Eosinophils in allergy and related diseases: proceedings of a workshop, Tokyo, Japan, June 21, 2003

Eosinophils in Allergy and Related Diseases Proceedings of a Workshop Tokyo, Japan, June 21, 2003

Guest Editors

Sohei Makino, Tokyo Takeru Ishikawa, Kumamoto Takeshi Fukuda, Tochigi Ken Ohta, Tokyo Itsuo Iwamoto, Chiba Takao Fujisawa, Mie Akio Mori, Kanagawa

23 figures, 1 in color and 3 tables, 2004

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Vol. 134, Suppl. 1, 2004

Contents

1 Preface Mori, A. (Kanagawa)

Reviews 30 Peroxisome Proliferator-Activated Receptor ␥

Regulates Eosinophil Functions: A New Therapeutic Target for Allergic Airway Inflammation

Original Papers 2 Expression of a Human SOCS Protein, HSOCP-1, in

Peripheral Blood Eosinophils from Patients with Atopic Dermatitis Ogawa, K.; Itoh, M.; Miyagawa, M.; Nagasu, T.; Sugita, Y. (Kawasaki); Katsunuma, T.; Akasawa, A.; Matsumoto, K.; Tsujimoto, G.; Saito, H. (Tokyo); Hashida, R. (Kawasaki) 7 Correlation of Allergen-Induced IL-5 and IL-13

Ueki, S.; Matsuwaki, Y.; Kayaba, H.; Oyamada, H.; Kanda, A.; Usami, A.; Saito, N.; Chihara, J. (Akita) 37 Dual Signaling and Effector Pathways Mediate

Human Eosinophil Activation by Platelet-Activating Factor Kato, M. (Gunma); Kita, H. (Rochester, Minn.); Tachibana, A. (Maebashi); Hayashi, Y.; Tsuchida, Y. (Gunma); Kimura, H. (Maebashi)

Production by Peripheral Blood T Cells of Asthma Patients Hashimoto, T.; Akiyama, K.; Kawaguchi, H.; Maeda, Y.; Taniguchi, M. (Sagamihara); Kobayashi, N. (Tokyo); Mori, A. (Sagamihara)

44 Abstracts 46 List of Lectures by Speakers Who Have Not

Submitted Their Manuscripts

12 Molecular Mechanisms of Repression of Eotaxin

Expression with Fluticasone Propionate in Airway Epithelial Cells Matsukura, S.; Kokubu, F.; Kurokawa, M.; Kawaguchi, M.; Kuga, H.; Ieki, K.; Odaka, M.; Suzuki, S.; Watanabe, S.; Takeuchi, H. (Tokyo); Schleimer, R.P. (Baltimore, Md.); Schindler, U. (South San Francisco, Calif.); Adachi, M. (Tokyo) 21 Immunotherapy Attenuates Eosinophil

Transendothelial Migration Induced by the Supernatants of Antigen-Stimulated Mononuclear Cells from Atopic Asthmatics Nagata, M.; Saito, K.; Kikuchi, I.; Tabe, K.; Hagiwara, K.; Kanazawa, M.; Sakamoto, Y. (Iruma-gun) 25 Tyk2 Is Essential for IFN-␣-Induced Gene Expression

in Mast Cells Mori, Y.; Hirose, K.; Suzuki, K.; Nakajima, H.; Seto, Y.; Ikeda, K. (Chiba); Shimoda, K.; Nakayama, K. (Fukuoka); Saito, Y.; Iwamoto, I. (Chiba)

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47 Author Index Vol. 134, Suppl. 1, 2004 48 Subject Index Vol. 134, Suppl. 1, 2004

Int Arch Allergy Immunol 2004;134(suppl 1):1 DOI: 10.1159/000077784

Preface Akio Mori, Kanagawa, Japan

Many cell types and molecules involved in allergic inflammation have been identified. The abundance of eosinophils at the site of inflammation and their relationship to the severity of disease have long supported the significance of this cell type in allergic disorders [1], although several recent investigations employing anti-IL-5 antibody failed to show a clinical benefit [2, 3]. The recent progress in eosinophil biology greatly improved our knowledge of the roles of cytokines, chemokines, their receptors, adhesion molecules, and signal transduction pathways. There is still a lot of work to be done to fully understand the mechanisms of allergy and manage severely ill patients. The 15th Annual Workshop on Eosinophils in Allergy and Related Diseases 2003 was held in Tokyo on June 21, 2003, with presentations in the fields of basic and clinical research on allergy. This workshop provided a forum for both basic and clinical investigators to exchange ideas on the recent advances in the field of eosinophils in relation to allergy. The current topics include migration, activation, degranulation, expression of surface markers, signal transduction molecules, and gene expression of eosinophils. The roles of T cell cytokines and the effect of new treatments were also discussed. The present proceedings include 5 original papers and 2 reviews. Four abstracts, some of which have been published elsewhere, are also included. The remaining 7 titles presented at the workshop are listed at the back of this issue. We hope that these investigations will contribute to a better understanding of allergic diseases. Finally, we would like to express our sincere appreciation to Novartis Pharma KK (Tokyo) for supporting this workshop and to the International Medical Publisher (Tokyo) for excellent assistance in publishing these proceedings.

References 1 Bousquet J, Chanez P, Lacoste JY, et al: Eosinophilic inflammation in asthma. N Engl J Med 1990;323: 1033. 2 Leckie MJ, Brinke A, Khan J, et al: Effects of an interleukin-5 blocking monoclonal antibody on eosinophils, airway hyper-responsiveness, and the late asthmatic response. Lancet 2000;356:2144–2148. 3 Kips JC, O’Connor BJ, Langley SJ, et al: Effect of SCH55700, a humanized anti-human interleukin-5 antibody, in severe persistent asthma. Am J Respir Crit Care Med 2003;167:1655–1659.

