Eotaxin 2 John R. White* Department of Molecular Immunology, Smithkline Beecham, 709 Swedeland Road, PO Box 1539, King of Prussia, PA 19406, USA * corresponding author tel: 610-270-4854, fax: 610-270-5114, e-mail:
[email protected] DOI: 10.1006/rwcy.2000.11009.
SUMMARY Eotaxin 2 is a CC chemokine that appears to bind exclusively to the chemokine receptor CCR3. As expected, eotaxin 2 acts on cells expressing the CCR3 receptor, including eosinophils (Sabroe et al., 1998), basophils (Uguccioni et al., 1997), TH2 T cells (Gerber et al., 1997), and certain subsets of dendritic cells (Sato et al., 1999) to promote chemotaxis, shape change, and inflammatory mediator production. Eotaxin 2 is therefore of particular interest to researchers studying allergy.
BACKGROUND
Discovery The discovery of eotaxin 2 was the result of an effort to clone the human genome by random sequencing of expressed sequence tags (ESTs) in a cDNA library from activated human monocytes. From this library, a single clone (HMSAF34) was identified that had the characteristic CC chemokine motif, i.e. four cysteines positioned at the correct distance from each other to establish this EST as a potential chemokine (Forssmann et al., 1997; White et al., 1997).
Alternative names Eotaxin 2 was first described in 1997 by three laboratories in a set of papers that reported the biological activity of this chemokine on eosinophils, basophils, and hematopoietic cells (Forssmann et al., 1997; Patel et al., 1997; White et al., 1997). It was referred to as eotaxin-2 (Forssmann et al., 1997;
White et al., 1997), MPIF-2 (myeloid progenitor inhibitory factor 2) (Patel et al., 1997), or CK -6 (White et al., 1997) and in the future will be known as CCL24 using the new nomenclature proposed at the 1999 Keystone Meeting on Chemokines. To date, eotaxin 2 remains the generally accepted name for this molecule.
Structure Eotaxin 2 is a member of the (CC) chemokine family and contains four cysteine residues at the same location noted for all other chemokines. Eotaxin 2 isolated from a baculovirus expression system is Nglycosylated with a fucosylated mannose (M3N2F) core at position 89. There are at least two different forms of expressed eotaxin 2 varying in C-terminal length (see Protein) which investigators have used. These different forms appear to have slightly different biological potency.
Main activities and pathophysiological roles Eotaxin 2 binds the CCR3 receptor with high affinity as a monogamous receptor pairing, therefore much of the information about the functional role for eotaxin 2 can be gleaned from the chapter on CCR3. The receptor for eotaxin 2, CCR3, is expressed in a number of cells including eosinophils (Forssmann et al., 1997; Heath et al., 1997; White et al., 1997), basophils (Forssmann et al., 1997; Uguccioni et al., 1997), TH2 Tcells (Sallusto et al., 1997), mast cells (Quackenbush et al., 1998), monocytes/macrophages (Ghorpade et al., 1998), microglia (Ghorpade et al., 1998), and activated neutrophils (Patel et al., 1997).
1232 John R. White Eotaxin 2 functions as a potent chemotactic factor for all of the above-mentioned cells.
HPP-CFC and LPP-CFC colonies and MPIF1 (CK 8) which only inhibits LPP-CFC (Patel et al., 1997).
Eosinophil Chemotaxis
Shape Change and Actin Polymerization in Human Eosinophils
Eotaxin 2 is an efficacious chemotactic factor for human eosinophils, resulting in a chemotactic index (CI) between 9.5 and 35 with an EC50 of 1±10 nM. Certain cloned molecular forms of eotaxin 2 appear to have slightly greater chemotactic potency than eotaxin (White et al., 1997), while other versions appear to be less efficacious (Forssmann et al., 1997). The reason for the potency difference is unclear but may be related to either the slightly longer C-terminal form of eotaxin 2 used by White et al. or to the expression system used, which combined with the longer C-terminal tail also includes an N-glycosylation site, which is mannose glycosylated. The exact role of chemokine glycosylation in vitro is unclear. Basophil Chemotaxis Along with eotaxin, eotaxin 2 also promotes the chemotaxis of human basophils (Forssmann et al., 1997) with a maximal CI of approximately 18 and an EC50 of 20±30 nM. As with human eosinophils, the chemotactic potency of certain molecular forms of eotaxin 2, appears to be slightly less than that of eotaxin. T Lymphocyte Chemotaxis Eotaxin 2 promoted the chemotaxis of resting (CD4, CD8) but not activated (anti-CD3 activation) T lymphocytes (Patel et al., 1997). The CI for this chemotaxis was 6 with an EC50 of 1±2 nM. Neutrophil Chemotaxis Eotaxin 2 has also been shown to be a weak chemotactic factor for human neutrophils with a CI of 2 (Patel et al., 1997), while others have not been able to demonstrate this activity (Forssmann et al., 1997; White et al., 1997). Inhibition of Hematopoietic Colony Formation Eotaxin 2 (MPIF-2) potently inhibits formation of murine HPP-CFC (high potential proliferativecolony forming cells) colonies (Patel et al., 1997). Approximately 80% of the colonies are inhibited from developing at a concentration of 80 ng/mL (IC50 10 ng/mL). Interestingly, eotaxin 2 does not inhibit the formation of murine low potential proliferative-colony forming cells (LPP-CFC) colonies. This contrasts with MIP-1 which inhibits both
