klickstein, L. B., Hassani, K., Cumplido, J. A., Lacouture, M. E., Austen, K. F., and Katz, H. R. (2001). gp49B1‐avb3 interaction inhibits antigen‐induced mast cell activation. Nat. Immunol. 2, 436–442. Castells, M. C., Wu, X., Arm, J. P., Austen, K. F., and Katz, H. R. (1994). Cloning of the gp49B gene of the immunoglobulin superfamily and demonstration that one of its two products is an early‐ expressed mast cell surface protein originally described as gp49. J. Biol. Chem. 269, 8393–8401. Cerwenka, A., and Lanier, L. L. (2001). Natural killer cells, viruses and cancer. Nat. Rev. Immunol. 1, 41–49. Chang, C. C., Ciubotariu, R., Manavalan, J. S., Yuan, J., Colovai, A. I., Piazza, F., Lederman, S., Colonna, M., Cortesini, R., Dalla‐Favera, R., and Suciu‐Foca, N. (2002). Tolerization of dendritic cells by TS cells: The crucial role of inhibitory receptors ILT3 and ILT4. Nat. Immunol. 3, 237–243. Chapman, T. L., Heikema, A. P., and Bjorkman, P. J. (1999). The inhibitory receptor LIR‐1 uses a common binding interaction to recognize class I MHC molecules and the viral MHC homolog UL18. Immunity 11, 603–611. Chen, C. C., Hurez, V., Brockenbrough, J. S., Kubagawa, H., and Cooper, M. D. (1999). Paternal monoallelic expression of the paired immunoglobulin‐like receptors PIR‐A and PIR‐B. Proc. Natl. Acad. Sci. USA 96, 6868–6872. Chen, C. C., Hurez, V., Brockenbrough, J. S., Kubagawa, H., and Cooper, M. D. (2000). Erratum: Paternal monoallelic expression of the paired immunoglobulin‐like receptors PIR‐A and PIR‐B. Proc. Natl. Acad. Sci. USA 97, 10294.

188

t o s h i y u k i tak a i

Clark, G. J., Green, B. J., and Hart, D. N. (2000). The CMRF‐35H gene structure predicts for an independently expressed member of an ITIM/ITAM pair of molecules localized to human chromosome 17. Tissue Antigens 55, 101–109. Clements, C. S., Kjer‐Nielsen, L., Kostenko, L., Hoare, H. L., Dunstone, M. A., Moses, E., Freed, K., Brooks, A. G., Rossjohn, J., and McCluskey, J. (2005). Crystal structure of HLA‐G: A nonclassical MHC class I molecule expressed at the fetal‐maternal interface. Proc. Natl. Acad. Sci. USA 102, 3360–3365. Colonna, M. (2003). TREMs in the immune system and beyond. Nat. Rev. Immunol. 3, 445–453. Colonna, M., Nakajima, H., and Cella, M. (1999a). Inhibitory and activating receptors involved in immune surveillance by human NK and myeloid cells. J. Leukoc. Biol. 66, 718–722. Colonna, M., Nakajima, H., Navarro, F., and Lopez‐Botet, M. (1999b). A novel family of Ig‐like receptors for HLA class I molecules that modulate function of lymphoid and myeloid cells. J. Leukoc. Biol. 66, 375–381. Colonna, M., Navarro, F., Bellon, T., Llano, M., Garcia, P., Samaridis, J., Angman, L., Cella, M., and Lopez‐Botet, M. (1997). A common inhibitory receptor for major histocompatibility complex class I molecules on human lymphoid and myelomonocytic cells. J. Exp. Med. 186, 1809–1818. Colonna, M., and Samaridis, J. (1995). Cloning of immunoglobulin‐superfamily members associated with HLA‐C and HLA‐B recognition by human natural killer cells. Science 268, 405–408. Colonna, M., Samaridis, J., Cella, M., Angman, L., Allen, R. L., O’Callaghan, C. A., Dunbar, R., Ogg, G. S., Cerundolo, V., and Rolink, A. (1998). Human myelomonocytic cells express an inhibitory receptor for classical and nonclassical MHC class I molecules. J. Immunol. 160, 3096–3100. Daheshia, M., Friend, D. S., Grusby, M. J., Austen, K. F., and Katz, H. R. (2001). Increased severity of local and systemic anaphylactic reactions in gp49B1‐deficient mice. J. Exp. Med. 194, 227–233. Dennis, Jr. G., Chen, C.‐C., Kubagawa, H., and Cooper, M. D. (2001). Phylogeny of paired Ig‐like receptors. In ‘‘Activating and Inhibitory Immunoglobulin‐Like Receptors’’ (Cooper, M. D., Takai, T., and Ravetch, J. V., Eds.), pp. 7–15. Springer‐Verlag, Tokyo. Dennis, Jr. G., Kubagawa, H., and Cooper, M. D. (2000). Paired Ig‐like receptor homologs in birds and mammals share a common ancestor with mammalian Fc receptors. Proc. Natl Acad. Sci. USA 21, 13245–13250. Dennis, Jr., G., Stephan, R. P., Kubagawa, H., and Cooper, M. D. (1999). Characterization of paired Ig‐like receptors in rats. J. Immunol. 163, 6371–6377. Dietrich, J., Cella, M., Seiffert, M., Buhring, H. J., and Colonna, M. (2000). Cutting edge: Signal‐ regulatory protein b1 is a DAP12‐associated activating receptor expressed in myeloid cells. J. Immunol. 164, 9–12. Doucey, M.‐A., Scarpellino, L., Zimmer, J., Guillaume, P., Luescher, I. F., Bron, C., and Held, W. (2004). Cis association of Ly49A with MHC class I restricts natural killer cell inhibition. Nat. Immunol. 5, 328–335. Fournier, N., Chalus, L., Durand, I., Garcia, E., Pin, J.‐J., Churakova, T., Patel, S., Zlot, C., Gorman, D., Zurawski, S., Abrams, J., Bates, E. E., and Garrone, P. (2000). FDF03, a novel inhibitory receptor of the immunoglobulin superfamily, is expressed by human dendritic and myeloid cells. J. Immunol. 165, 1197–1209. Gonzalez, L. C., Loyet, K. M., Calemine‐Fenaux, J., Chauhan, V., Wranik, B., Ouyang, W., and Eaton, D. L. (2005). A coreceptor interaction between the CD28 and TNF receptor family members B and T lymphocyte attenuator and herpesvirus entry mediator. Proc. Natl. Acad. Sci. USA 102, 1116–1121.

r ec o g n i t i o n sys t em o f m h c cl a s s i m o l e cu l e s

189

Greenwald, R. J., Freeman, G. J., and Sharpe, A. H. (2005). The B7 family revisited. Annu. Rev. Immunol. 23, 515–548. Hayami, K., Fukuta, D., Nishikawa, Y., Yamashita, Y., Inui, M., Ohyama, Y., Hikida, M., Ohmori, H., and Takai, T. (1997). Molecular cloning of a novel murine cell‐surface glycoprotein homologous to killer cell inhibitory receptors. J. Biol. Chem. 272, 7320–7327. Heeger, P. S. (2003). T‐cell allorecognition and transplant rejection: A summary and update. Am. J. Transplant. 3, 525–533. Ho, L. H., Uehara, T., Chen, C. C., Kubagawa, H., and Cooper, M. D. (1999). Constitutive tyrosine phosphorylation of the inhibitory paired Ig‐like receptor PIR‐B. Proc. Natl. Acad. Sci. USA 96, 15086–15090. Hoelsbrekken, S. E., Nylenna, O., Saether, P. C., Slettedal, I. O., Ryan, J. C., Fossum, S., and Dissen, E. (2003). Cutting edge: Molecular cloning of a killer cell Ig‐like receptor in the mouse and rat. J. Immunol. 170, 2259–2263. Hogarth, P. M. (2002). Fc receptors are major mediators of antibody‐based inflammation in autoimmunity. Curr. Opin. Immunol. 14, 798–802. Jensen, P. E., Sullivan, B. A., Reed‐Loisel, L. M., and Weber, D. A. (2004). Qa‐1, a nonclassical class I histocompatibility molecule with roles in innate and adaptive immunity. Immunol. Res. 29, 81–92. Ka¨ rre, K. (2002). NK cells, MHC class I molecules and the missing self. Scand. J. Immunol. 55, 221–228. Karsenty, G., and Wagner, E. F. (2002). Reaching a genetic and molecular understanding of skeletal development. Dev. Cell 2, 389–406. Kim, N., Takami, M., Rho, J., Josien, R., and Choi, Y. (2002). A novel member of the leukocyte receptor complex regulates osteoclast differentiation. J. Exp. Med. 195, 201–209. Koga, T., Inui, M., Inoue, K., Kim, S., Suematsu, A., Kobayashi, E., Iwata, T., Ohnishi, H., Matozaki, T., Kodama, T., Taniguchi, T., Takayanagi, H., and Takai, T. (2004). Costimulatory signals mediated by the ITAM motif cooperate with RANKL for bone homeostasis. Nature 428, 758–763. Kollnberger, S., Bird, L. A., Roddis, M., Hacquard‐Bouder, C., Kubagawa, H., Bodmer, H. C., Breban, M., McMichael, A. J., and Bowness, P. (2004). HLA‐B27 heavy chain homodimers are expressed in HLA‐B27 transgenic rodent models of spondyloarthritis and are ligands for paired Ig‐like receptors. J. Immunol. 173, 1699–1710. Kubagawa, H., Burrows, P. D., and Cooper, M. D. (1997). A novel pair of immunoglobulin‐like receptors expressed by B cells and myeloid cells. Proc. Natl. Acad. Sci. USA 94, 5261–5266. Kubagawa, H., Chen, C. C., Ho, L. H., Shimada, T. S., Gartland, L., Mashburn, C., Uehara, T., Ravetch, J. V., and Cooper, M. D. (1999). Biochemical nature and cellular distribution of the paired immunoglobulin‐like receptors, PIR‐A and PIR‐B. J. Exp. Med. 189, 309–318. Lanier, L. L., and Bakker, A. B. H. (2000). The ITAM‐bearing transmembrane adaptor DAP12 in lymphoid and myeloid cell function. Immunol. Today 21, 611–614. Lebbink, R. J., de Ruiter, T., Verbrugge, A., Bril, W. S., and Meyaard, L. (2004). The mouse homologue of the luekocyte‐associated Ig‐like receptor‐1 is an inhibitory receptor that recruits Src homology region 2‐containing protein tyrosine phosphatase (SHP)‐2 but not SHP‐1. J. Immunol. 172, 5535–5543. Lee, K. H., Ono, M., Inui, M., Yuasa, T., and Takai, T. (2000). Stimulatory function of gp49A, a murine Ig‐like receptor, in rat basophilic leukemia cells. J. Immunol. 165, 4970–4977. Liang, S., Baibakov, B., and Horuzsko, A. (2002). HLA‐G inhibits the functions of murine dendritic cells via the PIR‐B immune inhibitory receptor. Eur. J. Immunol. 32, 2418–2426. Litwin, V., Gumperz, J., Parham, P., Phillips, J. H., and Lanier, L. L. (1994). NKB1: A natural killer cell receptor involved in the recognition of polymorphic HLA‐B molecules. J. Exp. Med. 180, 537–543.

190

t o s h i y u k i tak a i

Liu, J., Liu, Z., Witkowski, P., Vlad, G., Manavalan, J. S., Scotto, L., Kim‐Schulze, S., Gortesini, R., Hardy, M. A., and Suciu‐Foca, N. (2004). Rat CD8 FOXP3 T suppressor cells mediate tolerance to allogeneic heart transplants, inducing PIR‐B in APC and rendering the graft invulnerable to rejection. Transplant. Immunol. 13, 239–247. Long, E. O. (1999). Regulation of immune responses through inhibitory receptors. Annu. Rev. Immunol. 17, 875–904. Maeda, A., Kurosaki, M., and Kurosaki, T. (1998a). Paired immunoglobulin‐like receptor (PIR)‐A is involved in activating mast cells through its association with Fc receptor g chain. J. Exp. Med. 188, 991–995. Maeda, A., Kurosaki, M., Ono, M., Takai, T., and Kurosaki, T. (1998b). Requirement of SH2‐ containing protein tyrosine phosphatases SHP‐1 and SHP‐2 for paired immunoglobulin‐like receptor B (PIR‐B)‐mediated inhibitory signal. J. Exp. Med. 187, 1355–1360. Magram, J., Connaughton, S. E., Warrier, R. R., Carvajal, D. M., Wu, C. Y., Ferrante, J., Stewart, C., Sarmiento, U., Faherty, D. A., and Gately, M. K. (1996). IL‐12‐deficient mice are defective in IFNg production and type 1 cytokine responses. Immunity 4, 471–481. Maliszewski, C. R., March, C. J., Schoenborn, M. A., Gimpel, S., and Shen, L. (1990). Expression cloning of a human Fc receptor for IgA. J. Exp. Med. 172, 1665–1672. Manavalan, J. S., Rossi, P. C., Vlad, G., Piazza, F., Yarilina, A., Cortesini, R., Mancini, D., and Suciu‐Foca, N. (2003). High expression of ILT3 and ILT4 is a general feature of tolerogenic dendritic cells. Transplant. Immunol. 11, 245–258. Mandelboim, O., Lieberman, N., Lev, M., Paul, L., Arnon, T. I., Bushkin, Y., Davis, D. M., Strominger, J. L., Yewdell, J. W., and Porgador, A. (2001). Recognition of haemagglutinins on virus‐infected cells by NKp46 activates lysis by human NK cells. Nature 409, 1055–1060. Mandelboim, O., and Porgador, A. (2001). NKp46. Int. J. Biochem. Cell Biol. 33, 1147–1150. Matsuda, Y., Harada, Y.‐N., Natsuume‐Sakai, S., Lee, K., Shiomi, T., and Chapman, V. M. (1992). Location of the mouse complement factor H gene (cfh) by FISH analysis and replication R‐ banding. Cytogenet. Cell Genet. 61, 282–285. Moretta, L., and Moretta, A. (2004). Unravelling natural killer cell function: Triggering and inhibitory human NK receptors. EMBO J. 23, 255–259. Mousseau, D. D., Banville, D., L’ Abbe, D., Bouchard., P., and Shen, S.‐H. (2000). PILRa, a novel immunoreceptor tyrosine‐based inhibitory motif‐bearing protein, recruits SHP‐1 upon tyrosine phosphorylation and is paired with the truncated counterpart PILRb. J. Biol. Chem. 275, 4467–4474. Nakajima, H., Samaridis, J., Angman, L., and Colonna, M. (1999). Human myeloid cells express an activating ILT receptor (ILT1) that associates with Fc receptor g‐chain. J. Immunol. 162, 5–8. Nakamura, A., Kobayashi, E., and Takai, T. (2004). Exacerbated graft‐versus‐host disease in  mice. Nat. Immunol. 5, 623–629. Pirb Ono, M., Yuasa, T., Ra, C., and Takai, T. (1999). Stimulatory function of paired immunoglobulin‐ like receptor‐A in mast cell line by associating with subunits common to Fc receptors. J. Biol. Chem. 274, 30288–30296. O’ Keefe, T. L., Williams, G. T., Davies, S. L., and Neuberger, M. S. (1996). Hyperresponsive B cells in CD22‐deficient mice. Science 274, 798–801. Oshima, K., Ruhul Amin, A. R., Suzuki, A., Hamaguchi, M., and Matsuda, S. (2002). SHPS‐1, a multifunctional transmembrane glycoprotein. FEBS Lett. 519, 1–7. Otipoby, K. L., Andersson, K. B., Draves, K. E., Klaus, S. J., Farr, A. G., Kerner, J. D., Perlmutter, R. M., Law, C. L., and Clark, E. A. (1996). CD22 regulates thymus‐independent responses and the lifespan of B cells. Nature 384, 634–637. Pereira, S., and Lowell, C. (2003). The Lyn tyrosine kinase negatively regulates neutrophil integrin signaling. J. Immunol. 171, 1319–1327.

r ec o g n i t i o n sys t em o f m h c cl a s s i m o l e cu l e s

191

Phillips, J. H., Gumperz, J. E., Parham, P., and Lanier, L. L. (1995). Superantigen‐dependent, cell‐ mediated cytotoxicity inhibited by MHC class I receptors on T lymphocytes. Science 268, 403–405. Ravetch, J. V., and Lanier, L. L. (2000). Immune inhibitory receptors. Science 290, 84–89. Ravetch, J. V., Luster, A. D., Weinshank, R., Kochan, J., Pavlovec, A., Portnoy, D. A., Hulmes, J., Pan, Y. C., and Unkeless, J. C. (1986). Structural heterogeneity and functional domains of murine immunoglobulin G Fc receptors. Science 234, 718–725. Rodan, G. A., and Martin, T. J. (2000). Therapeutic approaches to bone diseases. Science 289, 1508–1514. Rudge, E. U., Cutler, A. J., Pritchard, N. R., and Smith, K. G. C. (2002). Interleukin 4 reduces expression of inhibitory receptors on B cells and abolishes CD22 and FcgRII‐mediated B cell suppression. J. Exp. Med. 195, 1079–1085. Sedy, J. R., Gavrieli, M., Potter, K. G., Hurchla, M. A., Lindsley, R. C., Hildner, K., Scheu, S., Pfeffer, K., Ware, C. F., Murphy, T. L., and Murphy, K. M. (2005). B and T lymphocyte attenuator regulates T cell activation through interaction with herpesvirus entry mediator. Nat. Immunol. 6, 90–98. Shiratori, I., Ogasawara, K., Saito, T., Lanier, L. L., and Arase, H. (2004). Activation of natural killer cells and dendritic cells upon recognition of a novel CD99‐like ligand by paired immunoglobulin‐like type 2 receptor. J. Exp. Med. 199, 525–533. Shiroishi, M., Tsumoto, K., Amano, K., Shirakihara, Y., Colonna, M., Braud, V. M., Allan, D. S., Makadzange, A., Rowland‐Jones, S., Willcox, B., Jones, E. Y., van der Merwe, P. A., Kumagai, I., and Maenaka, K. (2003). Human inhibitory receptors Ig‐like transcript 2 (ILT2) and ILT4 compete with CD8 for MHC class I binding and bind preferentially to HLA‐G. Proc. Natl. Acad. Sci. USA 100, 8856–8861. Shlomchik, W. D., Couzens, M. S., Tang, C. B., McNiff, J., Robert, M. E., Liu, J., Shlomchik, M. J., and Emerson, S. G. (1999). Prevention of graft versus host disease by inactivation of host antigen‐presenting cells. Science 285, 412–415. Takai, T. (2002). Roles of Fc receptors in autoimmunity. Nat. Rev. Immunol. 8, 580–592. Takai, T., Li, M., Sylvestre, D., Clynes, R., and Ravetch, J. V. (1994). FcR g chain deletion results in pleiotrophic effector cell defects. Cell 76, 519–529. Takai, T., and Ono, M. (2001). Activating and inhibitory nature of the murine paired Ig‐like receptor (PIR) family. Immunol. Rev. 181, 215–222. Takayanagi, H., Kim, S., Koga, T., Nishina, H., Isshiki, M., Yoshida, H., Saiura, A., Isobe, M., Yokochi, T., Inoue, J., Wagner, E. F., Mak, T. W., Kodama, T., and Taniguchi, T. (2002). Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts. Dev. Cell 3, 889–901. Taylor, L. S., and McVicar, D. W. (1999). Functinal association of Fc
RIg with arginine632 of paired immunoglobulin‐like receptor (PIR)‐A3 in murine macrophages. Blood 94, 1790–1796. Teitelbaum, S. L., and Ross, F. P. (2003). Genetic regulation of osteoclast development and function. Nat. Rev. Genet. 4, 638–649. Timms, J. F., Carlberg, K., Gu, H., Chen, H., Kamatkar, S., Nadler, M. J., Rohrschneider, L. R., and Neel, B. G. (1998). Identification of major binding proteins and substrates for the SH2‐ containing protein tyrosine phosphatase SHP‐1 in macrophages. Mol. Cell. Biol. 18, 3838–3850. Tomasello, E., Cant, C., Buhring, HJ., Vely, F., Andre, P., Seiffert, M., Ullrich, A., and Vivier, E. (2000). Association of a signal‐regulatory proteins b with KARAP/DAP‐12. Eur. J. Immunol. 30, 2147–2156. Tun, T., Kubagawa, Y., Dennis, G., Burrows, P. D., Cooper, M. D., and Kubagawa, H. (2003). Genomic structure of mouse PIR‐A6, an activating member of the paired immunoglobulin‐like receptor gene family. Tissue Antigens 61, 220–230.

192

t o s h i y u k i tak a i

Uehara, T., Blery, M., Kang, D. W., Chen, C. C., Ho, L. H., Gartland, G. L., Liu, F. T., Vivier, E., Cooper, M. D., and Kubagawa, H. (2001). Inhibition of IgE‐mediated mast cell activation by the paired Ig‐like receptor PIR‐B. J. Clin. Invest. 108, 1041–1050. Ujike, A., Takeda, K., Nakamura, A., Ebihara, S., Akiyama, K., and Takai, T. (2002). Impaired dendritic cell maturation and increased TH2 responses in PIR‐B–/– mice. Nat. Immunol. 3, 542–548. van den Berg, T. K., Yoder, J. A., and Litman, G. W. (2004). On the origins of adaptive immunity: Innate immune receptors join the tale. Trends Immunol. 25, 11–16. Wagtmann, N., Biassoni, R., Cantoni, C., Verdiani, S., Malnati, M. S., Vitale, M., Bottino, C., Moretta, L., Moretta, A., and Long, E. O. (1995). Molecular clones of the p58 NK cell receptor reveal immunoglobulin‐related molecules with diversity in both the extra‐ and intracellular domains. Immunity 2, 439–449. Wang, Y., Subudhi, S. K., Anders, R. A., Lo, J., Sun, Y., Blink, S., Wang, Y., Wang, J., Liu, X., Mink, K., Degrandi, D., Pfeffer, K., and Fu, Y. X. (2005). The role of herpesvirus entry mediator as a negative regulator of T cell‐mediated responses. J. Clin. Invest. 115, 711–717. Wende, H., Colonna, M., Ziegler, A., and Volz, A. (1999). Organization of the leukocyte receptor cluster (LRC) on human chromosome 19q13.5. Mamm. Genome 10, 154–160. Wheadon, H., Paling, N. R. D., and Welham, M. J. (2002). Molecular interactions of SHP1 and SHP2 in IL‐3 signalling. Cell. Signalling 14, 219–229. Willcox, B. E., Thomas, L. M., and Bjorkman, P. J. (2003). Crystal structure of HLA‐A2 bound to LIR‐1, a host and viral major histocompatibility complex receptor. Nat. Immunol. 4, 913–919. Wilson, M. J., Torkar, M., Haude, A., Milne, S., Jones, T., Sheer, D., Beck, S., and Trowsdale, J. (2000). Plasticity in the organization and sequences of human KIR/ILT gene families. Proc. Natl Acad. Sci. USA 97, 4778–4783. Yamashita, Y., Fukuta, D., Tsuji, A., Nagabukuro, A., Matsuda, Y., Nishikawa, Y., Ohyama, Y., Ohmori, H., Ono, M., and Takai, T. (1998a). Genomic structures and chromosomal location of p91, a novel murine regulatory receptor family. J. Biochem. 123, 358–368. Yamashita, Y., Ono, M., and Takai, T. (1998b). Inhibitory and stimulatory functions of paired Ig‐like receptor (PIR) family in RBL‐2H3 cells. J. Immunol. 161, 4042–4047. Yotsumoto, K., Okoshi, Y., Shibuya, K., Yamazaki, S., Tahara‐Hanaoka, S., Honda, S., Osawa, M., Kuroiwa, A., Matsuda, Y., Tenen, D. G., Iwama, A., Nakauchi, H., and Shibuya, A. (2003). Paired activating and inhibitory immunoglobulin‐like receptors, MAIR‐I and MAIR‐II, regulate mast cell and macrophage activation. J. Exp. Med. 198, 223–233. Zhang, G., Young, J. R., Tregaskes, C. A., Sopp, P., and Howard, C. J. (1995). Identification of a novel class of mammalian Fc g receptor. J. Immunol. 155, 1534–1541. Zhang, H., Meng, F., Chu, C. L., Takai, T., and Lowell, C. A. (2005). The Src family kinases Hck and Fgr negatively regulate neutrophil and dendritic cell chemokine signaling via PIR‐B. Immunity 22, 235–246.

Dendritic Cell Biology Francesca Granucci, Maria Foti, and Paola Ricciardi‐Castagnoli Department of Biotechnology and Bioscience, University of Milano‐Bicocca, Piazza della Scienza 2, 20126 Milan, Italy

1. 2. 3. 4. 5. 6.

Abstract............................................................................................................. Introduction ....................................................................................................... DC Subtypes ...................................................................................................... Deciphering DC Biology with Genomic Approaches .................................................. DC Interactions with the Microbial World ............................................................... DC Functions..................................................................................................... Conclusions........................................................................................................ References .........................................................................................................

193 193 194 196 199 209 219 219

Abstract Dendritic cells (DCs) are a special type of leukocytes able to alert the immune system to the presence of infections. They play a central role in the initiation of both innate and adaptive immune responses. This particular DC feature is regulated by the activation of specific receptors at the cell surface called Toll‐ like receptors (TLRs) that bind a number of microbial products collectively referred to as microbial-associated molecular patterns (MAMP). TLRs initiate a cascade of events, which together define the process of DC maturation. This phenomenon allows DCs to progressively acquire varying specific functions. DC maturation depends on the nature of the perturbation and permits unique and efficient immune responses for each pathogen. In this review the discussion is focused on DCs in the context of interactions with pathogens and DC‐specific functions are highlighted. 1. Introduction During millions of years of coevolution, microorganisms have evolved mechanisms to infect the host by subverting the organism’s first lines of defense, the mucosal barrier and the innate response. The innate immune system is as old as the emergence of multicellular organisms and has been selected over evolutionary time rather than in individual cells. Indeed the receptors for innate immunity are found in all multicellular organisms, whereas adaptive immunity receptors are found uniquely in vertebrates. Because it is phylogenetically more ancient, the innate response has been regarded as a response broadly directed to the clearance of microorganisms. However, the discovery of a new class of receptors, involved in the

193 advances in immunology, vol. 88 # 2005 Elsevier Inc. All rights reserved.

0065-2776/05 $35.00 DOI: 10.1016/S0065-2776(05)88006-X

194

f ra n c e s c a g r a n uc c i

ET AL.

recognition of defined groups of microorganisms has more recently readdressed the role of innate immunity as a sophisticated discriminating system that uses a broad innate receptor repertoire to sense the nature of the environmental perturbation (Medzhitov and Janeway, 1997). Indeed, the induction of different types of effector responses seems to be dictated and conditioned by the type of innate response that follows microbial recognition. It was first suggested that the innate immune responses could be activated via receptors called pattern recognition receptors (PRRs) that are able to recognize microbial associated molecular patterns (Janeway, 1992). The identification of the Toll‐like receptor (TLR) family members capable of binding specific components of a wide variety of pathogens has provided evidence to this new theory (Medzhitov et al., 1997). DCs were first described as the ‘natural adjuvants’ inducing adaptive immune responses (Banchereau and Steinman, 1998; Ibrahim et al., 1995; Steinman, 1991). They represent a special type of leukocytes able to alert the immune system to the presence of infections and responsible for activation and control of both innate and adaptive immune responses (Steinman, 2001; Zitvogel, 2002). DCs are especially distributed in tissues that interface the external environment, such as the skin, the gut, and the lungs (Nelson et al., 1994; Nestle et al., 1993; Sertl et al., 1986), where they can perform a sentinel function for incoming pathogens, and have the capacity to recruit and activate cells of the innate immune system (Fernandez et al., 1999; Foti et al., 1999; Rescigno et al., 1998). After antigen uptake has occurred, DCs efficiently process antigens for their presentation in association with major histocompatibility complex (MHC) molecules. However, before DCs can prime the adaptive immune response they must complete a full maturation process that is initiated by direct exposure to TLR ligands or to other receptors of the innate receptor repertoire. Interaction with pathogens results in a DC activation state that leads to their migration to the T‐cell area of lymph nodes where antigen‐specific cells of the adaptive immune response can be primed. Given the high plasticity of DCs, the signals that determine a particular DC function, and consequently, the type of adaptive immune response, depend mostly on the local microenvironment and on the interaction between the DCs and the microbial signals. These interactions are complex and very different from one pathogen to the next. In this review the discussion is focused on DCs in the context of interactions with pathogens and DC‐specific functions are highlighted. 2. DC Subtypes DCs originate from hematopoietic precursors within the bone marrow. Different subtypes have been described according to the surface expression of

d en d r i t i c c e l l b i o l og y

195

particular markers and tissue distribution (Ardavin, 2003; Ardavin et al., 2001; Shortman and Liu, 2002). In mice, immature DCs are characterized by the expression of CD11c (the integrin a‐chain), low levels of the costimulatory molecules CD80 and CD86, and low levels of MHC class II (MHC II); these molecules can be upregulated at the cell surface upon activation (Henri et al., 2001). Interestingly, DCs can also express the T‐cell markers CD4 and CD8. This last molecule is expressed as aa‐homodimer, whereas in T cells are expressed as ab‐heterodimer. Another molecule that has been used to identify mouse DC subtypes is the CD11b; using CD4, CD8, and CD11b markers five distinct DC classes have been identified. Three DC classes have been observed in the spleen, CD4þCD8–, CD4–CD8þ, and CD4–CD8–. The CD8– DC reside mostly in the marginal zone while the CD8þ are mainly located in the T‐cell area (Shortman and Liu, 2002). CD8– DCs can migrate to T‐cell areas following stimulation with microbial stimuli (De Smedt et al., 1998). Another DC population has been identified in all lymph nodes. These lymph node DCs are CD4–CD8–CD11bþ and also express moderate levels of the scavenger receptor CD205 (Anjuere et al., 1999). Finally, in skin‐draining lymph nodes an additional DC subtype has been found. It expresses high levels of Langerin, a molecule typically produced by Langerhans cells (LCs), an immature population of DCs located in the skin (Henri et al., 2001). In humans, DC subtypes are less characterized. Human DCs do not express CD8 and in spleen and tonsils, DCs differentially positive for CD11b, CD11c, and CD4 have been identified (Shortman and Liu, 2002). Most of the information on human DC subtypes and their possible origin derive from in vitro studies. Blood monocytes are the most commonly used precursors to generate DC in culture. In the presence of granulocyte macrophage colony‐stimulating factor (GM‐CSF) and interleukin (IL)‐4, monocytes can differentiate to immature nonproliferating DCs, expressing low levels of CD86 and MHC II (Sallusto and Lanzavecchia, 1994). Following incubation with inflammatory products these DCs can reach the mature phenotype showing high levels of MHC II and costimulatory molecules (Sallusto and Lanzavecchia, 1994). A second human DC subtype, called interferon (IFN)‐ producing plasmacytoid DC was found in blood and many lymphoid tissues (Kadowaki and Liu, 2002). They are phenotypically characterized as CD11c–CD45RAþCD123þ. Recently the corresponding population of IFN‐ producing plasmacytoid DCs has been also identified in mouse blood and lymph nodes (Asselin‐Paturel et al., 2001). In this review, the discussion will focus on the functions of conventional myeloid CD8þCD11cþ, CD8–CD11cþCD11bþ mouse DCs, and human monocyte‐derived DCs.

196

f ra n c e s c a g r a n uc c i

ET AL.

3. Deciphering DC Biology with Genomic Approaches The immune response involves a complex network of dynamic interactions of a wide array of tissues, cells, and molecules. Each single cell of the immune system recruited in response to a perturbation induced by an invading microorganism undergoes a sophisticated activation process. This process transforms it from a quiescent cell to an effector functional cell able to communicate with other effectors. The final goal is to eliminate the perturbation and restore resting conditions. All these processes are extremely complex and their interpretation requires integrated approaches that, together with the classical reductionistic studies, help to reconstruct the complexity of immunological phenomena. Traditional approaches are based on analysis of a single parameter at a time and, though they provide detailed knowledge of a particular molecular entity or a particular isolated phenomenon, they cannot provide an exhaustive interpretation regarding how the immune system fights a particular pathogen, maintains self‐tolerance, or remembers past infections. The completion of draft sequences of the human and mouse genomes offers many opportunities for gene discovery in the field of immunology through the application of methods of computational genomics. In concert with emerging genomic and proteomic technologies, the biology of the immune system can be defined. Initiation and regulation of the immune response is complicated and occurs on many levels. Multicellular organisms have been obliged to develop multifaceted innate and adaptive immune systems to cope with the challenges to survival originating from microorganisms and their products. The diversity of innate immune mechanisms is in large part conserved in all multicellular organisms (Mushegian and Medzhitov, 2001). Some basic principles of microbial recognition and response are emerging, and recently, the application of computational genomics has played an important role in extending such observations from model organisms, such as Drosophila, to higher vertebrates, including humans (Burley and Bonanno, 2002). Analysis of gene expression in tissues, cells, and biological systems has evolved in the last decade from analysis of a selected set of genes to an efficient high‐throughput whole‐genome screening approach of potentially all genes expressed in a tissue or cell sample. Development of sophisticated methodologies such as microarray technology for gene expression studies allows an open‐ended survey to identify comprehensively the fraction of genes that are differentially expressed between samples and define the samples’ unique biology. This discovery‐based research provides the opportunity to characterize either new genes with unknown function or genes not previously known to be involved in a biological process, and can lead to unpredictable and unexpected results, which may advance new insights in immunology.

d en d r i t i c c e l l b i o l og y

197

Large‐scale gene expression analysis is of great relevance in the field of immunology to generate a global view of how the immune system integrates its resources to maintain the integrity of the organism. To this purpose, the study of host–pathogen interactions is instrumental in understanding how to control infectious diseases. Host eukaryotes are constantly exposed to attacks by microbes seeking to colonize and propagate in host cells. To counteract them, host cells utilize a whole battery of defense systems to combat microbes. However, in turn, successful microbes evolved sophisticated systems to evade host defense. As such, interactions between hosts and pathogens are perceived as evolutionary arms races between genes of the respective organisms (Bergelson et al., 2001; Kahn et al., 2002; Woolhouse et al., 2002). Any interaction between a host and its pathogen involves alterations in cell signaling cascades in both partners that may be mediated by transcriptional or posttranslational changes. Transcriptomics is one of the methodologies that can be used to select important genes to study in detail from among thousands of genes encoded in the genome and can help in defining complex gene networks. Many genomic studies have been performed to interpret how human and mouse DCs respond to microbial and nonmicrobial inflammatory stimuli. In kinetic experiments, gene expression profiles of immature in vitro‐derived mouse or human DCs have been compared with gene expression patterns of activated DCs at different times after challenge with the activation stimulus (Tang and Saltzman, 2004). Analysis of entire kinetic data sets has revealed that DCs undergo a profound reorganization of gene expression in the first few hours after activation and then they progress versus a new resting state that is clearly distinct from the original immature DC state (Aebischer et al., 2005; Granucci et al., 2001a); the process of DC maturation is almost complete 24 hours after activation (Granucci et al., 2001a). Moreover, the number of genes expressed at different stages of DC ontogeny remains similar, indicating that the same number of genes are induced and suppressed at different time‐points after activation. The diversity of transcripts expressed in immature, transitional, and mature DCs is, thus, similar in magnitude, as has already been suggested for resting and activated T cells (Marrack et al., 2000). Comparing the response of DCs to inflammatory microbial and nonmicrobial stimuli has shown clear differences in the activation of the DC maturation program induced by these two types of stimuli. In particular, when lipopolysaccharide (LPS) and Tumor Necrosis Factor (TNF)a have been compared, LPS has stimulated a more potent and more rapid stimulus for DC activation (Granucci et al., 2001b). The expression of many genes was modulated by LPS but not TNF, and most of the genes that are commonly modulated were upregulated or downregulated 2–5 times more with LPS than with TNF. Genes involved in the activation and control of inflammatory processes, such

198

f ra n c e s c a g r a n uc c i

ET AL.

as the complement component C1q, IL‐1b, IL‐1 receptor antagonist (IL‐ 1RA), and IL‐6, were markedly affected by DC exposure to LPS, but not to TNF. From this analysis, TNF emerged as a weak stimulus that could not drive DCs to a stage of maturation necessary for optimal immune‐response activation. This interpretation has been confirmed in a recent study showing that TNFa‐exposed DCs, although able to upregulate MHC II and costimulatory molecules, remain weak producers of proinflammatory cytokines and reach a semimature state that is tolerogenic rather than immunogenic (Lutz and Schuler, 2002; Menges et al., 2002). The tolerogenic activity of semimature DCs would be due to their capacity to induce IL‐10‐producing antigen‐ specific T cells (Menges et al., 2002). Besides in vitro‐derived DCs, the inefficacy of inflammatory nonmicrobial stimuli to induce full DC activation has also been demonstrated in vivo for naturally developing DCs. Indirect activation of DCs by inflammatory mediators generated antigen‐presenting DCs able to support T‐cell expansion but not the production of effector CD4þ T lymphocytes. In contrast, DC exposure to microbial stimuli resulted in generation of fully activated DCs able to sustain the differentiation of helper T‐cell responses (Sporri and Reise Sousa, 2005). These observations imply that DCs are extremely plastic and can adjust their responses depending on the nature of the stimulus they encounter. Therefore, DCs can distinguish between the actual presence of an infection and an inflammatory process mediated only by cytokines (Sporri and Reis e Sousa, 2005). This is particularly relevant for the activation of the appropriate responses. Genomic approaches for the study of DC maturation have emphasized this aspect revealing that DCs are able to activate distinct genetic reprogramming depending on the nature of the stimulus, even if the diverse stimuli are all of microbial origin. Thus, for distinct microorganisms it is always possible to identify a core and a pathogen‐specific DC response (Huang et al., 2001). An important aspect of using genomic approaches is the identification of molecules that regulate biological functions and, in this particular context, DC functions. One of the most unanticipated finding of global gene expression analysis applying to the study of mouse DC maturation was that they produce IL‐2 transiently at early time points after microbial encounter. The first observation was made for DCs stimulated with Gram‐negative bacteria (Granucci et al., 2001a). Afterward, it was shown that many different microbial stimuli are able to induce IL‐2 production by DCs while none of the inflammatory cytokine tested was able to elicit IL‐2 production by DCs (Granucci et al., 2003a), confirming the hypothesis that DCs are extremely plastic and can modulate their responses depending on the nature of the stimulus. DC‐ derived IL‐2 has been associated with regulatory functions in both innate and adaptive immune responses in mice (Granucci et al., 2001a; 2004).

d en d r i t i c c e l l b i o l og y

199

4. DC Interactions with the Microbial World 4.1. DC Receptor Repertoire 4.1.1. Toll‐like Receptor Family The receptor repertoire of DCs include a broad family of receptors that recognize microbial components which, whether pathogenic or not, serve as ligands to alert the immune response. One of the fastest growing families, collectively named TLRs, senses a distinct repertoire of conserved molecules expressed by fungi, viruses, bacteria, and protozoa. The TLR field was initiated thanks to the observation that Toll‐mutant flies were highly susceptible to fungal infection (Lemaitre et al., 1996; Poltorak et al., 1998). This study revealed for the first time that the innate immune system had a sophisticated means of detecting invasion by pathogens, such as Aspergillus fumigatus. Mammalian homologues were identified initially on the basis of their homology to the Drosophila Toll protein, the developmental protein required for antifungal immune responses in adult flies (Medzhitov et al., 1997). In 1998 one mammalian homologue named TLR4 was positionally identified as the LPS receptor, encoded by the Lps locus, known to be required in mice to respond to Gram‐negative bacteria (Poltorak et al., 1998). Since then, many other mammalian TLR have been identified: 10 in humans and 12 in mice, named TLR‐1 to TLR‐12 (Beutler, 2004). Their discovery is as fundamental as the discovery of antigen receptors on lymphocytes. Understanding TLR signaling pathways may allow us to dissect the complexity of immune responses in the balance between immunopathology and protective immunology. TLRs are expressed and modulated in myeloid mouse DCs and have a more selective distribution on human blood DCs (Degli‐Esposti and Smyth, 2005). Not all TLRs are expressed on the cell surface of DCs. TLR1, TLR2, and TLR4 are located at the cell surface; in contrast, TLR3, TLR7, and TLR9, all of which are involved in the recognition of microbial nucleic‐acid‐like structures, are not present on the cell surface (Ahmad‐Nejad et al., 2002; Heil et al., 2003; Matsumoto et al., 2003), but rather in the endoplasmic reticulum (ER). These receptors can be recruited from the ER to the endosomal compartments by microbial nucleic acid ligands (Latz et al., 2004). The TLR molecules are characterized by an extracellular domain with a leucine‐rich repeats (LRR) and a cytoplasmic domain (TIR domain) similar to that of the IL‐1R family (Akira and Takeda, 2004; Takeda et al., 2003). Functional analysis of each mammalian TLR member has revealed that they recognize different microbial ligands, such as LPS, lipoproteins, peptidoglycan, bacterial CpG DNA, single‐ and double‐stranded RNA, and bacterial flagellin, which are mostly conserved among pathogens and are not found in

200

f ra n c e s c a g r a n uc c i

ET AL.

mammals. In particular, the microbial compounds able to activate cellular responses mediated by TLR2, TLR3, TLR4, TLR5, TLR7, and TLR9 have been identified (Bendelac and Medzhitov, 2002; Diebold et al., 2004). In general, TLR2 is mainly involved in responses to microbial products from Gram‐positive bacteria (peptidoglycans or lipoproteins) as well as from yeast (Hirschfeld et al., 1999; Underhill et al., 1999; Yoshimura et al., 1999). By contrast, TLR4 mediates the interaction with Gram‐negative bacteria, by transducing the signals deriving from LPS. The activation of TLR signaling pathways originates from the cytoplasmic TIR domains. Mutations of the TIR domain of the tlr4 gene in C57BL/10ScCr or C3H/HeJ mice impede LPS signal transduction so that these mutant mice become resistant to endotoxin and highly susceptible to Gram‐negative infection (Poltorak et al., 1998). Downstream of the TIR domain, the adaptor protein Myeloid Differentiation Factor 88 (MyD88) has been shown to recruit the IL‐ 1 receptor‐associated kinase (IRAK) when activated. IRAK is then activated by phosphorylation and its association with TRAF6 (tumor‐necrosis‐factor‐ receptor‐associated‐factor 6) leads to the activation and the nuclear translocation of NF‐kB transcription factor or to the activation of c‐Jun N‐terminal kinase (JNK) (Takeda and Akira, 2004). In addition to the adaptor protein MyD88, other relevant adaptors have been identified: TRIF, TRAM, and TIRAP (Beutler, 2004). A MyD88‐independent pathway, typical of the TLR3 and TLR4 signaling pathways, has also been described (Akira et al., 2001; Takeuchi et al., 1999). This pathway activates IFN‐regulatory factor (IRF3) (Taniguchi and Takaoka, 2002) leading to the production of IFNb and the expression of a number of IFN‐inducible genes such as IRG1 (immunoresponsive gene1), GARG16 (glucocorticoid‐attenuated response gene 16), or the IFN‐inducible protein (IP)10 chemokine. Thus, myeloid DC activation can lead to type I IFN production. In particular, DC secretion of type I IFNs has been first shown after viral infections (Diebold et al., 2003). It was demonstrated that dsRNA, produced by most viruses, can elicit type I IFN production in MyD88‐ independent manner. Subsequently, it has been observed that type I IFN are rapidly produced by DCs also following exposure to many other infectious agents, including bacteria (Granucci et al., 2004), and play a key role in the control of immune responses. Interestingly, in myeloid DCs, it has been shown that helminths, such as the Schistosoma mansonii, trigger the MyD88‐ independent pathway leading to IFNb secretion (Trottein et al., 2004). Surprisingly, it has been found that Schistosoma eggs contain a TLR3 ligand (Aksoy et al., 2005). Thus, the engagement of TLRs in DCs leads to the production of type I IFNs (IFNa and b) that are responsible for the subsequent expression of chemokine genes in DCs and DC maturation

d en d r i t i c c e l l b i o l og y

201

(Luft et al., 1998). Type I IFNs produced by TLR‐activated DCs have been shown to elicit specific Ig production of all isotypes (Le Bon et al., 2001). There is an increased interest in studying the role of TLR signaling in host defense against live pathogens. Notably, MyD88‐deficient mice still generate an immune response against viruses and intracellular bacteria such as Mycobacterium tuberculosis or Listeria monocytogenes, and TLR‐independent mechanisms for their recognition have been proposed (Inohara and Nunez, 2003). TLRs recognize microbial ligands in the extracellular space, but many pathogens reach the cell cytoplasm so that hosts have had to evolve mechanisms to detect intracellular pathogens. For viral infections, a good example of this coevolution is the detection of dsRNA in the cell cytoplasm by the activated endogenous protein kinase R (PKR, IFN‐inducible dsRNA‐dependent protein kinase). The relevance of this host recognition is exemplified by the presence of the influenza virus protein NS1 that can mask the viral dsRNA genome and prevent PKR activation (Diebold et al., 2003). Intracellular receptors are also NOD1 and NOD2 (nucleotide‐binding oligomerization domain) that bind in the cytosol, the core structures of bacterial peptidoglycans. The NOD protein family has been shown to act as intracellular receptors of bacterial lipids (Girardin et al., 2003) and to play a major role in shaping the cell response to cytoplasmic invasion by L. monocytogenes (McCaffrey et al., 2004). NOD proteins may have evolved to complement the detection of pathogens through intracellular recognition also in DCs. DC maturation induced by TLRs seems to direct polarization of the effector cells of the adaptive response. In this regard, the so‐called ‘dirty trick of immunologists’ to mix the protein antigen with Freund adjuvants to achieve immunization, now has a molecular basis. The active component of the Freund adjuvant, heat‐killed M. tuberculosis, has several ligands for TLRs. It has been demonstrated that antigen mixed with adjuvant failed to trigger T‐cell responses in MyD88‐deficient mice, indicating that DC engagement of TLRs in vivo is necessary to induce T‐cell priming (Schnare et al., 2001). In addition to the Freund adjuvant, several other microbial components have potent immune‐stimulatory activity on DCs. CpG DNA, which is recognized by TLR9, is a potent adjuvant eliciting Th1 responses (Krieg, 2000; Lipford et al., 1998) and the outer membrane protein (the porin) of Neisseria, which is recognized by TLR2 is also an adjuvant with potent immunogenicity (Massari et al., 2002). The cell‐wall skeletal fraction (CWS) of M. bovis Calmette‐ Guerin strain (BCG) recognized by TLR2 and TLR4 and used as an adjuvant in anticancer therapy also has a potent immunostimulatory effect on DCs (Azuma and Seya, 2001; Tsuji et al., 2000).