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Original Paper Int Arch Allergy Immunol 2004;134(suppl 1):2–6 DOI: 10.1159/000077785

Expression of a Human SOCS Protein, HSOCP-1, in Peripheral Blood Eosinophils from Patients with Atopic Dermatitis Kaoru Ogawa a Mikito Itoh a Masami Miyagawa a Takeshi Nagasu a Yuji Sugita a Toshio Katsunuma b Akira Akasawa b Kenji Matsumoto b Gozoh Tsujimoto b Hirohisa Saito b Ryoichi Hashida a a Genox

Research Inc., Kawasaki, Kanagawa, and b National Research Institute for Child Health and Development, Setagaya, Tokyo, Japan

Key Words Apoptosis W Atopic dermatitis W Cell cycle W Differential display W Eosinophil W Gene expression W HSOCP-1 W Real-time RT-PCR W Yeast two-hybrid screening

Abstract To identify new genes related to atopic dermatitis (AD), we screened for differentially expressed genes in peripheral blood eosinophils derived from AD patients and healthy volunteers. RNA was prepared from peripheral blood eosinophils obtained from both AD patients and healthy volunteers, and the expression of various genes was monitored using fluorescent differential display and real-time RT-PCR. One of the expressed sequence tags (ESTs) was expressed at a significantly higher level in AD patients than in healthy volunteers. A full-length cDNA was identified that encoded a human suppressor of cytokine signaling (SOCS) protein, HSOCP-1, also named hASB-8. The expression of HSOCP-1 was increased in cultured peripheral blood eosinophils after IL-4 stimulation, and overexpression of HSOCP-1 caused cell death in an eosinophil cell line, AML14.3D10. p34SEI-1 was identified as a HSOCP-1-interacting protein by a yeast two-hybrid system. It is a protein that also interacts with the cyclindependent kinase inhibitor p16INK4, suggesting that HSOCP-1 is involved in cell cycle control and apoptosis.

Introduction

Allergic diseases are probably caused by multigene interactions and environmental factors, and eosinophils play important roles in conditions such as asthma [1, 2] and atopic dermatitis (AD) [3, 4]. Eosinophils are significantly involved in the incidence of tissue damage during the chronic phase of allergic diseases. Therefore, a comprehensive investigation of gene expression in eosinophils will be helpful to better understand the pathogenesis of allergic diseases. Recent developments in the technology of gene expression analysis, such as differential display [5, 6] and real-time RT-PCR [7, 8], make it possible to efficiently survey the expression of large numbers of genes in clinical samples. Therefore, we compared gene expression in peripheral blood eosinophils from both AD patients and healthy volunteers and then identified some genes that were differentially expressed in AD patients and healthy volunteers [9]. One of these encoded a human suppressor of cytokine signaling (SOCS) protein, HSOCP-1. SOCS proteins act as intracellular inhibitors of several cytokine signal transduction pathways. Members of the family of SOCS proteins contain a central SH2 domain and a C-terminal homology domain termed the SOCS box [10, 11]. HSOCP-1, which contains four ankyrin repeats and a SOCS box in the C-terminal region, is a gene with an

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Correspondence to: Dr. Kaoru Ogawa Laboratory of Seeds Finding Technology, Eisai Co., Ltd. 5-1-3 Tokodai Tsukuba, Ibaraki 300-2635 (Japan) Tel. +81 29 847 7196, Fax +81 29 847 7614, E-Mail [email protected]

unknown function. In this study, we tried to clarify the pathological importance of HSOCP-1 gene expression in eosinophils.

Materials and Methods Clinical Blood Samples for Fluorescent Differential Display and Real-Time RT-PCR We used 19 and 61 clinical blood samples for fluorescent differential display (FDD) and real-time RT-PCR, respectively [9]. Patients with AD were diagnosed according to the criteria of Hanifin [12], and the severity of AD was judged using a modified version of Leicester’s scoring system [13]. In this study, we obtained samples from 13 healthy volunteers, 15 mild AD (9 males and 6 females), 15 moderate AD (11 males and 4 females) and 18 severe AD (9 males and 9 females) patients for real-time RT-PCR. Purification of Peripheral Blood Eosinophils Granulocytes were isolated from heparinized venous blood of AD patients and healthy volunteers on Ficoll-Paque (Amersham Pharmacia Biotech). Red blood cells were removed by hypotonic lysis. The CD16-negative flow-through eosinophil fractions were collected with MACS separation columns (Miltenyi Biotec), and the purity was checked after Diff-Quick staining (purity was always greater than 99%). Total RNA was prepared using an RNA extraction kit (Isogen solution, Nippon Gene). Culture of Peripheral Blood Eosinophils after Stimulation by Cytokines Eosinophils from 100 ml of healthy volunteer peripheral blood with a purity of more than 99% were suspended in Iscove’s minimal essential medium supplemented with 10% immobilized fetal calf serum and 5 ! 10 –5 M 2-mercaptoethanol. The 24-well cell culture plates were coated with 1% bovine serum albumin in order to avoid binding of eosinophils to the plates. 1 ! 106 cells were incubated with 10-fold decreasing concentrations, ranging from 10 to 0.1 ng/ml, of the cytokines IL-5, IL-4, GM-CSF, IFN-Á and eotaxin. Incubation was for 3, 6 and 18 h, and the reaction was stopped by adding Isogen solution. FDD and Real-Time RT-PCR The FDD analysis of human peripheral blood eosinophils isolated from AD patients was described previously [9]. For quantification of gene expression, the real-time quantitative RT-PCR method was performed using an ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems) to analyze clinical samples as described previously [9]. Expression levels of the human housekeeping genes, ß-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were also assessed in each sample using the standard primers and the TaqMan probe (PE Applied Biosystems). Statistics The nonparametric Dunnet signed rank test was used to evaluate the changes of gene expression among each group of clinical samples. The results were expressed as means B standard error of the mean. p values of less than 0.05 were considered to be significant.