Eotaxin 2 potently promotes shape change in human eosinophils (Sabroe et al., 1999), which typically takes about 5 minutes to complete and occurs at lower concentrations of eotaxin 2 than does chemotaxis, EC50 0.2 nM (White et al., 1997). In addition to shape change, eotaxin 2 also promotes F-actin polymerization which was maximal by about 45 seconds, and thus precedes shape change (Elsner et al., 1998). Actin polymerization occurred over a broad range of eotaxin 2 concentrations (1±500 ng/mL). As for chemotaxis, shape change and actin polymerization appeared to be more sensitive to certain forms of eotaxin 2 than eotaxin stimulation, or some other CCR3 receptor-binding chemokines (Elsner et al., 1998; Sabroe et al., 1999). Presumably, shape change and actin polymerization are prerequisites for chemotaxis and diapedesis. Inflammatory Mediator Production Eotaxin 2 and other CCR3-associated chemokines cause the release of histamine and production of leukotriene C4 (LTC4) from human basophils primed with IL-3 (Forssmann et al., 1997). Eotaxin 2 released histamine and LTC4 with EC50 values of 1.1 and 20 nM, respectively. In addition to histamine and LTC4 production, eotaxin 2 is a weak promoter of superoxide production from human eosinophils at concentrations which are significantly higher than required for shape change or chemotaxis, EC50 50 nM (Elsner et al., 1998). Pathophysiological Role No direct evidence currently exists demonstrating that eotaxin 2 has a direct pathophysiological role. However, as its receptor utilization and expression pattern are similar to those of eotaxin, at least in nasal polyps, this chemokine may well have similar functions to eotaxin.
GENE AND GENE REGULATION
Accession numbers Human: AJ223461 Mouse: AF244367
Eotaxin 2 1233 Figure 1 Nucleotide sequence for human eotaxin 2. GGC GTC ATC ATT AGG AAG TGG AAG GGC
CTG TGT CCC CCT AGC AAG GTC AAG CCT
ATG GCC TCT GAG ACA GGC CAG GCT GTC
ACC CAC CCC AAC TGC CAG AGG TCC CAG
ATA CAC TGC CGA CTC CAG TAC CCT AGA
GTA ATC TGC GTG AAG TTC ATG AGG TAT
ACC ATC ATG GTC GCA TGT AAG GCC CCT
Sequence See Figure 1.
Chromosome location The gene for eotaxin 2 has been localized to chromosome 7q11.23-q21.1 by both somatic cell hybridization (SCH) and radiation hybrid (RH) mapping (Nomiyama et al., 1998).
Cells and tissues that express the gene There has been little work done to date to identify the specific expression of eotaxin 2. However, eotaxin 2 was cloned from an activated human monocyte library, which presumably represents a good source of eotaxin 2. Preliminary studies have shown that the messages for eotaxin and eotaxin 2 are expressed in nasal polyps (Jahnsen et al., 1998). Additionally, eotaxin 2 may also be expressed by skin monocytes/ macrophages as determined by histochemistry. TaqMan analysis indicates very low (0.5 copies/ cyclophilin) in multiple tissues to low expression in uterus, stomach, kidney, and bone marrow (2 copies/ cyclophilin). Recent work has shown that eotaxin 2 and other CCR3-binding chemokines, eotaxin and MCP-4, are produced in the lungs of both atopic and nonatopic asthmatics (Ying et al., 1999a). In addition, skin challenge of atopic patients with allergen (timothy grass pollen) resulted in an immediate (< 6 hours) production of eotaxin 2 message (Ying et al., 1999b).
PROTEIN
Accession numbers Human: CAA11383 Mouse: AAF61736
AGC CCT TTC AGC GGA GGC AAC AGG GGC
CTT ACG TTT TAC GTG GAC CTG GCA AAC
CTG GGC GTT CAG ATC CCC GAC GTG CAA
TTC TCT TCC CTG TTC AAG GCC GCT ACC
CTT GGT GTG GTC AAG AGA TCC AGC ACC ACC CAG GAG AAG CAG GTC AAG 345
39 78 117 156 195 234 273 312
Figure 2 Arrow indicates beginning of mature protein. Triangle indicates C-terminal end of Escherichia coli material produced by PeproTech and baculovirus expressed material used by Forssmann et al. (1997). Diamond indicates C-terminal end of Drosophilaexpressed material used by White et al. (1997), which contains an N-glycosylation site indicated by an asterisk. ↓
GLMTIVTSLL FLGVCAHHII PTGSVVIPSP CCMFFVSKRI 40 PENRVVSYQL SSRSTCLKAG VIFTTKKGQQ FCGDPKQEWV 80 * ♦ QRYMKNLDAK QKKASPRARA VAVKGPVQRY PGNQTTC 117
Sequence See Figure 2.