202

f ra n c e s c a g r a n uc c i

ET AL.

TLR engagement promotes uptake of bacteria through upregulation of expression of scavenger receptors (SR), such as SR‐1 and the Mannose Receptor (MMR) (Doyle et al., 2004) and through maturation of the phagosome (Blander and Medzhitov, 2004). In DCs, TLR signaling induces delivery of degraded antigen to MHC II loading compartments and interestingly, through the upregulation of the expression of the MARCO receptor, it induces a cytoskeletal rearrangement (Granucci et al., 2003b). Comparing the effects of the ligation of different TLRs in DCs has been a difficult task as the signals from the TLRs can be not only qualitative, but also quantitative and additive. For example, ligation of TLR4 on DCs can upregulate the expression of TLR2, TLR4, and TLR9 (An et al., 2002; Nilsen et al., 2004; Visintin et al., 2001), resulting in signaling amplification. Furthermore, when molecularly defined ligands are substituted with live pathogens that possess multiple TLR‐activating ligands, the conditioning of DCs responses might become very complex because the adaptation of pathogens might have evolved to circumvent effector immune responses and therefore TLR‐mediated signaling. Recent studies analyzing the association between polymorphism in TLR sequences and disease susceptibility have demonstrated the protective role of TLRs in vivo. For example, polymorphisms in the TLR4 gene have been associated with increased susceptibility to respiratory syncytial virus (RSV) and Gram‐negative bacterial infections (Agnese et al., 2002; Tal et al., 2004). Similarly, a role for TLR5 has been demonstrated by the increase in Legionella pneumophila infection in individuals with a common polymorphism that introduces a stop codon into the TLR5 gene (Hawn et al., 2003). In mice, infection with Yersinia enterocolitica, a pathogen that can activate TLR2, is cleared more efficiently in the absence of TLR2 (Sing et al., 2002), but no protective effect of TLR2 was observed in Borrelia burgdorferi and Staphylococcus aureus infections (Takeuchi et al., 2000; Wooten et al., 2002). It has also been suggested that pathogens could exploit TLRs to generate nonresolving Th2‐biased responses. 4.1.2. C‐Type Lectin Family: Their Targeting by Infectious Agents Might Support Pathogen Spreading This family of receptors recognizes a wide range of carbohydrate structures. These receptors include the MMR, Dectin‐1, DEC‐205, DC‐SIGN, BDCA‐2, and Langerin. They all possess at least one carbohydrate recognition domain and bind sugars in a variety of secondary and tertiary structures (Geijtenbeek et al., 2004). Most of these molecules are involved in receptor‐mediated phagocytosis or endocytosis of microbes but some of them, such as the

d en d r i t i c c e l l b i o l og y

203

DC‐SIGN, actually support pathogen spreading. Thus, the adaptation of pathogens to target DC‐SIGN has assured longer pathogen survival. The MMR is preferentially expressed on myeloid cells and in DC. Increasing evidence shows that the MMR is involved in the silent clearance of inflammatory molecules and in maintenance of homeostasis. The MMR is an endocytic and phagocytic receptor that binds carbohydrate moieties on several pathogens, such as bacteria, fungi, parasites, and viruses, in particular Influenza virus (Reading et al., 2000) and HSV‐1 (Milone and Fitzgerald‐ Bocarsly, 1998; Rong et al., 2003). The high mannan content in the bacterial oligosaccharide cell envelope in both Gram‐positive and Gram‐negative bacteria assures efficient molecular recognition. In addition, MMR also binds endogenous molecules (Allavena et al., 2004). DC‐SIGN, the nonintegrin DC‐specific intercellular adhesion molecule 3 (ICAM‐3) is a type II transmembrane protein expressed on DCs with a C‐type lectin extracellular domain, capable of binding endogenous ligands such as ICAM‐2 and ICAM‐3 on resting T cells (Geijtenbeek et al., 2000b), but also capable of recognizing bacteria, fungi, (Geijtenbeek et al., 2004) and viruses. In particular, DC‐SIGN has been shown to bind the Human Immunodeficiency Virus (HIV‐1) envelope glycoprotein gp120 but does not function as a receptor for viral entry. Instead, DC‐SIGN allows mucosal DCs to carry HIV‐1 through the lymphatics in a ‘Trojan horse’ fashion, where it is eventually delivered to the T cells. Thus, the period of infectivity of HIV‐1 is increased by several days as a result of DC‐SIGN‐gp120 binding (van Kooyk and Geijtenbeek, 2003). DCs that express DC‐SIGN are mostly located close to the mucosal barriers, such as lamina propria DCs and dermal DCs. In contrast, LCs of the skin do not express DC‐SIGN but rather the C‐type lectin, Langerin. The discovery that in the early phase of infection DCs are among the first target cells of HIV at mucosal sites and from there the virus is shuttled to the lymph nodes and transmitted to the T cells has led to a reexamination of the mechanisms underlying interactions between DCs and T cells and the pathogenesis of HIV‐1 infection. Other pathogens, such as M. tuberculosis, have been also shown to target the DC‐SIGN. Notably, these pathogens misuse DC‐SIGN by distinct mechanisms that either circumvent antigen processing or alter TLR‐mediated signaling. This implies that adaptation of pathogens to target DC‐SIGN might support pathogen survival. Recently, the signaling events downstream of C‐type lectin receptors have attracted attention. It has been shown that Dectin‐1 and TLR2 can cooperate to increase zymosan‐induced signaling and cytokine secretion (Gantner et al., 2003). Cross‐linking the MMR, despite inducing upregulation of costimulatory molecules, induces secretion of IL‐10 (Chieppa et al., 2003; Nigou et al., 2001).

204

f ra n c e s c a g r a n uc c i

ET AL.

Finally, the pentraxin protein, PTX3, which is produced by activated DCs and plays an important role in the clearance of Aspergillus infections, is associated with various TLRs and may be a host mechanism to expand the repertoire of microbial structures that are recognized (Doni et al., 2003; Garlanda et al., 2002). The receptor repertoire of DCs to sense microbes is, in fact, much broader and also includes phagocytic opsonic receptors such as the Fc receptors and the complement receptors. DCs express a moderate level of Fc receptors that are not modulated during maturation. DCs also express the Mac‐1 molecule (CD11b/CD18; aMb2 integrin), which is the CR3 complement receptor used for phagocytosis of complement‐coated bacteria. As with FcR, the surface expression of Mac‐1 molecules is not changed during activation of DCs. This is in contrast to monocytes and neutrophils that strongly upregulate Mac‐1 expression during differentiation, and in the presence of inflammatory stimuli. In addition to its role in receptor‐mediated internalization, the Mac‐1 molecule also mediates adhesion and chemotaxis (Anderson et al., 1986). Studies have shown that Mac‐1 is stored in intracellular vesicles, which are rapidly mobilized to the cell surface in response to chemoattractants (Miller et al., 1987). 4.2. DC Interaction with Bacteria Despite several early reports on the uptake of particulate material and cells by DCs (Austyn, 1996), the phagocytic capacity of DCs has long been denied. One reason for this was the technical difficulty of growing DCs in their immature state. Only in the past decade has it become possible to grow and maintain in vitro homogeneous immature DCs as long‐term growth‐factor‐ dependent lines (Winzler et al., 1997). This has allowed the investigation of DC biology in the absence of other contaminating cell populations. DCs have phagocytic activity that decreases with maturation. Indeed, several studies have shown that DCs can internalize Latex and zymosan beads (Austyn, 1996; Inaba et al., 1993; Matsuno et al., 1996; Reis e Sousa et al., 1993), but also apoptotic bodies (Parr et al., 1991), as well as microbes such as BCG (Henderson et al., 1997; Inaba et al., 1993), Saccaromyces cerevisiae, Corynebacterium parvum, S. aureus (Reis e Sousa et al., 1993), Leishmania spp. (Blank et al., 1993), and B. burgdorferi (Filgueira et al., 1996). The ability of DCs to phagocytose particulates or bacteria is greatest in immature DCs, whereas this capacity is reduced, but not eliminated, in mature DCs (Henderson et al., 1997). DCs are critical components of the innate immune response to bacterial pathogens such as Salmonella typhimurium. These cells can have several roles during the early stage of an infection including controlling bacterial

d en d r i t i c c e l l b i o l og y

205

replication and producing cytokines and chemokines that activate and recruit additional cells. In the lamina propria, DCs have been shown to take up bacteria across the mucosal epithelium (Rescigno et al., 2001). Recently, this process has been elucidated: DCs open the tight junctions between epithelial cells, send dendrites outside of the epithelium, and sample bacteria directly in the gut lumen. The molecular mechanism that allows it to preserve the integrity of the epithelial barrier is based on expression and modulation by DCs of tight junction proteins, such as occludin, claudin 1, Zonula occludens 1, and junctional adhesion molecule (JAM) (Rescigno et al., 2001). Upon attachment, DCs engulf the bacterium by actively surrounding it with pseudopodia. This process is facilitated by Fc‐type and complement‐type receptor‐mediated endocytosis. The movement of the pseudopodia in activated DCs involves actin‐binding proteins, and it can be blocked by the drug cytochalasin D, which stops the polymerization of actin and inhibits phagocytosis. The rearrangement of the cytoskeleton, associated with DC motility, involves depolymerization of the actin (Winzler et al., 1997). Once a bacterium has been fully internalized into the phagosome, fusion of the phagosome with other intracellular vacuoles or granules takes place. Processing bacterial molecules for antigen presentation occurs in lysosomes following their fusion with phagosomes. This process may take several hours, as antigen presentation of bacterial antigens is not observed earlier than 6 h following infection (Rescigno et al., 1998). Some bacteria prevent the normal maturation and trafficking of the phagosome and impair its normal bacteriolytic activities. Other bacteria escape from the vacuole and can replicate in the cytosol. After infection with live bacteria, DCs sense the intruders by reprogramming the transcription of about 1000 genes (Granucci et al., 2001a). Cytokine and chemokine genes as well as IFN‐inducible genes are differentially expressed in the first few hours, whereas at later time‐points, apoptosis and antiapoptosis genes as well as genes involved in T‐cell activation are induced. Model antigens expressed in recombinant Gram‐positive and Gram‐ negative bacteria are processed and can be directly presented on both MHC I and II molecules (Corinti et al., 1999; Rescigno et al., 1998; Svensson et al., 1997). Unlike macrophages, this exogenous pathway of MHC I presentation is transporter associated with antigen presentation (TAP)‐dependent (Rescigno et al., 1998). This implies that exogenous bacterial antigens introduced by phagocytosis are directed into the classical pathway of MHC I presentation. Indeed, transport of whole bacterial proteins from phagolysosome to the cytosol takes place after phagocytosis of bacteria as also shown with immune‐ complex internalization (Rodriguez et al., 1999). The capacity of DCs to

206

f ra n c e s c a g r a n uc c i

ET AL.

present bacterial antigens with very high efficiency on both MHC I and II molecules can be exploited to induce strong and long‐lasting immunity to bacteria as well as nominal antigens of interest. Several examples of partial protective immune responses achieved by injecting in vivo DCs loaded with microbes have been described against Chlamydiae trachomatis (Su et al., 1998), B. burgdorferi (Mbow et al., 1997) or M. tuberculosis (Demangel et al., 1999). A few hours after bacterial infection, DCs synthesize a number of cytokines and chemokines (Rescigno et al., 1998). TNFa and IL‐6 are readily detected in DCs infected with either Gram‐positive or Gram‐negative bacteria. Interestingly, activation of DCs is achieved when bacteria are alive. Indeed, heat‐inactivated bacteria, although able to induce maturation of DCs, fail to induce inflammatory cytokines (Rescigno et al., 2002). Biologically active p70 IL‐12 is produced by myeloid mouse DCs only in a very small amount after bacterial encounter, as compared to human monocyte‐derived DCs (Corinti et al., 1999). TNFa production is rapidly induced following infection. It is likely that the phenotypic and the functional maturation, which occurs in DCs within 24 h of bacterial uptake is the result of cytokine amplification during this response. Indeed, DC activation by TNFa alone mimics the phenotypic maturation observed after bacterial infection, although the addition of anti‐TNFa antibodies only partly inhibits this process. This is consistent with the finding that the pattern of genes induced after activation of DC by TNFa and LPS is very different (Granucci et al., 2001b). Moreover, DC maturation obtained by whole bacteria is quantitatively and qualitatively more pronounced indicating the induction of several transducing pathways, likely via receptors that recognize distinct bacterial components. Treatment of DCs with bacteria results in a clear modification of cell‐ surface DC‐activation markers. Consistent with acquisition of costimulatory activity during maturation is the upregulation of CD86 and CD40 molecules. Upregulation of CD86 and CD40 molecules has also been observed with BCG (Thurnher et al., 1997) and M. tuberculosis (Henderson et al., 1997), but it was not observed following the use of inert Latex beads of various sizes. Upregulation of the costimulatory molecules and the coordinated translocation of MHC molecules at the cell surface are essential molecular events for the subsequent antigen presentation and activation of both CD4þ and CD8þ T cells. The internalization of bacteria is also associated with increased stability of MHC class I‐ and class II‐peptide complexes. Indeed, the half‐lives of MHC class II‐ and class I‐peptide complexes change from 10 to 20 h and from 3 to 9 h, respectively. This has important consequences for T‐cell induction because it increases the chances of DCs to encounter antigen‐specific T cells in the draining lymph nodes (Rescigno et al., 1998).

d en d r i t i c c e l l b i o l og y

207

4.3. DC Interaction with Viruses Mucosal surfaces represent the main sites of interaction of DC with viruses. The ability of DCs in inducing a strong antiviral immunity suggests that it would be an evolutionary advantage for viruses to subvert this particular cell. In fact DCs often act as a portal for virus invasion and viruses have indeed evolved to trigger epithelial responses, via chemokine production that result in the recruitment of DCs at the mucosal sites (Reinhart, 2003). Viruses that can target DC receptors are also able to evade the host’s immune system and acquire a selective advantage. An interesting example is provided by a‐dystroglycan (a‐DG), a DC receptor for lymphocytic choriomeningitis virus (LCMV), and several other Arenaviruses. By selection, variants of LCMV that bind a‐DG with high affinity replicate in the majority of DC, causing a generalized immune‐suppression, and establish a persistent infection. In contrast, viral strains that bind with low affinity to a‐DG display minimal replication in DCs and generate a robust anti‐LCMV cytotoxic T‐lymphocyte response that clears the virus infection. Hence, receptor–virus interaction on DCs in vivo is an essential step in the initiation of virus‐induced immune‐suppression and viral persistence (Sevilla et al., 2003). The best example is represented by HIV‐1 which has, in fact, evolved mechanisms to subvert DC immune function. DCs promote replication of HIV by capture and infection of the cells themselves followed by transmission of the virions to T cells. HIV‐1 infection of DCs can occur either via CD4 or chemokine receptors, leading to full viral replication, or via binding to C‐type lectin receptors resulting in transfer and replication of the virus to T cells. DCs respond to this invasion by processing viral proteins through MHC class I and II pathways and undergoing a maturation that enhances their presentation of antigen to T cells for induction of adaptive antiviral immunity (Wilflingseder et al., 2005). In contrast to acute HIV‐1 infection, individuals in the chronic phase regularly show impaired HIV‐1‐specific CD4 helper responses and very low or no specific neutralizing antibodies. In addition to HIV‐1, DCs are likely to be responsible for virus‐induced immune‐suppression that has been observed with measles (MV) infections or with members of the herpes virus family, such as herpes simplex virus (HSV) or human cytomegalovirus (HCMV). HSV‐1 infects the epidermis, where the predominant cell type it encounters is keratinocytes. These cells allow efficient replication (Mikloska et al., 1996). In the epidermis, LCs can be infected at early stages, whereas dermal DCs seem to be unaffected by the virus and no detectable viruses are found in the draining lymph nodes (Mueller et al., 2002). Following HSV‐1 infection, LC functions are impaired allowing the virus sufficient time to replicate and infect

208

f ra n c e s c a g r a n uc c i

ET AL.

a second round of keratinocytes. This increases the local virus titre and favors an efficient infection of nerve endings (van Lint et al., 2004) that are in close anatomical association with LCs (Hosoi et al., 1993); retrograde migration of the virus to the dorsal root ganglions (DRG) allows the establishment of latency (Jones, 2003). It is still unclear whether a lytic lesion breaks through the epidermal‐dermal barrier. Infection of LCs/DCs with herpes viruses such as HSV‐1, HSV‐2, HCMV, VZV (varicella‐zoster virus), or HSV‐6 results in an impaired capacity to stimulate T‐cell proliferation (Kakimoto et al., 2002; Morrow et al., 2003; Moutaftsi et al., 2002). The ability of HCMV to bind DC‐SIGN may aid HCMV infection of T cells in trans, similarly to that seen with HIV‐1 (Geijtenbeek et al., 2000a; Halary et al., 2002). In addition, Epstein‐Barr virus (EBV) and HCMV also prevent the differentiation of DCs from their precursors (Gredmark and Soderberg‐Naucler, 2003; Guerreiro‐Cacais et al., 2004; Li et al., 2002) providing the establishment of latency, a characteristic of all herpes viruses. In fact it has been shown that HCMV reservoirs are myeloid DC progenitors (Hahn et al., 1998). Herpes viruses also modify the expression of relevant molecules in the DC–T cell interaction. HSV‐1‐ and HCMV‐infected DCs are unable to upregulate costimulatory molecule expression during maturation (Moutaftsi et al., 2002; Salio et al., 1999) and CD83 downregulation has been observed (Morrow et al., 2003). Herpes viruses may have evolved to specifically target DC–T cell interaction. In fact, HSV‐1 and HCMV infection of DCs induces upregulation of TRAIL and Fas, resulting in bystander killing of antigen‐ specific T cells (Muller et al., 2004). Other viruses such as Parainfluenza virus (PIV) and RSV also impair infected‐DC ability to activate T cells (Bartz et al., 2003; Plotnicky‐Gilquin et al., 2001; Vidalain et al., 2000) or to secrete cytokines, including IL‐2 (Andrews et al., 2001; Fugier‐Vivier et al., 1997). The downregulation of IL‐2 production by DCs likely accounts for the profound immune‐suppression observed in infected individuals (Klagge et al., 2000; Marie et al., 2001). Other microbes may avoid immunity by altering migration of DCs; examples include Poxvirus that mediates the release of chemokines antagonists. Some viruses also act on several signal transduction pathways known to modulate DC functions. For example, Vaccinia viruses express intracellular proteins that interfere with signal transduction from TLR or cytokine receptors (Bowie et al., 2000; Harte et al., 2003) whereas Paramyxoviruses target the CD40 signaling pathway (Fugier‐Vivier et al., 1997). Thus, viruses have evolved several strategies to evade the immune response. This could have resulted in uncontrolled microbial replication, which would have been deleterious for the host. One may imagine that to counteract such strategy the

d en d r i t i c c e l l b i o l og y

209

mammalian host has developed strategies to control the infection with minimal damage to self‐tissues that consist primarily in the coordination of the innate and the adaptive immune responses through DC activation and TLR signal transduction. 5. DC Functions 5.1. Role of DCs in Innate Immunity As highlighted before, innate immunity is the most ancient line of defense against microbial infection. It is present and conserved in all animals throughout evolution (Janeway and Medzhitov, 2002). Among the cells that are involved in innate responses, an important role is held by natural killer (NK) cells. NK cells are lymphocytes of the innate immune system that potently contribute to infection eradication (Moretta, 2002). NK cells comprise about 15% of circulating lymphocytes and are also found in peripheral tissues. Recently, the presence of NK cells in resting human lymph nodes and in lymph nodes of mice injected with mature DCs has also been described (Ferlazzo et al., 2004; Martin‐Fontecha et al., 2004). NK cells exert their activity by producing high amounts of IFNg, which activates a strong inflammatory response, and by having direct cytotoxic function (Moretta, 2002). The functions of NK cells are regulated by a balance of activating and inhibiting signals. These signals are transmitted by inhibitory receptors, which bind MHC I molecules, and activating receptors, which bind ligands on tumors and pathogen‐infected cells (Smyth et al., 2002). Other than surface receptors, cytokines such as IL‐2, IL‐12, IL‐18, and type I IFNs have been shown to promote NK cell priming (Smyth et al., 2002). Recent studies have focused on the function of DCs during the early phases of the immune response, and a predominant role for DCs in activation of NK cells has been described both in mice and in humans (Ferlazzo et al., 2002; Fernandez et al., 1999; Gerosa et al., 2002; Piccioli et al., 2002). The first place of contact between NK cells and DCs could be the site of infection where both resident and recruited DCs would be able to activate NK cells (Moretta, 2002). Activated DCs can migrate to the draining lymph nodes where they can probably stimulate resident and newly recruited NK cells. Indeed, in the T‐cell area of human lymph nodes NK cells have been described as colocalizing with DCs (Ferlazzo et al., 2004). Moreover, it has been found that a subpopulation of NK cells enriched in lymph nodes (CD16–CD56hi) is able to respond to DC‐derived stimuli, such as IL‐12, that elicits IFNg production by NK cells, and membrane‐bound IL‐15, which induces NK cell proliferation (Ferlazzo et al., 2004).

210

f ra n c e s c a g r a n uc c i

ET AL.

Maturing DCs can be recruited at the draining lymph nodes via the upregulation of the expression of the CC‐chemokine receptor CCR7 (Weninger and von Andrian, 2003). Concerning NK cell recruitment, it has been proposed that inflammatory stimuli can induce NK cell migration at the draining lymph nodes with a process partially dependent on the expression of CXCR3 (Martin‐Fontecha et al., 2004). Thus following activation with inflammatory microbial stimuli, DCs would produce CXCR3 ligands (CXCR3Ls) that would favor the recruitment of NK cells (Martin‐Fontecha et al., 2004). In accordance with this hypothesis, we have found that DCs, activated with microbial stimuli (LPS and CpG) able to guarantee NK‐cell accumulation at the draining lymph nodes, produce large amounts of two CXCR3Ls, CXCL9 and CXCL10. This suggests that the accumulation of NK cell at the draining lymph nodes might actually be due to DC‐mediated CXCR3‐dependent NK cell recruitment (Zanoni et al., 2005). Two pathways for DC‐mediated NK cell activation have been described in mice: one dependent on IL‐4 and the other dependent on microbial stimuli (Granucci et al., 2004; Terme et al., 2004). Therefore, an appropriate cytokine milieu containing IL‐4 renders DCs competent for NK‐cell activation independent of microbial stimuli, although the presence of microbial stimuli increases the efficiency of NK‐cell activation (Ferlazzo et al., 2003); alternatively, following a microbial encounter, DCs become capable of efficiently activating NK cells (Granucci et al., 2004). DCs differentiated in the presence of IL‐4 are strong producers of IL‐12 following activation with inflammatory stimuli (Macatonia et al., 1995). This cytokine has been shown to be required to obtain optimal IFNg production by NK cells (Smyth et al., 2002). In the context of viral infections, another DC‐derived cytokine able to promote efficient IFNg production by NK cells is IL‐18 (Andrews et al., 2003). In the absence of DC exposure to IL‐4 and in response to bacterial challenges or to bacterial cell products, DC‐derived IL‐2 plays a major role in eliciting IFNg production by NK cells (Granucci et al., 2004). The biological relevance of NK‐cell activation mediated by DCs during bacterial infections resides mainly in the secretion of IFNg (Ferlazzo et al., 2003), which represents the principal phagocyte‐activating factor (Boehm et al., 1997; Ferlazzo et al., 2003). The role of DC‐derived IL‐2 in inducing IFNg production by NK cells has been studied in the mouse system. Nevertheless, human monocyte‐ derived DCs cultured in vitro in the presence of IL‐15 and not in the presence of IL‐4 can produce IL‐2 (Feau et al., 2005); thus, it would be interesting to investigate whether, also in humans, IL‐2 could play a role in stimulating NK cells in a context in which IL‐15 is present. Bacterially activated DCs can also induce NK‐cell cytotoxic function. This phenomenon is type I IFN‐dependent and IL‐2‐independent. As stated

d en d r i t i c c e l l b i o l og y

211

previously, the production of type I IFNs by myeloid DCs has been shown in the context of viral infections (Diebold et al., 2003). In support of this observation, it has been found that myeloid DCs can also produce type I IFNs following bacterial activation (Granucci et al., 2004). The precise contribution of myeloid and plasmacytoid DCs in the production of type I IFNs in vivo in response to different types of infection remains to be determined. Results suggest that production of type I IFNs may represent a poorly recognized response of myeloid DCs to several infectious agents, including bacteria. The upregulation of NK‐cell cytotoxic function by bacterially activated DCs may not be directly related to NK‐cell antibacterial effects. The biological relevance of this response may reside in the fact that cytotoxicity could contribute to controlling the late phases of the immune response by limiting inflammation and restoring homeostatic balance after infection (Ferlazzo et al., 2003; Zitvogel, 2002). DCs are susceptible to NK cell‐mediated lysis, therefore the ability to elicit NK‐cell cytotoxicity may be a means for DCs to limit their own activity. Moreover, given the ability of NK cells to acquire strong cytolytic function following interaction with bacterially activated DCs, and given the fact that NK cell antitumor functions can be upregulated following contact with activated DCs (Fernandez et al., 1999; Van Den Broeke et al., 2003), it has been proposed that bacterial infections may contribute to maintaining a basal level of alert against tumors. It is well established that DC‐mediated NK‐cell activation requires cell–cell contact, although the relevant molecules have not been defined. In humans, the induction of IFNg production by NK cells occurs through the formation of an organized supramolecular structure called immunological synapse between DCs and NK cells (Borg et al., 2004; Grakoui et al., 1999). The formation of DC–NK cell contacts depends on cytoskeleton reorganization and lipid raft mobilization. Synapse formation allows the polarization and concentration of preformed IL‐12 at the contact site, a process required to elicit IFNg production (Borg et al., 2004). The outcome of DC–NK cell interaction is not univocal. Under appropriate conditions, NK cells can contribute to DC activation. Indeed, IL‐2‐activated NK cells can promote DC maturation measured in terms of upregulation of costimulatory and MHC molecules and production of inflammatory cytokines. As previously mentioned for DC‐mediated NK cell activation, NK‐mediated DC activation requires cell–cell contact and soluble mediators produced by NK cells and DCs, such as TNFa(Gerosa et al., 2002). 5.2. Role of DCs in Acquired Immunity Activated antigen‐loaded DCs migrate to the T‐cell zone of secondary lymphoid organs to prime T‐cell responses. The requirement of DCs for T‐cell

212

f ra n c e s c a g r a n uc c i

ET AL.

activation has been demonstrated in vivo for CD8þ T lymphocytes. Mice temporarily deprived of CD11cþ DCs are impaired in their ability to mount specific CD8þ T‐cell responses to infections with the intracellular bacterium Listeria monocytogenes, the parasite Plasmodium yoelii, the LCMV, or following antigen immunization (Jung et al., 2002; Probst and van den Broek, 2005). DCs can enter the lymph node either through blood or lymph (Cavanagh and Von Andrian, 2002). Initially they are concentrated in close vicinity to high endothelial venules (HEV), then progressively they change their intranodal distribution and, 1 day after reaching the lymph node, DCs are predominantly distributed throughout the T‐cell area (Mempel et al., 2004). The interaction of DCs with CD8þ T cells has been followed in vivo over a 48‐h period after T‐cell entry in the lymph node (Mempel et al., 2004). Experimental animals were injected in the footpad with immature DCs either pulsed or not with the peptide. To allow DC migration to draining lymph nodes, mice were also treated with LPS. Antigen‐specific T cells were then injected intravenously 18 h after DC administration. DC–T cell interactions were analyzed over time using intravital multiphoton microscopy (Sumen et al., 2004). It has been observed that during the initial 8 h after entering the lymph node, T cells scan many different DCs rapidly and establish only short interactions lasting no more than a few minutes. Subsequently, after 6–8 h, the motility of T cells decrease and the contact they form with DCs last longer than 1 h. Long‐lasting contacts between DCs and T cells continue for the first day after T‐cell entry in the lymph node and until they start to proliferate (Mempel et al., 2004). Stable DC–T cell interactions are necessary to induce T‐cell priming (Hugues et al., 2004). In vitro stable DC–T cell contacts are associated with the formation of an immunological synapse (Grakoui et al., 1999). Three‐dimensional (3D) analysis of the binding site between DCs and T cells by confocal microscopy shows that this specialized contact zone is a concentric structure with a central supramolecular activation cluster (cSMAC) enriched in TCR and CD28, which interact with peptideþMHC complexes and CD80 or CD86, respectively (Bromley et al., 2001), surrounded by a peripheral ring (pSMAC) enriched in lymphocyte function‐associated antigen (LFA‐1). The synapse depends on T‐cell cytoskeletal rearrangements that are required for receptor clustering (Friedl and Storim, 2004). The capacity of DCs to prime T‐cell responses has not been attributed to one particular DC‐specific surface or secreted molecule, but to a combination of factors such as the high level of expression of cell membrane costimulatory proteins, the secretion of specific cytokines, and the efficient antigen‐processing machinery, properties acquired by DCs during maturation (Steinman, 2000). Stimuli that induce DC activation and maturation increase the efficiency of antigen processing for both class I and class II pathways and the half‐life

d en d r i t i c c e l l b i o l og y

213

of peptideþMHC complexes at the cell surface that otherwise are rapidly internalized and recycled (Cella et al., 1997; Pierre et al., 1997; Rescigno et al., 1998). Regarding the class II pathway, DCs contain a large number of multivesicular bodies (MVBs), that store a presynthesized pool of MHC II molecules. The vesicles storing MHC II proteins are formed by invagination of the limiting MVB membrane. In MVBs the accessory molecules H2‐DM, required for MHC loading with peptides, are physically segregated into the MVB‐limiting membrane and cannot come in contact with class II molecules. This segregation prevents class II loading in immature DCs. Following DC activation by microbial or microbial product encounter, an extensive fusion of the MVB internal vesicles occurs, resulting in the formation of long tubular compartments where MHC II molecules and H2‐DM molecules become closely associated. This MVB reorganization process favors an efficient peptide‐ loading of MHC II molecules and allows massive export of peptide‐MHC complexes at the cell surface (Kleijmeer et al., 2001). In contrast with class II molecules, class I molecules are not stored inside immature DCs but are newly synthesized after activation stimuli encounter. The kinetics of new MHC I molecule biosynthesis, has a peak at 18 h after bacterial or LPS stimulation (Rescigno et al., 1998). Interestingly, DCs can delay the processing of internalized antigens by antigen retention in a storage compartment with a mildly acidic pH content (Lutz et al., 1997). In these vesicles, the internalized antigens are not immediately degraded and fusion with the lysosomes is delayed. This mechanism is apparently coordinated with the generation of newly synthesized MHC I molecules (Rescigno et al., 1998). Exclusively in DCs, class I molecules can cross‐present exogenous antigens. Thus, antigen retention could represent a good way to maximize the efficiency of class I presentation during DC maturation. The mechanism by which exogenous antigens internalized in phagosomes can access the MHC I loading compartment has been reported (Guermonprez et al., 2003). During or immediately after their formation, phagosomes can fuse with the endoplasmic reticulum. This permits a release of antigens in the cytosol and the consequent degradation in the proteasome. After degradation, peptides are transported into the lumen of the same phagosome (Guermonprez et al., 2003). In adaptive immunity DCs play the important role of directing the adequate type of immune response depending on the invading microorganism. DCs plasticity consists in their ability to express distinct polarizing signals on the basis of the type of PRR that is activated at the cell surface. The precise mechanisms that regulate this phenomenon have not been defined yet. In general, in response to most TLR stimuli, DCs skew T‐cell responses toward the Th1 type (Schnare et al., 2001). An exception is represented by TLR2 that, when stimulated on DCs by Pam3Cys, can mediate Th2 responses (Agrawal

214

f ra n c e s c a g r a n uc c i

ET AL.

et al., 2003). The activation of Th1 responses by DCs has been often associated with the production of cytokines of the IL‐12 family (Trinchieri et al., 2003). Surprisingly, the role of IL‐12 has been recently questioned and it seems to be redundant in many human infectious diseases (Fieschi and Casanova, 2003) and in many mouse models (Oxenius et al., 1999; Zhang et al., 2003). To this regard, a more important role for IL‐23 and IL‐27 has been proposed (Cua et al., 2003; Murphy et al., 2003). Concerning Th2 responses, although some DC‐expressed molecular mediators, such as monocyte chemoattractant protein (MCP‐1) (Chensue et al., 1996; Matsukawa et al., 2000) and OX40L (Akiba et al., 2000), have been proposed in particular infection models, crucial molecules still need to be identified. As will be discussed, DCs may also produce molecules involved in the peripheral differentiation of regulatory T cells (Lutz and Schuler, 2002). Besides their well‐established role in priming T lymphocytes, DC have the capacity to regulate B‐cell responses directly (Banchereau et al., 2000). Human tonsillar interdigitating DCs have been shown to interact with B cells in situ (Bjorck et al., 1997). Similarly, it has also been observed that, in rats, DCs can form T cell‐independent short‐lived clusters with B cells with a mechanism that depends on the LFA‐1 integrin (Kushnir et al., 1998). In vivo DCs have been shown to uptake the antigen, retain it unprocessed, and transfer it to naı¨ve B cells to initiate an antigen‐specific response (Wykes et al., 1998). The migration of DCs to the B‐cell zone in lymph nodes would be controlled by CXCR5 (Wu and Hwang, 2002). In vitro DCs are able to induce proliferation and IgM secretion of B cells activated through CD40 (Dubois et al., 1997). After 20 days of coculture approximately 20% of B cells differentiate to IgM‐secreting plasma cells with a mechanism that involve IL‐12 production by DCs (Dubois et al., 1998). DCs are also able to promote the differentiation of CD40‐activated memory B cells to Ig‐secreting cells (Dubois et al., 1997). This process is IL‐12 independent and requires secretion of soluble IL‐6R, a chain which can bind IL‐6 to form a complex that can interact with high affinity with the IL‐6R transducing chain and increase the biological activity of IL‐6 (Dubois et al., 1998). DCs are also involved in the induction of B‐cell class switch (Dubois et al., 1999). Upon exposure to different molecular mediators, such as IFNg, CD40 ligand, LPS, or IFNa, marginal zone DCs upregulate the expression of members of the TNF family, such as B lymphocyte stimulator protein (BlyS) and proliferation‐inducing ligand (APRIL). In the presence of IL‐4, IL‐10, or transforming growth factor‐b (TGFb activated DCs, expressing BlyS and APRIL, can induce B‐cell class switch recombination to Cg, Ca, and C in a CD40‐independent manner (Litinskiy et al., 2002). To acquire the ability to secrete immunoglobulins, BlyS‐ and APRIL‐stimulated B cells also require

d en d r i t i c c e l l b i o l og y

215

cross‐linking of the surface B‐cell receptor and exposure to IL‐15. Synergism of BlyS and APRIL with IL‐15 is needed for activation of transcription factors, including NF‐kB, that lead to efficient B‐cell proliferation and antibody production (Litinskiy et al., 2002). 5.3. Role of DCs in Central Tolerance The immune system of vertebrate animals has evolved to protect against perturbations provoked by incoming pathogens. Given the variety of pathogens, it is necessary that the T‐cell repertoire be extremely diverse to allow elimination of all possible invading microorganisms. However, the generation of an immense T‐cell repertoire increases the possibility of developing autoreactive T cells. To limit self‐tissue damages while maintaining T‐cell diversity, the immune system has therefore developed mechanisms for eliminating or rendering nonfunctional autoreactive T cells. Tolerance to tissue antigens is achieved through a combination of thymic and peripheral events that eliminate or inactivate potentially dangerous T cells (Stockinger, 1999). The thymus provides a very important initial step in eliminating potentially dangerous self‐specific T cells (Liston et al., 2003). Positive and negative selection of T lymphocytes are considered qualitatively distinct processes that depend on thymic compartmentalization and on the cellular context of TCR‐MHC interaction (Laufer et al., 1999). Different thymic cell types can give qualitatively different signals to T cells so that positive and negative selections can occur sequentially following T‐cell interaction with thymic stromal cells (Laufer et al., 1999). Three different stromal cell types are present in the thymus: cortical epithelial cells; medullary epithelial cells, and bone marrow‐derived cells that comprehend DCs, macrophages, and B cells. By developing mouse models in which the expression of MHC‐peptide complexes was limited to particular thymic cell types, it has been shown that cortical epithelium has the exclusive capacity to induce positive selection of both autoreactive and nonautoreactive thymocytes (Capone et al., 2001; Laufer et al., 1996). Negative selection is, then, consequent to positive selection, and mainly occurs in the corticomedullary junction and within the thymic medulla (Murphy et al., 1990; Surh and Sprent, 1994). In these regions, negative selection is mediated by medullary epithelial cells (Kyewski and Derbinski, 2004) and antigen presenting cells (APCs) (Marrack et al., 1988). The capacity of medullary epithelial cells to negatively select has been attributed to the fact that they are able to express tissue‐specific genes (Gotter and Kyewski, 2004). It has been proposed that the promiscuous gene expression of thymic epithelial cells is regulated by expression of a particular transcription factor called transcriptional regulator autoimmune regulator (Aire) (Gotter and Kyewski, 2004). Expression of

216

f ra n c e s c a g r a n uc c i

ET AL.

many tissue‐specific genes is, indeed, highly reduced in Aire‐deficient mice (Anderson et al., 2002) who develop tissue‐specific antibodies and lymphoid cell infiltration in many peripheral organs (Anderson et al., 2002). Moreover, the deletion of CD4þ T lymphocytes specific for a self‐antigen expressed under the rat insulin promoter is abrogated in Aire‐deficient mice (Liston et al., 2003). The function of DCs in central tolerance has been investigated in different experimental settings. DCs loaded with low doses of C5, the fifth component of the complement, or DCs spontaneously presenting the C5 are able, together with thymic epithelial cells, to delete C5‐specific T lymphocytes in culture (Volkmann et al., 1997; Zal et al., 1994). Moreover, in an in vivo model in which MHC II was selectively expressed by DCs under the CD11c promoter, efficient negative selection of I‐E reactive Vb5þ and Vb11þ CD4þ T cells has been described (Brocker et al., 1997). Recently, using a similar model in which MHC I was expressed exclusively by DCs the tolerizing role of these cells has also been shown in the context of CD8þ T cells (Cannarile et al., 2004). Thus, at present, it seems that thymic DCs are specialized in tolerance induction and cannot positively select either CD4þ or CD8þ T cells. 5.4. Role of DCs in Peripheral Tolerance Many tissue proteins are not expressed in the thymus at sufficient levels to induce clonal deletion or tolerization (Avery et al., 1995). For this reason, several mechanisms of peripheral T‐cell tolerization have evolved and it has been shown that autoreactive T cells that actively recognize the antigen in the periphery can undergo anergy (Schwartz, 2003), deletion (Burkly et al., 1990), or downregulation of T‐cell receptors (TCRs) (Schonrich et al., 1991) or coreceptors (Robbins and McMichael, 1991). Several models have been proposed to explain the induction of tolerance in peripheral autoreactive T cells. The first model (Bretscher and Cohn, 1970) hypothesized that the immune system is capable of responding only to nonself‐antigens, not to self‐antigens, and that antigen‐specific cells make the discrimination between self and nonself and make the decision to respond. According to this model, self is an invariant property of the individual and antigenic exposure early in ontogeny is tolerogenic because of the low frequency of effector T‐helper lymphocytes (Bretscher, 1999). Nevertheless, as emphasized previously, evidence accumulated in the last 20 years (Mueller et al., 1989) have led to a different assumption that the decision to initiate an adaptive immune response is not made by the antigen‐specific cells, but by the APCs. According to the infectious nonself and noninfectious self (INS) model, APCs are maintained in a resting state until they encounter microbes or microbial cell products that

d en d r i t i c c e l l b i o l og y

217

activate them and induce the upregulation of costimulatory molecules (signal two) necessary for peripheral T‐cell activation (Medzhitov et al., 1997). Thus, distinction between self and nonself in the periphery is made by the APCs. The INS model does not deal with the problem of peripheral tolerance and claims that self‐peptides presented by APC in the periphery are not recognized as nonself by T cells because self‐reactive T cells are eliminated during negative selection in the thymus (Medzhitov and Janeway, 2000). In contrast, the role of peripheral T‐cell tolerance is strongly emphasized in the Danger model (Matzinger, 1994). Like the INS model, the Danger theory proposes that T‐cell activation is mediated by activated APCs that deliver signals one and two to naı¨ve T cells, but that activation of APCs is mediated by danger signals derived from injured cells such as those exposed to pathogens or other possible stress signals. In the presence of danger, APCs are activated, expressing signal one and two and are capable of activating T cells; in the absence of danger, APCs are not activated and T cells (antigen‐experienced or naı¨ve) that interact with resting APCs die for lack of costimulation (Matzinger, 2002). Recently, the Danger model has been experimentally tested on antigen‐presenting DCs and it has been proposed that the activation state of DCs is relevant for the decision to suppress or activate an immune response (Steinman et al., 2003). In particular, immature or CD40‐activated DCs expressing the scavenger receptor CD205 have been targeted with the antigen in vivo and the fate of CD4þ and CD8þ antigen‐specific T cells followed over time (Bonifaz et al., 2002; Hawiger et al., 2001). T‐lymphocyte tolerization is observed when they encounter immature antigen‐loaded DCs, whereas T‐cell activation is described when they encounter activated antigen‐presenting DCs. The immature state of DCs, characterized by the absence of sufficient costimulation and sufficient signal two, has been interpreted as responsible for the tolerization process. A second evidence of the capacity of immature DCs to induce peripheral T‐cell tolerance relies on their ability to cross‐present peripheral tissue antigens and induce abortive T‐cell activation (Hernandez et al., 2001). Finally, a system that allows inducible antigen presentation by resting or activated DCs has been described. In this model, three distinct LCMV‐ derived CTL epitopes could be presented by 5% of the total DC population following induction (Probst et al., 2003). Presentation of LCMV‐derived CTL epitopes by resting DCs resulted in antigen‐specific tolerance, which could not be broken by subsequent infection with LCMV. Conversely, antigen presentation by activated DC-induced CTL activation and protective memory (Probst et al., 2003). The tolerization process depended on the synergistic effect of the PD‐1 and CTLA‐4 molecules (Probst et al., 2005). Although other explanations concerning all these observations may be possible, such as antigen persistence, they have been interpreted in the context of

218

f ra n c e s c a g r a n uc c i

ET AL.

the Danger model with a fundamental role exerted by immature DCs in maintaining peripheral tolerance. DCs have also been implicated in the functional control of regulatory T cells (Treg). These particular lymphocytes are involved in maintaining tolerance to self‐antigens by inhibiting responses mediated by effector CD4þ and CD8þ T cells (Sakaguchi, 2000). Some Treg cell populations [those expressing CD4 and IL‐2 receptor a chain (CD25)], originate in the thymus, while other Treg subtypes (those identified as IL‐10 producers) differentiate in the periphery (Roncarolo et al., 2003; Roncarolo and Levings, 2000). A role of DCs in the peripheral differentiation of Treg cells has been proposed. In particular, it has been observed that repetitive stimulation of naı¨ve T cells with allogeneic immature DCs may result in the generation of IL‐10‐producing anergic T cells able to suppress effector T‐cell functions (Jonuleit et al., 2000). The generation of Treg cell require production of IL‐10 by immature DC (Levings et al., 2005). Similar results have also been obtained in vivo in humans; injection of immature DCs pulsed with the influenza peptide induced the differentiation of peptide‐specific IL‐10‐producing T cells, and the disappearance of influenza‐specific effector CD8þ T cells. In contrast, a single injection of peptide‐pulsed mature DCs led to rapid expansion of specific T lymphocytes (Dhodapkar et al., 2001). Moreover, as mentioned previously, there are semimaturation stimuli, such as TNFa, that can induce a particular population of semimature DCs, able to sustain the peripheral differentiation of Treg cells. Repetitive injections of bone‐marrow‐derived TNFa‐activated DCs prevent the development of experimental autoimmune encephalomyelitis (EAE) by inducing IL‐10‐producing Treg cells (Menges et al., 2002). Thus, T‐cell interaction with antigen‐presenting immature or semimature DC could induce peripheral differentiation of Treg lymphocytes. Besides a role in peripheral Treg‐cell differentiation, DCs have been shown capable of influencing the function of thymus‐derived CD4þCD25þ Treg cells (Guiducci et al., 2005; Steinman et al., 2003). Mature DCs are, indeed, able to induce expansion of CD4þCD25þ Treg cells both in vitro and in vitro in the presence of a specific antigen or in the presence of IL‐2 (Yamazaki et al., 2003). Once expanded, following the DC encounter, the Treg cells show more of an increased suppressive activity in vitro than their ex vivo counterpart (Tarbell et al., 2004). Concerning the mechanism by which DCs would be able to control Treg‐ cell functions, it has been hypothesized that DC‐derived IL‐2 could play an important role (Malek and Bayer, 2004) because it has been observed that IL‐2 is necessary for Treg‐cell functionality. In this experiment, IL‐2‐ or CD25‐ deficient T cells were transferred in mice who harbor a monoclonal myelin basic protein (MBP)‐specific ab T‐cell repertoire and spontaneously develop

d en d r i t i c c e l l b i o l og y

219

EAE (Lafaille et al., 1994). Only IL‐2‐deficient T cells protected from the disease, whereas CD25‐negative lymphocytes did not, indicating that IL‐2 is not required for the generation of Treg cells in the thymus but is necessary for Treg cells in the periphery to exert their functions. Finally, DCs have been shown to be involved in the maintenance of CD4þCD25þ cell homeostasis through a mechanism involving cell–cell contact, CD40–CD40L interaction, and IL‐2 production (Guiducci et al., 2005). 6. Conclusions DCs are key regulators of immune responses. They react to infectious agents undergoing a complex reprogramming of their functions. On one hand, DCs are programmed to react to different microorganisms modulating a constant core of genes. On the other hand, they show a functional plasticity in their maturation process, which depends on the nature of the perturbation and which permits to arise unique and efficient immune responses for each pathogen. Different DC functions are segregated in time and in space, allowing these cells to control both innate and adaptive immune responses. Acknowledgments This work was supported by grants from AIRC (Italian Association Against Cancer), The European Commission 6th Framework Program (contracts DC VACC LSHB‐CT‐2003–503037, DC THERA LSHB‐CT‐2004–512074, and MICROBAN MRTN‐CT‐2003–504227), The Italian Ministry of Education and Research (Programs FIRB and COFIN), The Italian Ministry of Health, and The Foundation Cariplo.