HSOCP-1 Expression in Eosinophils

Cloning of the HSOCP-1 Gene A 189-bp DNA fragment was obtained by FDD, and then the ClonCapture cDNA Selection Kit (Clontech) [14] and human placenta ClonCapture cDNA library (Clontech) were used to elongate the sequence. Plasmid Construction and Transient Transfection into AML14.3D10 Cells For expression of an HSOCP-1-EGFP fusion protein, a fragment containing the HSOCP-1 sequence was amplified by PCR using cloned HSOCP-1 as a template; the primers were: 5)-CCCAAGCTTGGGCCACCATGAGTTCCAGTATG-3) and 5)-CGGGATCCCGCTGTTCTAAAAGTAACAG-3). The PCR product was digested with HindIII and BamHI, and cloned into pEGFP-N1 (Clontech). AML14.3D10 cells [15], obtained from Wright State University, were grown in RPMI 1640 containing 10% immobilized fetal calf serum, 5 ! 10 –5 M 2-mercaptoethanol and 1 mM sodium pyruvate. HSOCP-1 cDNA were transfected by electroporation into AML14.3D10 cells. 8 ! 106 cells were electroporated with 10 Ìg plasmid DNA at 800 ÌF and 300 V. Yeast Two-Hybrid Screening Yeast two-hybrid screening was performed using the MATCHMAKER GAL4 Two-Hybrid System (Clontech) according to the manufacturer’s instructions. The open reading frame (ORF) of the HSOCP-1 gene was fused to the GAL4 DNA-binding domain as bait. Approximately 1.0 ! 106 yeast colonies were screened with the human leukocyte MATCHMAKER cDNA library (Clontech), and positive colonies were selected by their expression of four reporter genes, ADE2, HIS3, MEL1 and lacZ. Positive colonies can grow in the SD medium without adenine and histidine, and have ·-galactosidase and ß-galactosidase activity, showing a blue color with X-·-gal and X-gal, respectively.

Results

We selected 18 sequences with enhanced mRNA expression in eosinophils from AD patients, and identified 6 ORFs among these 18 sequences [9]. After sequencing of the FDD bands and cDNA cloning with the ClonCapture cDNA Selection Kit, one of these ORFs was proved to be the human SOCS protein, HSOCP-1, named by Incyte Pharmaceuticals. The HSOCP-1 expression level, as determined by realtime RT-PCR, was significantly higher in eosinophils from AD patients than in those from healthy volunteers (fig. 1). On the other hand, the HSOCP-1 expression in eosinophils from healthy volunteers was increased by IL-4 stimulation in a time-dependent and dose-dependent manner (fig. 2), though the expression was not increased by IFN-Á, GM-CSF, eotaxin or IL-5. Among healthy human peripheral blood leukocytes, the basal HSOCP-1 expression level was dominant in granulocytes. The fractions consisting of T cells, B cells and monocytes showed little HSOCP-1 expression (data not shown).

Int Arch Allergy Immunol 2004;134(suppl 1):2–6

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To identify the function of HSOCP-1, the EGFP-fused HSOCP-1 gene was transiently introduced and expressed in AML14.3D10 eosinophilic cells. While the cells expressing control EGFP showed no morphological changes, the cells expressing EGFP-fused HSOCP-1 were sickly and appeared to be dying (fig. 3). We screened for proteins that interact with HSOCP-1 using a yeast twohybrid system, and identified p34SEI-1 as a protein that binds to it (data not shown).

Discussion

A novel SOCS protein with an unknown function, HSOCP-1, was cloned from eosinophil fractions of AD patients. HSOCP-1 contains four ankyrin repeats and a SOCS box at its C-terminus. It was expressed at a higher level in eosinophils of AD patients than in eosinophils of healthy volunteers (fig. 1). Although the ages of the AD patients and the healthy volunteers were not matched, age is unlikely to account for the differential expression because there were no differences between the younger and older AD patients [16, 17]. HSOCP-1 expression in eosinophils from healthy volunteers was increased by IL-4 stimulation in a time-dependent and dose-dependent

Fig. 1. Expression levels of HSOCP-1 mRNA in peripheral blood

eosinophils. The expression of HSOCP-1 in eosinophil samples from healthy volunteers and patients with mild, moderate and severe AD is shown. *p ! 0.05; **p ! 0.01; ***p ! 0.001. The copy numbers of each transcript per 1 ng RNA, standardized by levels of the GAPDH transcript, are shown on the ordinate.

Fig. 2. Expression levels of HSOCP-1 mRNA in peripheral blood eosinophils of healthy volunteers after cytokine stimulation. The dose-response (a) and the time course-response (b) of HSOCP-1 expression are shown. The copy

numbers of each transcript per 1 ng RNA, standardized by levels of the ß-actin transcript, are shown on the ordinate. The mean expression levels B SE are derived from duplicate experiments. Similar results were obtained in three independent experiments.

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Fig. 3. HSOCP-1-EGFP fusion protein expression in AML14.3D10 cells. Cells expressing EGFP are shown in bright field (b) and in fluorescent and bright field superimposed images (a). Cells expressing EGFP fused to HSOCP-1 are shown in bright field (d) and in fluorescent and bright field superimposed images (c). Arrowheads indicate the cells

that expressed the HSOCP-1-EGFP fusion protein or EGFP.

manner, but was not affected by IFN-Á, GM-CSF, eotaxin or IL-5 (fig. 2). These results showed that HSOCP-1, whose domain structure suggests that it is located in the cytoplasm, is part of an IL-4 signaling pathway. It has been reported that IL-5 and GM-CSF prolonged eosinophil survival and inhibited apoptosis [18]. In contrast, IL-4 induced apoptosis in peripheral blood eosinophils [18]. Therefore, our expression results suggest that HSOCP-1 might be involved in peripheral blood eosinophil apoptosis in pathological conditions. We reported previously that several genes including cytokine receptors were highly expressed in eosinophils from AD patients and in IL-4-stimulated eosinophils [19]. However, the relationships between these genes and HSOCP-1 remained unknown because the expression of almost all of these genes was also increased by IL-5 stimulation. HSOCP-1 is identical to the human ASB-8 protein. Its recently reported gene contains four ankyrin repeats and one SOCS box [20]. ASB-8 was expressed in human heart, brain, placenta, liver, kidney, pancreas, and was especially highly expressed in skeletal muscle [20]. We also examined HSOCP-1 expression by multiple-tissue Northern