Important homologies Eotaxin 2 is a member of the CC chemokine family; however, eotaxin 2 demonstrates surprisingly low homology with other chemokines including eotaxin and MCP-4, which utilizes the same receptor as eotaxin 2 (Table 1). The mouse homolog of eotaxin 2 has been cloned, homologs from other species have not been identified. The homology between mouse and human eotaxin 2 is 58% at the amino acid level.
CELLULAR SOURCES AND TISSUE EXPRESSION
Cellular sources that produce Little work has been done on the tissue or cell expression of eotaxin 2. Most of the work undertaken has been at the message level using Taqman analysis, which indicates that few tissues produce eotaxin 2 under resting conditions.
1234 John R. White Table 1 Homology table showing percentage identity for eotaxin 2 at the amino acid level Eotaxin Eotaxin 2
32
Eotaxin
MIP-1
LARC
MCP-1
MCP-2
MCP-3
MCP-4
29.3
21.9
32.0
30.3
29.4
35.4
32.6
24.0
84.0
67.0
67.0
54.4
22.8
36.0
31.5
29.3
32.9
28.0
24.0
24.0
21.5
92.0
84.0
32.0
62.4
53.2
MIP-1 LARC MCP-1 MCP-2 MCP-3
RECEPTOR UTILIZATION Unlike many other chemokines, eotaxin 2 appears to bind to a single seven transmembrane G proteinlinked receptor, CCR3. Eotaxin 2 does not appear to bind to any of the other chemokine receptors identified so far. The main evidence for this is the use of other CCR3-binding chemokines to promote homologous receptor cross-desensitization (Forssmann et al., 1997; Patel et al., 1997; White et al., 1997), or binding studies using the cloned CCR3 receptor (White et al., 1997) and the use of specific anti-CCR3 mAbs to prevent activation of the receptor in human eosinophils (Forssmann et al., 1997). In addition, eotaxin 2 couples to an intracellular signaling system through a pertussis toxin-sensitive G protein (Gi)coupled pathway (Elsner et al., 1998).
58.2
not the growth of LPP-CFC colonies (Patel et al., 1997).
Bioassays used The most frequently used bioassays are Ca2 mobilization (White et al., 1997) using fluorescent probes such as Fura-2 with human eosinophils or with cells stably expressing the CCR3 receptor. Eosinophil chemotaxis can also be used, with a modified Boyden chamber (White et al., 1997).
IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS
IN VITRO ACTIVITIES
Pharmacological effects
In vitro findings
Intradermal injection of eotaxin 2 (100 or 1000 pmol/ site) into the skin of rhesus monkeys leads to the infiltration of eosinophils compared with a control saline injection (Forssmann et al., 1997). Micrographs of skin, examined after 4 hours, showed the selective recruitment of eosinophils into the lumen; in addition eosinophil attachment to endothelial cells was also evident (Forssmann et al., 1997).
Eotaxin 2 has several main functions, including the chemotaxis of human eosinophils (Forssmann et al., 1997; White et al., 1997), resting T lymphocytes (Patel et al., 1997) and basophils (Forssmann et al., 1997). In addition, eotaxin 2 promotes shape change in human eosinophils, which is presumably the prerequisite for cell diapedesing across the endothelial cell layer (Sabroe et al., 1999). The chemokine can also induce inflammatory mediator production including the release of histamine and LTC4 from IL-3-primed human basophils (Forssmann et al., 1997) and the release of free radical oxygen species from eosinophils (Elsner et al., 1998). As with many other chemokines, eotaxin 2 regulates the development of murine hematopoietic cells. Eotaxin 2 leads to the selective suppression of HPP-CFC colonies but
Interactions with cytokine network No specific interactions with other cytokine networks have been demonstrated. However, cross-desensitization of the CCR3 receptor by other CCR3-binding chemokines could modulate the effects of eotaxin 2 on cell recruitment and activation, by leading to the internalization of the receptor.
Eotaxin 2 1235
PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY
Role in experiments of nature and disease states Disease association has not been extensively defined. However, there are a number of diseases associated with the infiltration of eosinophils, basophils, and T cells including asthma, rhinitis, allergy, contact dermatitis, and bronchitic exacerbations. Recent reports in atopic and nonatopic individuals indicate that eotaxin 2 is produced in skin and lung following challenge (Ying et al., 1999a, 1999b). The role of eotaxin 2 in contrast to or in association with the other CCR3-binding chemokines has not yet been elucidated.
IN THERAPY
Effects of therapy: Cytokine, antibody to cytokine inhibitors, etc. As with many other chemokine strategies, the therapeutic potential of this chemokine lies in the antagonism of the receptor rather than inhibition of the chemokine itself. Inhibitors of the CCR3 receptor could benefit a number of TH2, allergy-driven diseases, including asthma, contact dermatitis, chronic obstructive pulmonary disease (COPD), and bronchitic exacerbations.
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