References Aebischer, T., Bennett, C. L., Pelizzola, M., Vizzardelli, C., Pavelka, N., Urbano, M., Capozzoli, M., Luchini, A., Ilg, T., Granucci, F., Blackburn, C. C., and Ricciardi‐Castagnoli, P. (2005). A critical role for lipophosphoglycan in proinflammatory responses of dendritic cells to Leishmania mexicana. Eur. J. Immunol. 35, 476–486. Agnese, D. M., Calvano, J. E., Hahm, S. J., Coyle, S. M., Corbett, S. A., Calvano, S. E., and Lowry, S. F. (2002). Human Toll‐like receptor 4 mutations but not CD14 polymorphisms are associated with an increased risk of gram‐negative infections. J. Infect. Dis. 186, 1522–1525. Agrawal, S., Agrawal, A., Doughty, B., Gerwitz, A., Blenis, J., Van Dyke, T., and Pulendran, B. (2003). Cutting edge: different Toll‐like receptor agonists instruct dendritic cells to induce distinct Th responses via differential modulation of extracellular signal‐regulated kinase‐ mitogen‐activated protein kinase and c‐Fos. J. Immunol. 171, 4984–4989. Ahmad‐Nejad, P., Hacker, H., Rutz, M., Bauer, S., Vabulas, R. M., and Wagner, H. (2002). Bacterial CpG‐DNA and lipopolysaccharides activate Toll‐like receptors at distinct cellular compartments. Eur. J. Immunol. 32, 1958–1968.

220

f ra n c e s c a g r a n uc c i

ET AL.

Akiba, H., Miyahira, Y., Atsuta, M., Takeda, K., Nohara, C., Futagawa, T., Matsuda, H., Aoki, T., Yagita, H., and Okumura, K. (2000). Critical contribution of OX40 ligand to T helper cell type 2 differentiation in experimental leishmaniasis. J. Exp. Med. 191, 375–380. Akira, S., and Takeda, K. (2004). Toll‐like receptor signalling. Nat. Rev. Immunol. 4, 499–511. Akira, S., Takeda, K., and Kaisho, T. (2001). Toll‐like receptors: Critical proteins linking innate and acquired immunity. Nat. Immunol. 2, 675–680. Aksoy, E., Zouain, C. S., Vanhoutte, F., Fontaine, J., Pavelka, N., Thieblemont, N., Willems, F., Ricciardi‐Castagnoli, P., Goldman, M., Capron, M., Ryffel, B., and Trottein, F. (2005). Double‐ stranded RNAs from the helminth parasite Schistosoma activate TLR3 in dendritic cells. J. Biol. Chem. 280, 277–283. Allavena, P., Chieppa, M., Monti, P., and Piemonti, L. (2004). From pattern recognition receptor to regulator of homeostasis: The double‐faced macrophage mannose receptor. Crit. Rev. Immunol. 24, 179–192. An, H., Yu, Y., Zhang, M., Xu, H., Qi, R., Yan, X., Liu, S., Wang, W., Guo, Z., Guo, J., Qin, Z., and Cao, X. (2002). Involvement of ERK, p38 and NF‐kappaB signal transduction in regulation of TLR2, TLR4 and TLR9 gene expression induced by lipopolysaccharide in mouse dendritic cells. Immunology 106, 38–45. Anderson, D. C., Miller, L. J., Schmalstieg, F. C., Rothlein, R., and Springer, T. A. (1986). Contributions of the Mac‐1 glycoprotein family to adherence‐dependent granulocyte functions: Structure‐function assessments employing subunit‐specific monoclonal antibodies. J. Immunol. 137, 15–27. Anderson, M. S., Venanzi, E. S., Klein, L., Chen, Z., Berzins, S. P., Turley, S. J., von Boehmer, H., Bronson, R., Dierich, A., Benoist, C., and Mathis, D. (2002). Projection of an immunological self shadow within the thymus by the aire protein. Science 298, 1395–1401. Andrews, D. M., Andoniou, C. E., Granucci, F., Ricciardi‐Castagnoli, P., and Degli‐Esposti, M. A. (2001). Infection of dendritic cells by murine cytomegalovirus induces functional paralysis. Nat. Immunol. 2, 1077–1084. Andrews, D. M., Scalzo, A. A., Yokoyama, W. M., Smyth, M. J., and Degli‐Esposti, M. A. (2003). Functional interactions between dendritic cells and NK cells during viral infection. Nat. Immunol. 4, 175–181. Anjuere, F., Martin, P., Ferrero, I., Fraga, M. L., del Hoyo, G. M., Wright, N., and Ardavin, C. (1999). Definition of dendritic cell subpopulations present in the spleen, Peyer’s patches, lymph nodes, and skin of the mouse. Blood 93, 590–598. Ardavin, C. (2003). Origin, precursors and differentiation of mouse dendritic cells. Nat. Rev. Immunol. 3, 582–590. Ardavin, C., Martinez del Hoyo, G., Martin, P., Anjuere, F., Arias, C. F., Marin, A. R., Ruiz, S., Parrillas, V., and Hernandez, H. (2001). Origin and differentiation of dendritic cells. Trends Immunol. 22, 691–700. Asselin‐Paturel, C., Boonstra, A., Dalod, M., Durand, I., Yessaad, N., Dezutter‐Dambuyant, C., Vicari, A., O’Garra, A., Biron, C., Briere, F., and Trinchieri, G. (2001). Mouse type I IFN‐ producing cells are immature APCs with plasmacytoid morphology. Nat. Immunol. 2, 1144–1150. Austyn, J. M. (1996). New insights into the mobilization and phagocytic activity of dendritic cells. J. Exp. Med. 183, 1287–1292. Avery, A. C., Zhao, Z. S., Rodriguez, A., Bikoff, E. K., Soheilian, M., Foster, C. S., and Cantor, H. (1995). Resistance to herpes stromal keratitis conferred by an IgG2a‐derived peptide. Nature 376, 431–434. Azuma, I., and Seya, T. (2001). Development of immunoadjuvants for immunotherapy of cancer. Int. Immunopharmacol. 1, 1249–1259.

d en d r i t i c c e l l b i o l og y

221

Banchereau, J., Briere, F., Caux, C., Davoust, J., Lebecque, S., Liu, Y. J., Pulendran, B., and Palucka, K. (2000). Immunobiology of dendritic cells. Annu. Rev. Immunol. 18, 767–811. Banchereau, J., and Steinman, R. M. (1998). Dendritic cells and the control of immunity. Nature 392, 245–252. Bartz, H., Turkel, O., Hoffjan, S., Rothoeft, T., Gonschorek, A., and Schauer, U. (2003). Respiratory syncytial virus decreases the capacity of myeloid dendritic cells to induce interferon‐gamma in naive T cells. Immunology 109, 49–57. Bendelac, A., and Medzhitov, R. (2002). Adjuvants of immunity: Harnessing innate immunity to promote adaptive immunity. J. Exp. Med. 195, F19–23. Bergelson, J., Kreitman, M., Stahl, E. A., and Tian, D. (2001). Evolutionary dynamics of plant R‐genes. Science 292, 2281–2285. Beutler, B. (2004). Inferences, questions and possibilities in Toll‐like receptor signalling. Nature 430, 257–263. Bjorck, P., Flores‐Romo, L., and Liu, Y. J. (1997). Human interdigitating dendritic cells directly stimulate CD40‐activated naive B cells. Eur. J. Immunol. 27, 1266–1274. Blander, J. M., and Medzhitov, R. (2004). Regulation of phagosome maturation by signals from Toll‐like receptors. Science 304, 1014–1018. Blank, C., Fuchs, H., Rappersberger, K., Rollinghoff, M., and Moll, H. (1993). Parasitism of epidermal Langerhans cells in experimental cutaneous leishmaniasis with Leishmania major. J. Infect. Dis. 167, 418–425. Boehm, U., Klamp, T., Groot, M., and Howard, J. C. (1997). Cellular responses to interferon‐ gamma. Annu. Rev. Immunol. 15, 749–795. Bonifaz, L., Bonnyay, D., Mahnke, K., Rivera, M., Nussenzweig, M. C., and Steinman, R. M. (2002). Efficient targeting of protein antigen to the dendritic cell receptor DEC‐205 in the steady state leads to antigen presentation on major histocompatibility complex class I products and peripheral CD8þ T cell tolerance. J. Exp. Med. 196, 1627–1638. Borg, C., Jalil, A., Laderach, D., Maruyama, K., Wakasugi, H., Charrier, S., Ryffel, B., Cambi, A., Figdor, C., Vainchenker, W., Galy, A., Caignard, A., and Zitvogel, L. (2004). NK cell activation by dendritic cells (DCs) requires the formation of a synapse leading to IL‐12 polarization in DCs. Blood 104, 3267–3275. Bowie, A., Kiss‐Toth, E., Symons, J. A., Smith, G. L., Dower, S. K., and O’Neill, L. A. (2000). A46R and A52R from vaccinia virus are antagonists of host IL‐1 and Toll‐like receptor signaling. Proc. Natl. Acad. Sci. USA 97, 10162–10167. Bretscher, P., and Cohn, M. (1970). A theory of self‐nonself discrimination. Science 169, 1042–1049. Bretscher, P. A. (1999). A two‐step, two‐signal model for the primary activation of precursor helper T cells. Proc. Natl. Acad. Sci. USA 96, 185–190. Brocker, T., Riedinger, M., and Karjalainen, K. (1997). Targeted expression of major histocompatibility complex (MHC) class II molecules demonstrates that dendritic cells can induce negative but not positive selection of thymocytes in vivo. J. Exp. Med. 185, 541–550. Bromley, S. K., Iaboni, A., Davis, S. J., Whitty, A., Green, J. M., Shaw, A. S., Weiss, A., and Dustin, M. L. (2001). The immunological synapse and CD28–CD80 interactions. Nat. Immunol. 2, 1159–1166. Burkly, L. C., Lo, D., and Flavell, R. A. (1990). Tolerance in transgenic mice expressing major histocompatibility molecules extrathymically on pancreatic cells. Science 248, 1364–1368. Burley, S. K., and Bonanno, J. B. (2002). Structuring the universe of proteins. Annu. Rev. Genomics Hum. Genet. 3, 243–262. Cannarile, M. A., Decanis, N., van Meerwijk, J. P., and Brocker, T. (2004). The role of dendritic cells in selection of classical and nonclassical CD8þ T cells in vivo. J. Immunol. 173, 4799–4805.

222

f ra n c e s c a g r a n uc c i

ET AL.

Capone, M., Romagnoli, P., Beermann, F., Mac Donald, H. R., and van Meerwijk, J. P. (2001). Dissociation of thymic positive and negative selection in transgenic mice expressing major histocompatibility complex class I molecules exclusively on thymic cortical epithelial cells. Blood 97, 1336–1342. Cavanagh, L. L., and Von Andrian, U. H. (2002). Travellers in many guises: The origins and destinations of dendritic cells. Immunol. Cell Biol. 80, 448–462. Cella, M., Engering, A., Pinet, V., Pieters, J., and Lanzavecchia, A. (1997). Inflammatory stimuli induce accumulation of MHC class II complexes on dendritic cells. Nature 388, 782–787. Chensue, S. W., Warmington, K. S., Ruth, J. H., Sanghi, P. S., Lincoln, P., and Kunkel, S. L. (1996). Role of monocyte chemoattractant protein‐1 (MCP‐1) in Th1 (mycobacterial) and Th2 (schistosomal) antigen‐induced granuloma formation: Relationship to local inflammation, Th cell expression, and IL‐12 production. J. Immunol. 157, 4602–4608. Chieppa, M., Bianchi, G., Doni, A., Del Prete, A., Sironi, M., Laskarin, G., Monti, P., Piemonti, L., Biondi, A., Mantovani, A., Introna, M., and Allavena, P. (2003). Cross‐linking of the mannose receptor on monocyte‐derived dendritic cells activates an anti‐inflammatory immunosuppressive program. J. Immunol. 171, 4552–4560. Corinti, S., Medaglini, D., Cavani, A., Rescigno, M., Pozzi, G., Ricciardi‐Castagnoli, P., and Girolomoni, G. (1999). Human dendritic cells very efficiently present a heterologous antigen expressed on the surface of recombinant gram‐positive bacteria to CD4þ T lymphocytes. J. Immunol. 163, 3029–3036. Cua, D. J., Sherlock, J., Chen, Y., Murphy, C. A., Joyce, B., Seymour, B., Lucian, L., To, W., Kwan, S., Churakova, T., Zurawski, S., Wiekowski, M., Lira, S. A., Gorman, D., Kastelein, R. A., and Sedgwick, J. D. (2003). Interleukin‐23 rather than interleukin‐12 is the critical cytokine for autoimmune inflammation of the brain. Nature 421, 744–748. De Smedt, T., Pajak, B., Klaus, G. G., Noelle, R. J., Urbain, J., Leo, O., and Moser, M. (1998). Antigen‐specific T lymphocytes regulate lipopolysaccharide‐induced apoptosis of dendritic cells in vivo. J. Immunol. 161, 4476–4479. Degli‐Esposti, M. A., and Smyth, M. J. (2005). Close encounters of different kinds: dendritic cells and NK cells take centre stage. Nat. Rev. Immunol. 5, 112–124. Demangel, C., Bean, A. G., Martin, E., Feng, C. G., Kamath, A. T., and Britton, W. J. (1999). Protection against aerosol Mycobacterium tuberculosis infection using Mycobacterium bovis Bacillus Calmette Guerin‐infected dendritic cells. Eur. J. Immunol. 29, 1972–1979. Dhodapkar, M. V., Steinman, R. M., Krasovsky, J., Munz, C., and Bhardwaj, N. (2001). Antigen‐ specific inhibition of effector T cell function in humans after injection of immature dendritic cells. J. Exp. Med. 193, 233–238. Diebold, S. S., Kaisho, T., Hemmi, H., Akira, S., and Reis e Sousa, C. (2004). Innate antiviral responses by means of TLR7‐mediated recognition of single‐stranded RNA. Science 303, 1529–1531. Diebold, S. S., Montoya, M., Unger, H., Alexopoulou, L., Roy, P., Haswell, L. E., Al‐Shamkhani, A., Flavell, R., Borrow, P., and Reis e Sousa, C. (2003). Viral infection switches non‐plasmacytoid dendritic cells into high interferon producers. Nature 424, 324–328. Doni, A., Peri, G., Chieppa, M., Allavena, P., Pasqualini, F., Vago, L., Romani, L., Garlanda, C., and Mantovani, A. (2003). Production of the soluble pattern recognition receptor PTX3 by myeloid, but not plasmacytoid, dendritic cells. Eur. J. Immunol. 33, 2886–2893. Doyle, S. E., O’Connell, R. M., Miranda, G. A., Vaidya, S. A., Chow, E. K., Liu, P. T., Suzuki, S., Suzuki, N., Modlin, R. L., Yeh, W. C., Lane, T. F., and Cheng, G. (2004). Toll‐like receptors induce a phagocytic gene program through p38. J. Exp. Med. 199, 81–90. Dubois, B., Bridon, J. M., Fayette, J., Barthelemy, C., Banchereau, J., Caux, C., and Briere, F. (1999). Dendritic cells directly modulate B cell growth and differentiation. J. Leukoc. Biol. 66, 224–230.

d en d r i t i c c e l l b i o l og y

223

Dubois, B., Massacrier, C., Vanbervliet, B., Fayette, J., Briere, F., Banchereau, J., and Caux, C. (1998). Critical role of IL‐12 in dendritic cell‐induced differentiation of naive B lymphocytes. J. Immunol. 161, 2223–2231. Dubois, B., Vanbervliet, B., Fayette, J., Massacrier, C., Van Kooten, C., Briere, F., Banchereau, J., and Caux, C. (1997). Dendritic cells enhance growth and differentiation of CD40‐activated B lymphocytes. J. Exp. Med. 185, 941–951. Feau, S., Facchinetti, V., Granucci, F., Citterio, S., Jarrossay, D., Seresini, S., Protti, M. P., Lanzavecchia, A., and Ricciardi‐Castagnoli, P. (2005). Dendritic cell‐derived IL‐2 production is regulated by IL‐15 in humans and in mice. Blood 105, 697–702. Ferlazzo, G., Morandi, B., D’Agostino, A., Meazza, R., Melioli, G., Moretta, A., and Moretta, L. (2003). The interaction between NK cells and dendritic cells in bacterial infections results in rapid induction of NK cell activation and in the lysis of uninfected dendritic cells. Eur. J. Immunol. 33, 306–313. Ferlazzo, G., Pack, M., Thomas, D., Paludan, C., Schmid, D., Strowig, T., Bougras, G., Muller, W. A., Moretta, L., and Munz, C. (2004). Distinct roles of IL‐12 and IL‐15 in human natural killer cell activation by dendritic cells from secondary lymphoid organs. Proc. Natl. Acad. Sci. USA 101, 16606–16611. Ferlazzo, G., Tsang, M. L., Moretta, L., Melioli, G., Steinman, R. M., and Munz, C. (2002). Human dendritic cells activate resting natural killer (NK) cells and are recognized via the NKp30 receptor by activated NK cells. J. Exp. Med. 195, 343–351. Fernandez, N. C., Lozier, A., Flament, C., Ricciardi‐Castagnoli, P., Bellet, D., Suter, M., Perricaudet, M., Tursz, T., Maraskovsky, E., and Zitvogel, L. (1999). Dendritic cells directly trigger NK cell functions: cross‐talk relevant in innate anti‐tumor immune responses in vivo. Nat. Med. 5, 405–411. Fieschi, C., and Casanova, J. L. (2003). The role of interleukin‐12 in human infectious diseases: Only a faint signature. Eur. J. Immunol. 33, 1461–1464. Filgueira, L., Nestle, F. O., Rittig, M., Joller, H. I., and Groscurth, P. (1996). Human dendritic cells phagocytose and process Borrelia burgdorferi. J. Immunol. 157, 2998–3005. Foti, M., Granucci, F., Aggujaro, D., Liboi, E., Luini, W., Minardi, S., Mantovani, A., Sozzani, S., and Ricciardi‐Castagnoli, P. (1999). Upon dendritic cell (DC) activation chemokines and chemokine receptor expression are rapidly regulated for recruitment and maintenance of DC at the inflammatory site. Int. Immunol. 11, 979–986. Friedl, P., and Storim, J. (2004). Diversity in immune‐cell interactions: states and functions of the immunological synapse. Trends Cell Biol. 14, 557–567. Fugier‐Vivier, I., Servet‐Delprat, C., Rivailler, P., Rissoan, M. C., Liu, Y. J., and Rabourdin‐Combe, C. (1997). Measles virus suppresses cell‐mediated immunity by interfering with the survival and functions of dendritic and T cells. J. Exp. Med. 186, 813–823. Gantner, B. N., Simmons, R. M., Canavera, S. J., Akira, S., and Underhill, D. M. (2003). Collaborative induction of inflammatory responses by dectin‐1 and Toll‐like receptor 2. J. Exp. Med. 197, 1107–1117. Garlanda, C., Hirsch, E., Bozza, S., Salustri, A., De Acetis, M., Nota, R., Maccagno, A., Riva, F., Bottazzi, B., Peri, G., Doni, A., Vago, L., Botto, M., De Santis, R., Carminati, P., Siracusa, G., Altruda, F., Vecchi, A., Romani, L., and Mantovani, A. (2002). Non‐redundant role of the long pentraxin PTX3 in anti‐fungal innate immune response. Nature 420, 182–186. Geijtenbeek, T. B., Kwon, D. S., Torensma, R., van Vliet, S. J., van Duijnhoven, G. C., Middel, J., Cornelissen, I. L., Nottet, H. S., Kewal Ramani, V. N., Littman, D. R., Figdor, C. G., and van Kooyk, Y. (2000a). DC‐SIGN, a dendritic cell‐specific HIV‐1‐binding protein that enhances trans‐infection of T cells. Cell 100, 587–597.

224

f ra n c e s c a g r a n uc c i

ET AL.

Geijtenbeek, T. B., Torensma, R., van Vliet, S. J., van Duijnhoven, G. C., Adema, G. J., van Kooyk, Y., and Figdor, C. G. (2000b). Identification of DC‐SIGN, a novel dendritic cell‐specific ICAM‐3 receptor that supports primary immune responses. Cell 100, 575–585. Geijtenbeek, T. B., van Vliet, S. J., Engering, A., t Hart, B. A., and van Kooyk, Y. (2004). Self‐ and nonself‐recognition by C‐type lectins on dendritic cells. Annu. Rev. Immunol. 22, 33–54. Gerosa, F., Baldani‐Guerra, B., Nisii, C., Marchesini, V., Carra, G., and Trinchieri, G. (2002). Reciprocal activating interaction between natural killer cells and dendritic cells. J. Exp. Med. 195, 327–333. Girardin, S. E., Boneca, I. G., Viala, J., Chamaillard, M., Labigne, A., Thomas, G., Philpott, D. J., and Sansonetti, P. J. (2003). Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J. Biol. Chem. 278, 8869–8872. Gotter, J., and Kyewski, B. (2004). Regulating self‐tolerance by deregulating gene expression. Curr. Opin. Immunol. 16, 741–745. Grakoui, A., Bromley, S. K., Sumen, C., Davis, M. M., Shaw, A. S., Allen, P. M., and Dustin, M. L. (1999). The immunological synapse: a molecular machine controlling T‐cell activation. Science 285, 221–227. Granucci, F., Feau, S., Angeli, V., Trottein, F., and Ricciardi‐Castagnoli, P. (2003a). Early IL‐2 production by mouse dendritic cells is the result of microbial‐induced priming. J. Immunol. 170, 5075–5081. Granucci, F., Petralia, F., Urbano, M., Citterio, S., Di Tota, F., Santambrogio, L., and Ricciardi‐ Castagnoli, P. (2003b). The scavenger receptor MARCO mediates cytoskeleton rearrangements in dendritic cells and microglia. Blood 102, 2940–2947. Granucci, F., Vizzardelli, C., Pavelka, N., Feau, S., Persico, M., Virzi, E., Rescigno, M., Moro, G., and Ricciardi‐Castagnoli, P. (2001a). Inducible IL‐2 production by dendritic cells revealed by global gene expression analysis. Nat. Immunol. 2, 882–888. Granucci, F., Vizzardelli, C., Virzi, E., Rescigno, M., and Ricciardi‐Castagnoli, P. (2001b). Transcriptional reprogramming of dendritic cells by differentiation stimuli. Eur. J. Immunol. 31, 2539–2546. Granucci, F., Zanoni, I., Pavelka, N., Van Dommelen, S. L., Andoniou, C. E., Belardelli, F., Degli Esposti, M. A., and Ricciardi‐Castagnoli, P. (2004). A contribution of mouse dendritic cell‐ derived IL‐2 for NK cell activation. J. Exp. Med. 200, 287–295. Gredmark, S., and Soderberg‐Naucler, C. (2003). Human cytomegalovirus inhibits differentiation of monocytes into dendritic cells with the consequence of depressed immunological functions. J. Virol. 77, 10943–10956. Guermonprez, P., Saveanu, L., Kleijmeer, M., Davoust, J., Van Endert, P., and Amigorena, S. (2003). ER‐phagosome fusion defines an MHC class I cross‐presentation compartment in dendritic cells. Nature 425, 397–402. Guerreiro‐Cacais, A. O., Li, L., Donati, D., Bejarano, M. T., Morgan, A., Masucci, M. G., Hutt‐ Fletcher, L., and Levitsky, V. (2004). Capacity of Epstein‐Barr virus to infect monocytes and inhibit their development into dendritic cells is affected by the cell type supporting virus replication. J. Gen. Virol. 85, 2767–2778. Guiducci, C., Valzasina, B., Dislich, H., and Colombo, M. P. (2005). CD40/CD40L interaction regulates CD4(þ)CD25(þ) T reg homeostasis through dendritic cell‐produced IL‐2. Eur. J. Immunol. 35, 557–567. Hahn, G., Jores, R., and Mocarski, E. S. (1998). Cytomegalovirus remains latent in a common precursor of dendritic and myeloid cells. Proc. Natl. Acad. Sc.i USA 95, 3937–3942. Halary, F., Amara, A., Lortat‐Jacob, H., Messerle, M., Delaunay, T., Houles, C., Fieschi, F., Arenzana‐Seisdedos, F., Moreau, J. F., and Dechanet‐Merville, J. (2002). Human cytomegalovirus

d en d r i t i c c e l l b i o l og y

225

binding to DC‐SIGN is required for dendritic cell infection and target cell trans‐infection. Immunity 17, 653–664. Harte, M. T., Haga, I. R., Maloney, G., Gray, P., Reading, P. C., Bartlett, N. W., Smith, G. L., Bowie, A., and O’Neill, L. A. (2003). The poxvirus protein A52R targets Toll‐like receptor signaling complexes to suppress host defense. J. Exp. Med. 197, 343–351. Hawiger, D., Inaba, K., Dorsett, Y., Guo, M., Mahnke, K., Rivera, M., Ravetch, J. V., Steinman, R. M., and Nussenzweig, M. C. (2001). Dendritic cells induce peripheral T‐cell unresponsiveness under steady state conditions in vivo. J. Exp. Med. 194, 769–779. Hawn, T. R., Verbon, A., Lettinga, K. D., Zhao, L. P., Li, S. S., Laws, R. J., Skerrett, S. J., Beutler, B., Schroeder, L., Nachman, A., Ozinsky, A., Smith, K. D., and Aderem, A. (2003). A common dominant TLR5 stop codon polymorphism abolishes flagellin signaling and is associated with susceptibility to legionnaires’ disease. J. Exp. Med. 198, 1563–1572. Heil, F., Ahmad‐Nejad, P., Hemmi, H., Hochrein, H., Ampenberger, F., Gellert, T., Dietrich, H., Lipford, G., Takeda, K., Akira, S., Wagner, H., and Bauer, S. (2003). The Toll‐like receptor 7 (TLR7)‐specific stimulus loxoribine uncovers a strong relationship within the TLR7, 8 and 9 subfamily. Eur. J. Immunol. 33, 2987–2997. Henderson, R. A., Watkins, S. C., and Flynn, J. L. (1997). Activation of human dendritic cells following infection with Mycobacterium tuberculosis. J. Immunol. 159, 635–643. Henri, S., Vremec, D., Kamath, A., Waithman, J., Williams, S., Benoist, C., Burnham, K., Saeland, S., Handman, E., and Shortman, K. (2001). The dendritic cell populations of mouse lymph nodes. J. Immunol. 167, 741–748. Hernandez, J., Aung, S., Redmond, W. L., and Sherman, L. A. (2001). Phenotypic and functional analysis of CD8(þ) T cells undergoing peripheral deletion in response to cross‐presentation of self‐antigen. J. Exp. Med. 194, 707–717. Hirschfeld, M., Kirschning, C. J., Schwandner, R., Wesche, H., Weis, J. H., Wooten, R. M., and Weis, J. J. (1999). Cutting edge: Inflammatory signaling by Borrelia burgdorferi lipoproteins is mediated by Toll‐like receptor 2. J. Immunol. 163, 2382–2386. Hosoi, J., Murphy, G. F., Egan, C. L., Lerner, E. A., Grabbe, S., Asahina, A., and Granstein, R. D. (1993). Regulation of Langerhans cell function by nerves containing calcitonin gene‐related peptide. Nature 363, 159–163. Huang, Q., Liu, D., Majewski, P., Schulte, L. C., Korn, J. M., Young, R. A., Lander, E. S., and Hacohen, N. (2001). The plasticity of dendritic cell responses to pathogens and their components. Science 294, 870–875. Hugues, S., Fetler, L., Bonifaz, L., Helft, J., Amblard, F., and Amigorena, S. (2004). Distinct T‐cell dynamics in lymph nodes during the induction of tolerance and immunity. Nat. Immunol. 5, 1235–1242. Ibrahim, M. A., Chain, B. M., and Katz, D. R. (1995). The injured cell: The role of the dendritic cell system as a sentinel receptor pathway. Immunol. Today 16, 181–186. Inaba, K., Inaba, M., Naito, M., and Steinman, R. M. (1993). Dendritic cell progenitors phagocytose particulates, including bacillus Calmette‐Guerin organisms, and sensitize mice to mycobacterial antigens in vivo. J. Exp. Med. 178, 479–488. Inohara, N., and Nunez, G. (2003). NODs: intracellular proteins involved in inflammation and apoptosis. Nat. Rev. Immunol. 3, 371–382. Janeway, C. A., Jr. (1992). The immune system evolved to discriminate infectious nonself from noninfectious self. Immunol. Today. 13, 11–16. Janeway, C. A., Jr., and Medzhitov, R. (2002). Innate immune recognition. Annu. Rev. Immunol. 20, 197–216. Jones, C. (2003). Herpes simplex virus type 1 and bovine herpesvirus 1 latency. Clin. Microbiol. Rev. 16, 79–95.

226

f ra n c e s c a g r a n uc c i

ET AL.

Jonuleit, H., Schmitt, E., Schuler, G., Knop, J., and Enk, A. H. (2000). Induction of interleukin 10‐producing, nonproliferating CD4(þ) T cells with regulatory properties by repetitive stimulation with allogeneic immature human dendritic cells. J. Exp. Med. 192, 1213–1222. Jung, S., Unutmaz, D., Wong, P., Sano, G., De los Santos, K., Sparwasser, T., Wu, S., Vuthoori, S., Ko, K., Zavala, F., Pamer, E. G., Littman, D. R., and Lang, R. A. (2002). In vivo depletion of CD11c(þ) dendritic cells abrogates priming of CD8(þ) T cells by exogenous cell‐associated antigens. Immunity 17, 211–220. Kadowaki, N., and Liu, Y. J. (2002). Natural type I interferon‐producing cells as a link between innate and adaptive immunity. Hum. Immunol. 63, 1126–1132. Kahn, R. A., Fu, H., and Roy, C. R. (2002). Cellular hijacking: a common strategy for microbial infection. Trends Biochem. Sci. 27, 308–314. Kakimoto, M., Hasegawa, A., Fujita, S., and Yasukawa, M. (2002). Phenotypic and functional alterations of dendritic cells induced by human herpesvirus 6 infection. J. Virol. 76, 10338–10345. Klagge, I. M., ter Meulen, V., and Schneider‐Schaulies, S. (2000). Measles virus‐induced promotion of dendritic cell maturation by soluble mediators does not overcome the immunosuppressive activity of viral glycoproteins on the cell surface. Eur. J. Immunol. 30, 2741–2750. Kleijmeer, M., Ramm, G., Schuurhuis, D., Griffith, J., Rescigno, M., Ricciardi‐Castagnoli, P., Rudensky, A. Y., Ossendorp, F., Melief, C. J., Stoorvogel, W., and Geuze, H. J. (2001). Reorganization of multivesicular bodies regulates MHC class II antigen presentation by dendritic cells. J. Cell Biol. 155, 53–63. Krieg, A. M. (2000). The role of CpG motifs in innate immunity. Curr. Opin. Immunol. 12, 35–43. Kushnir, N., Liu, L., and Mac Pherson, G. G. (1998). Dendritic cells and resting B cells form clusters in vitro and in vivo: T‐cell independence, partial LFA‐1 dependence, and regulation by cross‐linking surface molecules. J. Immunol. 160, 1774–1781. Kyewski, B., and Derbinski, J. (2004). Self‐representation in the thymus: An extended view. Nat. Rev. Immunol. 4, 688–698. Lafaille, J. J., Nagashima, K., Katsuki, M., and Tonegawa, S. (1994). High incidence of spontaneous autoimmune encephalomyelitis in immunodeficient antimyelin basic protein T‐cell receptor transgenic mice. Cell 78, 399–408. Latz, E., Schoenemeyer, A., Visintin, A., Fitzgerald, K. A., Monks, B. G., Knetter, C. F., Lien, E., Nilsen, N. J., Espevik, T., and Golenbock, D. T. (2004). TLR9 signals after translocating from the ER to CpG DNA in the lysosome. Nat. Immunol. 5, 190–198. Laufer, T. M., De Koning, J., Markowitz, J. S., Lo, D., and Glimcher, L. H. (1996). Unopposed positive selection and autoreactivity in mice expressing class II MHC only on thymic cortex. Nature 383, 81–85. Laufer, T. M., Glimcher, L. H., and Lo, D. (1999). Using thymus anatomy to dissect T cell repertoire selection. Semin. Immunol. 11, 65–70. Le Bon, A., Schiavoni, G., D’Agostino, G., Gresser, I., Belardelli, F., and Tough, D. F. (2001). Type I interferons potently enhance humoral immunity and can promote isotype switching by stimulating dendritic cells in vivo. Immunity 14, 461–470. Lemaitre, B., Nicolas, E., Michaut, L., Reichhart, J. M., and Hoffmann, J. A. (1996). The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 86, 973–983. Levings, M. K., Gregori, S., Tresoldi, E., Cazzaniga, S., Bonini, C., and Roncarolo, M. G. (2005). Differentiation of Tr1 cells by immature dendritic cells requires IL‐10 but not CD25þCD4þ Tr cells. Blood 105, 1162–1169. Li, L., Liu, D., Hutt‐Fletcher, L., Morgan, A., Masucci, M. G., and Levitsky, V. (2002). Epstein‐ Barr virus inhibits the development of dendritic cells by promoting apoptosis of their monocyte

d en d r i t i c c e l l b i o l og y

227

precursors in the presence of granulocyte macrophage‐colony‐stimulating factor and interleukin‐4. Blood 99, 3725–3734. Lipford, G. B., Heeg, K., and Wagner, H. (1998). Bacterial DNA as immune cell activator. Trends Microbiol. 6, 496–500. Liston, A., Lesage, S., Wilson, J., Peltonen, L., and Goodnow, C. C. (2003). Aire regulates negative selection of organ‐specific T cells. Nat. Immunol. 4, 350–354. Litinskiy, M. B., Nardelli, B., Hilbert, D. M., He, B., Schaffer, A., Casali, P., and Cerutti, A. (2002). DCs induce CD40‐independent immunoglobulin class switching through BLyS and APRIL. Nat. Immunol. 3, 822–829. Luft, T., Pang, K. C., Thomas, E., Hertzog, P., Hart, D. N., Trapani, J., and Cebon, J. (1998). Type I IFNs enhance the terminal differentiation of dendritic cells. J. Immunol. 161, 1947–1953. Lutz, M. B., Rovere, P., Kleijmeer, M. J., Rescigno, M., Assmann, C. U., Oorschot, V. M., Geuze, H. J., Trucy, J., Demandolx, D., Davoust, J., and Ricciardi‐Castagnoli, P. (1997). Intracellular routes and selective retention of antigens in mildly acidic cathepsin D/lysosome‐associated membrane protein‐1/MHC class II‐positive vesicles in immature dendritic cells. J. Immunol. 159, 3707–3716. Lutz, M. B., and Schuler, G. (2002). Immature, semi‐mature and fully mature dendritic cells: Which signals induce tolerance or immunity? Trends Immunol. 23, 445–449. Macatonia, S. E., Hosken, N. A., Litton, M., Vieira, P., Hsieh, C. S., Culpepper, J. A., Wysocka, M., Trinchieri, G., Murphy, K. M., and O’Garra, A. (1995). Dendritic cells produce IL‐12 and direct the development of Th1 cells from naive CD4þ T cells. J. Immunol. 154, 5071–5079. Malek, T. R., and Bayer, A. L. (2004). Tolerance, not immunity, crucially depends on IL‐2. Nat. Rev. Immunol. 4, 665–674. Marie, J. C., Kehren, J., Trescol‐Biemont, M. C., Evlashev, A., Valentin, H., Walzer, T., Tedone, R., Loveland, B., Nicolas, J. F., Rabourdin‐Combe, C., and Horvat, B. (2001). Mechanism of measles virus‐induced suppression of inflammatory immune responses. Immunity 14, 69–79. Marrack, P., Lo, D., Brinster, R., Palmiter, R., Burkly, L., Flavell, R. H., and Kappler, J. (1988). The effect of thymus environment on T‐cell development and tolerance. Cell 53, 627–634. Marrack, P., Mitchell, T., Hildeman, D., Kedl, R., Teague, T. K., Bender, J., Rees, W., Schaefer, B. C., and Kappler, J. (2000). Genomic‐scale analysis of gene expression in resting and activated T cells. Curr. Opin. Immunol. 12, 206–209. Martin‐Fontecha, A., Thomsen, L. L., Brett, S., Gerard, C., Lipp, M., Lanzavecchia, A., and Sallusto, F. (2004). Induced recruitment of NK cells to lymph nodes provides IFN‐gamma for T(H)1 priming. Nat. Immunol. 5, 1260–1265. Massari, P., Henneke, P., Ho, Y., Latz, E., Golenbock, D. T., and Wetzler, L. M. (2002). Cutting edge: Immune stimulation by neisserial porins is Toll‐like receptor 2 and MyD88 dependent. J. Immunol. 168, 1533–1537. Matsukawa, A., Lukacs, N. W., Standiford, T. J., Chensue, S. W., and Kunkel, S. L. (2000). Adenoviral‐mediated overexpression of monocyte chemoattractant protein‐1 differentially alters the development of Th1 and Th2 type responses in vivo. J. Immunol. 164, 1699–1704. Matsumoto, M., Funami, K., Tanabe, M., Oshiumi, H., Shingai, M., Seto, Y., Yamamoto, A., and Seya, T. (2003). Subcellular localization of Toll‐like receptor 3 in human dendritic cells. J. Immunol. 171, 3154–3162. Matsuno, K., Ezaki, T., Kudo, S., and Uehara, Y. (1996). A life stage of particle‐laden rat dendritic cells in vivo: Their terminal division, active phagocytosis, and translocation from the liver to the draining lymph. J. Exp. Med. 183, 1865–1878. Matzinger, P. (1994). Tolerance, danger, and the extended family. Annu. Rev. Immunol. 12, 991–1045. Matzinger, P. (2002). An innate sense of danger. Ann. NY Acad. Sci. 961, 341–342.

228

f ra n c e s c a g r a n uc c i

ET AL.