blot (Clontech) and detected transcripts in spleen, prostate, testis, ovary, colon and peripheral blood leukocytes (data not shown), indicating that HSOCP-1 is expressed in many tissues including immune tissues. Although it was reported that there were several proteins besides those in the SOCS family that contain a SOCS box, the function of these SOCS box-containing proteins that contain ankyrin repeats is unknown [11]. To clarify the functional role of HSOCP-1, HSOCP-1 DNA was transfected into an eosinophilic cell line, AML14.3D10. After transfection, the GFP-fused HSOCP-1-expressed cells underwent dramatic morphological changes and began to die (fig. 3). These results suggest that overexpression of HSOCP-1 promotes apoptosis in AML14.3D10 cells. The yeast two-hybrid system identified p34SEI-1 as a HSOCP-1 interacting protein. p34SEI-1 was previously reported to antagonize the function of p16INK4a, a member of the INK4 family that inhibits cyclin-dependent kinase CDK4/CDK6 [21]. In mammalian cells, the cyclin D-CDK4 complex regulates entry into the cell cycle and passage through the restriction point in late G1 [22]. p34SEI-1 and p16INK4a regulate cyclin D-

HSOCP-1 Expression in Eosinophils

Int Arch Allergy Immunol 2004;134(suppl 1):2–6

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CDK4 complex activity and the cell cycle, suggesting that HSOCP-1 may be involved in cell cycle progression or cell cycle arrest, leading to apoptosis. Since HSOCP-1 is expressed in many tissues, especially in skeletal muscle, it may also be involved in other functions besides apoptosis in various tissues. In this paper, we report that HSOCP-1 is highly expressed in human peripheral eosinophils, and we suggest that it plays a spe-

cific role in apoptosis. Although the exact function of HSOCP-1 is still not clear, we suggest that it might be involved in the pathology of AD because of its high expression in eosinophils obtained from AD patients. Therefore, it is hoped that these results will be helpful in the search for new therapeutic targets to treat allergic diseases such as AD.

References 1 Gleich GJ: Mechanisms of eosinophil-associated inflammation. J Allergy Clin Immunol 2000;105:651–663. 2 Holt PG, Macaubas C, Stumbles PA, Sly PD: The role of allergy in the development of asthma. Nature 1999;402(suppl):B12–17. 3 Kristin M, Leiferman MD: Eosinophils in atopic dermatitis. J Allergy Clin Immunol 1994;94:1310–1317. 4 Kapp A: The role of eosonophils in the pathogenesis of atopic dermatitis – Eosinophil granule proteins as markers of disease activity. Allergy 1993;48:1–5. 5 Liang P, Pardee AB: Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 1992;257: 967–971. 6 Ito T, Kito K, Adati N, Mitsui Y, Hagiwara H, Sakaki Y: Fluorescent differential display: Arbitrary primed RT-PCR fingerprinting on an automatic DNA sequencer. FEBS Lett 1994; 351:231–236. 7 Lie YS, Petropoulos CJ: Advances in quantitative PCR technology: 5)-Nuclease assays. Curr Opin Biotechnol 1998;9:43–48. 8 Kafert S, Krauter J, Ganser A, Eder M: Differential quantitation of alternatively spliced messenger RNAs using isoform-specific real-time RT-PCR. Anal Biochem 1999;269:210–213. 9 Hashida R, Ogawa K, Miyagawa M, Sugita Y, Takahashi E, Nagasu T, Katsunuma T, Akasawa A, Tsujimoto G, Matsumoto K, Saito H: Analysis of gene expression in peripheral blood eosinophils from patients with atopic dermatitis by differential display. Int Arch Allergy Immunol 2003;131(suppl 1):26–33.

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10 Starr R, Willson TA, Viney EM, Murray LJ, Rayner JR, Jenkins BJ, Gonda TJ, Alexander WS, Metcalf D, Nicola NA, Hilton DJ: A family of cytokine-inducible inhibitors of signalling. Nature 1997;387:917–921. 11 Hilton DJ, Richardson RT, Alexander WS, Viney EM, Willson TA, Sprigg NS, Starr R, Nicholson SE, Metcalf D, Nicola NA: Twenty proteins containing a C-terminal SOCS box form five structural classes. Proc Natl Acad Sci USA 1998;95:114–119. 12 Hanifin JM: Basic and clinical aspects of atopic dermatitis. Ann Allergy 1984;52:386–395. 13 Nomura I, Tanaka K, Tomita H, Katsunuma T, Ohya Y, Ikeda N, Takeda T, Saito H, Akasawa A: Evaluation of the staphylococcal exotoxins and their specific IgE in childhood atopic dermatitis. J Allergy Clin Immunol 1999;104: 441–446. 14 Teintze M, Arzimanoglou II, Lovelace C, Xu Z, Rigas B: RecA-assisted rapid enrichment of specific clones from model DNA libraries. Biochem Biophys Res Commun 1995;211:804– 811. 15 Baumann MA, Paul CC: The AML14 and AML14.3D10 cell lines: A long-overdue model for the study of eosinophils and more. Stem Cells 1998;16/1:16–24. 16 Matsumoto Y, Oshida T, Obayashi I, Imai Y, Matsui K, Yoshida NL, Nagata N, Ogawa K, Obayashi M, Kashiwabara T, Gunji S, Nagasu T, Sugita Y, Tanaka T, Tsujimoto G, Katsunuma T, Akasawa A, Saito H: Identification of highly expressed genes in peripheral blood T cells from patients with atopic dermatitis. Int Arch Allergy Immunol 2002;129:327–340.

17 Heishi M, Kagaya S, Katsunuma T, Nakajima T, Yuki K, Akasawa A, Maeda M, Gunji S, Sugita Y, Tsujimoto G, Saito H: High-density oligonucleotide array analysis of mRNA transcripts in peripheral blood cells of severe atopic dermatitis patients. Int Arch Allergy Immunol 2002;129:57–66. 18 Wedi B, Raap U, Lewrick H, Kapp A: IL-4induced apoptosis in peripheral blood eosinophils. J Allergy Clin Immunol 1998;102:1013– 1020. 19 Ogawa K, Hashida R, Miyagawa M, Kagaya S, Sugita Y, Matsumoto K, Katsunuma T, Akasawa A, Tsujimoto G, Saito H: Analysis of gene expression in peripheral blood eosinophils from patients with atopic dermatitis and in vitro cytokine-stimulated blood eosinophils. Clin Exp Immunol 2003;131:436–445. 20 Liu Y, Li J, Zhang F, Qin W, Yao G, He X, Xue P, Wan D, Gu J: Molecular cloning and characterization of the human ASB-8 gene encoding a novel member of ankyrin repeat and SOCS box containing protein family. Biochem Biophys Res Commun 2003;300:972–979. 21 Sugimoto M, Nakamura T, Ohtani N, Hampson L, Hampson IN, Shimamoto A, Furuichi Y, Okumura K, Niwa S, Taya Y, Hara E: Regulation of CDK4 activity by a novel CDK4binding protein, p34SEI-1. Genes Dev 1999;13: 3027–3033. 22 Sherr CJ: Mammalian G1 cyclins. Cell 1993; 73:1059–1065.

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Original Paper Int Arch Allergy Immunol 2004;134(suppl 1):7–11 DOI: 10.1159/000077786

Correlation of Allergen-Induced IL-5 and IL-13 Production by Peripheral Blood T Cells of Asthma Patients Tomomi Hashimoto a Kazuo Akiyama a Hiroshi Kawaguchi a Yuji Maeda a Masami Taniguchi a Noriaki Kobayashi b Akio Mori a a Clinical

Research Center for Allergy and Rheumatology, National Sagamihara Hospital, Kanagawa, and of Biotechnology, Tokyo Technical College, Kunitachi, Tokyo, Japan

b Department

Key Words Atopic dermatitis W Bronchial asthma W IL-5 W IL-13 W T cell

Abstract Background: Helper T cells and T cell cytokines are implicated in allergic disorders such as asthma and atopic dermatitis (AD). We reported enhanced interleukin-5 (IL5) production by peripheral blood T cells of asthmatic patients. Production of cytokines, particularly IL-5 and IL13, by peripheral blood mononuclear cells (PBMC) obtained from bronchial asthma (BA) and AD patients was investigated in the present study. Methods: PBMC were cultured in the presence of either polyclonal activator, phorbol ester plus Ca2+ ionophore or Dermatophagoides farinae (Df) antigen. The resulting supernatants were assayed for IL-4, IL-5, and IL-13 by specific ELISAs. Results: IL-5 and IL-13 production in response to Df antigen is significantly higher in allergic groups compared to control subjects. IL-5 responses induced by Df antigen were strongly correlated with IL-13 responses. Conclusion: In spite of IL-13 gene proximity to IL-4 gene, IL-5 production was more strongly correlated to IL-13 production in the BA group, suggesting a common control mechanism that regulates the IL-5 and IL-13 gene. Copyright © 2004 S. Karger AG, Basel

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Introduction

The critical role of activated T cells and T cell cytokines in bronchial asthma (BA) and atopic dermatitis (AD) has been well established [1–3]. Th2 cells influence allergic diseases through the secretion of an array of cytokines (IL-4, IL-5, IL-13) that activate inflammatory and residential cells both directly and indirectly. IL-5 is a key factor for eosinophilia and thought to be responsible for the tissue damage observed in chronic asthma [2, 3]. IL-4 is essential for IgE synthesis by B cells. Increased IL-4 and decreased interferon gamma production have been postulated to promote IgE production in AD [4]. IL-13 shares many of the properties of IL-4. IL-13 and IL-4 genes are located a short distance apart and tend to be coordinately expressed [5]. On the other hand, other investigators reported that IL-4 and IL-13 production was independently regulated [6, 7]. We previously reported that IL-5 production by peripheral blood T cells of asthmatic patients in response to Dermatophagoides farinae (Df) antigen was highly enhanced compared to healthy subjects [8]. IL-13 production in response to Df antigen has never been examined so far. Therefore, in this study we investigated cytokine production by peripheral blood mononuclear cells (PBMC) obtained from AD and BA patients, and a possible correlation between IL-5 and IL-13 synthesis was explored.

Correspondence to: Dr. A. Mori 18-1 Sakuradai Sagamihara, Kanagawa 228-8522 (Japan) Tel. +81 42 742 8311, Fax +81 42 742 7990 E-Mail [email protected]

Material and Methods Subjects Adult patients with AD and BA referred to the Sagamihara National Hospital were enrolled in this study. Informed consent was obtained from each subject. The study protocol was approved by the local ethics committee of the institute. 14 AD patients [mean age B standard error of the mean (SEM): 30.2 B 2.2 years] and 27 BA patients (aged 37.8 B 2.9 years) were selected on the basis of disease phenotypes, showing either AD or BA alone. All the BA patients were mild asthmatics and did not receive inhaled corticosteroids at the time of the study. 7 healthy control subjects (aged 58.1 B 4.3 years) were also recruited. All the patients with AD and BA showed a positive radioallergosorbent test against house dust mite (score more than 2). Total serum IgE level (mean B SEM) was 3,189.1 B 1,203.1, 706.9 B 281.9, and 90.1 B 29.2 IU for AD, BA, and control subjects, respectively. Peripheral blood eosinophils (mean B SEM) were 517.9 B 82.3, 408.6 B 63.7, and 214.3 B 42.5 cells/Ìl for AD, BA, and control subjects, respectively. Cell Cultures All blood samples were collected at the time of initial examination. PBMC were prepared by Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden) density gradient centrifugation as described previously [8]. Cells were washed and suspended in AIM-V medium (Invitrogen, Carlsbad, Calif., USA) at a density of 2 ! 106 cells/ml. PBMC were cultured in the presence of 20 nM phorbol 12-myristate 13-acetate (PMA; Sigma, St. Louis, Mo., USA) plus 1 ÌM ionomycin (Calbiochem, La Jolla, Calif., USA) for 24 h. In some experiments, PBMC were cultured with Df mite extract (Torii Pharmaceutical, Chiba, Japan) for 6 days. The resulting supernatants were harvested and frozen at –30 ° C until use. Quantitation of Cytokines in the Culture Supernatants The resulting supernatants were assayed for IL-4 and IL-13 using OptEIATM ELISA sets (BD Biosciences, San Diego, Calif., USA), according to the manufacturer’s directions. IL-5 was measured by a sandwich ELISA using monoclonal anti-human IL-5 (D138) as the capture antibody and biotinylated purified rabbit anti-human IL-5 as the second antibody as described previously [9]. Statistical Analysis Statistical analysis was performed with Welch’s t test, and a p value ! 0.05 was considered statistically significant. Results are presented as the mean B SEM. Correlations were determined using nonparametric statistics (Spearman’s rank sum).