Mbow, M. L., Zeidner, N., Panella, N., Titus, R. G., and Piesman, J. (1997). Borrelia burgdorferi‐ pulsed dendritic cells induce a protective immune response against tick‐transmitted spirochetes. Infect. Immun. 65, 3386–3390. McCaffrey, R. L., Fawcett, P., O’ Riordan, M., Lee, K. D., Havell, E. A., Brown, P. O., and Portnoy, D. A. (2004). A specific gene expression program triggered by Gram‐positive bacteria in the cytosol. Proc. Natl. Acad. Sci. USA 101, 11386–11391. Medzhitov, R., and Janeway, C. A., Jr. (1997). Innate immunity: The virtues of a nonclonal system of recognition. Cell 91, 295–298. Medzhitov, R., and Janeway, C. A., Jr. (2000). How does the immune system distinguish self from nonself? Semin. Immunol. 12, 185–188, discussion 257–344. Medzhitov, R., Preston‐Hurlburt, P., and Janeway, C. A., Jr. (1997). A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 388, 394–397. Mempel, T. R., Henrickson, S. E., and Von Andrian, U. H. (2004). T‐cell priming by dendritic cells in lymph nodes occurs in three distinct phases. Nature 427, 154–159. Menges, M., Rossner, S., Voigtlander, C., Schindler, H., Kukutsch, N. A., Bogdan, C., Erb, K., Schuler, G., and Lutz, M. B. (2002). Repetitive injections of dendritic cells matured with tumor necrosis factor alpha induce antigen‐specific protection of mice from autoimmunity. J. Exp. Med. 195, 15–21. Mikloska, Z., Kesson, A. M., Penfold, M. E., and Cunningham, A. L. (1996). Herpes simplex virus protein targets for CD4 and CD8 lymphocyte cytotoxicity in cultured epidermal keratinocytes treated with interferon‐gamma. J. Infect. Dis. 173, 7–17. Miller, L. J., Bainton, D. F., Borregaard, N., and Springer, T. A. (1987). Stimulated mobilization of monocyte Mac‐1 and p150,95 adhesion proteins from an intracellular vesicular compartment to the cell surface. J. Clin. Invest. 80, 535–544. Milone, M. C., and Fitzgerald‐Bocarsly, P. (1998). The mannose receptor mediates induction of IFN‐alpha in peripheral blood dendritic cells by enveloped RNA and DNA viruses. J. Immunol. 161, 2391–2399. Moretta, A. (2002). Natural killer cells and dendritic cells: Rendezvous in abused tissues. Nat. Rev. Immunol. 2, 957–964. Morrow, G., Slobedman, B., Cunningham, A. L., and Abendroth, A. (2003). Varicella‐zoster virus productively infects mature dendritic cells and alters their immune function. J. Virol. 77, 4950–4959. Moutaftsi, M., Mehl, A. M., Borysiewicz, L. K., and Tabi, Z. (2002). Human cytomegalovirus inhibits maturation and impairs function of monocyte‐derived dendritic cells. Blood 99, 2913–2921. Mueller, D. L., Jenkins, M. K., and Schwartz, R. H. (1989). Clonal expansion versus functional clonal inactivation: A costimulatory signalling pathway determines the outcome of T‐cell antigen receptor occupancy. Annu. Rev. Immunol. 7, 445–480. Mueller, S. N., Jones, C. M., Smith, C. M., Heath, W. R., and Carbone, F. R. (2002). Rapid cytotoxic T lymphocyte activation occurs in the draining lymph nodes after cutaneous herpes simplex virus infection as a result of early antigen presentation and not the presence of virus. J. Exp. Med. 195, 651–656. Muller, D. B., Raftery, M. J., Kather, A., Giese, T., and Schonrich, G. (2004). Frontline: Induction of apoptosis and modulation of c‐FLIPL and p53 in immature dendritic cells infected with herpes simplex virus. Eur. J. Immunol. 34, 941–951. Murphy, C. A., Langrish, C. L., Chen, Y., Blumenschein, W., McClanahan, T., Kastelein, R. A., Sedgwick, J. D., and Cua, D. J. (2003). Divergent pro‐ and antiinflammatory roles for IL‐23 and IL‐12 in joint autoimmune inflammation. J. Exp. Med. 198, 1951–1957. Murphy, K. M., Heimberger, A. B., and Loh, D. Y. (1990). Induction by antigen of intrathymic apoptosis of CD4þCD8þTCRlo thymocytes in vivo. Science 250, 1720–1723.

d en d r i t i c c e l l b i o l og y

229

Mushegian, A., and Medzhitov, R. (2001). Evolutionary perspective on innate immune recognition. J. Cell Biol. 155, 705–710. Nelson, D. J., McMenamin, C., McWilliam, A. S., Brenan, M., and Holt, P. G. (1994). Development of the airway intraepithelial dendritic cell network in the rat from class II major histocompatibility (Ia)‐negative precursors: Differential regulation of Ia expression at different levels of the respiratory tract. J. Exp. Med. 179, 203–212. Nestle, F. O., Zheng, X. G., Thompson, C. B., Turka, L. A., and Nickoloff, B. J. (1993). Characterization of dermal dendritic cells obtained from normal human skin reveals phenotypic and functionally distinctive subsets. J. Immunol. 151, 6535–6545. Nigou, J., Zelle‐Rieser, C., Gilleron, M., Thurnher, M., and Puzo, G. (2001). Mannosylated lipoarabinomannans inhibit IL‐12 production by human dendritic cells: Evidence for a negative signal delivered through the mannose receptor. J. Immunol. 166, 7477–7485. Nilsen, N., Nonstad, U., Khan, N., Knetter, C. F., Akira, S., Sundan, A., Espevik, T., and Lien, E. (2004). Lipopolysaccharide and double‐stranded RNA upregulate Toll‐like receptor 2 independently of myeloid differentiation factor 88. J. Biol. Chem. 279, 39727–39735. Oxenius, A., Karrer, U., Zinkernagel, R. M., and Hengartner, H. (1999). IL‐12 is not required for induction of type 1 cytokine responses in viral infections. J. Immunol. 162, 965–973. Parr, M. B., Kepple, L., and Parr, E. L. (1991). Langerhans cells phagocytose vaginal epithelial cells undergoing apoptosis during the murine estrous cycle. Biol. Reprod. 45, 252–260. Piccioli, D., Sbrana, S., Melandri, E., and Valiante, N. M. (2002). Contact‐dependent stimulation and inhibition of dendritic cells by natural killer cells. J. Exp. Med. 195, 335–341. Pierre, P., Turley, S. J., Gatti, E., Hull, M., Meltzer, J., Mirza, A., Inaba, K., Steinman, R. M., and Mellman, I. (1997). Developmental regulation of MHC class II transport in mouse dendritic cells. Nature 388, 787–792. Plotnicky‐Gilquin, H., Cyblat, D., Aubry, J. P., Delneste, Y., Blaecke, A., Bonnefoy, J. Y., Corvaia, N., and Jeannin, P. (2001). Differential effects of parainfluenza virus type 3 on human monocytes and dendritic cells. Virology 285, 82–90. Poltorak, A., He, X., Smirnova, I., Liu, M. Y., Van Huffel, C., Du, X., Birdwell, D., Alejos, E., Silva, M., Galanos, C., Freudenberg, M., Ricciardi‐Castagnoli, P., Layton, B., and Beutler, B. (1998). Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282, 2085–2088. Probst, H. C., Lagnel, J., Kollias, G., and van den Broek, M. (2003). Inducible transgenic mice reveal resting dendritic cells as potent inducers of CD8þ T cell tolerance. Immunity 18, 713–720. Probst, H. C., McCoy, K., Okazaki, T., Honjo, T., and van den Broek, M. (2005). Resting dendritic cells induce peripheral CD8þ T cell tolerance through PD‐1 and CTLA‐4. Nat. Immunol. 6, 280–286. Probst, H. C., and van den Broek, M. (2005). Priming of CTLs by Lymphocytic Choriomeningitis Virus Depends on Dendritic Cells. J. Immunol. 174, 3920–3924. Reading, P. C., Miller, J. L., and Anders, E. M. (2000). Involvement of the mannose receptor in infection of macrophages by influenza virus. J. Virol. 74, 5190–5197. Reinhart, T. A. (2003). Chemokine induction by HIV‐1: Recruitment to the cause. Trends Immunol. 24, 351–353. Reis e Sousa, C., Stahl, P. D., and Austyn, J. M. (1993). Phagocytosis of antigens by Langerhans cells in vitro. J. Exp. Med. 178, 509–519. Rescigno, M., Citterio, S., Thery, C., Rittig, M., Medaglini, D., Pozzi, G., Amigorena, S., and Ricciardi‐Castagnoli, P. (1998). Bacteria‐induced neo‐biosynthesis, stabilization, and surface expression of functional class I molecules in mouse dendritic cells. Proc. Natl. Acad. Sci. USA 95, 5229–5234.

230

f ra n c e s c a g r a n uc c i

ET AL.

Rescigno, M., Urbano, M., Rimoldi, M., Valzasina, B., Rotta, G., Granucci, F., and Ricciardi‐ Castagnoli, P. (2002). Toll‐like receptor 4 is not required for the full maturation of dendritic cells or for the degradation of Gram‐negative bacteria. Eur. J. Immunol. 32, 2800–2806. Rescigno, M., Urbano, M., Valzasina, B., Francolini, M., Rotta, G., Bonasio, R., Granucci, F., Kraehenbuhl, J. P., and Ricciardi‐Castagnoli, P. (2001). Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nat. Immunol. 2, 361–367. Robbins, P. A., and McMichael, A. J. (1991). Immune recognition of HLA molecules downmodulates CD8 expression on cytotoxic T lymphocytes. J. Exp. Med. 173, 221–230. Rodriguez, A., Regnault, A., Kleijmeer, M., Ricciardi‐Castagnoli, P., and Amigorena, S. (1999). Selective transport of internalized antigens to the cytosol for MHC class I presentation in dendritic cells. Nat. Cell Biol. 1, 362–368. Roncarolo, M. G., Gregori, S., and Levings, M. (2003). Type 1 T regulatory cells and their relationship with CD4þCD25þ T regulatory cells. Novartis Found. Symp. 252, 115–127 discussion 127–131, 203–210.. Roncarolo, M. G., and Levings, M. K. (2000). The role of different subsets of T regulatory cells in controlling autoimmunity. Curr. Opin. Immunol. 12, 676–683. Rong, Q., Alexander, T. S., Koski, G. K., and Rosenthal, K. S. (2003). Multiple mechanisms for HSV‐1 induction of interferon alpha production by peripheral blood mononuclear cells. Arch. Virol. 148, 329–344. Sakaguchi, S. (2000). Regulatory T cells: Key controllers of immunologic self‐tolerance. Cell 101, 455–458. Salio, M., Cella, M., Suter, M., and Lanzavecchia, A. (1999). Inhibition of dendritic cell maturation by herpes simplex virus. Eur. J. Immunol. 29, 3245–3253. Sallusto, F., and Lanzavecchia, A. (1994). Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony‐stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J. Exp. Med. 179, 1109–1118. Schnare, M., Barton, G. M., Holt, A. C., Takeda, K., Akira, S., and Medzhitov, R. (2001). Toll‐like receptors control activation of adaptive immune responses. Nat. Immunol. 2, 947–950. Schonrich, G., Kalinke, U., Momburg, F., Malissen, M., Schmitt‐Verhulst, A. M., Malissen, B., Hammerling, G. J., and Arnold, B. (1991). Downregulation of T‐cell receptors on self‐reactive T cells as a novel mechanism for extrathymic tolerance induction. Cell 65, 293–304. Schwartz, R. H. (2003). T cell anergy. Annu. Rev. Immunol. 21, 305–334. Sertl, K., Takemura, T., Tschachler, E., Ferrans, V. J., Kaliner, M. A., and Shevach, E. M. (1986). Dendritic cells with antigen‐presenting capability reside in airway epithelium, lung parenchyma, and visceral pleura. J. Exp. Med. 163, 436–451. Sevilla, N., Kunz, S., McGavern, D., and Oldstone, M. B. (2003). Infection of dendritic cells by lymphocytic choriomeningitis virus. Curr. Top. Microbiol. Immunol. 276, 125–144. Shortman, K., and Liu, Y. J. (2002). Mouse and human dendritic cell subtypes. Nat. Rev. Immunol. 2, 151–161. Sing, A., Rost, D., Tvardovskaia, N., Roggenkamp, A., Wiedemann, A., Kirschning, C. J., Aepfelbacher, M., and Heesemann, J. (2002). Yersinia V‐antigen exploits Toll‐like receptor 2 and CD14 for interleukin 10‐mediated immunosuppression. J. Exp. Med. 196, 1017–1024. Smyth, M. J., Hayakawa, Y., Takeda, K., and Yagita, H. (2002). New aspects of natural‐killer‐cell surveillance and therapy of cancer. Nat. Rev. Cancer 2, 850–861. Sporri, R., and Reis e Sousa, C. (2005). Inflammatory mediators are insufficient for full dendritic cell activation and promote expansion of CD4þ T cell populations lacking helper function. Nat. Immunol. 6, 163–170. Steinman, R. M. (1991). The dendritic cell system and its role in immunogenicity. Annu. Rev. Immunol. 9, 271–296.

d en d r i t i c c e l l b i o l og y

231

Steinman, R. M. (2000). DC‐SIGN: A guide to some mysteries of dendritic cells. Cell 100, 491–494. Steinman, R. M. (2001). Dendritic cells and the control of immunity: Enhancing the efficiency of antigen presentation. Mt Sinai J. Med. 68, 106–166. Steinman, R. M., Hawiger, D., and Nussenzweig, M. C. (2003). Tolerogenic dendritic cells. Annu. Rev. Immunol. 21, 685–711. Stockinger, B. (1999). T lymphocyte tolerance: From thymic deletion to peripheral control mechanisms. Adv. Immunol. 71, 229–265. Su, H., Messer, R., Whitmire, W., Fischer, E., Portis, J. C., and Caldwell, H. D. (1998). Vaccination against chlamydial genital tract infection after immunization with dendritic cells pulsed ex vivo with nonviable Chlamydiae. J. Exp. Med. 188, 809–818. Sumen, C., Mempel, T. R., Mazo, I. B., and von Andrian, U. H. (2004). Intravital microscopy: Visualizing immunity in context. Immunity 21, 315–329. Surh, C. D., and Sprent, J. (1994). T‐cell apoptosis detected in situ during positive and negative selection in the thymus. Nature 372, 100–103. Svensson, M., Stockinger, B., and Wick, M. J. (1997). Bone marrow‐derived dendritic cells can process bacteria for MHC‐I and MHC‐II presentation to T cells. J. Immunol. 158, 4229–4236. Takeda, K., and Akira, S. (2004). TLR signaling pathways. Semin. Immunol. 16, 3–9. Takeda, K., Kaisho, T., and Akira, S. (2003). Toll‐like receptors. Annu. Rev. Immunol. 21, 335–376. Takeuchi, O., Hoshino, K., and Akira, S. (2000). Cutting edge: TLR2‐deficient and MyD88‐ deficient mice are highly susceptible to Staphylococcus aureus infection. J. Immunol. 165, 5392–5396. Takeuchi, O., Hoshino, K., Kawai, T., Sanjo, H., Takada, H., Ogawa, T., Takeda, K., and Akira, S. (1999). Differential roles of TLR2 and TLR4 in recognition of gram‐negative and gram‐positive bacterial cell wall components. Immunity 11, 443–451. Tal, G., Mandelberg, A., Dalal, I., Cesar, K., Somekh, E., Tal, A., Oron, A., Itskovich, S., Ballin, A., Houri, S., Beigelman, A., Lider, O., Rechavi, G., and Amariglio, N. (2004). Association between common Toll‐like receptor 4 mutations and severe respiratory syncytial virus disease. J. Infect. Dis. 189, 2057–2063. Tang, Z., and Saltzman, A. (2004). Understanding human dendritic cell biology through gene profiling. Inflamm. Res. 53, 424–441. Taniguchi, T., and Takaoka, A. (2002). The interferon‐alpha/beta system in antiviral responses: A multimodal machinery of gene regulation by the IRF family of transcription factors. Curr. Opin. Immunol. 14, 111–116. Tarbell, K. V., Yamazaki, S., Olson, K., Toy, P., and Steinman, R. M. (2004). CD25þ CD4þ T cells, expanded with dendritic cells presenting a single autoantigenic peptide, suppress autoimmune diabetes. J. Exp. Med. 199, 1467–1477. Terme, M., Tomasello, E., Maruyama, K., Crepineau, F., Chaput, N., Flament, C., Marolleau, J. P., Angevin, E., Wagner, E. F., Salomon, B., Lemonnier, F. A., Wakasugi, H., Colonna, M., Vivier, E., and Zitvogel, L. (2004). IL‐4 confers NK stimulatory capacity to murine dendritic cells: A signaling pathway involving KARAP/DAP12‐triggering receptor expressed on myeloid cell 2 molecules. J. Immunol. 172, 5957–5966. Thurnher, M., Ramoner, R., Gastl, G., Radmayr, C., Bock, G., Herold, M., Klocker, H., and Bartsch, G. (1997). Bacillus Calmette‐Guerin mycobacteria stimulate human blood dendritic cells. Int. J. Cancer 70, 128–134. Trinchieri, G., Pflanz, S., and Kastelein, R. A. (2003). The IL‐12 family of heterodimeric cytokines: New players in the regulation of T cell responses. Immunity 19, 641–644. Trottein, F., Pavelka, N., Vizzardelli, C., Angeli, V., Zouain, C. S., Pelizzola, M., Capozzoli, M., Urbano, M., Capron, M., Belardelli, F., Granucci, F., and Ricciardi‐Castagnoli, P. (2004). A type

232

f ra n c e s c a g r a n uc c i

ET AL.

I IFN‐dependent pathway induced by Schistosoma mansoni eggs in mouse myeloid dendritic cells generates an inflammatory signature. J. Immunol. 172, 3011–3017. Tsuji, S., Matsumoto, M., Takeuchi, O., Akira, S., Azuma, I., Hayashi, A., Toyoshima, K., and Seya, T. (2000). Maturation of human dendritic cells by cell wall skeleton of Mycobacterium bovis bacillus Calmette‐Guerin: Involvement of Toll‐like receptors. Infect. Immun. 68, 6883–6890. Underhill, D. M., Ozinsky, A., Hajjar, A. M., Stevens, A., Wilson, C. B., Bassetti, M., and Aderem, A. (1999). The Toll‐like receptor 2 is recruited to macrophage phagosomes and discriminates between pathogens. Nature 401, 811–815. Van Den Broeke, L. T., Daschbach, E., Thomas, E. K., Andringa, G., and Berzofsky, J. A. (2003). Dendritic Cell‐Induced Activation of Adaptive and Innate Antitumor Immunity. J. Immunol. 171, 5842–5852. van Kooyk, Y., and Geijtenbeek, T. B. (2003). DC‐SIGN: escape mechanism for pathogens. Nat. Rev. Immunol. 3, 697–709. van Lint, A., Ayers, M., Brooks, A. G., Coles, R. M., Heath, W. R., and Carbone, F. R. (2004). Herpes simplex virus‐specific CD8þ T cells can clear established lytic infections from skin and nerves and can partially limit the early spread of virus after cutaneous inoculation. J. Immunol. 172, 392–397. Vidalain, P. O., Azocar, O., Lamouille, B., Astier, A., Rabourdin‐Combe, C., and Servet‐Delprat, C. (2000). Measles virus induces functional TRAIL production by human dendritic cells. J. Virol. 74, 556–559. Visintin, A., Mazzoni, A., Spitzer, J. H., Wyllie, D. H., Dower, S. K., and Segal, D. M. (2001). Regulation of Toll‐like receptors in human monocytes and dendritic cells. J. Immunol. 166, 249–255. Volkmann, A., Zal, T., and Stockinger, B. (1997). Antigen‐presenting cells in the thymus that can negatively select MHC class II‐restricted T cells recognizing a circulating self antigen. J. Immunol. 158, 693–706. Weninger, W., and von Andrian, U. H. (2003). Chemokine regulation of naive T‐cell traffic in health and disease. Semin. Immunol. 15, 257–270. Wilflingseder, D., Banki, Z., Dierich, M. P., and Stoiber, H. (2005). Mechanisms promoting dendritic cell‐mediated transmission of HIV. Mol. Immunol. 42, 229–237. Winzler, C., Rovere, P., Rescigno, M., Granucci, F., Penna, G., Adorini, L., Zimmermann, V. S., Davoust, J., and Ricciardi‐Castagnoli, P. (1997). Maturation stages of mouse dendritic cells in growth factor‐dependent long‐term cultures. J. Exp. Med. 185, 317–328. Woolhouse, M. E., Webster, J. P., Domingo, E., Charlesworth, B., and Levin, B. R. (2002). Biological and biomedical implications of the co‐evolution of pathogens and their hosts. Nat. Genet. 32, 569–577. Wooten, R. M., Ma, Y., Yoder, R. A., Brown, J. P., Weis, J. H., Zachary, J. F., Kirschning, C. J., and Weis, J. J. (2002). Toll‐like receptor 2 is required for innate, but not acquired, host defense to Borrelia burgdorferi. J. Immunol. 168, 348–355. Wu, M. T., and Hwang, S. T. (2002). CXCR5‐transduced bone marrow‐derived dendritic cells traffic to B cell zones of lymph nodes and modify antigen‐specific immune responses. J. Immunol. 168, 5096–5102. Wykes, M., Pombo, A., Jenkins, C., and Mac Pherson, G. G. (1998). Dendritic cells interact directly with naive B lymphocytes to transfer antigen and initiate class switching in a primary T‐dependent response. J. Immunol. 161, 1313–1319. Yamazaki, S., Iyoda, T., Tarbell, K., Olson, K., Velinzon, K., Inaba, K., and Steinman, R. M. (2003). Direct expansion of functional CD25þ CD4þ regulatory T cells by antigen‐processing dendritic cells. J. Exp. Med. 198, 235–247.

d en d r i t i c c e l l b i o l og y

233

Yoshimura, A., Lien, E., Ingalls, R. R., Tuomanen, E., Dziarski, R., and Golenbock, D. (1999). Cutting edge: Recognition of Gram‐positive bacterial cell wall components by the innate immune system occurs via Toll‐like receptor 2. J. Immunol. 163, 1–5. Zal, T., Volkmann, A., and Stockinger, B. (1994). Mechanisms of tolerance induction in major histocompatibility complex class II‐restricted T cells specific for a blood‐borne self‐antigen. J. Exp. Med. 180, 2089–2099. Zanoni, I., Foti, M., Ricciardi-Castagnoli, P., and Granucci, F. (2005). TLR-dependent activation stimuli associated with Th1 responses confer NK-cell stimulatory capacity to mouse dendritic cells. J. Immunol. 175, 286–292. Zhang, G. X., Gran, B., Yu, S., Li, J., Siglienti, I., Chen, X., Kamoun, M., and Rostami, A. (2003). Induction of experimental autoimmune encephalomyelitis in IL‐12 receptor‐beta 2‐deficient mice: IL‐12 responsiveness is not required in the pathogenesis of inflammatory demyelination in the central nervous system. J. Immunol. 170, 2153–2160. Zitvogel, L. (2002). Dendritic and natural killer cells cooperate in the control/switch of innate immunity. J. Exp. Med. 195, F9–14.

The Murine Diabetogenic Class II Histocompatibility Molecule I‐Ag7: Structural and Functional Properties and Specificity of Peptide Selection Anish Suri and Emil R. Unanue Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri 63110 Abstract............................................................................................................. Introduction ....................................................................................................... Structural Features of I‐Ag7 and DQ8 ..................................................................... Biochemical Basis of Peptide Selection by I‐Ag7 ........................................................ Why Is There a Difference in Results Between Binding and Peptide Selection? ............................................................................................... 5. The Biological Role of I‐Ag7 .................................................................................. References .........................................................................................................

1. 2. 3. 4.

235 235 236 241 254 256 259

Abstract The onset of type 1 diabetes mellitus (T1DM) is directly linked to the expression of class II MHC molecules. The NOD mouse, which is an excellent animal model for the human disease, expresses the I‐Ag7 molecule that shares many features with the human diabetogenic class II MHC alleles. In this review, the structural, biochemical, and biological properties of the I‐Ag7 molecules and how they relate to onset of diabetes is discussed. In particular, the focus is on the unique properties of peptide selection by I‐Ag7 that reveal the preferred binding motif of diabetogenic MHC molecules and its role in display of peptides derived from islet b cells. 1. Introduction Type 1 diabetes mellitus (T1DM) is an organ‐specific autoimmune disorder characterized by T cell‐mediated destruction of the b cells of the pancreatic islets of Langerhans, resulting in insulin deficiency (Bach, 1994; Castano and Eisenbarth, 1990; Tisch and McDevitt, 1996). The nonobese diabetic (NOD) mouse is an excellent animal model for T1DM especially for exhibiting histopathological and immunological features of the human disease (Kikutani and Makino, 1992). Numerous studies demonstrated that the class II MHC alleles are the single‐most important genetic elements that determined susceptibility to T1DM (Acha‐Orbea and McDevitt, 1987; Morel et al., 1988; Todd et al., 1987). HLA‐DQ2 and DQ8 (from here on referred to as DQ2 and DQ8, respectively) in humans, and I‐Ag7 molecule in the NOD mice are

235 advances in immunology, vol. 88 # 2005 Elsevier Inc. All rights reserved.

0065-2776/05 $35.00 DOI: 10.1016/S0065-2776(05)88007-1

236

a n i s h s u ri a n d e m i l r . u na n u e

most critical for the onset of diabetes (Castano and Eisenbarth, 1990; Wicker et al., 1995). What is the primary role of the class II MHC molecule in predisposing to T1DM? Several studies in the past suggested the involvement of I‐Ag7 in generating a thymic environment that allows for escape of self‐reactive T cells, while many others implicated the same molecule in the activation of diabetogenic T cells in the periphery. The analysis of these two functions is complicated by the fact that several other components influence the development of diabetes in the NOD mouse. For example, whether thymic‐negative selection is only caused by structural features of I‐Ag7 or other components is a fundamental issue not yet resolved. Rather than analyzing the many cellular and biochemical components in NOD diabetes, this review has narrowed the focus to the properties of I‐Ag7. Because the only known function of class II MHC molecules is to present peptides to CD4 T cells, it becomes important to identify the structural and biochemical parameters that determine the nature of peptides displayed by a given allele of interest, and how these peptides influence the development of autoimmunity. This is especially crucial in cases of MHC‐linked autoimmune disorders, such as T1DM, where the activation of diabetogenic T cells is directly dependent upon the peptides selected by the APCs. Here the structural, biological, and biochemical features of the murine diabetogenic class II MHC molecules are reviewed and emphasis is placed on recent findings from the laboratory. 2. Structural Features of I‐Ag7 and DQ8 The seminal analysis by Todd, Bell, and McDevitt (1987) extended several genetic observations by relating T1DM to HLA‐DR alleles (DR3 and DR4), and particularly HLA‐DQ alleles, in linkage disequilibrium with HLA‐DR. Researchers came to several conclusions: the DQ b‐chain sequence correlated with the susceptibility to T1DM, and that the sequence differences were allelic and not resulting from mutations; that the differences resided particularly on the residue 57 of the b chain, a nonaspartic acid, and that the NOD I‐Ag7 strain also had such a non‐Asp residue at b57 (Acha‐Orbea and McDevitt, 1987; Morel et al., 1988; Todd et al., 1987). The important issue of homozygosity of the non‐Asp forms was also an important component of this genetic analysis. This conclusion has served as a strong incentive for investigating the relationship between an HLA genotype and autoimmunity, that is, ‘‘We suggest that DQb allelic polymorphisms particularly at position 57, determine the specificity and extent of the autoimmune response against islet cell antigens through T cell help and/or suppression’’ (Todd et al., 1987). Subsequently, studies in NOD mice indicated that transgenic or F1 NOD mice that expressed class II

c la s s ii m h c m o l e cu l e i ‐a g 7

237

MHC alleles with a b57Asp exhibited marked protection from diabetes (Hattori et al., 1986; Kanagawa, et al., 2000; Lund et al., 1990; Miyazaki et al., 1990; Nishimoto et al., 1987; Quartey‐Papafio et al., 1995; Singer et al., 1993, 1998; Slattery et al., 1990). A key issue is to identify the structural characteristics of the diabetogenic class II MHC molecules, particularly to analyze the effect of the presence of a non‐Asp amino acid at b57. Past studies on I‐Ag7 and DQ8 suggested structural and unique conformational features that could contribute to these molecules being unstable and poor peptide binders (Arneson et al., 2000; Bhatnagar et al., 2001; Carrasco‐Marin et al., 1996; Nabavieh et al., 1998; Peterson and Sant, 1998; Reizis et al., 1997a,b). Class II MHC molecules can be examined in sodium dodecyl sulphate‐ polyacrylamide gel electrophoresis (SDS‐PAGE) under conditions where the proteins are not boiled. With many different haplotypes, two molecular forms are discerned: the stable form contains the ab dimer, the unstable contains the separated a and b chains (Germain and Hendrix, 1991; Nelson et al., 1993, 1994; Sadegh‐Nasseri and Germain, 1991). With haplotypes like I‐Ak in particular, the presence of the stable form correlates very well with their content of strong binding peptides, and with its half‐life on the surface of APC (Nelson et al., 1993, 1994, 1996). For example, the dominant peptide from HEL residues 48–62 has a main anchor residue, Asp52, at P1 (DGSTDYGILQINSRW). In complex with I‐Ak it forms an SDS stable complex that is long‐lived and highly resistant to proteolytic enzymes. However, changing its P1 anchor to an alanine reduced binding affinity for I‐Ak, the complex became SDS unstable, short‐lived, and sensitive to proteolytic enzymes (Carrasco‐Marin et al., 1999b). These sets of results, also evaluated with other haplotypes, led researchers to correlate SDS stability of class II MHC with their content of strong binding peptides. Most I‐Ag7 molecules obtained from APC, pulsed or not pulsed with protein antigens, were overwhelmingly SDS‐unstable (Carrasco‐Marin et al., 1996). Metabolically labeled I‐Ag7 in APC also had a shorter life than I‐Ak (Carrasco‐Marin et al., 1996). Moreover, I‐Ag7 molecules were unstable when isolated by detergent solubilization of cells (Carrasco‐Marin et al., 1996; Reizis et al., 1997b). These findings led to the conclusion that I‐Ag7 contained weak binding peptides and that this feature could be relevant in terms of its biological context. The preceding findings were tempered with the observations that NOD mice mounted efficient antigen‐specific immune responses and did not exhibit any immunodeficient characteristics when challenged with various pathogens. Hence to settle many of these issues, it became imperative that the structure of diabetogenic class II MHC molecules be determined.

238

a n i s h s u ri a n d e m i l r . u na n u e

Subsequent studies tested proteins expressed with fos‐jun leucine zippers mainly from insect cells (Corper et al., 2000; Hausmann et al., 1999; Latek et al., 2000). These proteins were stable in solution, bound to a variety of peptides with varying koff s depending on structural features, maintained SDS‐ instability, and could be crystallized. The explanation, therefore, that I‐Ag7 holds weak peptides based solely on SDS‐unstable criteria is not warranted (Hausmann et al., 1999). Regardless, the structural basis of SDS instability of I‐Ag7 molecules remains unexplained. Work from Teyton, Wilson, and their associates, as well as from our own group in collaboration with David Fremont, resolved the protein structure of I‐Ag7 (Corper et al., 2000; Latek et al., 2000). Corper et al. (2000) resolved the structure of a peptide from the glutamic acid decarboxylase (GAD) 65 protein, from residues 207–220. We resolved the structure of the hen egg white lysozyme (HEL) peptide from residues 11–25 (Latek et al., 2000). Wiley’s group later solved the structure of HLA‐DQ8 containing an insulin peptide (Lee et al., 2001). Both I‐Ag7 structures indicated that the a‐ and b‐chains of I‐Ag7 interacted productively to generate a heterodimer whose overall topology was similar to the previously known structures of other class II MHC molecules (Corper et al., 2000; Latek et al., 2000). The peptide binding groove of I‐Ag7 contained four to five major anchor pockets (P1, P4, P6, P7, and P9) that interacted with side chains derived from the peptide, which lay in the binding groove in an extended conformation (Fig. 1). In most other class II MHC molecules there is a conserved b57Asp forming an ion‐pair with the opposing a76Arg, which defines the rim of the P9 pocket (Brown et al., 1993; Dessen et al., 1997; Fremont et al., 1998; Jardetzky et al., 1996; Scott et al., 1998). The unique structural feature in I‐Ag7 is this P9 pocket being composed of a b57Ser residue instead. Another feature of I‐Ag7 is the substitution of b56Pro by a histidine. The absence of the salt bridge with a76 generates a P9 pocket where the b57Ser moves away from the binding groove which then becomes wide, shallow, and more open toward the C‐terminus of the bound peptide (Corper et al. 2000; Latek et al., 2000) (Fig. 1). In addition, the b56His favors the alteration of the C‐terminal portion of the b chain—the bH1 helix. This reconfigured P9 anchor site is predicted to favor small side chains like alanine, glycine, or negatively charged residues. In the structure resolved by Corper et al. (2000), the P9 residue from the GAD65 peptide was Glu217 which formed an ion pair with aArg76 and also a hydrogen bond with the bSer57, filling in the reconfigured anchor site. In the structure solved by Latek et al. (2000) the P9 peptide residue was glycine, which interacted with the a76Arg indirectly via a water molecule buried in the P9 pocket. In addition, we noticed a solvent‐exposed channel at the base of

c la s s ii m h c m o l e cu l e i ‐a g 7

239

Figure 1 I‐Ag7 Pockets, anchors, and binding. (A) Top‐down view of the peptide‐binding groove of I‐Ag7. The acidic and basic regions of the binding groove are shown in red and blue, respectively. (B) The solvent‐accessible surface of the I‐Ag7 peptide‐binding groove (blue) viewed from the side as a cross‐section. In addition to the usual peptide anchor side chains (P1, P4, P6, P7, and P9), the

240

a n i s h s u ri a n d e m i l r . u na n u e

the P1 pocket generated most likely from the presence of a basic peptide P1Arg anchor buried deep into the pocket. This arginine was critical for the T‐cell recognition of the peptide‐I‐Ag7 complex (Latek et al., 2000). The P1 pocket anchor of I‐Ag7 was made up of hydrophilic residues and was predicted to show a broad specificity for peptides. In the HEL peptide, P1 was an arginine and substitution of the peptides by alanine, or bulky residues like phenylalanine, did not alter binding affinity. However, changing it to aspartic acid markedly reduced binding (Fig. 1) (Latek et al., 2000). The P4 pocket was small and rich in hydrophobic residues, predicted to accommodate small and medium hydrophobic amino acids and to disfavor large residues (Fig. 1). The P6 contained hydrophobic residues with some polar residues and could take either hydrophobic or hydrophilic small to medium amino acids (Fig. 1). I‐Ag7 also has a side P7 pocket that can receive a variety of residues. The DQ8 structure was comparable to I‐Ag7 with respect to the P9 pocket, that is, both were similar in their spatial features, as previously detailed. However, the other binding pockets of DQ8 were unique—for example, the P4 pocket of DQ8 is the largest and can easily accommodate large bulky residues (Lee et al., 2001). Similarly, the P1 pocket of DQ8 contains an arginine at a52 (in contrast to an isoleucine in the case of I‐Ag7) which forms an ion‐pair with an acidic amino acid from the peptide (Lee et al., 2001). In studies with I‐Ak, which also has a52Arg, it was noticed that most of the high‐affinity peptides selected by this molecule contained an acidic P1 anchor residue (Nelson et al., 1996). Hence, at the P1 position DQ8 resembled I‐Ak more than I‐Ag7. In their paper, Lee et al. (2001) commented on the DQ structure that gives susceptibility to diabetes in the Japanese population: it shares the same a chain but the b chain has a b57Asp. Their point is that the b chain has a Leu at b56 (instead of proline) and these additional atoms would distort the P9 position by forcing the a76Arg away from the b57Asp and leaving it unpaired. It would be interesting to examine the peptides selected from it to determine whether terminal acidic residues are also favored. Several additional points were made in the structural reports. The point was made that an acidic residue in P10 of the peptide could also establish a salt bridge with a76Arg, following some rearrangements of the peptide backbone (Corper et al., 2000). It is noteworthy that some natural peptides show runs of

P10 side chain also makes extensive contact with the I‐Ag7 molecule. (C) A set of single amino acid substitutions made at HEL11–25 anchor positions. Listed beside each peptide sequence is an IC50 value, the concentration of peptide required to inhibit 50% of the binding of a reference peptide to I‐Ag7. NB, no binding. (Adapted from Latek et al., 2000.)

c la s s ii m h c m o l e cu l e i ‐a g 7

241

two or three acidic residues at the C‐terminus which cooperate in binding to I‐Ag7 (Suri et al., 2002). All of the preceding studies taken together established the structural features of the murine and human diabetogenic class II MHC molecules. As mentioned before, the general features of these molecules did not reveal any structural defects or any major flaws in peptide interactions with the binding groove. The structural studies highlighted the commonalities and uniqueness of murine and human diabetogenic class II MHC molecules, and the precise features of each of the binding pockets in selecting the repertoire of peptides. 3. Biochemical Basis of Peptide Selection by I‐Ag7 3.1. Binding Motifs for I‐Ag7 Four approaches were taken to study peptide binding motifs for I‐Ag7: (1) motifs were estimated based on the X‐ray crystallographic examination of the bound peptides; (2) peptides from diabetogenic antigens (e.g., GAD or insulin) or other model antigens were studied, their sequences aligned and compared mainly following responses of T cells (Carrasco‐Marin et al., 1999a; Chao and McDevitt, 1997; Chao et al., 1999; Griffin et al., 1995; Harrison et al., 1997; Herman et al., 1999; Patel et al., 1997); (3) synthetic peptide libraries or phage display peptide libraries were examined for binding to I‐Ag7 (Gregori et al., 2000; Harfouch‐Hammoud et al., 1999; Hausmann et al., 1999; Oiso et al., 1998; Stratmann et al., 2000); and (4) libraries of naturally selected peptides were examined (Carrasco‐Marin et al., 1999a; Munz et al., 2002; Reich et al., 1994; Suri et al., 2002). The issues that are for discussion are whether there are dominant or minor sequences participating in binding to I‐Ag7; that is to say, how broad or narrow, or ‘‘promiscuous,’’ is the repertoire of peptides? And, knowing the peptide repertoire, does it help in understanding the pathophysiology of the diabetes‐propensity class II MHC molecules? The two reports on the structure of I‐Ag7 made notable progress in establishing features of the binding sites that favored particular amino acid side chains. In the study by Teyton, Wilson, and their group, much was made of sequence analysis among several diabetogenic antigens comparing them to the crystal structure of the bound GAD65 peptide (Corper et al., 2000). In Fremont’s analysis, we complemented the structure with mutagenesis analysis of the peptide, together with binding and T‐cell responses (Latek et al., 2000). In general agreement, the following features were defined: P1: tolerated many residues; no single residue was preferred, although in the HEL peptide, acidic residues decreased binding;

242

a n i s h s u ri a n d e m i l r . u na n u e

P4: small to medium hydrophobic residues with a preference for leucine, isoleucine, and valine; charged residues were not favored; P6: favored small hydrophobic and hydrophilic residues; residues with large side chains were not favored; P7: hydrophobic residues were favored; P9: acidic residues and glycine, alanine, and perhaps serine, were tolerated but basic residues were clearly hindering. Random peptide libraries have been examined and their sequences aligned to determine binding motifs. In a very detailed analysis, random phage display libraries made up of 12 residues that were bound and selected by empty I‐Ag7 molecules were made (Stratmann et al., 2000). A similar library of peptides from GAD 65 and ovalbumin was evaluated. Some of the isolated peptides were tested for their binding affinity. Comparisons were made with I‐Ad, which shares a common a chain with I‐Ag7. Their conclusions were that ‘‘. . . I‐Ag7. . . appears to be very promiscuous in terms of peptide binding. Their binding motifs are degenerate. . . the degree of promiscuity is increased for I‐Ag7 over I‐Ad as a consequence of a larger P9 pocket that can specifically accommodate negatively charged residues, as well as possibly residues with bulky side chains’’ (Stratmann et al., 2000). The contrasting result between the binding analysis of peptide libraries and the identification of naturally selected peptides from processing of proteins is that, in the latter there is a striking specificity of peptides bearing acidic residues at the carboxy end of the molecule, an issue discussed in the next section. The problem comes from the fact that for many peptides tested for binding, an acidic residue at the P9 position was not required. Note the crystal structure of the HEL11–25 peptide where P9 has a glycine (Latek et al., 2000). Laboratory‐tested peptides from the Ea chain on both binding and T‐cell responses revealed: a peptide backbone containing alanines in which the T‐cell receptor contact residues were displayed in the correct spacing, stimulated the T cells (Carrasco‐Marin et al., 1999a). Thus, there were no requirements with this peptide of particular MHC anchor amino acids. A polyalanine peptide was studied and also found to bind to I‐Ag7 (Corper et al., 2000). However, in both studies placing an acidic residue at the P9 position increased binding (Corper et al., 2000; Stratmann et al., 2000). Along these lines in the study from Wucherpfennig laboratory (Hausmann et al., 1999), peptides like that from the CLIP segment of the invariant chain bound poorly and readily dissociated, but replacing the unfavorable methionine at P9 with acidic residues resulted in higher affinity binding with a long dissociation rate. Therefore, there is general consensus that (1) peptides can bind to I‐Ag7 without a major need of acidic residues to occupy the P9 anchor pocket;

c la s s ii m h c m o l e cu l e i ‐a g 7

243

(2) peptides with acidic residues at P9 tend to bind better to I‐Ag7; (3) peptides’ affinities and dissociation times vary greatly; (4) no correlation exists between binding and SDS‐stability; and (5) the peptides in general tend to have low binding affinities for I‐Ag7 relative to that of other peptides to their respective class II MHC molecules. The same issues also exist for the human DQ2 and DQ8 class II MHC molecules in which some reports suggested that the P9 pocket prefers an acidic amino acid, whereas others have indicated no such preference (Chicz et al., 1994; Godkin et al., 1997; Johansen et al., 1996; Kwok et al., 1996; Lee et al., 2001; Oiso et al., 1997; Quarsten et al., 1998; Vartdal et al., 1996; Verreck et al., 1994). 3.2. Naturally Processed Peptides Selected by I‐Ag7: Evidence for Exquisite Specificity Dependent on the P9 Pocket The main advantage of the approach of analyzing libraries of natural peptides over other approaches is that the peptides identified are the physiological products of the antigen processing and presentation pathways. From the earliest seminal work of Rammensee and colleagues, the identification of naturally selected peptides have offered invaluable insights into allele‐ specific motifs for various MHC molecules (Falk et al., 1991; Rammensee et al., 1995). There are four reports on naturally processed peptides obtained from I‐Ag7 molecules isolated from APC, encompassing a total of over 100 different peptides (Munz et al., 2002; Reich et al., 1994; Suri et al., 2002, 2003). A limited study by Reich et al. first reported naturally processed peptides selected by I‐Ag7 from NOD splenocytes. With only five peptides identified, it left unclear the sequence preferences in selection. With the introduction of electrospray tandem mass spectrometry, particularly by the Hunt laboratory (Hunt et al., 1992), sequence analysis of peptides was made much easier and results with hundreds of peptides could be obtained. In other studies, APC lines were engineered that expressed either wild‐type I‐Ag7 or a modified I‐Ag7 molecule wherein the b56 and b57 were changed from histidine and serine to a proline and aspartic acid, respectively (called I‐Ag7PD). These cell lines are shown in Table 1. To note is that C3.G7 (I‐Ag7þ) and C3.G7PD (I‐Ag7PDþ) were genetically identical cells except for the expression of the class II MHC molecules (Table 1). The APC lines were expanded in bulk cultures and naturally processed peptides were isolated and sequenced by mass spectrometry (MS). Peptides isolated from I‐Ag7 followed the same general features as many class II peptides, particularly their presence in ‘‘peptide families’’ wherein all

a n i s h s u ri a n d e m i l r . u na n u e

244

Table 1 Generation of APC Lines Expressing I‐Ag7 or I‐Ag7PD* APC Line C3G7 M12G7 C3G7PD

b56

b57

I‐Ag7

His

Ser

I‐Ag7 I‐Ag7PD

His Pro

Ser Asp

Class II MHC

*The M12.C3 B‐cell lymphoma was transfected with plasmids encoding I‐Aad along with either I‐Abg7 or I‐Abg7PD to generate C3.G7 or C3.G7PD, respectively (Suri et al., 2002). The M12G7 line was generated by fusing B cells from LPS‐activated NOD splenocytes to the M12.C3 B‐cell lymphoma (Suri et al., 2002).

the members share a common binding core that spans from the P1 to P9 residues along with varying flanking residues along the C‐ and/or N‐terminus (Suri et al., 2002, 2003). The heterogeneity of flanking residues is reflective of the antigen processing steps wherein the earliest event involves protein unfolding in acidic vesicles followed by binding of the preferred 9‐mer core by the class II MHC molecule, which also protects the core from proteolysis. The bound polypeptides are trimmed from both termini by various proteases and peptidases, which then give rise to the presence of varying flanks. An example of this was evident in studies by Nelson et al. (1997), wherein the lengths of a naturally selected HEL peptide family varied when APCs processed wild‐type HEL versus a mutant HEL that contained prolines in the flanking region of the binding core. Prolines blocked the amino peptidases, and hence the family members on such mutant HEL were extended at their N‐terminal flanks (Nelson et al., 1997). The flanking residues were shown to contribute toward peptide binding affinity to MHC, in display of TCR contact residues, and at times have themselves served as direct TCR‐contact residues (Carson et al., 1997; Latek and Unanue, 1999; Pu et al., 2002). In our studies with I‐Ag7, we noticed that the presence of acidic amino acids in the C‐terminal flanking residues enhanced peptide‐binding affinity to I‐Ag7, in consensus with the structural prediction by Corper et al. (Corper et al., 2000; Suri et al., 2002). Analysis of large numbers of naturally processed peptides demonstrated that peptides selected by I‐Ag7 exhibited a biased presence of acidic amino acids toward their C‐termini (Fig. 2) (Suri et al., 2002). In addition, many peptides selected by I‐Ag7 contained runs of double or triple acidic amino acids toward their C‐termini—an excellent example of one such peptide family derived from the E2B protein is shown in Figure 3. As mentioned previously, note that all 12 members of this peptide family share a common 9‐mer binding core (shown underlined and in bold) along with varying flanks. Also of note is the

c la s s ii m h c m o l e cu l e i ‐a g 7

245

246

a n i s h s u ri a n d e m i l r . u na n u e

presence of triple acidic amino acids (123E, 124E, and 125D) toward the C‐terminus of this peptide family (Fig. 3). Alignment of over 100 naturally processed peptides examined indicated the following preferences for the MHC binding pockets (Fig. 4) (Suri et al., 2003): a wide variety of residues that included small to medium hydrophobic amino acids, as well as charged and polar amino acids were found at the P1 position of the peptide, in accordance with the results of the structural analysis; hydrophobic amino acids (>75%) along with some polar residues were at the P4 position; the P6‐peptide position preferred mostly hydrophobic and polar residues; and acidic amino acids (>80%) dominated at P9 although some hydrophobic and small polar residues could also be accommodated (Fig. 4). In more recent analysis with larger data sets, it was ascertained that >90% of peptides selected by I‐Ag7‐expressing APCs contained an acidic amino acid at the P9 position (unpublished observations). In binding and mutational analyses, studies confirmed that the C‐terminal acidic residues interacted with the P9 pocket of I‐Ag7 (Fig. 5) (Suri et al., 2002). An example is shown in Fig. 5 with a peptide derived from the E2B protein (note: the entire E2B peptide family is shown in Fig. 3). The E2B 112– 126 peptide contained three acidic amino acids toward its C‐terminus: 123E, 124E, and 125D (Fig. 5). To identify which of these was being used as the P9 anchor, each acidic residue was singly changed to a lysine, a residue that is hindering at this position. Changing the 123E or 125D did not affect binding; however, mutating the 124E to 124K resulted in a 30‐fold loss, which suggested that this residue was being preferentially used as the P9 anchor (Fig. 5). To confirm the binding register, the putative P4 (119I) or P6 (121I) was also mutated to a hindering lysine—here again these changes drastically reduced binding which validated the binding register for this peptide (Fig. 5). Why was the 124E used as the P9 anchor and not 123E or 125D? The answer to this question is evident in the binding register of the peptide: if 123E was P9, then 120K would be P6, which is an extremely unfavorable residue at this position

Figure 2 Distribution of acidic amino acids along the C termini of peptides isolated from I‐Ag7 or I‐Ag7PD. I‐Ag7‐associated peptides were isolated from C3.G7 and M12.G7 APC lines, whereas I‐Ag7PD‐associated peptides were isolated from the C3G7PD APC line (Suri et al., 2002). All peptides are included in the analyses. The last 10 C‐termini residues of each peptide were scored for the presence of either Glu or Asp at each position. The bar graphs depict the presence of acidic residues, shown as percent positive, for each position starting at the most C‐terminal residue. The most C‐terminal residue was assigned position 10; similarly, the second‐most C‐terminal residue was assigned position 9, and so on. As shown, peptides isolated from I‐Ag7 exhibit the biased presence of acidic amino acids among the C‐terminal residues when compared with peptides isolated from I‐Ag7PD (especially at positions 10, 9, 8, 7, and 6).