Results and Discussion

Cytokine production stimulated by a polyclonal activator, PMA plus IOM, was first compared between the BA patients, AD patients, and control subjects. Supernatants were collected after 24 h of culture and assayed for IL-4, IL-5, and IL-13. IL-5 production by PBMC obtained from BA and AD patients was significantly higher than that from control subjects (fig. 1a), whereas there was no significant difference in IL-5 production between BA and

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AD patients. IL-13 production by PBMC obtained from BA patients was significantly higher than that of AD patients and control subjects, while IL-13 production by PBMC of AD patients was not significantly different from that of control subjects (fig. 1b). IL-4 production by PBMC of AD patients was not different from that of control subjects (data not shown), although the total serum IgE level of AD patients was significantly higher than that of BA patients and control subjects. Arguing that antigen-mediated activation would provide a biologically more relevant comparison of responses in the three groups, we next evaluated cytokine synthesis induced by Df antigen. IL-5 production by PBMC obtained from BA and AD patients was significantly higher than that of control subjects (fig. 2a). IL-5 production of BA patients was significantly higher than that of AD patients. Df antigen-induced IL-13 production by PBMC of BA and AD patients was significantly higher than that of control subjects. IL-13 production of BA patients was significantly higher than that of AD patients (fig. 2b). A possible relationship between IL-4, IL-5, and IL-13 production in response to Df antigen was examined next. As shown in figure 3, the amounts of IL-5 and IL-13 produced by PBMC obtained from BA patients were significantly correlated, whereas those from AD patients were not significantly correlated. In addition, there was no correlation between IL-4 and IL-5 production in the AD and BA groups. Our present finding that IL-5 production by PBMC obtained from BA and AD patients in response to PMA plus IOM was significantly higher than that of control subjects (fig. 1a) is consistent with our previous report that IL-5 production by PBMC obtained from BA patients is significantly enhanced compared to control subjects [9] and further extends the notion of IL-5 dysregulation into AD status. As eosinophilic inflammation is a common pathological feature both in AD and BA, IL-5producing T cells might be involved in eosinophil activation in AD as well. IL-13 production in response to PMA plus IOM was also upregulated in BA and AD patients (fig. 1b). Li et al. [10] reported that PHA-induced IL-13 production was not significantly different between allergic patients and control subjects. The discrepancy might stem from the fact that they enrolled rhinitis patients allergic to grass pollens, while asthma and eczema patients were enrolled in the current investigation. IL-5 production in response to Df antigen was highly elevated in BA patients, consistent with our previous reports [11]. The finding that IL-5 production in response to Df antigen by AD patients was also higher than that of

Hashimoto/Akiyama/Kawaguchi/Maeda/ Taniguchi/Kobayashi/Mori

Fig. 1. IL-5 (a) and IL-13 (b) production by PBMC of AD, BA, and control subjects in response to polyclonal activator. PBMC were obtained from 27 BA, 14 AD and 7 control subjects, and stimulated with PMA (20 nM ) + IOM (1 ÌM ) for 24 h. IL-5 and IL-13 concentrations in the supernatants were assayed by ELISA. Each point represents an individual subject. *p ! 0.05 is considered to be statistically significant (t test).

Fig. 2. IL-5 (a) and IL-13 (b) production by PBMC of AD, BA, and control subjects in response to Df antigen. PBMC

obtained from 14 AD, 27 BA, and 7 control subjects were cultured for 6 days in the presence of Df mite extract (10 Ìg/ml). IL-5 and IL-13 levels in the culture supernatants were determined by ELISA. Each point represents an individual subject. *p ! 0.05 is considered to be statistically significant (t test).

Coordinate Synthesis of IL-5 and IL-13

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Fig. 3. Correlation among IL-4, IL-5, and IL-13 in response to Df antigen. Data were evaluated using Spearman’s

rank sum test for each subject participating. rS = Spearman’s rank correlation.

control subjects, but significantly lower than that of BA patients might suggest a different distribution of miteresponsive T cells, or different antigen specificity of IL5-producing T cells between AD and BA. Heightened IL13 production in response to Df antigen by BA and AD patients is consistent with the report of Li et al. [10]. As IL-13 enhances IgE production by human B cells [12], our finding is consistent with the view that IL-13 overproduction by Th cells causes the exaggerated IgE response in AD patients. The reason why IL-13 production by the BA group is much higher than that by the AD group is not clear at this moment, considering that mitespecific IgE titer of AD patients was by far higher than that of BA patients. The most impressive finding in the

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current study is that the amounts of IL-5 and IL-13 produced in response to Df antigen were highly correlated, suggesting that IL-5 and IL-13 were produced by the same T cell populations or controlled under similar signal requirements. In this study, we have found that IL-5 production is induced by IL-2 and at the same time dependent on autologically produced IL-2 [13]. IL-13 production was also reportedly induced by IL-2 [14], although IL-2 dependence of IL-13 synthesis has not yet been proven. The close regulation between IL-5 and IL-13 production may suggest a common trans-regulatory mechanism between the two cytokine genes, considering that relatively less proximity between the two genes is noted in comparison to that between IL-13 and IL-4 genes.

Hashimoto/Akiyama/Kawaguchi/Maeda/ Taniguchi/Kobayashi/Mori

Enhanced cytokine responses of PBMC of AD and BA patients may imply that both diseases are considered system disorders rather than local dysfunctions. The lack of the association in the IL-5 and IL-13 production in AD patients is intriguing considering the close relationship between the two cytokines in the BA group. This finding may suggest that the sensitization process of cellular immunity by Df antigen might be different in some aspects among diseases, i.e. routes of sensitization or more precise antigen specificities.

Acknowledgments The authors gratefully acknowledge the technical assistance of Ms. Minako Chujo and the secretarial assistance of Mr. Akira Fujii. This work was supported in part by Hearth and Labour Sciences Research Grants for Research on Allergic Diseases and Immunology (A.M.) and a grant from the Takeda Science Foundation (A.M.).

References 1 Renauld JC: New insight into the role of cytokines in asthma. J Clin Pathol 2001;54:577– 589. 2 Hamelmann E, Gelfand EW: IL-5-induced airway eosinophilia – The key to asthma? Immunol Rev 2001;179:182–191. 3 Kay AB: ‘Helper’ (CD4+) T cells and eosinophils in allergy and asthma. Am Rev Respir Dis 1992;145:S22–S26. 4 Kristin M: Eosinophils in atopic dermatitis. J Allergy Clin Immunol 1994;4:310–317. 5 Dolganov G, Bort S, Lovett M, Burr J, Schubert L, Short D, McGurn M, Gibson C, Lewis DB: Coexpression of the interleukin-13 and interleukin-4 genes correlates with their physical linkage in the cytokine gene cluster on human chromosome 5q23–31. Blood 1996;87: 3316–3326. 6 Van der Pouw Kraan TC, Boeije LC, Troon JT, Rutschmann SK, Wijdenes J, Aardenman LA: Human IL-13 production is negatively influenced by CD3 engagement: Enhancement of IL-13 production by cyclosporin A. J Immunol 1996;156:1818–1823.

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7 Minty A, Asselin S, Bensussan A, Shire D, Vita N, Vyakarmam A, Wijdenes J, Ferrara P, Caput D: The related cytokines interleukin 13 and interleukin 4 are distinguished by differential production and differential effects on T lymphocytes. Eur Cytokine Netw 1997;8:203–213. 8 Mori A, Yamamoto K, Dohi M, Suko M, Okudaira H: Interleukin-4 gene expression in human peripheral blood mononuclear cells. Int Arch Allergy Appl Immunol 1995;95:282–284. 9 Mori A, Suko M, Nishizaki Y, Kaminuma O, Kobayashi S, Matsuzaki G, Yamamoto K, Ito K, Tsuruoka N, Okudaira H: IL-5 production by CD4+ T cells of asthmatic patients is suppressed by glucocorticoids and immunosuppressants FK506 and cyclosporin A. Int Immunol 1995;7:449–457. 10 Li Y, Simons FE, HayGlass KT: Environmental antigen-induced IL-13 responses are elevated among subjects with allergic rhinitis, are independent of IL-4, and are inhibited by endogenous IFN-gamma synthesis. J Immunol 1998;161:7007–7014.

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11 Mori A, Kaminuma O, Suko M, Mikami T, Nishizaki Y, Ohmura T, Hoshino A, Asakura Y, Miyazawa K, Ando T, Okumura Y, Yamamoto K, Okudaira H: Cellular and molecular mechanisms of IL-5 synthesis in atopic diseases: A study with allergen-specific human helper T cells. J Allergy Clin Immunol 1997; 100:S56–S64. 12 Wynn TA: IL-13 effector functions. Annu Rev Immunol 2003;21:425–456. 13 Mori A, Suko M, Kaminuma O, Nishizaki Y, Mikami T, Ohmura T, Hoshino A, Inoue S, Tsuruoka N, Okumura Y, Sato G, Ito K, Okudaira H: A critical role of IL-2 for the production and gene transcription of IL-5 in allergenspecific human T cell clones. Int Immunol 1996;8:1889–1895. 14 Verheyen J, Bonig H, Kim TM, Banning U, Mauz-Korholz C, Kramm C, Korholz D: Regulation of interleukin-2 induced interleukin-5 and interleukin-13 production in human peripheral blood mononuclear cells. Scand J Immunol 2000;51/1:45–53.

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Original Paper Int Arch Allergy Immunol 2004;134(suppl 1):12–20 DOI: 10.1159/000077787

Molecular Mechanisms of Repression of Eotaxin Expression with Fluticasone Propionate in Airway Epithelial Cells Satoshi Matsukura a Fumio Kokubu a Masatsugu Kurokawa a Mio Kawaguchi a Hideki Kuga a Koushi Ieki a Miho Odaka a Shintaro Suzuki a Shin Watanabe a Hiroko Takeuchi a Robert P. Schleimer b Ulrike Schindler c Mitsuru Adachi a a First

Department of Internal Medicine, Showa University School of Medicine, Tokyo, Japan; Hopkins Asthma and Allergy Center, Baltimore, Md., and c Tularik, Inc., South San Francisco, Calif., USA

b Johns

Key Words Asthma W Airway epithelial cells W Eotaxin W Fluticasone W Glucocorticoid W Nuclear factor-kappa B W Signal transducers and activators of transcription 6

Abstract Background: Glucocorticoids are known to repress the expression of CC chemokine eotaxin in airway epithelial cells. We focused our study on the molecular mechanisms of the glucocorticoid, fluticasone, in the inhibition of the expression of the eotaxin gene in the cells. Methods: The airway epithelial cell line, BEAS-2B, was stably transfected with signal transducers and activators of transcription 6 (STAT6)-expressing vector and used in the following experiments to clarify the function of STAT6. Levels of eotaxin mRNA and protein expression were determined with RT-PCR and ELISA. Mechanisms of transcriptional regulation were assessed by the electrophoretic mobility shift assay and dual luciferase assay using eotaxin promoter-luciferase reporter plasmids. Results: Fluticasone significantly inhibited the induction of eotaxin protein stimulated with TNF-· and IL-4 in the

ABC

© 2004 S. Karger AG, Basel 1018–2438/04/1345–0012$21.00/0

Fax + 41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/iaa

cells. Fluticasone also repressed the induction of eotaxin mRNA with these stimuli. It partially inhibited the activity of eotaxin promoter; however, it did not inhibit the nuclear translocation and binding of transcription factors, nuclear factor-kappa B (NF-ÎB) or STAT6, to the DNA derived from the proximal promoter region of the eotaxin gene. Moreover, the inhibitory effect was also conserved in the experiments using the reporter plasmid of which the putative glucocorticoid-responsive element was mutated. Conclusions: Fluticasone inhibits the expression of eotaxin gene in airway epithelial cells in part through repression of the transcription. However, the mechanisms depend neither on the inhibition of transcription factors’ translocation into nuclei nor the function of the putative glucocorticoid-responsive element in the promoter, indicating that other mechanisms would be related to the transcriptional repression of the eotaxin gene in airway epithelial cells. Copyright © 2004 S. Karger AG, Basel