247 Figure 3 E2B peptide family isolated from I‐Ag7‐expressing APCs. The E2B peptide family was present in high abundance among the naturally processed peptides isolated from I‐Ag7 APCs (Suri et al., 2002, 2003). Note that all the members of this peptide family share a common binding core that extends from P1 to P9 residues (shown in bold and underlined) along with varying flanking residues at the N‐ and C‐terminus. Note also the presence of multiple C‐termini acidic residues (at positions 123, 124, and 125), which was characteristic of large numbers of peptides isolated from I‐Ag7.

248 Figure 4 Amino acid frequencies in the bound peptides. Identification of preferred amino acids at P1, P4, P6, and P9 pockets from alignment of over 100 naturally processed peptides isolated from I‐Ag7 (Suri et al., 2003).

249 Figure 5 Mapping of preferred P9 anchors using Lys‐mutants of C‐termini acidic residues: E2B 112–126 peptide. Each of the C‐terminal acidic amino acid was mutated to a lysine to identify the preferred P9 anchor (Suri et al., 2002). The binding register was further confirmed by mutating the putative P4 and P6 to a lysine, which also resulted in a noticeable loss of binding. For I‐Ag7 peptides, basic residues are hindering at the P4, P6, and P9 positions. Binding 9‐mer core is shown in bold and underlined.

250

a n i s h s u ri a n d e m i l r . u na n u e

(Fig. 5). Similarly, 125D as an acidic P9 anchor would place 120K at P4 and 122F at P6—both of which are very hindering at their respective positions (Fig. 5). Similar mutational approaches with many other naturally processed peptides identified their preferred P9 anchor residues and binding registers (Suri et al., 2002). A second issue with binding analysis pertained to the cooperative role of multiple C‐terminal acidic residues in increasing the peptide‐binding affinity to I‐Ag7 (Suri et al., 2002). As mentioned before, in their report of I‐Ag7 structure, Corper et al. commented that acidic residues at P10 or P11 could have productive interactions with the unpaired Arga76 residue; however the precise mode of such interactions remained unclear (Corper et al., 2000). Because the HEL 11–25 peptide bound to I‐Ag7 had previously been crystallized and hence its mode of binding known, this same peptide was utilized in mutational analysis to examine the role of multiple C‐termini acidic residues. The wild‐type HEL 11–25 peptide used Gly22 as its P9 anchor, which when changed singly to either an aspartic acid or glutamic acid increased binding moderately (Fig. 6). However, when the P9, P10, and P11 residues were replaced by acidic amino acids from the E2B peptide (22G–>E, 23Y–>E and 24S–>D), the resulting HEL 11–25EED peptide bound 10‐fold better than the wild‐type peptide (Fig. 6). To ensure that this enhanced binding was not due to a shift in the binding register, the Lys‐scan mutations were again performed to identify the P9 anchor; in this case, if the binding register was still the same then the acidic residue at position 22 should be the P9 anchor. Results from these analyses confirmed that the binding register of the HEL 11–25EED peptide was unaffected; mutating the 22E to a lysine abrogated binding while changing either 23E or 24D, had no effect (Fig. 6). Evidence of the role of a P10 acidic residue was evident when testing a HEL 11–25 peptide that retained the Gly as its P9 anchor at position 22 but acidic amino acids were replaced at P10 (23E) and P11 (24D). This HEL 11–25GED peptide still bound 10‐fold better than the wild‐type peptide and was similar to the HEL 11–25EED peptide (Fig. 6). The HEL 11–25GED peptide was tested with CD4þ T‐cell clones that had been raised against the wild‐type HEL 11–25 peptide; all T cells responded to the HEL 11–25GED peptide with the same vigor as the wild‐type peptide, which confirmed that the binding register of this peptide remained unchanged. Thus, taken together, these results indicated that in cases in which the P9 anchor was a nonacidic anchor, acidic amino acids at the P10 and P11 positions cooperated to increased binding affinity to I‐Ag7. Further structural studies of peptides with multiple C‐termini acidic residues bound to I‐Ag7 molecules are needed to resolve the basis of this synergistic binding.

251 Figure 6 Multiple C‐termini acidic residues cooperate to increase peptide‐binding affinity to I‐Ag7. The P9, P10, and P11 residues of HEL 11–25 peptide were replaced by acidic amino acids, which increased binding by 10‐fold (see HEL 11–25EED peptide). Acidic amino acids at P10 and P11 also cooperated to increase peptide‐binding affinity (see HEL 11–25GED peptide).

252

a n i s h s u ri a n d e m i l r . u na n u e

A subsequent study confirmed the findings of C‐termini acidic residues among naturally processed peptides from I‐Ag7 (Munz et al., 2002). In their experiments researchers identified a total of seven peptides obtained from an I‐Ag7‐transfected B‐cell lymphoma line, akin to the APC line generated previously (Table 1 and Fig. 2) (Suri et al., 2002; 2003). These peptides were shown to bind to I‐Ag7 via an acidic P9 anchor (Munz et al., 2002). Incidentally, five out of the seven peptides identified in this study had also been identified in the early analysis of naturally processed I‐Ag7 peptides (Suri et al., 2002, 2003). Although Munz et al. (2002) pointed out by researchers that the peptide yield from I‐Ag7 was poor compared to other class II MHC alleles, they did not notice any such correlation in analysis where they were able to identify over 100 peptides (Suri et al., 2002, 2003). Perhaps such differences can account for the levels of class II MHC molecules on the cell surface of APCs (the transfected APC line did express higher levels of cell surface I‐Ag7, when compared to the one generated by Munz et al.) and/or the affinity of antibodies that are used for immunoprecipitating cell surface I‐Ag7‐peptide complexes. When peptides were isolated from I‐Ag7PD molecules no such bias for C‐termini acidic residues was evident, which indicated that the specificity of the P9 pocket was the single‐most important factor influencing peptide selection by I‐Ag7 (Fig. 2). To ensure that peptide selection was unique to each haplotype, we focused our MS analysis specifically on searching for peptides previously found bound to I‐Ag7 among the peptides isolated from I‐Ag7PD. None of the I‐Ag7 peptides searched were ever detected among the peptides identified from I‐Ag7PD (Suri et al., 2002). Other evidence for the role of the P9 pocket emerged from the analysis of naturally processed peptides from I‐Ad, and comparing those to I‐Ag7 (Suri et al., 2003). I‐Ag7 and I‐Ad are the two closest naturally occurring murine class II alleles: both share the same I‐Ada chain and differ by 17 amino acids in their b chain (2 of these 17 amino acid differences are at b56 and b57). In addition, structural studies demonstrate that besides the P9 pocket, I‐Ag7 and I‐Ad share similar features at the other anchoring sites (Corper et al., 2000; Latek et al., 2000; Scott et al., 1998). Moreover, past studies with T‐cell responses indicated that both I‐Ag7 and I‐Ad APCs can bind to many common synthetic peptides (Deng et al., 1993; Kanagawa et al., 1997; Moudgil et al., 1998; Mukherjee et al., 2003). Despite these similarities, the identification of naturally processed peptides from I‐Ag7 and I‐Ad established that each allele selected for a unique repertoire of peptides: while both molecules selected peptides that contained very similar residues at P4 and P6 (mostly hydrophobic and some polar amino acids), the ones selected by I‐Ag7 contained acidic amino acids at P9 while those selected by I‐Ad contained mostly hydrophobic and polar residues at the same position (Suri et al., 2003).

c la s s ii m h c m o l e cu l e i ‐a g 7

253

Another important observation gained from analyses was the noticeable lack of relationship between peptide‐binding affinity and cell‐surface abundance of naturally processed peptides (Suri et al., 2002). Similar results were obtained from earlier studies from Engelhard’s laboratory on naturally processed peptides from class I MHC molecules (Engelhard et al., 2002). Indeed, other parameters such as expression levels of proteins, their turnover rates, and their accessibility to antigen processing and presentation pathways must affect the final display on the cell surface, and may play an important role in autoimmune or pathogen‐specific immune responses. On a related note, similar extensive analyses need to be performed with the human DQ2 and/or DQ8 molecules to establish their binding motifs and bias with respect to peptide selection. Two previous reports generated limited data sets of naturally processed peptides isolated from DQ8 APCs to identify the preferred binding motif. The first study by Chicz et al. (1994) identified a few peptides that ranged from 13 to 74 residues in length; however, none of these exhibited a consensus binding motif. A second analysis on naturally processed peptides from DQ8 APCs utilized pool sequencing to suggest the following binding motif: acidic amino acids at P4, basic amino acids at P6, and both acidic residues and basic residues at the P9 position (Godkin et al., 1997). Similarly, the two past reports concerning natural peptides of DQ2 used pool sequencing to suggest that bulky hydrophobic residues, such as phenylalanine, were preferred at the P9 position (Vartdal et al., 1996; Verreck et al., 1994). Of note is the fact that for I‐Ag7 basic amino acids at P4 and P9 were hindering and resulted in a noticeable loss of binding (Figs. 4 and 5) (Latek et al., 2000; Suri et al., 2002). Thus, the anomalies that existed for the binding motif preferences for I‐Ag7 also are evident in the case of human diabetogenic class II MHC molecules. In a recent study, we isolated and identified I‐Ag7‐ or DQ8‐bound peptides from genetically identical APC lines. Extensive numbers of naturally processed peptides demonstrated that both murine and human diabetogenic class II MHC molecules display peptides with common binding motifs. Both I‐Ag7 and DQ8 selected for identical peptide families that bound to each exactly in the same binding register. This report provides the first direct evidence of a common outcome of antigen processing events between the murine and human diabetogenic class II MHC molecules (Suri et al., 2005). A final issue pertained to the effects of the presence of additional class II MHC molecules on the repertoire of peptides selected by I‐Ag7. The underlying basis for these experiments were past observations with transgenic NOD mice wherein expression of additional b57 Asp‐bearing class II MHC molecules resulted in a marked protection from diabetes (Kanagawa et al., 2000; Lund et al., 1990; Miyazaki et al., 1990; Nishimoto et al., 1987; Quartey‐Papafio et al., 1995; Singer et al., 1993, 1998; Slattery et al., 1990). Although the mechanism

254

a n i s h s u ri a n d e m i l r . u na n u e

for this protection remains unknown, three primary possibilities have been suggested. First, the presence of additional class II MHC molecules could generate a thymic milieu that allows for more efficient negative selection of self‐reactive T cells (Schmidt et al., 1997, 1999; Verdaguer et al., 1997). Second, additional class II MHC molecules could select for T cells that are regulatory in nature and hence control diabetogenic T cells in the periphery (Luhder et al., 1998; Singer et al., 1993). And third, the presence of other class II MHC molecules could alter the repertoire of peptides selected by I‐Ag7 via a process termed ‘‘determinant capture’’ (Deng et al., 1993; Moudgil et al., 1998). The evidence for the ‘‘determinant capture’’ hypothesis stemmed from indirect analyses of T cells to model antigens (Deng et al., 1993; Moudgil et al., 1998). We designed experiments with a direct biochemical approach to test whether the presence of other class II alleles affected peptide selection by I‐Ag7. Naturally processed peptides were isolated and identified from APC lines that were either homozygous for I‐Ag7 or expressed I‐Ag7 in conjunction with I‐Ag7PD or I‐Ad (Suri et al., 2003). Results from these analyses of large data sets demonstrated that despite being structurally related, the peptides selected by each allele were unique and were minimally affected by the presence of additional class II MHC molecules (Suri et al., 2003). Hence MHC‐heterozygosity‐linked protection from diabetes is likely due to factors that affect T‐cell development and/or regulatory T cells, and is unlikely due to competition that may exist between MHC molecules at the level of peptide selection. Finally, do the autoantigenic T cell epitopes in NOD mice reflect the highly specific nature of peptide selection by I‐Ag7? Table 2 shows the alignment of a few peptides derived from self‐proteins, such as insulin and GAD, involved in diabetes. Note that the motif exhibited by these peptides is dominated by the presence of an acidic P9 anchor along with favorable residues at other positions (Table 2). Moreover, in our prior analysis we determined that the HSP‐60 164–183 peptide family was selected by APCs expressing I‐Ag7 and was noticeably absent when the same APCs expressed I‐Ag7PD (Table 2) (Suri et al., 2002). Thus, a critical role for I‐Ag7 in diabetes may be its ability to efficiently select and display islet b‐cell peptides, many of which should contain the binding motif described by analyses of naturally processed peptides. 4. Why Is There a Difference in Results Between Binding and Peptide Selection? To summarize, upon aligning the large data sets of naturally processed peptides, an unequivocal preference for acidic amino acids at the P9 position was evident (~90% of natural peptides had an Asp or Glu at P9) (Suri et al., 2002, 2003). This striking bias was also in stark contrast to previous reports on I‐Ag7’s

Table 2 Alignment of I‐Ag7‐Restricted Islet b‐Cell Epitopes Recognized by T Cells in NOD Mice 1

Peptide

255

INSULIN 9–23* GAD65 206–220* GAD65 222–235 GAD65 247–266* GAD65 286–300 GAD65 401–415 GAD65 510–526 GAD65 561–575 HSP‐60 164–183 HSP‐60 437–460* I‐A2b 755–777 ICA69 36–47*

P

N

Q

R

T V E

P L E

M

E G N

Y

E G A

4

6

9

H

L

V

E

A

L

Y

L

V

C

G

E

R

G

T

Y K M K L P S A C K

E K L G Q S N Q A N A

I M I A C L P V L R F

A R A A S R A A L S I

P E R A A T A T R L K

V I Y L L L T I C A A

F I K G L E H S I V T

V G M I V D Q A P L G

L W S G R N D N A T K

L P P T E E I G L Y K

E G E D E E D D D D E

Y G V S G R F K S H D

V S K V L M L D L A E

A K P P I I G P

T G E I M S I I K S

K

G

R

L

G P R

A I

N

E

D

Note the peptides aligned above exhibit the consensus binding motif for I‐Ag7 as identified by the analysis of naturally processed peptides. Asterisk (*) indicates peptides that are recognized in humans. Data adapted from the report by Lieberman and DiLorenzo (2003).

256

a n i s h s u ri a n d e m i l r . u na n u e

binding motif that utilized peptide libraries or phage display peptides to suggest basic or bulky residues were preferred at this position (Gregori et al., 2000; Harrison et al., 1997; Stratmann et al., 2000). The anchor pockets of I‐Ag7 are tolerant to many amino acids and hence binding analyses with synthetic peptides could be misleading when extrapolating to the repertoire of naturally selected peptides. As noted, the P9 pocket of I‐Ag7 accommodated numerous amino acids. The binding motif described by the naturally processed peptides was in agreement with the structural features reported earlier (Corper et al., 2000; Latek et al., 2000). Hence all of the data on identification of natural epitopes of I‐Ag7 demonstrates the exquisite specificity of peptide selection exhibited by this diabetogenic class II MHC molecule (Munz et al., 2002; Suri et al., 2002, 2003). We believe that the intracellular events eventually dictate which peptides survive the traffic through the highly proteolytic rich and acidic vesicular environment. It is possible that during intracellular events of antigen‐processing peptides with various motifs may bind to I‐Ag7, however only those containing the terminal acidic amino acids have a sustained stable interaction, survive, and hence are displayed on the cell surface for their interaction with T cells. Although not found with all, peptides with acidic terminal residues tend to bind better, particularly at low pH. Consistent with this interpretation, at pH5.5, peptides with acidic P9 anchors form long‐lived complexes with I‐Ag7 and I‐A g 7 P D , however at pH7.0, the same peptides rapidly dissociated from I‐Ag7PD while still remaining stably bound to I‐Ag7 (Suri and Unanue, unpublished observations). Although further studies should clarify these findings, the take‐home message is clear that there is an important dichotomy between straight binding result with peptides and what peptide the APC likes eventually to select for presentation. It is the latter that at the end counts because it establishes the T‐cell repertoire that is activated. 5. The Biological Role of I‐Ag7 This discussion focuses on the features of I‐Ag7 as a peptide‐binding molecule that favors the development of autoimmunity. Undoubtedly, its chemical features are a major element in the susceptibility to autoimmune diabetes of both humans and the NOD mouse. But, the chemical features of I‐Ag7 are not the sole features that lead to diabetic autoimmunity; indeed, there are many genes involved in creating the diabetic phenotype of the NOD mouse (Wicker et al., 1995). These are the subject of past evaluations by different laboratories. The diabetic process is a long one with various stages in its development, first starting with the peri‐insulitis lesion, which progresses with time into the aggressive stage in which b cells die and diabetes develops. The autoimmune process involves cooperativity among CD4 and CD8 T cells,

c la s s ii m h c m o l e cu l e i ‐a g 7

257

with participation of various effector cells like macrophages and NK cells. The autoimmune process is regulated at the various stages: regulatory T cells, NKT cells, as well as others, can participate in modulating the aggressiveness of the process (Gonzalez et al., 2001; Sakaguchi, 2004; Sakaguchi et al., 1982). The results suggest that many components have to be placed into action in order for the autoimmune process to move forward. And that any manipulation that slightly tilts away from any given component modulates the NOD diabetogenic process. The features of I‐Ag7 molecules of the NOD mouse that are more relevant in the perspective of autoimmunity are those dealing with its peptide‐binding features and that translate into: (1) the process of positive or negative selection in the thymus; and (2) the presentation of b‐cell peptides. An important feature of the NOD mouse is its tendency to develop more than one autoimmune disorder, implicating a more generalized, ‘‘central’’ defect. For example, adrenalitis, sialitis, thyroiditis, neuritis, and prostatitis of an autoimmune nature have been reported (Beales et al., 2002; Boulard et al., 2002; Braley‐Mullen et al., 1999; Many et al., 1996; Rivero et al., 1998; Salomon et al., 2001). So the first explanation is whether the NOD mice show a defect in thymic negative selection that allows for a high number of T cells to escape into the periphery. Our own group, Kanagawa et al., examined the number of CD4 T cells in NOD mice that are reactive in ‘‘autologous MLR’’ by limiting dilution cloning. The conclusions were quite striking: NOD had a large number of autoreactive T cells; this high number correlated with the expression of the I‐Ag7 molecule and not with the background genes of the strain (Kanagawa et al., 1998a). Thus a C57Bl mouse bearing class II MHC genes encoding for I‐Ag7 also contained a high number of self‐reactive T cells in the periphery (but such mice did not develop disease). Using a different approach, Fathman’s laboratory also claimed a high level of autoreactivity: they immunized mice with autologous proteins and found a striking degree of proliferation in cells from the draining lymph node (Ridgway et al., 1998). A third study to evaluate is that of Teyton’s group. The group searched for the presence of the TCR idiotype from the BDC2.5 CD4 T cells (Stratmann et al., 2003). BDC2.5 T cells (Haskins et al., 1988) were developed into a T‐cell receptor transgenic and have been so useful in the study of diabetogenesis (Katz et al., 1993). Using peptide mimics of the T cells, as an MHC‐tracking reagent, they found a high level of T cells in lymph nodes of NOD mice. Their presence correlated with the expression of I‐Ag7 molecule and, as in the Kanagawa et al. study (1998a), not with the background genes. The fascinating and very puzzling finding is that the positive selection of the BDC2.5 T cells can take place from a thymus in which the class II MHC is I‐Ag7PD (Kanagawa et al., 1998b). BDC2.5 is not activated by the I‐Ag7PD variant. Here the situation is

258

a n i s h s u ri a n d e m i l r . u na n u e

one that the T cells are positively selected, are peripheralized, but are never activated in the peripheral lymphoid organs (Kanagawa et al., 1998b). Other claims have been made on the relative resistance of NOD mice to negative selection, but in contrast to the papers just mentioned, the results were independent of the expression of the I‐Ag7 molecule. Sprent’s laboratory based their findings on an in vitro assay where a subset of thymocytes were treated with antibodies to the TCR (Kishimoto and Sprent, 2001). Goodnow’s group claimed a failure of apoptosis based on a failure to express the proapoptotic Bim protein (Liston et al., 2004). More recently, data from Mathis and Benoist’s group indicated that the BDC T cell is more resistant to thymic deletion on the NOD versus B6 background (Zucchelli et al., 2005). In these experiments peptide mimics (as mentioned previously) were used in fetal‐ thymic organ cultures (FTOCs) for their ability to delete double‐positive transgenic thymocytes (Zucchelli et al., 2005). At face value one would conclude that more than one component is involved in thymic selection involving MHC and non‐MHC genes. Does the I‐Ag7 molecule then function in favoring escape from the thymus? We go back to some of the chemical features of I‐Ag7 with a relatively short life in APC, and a selection of a relatively limited family of peptides. These two components could conceivably result in the observed escape of more autoreactive T cells akin to results showing that limiting the repertoire of peptides favor release of autoreactive T cells (Liu et al., 1997; Martin et al., 1996; Miyazaki et al., 1996). Indeed, one facet of MHC‐heterozygosity‐linked protection from diabetes is attributed to the ability of additional alleles in deletion of self‐reactive T cells (Schmidt et al., 1997, 1999). However, to directly test this one would need to evaluate the diversity of thymic peptides presented by mice expressing I‐Ag7 in contrast to other haplotypes. Another component of I‐Ag7‐linked predisposition may involve the generation and maintenance of regulatory T cells. Although the thymus is required for generation of regulatory T cells (Sakaguchi, Takahashi, and Nishizuka, 1982), the precise role of self‐peptides in promoting differentiation toward this lineage is unknown. There are indications that varying levels of self‐ antigen in the thymus may affect the development of regulatory T cells (Liston et al., 2005). With regard to this observation, the unique peptide‐selection properties of I‐Ag7 could hamper the expression levels and/or repertoire of thymic self‐ligands, which may consequently affect development of regulatory T cells. Past reports have demonstrated that NOD mice expressing additional class II MHC molecules exhibit an increased presence of regulatory T cells and are protected from diabetes (Luhder et al., 1998; Singer et al., 1993). Regardless of the thymic events that allow or do not allow peripheralization of autoreactive CD4 T cells, the class II MHC molecules are critically involved

c la s s ii m h c m o l e cu l e i ‐a g 7

259

in the activation of CD4 T cells in the pancreatic draining node that starts the process. Whether particular chemical features of b‐cell proteins favor a productive interaction with I‐Ag7 is an important consideration that will be determined by the direct analysis of the b‐cell peptides that are naturally selected. Acknowledgments We thank the National Institutes of Health and the Kilo Diabetes and Vascular Research Foundation for the support of our work cited here, and we acknowledge our colleagues who have participated in this work: Michael Gross and James Walters who were in charge of the mass spectrometry work; Daved Fremont and Robert Latek who were responsible for the structural analysis; and our laboratory colleagues Osami Kanagawa, Boris Calderon, Eugenio Carrasco‐ Marin, Matteo Levisetti, and Shirley Petzold.

References Acha‐Orbea, H., and McDevitt, H. O. (1987). The first external domain of the nonobese diabetic mouse class II I‐A beta chain is unique. Proc. Natl. Acad. Sci. USA 84, 2435–2439. Arneson, L. S., Peterson, M., and Sant, A. J. (2000). The MHC class II molecule I‐Ag7 exists in alternate conformations that are peptide dependent. J. Immunol. 165, 2059–2067. Bach, J. F. (1994). Insulin‐dependent diabetes mellitus as an autoimmune disease. Endocr. Rev. 15, 516–542. Beales, P. E., Castri, F., Valiant, A., Rosignoli, G., Buckley, L., and Pozzilli, P. (2002). Adrenalitis in the non‐obese diabetic mouse. Autoimmunity 35, 329–333. Bhatnagar, A., Milburn, P. J., Lobigs, M., Blanden, R. V., and Gautam, A. M. (2001). Nonobese diabetic mice display elevated levels of class II‐associated invariant chain peptide associated with I‐Ag7 on the cell surface. J. Immunol. 166, 4490–4497. Boulard, O., Fluteau, G., Eloy, L., Damotte, D., Bedossa, P., and Garchon, H. J. (2002). Genetic analysis of autoimmune sialadenitis in nonobese diabetic mice: A major susceptibility region on chromosome 1. J. Immunol. 168, 4192–4201. Braley‐Mullen, H., Sharp, G. C., Medling, B., and Tang, H. (1999). Spontaneous autoimmune thyroiditis in NOD.H‐2h4 mice. J. Autoimmun. 12, 157–165. Brown, J. H., Jardetzky, T. S., Gorga, J. C., Stern, L. J., Urban, R. G., Strominger, J. L., and Wiley, D. C. (1993). Three‐dimensional structure of the human class II histocompatibility antigen HLA‐DR1. Nature 364, 33–39. Carrasco‐Marin, E., Kanagawa, O., and Unanue, E. R. (1999a). The lack of consensus for I‐A(g7)‐ peptide binding motifs: Is there a requirement for anchor amino acid side chains? Proc. Natl. Acad. Sci. USA 96, 8621–8626. Carrasco‐Marin, E., Petzold, S., and Unanue, E. R. (1999b). Two structural states of complexes of peptide and class II major histocompatibility complex revealed by photoaffinity‐labeled peptides. J. Biol. Chem. 274, 31333–31340. Carrasco‐Marin, E., Shimizu, J., Kanagawa, O., and Unanue, E. R. (1996). The class II MHC I‐Ag7 molecules from non‐obese diabetic mice are poor peptide binders. J. Immunol. 156, 450–458.

260

a n i s h s u ri a n d e m i l r . u na n u e

Carson, R. T., Vignali, K. M., Woodland, D. L., and Vignali, D. A. (1997). T‐cell receptor recognition of MHC class II‐bound peptide flanking residues enhances immunogenicity and results in altered TCR V region usage. Immunity 7, 387–399. Castano, L., and Eisenbarth, G. S. (1990). Type‐I diabetes: A chronic autoimmune disease of human, mouse, and rat. Annu. Rev. Immunol. 8, 647–649. Chao, C. C., and McDevitt, H. O. (1997). Identification of immunogenic epitopes of GAD 65 presented by Ag7 in non‐obese diabetic mice. Immunogenetics 46, 29–34. Chao, C. C., Sytwu, H. K., Chen, E. L., Toma, J., and McDevitt, H. O. (1999). The role of MHC class II molecules in susceptibility to type I diabetes: Identification of peptide epitopes and characterization of the T‐cell repertoire [published erratum appears in Proc. Natl. Acad. Sci. USA 1999 Oct 26;96(22):12970]. Proc. Natl. Acad. Sci. USA 96, 9299–9304. Chicz, R. M., Lane, W. S., Robinson, R. A., Trucco, M., Strominger, J. L., and Gorga, J. C. (1994). Self‐peptides bound to the type I diabetes associated class II MHC molecules HLA‐DQ1 and HLA‐DQ8. Int. Immunol. 6, 1639–1649. Corper, A. L., Stratmann, T., Apostolopoulos, V., Scott, C. A., Garcia, K. C., Kang, A. S., Wilson, I. A., and Teyton, L. (2000). A structural framework for deciphering the link between I‐Ag7 and autoimmune diabetes. Science 288, 505–511. Deng, H., Apple, R., Clare‐Salzler, M., Trembleau, S., Mathis, D., Adorini, L., and Sercarz, E. (1993). Determinant capture as a possible mechanism of protection afforded by major histocompatibility complex class II molecules in autoimmune disease. J. Exp. Med. 178, 1675–1680. Dessen, A., Lawrence, C. M., Cupo, S., Zaller, D. M., and Wiley, D. C. (1997). X‐ray crystal structure of HLA‐DR4 (DRA*0101, DRB1*0401) complexed with a peptide from human collagen II. Immunity 7, 473–481. Engelhard, V. H., Brickner, A. G., and Zarling, A. L. (2002). Insights into antigen processing gained by direct analysis of the naturally processed class I MHC associated peptide repertoire. Mol. Immunol. 39, 127–137. Falk, K., Rotzschke, O., Stevanovic, S., Jung, G., and Rammensee, H. G. (1991). Allele‐specific motifs revealed by sequencing of self‐peptides eluted from MHC molecules. Nature 351, 290–296. Fremont, D. H., Monnaie, D., Nelson, C. A., Hendrickson, W. A., and Unanue, E. R. (1998). Crystal structure of I‐Ak in complex with a dominant epitope of lysozyme. Immunity 8, 305–317. Germain, R. N., and Hendrix, L. R. (1991). MHC class II structure, occupancy and surface expression determined by post‐endoplasmic reticulum antigen binding. Nature 353, 134–139. Godkin, A., Friede, T., Davenport, M., Stevanovic, S., Willis, A., Jewell, D., Hill, A., and Rammensee, H. G. (1997). Use of eluted peptide sequence data to identify the binding characteristics of peptides to the insulin‐dependent diabetes susceptibility allele HLA‐DQ8 (DQ 3.2). Int. Immunol. 9, 905–911. Gonzalez, A., Andre‐Schmutz, I., Carnaud, C., Mathis, D., and Benoist, C. (2001). Damage control, rather than unresponsiveness, effected by protective DX5þ T cells in autoimmune diabetes. Nat. Immunol. 2, 1117–1125. Gregori, S., Bono, E., Gallazzi, F., Hammer, J., Harrison, L. C., and Adorini, L. (2000). The motif for peptide binding to the insulin‐dependent diabetes mellitus‐associated class II MHC molecule I‐A(g7) validated by phage display library. Int. Immunol. 12, 493–503. Griffin, A. C., Zhao, W., Wegmann, K. W., and Hickley, W. F. (1995). Experimental autoimmune insulitis. Induction by T lymphocytes specific for a peptide of proinsulin. Am. J. Pathol. 147, 845–857. Harfouch‐Hammoud, E., Walk, T., Otto, H., Jung, G., Bach, J. F., van Endert, P. M., and Caillat‐ Zucman, S. (1999). Identification of peptides from autoantigens GAD65 and IA‐2 that bind to HLA class II molecules predisposing to or protecting from type 1 diabetes. Diabetes 48, 1937–1947.

c la s s ii m h c m o l e cu l e i ‐a g 7

261

Harrison, L. C., Honeyman, M. C., Trembleau, S., Gregori, S., Gallazzi, F., Augstein, P., Brusic, V., Hammer, J., and Adorini, L. (1997). A peptide‐binding motif for I‐A(g7), the class II major histocompatibility complex (MHC) molecule of NOD and Biozzi AB/H mice. J. Exp. Med. 185, 1013–1021. Haskins, K., Portas, M., Bradley, B., Wegmann, D., and Lafferty, K. (1988). T‐lymphocyte clone specific for pancreatic islet antigen. Diabetes 37, 1444–1448. Hattori, M., Buse, J. B., Jackson, R. A., Glimcher, L., Dorf, M. E., Minami, M., Makino, S., Moriwaki, K., Kuzuya, H., and Imura, H. (1986). The NOD mouse: Recessive diabetogenic gene in the major histocompatibility complex. Science 231, 733–735. Hausmann, D. H., Yu, B., Hausmann, S., and Wucherpfennig, K. W. (1999). pH‐dependent peptide binding properties of the type I diabetes‐ associated I‐Ag7 molecule: Rapid release of CLIP at an endosomal pH. J. Exp. Med. 189, 1723–1734. Herman, A. E., Tisch, R. M., Patel, S. D., Parry, S. L., Olson, J., Noble, J. A., Cope, A. P., Cox, B., Congia, M., and McDevitt, H. O. (1999). Determination of glutamic acid decarboxylase 65 peptides presented by the type I diabetes‐associated HLA‐DQ8 class II molecule identifies an immunogenic peptide motif. J. Immunol. 163, 6275–6282. Hunt, D. F., Michel, H., Dickinson, T. A., Shabanowitz, J., Cox, A. L., Sakaguchi, K., Appella, E., Grey, H. M., and Sette, A. (1992). Peptides presented to the immune system by the murine class II major histocompatibility complex molecule I‐Ad. Science 256, 1817–1820. Jardetzky, T. S., Brown, J. H., Gorga, J. C., Stern, L. J., Urban, R. G., Strominger, J. L., and Wiley, D. C. (1996). Crystallographic analysis of endogenous peptides associated with HLA‐DR1 suggests a common, polyproline II‐like conformation for bound peptides. Proc. Natl. Acad. Sci. USA 93, 734–738. Johansen, B. H., Vartdal, F., Eriksen, J. A., Thorsby, E., and Sollid, L. M. (1996). Identification of a putative motif for binding of peptides to HLA‐DQ2. Int Immunol. 8, 177–182. Kanagawa, O., Martin, S. M., Vaupel, B. A., Carrasco‐Marin, E., and Unanue, E. R. (1998a). Autoreactivity of T cells from nonobese diabetic mice: An I‐Ag7‐ dependent reaction. Proc. Natl. Acad. Sci. USA 95, 1721–1724. Kanagawa, O., Shimizu, J., and Unanue, E. R. (1997). The role of I‐Ag7 beta chain in peptide binding and antigen recognition by T cells. Int. Immunol. 9, 1523–1526. Kanagawa, O., Shimizu, J., and Vaupel, B. A. (2000). Thymic and postthymic regulation of diabetogenic CD8 T‐cell development in TCR transgenic nonobese diabetic (NOD) mice. J. Immunol. 164, 5466–5473. Kanagawa, O., Vaupel, B. A., Xu, G., Unanue, E. R., and Katz, J. D. (1998b). Thymic positive selection and peripheral activation of islet antigen‐ specific T cells: Separation of two diabetogenic steps by an I‐A(g7) class II MHC beta‐chain mutant. J. Immunol. 161, 4489–4492. Katz, J. D., Wang, B., Haskins, K., Benoist, C., and Mathis, D. (1993). Following a diabetogenic T cell from genesis through pathogenesis. Cell 74, 1089–1100. Kikutani, H., and Makino, S. (1992). The murine autoimmune diabetes model: NOD and related strains. Adv. Immunol. 51, 285–322. Kishimoto, H., and Sprent, J. (2001). A defect in central tolerance in NOD mice. Nat. Immunol. 2, 1025–1031. Kwok, W. W., Domeier, M. L., Raymond, F. C., Byers, P., and Nepom, G. T. (1996). Allele‐specific motifs characterize HLA‐DQ interactions with a diabetes‐associated peptide derived from glutamic acid decarboxylase. J. Immunol. 156, 2171–2177. Latek, R. R., Suri, A., Petzold, S. J., Nelson, C. A., Kanagawa, O., Unanue, E. R., and Fremont, D. H. (2000). Structural basis of peptide binding and presentation by the type I diabetes‐ associated MHC class II molecule of NOD mice. Immunity 12, 699–710.

262

a n i s h s u ri a n d e m i l r . u na n u e

Latek, R. R., and Unanue, E. R. (1999). Mechanisms and consequences of peptide selection by the I‐Ak class II molecule. Immunol. Rev. 172, 209–228. Lee, K. H., Wucherpfennig, K. W., and Wiley, D. C. (2001). Structure of a human insulin peptide‐ HLA‐DQ8 complex and susceptibility to type 1 diabetes. Nat. Immunol. 2, 501–507. Lieberman, S. M., and Di Lorenzo, T. P. (2003). A comprehensive guide to antibody and T‐cell responses in type 1 diabetes. Tissue Antigens 62, 359–377. Liston, A., Lesage, S., Gray, D. H., Boyd, R. L., and Goodnow, C. C. (2005). Genetic lesions in T‐cell tolerance and thresholds for autoimmunity. Immunol. Rev. 204, 87–101. Liston, A., Lesage, S., Gray, D. H., O’Reilly, L. A., Strasser, A., Fahrer, A. M., Boyd, R. L., Wilson, J., Baxter, A. G., Gallo, E. M., Crabtree, G. R., Peng, K., Wilson, S. R., and Goodnow, C. C. (2004). Generalized resistance to thymic deletion in the NOD mouse; a polygenic trait characterized by defective induction of Bim. Immunity 21, 817–830. Liu, C. P., Parker, D., Kappler, J., and Marrack, P. (1997). Selection of antigen‐specific T cells by a single IEk peptide combination. J. Exp. Med. 186, 1441–1450. Luhder, F., Katz, J., Benoist, C., and Mathis, D. (1998). Major histocompatibility complex class II molecules can protect from diabetes by positively selecting T cells with additional specificities. J. Exp. Med. 187, 379–387. Lund, T., O’Reilly, L., Hutchings, P., Kanagawa, O., Simpson, E., Gravely, R., Chandler, P., Dyson, J., Picard, J. K., Edwards, A., Kioussis, D., and Cooke, A. (1990). Prevention of insulin‐ dependent diabetes mellitus in non‐obese diabetic mice by transgenes encoding modified I‐A beta‐chain or normal I‐E alpha‐ chain. Nature 345, 727–729. Many, M. C., Maniratunga, S., and Denef, J. F. (1996). The non‐obese diabetic (NOD) mouse: An animal model for autoimmune thyroiditis. Exp. Clin. Endocrinol. Diabetes 104(Suppl 3), 17–20. Martin, W. D., Hicks, G. G., Mendiratta, S. K., Leva, H. I., Ruley, H. E., and Van Kaer, L. (1996). H2‐M mutant mice are defective in the peptide loading of class II molecules, antigen presentation, and T‐cell repertoire selection. Cell 84, 543–540. Miyazaki, T., Uno, M., Uehira, M., Kikutani, H., Kishimoto, T., Kimoto, M., Nishimoto, H., Miyazaki, J., and Yamamura, K. (1990). Direct evidence for the contribution of the unique I‐ANOD to the development of insulitis in non‐obese diabetic mice. Nature 345, 722–724. Miyazaki, T., Wolf, P., Tourne, S., Waltzinger, C., Dierich, A., Barois, N., Ploegh, H., Benoist, C., and Mathis, D. (1996). Mice lacking H2‐M complexes, enigmatic elements of the MHC class II peptide‐loading pathway. Cell 84, 531–541. Morel, P. A., Dorman, J. S., Todd, J. A., McDevitt, H. O., and Trucco, M. (1988). Aspartic acid at position 57 of the HLA‐DQ beta chain protects against type I diabetes: A family study [published erratum appears in Proc. Natl. Acad. Sci. USA 1989 Feb;86(4):1317]. Proc. Natl. Acad. Sci. USA 85, 8111–8115. Moudgil, K. D., Wang, J., Yeung, V. P., and Sercarz, E. E. (1998). Heterogeneity of the T cell response to immunodominant determinants within hen egg‐white lysozyme of individual syngeneic hybrid F1 mice: Implications for autoimmunity and infection. J. Immunol. 161, 6046–6053. Mukherjee, R., Chaturvedi, P., Qin, H. Y., and Singh, B. (2003). CD4þCD25þ regulatory T cells generated in response to insulin B:9–23 peptide prevent adoptive transfer of diabetes by diabetogenic T cells. J. Autoimmun. 21, 221–237. Munz, C., Hofmann, M., Yoshida, K., Moustakas, A. K., Kikutani, H., Stevanovic, S., Papadopoulos, G. K., and Rammensee, H. G. (2002). Peptide analysis, stability studies, and structural modeling explain contradictory peptide motifs and unique properties of the NOD mouse MHC class II molecule H2‐A(g7). Eur. J. Immunol. 32, 2105–2116.

c la s s ii m h c m o l e cu l e i ‐a g 7

263

Nabavieh, A., Chou, H., Volokhov, I., Lee, J. E., Purdy, L. E., Elliott, J. F., Singh, B., and Madrenas, J. (1998). Development of an I‐Ag7‐expressing antigen‐presenting cell line: Intrinsic molecular defect in compact I‐Ag7 dimer generation. J. Autoimmun. 11, 63–71. Nelson, C. A., Petzold, S. J., and Unanue, E. R. (1993). Identification of two distinct properties of class II major histocompatibility complex‐associated peptides. Proc. Natl. Acad. Sci. USA 90, 1227–1231. Nelson, C. A., Petzold, S. J., and Unanue, E. R. (1994). Peptides determine the lifespan of MHC class II molecules in the antigen‐presenting cell. Nature 371, 250–252. Nelson, C. A., Vidavsky, I., Viner, N. J., Gross, M. L., and Unanue, E. R. (1997). Amino‐terminal trimming of peptides for presentation on major histocompatibility complex class II molecules. Proc. Natl. Acad. Sci. USA 94, 628–633. Nelson, C. A., Viner, N. J., Young, S. P., Petzold, S. J., and Unanue, E. R. (1996). A negatively charged anchor residue promotes high affinity binding to the MHC class II molecule I‐Ak. J. Immunol. 157, 755–762. Nishimoto, H., Kikutani, H., Yamamura, K., and Kishimoto, T. (1987). Prevention of autoimmune insulitis by expression of I‐E molecules in NOD. Nature 328, 432–434. Oiso, M., Matsushita, S., Nishi, T., Ishikawa, T., Nakano, N., Yoshida, K., Kikutani, H., and Nishimura, Y. (1998). Differential binding of peptides substituted at a putative C‐terminal anchor residue to I‐Ag7beta56Hisbeta57Ser and I‐Ag7beta56Probeta57Asp. Immunogenetics 47, 411–414. Oiso, M., Nishi, T., Ishikawa, T., Nishimura, Y., and Matsushita, S. (1997). Differential binding of peptides substituted at putative C‐terminal anchor residue to HLA‐DQ8 and DQ9 differing only at beta 57. Hum. Immunol. 52, 47–53. Patel, S. D., Cope, A. P., Congia, M., Chen, T. T., Kim, E., Fugger, L., Wherrett, D., and Sonderstrup‐McDevitt, G. (1997). Identification of immunodominant T‐cell epitopes of human glutamic acid decarboxylase 65 by using HLA‐DR(alpha1*0101,beta1*0401) transgenic mice. Proc. Natl. Acad. Sci. USA 94, 8082–8087. Peterson, M., and Sant, A. J. (1998). The inability of the nonobese diabetic class II molecule to form stable peptide complexes does not reflect a failure to interact productively with DM. J. Immunol. 161, 2961–2967. Pu, Z., Carrero, J. A., and Unanue, E. R. (2002). Distinct recognition by two subsets of T cells of an MHC class II‐peptide complex. Proc. Natl. Acad. Sci. USA 99, 8844–8849. Quarsten, H., Paulsen, G., Johansen, B. H., Thorpe, C. J., Holm, A., Buus, S., and Sollid, L. M. (1998). The P9 pocket of HLA‐DQ2 (non‐Aspbeta57) has no particular preference for negatively charged anchor residues found in other type 1 diabetes‐ predisposing non‐Aspbeta57 MHC class II molecules. Int. Immunol. 10, 1229–1236. Quartey‐Papafio, R., Lund, T., Chandler, P., Picard, J., Ozegbe, P., Day, S., Hutchings, P. R., O’Reilly, L., Kioussis, D., and Simpson, E. (1995). Aspartate at position 57 of nonobese diabetic I‐Ag7 beta‐chain diminishes the spontaneous incidence of insulin‐dependent diabetes mellitus. J. Immunol. 154, 5567–5576. Rammensee, H. G., Friede, T., and Stevanoviic, S. (1995). MHC ligands and peptide motifs: First listing. Immunogenetics 41, 178–228. Reich, E. P., von Grafenstein, H., Barlow, A., Swenson, K. E., Williams, K., and Janeway, C. A., Jr. (1994). Self peptides isolated from MHC glycoproteins of non‐obese diabetic mice. J. Immunol. 152, 2279–2288. Reizis, B., Altmann, D. M., and Cohen, I. R. (1997a). Biochemical characterization of the human diabetes‐associated HLA‐DQ8 allelic product: Similarity to the major histocompatibility complex class II I‐A(g)7 protein of non‐obese diabetic mice. Eur J. Immunol. 27, 2478–2483.

264

a n i s h s u ri a n d e m i l r . u na n u e

Reizis, B., Eisenstein, M., Bockova, J., Konen‐Waisman, S., Mor, F., Elias, D., and Cohen, I. R. (1997b). Molecular characterization of the diabetes‐associated mouse MHC class II protein, I‐Ag7. Int. Immunol. 9, 43–51. Ridgway, W. M., Ito, H., Fasso, M., Yu, C., and Fathman, C. G. (1998). Analysis of the role of variation of major histocompatibility complex class II expression on nonobese diabetic (NOD) peripheral T‐cell response. J. Exp. Med. 188, 2267–2275. Rivero, V. E., Cailleau, C., Depiante‐Depaoli, M., Riera, C. M., and Carnaud, C. (1998). Non‐ obese diabetic (NOD) mice are genetically susceptible to experimental autoimmune prostatitis (EAP). J. Autoimmun. 11, 603–610. Sadegh‐Nasseri, S., and Germain, R. N. (1991). A role for peptide in determining MHC class II structure. Nature 353, 167–170. Sakaguchi, S. (2004). Naturally arising CD4þ regulatory T cells for immunologic self‐tolerance and negative control of immune responses. Annu. Rev. Immunol. 22, 531–562. Sakaguchi, S., Takahashi, T., and Nishizuka, Y. (1982). Study on cellular events in post‐thymectomy autoimmune oophoritis in mice. II. Requirement of Lyt‐1 cells in normal female mice for the prevention of oophoritis. J. Exp. Med. 156, 1577–1586. Salomon, B., Rhee, L., Bour‐Jordan, H., Hsin, H., Montag, A., Soliven, B., Arcella, J., Girvin, A. M., Padilla, J., Miller, S. D., and Bluestone, J. A. (2001). Development of spontaneous autoimmune peripheral polyneuropathy in B7–2‐deficient NOD mice. J. Exp. Med. 194, 677–684. Schmidt, D., Amrani, A., Verdaguer, J., Bou, S., and Santamaria, P. (1999). Autoantigen‐independent deletion of diabetogenic CD4þ thymocytes by protective MHC class II molecules. J. Immunol. 162, 4627–4636. Schmidt, D., Verdaguer, J., Averill, N., and Santamaria, P. (1997). A mechanism for the major histocompatibility complex‐linked resistance to autoimmunity. J. Exp. Med. 186, 1059–1075. Scott, C. A., Peterson, P. A., Teyton, L., and Wilson, I. A. (1998). Crystal structures of two I‐Ad‐ peptide complexes reveal that high affinity can be achieved without large anchor residues [published erratum appears in Immunity 1998 Apr;8(4):531]. Immunity 8, 319–329. Singer, S. M., Tisch, R., Yang, X. D., and McDevitt, H. O. (1993). An Abd transgene prevents diabetes in nonobese diabetic mice by inducing regulatory T cells. Proc Natl. Acad. Sci. USA 90, 9566–9570. Singer, S. M., Tisch, R., Yang, X. D., Sytwu, H. K., Liblau, R., and McDevitt, H. O. (1998). Prevention of diabetes in NOD mice by a mutated I‐Ab transgene. Diabetes 47, 1570–1577. Slattery, R. M., Kjer‐Nielsen, L., Allison, J., Charlton, B., Mandel, T. E., and Miller, J. F. (1990). Prevention of diabetes in non‐obese diabetic I‐Ak transgenic mice. Nature 345, 724–726. Stratmann, T., Apostolopoulos, V., Mallet‐Designe, V., Corper, A. L., Scott, C. A., Wilson, I. A., Kang, A. S., and Teyton, L. (2000). The I‐Ag7 MHC class II molecule linked to murine diabetes is a promiscuous peptide binder. J. Immunol. 165, 3214–3225. Stratmann, T., Martin‐Orozco, N., Mallet‐Designe, V., Poirot, L., McGavern, D., Losyev, G., Dobbs, C. M., Oldstone, M. B., Yoshida, K., Kikutani, H., Mathis, D., Benoist, C., Haskins, K., and Teyton, L. (2003). Susceptible MHC alleles, not background genes, select an autoimmune T cell reactivity. J. Clin. Invest. 112, 902–914. Suri, A., Vidavsky, I., van der Drift, K., Kanagawa, O., Gross, M. L., and Unanue, E. R. (2002). In APCs, the autologous peptides selected by the diabetogenic I‐Ag7 molecule are unique and determined by the amino acid changes in the P9. J. Immunol. 168, 1235–1243. Suri, A., Walters, J. W., Gross, M. L., and Unanue, E. R. (2005). Natural peptides selected by diabetogenic DQ8 and murine I‐Ag7 molecules show common sequence specificity. J. Clin. Invest. 115 (in press).

c la s s ii m h c m o l e cu l e i ‐a g 7

265

Suri, A., Walters, J. J., Kanagawa, O., Gross, M. L., and Unanue, E. R. (2003). Specificity of peptide selection by antigen‐presenting cells homozygous or heterozygous for expression of class II MHC molecules: The lack of competition. Proc. Natl. Acad. Sci. USA 100, 5330–5335. Tisch, R., and McDevitt, H. (1996). Insulin‐dependent diabetes mellitus. Cell 85, 291–297. Todd, J. A., Bell, J. I., and McDevitt, H. O. (1987). HLA‐DQ beta gene contributes to susceptibility and resistance to insulin‐dependent diabetes mellitus. Nature 329, 599–604. Vartdal, F., Johansen, B. H., Friede, T., Thorpe, C. J., Stevanovic, S., Eriksen, J. E., Sletten, K., Thorsby, E., Rammensee, H. G., and Sollid, L. M. (1996). The peptide binding motif of the disease associated HLA‐DQ (alpha 1* 0501, beta 1* 0201) molecule. Eur. J. Immunol. 26, 2764–2772. Verdaguer, J., Schmidt, D., Amrani, A., Anderson, B., Averill, N., and Santamaria, P. (1997). Spontaneous autoimmune diabetes in monoclonal T cell nonobese diabetic mice. J. Exp. Med. 186, 1663–1676. Verreck, F. A., van de Poel, A., Termijtelen, A., Amons, R., Drijfhout, J. W., and Koning, F. (1994). Identification of an HLA‐DQ2 peptide binding motif and HLA‐DPw3‐bound self‐peptide by pool sequencing. Eur. J. Immunol. 24, 375–379. Wicker, L. S., Todd, J. A., and Peterson, L. B. (1995). Genetic control of autoimmune diabetes in the NOD mouse. Annu. Rev. Immunol. 13, 179–200. Zucchelli, S., Holler, P., Yamagata, T., Roy, M., Benoist, C., and Mathis, D. (2005). Defective central tolerance induction in NOD mice: Genomics and genetics. Immunity 22, 385–396.

RNAi and RNA‐Based Regulation of Immune System Function Dipanjan Chowdhury
and Carl D. Novina{,z


Center for Blood Research and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115 { Cancer Immunology and AIDS, Dana‐Farber Cancer Institute, Boston, Massachusetts 02115 z Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115

1. 2. 3. 4. 5. 6. 7. 8.

Abstract............................................................................................................. Introduction ....................................................................................................... Short RNAs in the Mammalian Immune System ....................................................... miRNAs in Mammalian Virus Infection ................................................................... RNAi, NMD, and TCR ........................................................................................ NMD and TCR Expression ................................................................................... RNAi and NMD Are Genetically Linked ................................................................. RNAi, ADARs, and Viruses ................................................................................... Conclusions........................................................................................................ References .........................................................................................................

267 267 271 273 274 276 278 280 284 286

Abstract Gene regulation by short RNAs is a ubiquitous and important mode of control. MicroRNAs are short, single‐strand RNAs that bind with partial complementarity to the 30 untranslated region of several genes to silence their expression. This expanding class of endogenous short RNAs are evolutionarily conserved and participate in control of development and cell‐specific gene function. Several of these microRNAs have been cloned uniquely from mammalian lymphocytes suggesting specialized roles in lymphocyte development and function. In addition, several genes linked to RNAi in lower eukaryotes have mammalian homologs with specialized roles in adaptive immunity. For example, in worms, the nonsense‐mediated decay (NMD) and RNAi pathways appear to be intricately linked. NMD plays a key role in regulating antigen‐receptor expression in lymphocytes and there are mammalian homologs for factors identified in worms that appear to be common in both RNAi and NMD pathways. On the other hand, RNA editing and RNAi have an inverse relationship and RNA editing has an important role in viral immunity. These observations indicate unique roles for dsRNAs in the mammalian immune system. 1. Introduction In 1998, researchers described a new process of gene silencing triggered by double‐strand (ds) RNAs called RNA interference (RNAi) (Fire et al., 1998).

267 advances in immunology, vol. 88 # 2005 Elsevier Inc. All rights reserved.

0065-2776/05 $35.00 DOI: 10.1016/S0065-2776(05)88008-3

268

d i pa n j a n c h o w d h ur y an d ca r l d. no v i na

Simply, introduction of dsRNAs into the nematode worm, Caenorhabditis elegans caused potent silencing of a gene containing sequences complementary to the dsRNAs. The discovery of RNAi resonated around the world and sparked a revolution in how we think about regulation of gene expression. RNAi is an evolutionarily ancient and highly conserved pathway of gene silencing mediated by short RNAs (reviewed in Novina and Sharp, 2004). An impacting result of the RNAi revolution was the surfacing of several biological processes across phyla, which had been incompletely understood. Viral gene silencing in plants, quelling in fungi, and cosuppression in multiple organisms subsequently emerged as processes mediated by short RNAs derived from dsRNA (reviewed in Tomari and Zamore, 2005). RNAi has been genetically linked to other pathways of short RNA‐directed gene silencing (reviewed in He and Hannon, 2004). In the process of RNAi, long dsRNAs are cleaved by an endoribonuclease called Dicer into short dsRNAs called short interfering RNAs (siRNAs). An endogenous class of genes expressing hairpin RNAs are also cleaved by Dicer into short, single‐ strand RNAs called microRNAs (miRNAs). siRNAs and miRNAs are incorporated into a microribonucleoprotein complex (miRNP) that contains members expressed from an evolutionarily ancient family of Argonaute genes (reviewed in Carmell et al., 2002; Schwarz and Zamore, 2002). The short RNAs recruit the miRNP to mRNAs containing the complementary sequence. The fate of the miRNP‐loaded mRNA depends partly upon the degree of complementarity between the targeted mRNA and the short RNA. The short RNA may block protein production in two ways. Perfectly complementary base pairing between the short RNA and the targeted mRNA will lead to mRNA cleavage and degradation. Alternatively, partially complementary base pairing between the short RNA and the targeted mRNA will lead to repression of translation of the targeted mRNA (Fig. 1). Thus, gene expression may be repressed or derepressed at a distal point in the gene expression pathway with rapid kinetics that would not require de novo transcription, mRNA splicing, processing, or export. Though short noncoding RNAs that control the precise timing of developmental transitions were first described in worms (Lee et al., 1993; Reinhart et al., 2000), miRNAs have since been implicated in developmental transitions in virtually every multicellular organism. There are more than 250 miRNAs expressed in humans (Lim et al., 2003); however, very few have been assigned a biological function. The importance of miRNA function is underscored by recent reports that predict that miRNAs target one fifth (Xie et al., 2005) to more than one third (Lewis et al., 2005) of genes in the human genome. A more complete understanding of the processes regulated by miRNAs has been confounded by imperfect base pairing between the miRNA and its cognate mRNA. Typically, miRNAs control cell‐fate

r na i i n t h e i m m u ne sy s t e m

269

Figure 1 The miRNA pathway. The processing step that generates miRNAs and siRNAs requires the endoribonuclease, Dicer. The effector step of short RNA‐directed silencing requires a highly conserved Argonaute family of proteins. If the short RNA binds to its target with partial sequence complementarity, the Argonaute‐containing microribonucleoprotein complex (miRNP) may be recruited for translational inhibition of the mRNA. If the short RNA binds with perfect sequence complementarity, the miRNP may be recruited for mRNA cleavage.

decisions and are involved in cell‐specific gene expression (reviewed in Bartel, 2004). For example, the pancreatic islet‐specific miRNA, miR375, regulates insulin secretion (Poy et al., 2004) and miR143 regulates adipocyte differentiation (Esau et al., 2004). Several miRNAs have been cloned selectively from different populations of immune cells (Chen et al., 2004). The importance of some of these miRNA functions in lymphocytes is highlighted by dysregulated miRNA activities that are correlated with leukemias (Calin et al., 2002; 2004a,b; Gauwerky et al., 1989). It has become clear that RNAi is a mechanism of genome defense in several organisms, in which short RNAs perform adaptive immune‐effector functions. In worms, mutations in RNAi pathway genes increase susceptibility to mutations by transposons (Ketting et al., 1999; Tabara et al., 1999). In plants, the RNAi pathway serves as an immune system protecting against virus attack.

270

d i pa n j a n c h o w d h ur y an d ca r l d. no v i na

siRNAs play very important roles as the effector arm of the antivirus response (Hamilton and Baulcombe, 1999). Short RNAs associated with specific gene‐ silencing phenomena were first reported in virus‐infected or transgenic plants (Hamilton and Baulcombe, 1999). Conversely, plant viruses have evolved counter‐defense mechanisms against the plant RNAi response, thereby promoting infection (reviewed in Lecellier and Voinnet, 2004). Recently, miRNAs were cloned from B cells infected with Epstein‐Barr Virus (EBV) (Pfeffer et al., 2004), indicating that there may be very specialized roles for short RNAs in immune system functions. It is not surprising that the war between host cell and parasitic genetic elements is being waged on the RNA battlefields. Short RNA‐directed gene silencing also occurs in the nucleus at the transcriptional level. In plants, short RNAs direct transcriptional gene silencing (TGS) through chromatin modifications (reviewed in Matzke and Matzke, 2004; Matzke et al., 2004). The most efficient TGS was observed in plants when promoter sequences were inserted into a replicating virus which resulted in methylation of the endogenous promoter sequences (Lindroth et al., 2001; Wassenegger et al., 1994). In mammals, introduction of siRNAs complementary to promoter sequences led to promoter methylation and TGS (Kawasaki and Taira, 2004; Morris et al., 2004). Short RNAs have been proposed to play a role in nuclear events in the mammalian immune system such as regulating the chromatin structure at antigen receptor and cytokine loci (Chowdhury and Novina, 2005). Consistent with the notion that RNAi constitutes an intracellular immune system that functions in genome defense, yeast with mutations in RNAi genes demonstrate desilencing of centromeric repeat DNA (Griffths‐Jones, 2004; Grishok et al., 2001; Hutvagner et al., 2001; Lee et al., 2003; Schramke and Allshire, 2003; Verdel et al., 2004). Similarly, mammals with homologous disruption in the gene coding for Dicer demonstrate defects in heterochromatin formation (Lewis et al., 2003) and desilencing of centromeric repeat DNA (Fukagawa et al., 2004; Kanellopoulou et al., 2005). In flies, short RNAs promote TGS of high copy number transgenes through chromatin modifications (Pal‐Bhadra et al., 2004) and in Tetrahymena thermophila, short RNAs specify sites of genome fragmentation and DNA deletion (Mochizuki et al., 2002). Paradigms of short RNA‐directed gene silencing established in other eukaryotes may have homologous functions in mammalian lymphocytes. A defined role for RNAi in mammals has yet to be described. Much of our understanding about RNAi and short‐RNA function is derived from worms. RNAi is linked to RNA editing and nonsense‐mediated decay in worms. Several of the PAZ‐PIWI‐Domain (PPD) proteins implicated in RNAi‐related pathways in worms and other eukaryotes have homologs with specialized functions in lymphocytes which require dsRNA for proper function and which are

r na i i n t h e i m m u ne sy s t e m

271

distinct from their functions in other mammalian cell types. This review describes recent data on short RNAs in mammalian lymphocyte development and function and explores the unique relationships between RNAi genes and dsRNA intermediates in processes required for antigen‐receptor expression, indicating that lymphocyte‐specific gene expression may require RNAi. 2. Short RNAs in the Mammalian Immune System Short RNAs have been cloned from cells of the hematopoietic lineages. Identification of these short RNAs unique to these cell lineages and from a variety of cellular contexts suggests their importance in the generation and maintenance of immune system functions. In addition to the evolving roles for short RNAs in the nucleus, short RNAs in the cytoplasm may play pivotal roles in lymphocyte‐restricted gene expression. miRNAs are conserved and have been implicated in developmental transitions in numerous organisms (reviewed in Bartel, 2004). miRNAs are expressed as long polII transcripts that are processed by the RNaseIII enzyme Drosha (Lee et al., 2003) to produce shorter hairpin RNAs that are exported to the cytoplasm where they are further processed by Dicer (Grishok et al., 2001; Hutvagner et al., 2001) into mature miRNAs. Therefore, miRNAs are related to siRNAs in the Dicer processing step (Fig. 1). Unlike siRNAs that are derived from long dsRNAs typically from exogenous sources such as transgenes, miRNAs are expressed from endogenous genes to silence target genes expressed in trans (reviewed in Bartel, 2004). A comprehensive list of known miRNAs and their predicted targets can be found in the miRNA registry (Griffiths-Jones, 2004). To better understand the roles for miRNAs in mammalian development, researchers cloned 100 unique miRNAs from mouse bone marrow (Chen et al., 2004). Three of these miRNAs (miR181, miR223, and miR142) were preferentially or uniquely expressed in hematopoietic tissues. To characterize the role of these miRNAs in hematopoiesis and to investigate their pattern of expression during differentiation, these miRNAs were ectopically expressed in hematopoietic progenitor cells cultured in a cocktail of cytokines. Expression of miR181 doubled the number of B cells with no effect on the number of T cells. Conversely, ectopic expression of miR142 and miR233 had a modest but opposite effect that increased T-cell but not B‐cell number. The effect of miR181 in cultured cells was reproduced in vivo by adoptive transfer of miR181‐expressing bone marrow cells in lethally irradiated mice. Though the target gene(s) of miR-181 have not been determined, these observations implicate miRNAs in regulation of mammalian immune system gene expression.

272

d i pa n j a n c h o w d h ur y an d ca r l d. no v i na

To gain insight into the roles of miRNAs in mammalian immune system function, several laboratories are attempting to identify the target genes through genetic and biochemical approaches while other laboratories are using a bioinformatics approach to predict the mRNA target genes for the miRNAs. More than one-third of human genes appear to the conserved miRNA targets (Lewis et al., 2005). miRNA-directed silencing is specified by perfect Watson-Crick complementarity among nucleotides 2–7 relative to the 50 end of the miRNA (Doench and Sharp, 2004; Lewis et al., 2003, 2005). Using this and other parameters, mRNAs with conserved pairing to the 50 region of the miRNA can be identified as candidate genes regulated by cloned miRNAs. Translational inhibition requires imperfect nucleotide base pairing between the mRNA and the miRNA and the degree of translational inhibition is thought to depend on the number of miRNAs bound to the mRNA. Approximately 3% of these targets are genes involved in the immune system suggesting cell‐type‐ specific roles for these short RNAs. The identities of some of these targets are listed in Table 1. miRNAs have been implicated in cell‐cycle control, and dysregulation of miRNA pathways can lead to initiation of cancer. For example, in flies, bantam encodes a miRNA required for cell‐cycle progression and activation of the proapoptotic pathway (Brennecke et al., 2003). In lymphocytes, there are examples in which loss of miRNA genes and oncogene dysregulation correlates directly with cancer formation. Chronic lymphocyte leukemia (CLL) is the most common form of adult leukemia in the western world. Croce and colleagues demonstrated that miR15 and miR16 are located at chromosome 13q14 in a region that is deleted in most patients with CLL (Calin et al., 2002). Genome‐wide expression profiling of miRNAs in human CD5þB cells from CLL patients reveal distinct expression patterns compared to normal controls (Calin et al., 2004a). One way of interpreting these observations is that these miRNAs have tumor suppressor roles. As many as 98 of 186 miRNA genes are located in genomic regions or in fragile sites, suggesting that miRNA loss‐of‐ function may be involved in cancer progression (Calin et al., 2004b). Alternatively, miRNAs could promote cancer if an oncogene is translocated 30 relative to a miRNA promoter. An example of this mechanism is the translocation of the myc gene to a site 4 nucleotides 30 relative to miR142, causing an aggressive B‐cell leukemia due to upregulated MYC expression (Gauwerky et al., 1989). One consequence of myc translocation is loss of a conserved 20 nucleotide element 30 relative to miR142 (Gauwerky et al., 1989). It is possible that loss of this element causes inefficient processing of the miR142/MYC fusion transcript resulting in a reduction of miR142, which leads to increased translation and higher levels of MYC protein.

r na i i n t h e i m m u ne sy s t e m

273

Table 1 Predicted miRNA Targets in Mammalian Immune Systems microRNA

Families

Target gene

miR17/20/106 miR17/20/106 miR17/20/106 miR25/32/92/367 miR15/16/195, miR-181, miR182 miR181 miR181 miR30

IL17E

Interleukin 17E

PTEN RUNX1 CD69 BCL2 BCLAF1 CD4 PRDM1

miR30 miR30 miR30 miR30 miR200b miR200b miR130/301 miR130/301

STIM1 NFAT5 CBFB BCL9 BCL11B TCF8 BTG1 IRF1

Phosphatase and tensin homolog Runt-related transcription factor 1 CD69 antigen (p60, early T-cell activation) B-cell lymphoma 2 protein BCL2-associated transcription factor 1 CD4 antigen (p55) PR domain containing 1, with ZNF domain isoform (BLIMP1) Stromal interaction molecule 2 Nuclear factor of activated T cells 5 Core-binding factor, beta subunit B-cell CLL/lymphoma 9 B-cell CLL/lymphoma 11B Ttranscription factor 8 (represses interleukin 2) B-cell translocation protein 1 Interferon regulatory factor 1

Several of the known miRNAs (column 1) have predicted target sites in the 30 UTR of genes involved in immune‐specific functions. Based partly upon exact nucleotide complementarity between the 50 end of the miRNA and the target mRNA, miRNAs have predicted targets in the immune system (column 2). Targets include transcription factors required for B-cell‐specific transcriptional activation and differentiation markers for B- and T-cell lineages. As observed in Table 1, one particular miRNA may target more than one gene, and one gene may have binding sites for more than one particular miRNA. Note that some microRNAs, such as miR16, may be expressed in nonhematopoietic tissues but may have regulatory targets that are immune-restricted. A more comprehensive listing of miRNA target predictions can be found at http://genes.mit.edu/ targetscan/. (Adapted from Lewis et al., 2003.)

3. miRNAs in Mammalian Virus Infection Mammals produce short RNAs during viral infections. Researchers cloned short RNAs from a Burkitt’s lymphoma cell line latently infected with EBV (Pfeffer et al., 2004). The EBV miRNAs were produced from two regions of the EBV genome. The first cluster is located within the mRNA of the BHRF1 gene, encoding a distant Bcl‐2 homolog. The other EBV miRNAs cluster in the intronic region of the BART gene whose function is still unknown. Researchers examined the expression of the miRNAs in cell lines infected with the EBV virus in different stages of latency. Interestingly, BART miRNAs were detected in every stage but were significantly higher in cells in the lytic phase. BHRF miRNAs appeared to be downregulated at the early stages of latency but were present during the later stages of latency and the lytic phase. Although BHRF1 is a lytic‐stage protein, earlier studies have shown that

274

d i pa n j a n c h o w d h ur y an d ca r l d. no v i na

latent‐stage EBV seems to transcribe this gene (Murray et al., 1996; Oudejans et al., 1995). It is possible that these transcripts are processed to miRNAs during this stage and there is no BHRF protein production. However, it remains unclear how the virus overcomes this block during the lytic phase and produces the BHRF protein. The authors speculate that during the lytic phase, the expression level of the BHRF genes increases and high transcript levels exceed cellular miRNA processing capacity and the unprocessed transcripts then get translated. The BART miRNAs target the mRNA of virally encoded DNA polymerase BALF5 for degradation. This could be a key step in initiating the lytic phase of the virus. Therefore, the working model for EBV‐ derived miRNA function is regulation of the transition between latent and lytic phases of the replication cycle. One surprising observation was that the noncoding hairpins EBERs1 and 2 (Glickman et al., 1988), which are the most abundant transcripts in the latent phase and are absent in the lytic phases, did not demonstrate detectable miRNA production. Though miRNAs appear to be a mechanism used by the virus to subvert the molecular immune system, there could be another intended function for miRNAs used by mammalian cells. Similar to the paradigm established in plants, short RNAs produced by mammalian cells in response to virus infection could be used to prevent virus infection and establish intracellular immunity against infecting viruses. These apparently paradoxical ‘‘proviral’’ versus ‘‘antiviral’’ effects of short RNA may be explained by the idea that large DNA viruses such as EBV have evolved mechanisms to co‐opt the virally derived short RNA, produced by the host and intended to cleave viral transcripts, and instead use these short RNAs to silence genes that effect the transition between latent and lytic stages of the virus replication cycle. 4. RNAi, NMD, and TCR In addition to miRNA‐directed translational repression, RNAi may play important roles in dsRNA‐directed surveillance events in lymphocytes. Antigen‐ receptor expression requires successful recombination of several repetitive and highly related genetic elements including variable (V) diversity (D) and joining (J) region gene segments. Recombination leading to premature termination codons creates a cellular stress that could lead to lymphocyte death. During V(D)J recombination, there are two intermediate steps that add to antigen‐receptor diversity. Recombinase Activating Genes (rag)1 and 2 initiate V(D)J recombination by introducing a DNA double‐strand break at the border of the recombination signal sequence, leaving hairpin‐sealed coding ends and blunt signal ends that are usually excised from the chromosome (reviewed in Gellert, 2002). The hairpins generated at the end of the

r na i i n t h e i m m u ne sy s t e m

275

coding segment are resolved by endonucleolytic cleavage by the newly identified factor Artemis, and there is transfer of nucleotides (P‐addition) from the complementary strand to the coding strand, thereby adding to the diversity (Ma et al., 2002; Pannicke et al., 2004). Secondly, during repair of intermediates in V(D)J recombination, nongermline-encoded sequence (N‐addition) is added to broken ends before joining through the action of terminal deoxynucleotidyl transferase (TdT). TdT is a template‐independent DNA polymerase expressed only in cells actively undergoing V(D)J recombination (Gilfillan et al., 1995). Although these mechanisms for the addition of nontemplated nucleotide addition to antigen‐receptor transcripts lead to variability in nucleotides at the junction of V, D, and J segments and increase the receptor repertoire, they also change the reading frame and therefore two of three rearrangement events can generate premature termination codons (PTCs). If the truncated proteins encoded by PTC‐bearing antigen‐receptor genes were translated, they could be deleterious as they act as dominant‐negative mutants that inhibit the function of the wild‐type immunoglobulin (Ig) and T‐cell receptor (TCR) proteins (Herskowitz, 1987). Earlier work demonstrated that truncated Ig proteins also induce a stress response in the endoplasmic reticulum (ER) if they are overexpressed or misfolded (Pahl and Baeuerle, 1997). Recent work shows that the unfolded protein response (UPR), a multifaceted signaling pathway emanating from the ER membrane, plays a key role in the differentiation of mature B cells to plasma cells. UPR upregulates the expression of ER chaperones, folding enzymes and factors like X‐box binding protein 1 (XBP1) which are known to be crucial for the terminal differentiation of B cells (reviewed in Gass et al., 2004). One can envisage a scenario in which in the absence of nonsense-mediated decay (NMD) there would be an overload of truncated Ig molecules in the ER leading to UPR signaling which could adversely affect B‐cell development. How does the adaptive immune system get around this problem? It was observed over a decade ago that the steady‐state level of PTC containing Ig and TCR mRNAs (Aoufouchi et al., 1996; Baumann et al., 1985; Carter et al., 1995; 1996; Connor et al., 1994; Jack et al., 1989; Li et al., 1997; Lozano et al., 1994) is dramatically lower than that of productively rearranged counterparts, although the transcriptional rates of these genes are comparable. This posttranscriptional quality‐control mechanism is termed nonsense‐mediated mRNA decay. NMD distinguishes the product of a successful rearrangement and selectively allows its translation. It is important to note that this selection is being made at the level of RNA, thus allowing the system to be promiscuous at the level of DNA and generate greater diversity, but then imposes a conservative checkpoint at the level of protein.

276

d i pa n j a n c h o w d h ur y an d ca r l d. no v i na

In worms, regulation of premature termination codons by nonsense‐ mediated decay is genetically linked to RNAi. Worms that are mutated in certain genes required for nonsense‐mediated decay are also defective in RNAi. The following sections explore the unique relationships between RNAi genes and processes required for antigen receptor expression. 5. NMD and TCR Expression In mammalian cells, NMD is triggered by pre‐mRNA processing (Maquat, 2004). Pre‐mRNA is bound by cap‐binding proteins CBP80 and CBP20 at the 50 cap and by poly(A) binding protein PABP2 at the 30 poly(A) tail (Fig. 2). Splicing deposits an exon junction complex (EJC) 20–24 nucleotides 50 of the exon–exon junction. Minimally, the EJC consists of RNPS1, SRm160, UAP56, Y14, REF, and TAP‐P15, (Lejeune et al., 2002; Reichert et al., 2002) proteins involved in splicing and mRNA transport. Then this complex acquires proteins involved in NMD starting with Upf3/3X, a nuclear protein that is exported to the cytoplasm with the mRNA (Gehring et al., 2003; Kim et al., 2001). A perinuclear protein Upf2 is then recruited to the complex forming the pioneer translation initiation complex (Lykke‐Andersen et al., 2000; Serin et al., 2001). NMD is initiated during this preliminary round of translation when a PTC resides more than 50–55 nucleotides away from the 50 end of an exon–exon junction (Ishigaki et al., 2001). The EJC‐Upf3/3X‐Upf2 complex together with Upf1 triggers NMD. Upf1 is a RNA‐dependent ATPase and 50 to 30 helicase, which unlike other Upfs, does not appear to form a stable complex with EJC (Mendell et al., 2002; Sun et al., 1998). Upf1 phosphorylation by SMG1 (phosphatidylinositol kinase‐related protein kinase) is vital for NMD (Denning et al., 2001; Yamashita et al., 2001). The RNA degradation involves decapping followed by 50 to 30 decay, as well as deadenylation followed by 30 to 50 decay (Lejeune et al., 2003). In order for normal translation to proceed, the initial translation initiation complex must be remodeled. The EJC and the Upf proteins are removed, CBP80 and CBP20 are replaced by eukaryotic initiation factor 4E, and at the 30 end, PAPB2 is replaced by PABP1 (Lejeune et al., 2002). Intron position within a pre‐mRNA is an important determinant of NMD. According to the established rule, PTCs followed by an intron located 50–55 nucleotides downstream usually elicit NMD (Nagy and Maquat, 1998). The exception is provided by both the TCRb and IgH transcripts that are susceptible to NMD even when the distance is less than 50–55 nucleotides (Carter et al., 1996; Wang et al., 2002). The reason for this exception to the rule is unknown. One possibility is the presence of cis‐elements within these transcripts that catalyze NMD, other than an exon–exon junction. This possibility

r na i i n t h e i m m u ne sy s t e m

277

Figure 2 NMD and RNAi are directly related. Pre‐mRNA consists of the exons (colored boxes) and the introns (line between boxes). Splicing results in the removal of introns and deposition of a complex of proteins called the exon junction complex (EJC) 20–25 nucleotides (nt) 50 relative to the exon–exon junction. The processed mRNA is bound by cap-binding proteins (CBP20 and CBP80) at the 50 cap and by the polyA binding protein (PABP2) at the 30 poly A tail. The EJC recruits Upf3 (ortholog of SMG4) or Upf3X, the first factors involved in NMD that associate with mature mRNA. Upf3 or Upf3X is thought to recruit Upf2 (ortholog of SMG3) leading to the first (pioneer) round of translation. It is not clear whether multiple ribosomes are involved in this process, although the substrate for this event is a messenger ribonucleoprotein (mRNP) complex that is very different from the mRNPs during subsequent rounds of translation. Events during the pioneer round of translation determine whether the mRNA is going to be translated or destroyed. In the absence of a premature termination codon (PTC), the pioneer translation initiation complex is modified to the steady‐state translation initiation complex. The events numbered 1–3 lead to translation of mRNA. (1) The EJC complex and Upf proteins are removed. (2) CBP20 and CBP80 are replaced by the eukaryotic initiation factor eIF4E. (3) The poly‐A binding protein PABP2 is replaced by PABP1. When a PTC is located 50–55 nucleotides (nt) 50 relative to the exon–exon junction, a series of events are triggered (numbered 4–6) that lead to decay of mRNA. (4) Upf1 (ortholog of SMG2) is recruited to the EJC and likely functions in translation termination. (5) Decapping of the mRNA occurs, followed by 50 ‐to‐30 exonucleolytic decay. (6) Deadenylation of the mRNA occurs, followed by 30 ‐to‐50 exonucleolytic decay. Several of the SMG proteins (orthologs of Upfs) have been implicated in the siRNA pathway.

278

d i pa n j a n c h o w d h ur y an d ca r l d. no v i na

is supported by the identification of regulatory sequences from two different TCRb transcripts that were localized to a region composed of the rearranged V(D)J exon and the immediately flanking intron sequences (Gudikote and Wilkinson, 2002). Typically, NMD is 10‐ to 100‐fold more efficient in TCR and Ig transcripts relative to other mammalian transcripts such as b‐globin, v‐src, or triosephosphateisomerase (TPI) (Baumann et al., 1985; Carter et al., 1996; Cheng and Maquat, 1993; Jack et al., 1989; Naeger et al., 1992; Simpson and Stoltzfus, 1994). Introduction of the VDJ exon, together with immediately flanking introns of TCRb, was sufficient to trigger robust downregulation in response to nonsense codons in other mammalian genes like TPI (Gudikote and Wilkinson, 2002). Importantly, PTCs located within 30 , but not 50 of the VDJ exon triggered strong downregulation of gene expression by NMD (Gudikote and Wilkinson, 2002). The same holds true for IgH transcripts, where a 177long sequence comprising the 50 half within the VDJ exon is necessary for efficient NMD (Buhler et al., 2004). Another distinctive feature of NMD in lymphoid cells is the cell specificity. Transcripts from a TCRb construct with a PTC in the penultimate exon is only downregulated in T cells and not in a nonlymphoid cell line (Carter et al., 1996). Biochemical assays using B‐cell nuclear extracts showed a decreased level of PTC‐bearing processed Igk transcripts, a result not observed in nuclear extracts from other cell types (Aoufouchi et al., 1996). In the same system, the B‐cell nuclear extracts preferentially downregulated PTC‐bearing Igk transcripts compared to b‐globin transcripts. This lymphoid specificity might be a consequence of quantitative differences in trans‐acting factors present rather than a fundamental difference in the mechanism of NMD. 6. RNAi and NMD Are Genetically Linked The NMD and RNAi mRNA degradation pathways are linked in worms. SMG (suppressor with morphogenic effect on genitalia) factors were shown to be involved in NMD in worms. A study in C. elegans revealed that three smg genes, smg‐2, smg‐5, and smg‐6, were required for persistence of RNAi as long as the target mRNA was continuously transcribed (Domeier et al., 2000). Animals with a mutation in either of these genes initially demonstrated silencing at levels comparable to wild‐type worms but rapidly recovered from RNAi. These results suggest that these proteins do not have a role in initiating RNAi‐mediated silencing but function in maintenance of the silencing signal. The SMG proteins are required for NMD and mutations that block the phosphorylation of SMG2 by SMG1, and those that impair the subsequent dephosphorylation by SMG5, SMG6, and SMG7, eliminate NMD. Interest-

r na i i n t h e i m m u ne sy s t e m

279

ingly, smg‐2, smg‐3, and smg‐4 in C. elegans have orthologs Upf1, Upf2, and Upf3, respectively, in mammals. Based upon sequence conservation, all the other SMG proteins also have mammalian orthologs. Posttranscriptional gene silencing by RNAi and mRNA surveillance by NMD was also connected in the green algae, Chlamydomonas reinhardtii. Researchers showed that the DEAH‐box RNA helicase, Mut6 is involved in both processes (Wu‐Scharf et al., 2000). Mut6 appears to be part of a nuclear complex comprised of a single‐stranded RNA‐binding protein and a ribonuclease containing a staphylococcal nuclease‐like domain. There is speculation that Mut6 might be part of a nuclear RISC‐like complex. Studies in C. elegans suggest that RNAi does not target pre‐mRNA. But they do not rule out the possibility that a nuclear RISC‐like complex might selectively target nonpolyadenylated transcripts from transposons. In mammals, RNAi has been shown to be restricted to the cytoplasm (Hutvagner and Zamore, 2002; Martinez et al., 2002) although there are alternate possibilities. A fraction of the RISC‐complex may be located at the nuclear pore, potentially accessing the transcripts prior to complete export from the nucleus. This mechanism would allow the RISC complex to scan the RNA that is being exported and function as a quality‐control checkpoint. The subcellular location of NMD of the TCR and Ig transcripts is controversial (Wilkinson and Shyu, 2002). Two independent approaches were used to demonstrate that inhibition of mRNA export did not affect the downregulation of PTC‐containing TCRb transcripts in the nuclear fraction of mammalian cells (Buhler et al., 2002; Li et al., 1997). These observations provide strong evidence for intranuclear NMD. Translation is a necessary event for NMD and whether translation can occur in the nucleus is also a matter of debate (Kapp and Lorsch, 2004; Strudwick and Borden, 2002). By one estimate, nuclear translation may constitute as much as 10–15% of reporter gene‐translation activity in mammals (Iborra et al., 2001). RNAi and NMD are distinct but genetically related RNA degradation pathways that serve the common function of removing potentially deleterious RNA from the cell. Though there are obvious mechanistic differences between these two pathways, there are likely some overlapping mechanisms. In vitro studies revealed that RISC is bound to ribosomes in cell‐free extracts (Hammond et al., 2000) potentially connecting RNAi to translation and as discussed earlier, initiation of translation is an integral part of NMD. Identification of common factors in lower organisms that are required for both RNAi and NMD has raised the tantalizing possibility that RNAi and NMD may be linked in the mammalian adaptive immune system. Because antigen receptor genes acquire PTCs at very high rates and the NMD pathway functions at its highest efficiency in lymphocytes relative to other cell types

280

d i pa n j a n c h o w d h ur y an d ca r l d. no v i na

(Baumann et al., 1985; Carter et al., 1996; Cheng and Maquat, 1993; Jack et al., 1989; Naeger et al., 1992; Simpson and Stoltzfus, 1994), it is logical to investigate potential links between RNAi and NMD in the adaptive immune system. 7. RNAi, ADARs, and Viruses Although RNAi and NMD are two pathways acting synergistically to affect RNA degradation using some common factors; in worms, RNAi has an inverse relationship with other RNA‐directed processes such as the RNA‐editing pathway. Changes in mRNA sequence may result in diversifying or correcting information encoded by the genome. RNA editing is another posttranscriptional mechanism to alter RNA sequence important for cellular and viral genes. Two types of RNA editing have been described in nuclear‐encoded mRNA. Inosine formed by adenosine deamination (A‐I editing) is the most frequent form of editing (Bass, 2002; Seeburg, 2002) while uridine formed by cytidine deamination (C‐U editing) (Balnc and Davidson, 2003) is relatively rare. An A‐I editing enzyme was first discovered in Xenopus through its ability to unwind long dsRNA by deaminating multiple A’s to I’s, which result in unstable I:U base pairs (Bass and Weintraub, 1987; 1988) (Fig. 3A). A‐I editing is catalyzed by adenosine deaminases that act on RNA (ADARs). 7.1. RNAi Versus ADAR Activities The substrate for ADAR activity is dsRNA and in worms, RNA editing is competitively related to RNAi. Worms with high‐ADAR activity are poor at RNAi. Conversely, worms with low‐ADAR activity are efficient at RNAi. Wild‐type and adr‐1;adr‐2 mutant worms trigger RNAi in response to injected long dsRNA directed against a reporter. Only in somatic tissues of adr‐1;adr‐2 mutant worms but not in somatic tissues of wild‐type worms, long dsRNAs produced from a transgene expressing repetitive arrays triggered RNAi. In these experiments, ADAR’s ability to antagonize RNAi triggered by a transgene required a dsRNA intermediate (Knight and Bass, 2002) and worms were capable of editing injected, long dsRNA (Domeier et al., 2000). Furthermore, adr‐1;adr‐2 double‐mutant worms demonstrated a reduced ability to silence transgene‐triggered RNAi when the complexity of the repetitive transgene was reduced (when random DNA was inserted in the repetitive array) (Knight and Bass, 2002). These observations suggest host cells are capable of discerning the origin of the trigger dsRNA. In another study, it had been observed that deletions in each or both of the C. elegans adr genes (adr‐1 and adr‐2) produced animals with defects in

r na i i n t h e i m m u ne sy s t e m

281

Figure 3 Competition between ADAR activity and RNAi. (A) ADARs convert adenosines to inosines by hydrolytic cleavage. Inosine is translated as guanosine, which base pairs with cytidine. (B) dsRNA is the substrate for both ADARs and the RNAi machinery. RNAi and ADAR activities appear to function in a mutually exclusive fashion suggesting a model of noncompetitive inhibition. Editing could change the secondary structure of long dsRNA making it a poor substrate for Dicer processing of long dsRNA into siRNAs (left panel). Conversion of long dsRNA into siRNA could antagonize the activity of ADARs because siRNA duplexes are expected to be too short for adenosine deamination (right panel). Viral dsRNA and cellular single strand RNAs that form intramolecular dsRNA duplexes may be targeted by different ADARs in the nucleus prior to export to the cytoplasm to prevent Dicer activity. Conversely, ADAR1(p150) and Dicer may directly compete for long dsRNA binding in the cytoplasm. These mechanisms are not well understood.

282

d i pa n j a n c h o w d h ur y an d ca r l d. no v i na

chemotaxis (Tonkin et al., 2002). When C. elegans strains lacking ADARs were crossed with RNAi‐defective mutants rde1 and rde4, the rde‐1 and rde‐4 alleles rescued the chemotaxis defects of the adr‐1;adr‐2 animals (Tonkin and Bass, 2003). In flies, ADAR activity competes with RNAi in vitro (Scadden and Smith, 2001). Also, subcellular localization or alternate contexts may be important determinants in the relationship between ADAR activity and RNAi. ADARs have the ability to create sequence and structural changes in dsRNA, which could affect the RNAi pathway in several ways. First, editing could prevent dsRNA from being recognized by Dicer and make it a poor substrate for siRNA production. Second, the mRNA targeting step of RNAi could be inhibited by the substitution of I:U for A:U base pairs between the siRNAs and the target mRNA. Finally, I:U base pairs within the siRNA duplexes could interfere with the ability of RNA‐dependent RNA polymerase (RdRP) to recognize the siRNA as a primer. Conversely, RNAi could also impede ADAR activity. Conversion of dsRNA to siRNA would antagonize the activity of ADARs, because siRNA duplexes are expected to be too short for adenosine deamination. Thus, in the absence of ADAR activity, the RNAi pathway should be more effective, whereas any deficiency in the RNAi pathway should enhance ADAR activity. Three ADAR family members are expressed in mammals. ADARs from all organisms have multiple dsRNA binding (DSRB) domains at varying distances from each other and a highly conserved C‐terminal catalytic domain (reviewed in Bass, 2002). ADAR1 has two forms: a long form (150 kDa) produced in response to interferons, which is localized in the cytoplasm and a short form (110 kDa) which is constitutively nuclear (George and Samuel, 1999b). Because RNAi is restricted to the cytoplasm and RNA editing is both nuclear and cytoplasmic depending upon the ADAR activity, RNA editing may have a noncompetitive relationship with RNAi for dsRNA binding in the nucleus (Fig. 3B). ADAR activity may be prevented from antagonizing RNAi and the relationship between ADAR activity and RNAi may differ from tissue to tissue depending upon the triggering source of the dsRNA and other factors present in a particular cell type. ADARs target mRNAs in coding regions, as well as in noncoding sequences such as 50 and 30 UTRs. Most of the known cellular substrates of the ADARs are in the nervous system (Bass, 2002; Sanders‐Bush et al., 2003). ADARs target mRNAs of five glutamate receptor subunits and all the editing occurs within the dsRNA that forms between an exon sequence and an intron with an exon‐complementary sequence (Seeburg et al., 1998). ADARs also target the mRNA encoding the 2C subtype of the serotonin receptor (Niswender et al., 2001). Other examples of neuronal substrates (calcium and sodium channel

r na i i n t h e i m m u ne sy s t e m

283

forming proteins) of ADARs are found in flies (Hanrahan et al., 2000; Palladino et al., 2000; Reenan et al., 2000). The ADAR1 knockout mouse revealed a heterozygous embryonic lethal phenotype and though most of the heterozygous embryos died before embryonic day 14 (E14), each had defects in hematopoiesis (Wang et al., 2000). The expression level of ADAR1 was reported to increase in the liver from day E13 to E14. It is feasible that ADAR1 targets in the liver need to be edited at this point for further embryonic development. Because early development of the adaptive immune system occurs in the fetal liver, one may speculate that ADAR substrates are involved in this developmental process. 7.2. ADARs and Viruses Although there are no known targets of the ADARs in the immune system, ADARs play a significant role in the cellular response to viral pathogens. ADAR activity on transcripts of measles virus, polyoma virus, and hepatitis delta virus (HDV) has been well characterized. Soon after the discovery of ADARs, a viral RNA isolated from the brain of a patient who died of a persistent measles virus infection corresponded to changes resembling an ADAR substrate (Cattaneo et al., 1988). The functions of these mutations are still unclear, but there is speculation that these mutations somehow lead to viral persistence. ADAR1 edits HDV in vitro, an event reportedly required for the virus life cycle. HDV encodes two forms of a single protein (HD‐Ag). RNA editing converts a stop codon to a tryptophan resulting in the long form (HD‐Ag‐L) as opposed to the short form (HD‐Ag‐ S) of the viral protein (Casey and Gerin, 1995; Luo et al., 1990; Polson et al., 1996). Both proteins are essential for the viral cycle: the short form is necessary for replication (Kuo et al., 1989) and the long form for assembly (Chang et al., 1991). In the case of polyoma virus, conversion of adenosines to inosines serves a novel purpose. Transcripts with a large number of inosines are retained in the nucleus by a complex containing an inosine‐ specific RNA‐binding protein (Zhang and Carmichael, 2001). These transcripts are not translated, leading to downregulation of the protein products. The early transcripts of the polyoma virus are regulated via ADARs in this fashion allowing the late transcripts to be selectively exported (Kumar and Carmichael, 1997). Several other observations provide indirect evidence that A‐I editing plays a role in inhibiting viral replication and helps modulate cellular responses to virus infection (Jayan, 2004; Jayan and Casey, 2002; Macnaughton et al., 2003). Genes induced by interferons are known to function in viral defense. Although there is no direct evidence to support this idea, unlike other ADARs,

284

d i pa n j a n c h o w d h ur y an d ca r l d. no v i na

the cytoplasmic localization of the long form of ADAR1, and its production from an interferon‐inducible promoter (George and Samuel, 1999a) gives some credence to the theory that the long form of ADAR1 functions in viral defense. The other type of RNA editing which is less frequent in the mammalian system is C to U. The best‐characterized example of this form of RNA editing involves the nuclear transcript encoding intestinal apolipoprotein B (apo B) (Lau et al., 1991). The enzyme responsible for this editing activity is APOBEC‐1 (apo‐B editing catalytic subunit 1). Recently, two members of the APOBEC family, APOBEC3G and AID (activation induced cytidine‐ deaminase) have been the focus of intensive investigation by immunologists (Wedekind et al., 2003). APOBEC3G induces hypermutations on newly synthesized viral DNA, acting as an antiviral agent. A virus like HIV‐1 has developed a mechanism to counter APOBEC3G activity. The viral protein called viral infectivity factor (Vif) binds to APOBEC3G triggering its polyubiquitination and rapid degradation preventing its entry into progeny virions. Without Vif, the encapsidated APOBEC3G would damage the viral reverse transcripts causing their degradation (Bhagwat, 2004; Schrofelbauer et al., 2004). AID is necessary for both class switching and somatic hypermutation in B cells. These proteins have RNA‐ and DNA‐editing activity in vitro though their principle substrate may be DNA in vivo. There are two distinct schools of thought regarding how AID functions. The more popular idea is that AID edits DNA, causing C‐to‐U conversions which leads to AP‐endonuclease‐mediated breaks necessary for class switching (Chaudhuri and Alt, 2004; Conticello et al., 2004). The alternative view suggested, is that AID edits an unidentified mRNA and the edited form of mRNA codes for a DNA endonuclease (Honjo et al., 2004). However, there has been no reported link between C‐to‐U RNA editing and RNAi. 8. Conclusions Recent observations in the studies of RNAi and short‐RNA function provide evidence that RNAi and related pathways could be critical for lymphocyte‐ specific gene expression and development. microRNAs are expressed from endogenous genes that are conserved (reviewed in Bartel, 2004). The common theme for miRNA function across phyla is their participation in developmental transitions and cell‐specific gene expression. When small RNAs are derived from transgenes or mobile genetic elements, the common theme that emerges is their participation in genome defense. In plants, short RNAs act as an antivirus ‘‘immune system’’ (reviewed in Lecellier and Voinnet, 2004). Recent evidence indicates that the RNA arms race may have reached

r na i i n t h e i m m u ne sy s t e m

285

mammals. Mammalian B cells may possess similar short RNA‐directed antiviral defense mechanisms observed in plants (Pfeffer et al., 2004). Conversely, influenza and vaccinia viruses express factors that inhibit mammalian RNAi (Li et al., 2004). RNAi has not yet been implicated in antigen‐receptor expression in lymphocytes, directly. However, there are several posttranscriptional quality‐ control mechanisms in place that may perform a surveillance function in antigen‐receptor expression in addition to RNA editing and NMD. Curiously, extensive antisense transcripts have been identified in immunoglobulin heavy chain variable (IgHv) region genes. It may be possible that long dsRNA complementary to promoter regions in IgHv is processed by Dicer and leads to short RNAs that affect accessibility at the IgHv promoters. Indeed, antisense transcription has been implicated in methylation, TGS, and human disease (Tufarelli et al., 2003). Observations such as these could provide the link between RNAi and control of antigen receptor expression. An intracellular immune system helps an organism defend against genetic challenges. Ruthless evolutionary pressure selects for genetic outliers, that is, rare organisms that possess advantageous genetic changes. However, the majority of challenges resulting in genetic changes are disadvantageous and lead to the death of the organism. In one model, an RNA‐directed silencing pathway that potentially helps maintain genomic integrity may constitute an intracellular ‘‘immune system’’ with parallel functions to the cellular adaptive immune system (Chowdhury and Novina, 2005). In another model, an RNA‐ directed silencing pathway has been compared to the activation kinetics of the adaptive immune response (McManus, 2004). Because it plays a central role in preservation of the integrity of an organism, the intended (immune) functions of RNAi may have been conserved through evolution, even if specific RNAi gene functions have been adapted to other processes. Unique relationships exist between dsRNAs and the genes mediating short‐RNA function in lower eukaryotes. It is tempting to speculate that the homologs of genes implicated in RNAi in lower eukaryotes may be conserved in mammals. A better understanding of their roles in mammals may illuminate the biological roles of RNAi in lymphocytes. Acknowledgments We wish to thank Alla Grishok, Ananda Roy, Matthew Call, Tara Love, Rachel Gerstein, Klaus Rajewsky, and Chris Burge for thoughtful comments and discussion. We wish to thank Benjamin Lewis for his assistance verifying predicted miRNA targets in the immune system presented in Table 1.

286

d i pa n j a n c h o w d h ur y an d ca r l d. no v i na

References Aoufouchi, S., Yelamos, J., and Milstein, C. (1996). Nonsense mutations inhibit RNA splicing in a cell‐free system: Recognition of mutant codon is independent of protein synthesis. Cell 85, 415–422. Bartel, D. P. (2004). MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell 116, 281–297. Bass, B. L. (2002). RNA editing by adenosine deaminases that act on RNA. Annu. Rev. Biochem. 71, 817–846. Bass, B. L., and Weintraub, H. (1987). A developmentally regulated activity that unwinds RNA duplexes. Cell 48, 607–613. Bass, B. L., and Weintraub, H. (1988). An unwinding activity that covalently modifies its double‐ stranded RNA substrate. Cell 55, 1089–1098. Baumann, B., Potash, M. J., and Kohler, G. (1985). Consequences of frameshift mutations at the immunoglobulin heavy chain locus of the mouse. EMBO J 4, 351–359. Bhagwat, A. S. (2004). DNA‐cytosine deaminases: From antibody maturation to antiviral defense. DNA Repair (Amst) 3, 85–89. Brennecke, J., Hipfner, D. R., Stark, A., Russell, R. B., and Cohen, S. M. (2003). Bantam encodes a developmentally regulated microRNA that controls cell proliferation and regulates the proapoptotic gene hid in Drosophila. Cell 113, 25–36. Buhler, M., Paillusson, A., and Muhlemann, O. (2004). Efficient downregulation of immunoglobulin mu mRNA with premature translation‐termination codons requires the 50 ‐half of the VDJ exon. Nucleic Acids Res. 32, 3304–3315. Buhler, M., Wilkinson, M. F., and Muhlemann, O. (2002). Intranuclear degradation of nonsense codon‐containing mRNA. EMBO Rep. 3, 646–651. Calin, G. A., Dumitru, C. D., Shimizu, M., Bichi, R., Zupo, S., Noch, E., Aldler, H., Rattan, S., Keating, M., Rai, K., Rassenti, L., Kipps, T., Negrini, M., Bullrich, F., and Croce, C. M. (2002). Frequent deletions and down‐regulation of micro‐ RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc. Natl. Acad. Sci. USA 99, 15524–15529. Calin, G. A., Liu, C. G., Sevignani, C., Ferracin, M., Felli, N., Dumitru, C. D., Shimizu, M., Cimmino, A., Zupo, S., Dono, M., Dell’Aquila, M. L., Alder, H, Rassenti, L., Kipps, T. J., Bullrich, F., Negrini, M., and Croce, C. M. (2004a). MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias. Proc. Natl. Acad. Sci. USA 101, 11755–11760. Calin, G. A., Sevignani, C., Dumitru, C. D., Hyslop, T., Noch, E., Yendamuri, S., Shimizu, M., Rattan, S., Bullrich, F., Negrini, M., and Croce, C. M. (2004b). Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc. Natl. Acad. Sci. USA 101, 2999–3004. Carmell, M. A., Xuan, Z., Zhang, M. Q., and Hannon, G. J. (2002). The Argonaute family: Tentacles that reach into RNAi, developmental control, stem cell maintenance, and tumorigenesis. Genes Dev. 16, 2733–2742. Carter, M. S., Doskow, J., Morris, P., Li, S., Nhim, R. P., Sandstedt, S., and Wilkinson, M. F. (1995). A regulatory mechanism that detects premature nonsense codons in T‐cell receptor transcripts in vivo is reversed by protein synthesis inhibitors in vitro. J. Biol. Chem. 270, 28995–29003. Carter, M. S., Li, S., and Wilkinson, M. F. (1996). A splicing‐dependent regulatory mechanism that detects translation signals. EMBO J. 15, 5965–5975. Casey, J. L., and Gerin, J. L. (1995). Hepatitis D virus RNA editing: Specific modification of adenosine in the antigenomic RNA. J. Virol. 69, 7593–7600.

r na i i n t h e i m m u ne sy s t e m

287

Cattaneo, R., Schmid, A., Eschle, D., Baczko, K., ter Meulen, V., and Billeter, M. A. (1988). Biased hypermutation and other genetic changes in defective measles viruses in human brain infections. Cell 55, 255–265. Chang, F. L., Chen, P. J., Tu, S. J., Wang, C. J., and Chen, D. S. (1991). The large form of hepatitis delta antigen is crucial for assembly of hepatitis delta virus. Proc. Natl. Acad. Sci. USA 88, 8490–8494. Chaudhuri, J., and Alt, F. W. (2004). Class‐switch recombination: Interplay of transcription, DNA deamination and DNA repair. Nat. Rev. Immunol. 4, 541–552. Chen, C. Z., Li, L., Lodish, H. F., and Bartel, D. P. (2004). MicroRNAs modulate hematopoietic lineage differentiation. Science 303, 83–86. Cheng, J., and Maquat, L. E. (1993). Nonsense codons can reduce the abundance of nuclear mRNA without affecting the abundance of pre‐mRNA or the half‐life of cytoplasmic mRNA. Mol. Cell. Biol. 13, 1892–1902. Chowdhury, D., and Novina, C. D. (2005). Potential roles for short RNAs in lymphocytes. Immunol. Cell Biol. 83, 201–210. Connor, A., Wiersma, E., and Shulman, M. J. (1994). On the linkage between RNA processing and RNA translatability. J. Biol. Chem. 269, 25178–25184. Conticello, S. G., Thomas, C. J., Petersen‐Mahrt, S., and Neuberger, M. S. (2004). Evolution of the AID/APOBEC family of polynucleotide (deoxy)cytidine deaminases. Mol. Biol. Evol. 22, 367–377. Denning, G., Jamieson, L., Maquat, L. E., Thompson, E. A., and Fields, A. P. (2001). Cloning of a novel phosphatidylinositol kinase‐related kinase: Characterization of the human SMG‐1 RNA surveillance protein. J. Biol. Chem. 276, 22709–22714. Doench, J. G., and Sharp, P. A. (2004). Specificity of microRNA target selection in translational repression. Genes Dev. 18, 504–511. Domeier, M. E., Morse, D. P., Knight, S. W., Portereiko, M., Bass, B. L., and Mango, S. E. (2000). A link between RNA interference and nonsense‐mediated decay in Caenorhabditis elegans. Science 289, 1928–1931. Esau, C., Kang, X., Peralta, E., Hanson, E., Marcusson, E. G., Ravichandran, L. V., Sun, Y., Koo, S., Perera, R. J., Jain, R., Dean, N. M., Freier, S. M., Bennett, C. F., Lollo, B., and Griffey, R. (2004). MicroRNA‐143 Regulates Adipocyte Differentiation. J. Biol. Chem. 279, 52361–52365. Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., and Mello, C. C. (1998). Potent and specific genetic interference by double‐stranded RNA in Caenorhabditis elegans. Nature 391, 806–811. Fukugawa, T., Nogami, M., Yoshikawa, M., Ikeno, M., Okazaki, T., Takami, Y., Nakayama, T., and Oshimura, M. (2004). Dicer is essential for formation of the heterochromatin structure in vertebrate cells. Nat. Cell Biol. 6, 784–791. Gass, J. N., Gunn, K. E., Sriburi, R., and Brewer, J. W. (2004). Stressed‐out B cells? Plasma‐cell differentiation and the unfolded protein response. Trends Immunol. 25, 17–24. Gauwerky, C. E., Huebner, K., Isobe, M., Nowell, P. C., and Croce, C. M. (1989). Activation of MYC in a masked t(8;17) translocation results in an aggressive B‐cell leukemia. Proc. Natl. Acad. Sci. USA 86, 8867–8871. Gehring, N. H., Neu‐Yilik, G., Schell, T., Hentze, M. W., and Kulozik, A. E. (2003). Y14 and hUpf3b form an NMD‐activating complex. Mol. Cell 11, 939–949. Gellert, M. (2002). V(D)J recombination: RAG proteins, repair factors, and regulation. Annu. Rev. Biochem. 71, 101–132. George, C. X., and Samuel, C. E. (1999a). Characterization of the 50 ‐flanking region of the human RNA‐specific adenosine deaminase ADAR1 gene and identification of an interferon‐inducible ADAR1 promoter. Gene 229, 203–213.

288

d i pa n j a n c h o w d h ur y an d ca r l d. no v i na

George, C. X., and Samuel, C. E. (1999b). Human RNA‐specific adenosine deaminase ADAR1 transcripts possess alternative exon 1 structures that initiate from different promoters, one constitutively active and the other interferon inducible. Proc. Natl. Acad. Sci. USA 96, 4621–4626. Griffiths‐Jones, S. (2004). The microRNA Registry. Nucl. Acids Res. 32, D109–D111. Gilfillan, S., Benoist, C., and Mathis, D. (1995). Mice lacking terminal deoxynucleotidyl transferase: Adult mice with a fetal antigen receptor repertoire. Immunol. Rev. 148, 201–219. Glickman, J. N., Howe, J. G., and Steitz, J. A. (1988). Structural analyses of EBER1 and EBER2 ribonucleoprotein particles present in Epstein‐Barr virus‐infected cells. J. Virol. 62, 902–911. Grishok, A., Pasquinelli, A. E., Conte, D., Li, N., Parrish, S., Ha, I., Baillie, D. L., Fire, A., Ruvkun, G., and Mello, C. C. (2001). Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106, 23–34. Gudikote, J. P., and Wilkinson, M. F. (2002). T‐cell receptor sequences that elicit strong down‐ regulation of premature termination codon‐bearing transcripts. EMBO J. 21, 125–134. Hamilton, A. J., and Baulcombe, D. C. (1999). A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286, 950–952. Hammond, S. M., Bernstein, E., Beach, D., and Hannon, G. J. (2000). An RNA‐directed nuclease mediates post‐transcriptional gene silencing in Drosophila cells. Nature 404, 293–296. Hanrahan, C. J., Palladino, M. J., Ganetzky, B., and Reenan, R. A. (2000). RNA editing of the Drosophila para Na(þ) channel transcript. Evolutionary conservation and developmental regulation. Genetics 155, 1149–1160. He, L., and Hannon, G. J. (2004). MicroRNAs: Small RNAs with a big role in gene regulation. Nat. Rev. Genet 5, 522–531. Herskowitz, I. (1987). Functional inactivation of genes by dominant negative mutations. Nature 329, 219–222. Honjo, T., Muramatsu, M., and Fagarasan, S. (2004). AID: How does it aid antibody diversity? Immunity 20, 659–668. Hutvagner, G., McLachlan, J., Pasquinelli, A. E., Balint, E., Tuschl, T., and Zamore, P. D. (2001). A cellular function for the RNA‐interference enzyme Dicer in the maturation of the let‐7 small temporal RNA. Science 293, 834–838. Hutvagner, G., and Zamore, P. D. (2002). A microRNA in a multiple‐turnover RNAi enzyme complex. Science 297, 2056–2060. Iborra, F. J., Jackson, D. A., and Cook, P. R. (2001). Coupled transcription and translation within nuclei of mammalian cells. Science 293, 1139–1142. Ishigaki, Y., Li, X., Serin, G., and Maquat, L. E. (2001). Evidence for a pioneer round of mRNA translation: mRNAs subject to nonsense‐mediated decay in mammalian cells are bound by CBP80 and CBP20. Cell 106, 607–617. Jack, H. M., Berg, J., and Wabl, M. (1989). Translation affects immunoglobulin mRNA stability. Eur. J. Immunol. 19, 843–847. Jayan, G. C. (2004). RNA editing in hepatitis delta virus: Unsolved puzzles. Scient. World J. 4, 628–637. Jayan, G. C., and Casey, J. L. (2002). Increased RNA editing and inhibition of hepatitis delta virus replication by high‐level expression of ADAR1 and ADAR2. J. Virol. 76, 3819–3827. Kanellopoulou, C., Muljo, S. A., Kung, A. L., Ganesan, S., Drapkin, R., Jenuwein, T., Livingston, D. M., and Rajewsky, K. (2005). Dicer‐deficient mouse embryonic stem cells are defective in differentiation and centrometric silencing. Genes Dev. 19, 489–501. Kapp, L. D., and Lorsch, J. R. (2004). The molecular mechanics of eukaryotic translation. Annu. Rev. Biochem. 73, 657–704. Kawasaki, H., and Taira, K. (2004). Induction of DNA methylation and gene silencing by short interfering RNAs in human cells. Nature 431, 211–217.

r na i i n t h e i m m u ne sy s t e m

289

Ketting, R. F., Haverkamp, T. H., van Luenen, H. G., and Plasterk, R. H. (1999). Mut‐7 of C. elegans, required for transposon silencing and RNA interference, is a homolog of Werner syndrome leicase and RNaseD. Cell 99, 133–141. Kim, V. N., Kataoka, N, and Dreyfuss, G (2001). Role of the nonsense‐mediated decay factor hUpf3 in the splicing‐dependent exon‐exon junction complex. Science 293, 1832–1836. Knight, S. W., and Bass, B. L. (2002). The role of RNA editing by ADARs in RNAi. Mol. Cell 10, 809–817. Kumar, M., and Carmichael, G. G. (1997). Nuclear antisense RNA induces extensive adenosine modifications and nuclear retention of target transcripts. Proc. Natl. Acad. Sci. USA 94, 3542–3547. Kuo, M. Y., Chao, M., and Taylor, J. (1989). Initiation of replication of the human hepatitis delta virus genome from cloned DNA: Role of delta antigen. J. Virol. 63, 1945–1950. Lau, P. P., Xiong, W. J., Zhu, H. J., Chen, S. H., and Chan, L. (1991). Apolipoprotein B mRNA editing is an intranuclear event that occurs posttranscriptionally coincident with splicing and polyadenylation. J. Biol. Chem. 266, 20550–20554. Lecellier, C. H., and Voinnet, O. (2004). RNA silencing: No mercy for viruses? Immunol. Rev. 198, 285–303. Lee, R. C., Feinbaum, R. L., and Ambros, V. (1993). The C. elegans heterochronic gene lin‐4 encodes small RNAs with antisense complementarity to lin‐14. Cell 75, 843–854. Lee, Y., Ahn, C., Han, J., Choi, H., Kim, J., Yim, J., Lee, J., Provost, P., Radmark, O., Kim, S., and Kim, V. N. (2003). The nuclear RNase III Drosha initiates microRNA processing. Nature 425, 415–419. Lejeune, F., Li, X., and Maquat, L. E. (2003). Nonsense‐mediated mRNA decay in mammalian cells involves decapping, deadenylating, and exonucleolytic activities. Mol. Cell 12, 675–687. Lejeune, F., Ishigaki, Y., Li, X., and Maquat, L. E. (2002). The exon junction complex is detected on CBP80‐bound but not eIF4E‐bound mRNA in mammalian cells: Dynamics of mRNP remodeling. EMBO J. 21, 3536–3545. Lewis, B. P., Burge, C. B., and Bartel, D. P. (2005). Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120, 15–20. Lewis, B. P., Shih, I. H., Jones‐Rhoades, M. W., Bartel, D. P., and Burge, C. B. (2003). Prediction of mammalian microRNA targets. Cell 115, 787–798. Li, S., Leonard, D., and Wilkinson, M. F. (1997). T cell receptor (TCR) mini‐gene mRNA expression regulated by nonsense codons: A nuclear‐associated translation‐like mechanism. J. Exp. Med. 185, 985–992. Li, W. X., Li, H., Lu, R., Li, F., Dus, M., Atkinson, P., Brydon, E. W., Johnson, K. L., Garcia‐Sastre, A., Ball, L. A., Palese, P., and Ding, S. W. (2004). Interferon antagonist proteins of influenza and vaccinia viruses are suppressors of RNA silencing. Proc. Natl. Acad. Sci. USA 101, 1350–1355. Lindroth, A. M., Cao, X., Jackson, J. P., Zilberman, D., McCallum, C. M., Henikoff, S., and Jacobsen, S. E. (2001). Requirement of CHROMOMETHYLASE3 for maintenance of CpXpG methylation. Science 292, 2077–2080. Lozano, F., Maertzdorf, B., Pannell, R., and Milstein, C. (1994). Low cytoplasmic mRNA levels of immunoglobulin kappa light chain genes containing nonsense codons correlate with inefficient splicing. EMBO J. 13, 4617–4622. Luo, G. X., Chao, M., Hsieh, S. Y., Sureau, C., Nishikura, K., and Taylor, J. (1990). A specific base transition occurs on replicating hepatitis delta virus RNA. J. Virol. 64, 1021–1027. Lykke‐Andersen, J., Shu, M. D., and Steitz, J. A. (2000). Human Upf proteins target an mRNA for nonsense‐mediated decay when bound downstream of a termination codon. Cell 103, 1121–1131.

290

d i pa n j a n c h o w d h ur y an d ca r l d. no v i na

Ma, Y., Pannicke, U., Schwarz, K., and Lieber, M. R. (2002). Hairpin opening and overhang processing by an Artemis/DNA‐dependent protein kinase complex in nonhomologous end joining and V(D)J recombination. Cell 108, 781–794. Macnaughton, T. B., Li, Y. I., Doughty, A. L., and Lai, M. M. (2003). Hepatitis delta virus RNA encoding the large delta antigen cannot sustain replication due to rapid accumulation of mutations associated with RNA editing. J. Virol. 77, 12048–12056. Maquat, L. E. (2004). Nonsense‐mediated mRNA decay: Splicing, translation and mRNP dynamics. Nat. Rev. Mol. Cell Biol. 5, 89–99. Martinez, J., Patkaniowska, A., Urlaub, H., Luhrmann, R., and Tuschl, T. (2002). Single‐stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell 110, 563–574. Matzke, M., Aufsatz, W., Kanno, T., Daxinger, L., Papp, I., Mette, M. F., and Matzke, A. J. (2004). Genetic analysis of RNA‐mediated transcriptional gene silencing. Biochem. Biophys. Acta 1677, 129–141. Matzke, M. A., and Matzke, A. J. (2004). Planting the seeds of a new paradigm. PLoS Biol. 2, E133. McManus, M. T. (2004). Small RNAs and immunity. Immunity 21(6), 747–756. Mendell, J. T., Ap Rhys, C. M., and Dietz, H. C. (2002). Separable roles for rent1/hUpf1 in altered splicing and decay of nonsense transcripts. Science 298, 419–422. Mochizuki, K., Fine, N. A., Fujisawa, T., and Gorovsky, M. A. (2002). Analysis of a piwi‐related gene implicates small RNAs in genome rearrangement in tetrahymena. Cell 110, 689–699. Morris, K. V., Chan, S. W., Jacobsen, S. E., and Looney, D. J. (2004). Small interfering RNA‐ induced transcriptional gene silencing in human cells. Science 305, 1289–1292. Murray, P. G., Swinnen, L. J., Constandinou, C. M., Pyle, J. M., Carr, T. J., Hardwick, J. M., and Ambinder, R. F. (1996). BCL‐2 but not its Epstein‐Barr virus‐encoded homologue, BHRF1, is commonly expressed in posttransplantation lymphoproliferative disorders. Blood 87, 706–711. Naeger, L. K., Schoborg, R. V., Zhao, Q., Tullis, G. E., and Pintel, D. J. (1992). Nonsense mutations inhibit splicing of MVM RNA in cis when they interrupt the reading frame of either exon of the final spliced product. Genes Dev. 6, 1107–1119. Nagy, E., and Maquat, L. E. (1998). A rule for termination‐codon position within intron‐containing genes: When nonsense affects RNA abundance. Trends Biochem. Sci. 23, 198–199. Niswender, C. M., Herrick‐Davis, K., Dilley, G. E., Meltzer, H. Y., Overholser, J. C., Stockmeier, C. A., Emeson, R. B., and Sanders‐Bush, E. (2001). RNA editing of the human serotonin 5‐HT2C receptor. Alterations in suicide and implications for serotonergic pharmacotherapy. Neuropsychopharmacology 24, 478–491. Novina, C. D., and Sharp, P. A. (2004). The RNAi revolution. Nature 430, 161–164. Oudejans, J. J., van den Brule, A. J., Jiwa, N. M., de Bruin, P. C., Ossenkoppele, G. J., van der Valk, P., Walboomers, J. M., and Meijer, C. J. (1995). BHRF1, the Epstein‐Barr virus (EBV) homologue of the BCL‐2 protooncogene, is transcribed in EBV‐associated B‐cell lymphomas and in reactive lymphocytes. Blood 86, 1893–1902. Pahl, H. L., and Baeuerle, P. A. (1997). The ER‐overload response: Activation of NF‐kappa B. Trends Biochem. Sci. 22, 63–67. Pal‐Bhadra, M., Leibovitch, B. A., Gandhi, S. G., Rao, M., Bhadra, U., Birchler, J. A., and Elgin, S. C. (2004). Heterochromatic silencing and HP1 localization in Drosophila are dependent on the RNAi machinery. Science 303, 669–672. Palladino, M. J., Keegan, L. P., O’Connell, M. A., and Reenan, R. A. (2000). A‐to‐I pre‐mRNA editing in Drosophila is primarily involved in adult nervous system function and integrity. Cell 102, 437–449. Pannicke, U., Ma, Y., Hopfner, K. P., Niewolik, D., Lieber, M. R., and Schwarz, K. (2004). Functional and biochemical dissection of the structure‐specific nuclease ARTEMIS. EMBO J. 23, 1987–1997.

r na i i n t h e i m m u ne sy s t e m

291

Pfeffer, S., Zavolan, M., Grasser, F. A., Chien, M., Russo, J. J., Ju, J., John, B., Enright, A. J., Marks, D., Sander, C., and Tuschl, T. (2004). Identification of virus‐encoded microRNAs. Science 304, 734–736. Polson, A. G., Bass, B. L., and Casey, J. L. (1996). RNA editing of hepatitis delta virus antigenome by dsRNA‐adenosine deaminase. Nature 380, 454–456. Poy, M. N., Eliasson, L., Krutzfeldt, J., Kuwajima, S., Ma, X., Macdonald, P. E., Pfeffer, S., Tuschl, T., Rajewsky, N., Rorsman, P., and Stoffel, M. (2004). A pancreatic islet‐specific microRNA regulates insulin secretion. Nature 432, 226–230. Reenan, R. A., Hanrahan, C. J., and Barry, G. (2000). The mle(napts) RNA helicase mutation in Drosophila results in a splicing catastrophe of the para Naþ channel transcript in a region of RNA editing. Neuron 25, 139–149. Reichert, V. L., Le Hir, H., Jurica, M. S., and Moore, M. J. (2002). 50 exon interactions within the human spliceosome establish a framework for exon junction complex structure and assembly. Genes Dev. 16, 2778–2791. Reinhart, B. J., Slack, F. J., Basson, M., Pasquinelli, A. E., Bettinger, J. C., Rougvie, A. E., Horvitz, H. R., and Ruvkun, G. (2000). The 21‐nucleotide let‐7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403, 901–906. Sanders‐Bush, E., Fentress, H., and Hazelwood, L. (2003). Serotonin 5‐ht2 receptors: Molecular and genomic diversity. Mol. Interv. 3, 319–330. Scadden, A. D., and Smith, C. W. (2001). RNAi is antagonized by A––>I hyper‐editing. EMBO Rep. 2, 1107–1111. Schramke, V., and Allshire, R. (2003). Hairpin RNAs and retrotransposon LTRs effect RNAi and chromatin‐based gene silencing. Science 301, 1069–1074. Schrofelbauer, B., Yu, Q., and Landau, N. R. (2004). New insights into the role of Vif in HIV‐1 replication. AIDS Rev. 6, 34–39. Schwarz, D. S., and Zamore, P. D. (2002). Why do miRNAs live in the miRNP? Genes Dev. 16, 1025–1031. Seeburg, P. H. (2002). A‐to‐I editing: New and old sites, functions and speculations. Neuron 35, 17–20. Seeburg, P. H., Higuchi, M., and Sprengel, R. (1998). RNA editing of brain glutamate receptor channels: Mechanism and physiology. Brain Res. Rev. 26, 217–229. Serin, G., Gersappe, A., Black, J. D., Aronoff, R., and Maquat, L. E. (2001). Identification and characterization of human orthologues to Saccharomyces cerevisiae Upf2 protein and Upf3 protein (Caenorhabditis elegans SMG‐4). Mol. Cell. Biol. 21, 209–223. Simpson, S. B., and Stoltzfus, C. M. (1994). Frameshift mutations in the v‐src gene of avian sarcoma virus act in cis to specifically reduce v‐src mRNA levels. Mol. Cell. Biol. 14, 1835–1844. Strudwick, S., and Borden, K. L. (2002). The emerging roles of translation factor eIF4E in the nucleus. Differentiation 70, 10–22. Sun, X., Perlick, H. A., Dietz, H. C., and Maquat, L. E. (1998). A mutated human homologue to yeast Upf1 protein has a dominant‐negative effect on the decay of nonsense‐containing mRNAs in mammalian cells. Proc. Natl. Acad. Sci. USA 95, 10009–10014. Tabara, H., Sarkissian, M., Kelly, W. G., Fleenor, J., Grishok, A., Timmons, L., Fire, A., and Mello, C. C. (1999). The rde‐1 gene, RNA interference, and transposon silencing in C. elegans. Cell 99, 123–132. Tomari, Y., and Zamore, P. D. (2005). Perspective: Machines for RNAi. Genes Dev. 19, 517–529. Tonkin, L. A., and Bass, B. L. (2003). Mutations in RNAi rescue aberrant chemotaxis of ADAR mutants. Science 302, 1725.

292

d i pa n j a n c h o w d h ur y an d ca r l d. no v i na

Tonkin, L. A., Saccomanno, L., Morse, D. P., Brodigan, T., Krause., M., and Bass, B. L. (2002). RNA editing by ADARs is important for normal behavior in Caenorhabditis elegans. EMBO. J. 21, 6025–6035. Tufarelli, C., Stanley, J. A., Garrick, D., Sharpe, J. A., Ayyub, H., Wood, W. G., and Higgs, D. R. (2003). Transcription of antisense RNA leading to gene silencing and methylation as a novel cause of human genetic disease. Nat. Genet. 34, 157–165. Verdel, A., Jia, S., Gerber, S., Sugiyama, T., Gygi, S., Grewal, S. I., and Moazed, D. (2004). RNAi‐ mediated targeting of heterochromatin by the RITS complex. Science 303, 672–676. Wang, J., Gudikote, J. P., Olivas, O. R., and Wilkinson, M. F. (2002). Boundary‐independent polar nonsense‐mediated decay. EMBO Rep. 3, 274–279. Wang, Q., Khillan, J., Gadue, P., and Nishikura, K. (2000). Requirement of the RNA editing deaminase ADAR1 gene for embryonic erythropoiesis. Science 290, 1765–1768. Wassenegger, M., Heimes, S., Riedel, L., and Sanger, H. L. (1994). RNA‐directed de novo methylation of genomic sequences in plants. Cell 76, 567–576. Wedekind, J. E., Dance, G. S., Sowden, M. P., and Smith, H. C. (2003). Messenger RNA editing in mammals: New members of the APOBEC family seeking roles in the family business. Trends Genet. 19, 207–216. Wilkinson, M. F., and Shyu, A. B. (2002). RNA surveillance by nuclear scanning? Nat. Cell Biol. 4, E144–147. Wu‐Scharf, D., Jeong, B., Zhang, C., and Cerutti, H. (2000). Transgene and transposon silencing in Chlamydomonas reinhardtii by a DEAH‐box RNA helicase. Science 290, 1159–1162. Xie, X., Lu, J., Kulbokas, E. J., Golub, T. R., Mootha, V., Lindblad‐Toh, K., Lander, E. S., and Kellis, M. (2005). Systematic discovery of regulatory motifs in human promoters and 30 UTRs by comparison of several mammals. Nature 434, 338–345. Yamashita, A., Ohnishi, T., Kashima, I., Taya, Y., and Ohno, S. (2001). Human SMG‐1, a novel phosphatidylinositol 3‐kinase‐related protein kinase, associates with components of the mRNA surveillance complex and is involved in the regulation of nonsense‐mediated mRNA decay. Genes Dev. 15, 2215–2228. Zhang, Z., and Carmichael, G. G. (2001). The fate of dsRNA in the nucleus: A p54(nrb)‐containing complex mediates the nuclear retention of promiscuously A‐to‐I edited RNAs. Cell 106, 465–475.

Further Readings Almeida, R., and Allshire, R. C. (2005). RNA silencing and genome regulation. Trends Cell Biol. 15, 251–258. Blanc., V., and Davidson, N. O. (2003). C‐to‐U RNA editing: Mechanisms leading to genetic diversity. J. Biol. Chem. 278, 1395–1398. Buhler, M., Mohn, F., Stalder, L., and Muhlemann, O. (2005). Transcriptional silencing of nonsense codon‐containing immunoglobulin minigenes. Mol. Cell 18, 307–317. Fernandez, H. R., Kavi, H. H., Xie, W., and Birchler, J. A. (2005). Heterochromatin: on the ADAR radar? Curr. Biol. 15, R132–R134. Matzke, M. A., and Birchler, J. A. (2005). RNAi‐mediated pathways in the nucleus. Nat. Rev. Genet. 6, 24–35.

Index

A Activation, in PLC-g2 network regulation, 74–75 Activation-induced cell death (AICD), in tetramer analysis/autoreactive T cells, 64, 65–66 Adenosine deaminases that act on RNA (ADAR) in RNA-based regulation, 280–283 viruses v., 283–284 Adenylate cyclase activators, in asthma/mast cells/basophils, 131–132 Adhesion, in CD22 ligand binding, 5–7, 14–15 Ag. See Antigens AICD. See Activation-induced cell death Airway smooth muscle (ASM), in asthma/mast cells/basophils, 99, 115, 118–119, 128 Anatomical/functional evidence, in asthma/mast cells/basophils, 119–120 Anchor pockets, in MHC class II molecule I-Ag7, 239–241, 244, 256 Angiogenesis, asthma/mast cells/basophils and, 127–128 Antigen cross-linking, in asthma/mast cells/basophils, 99–100 Antigen presenting cells (APC) in dendritic cell biology, 215, 216–217 in MHC class II molecule I-Ag7, 236, 237, 243, 252, 253, 254 Antigens (Ag), in CD22 ligand binding, 2, 8, 9, 13, 14 Antimicrobial peptides, in asthma/mast cells/basophils, 121, 125 APC. See Antigen presenting cells Apoptosis asthma treatment and, 135–136 CD22 ligand binding and, 2, 7, 11, 30, 36 ASM. See Airway smooth muscle

Asthma, role of mast cells/basophils adenylate cyclase activators in, 131–132 anatomical/functional evidence in, 119–120 angiogenesis in, 127–128 antigen cross-linking in, 99–100 antimicrobial peptides in, 121, 125 ASM in, 99, 115, 118–119, 128 atopic disorders and, 98, 126–127, 133–134 autocrine/paracrine effects in, 115–116, 137–138 basophils mediators and, 111–112 morphology-ultrastructure and, 109–110 origins and, 109 role in, 117–118 surface markers and, 112–115 cAMP in, 112, 127, 131–132 chemoattractants in, 114–115 chemokines in, 103, 104–105, 107, 112, 114, 130–131 chemotactic receptors in, 99, 105 c-kit in, 99, 101, 136 collagen synthesis in, 129–130 COX in, 104, 111–112 CysLT in, 105, 107–108, 120, 135 cytokines in, 98, 103, 104, 105, 109, 112, 115–116, 121, 122, 125, 126, 129, 130 DP receptors in, 108–109 environmental factors in, 98–99 eotaxins in, 118–119 FceRI activation in, 121–123, 127, 130–131, 136–137 cell recruitment in, 118–119 expression in, 97, 105–106, 113, 115

293

294 Asthma, role of mast cells/basophils (continued) genetic factors in, 98–99, 136–137 GM-CSF in, 113, 114, 121 histamine in, 97, 107–109, 111–112, 115, 126 histamine synthesis in, 97, 126 HUVECs in, 118–119 IGFs in, 119 ILs in, 107–109, 112, 113–116, 117, 118, 124, 126, 129, 134 5-LO in, 105, 135 LTC4 in, 105, 111, 132, 134 mast cells activation in, 120 eicosanoid metabolism and, 102–103 eicosanoid receptors and, 107–109 mediators and, 103–105 morphology-ultrastructure and, 102–103 origins and, 101–102 role in, 116–117 surface markers in, 105–107 mediator release in, 99–100, 110 NF-kB in, 133 NGF in, 113, 116 pathogenesis of, 97–98, 99, 105, 136–138 PGD2 in, 112, 116, 132, 134, 135 phosphodiesterase inhibitors in, 131–132 SCF in, 101–102, 116 superallergens in, 121–123, 125 HIV-1 gp120 in, 124–125 protein A in, 126 protein Fv in, 123–124 protein L in, 126–127 tissue injury in, 125 superantigens in, 122–123 TGF-b in, 129 tissue remodeling in, 128–130 TLR system in, 106–107, 121–122 TNF in, 107, 112, 118, 129 treatment apoptosis inducers in, 135–136 glucocorticoids in, 132–133 immunophilin ligands in, 133–134 proliferation inhibition in, 135–136 receptor antagonists in, 130–131, 134–135 receptor blockades in, 130–131 signal inhibition in, 131–134

index VGEF in, 112, 115, 127–128, 131, 135 Atopic disorders, asthma/mast cells/basophils and, 98, 126–127, 133–134 Autocrine/paracrine effects, in asthma/mast cells/basophils, 115–116, 137–138 Autoimmunity. See also Experimental autoimmune encephalomyelitis B-cell activation and, 31–33 BAFF and, 32–33, 35, 36 mABs and, 36–37 in MHC class II molecule I-Ag7, 235, 236, 256–257 MZ B cells and, 35–36 SLE and, 32–33 SSc and, 31 T cell activation and, 54–55, 57 expansion and, 54–55 selection and, 55, 57 triggering and, 55, 57–58 therapies for, 1–2, 30–36 Autoreactivity, in tetramer analysis/autoreactive T cells, 65–67 B B cell activating factor (BAFF), CD22 ligand binding and, 32–33, 35, 36 activation in CD22 ligand binding, 6–8, 31–33 differentiation in CD22 ligand binding, 7–8, 9 proliferation in CD22 ligand binding, 1, 7, 8–15 receptors (BCR) in PLC-g2 network regulation, 73–74, 78, 79, 82, 83, 85, 88 Bacterial interactions, in dendritic cell biology, 204–206 Basophil mediators, in asthma/mast cells/ basophils, 111–112 Basophils. See Asthma, role of mast cells/ basophils morphology-ultrastructure, asthma/mast cells/basophils and, 109–110 origins, asthma/mast cells/basophils and, 109 role, in asthma/mast cells/basophils, 117–118 surface markers, asthma/mast cells/basophils and, 112–115

i n d ex BCR. See B-cell receptors cross-linking, CD22 ligand binding and, 15, 16–17, 20, 22 ligation, CD22 ligand binding and, 7, 8, 11–12, 16, 22, 23–25 signaling, CD22 ligand binding and, 1, 2, 4, 7, 9, 11, 13, 15–23, 27–28 Beta cell antigen receptors (BCR), in MHC class I molecule regulation, 163, 176, 178 BHRF protein, RNA-based regulation and, 274–275 Binding motifs, for MHC class II molecule I-Ag7, 241–243, 254–255, 256 Biological role, of MHC class II molecule I-Ag7, 256–259 BLINK, in PLC-g2 network regulation, 77–78, 89 Btk, in PLC-g2 network regulation, 74, 76, 77, 78 C [Ca2þ]i. See Calcium, intracellular Calcineurin, in PLC-g2 network regulation, 87, 88 Calcium entry channels, in PLC-g2 network regulation, 73, 74, 83–86, 89–90 influx factor (CIF), in PLC-g2 network regulation, 80–81, 82 intracellular ([Ca2þ]i), in CD22 ligand binding, 8, 15, 17, 18–19, 21, 22, 23–28 mobilization, in PLC-g2 network regulation, 78–79 release/influx, in PLC-g2 network regulation, 80–81, 83, 89–90 signal modulation, in PLC-g2 network regulation, 88–89 cAMP, in asthma/mast cells/basophils, 112, 127, 131–132 Cancer, RNA-based regulation and, 272, 274 CD4þ T cells, tetramer analysis/autoreactive T cells and, 52, 53, 54, 57, 61–63 CD8þ T cells, tetramer analysis/autoreactive T cells and, 52, 60, 61, 66 CD19 coreceptors, in CD22 ligand binding, 2, 11, 15 CD19 regulatory loop, in CD22 ligand binding, 23–25 CD22 domains

295 CD22 ligand binding and, 3, 4 cytoplasmic, 15–28CD22 expression, in CD22 ligand binding, 3–4, 12–13 CD22 glycoprotein mutants of, 5, 8, 10, 11, 12–13 structure of, 4–5 CD22 ligand binding, in B-cell survival, 9–12 adhesion in, 5–7, 14–15 Ags in, 2, 8, 9, 13, 14 apoptosis and, 2, 7, 11, 30, 36 autoimmunity B-cell activation and, 31–33 BAFF and, 32–33, 35, 36 mABs and, 36–37 MZ B cells and, 35–36 SLE and, 32–33 SSc and, 31 therapies and, 1–2, 30–36 B cell activation in, 6–7, 8, 31–33 differentiation in, 7–8, 9 proliferation in, 1, 7, 8–15 survival in, 9–12 BCR cross-linking and, 15, 16–17, 20, 22 ligation and, 7, 8, 11–12, 16, 22, 23–25 signaling and, 1, 2, 4, 7, 9, 11, 13, 15–23, 27–28 [Ca2þ]i in, 7, 8, 15, 17, 18–19, 21, 22, 23–28 CD19 coreceptors in, 2, 11, 15 CD19 regulatory loop in, 23–25 CD22
/
mice in, 9–11, 13–14, 15, 23, 26, 31, 34 CD40 signaling in, 1, 2, 6–7, 9, 10, 11–12, 36 CD79a/CD79b heterodimers in, 2, 15, 23 Cul1 expression and, 11 domains of, 3, 4 cytoplasmic in, 15–28 ERK in, 20, 22, 23 expression in, 3–4, 12–13 follicular mantle B cells in, 3–4, 14 hCD22/mCD22 expression in, 3–4, 5, 6, 19, 33 Ig fusion proteins in, 5, 6, 14, 36 Ig superfamily in, 1, 2, 6 IgM cell surface expression in, 11, 13, 15, 25 ILs and, 4, 7, 15

296 CD22 ligand binding, in B-cell survival, (continued) immunoprecipitation and, 4–5, 6 ITAMs in, 15, 19, 20 ITIMs in, 16, 19, 21 JNK in, 20, 23 Lyn in, 7–8, 15, 16, 20, 27, 31–32 mABs in, 4, 7, 22, 28–29, 36 MAPK in, 20, 21, 23, 24 masking/unmasking in, 6–7 mutants in, 5, 8, 10, 11, 12–13 MZ B cells in, 3–4, 11, 14, 35–36 phosphatases in, 1, 2 PI 3-kinase in, 22–23, 25 PLC in, 19, 20, 22, 23, 25–26 PTK amplification in, 1, 2, 7, 15–16, 17, 19, 22 Rituximab and, 28–29 SHIP in, 21–22, 23, 25–26 SHP1 recruitment in, 8, 13, 17–18, 19–22, 23, 26, 27, 31–32, 34 sialylated ligands in, 5–7, 8 SIgLec in, 6 Src-family in, 1, 2, 16, 17, 19, 23 structure of, 4–5 Syk1 phosphorylation in, 25–26, 27 T cell proliferation in, 8, 36 therapies clinical trials and, 1–2, 28–30 immunotoxins and, 29 Vav in, 23–25 CD22
/
mice, CD22 ligand binding and, 9–11, 13–14, 15, 23, 26, 31, 34 CD40 signaling, in CD22 ligand binding, 1, 2, 6–7, 9, 10, 11–12, 36 CD79a/CD79b heterodimers, in CD22 ligand binding, 2, 15, 23 Chemoattractants, in asthma/mast cells/ basophils, 114–115 Chemokines in asthma/mast cells/basophils, 103, 104– 105, 107, 112, 114, 130–131 in MHC class I molecule regulation, 179 Chemotactic receptors, in asthma/mast cells/ basophils, 99, 105 CIA. See Collagen-induced arthritis CIF. See Calcium influx factor C-kit, in asthma/mast cells/basophils, 99, 101, 136

index Collagen synthesis, in asthma/mast cells/ basophils, 129–130 Collagen-induced arthritis (CIA), tetramer analysis/autoreactive T cells and, 59, 63 CTL. See Cytotoxic T-lymphocytes C-type lectins, in dendritic cell biology, 202–204 Cul1 expression, CD22 ligand binding and, 11 Cyclooxygenase (COX), in asthma/mast cells/ basophils, 104, 111–112 CysLT, in asthma/mast cells/basophils, 105, 107–108, 120, 135 Cytokine signal inhibition, in MHC class I molecule regulation, 180–182 Cytokines in asthma/mast cells/basophils, 98, 103, 104, 105, 109, 112, 115–116, 121, 122, 125, 126, 129, 130 in dendritic cell biology, 198, 206, 208, 212 in MHC class I molecule regulation, 165, 180–182 tetramer analysis/autoreactive T cells and, 57–58, 64, 65 Cytotoxic T-lymphocytes (CTL) in MHC class I molecule regulation, 182–183, 184, 185 tetramer analysis/autoreactive T cells and, 52 D DAG. See Diacylglycerol Danger model, in dendritic cell biology, 217–218 DC. See Dendritic cell biology activation, in dendritic cell biology, 197–198, 211–212, 213 maturation, in dendritic cell biology, 193–194, 195, 201, 212–213 subtypes, in dendritic cell biology, 194–195 DC-SIGN adhesion molecule, in dendritic cell biology, 202–203 Deficient mouse studies, in MHC class I molecule regulation, 171, 176, 182–186 Dendritic cell biology activation in, 197–198, 211–212, 213 APCs in, 215, 216–217 bacterial interactions in, 204–206 complexity of, 196 C-type lectins in, 202–204

i n d ex cytokines in, 198, 206, 208, 212 Danger model in, 217–218 DC subtypes in, 194–195 DC-SIGN in, 202–203 EAE in, 218–219 endocytosis in, 202–203, 205 expression analysis in, 197–198 genomic methods in, 196–198 GM-CSF in, 195 HCMV in, 207–208 HIV-1 in, 203, 207–208 HSV in, 203, 207–208 IFNs in, 195, 200–201, 205, 209–211, 214 ILs in, 195, 206, 209–211, 215, 218, 219 immunity acquired in, 212–215 innate in, 193–194, 196, 209–211 inflammatory stimuli in, 197–198, 201, 204, 212–213 INS model in, 216–217 LCMV in, 207, 212 LCMV-derived CTL epitope model in, 217 LPS and, 197–198, 200, 206, 212–214 LRRs in, 199 MAMPs in, 193–194 maturation in, 193–194, 195, 201, 212–213 MHC in, 194, 195, 202, 205–206, 209, 211, 213, 215, 216 MMRs in, 202–203 NK cells in, 209–211 phagocytosis in, 202–203, 204 PRRs in, 194, 213 PTX3 in, 204 receptor repertoire in, 199–204 T-cell repertoire in, 215–216 TIR domains in, 199–200 TLRs in, 193–194, 199–202, 208, 213 TNF and, 197–198, 206, 214, 218 tolerance central in, 215–216 peripheral in, 216–219 Tregs in, 218–219 viral interactions in, 207–209

297 Determinant capture hypothesis, MHC class II molecule I-Ag7 and, 254 Diabetes. See Type 1 diabetes MHC class II molecule I-Ag7 and, 236, 254, 256 Diacylglycerol (DAG), PLC-g2 network regulation, 74, 77, 85, 86 Dicer, RNA-based regulation and, 269, 271, 281, 285 Double strand RNA (dsRNA), in RNA-based regulation, 267–268, 270–271, 280, 282, 283, 285 DP receptors, in asthma/mast cells/basophils, 108–109 DQ2 molecules, MHC class II molecule I-Ag7 and, 253 DQ8 molecules, MHC class II molecule I-Ag7 and, 235–236, 237, 238, 240, 253 dsRNA. See Double strand RNA E E3 ligases, in PLC-g2 network regulation, 88–89 EAE. See Experimental autoimmune encephalomyelitis EBV. See Epstein-Barr virus Endocytosis, in dendritic cell biology, 202–203, 205 Eotaxins, in asthma/mast cells/basophils, 118–119 Epitope length, tetramer analysis/autoreactive T cells and, 63–64 Epstein-Barr virus (EBV), RNA-based regulation and, 274–275 ERK. See Extracellular signal-regulated kinase Experimental autoimmune encephalomyelitis (EAE) in dendritic cell biology, 218–219 tetramer analysis/autoreactive T cells and, 59, 61 Expression analysis, in dendritic cell biology, 197–198 Extracellular signal-regulated kinase (ERK) in CD22 ligand binding, 20, 22, 23 in PLC-g2 network regulation, 88

index

298 F FceRI activation, in asthma/mast cells/basophils, 121–123, 127, 130–131, 136–137 cell recruitment, in asthma/mast cells/ basophils, 118–119 expression, in asthma/mast cells/basophils, 97, 105–106, 113, 115 FcRg signaling, in MHC class I molecule regulation, 174–176, 178, 181 Feedback loops, in PLC-g2 network regulation, 74, 75, 76–77 Follicular mantle B cells, in CD22 ligand binding, 3–4, 14 G GAD. See Glutamic acid decarboxylase GAD65 peptides, in MHC class II molecule I-Ag7, 238, 241, 242, 254 Gene silencing, in RNA-based regulation, 270, 279, 282, 285 Genetic factors, in asthma/mast cells/basophils, 98–99, 136–137 Genome defense, in RNA-based regulation, 268–271 Genomic methods, in dendritic cell biology, 196–198 Glucocorticoids, in asthma treatment, 132–133 Glutamic acid decarboxylase (GAD), in tetramer analysis/autoreactive T cells, 52, 56–58, 60–63, 65 Grafting-versus host disease (GVHD), in MHC class I molecule regulation, 178, 182–186 Granulocyte macrophage colony stimulating factor (GM-CSF) in asthma/mast cells/basophils, 113, 114, 121 in dendritic cell biology, 195 in MHC class I molecule regulation, 180–181 GVHD. See Grafting-versus host disease H hCD22/mCD22 expression, in CD22 ligand binding, 3–4, 5, 6, 19, 33 HEL peptides, in MHC class II molecule I-Ag7, 238, 240, 241–242, 244, 250, 251

Herpes simplex virus (HSV), in dendritic cell biology, 203, 207–208 Histamine, in asthma/mast cells/basophils, 107–109, 111–112, 115 Histocompatibility complex (MHC), in dendritic cell biology, 194, 195, 202, 205–206, 209, 211, 213, 215, 216 HIV-1, in dendritic cell biology, 203, 207–208 HSV. See Herpes simplex virus Human cytomegalovirus (HCMV), in dendritic cell biology, 207–208 Human umbilical vein endothelial cells (HUVEC), in asthma/mast cells/basophils, 118–119 HUVEC. See Human umbilical vein endothelial cells Hyperresponsiveness, in MHC class I molecule regulation, 178–180 I IAg7 antigen, in tetramer analysis/autoreactive T cells, 54, 59, 60 IFN. See Interferons IFN-g, in MHC class I molecule regulation, 180, 183, 184, 185 Ig fusion proteins, in CD22 ligand binding, 5, 6, 14, 36 Ig superfamily, in CD22 ligand binding, 1, 2, 6 IGF. See Insulin-like growth factor Ig-like receptor family, in MHC class I molecule regulation, 163–166 IgM cell surface expression, in CD22 ligand binding, 11, 13, 15, 25 IKK inhibitors, PLC-g2 network regulation and, 86–88 IL. See Interleukins Immune system RNAi/RNA-based regulation ADARs v. RNAis in, 280–283 ADARs v. viruses in, 283–284 cancer and, 272, 274 Dicer and, 269, 271, 281, 285 dsRNA in, 267–268, 270–271, 280, 282, 283, 285 gene silencing in, 270, 279, 282, 285 genome defense in, 268–271 INFs in, 284

i n d ex miRNAs in, 267–269, 271–272, 273 BHRF and, 273–275 EBV and, 273–275 viral infections and, 273–275 miRNPs in, 268 MYC expression in, 272, 274 NMD expression in, 267, 274–276, 278–280 PTCs in, 275, 276, 278, 279–280 RISC complex in, 279–280 RNAis in, 267–271, 276, 277, 278–280 short RNAs in, 271–274, 285–286 siRNAs in, 268–271 TCR expression in, 274–276, 278, 279 UPR in, 275–276 VDJ expression in, 275, 278 Immunity, in dendritic cell biology acquired, 212–215 innate, 193–194, 196, 209–211 Immunophilin ligands, in asthma treatment, 133–134 Immunoprecipitation, CD22 ligand binding and, 4–5, 6 Immunoreceptor tyrosine-based activation motif (ITAM) in CD22 ligand binding, 15, 19, 20 in MHC class I molecule regulation, 165, 169, 174, 181 Immunoreceptor tyrosine-based inhibition motif (ITIM) in CD22 ligand binding, 16, 19, 21 in MHC class I molecule regulation, 165, 168–169, 174, 176–177, 179 Immunotherapy, tetramer analysis/autoreactive T cells and, 52, 54, 67 Infectious nonself (INS) model, in dendritic cell biology, 216–217 Inflammatory stimuli, in dendritic cell biology, 197–198, 201, 204, 212–213 Inositol-1,4,5-triphosphate (IP3), in PLC-g 2 network regulation, 73, 74, 77, 83, 85 Insulin-like growth factor (IGF), in asthma/ mast cells/basophils, 119 Interferons (IFN) in dendritic cell biology, 195, 200–201, 205, 209–211, 214 in RNA-based regulation, 284 Interleukins (IL) in asthma/mast cells/basophils, 107–109, 112–116, 117, 118, 124, 126, 129, 134

299 CD22 ligand binding and, 4, 7, 15 in dendritic cell biology, 195, 206, 209–211, 215, 218, 219 IP3. See Inositol-1,4,5-triphosphate IP3 receptors, in PLC-g2 network regulation, 79–81 ITAM. See Immunoreceptor tyrosine-based activation motif ITIM. See Immunoreceptor tyrosine-based inhibition motif J JNK kinases in CD22 ligand binding, 20, 23 in PLC-g2 network regulation, 88 K Killer immunoglobulin-like receptor (KIR), in MHC class I molecule regulation, 161–163, 182 L LCMV. See Lymphocytic choriomeningitis LCMV-derived CTL epitope model, in dendritic cell biology, 217 Leukocyte Ig-like receptors (LILR), in MHC class I molecule regulation, 162, 165–166, 182, 186 LILR. See Leukocyte Ig-like receptors LILR protein structure, in MHC class I molecule regulation, 171–174 Lipopolysaccharide (LPS), dendritic cell biology and, 197–198, 200, 206, 212–214 5-Lipoxygenase (5-LO), in asthma/mast cells/ basophils, 104, 105, 135 LTC4, in asthma/mast cells/basophils, 105, 111, 132, 134 Lymphocytic choriomeningitis (LCMV), in dendritic cell biology, 207, 212 Lyn in CD22 ligand binding, 7–8, 15, 16, 20, 27, 31–32 in PLC-g2 network regulation, 74 M mABs. See Monoclonal antibodies Major histocompatibility complex. See MHC MAMP. See Microbial associated molecular patterns

300 Mannose receptor (MMR), in dendritic cell biology, 202–203 MAPK. See Mitogen-activated protein kinases Marginal zone. See MZ Masking/unmasking, in CD22 ligand binding, 6–7 Mast cells. See Asthma, role of mast cells/ basophils activation of, 120 eicosanoid metabolism and, 102–103 eicosanoid receptors and, 107–109 mediators and, 103–105 morphology-ultrastructure and, 102–103 origins of, 101–102 role of, 116–117 surface markers in, 105–107 Mediator release, in asthma/mast cells/ basophils, 99–100, 110 MHC class I molecule regulation BCRs in, 163, 176, 178 chemokines in, 179 CTLs in, 182–183, 184, 185 cytokines in, 165, 180–182 deficient mouse studies in, 171, 176, 182–186 FcRg signaling in, 174–176, 178, 181 GM-CSF in, 180–181 GVHD in, 178, 182–186 hyperresponsiveness in, 178–180 IFN-g in, 180, 183, 184, 185 Ig-like receptor family in, 163–166 ITAMs in, 165, 169, 174, 181 ITIMs in, 165, 168–169, 174, 176–177, 179 KIRs in, 161–163, 182 LILR protein structure in, 171–174 LILRs in, 162, 165–166, 182, 186 MAPK in, 179–180 NK-cell activation/inhibition in, 173–174 PIR domains in, 169, 170, 171 ligands in, 169–171, 177 protein structure in, 166–169, 171–174 PIR-B expression in, 177–178, 186 PIRs in, 161–163, 166 RANKL in, 181–182 schematic structures in, 162, 167–168 signal activation/PIR-A in, 174–176 signal inhibition cytokines in, 180–182 PIR-B in, 175, 176–177, 178, 180

index Src kinase family in, 179–180 TCRs in, 161–163 transplantation and, 182–186 MHC class II molecule I-Ag7 anchor pockets in, 239–241, 244, 256 APCs in, 236, 237, 243, 252, 253, 254 autoimmunity in, 235, 236, 256–257 binding motifs for, 241–243, 254–255, 256 biological role of, 256–259 determinant capture hypothesis, 254 diabetes and, 236, 254, 256 DQ2 molecules and, 253 DQ8 molecules and, 235–236, 237, 238, 240, 253 GAD65 peptides in, 238, 241, 242, 254 HEL peptides in, 238, 240, 241–242, 244, 250, 251 naturally processed peptides and, 243–254, 255, 256 NOD mice and, 235–237, 253, 254, 256–258 P1 pocket in, 239–240, 241 P4 pocket in, 239–240, 242, 246, 249, 252, 253 P6 pocket in, 239–240, 242, 246, 249, 250 P9 pocket in, 238, 239–240, 242–243, 246, 249, 250, 252, 253, 254, 256 P10 pocket in, 240, 250, 251 P11 pocket in, 250, 251 structure of, 236–241 T-cell epitopes in, 254, 255 T cells and, 236, 254, 256–257, 258–259 T1DM and, 235, 236 TCR contact residues in, 244, 258 Microbial associated molecular patterns (MAMP), in dendritic cell biology, 193–194 Microribonucleoprotein complex (MiRNP), in RNA-based regulation, 268 MicroRNAs (miRNA), in RNA-based regulation, 267–269, 271–272, 273 Mitogen-activated protein kinases (MAPK) in CD22 ligand binding, 20, 21, 23, 24 in MHC class I molecule regulation, 179–180 MMR. See Mannose receptor

i n d ex Monoclonal antibodies (mAB), in CD22 ligand binding, 4, 7, 22, 28–29, 36 Mutational analysis, in PLC-g2 network regulation, 80–81 MYC expression, in RNA-based regulation, 272, 274 MZ B cells, in CD22 ligand binding, 3–4, 11, 14, 35–36 N Natural killer (NK) cells, in dendritic cell biology, 209–211 Naturally processed peptides, MHC class II molecule I-Ag7 and, 243–254, 255, 256 Nerve growth factor (NGF), in asthma/mast cells/basophils, 113, 116 NFAT. See Nuclear factor of activated T cells NF-kB in asthma/mast cells/basophils, 133 in PLC-g2 network regulation, 74, 79, 86–88 NGF. See Nerve growth factor NK. See Natural killer cells NK-cell activation/inhibition, in MHC class I molecule regulation, 173–174 NMD. See Nonsense-mediated decay NOD mouse studies MHC class II molecule I-Ag7 and, 235–237, 253, 254, 256–258 in tetramer analysis/autoreactive T cells, 54–55, 59–63 Nonsense-mediated decay (NMD) expression, in RNA-based regulation, 267, 274–276, 278–280 Nuclear factor of activated T cells (NFAT), in PLC-g2 network regulation, 74, 79, 86–88 P P1 pocket, in MHC class II molecule I-Ag7, 239–240, 241 P4 pocket, in MHC class II molecule I-Ag7, 239–240, 242, 246, 249, 252, 253 P6 pocket, in MHC class II molecule I-Ag7, 239–240, 242, 246, 249, 250 P9 pocket, in MHC class II molecule I-Ag7, 238, 239–240, 242–243, 246, 249, 250, 252–254, 256 P10 pocket, in MHC class II molecule I-Ag7, 240, 250, 251

301 P11 pocket, in MHC class II molecule I-Ag7, 250, 251 Paired Ig-like receptors (PIR) ligands, 169–171, 177 MHC class I molecule regulation, 161–163, 166, 169, 170, 171 PIR-A signal activation, 174–176 PIR-B expression, 177–178, 186 signal inhibition and, 175, 176–177, 178, 180 protein structure of, 166–169, 171–174 Pathogenesis, of asthma/mast cells/basophils role in, 97–98, 99, 105, 136–138 Pattern recognition receptors (PRR), in dendritic cell biology, 194, 213 PBL. See Peripheral blood lymphocytes Peptide-MHC (pMHC), 51–59 recognition of, 55–58, 59 Peripheral blood lymphocytes (PBL), in tetramer analysis/autoreactive T cells, 52, 53 PGD2. See Prostaglandin-2 Phagocytosis, in dendritic cell biology, 202–203, 204 Phosphodiesterase inhibitors, in asthma/mast cells/basophils, 131–132 Phospholipase C (PLC), in CD22 ligand binding, 19, 20, 22, 23, 25–26 PI3-kinase in CD22 ligand binding, 22–23, 25 in PLC-g2 network regulation, 77, 78, 83 PKC. See Protein kinase C PLC. See Phospholipase C PLC-g2 network regulation activation in, 74–75 BCRs in, 73–74, 78, 79, 82, 83, 85, 88 BLINK in, 77–78, 89 Btk in, 74, 76, 77, 78 calcineurin in, 87, 88 calcium entry channels in, 73, 74, 83–86, 89–90 mobilization in, 78–79 release/influx in, 80–81, 83, 89–90 signal modulation in, 88–89 CIF in, 80–81, 82 DAG in, 74, 77, 85, 86

302 PLC-g2 network regulation (continued) E3 ligases in, 88–89 ERK in, 88 feedback loops in, 74, 75, 76–77 IKK inhibitors and, 86–88 IP3 in, 73, 74, 77, 83, 85 IP3 receptors in, 79–81 JNK in, 88 Lyn in, 74 mutational analysis in, 80–81 NFAT in, 74, 79, 86–88 NF-kB in, 74, 79, 86–88 PI3K activation in, 77, 78, 83 PKC in, 86 PMCAs in, 86 SHIP in, 78 SOCs in, 79, 80–81, 83 Syk in, 74, 75, 76–77, 78, 89 TCR stimulation in, 89 TRP ion channels in, 81–83, 86, 84, 89 Vav in, 78 PMCA calcium pumps, PLC-g2 network regulation, 86 pMHC. see Peptide-MHC Premature termination codons (PTC), in RNAbased regulation, 275, 276, 278, 279–280 Progression model, in tetramer analysis/ autoreactive T cells and, 55, 60 Proliferation inhibition, in asthma treatment, 135–136 Prostaglandin-2 (PGD2), in asthma/mast cells/ basophils, 112, 116, 132, 134, 135 Protein kinase C (PKC), in PLC-g2 network regulation, 86 Protein tyrosine kinase (PTK) amplification, in CD22 ligand binding, 1, 2, 7, 15–16, 17, 19, 22 PRR. See Pattern recognition receptors PTX3, in dendritic cell biology, 204 R RANKL, in MHC class I molecule regulation, 181–182 Receptor antagonists, in asthma treatment, 130–131, 134–135 Receptor blockades, in asthma treatment, 130–131 Receptor repertoire, in dendritic cell biology, 199–204

index Regulatory T cells (Treg) in dendritic cell biology, 218–219 tetramer analysis/autoreactive T cells and, 54, 58–59 RISC complex, RNA-based regulation and, 279–280 Rituximab, CD22 ligand binding and, 28–29 RNA interference (RNAi), in RNA-based regulation, 267–271, 276, 277, 278–280 S SCF. See Stem cell factor Schematic structures, in MHC class I molecule regulation, 162, 167–168 See Premature termination codons, PTC Self antigens, in tetramer analysis/autoreactive T cells, 55–58 SHIP in CD22 ligand binding, 21–22, 23, 25–26 in PLC-g2 network regulation, 78 Short interfering RNAs (SiRNA), in RNA-based regulation, 268–271 Short RNAs, in RNA-based regulation, 271–274, 285–286 SHP1 recruitment, in CD22 ligand binding, 8, 13, 17–18, 19–22, 23, 26, 27, 31–32, 34 Sialylated ligands, in CD22 ligand binding, 5–7, 8 SIgLec, in CD22 ligand binding, 6 Signal inhibition, in asthma treatment, 131–134 siRNAs. See Short interfering RNAs SLE. See Systemic lupus erythematosus SOC. See Store-operated calcium channels Src kinase family in CD22 ligand binding, 1, 2, 16, 17, 19, 23 in MHC class I molecule regulation, 179–180 SSc. See Systemic sclerosis Stem cell factor (SCF), in asthma/mast cells/ basophils, 101–102, 116 Store-operated calcium channels (SOC), in PLC-g2 network regulation, 79, 80–81, 83 Structure of MHC class II molecule I-Ag7, 236–241 in tetramer analysis/autoreactive T cells, 51

i n d ex Superallergens, in asthma, 121–123, 125 HIV-1 gp120, 124–125 protein A and, 126 protein Fv and, 123–124 protein L and, 126–127 tissue injury and, 125 Superantigens, in asthma/mast cells/basophils, 122–123 Syk phosphorylation in CD22 ligand binding, 25–26, 27 in PLC-g2 network regulation, 74, 75, 76–77, 78, 89 Systemic lupus erythematosus (SLE), CD22 ligand binding and, 32–33 Systemic sclerosis (SSc), CD22 ligand binding and, 31 T T cells activation, autoimmunity and, 54–55, 57 epitopes, in MHC class II molecule I-Ag7, 254, 255 expansion, autoimmunity and, 54–55 proliferation, in CD22 ligand binding, 8, 36 receptors (TCR) expression/RNA-based regulation and, 274–276, 278, 279 in MHC class I molecule regulation, 161– 163 MHC class II molecule I-Ag7 and, 236, 254, 256–257, 258–259 repertoire, in dendritic cell biology, 215–216 selection, autoimmunity and, 55, 57 triggering, autoimmunity and, 55, 57–58 T1D. See Type 1 diabetes TCR expression. See T cell receptor expression interaction/activation, in tetramer analysis/ autoreactive T cells, 55–56, 63, 64, 65–67 stimulation, in PLC-g2 network regulation, 89 Tetramer analysis, of human autoreactive T cells AICD in, 64, 65–66 autoimmunity/ T cell

303 activation in, 54–55, 57 expansion in, 54–55 selection in, 55, 57 triggering in, 55, 57–58 autoreactivity in, 65–67 CD4þ T cells and, 52, 53, 54, 57, 61–63 CD8þ T cells and, 52, 60, 61, 66 CIA and, 59, 63 CTLs and, 52 cytokines and, 57–58, 64, 65 EAE and, 59, 61 epitope length and, 63–64 GAD in, 52, 56–58, 60–63, 65 IAg7 antigen and, 54, 59, 60 immunotherapy and, 52, 54, 67 NOD mouse studies in, 54–55, 59–63 PBLs and, 52, 53 pMHC recognition in, 55–58, 59 pMHCs in, 51–59 progression model and, 55, 60 self antigens in, 55–58 structural studies in, 51 T1D studies in, 54–55, 59, 61–64, 65–66 TCR interaction/activation and, 55–56, 63, 64, 65–67 Treg and, 54, 58–59 TGF-b. See Tumor growth factor-b Therapies CD22 ligand binding and, 1–2, 28–30 immunotoxins, CD22 ligand binding and, 29 TIR domains, in dendritic cell biology, 199–200 Tissue remodeling, in asthma/mast cells/ basophils, 128–130 TLR. See Toll-like receptor Tolerance, in DCs central, 215–216 peripheral, 216–219 Toll-like receptor (TLR) system in asthma/mast cells/basophils, 106–107, 121–122 in dendritic cell biology, 193–194, 199–202, 208, 213 Transplantation, MHC class I molecule regulation and, 182–186 Treg. See Regulatory T cells TRP ion channels, in PLC-g2 network regulation, 81–83, 86, 89

304 Tumor growth factor-b (TGF-b), in asthma/ mast cells/basophils, 129 Tumor necrosis factor (TNF) in asthma/mast cells/basophils, 107, 112, 118, 129 dendritic cell biology and, 197–198, 206, 214, 218 Type 1 diabetes (T1D) MHC class II molecule I-Ag7 and, 235, 236 in tetramer analysis/autoreactive T cells, 54–55, 59, 61–64, 65–66 U Unfolded protein response (UPR), in RNA-based regulation, 275–276

index V Vascular endothelial growth factor (VEGF), in asthma/mast cells/basophils, 111, 115, 127–128, 131, 135 Vav in CD22 ligand binding, 23–25 in PLC-g2 network regulation, 78 VDJ expression, in RNA-based regulation, 275, 278 VEGF. See Vascular endothelial growth factor Viral interactions in dendritic cell biology, 207–209 RNA-based regulation and, 274–275

Contents of Recent Volumes

Anthony J. Coyle, and Jose-Carlos Gutierrez-Ramos

Volume 77 The Actin Cytoskeleton, Membrane Lipid Microdomains, and T Cell Signal Transduction S. Celeste Posey Morley and Barbara E. Bierer

Selected Comparison of Immune and Nervous System Development Jerold Chun Index

Raft Membrane Domains and Immunoreceptor Functions Thomas Harder

Volume 78

Human Basophils: Mediator Release and Cytokine Production John T. Schroeder, Donald W. MacGlashan, Jr., and Lawrence M. Lichtenstein

Toll-like Receptors and Innate Immunity Shizuo Akira Chemokines in Immunity Osamu Yoshie, Toshio Imai, and Hisayuki Nomiyama

Btk and BLNK in B Cell Development Satoshi Tsukada, Yoshihiro Baba, and Dai Watanabe Diversity and Regulatory Functions of Mammalian Secretory Phospholipase A2s Makoto Murakami and Ichiro Kudo

Attractions and Migrations of Lymphoid Cells in the Organization of Humoral Immune Responses Christoph Schaniel, Antonius G. Rolink, and Fritz Melchers Factors and Forces Controlling V(D)J Recombination David G. T. Hesslein and David G. Schatz

The Antiviral Activity of Antibodies in Vitro and in Vivo Paul W. H. I. Parren and Dennis R. Burton

T Cell Effector Subsets: Extending the Th1/Th2 Paradigm Tatyana Chtanova and Charles R. Mackay

Mouse Models of Allergic Airway Disease Clare M. Lloyd, Jose-Angel Gonzalo,

305

306 MHC-Restricted T Cell Responses against Posttranslationally Modified Peptide Antigens Ingelise Bjerring Kastrup, Mads Hald Andersen, Tim Elliot, and John S. Haurum Gastrointestinal Eosinophils in Health and Disease Marc E. Rothenberg, Anil Mishra, Eric B. Brandt, and Simon P. Hogan Index

Volume 79 Neutralizing Antiviral Antibody Responses Rolf M. Zinkernagel, Alain Lamarre, Adrian Ciurea, Lukas Hunziker, Adrian F. Ochsenbein, Kathy D. McCoy, Thomas Fehr, Martin F. Bachmann, Ulrich Kalinke, and Hans Hengartner Regulation of Interleukin-12 Production in Antigen-Presenting Cells Xiaojing Ma and Giorgio Trinchieri Mechanisms of Signaling by the Hematopoietic-Specific Adaptor Proteins, SLP-76 and LAT and Their B Cell Counterpart, BLNK/SLP-65 Deborah Yablonski and Arthur Weiss Xenotransplantation David H. Sachs, Megan Sykes, Simon C. Robson, and David K. C. Cooper Regulation of Antibacterial and Antifungal Innate Immunity in Fruitflies and Humans Michael J. Williams Functional Heavy-Chain Antibodies in Camelidae Viet Khong Nguyen, Aline Desmyter, and Serge Muyldermans

co n t e nt s o f re c e nt vo l um es Uterine Natural Killer Cells in the Pregnant Uterus Chau-Ching Liu and John Ding-E Young Index

Volume 80 Protein Degradation and the Generation of MHC Class I-Presented Peptides Kenneth L. Rock, Ian A. York, Tomo Saric, and Alfred L. Goldberg Proteoanalysis and Antigen Presentation by MHC Class II Molecules Paula Wolf Bryant, Ana-Maria Lennon-DumA˚nil, Edda Fiebiger, CA˚cile LagaudriA˚re-Gesbert, and Hidde L. Ploegh Cytokine Memory of T Helper Lymphocytes Max Lo«hning, Anne Richter, and Andreas Radbruch Ig Gene Hypermutation: A Mechanism Is Due Jean-Claude Weill, Barbara Bertocci, Ahmad Faili, Said Aoufouchi, StA˚phane Frey, Annie De Smet, SA˚bastian Storck, Auriel Dahan, FrA˚dA˚ric Delbos, Sandra Weller, Eric Flatter, and Claude-AgnA˚s Reynaud Generalization of Single Immunological Experiences by Idiotypically Mediated Clonal Connections Hilmar Lemke and Hans Lange The Aging of the Immune System B. Grubeck-Loebenstein and G. Wick Index

c o nt e n t s of re c e n t vo l u m es

Volume 81 Regulation of the Immune Response by the Interaction of Chemokines and Proteases Jo Van Damme and Sofie Struyf Molecular Mechanisms of Host-Pathogen Interaction: Entry and Survival of Mycobacteria in Macrophages Jean Pieters and John Gatfield B Lymphoid Neoplasms of Mice: Characteristics of Naturally Occurring and Engineered Diseasse and Relationships to Human disorders Herbert Morse et al. Prions and the Immune System: A Journey Through Gut Spleen, and Nerves Adriano Aguzzi Roles of the Semaphorin Family in Immune Regulation H. Kikutani and A. Kumanogoh HLA-G Molecules: from Maternal-Fetal Tolerance to Tissue Acceptance Edgardo Carosella et al. The Zebrafish as a Model Organism to Study Development of the Immune System Nick Trede et al. Control of Autoimmunity by Naturally Arising Regulatory CD4þ T Cells S. Sakaguchi

307 Tumor Vaccines Freda K. Stevenson, Jason Rice, and Delin Zhu Immunotherapy of Allergic Disease R. Valenta, T. Ball, M. Focke, B. Linhart, N. Mothes, V. Niederberger, S. Spitzauer, I. Swoboda, S.Vrtala, K. Westritschnic, and D. Kraft Interactions of Immunoglobulins Outside the Antigen-Combining Site Roald Nezlin and Victor Ghetie The Roles of Antibodies in Mouse Models of Rheumatoid Arthritis, and Relevance to Human Disease Paul A. Monach, Christophe Benoist, and Diane Mathis MUC1 Immunology: From Discovery to Clinical Applications Anda M. Vlad, Jessica C. Kettel, Nehad M. Alajez, Casey A. Carlos, and Olivera J. Finn Human Models of Inherited Immunoglobulin Class Switch Recombination and Somatic Hypermutation Defects (Hyper-IgM Syndromes) Anne Durandy, Patrick Revy, and Alain Fischer The Biological Role of the C1 Inhibitor in Regulation of Vascular Permeability and Modulation of Inflammation Alvin E. Davis, III, Shenghe Cai, and Dongxu Liu Index

Index

Volume 82 Transcriptional Regulation in Neutrophils: Teaching Old Cells New Tricks Patrick P. McDonald

Volume 83 Lineage Commitment and Developmental Plasticity in Early Lymphoid Progenitor Subsets David Traver and Koichi Akashi

308

co n t e nt s o f re c e nt vo l um es

The CD4/CD8 Lineage Choice: New Insights into Epigenetic Regulation during T Cell Development Ichiro Taniuchi, Wilfried Ellmeier, and Dan R. Littman

Customized Antigens for Desensitizing Allergic Patients Fa¨tima Ferreira, Michael Wallner, and Josef Thalhamer

CD4/CD8 Coreceptors in Thymocyte Development, Selection, and Lineage Commitment: Analysis of the CD4/CD8 Lineage Decision Alfred Singer and Remy Bosselut

Immune Response Against Dying Tumor Cells Laurence Zitvogel, Noelia Casares, Marie O. Pe¨quignot, Nathalie Chaput, Mathew L. Albert, and Guido Kroemer

Development and Function of T Helper 1 Cells Anne O’Garra and Douglas Robinson Th2 Cells: Orchestrating Barrier Immunity Daniel B. Stetson, David Voehringer, Jane L. Grogan, Min Xu, R. Lee Reinhardt, Stefanie Scheu, Ben L. Kelly, and Richard M. Locksley Generation, Maintenance, and Function of Memory T Cells Patrick R. Burkett, Rima Koka, Marcia Chien, David L. Boone, and Averil Ma CD8þ Effector Cells Pierre A. Henkart and Marta Catalfamo An Integrated Model of Immunoregulation Mediated by Regulatory T Cell Subsets Hong Jiang and Leonard Chess Index

HMGB1 in the Immunology of Sepsis (Not Septic Shock) and Arthritis Christopher J. Czura, Huan Yang, Carol Ann Amella, and Kevin J. Tracey Selection of the T-Cell Repertoire: Receptor-Controlled Checkpoints in T-Cell Development Harald Von Boehmer The Pathogenesis of Diabetes in the NOD Mouse Michelle Solomon and Nora Sarvetnick Index

Volume 85 Cumulative Subject Index Volumes 66–82

Volume 84 Interactions Between NK Cells and B Lymphocytes Dorothy Yuan Multitasking of Helix-Loop-Helix Proteins in Lymphopoiesis Xiao-Hong Sun

Volume 86 Adenosine Deaminase Deficiency: Metabolic Basis of Immune Deficiency and Pulmonary Inflammation Michael R. Blackburn and Rodney E. Kellems

c o nt e n t s of re c e n t vo l u m es

309

Mechanism and Control of V(D)J Recombination Versus Class Switch Recombination: Similarities and Differences Darryll D. Dudley, Jayanta Chaudhuri, Craig H. Bassing, and Frederick W. Alt

The Integration of Conventional and Unconventional T Cells that Characterizes Cell-Mediated Responses Daniel J. Pennington, David Vermijlen, Emma L. Wise, Sarah L. Clarke, Robert E. Tigelaar, and Adrian C. Hayday

Isoforms of Terminal Deoxynucleotidyltransferase: Developmental Aspects and Function To-Ha Thai and John F. Kearney

Negative Regulation of Cytokine and TLR Signalings by SOCS and Others Tetsuji Naka, Minoru Fujimoto, Hiroko Tsutsui, and Akihiko Yoshimura

Innate Autoimmunity Michael C. Carroll and V. Michael Holers

Pathogenic T-Cell Clones in Autoimmune Diabetes: More Lessons from the NOD Mouse Kathryn Haskins

Formation of Bradykinin: A Major Contributor to the Innate Inflammatory Response Kusumam Joseph and Allen P. Kaplan Interleukin-2, Interleukin-15, and Their Roles in Human Natural Killer Cells Brian Becknell and Michael A. Caligiuri Regulation of Antigen Presentation and Cross-Presentation in the Dendritic Cell Network: Facts, Hypothesis, and Immunological Implications Nicholas S. Wilson and Jose A. Villadangos Index

Volume 87 Role of the LAT Adaptor in T-Cell Development and Th2 Differentiation Bernard Malissen, Enrique Aguado, and Marie Malissen

The Biology of Human Lymphoid Malignancies Revealed by Gene Expression Profiling Louis M. Staudt and Sandeep Dave New Insights into Alternative Mechanisms of Immune Receptor Diversification Gary W. Litman, John P. Cannon, and Jonathan P. Rast The Repair of DNA Damages/Modifications During the Maturation of the Immune System: Lessons from Human Primary Immunodeficiency Disorders and Animal Models Patrick Revy, Dietke Buck, Franc,oise le Deist, and Jean-Pierre de Villartay Antibody Class Switch Recombination: Roles for Switch Sequences and Mismatch Repair Proteins Irene M. Min and Erik Selsing Index