Correspondence to: Dr. Satoshi Matsukura First Department of Internal Medicine Showa University School of Medicine 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8666 (Japan) Tel. +81 3 3784 8532, Fax +81 3 3784 8742, E-Mail [email protected]

Introduction

Asthma is a disease characterized by variable airflow obstruction, chronic airway inflammation and bronchial hyperresponsiveness. Main cells which infiltrate into the airway are Th2 lymphocytes and eosinophils [1–3]. Th2 cells are known to be a key factor in causing allergic responses, such as synthesis of IgE and subsequent release of chemical mediators from basophils and mast cells. The Th2 cytokines, IL-4 and IL-13, are known to stimulate the expression of several chemokines, such as eotaxin, eotaxin-2, eotaxin-3, MCP-4, TARC and adhesion molecule VCAM-1 [4–10]. These chemokines and adhesion molecules play a pivotal role in the infiltration of inflammatory cells into the epithelium. Th2 cells also produce IL-5 which activates eosinophils. Activated eosinophils infiltrate airway epithelium and cause epithelial damage and inflammation by releasing major basic protein and eosinophil cationic protein. Glucocorticoids (GC) are potent drugs used to treat inflammatory diseases. Inhaled corticosteroid is known to be effective in the treatment of asthma. It reduces airway inflammation and bronchial hyperresponsiveness of asthmatics. While there have been reports on many of the mechanisms of how inhaled corticosteroid acts in the airway, one of them may be inhibition of inflammatory function of airway epithelial cells. Airway epithelial cells have been classically thought of as barrier cells of the airway wall. However, recently accumulated data suggest that airway epithelial cells also act as effector cells and contribute to the pathogenesis of asthma. Epithelial cells produce and secrete several inflammatory mediators, including oxygen radicals, lipid mediators, and cytokines [11]. Among of a number of cytokines produced by epithelial cells, we focused our study on the molecular mechanisms of the regulation of eotaxin expression. The CC chemokine, eotaxin, was originally discovered as the predominant eosinophil chemoattractive cytokine present in bronchoalveolar lavage fluids of antigen-challenged guinea pigs [12]. Accumulated data have suggested an important role of eotaxin in allergic inflammatory diseases. Upregulation of eotaxin expression in the airway has been reported in asthmatics and animal models of asthma [13–18]. It mediates eosinophil migration via the CCR3 receptor and also has chemotactic activity for basophils, mast cells and T cells, especially Th2 cells. Histological studies have shown that a primary cell type expressing eotaxin is airway epithelial cells [19, 20]. Investigators have reported that GC repress eotaxin expression in epithelium and this effect correlates with

Mechanisms of Inhibition of Eotaxin Expression with Fluticasone

decreased allergic inflammation of airway in asthma and allergic rhinitis [21, 22]. These inhibitory effects of GC on the eotaxin expression have also been confirmed in in vitro studies, while these molecular mechanisms in the regulation of eotaxin transcription have not yet been well understood [5, 23]. Previous reports have demonstrated that tumor necrosis factor alpha (TNF-·) and IL-4 synergistically stimulated eotaxin expression in epithelial cells and fibroblasts [4, 5]. A similar synergistic effect on the induction of eotaxin expression in airway epithelial cells has been observed with TNF-· and IL-13 [24]. We have demonstrated that the eotaxin promoter has adjacent binding sites for nuclear factor-kappa B (NF-ÎB) and signal transducers and activators of transcription 6 (STAT6). These two transcription factors activate eotaxin transcription and subsequent mRNA and protein induction in response to TNF-· and IL-4/IL-13 stimulation, respectively, in airway epithelial cells [24, 25]. The importance of these two transcription factors in the regulation of eotaxin expression was recently indicated in other cell types in in vitro studies and in animal models of asthma [26–29]. In the present study, we show that the GC, fluticasone propionate, represses the expression of eotaxin protein and mRNA induced by TNF-· and IL-4, in airway epithelial cells. We next focused our study on the possible mechanisms of fluticasone in the repression of eotaxin transcription in the cells.

Materials and Methods Cell Culture and Stable Transfection BEAS-2B is a human airway epithelial cell line transformed with the adenovirus 12-SV40 hybrid virus (a kind gift from Dr. Curtis Harris) [30]. This cell line is reported to express eotaxin in response to stimulation with cytokines, but expresses a minimum level of STAT6 [24, 25]. To observe the action of STAT6 in epithelial cells using electrophoretic mobility shift assay (EMSA), BEAS-2B cells were stably transfected with STAT6-expressing vector [31]. Transfected cells were selected with G418 (Invitrogen, Tokyo, Japan) and referred to as STAT6-BEAS cells. The cells were cultured in DMEM/ F12 with 10% FBS, 100 U/ml penicillin and 100 ng/ml streptomycin (Invitrogen) at 37 ° C with 5% CO2 in humidified air. In some experiments, cells were preincubated with fluticasone propionate (GlaxoSmithKline) or its diluent, dimethyl acetamide (DMA, Sigma-RBL, Tokyo, Japan) and then stimulated with TNF-· and/or IL-4 (R&D Systems, Tokyo, Japan). Assay of Eotaxin Protein Release into the Culture Medium Concentrations of eotaxin in the collected culture medium were determined with a commercially available system for ELISA (R&D Systems). The standards and samples were added to 96-well micro-

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titer plates coated with anti-eotaxin antibody. After incubation at room temperature for 2 h, each well was washed 5 times with wash buffer. Biotinylated antibody against eotaxin was then added to each well. After incubation at room temperature for 2 h, each well was washed 7 times with wash buffer. Substrate solution (stabilized hydrogen peroxide and tetramethylbenzidine) was added to each well, after which the plate was incubated at room temperature for 20 min. Sulfuric acid was then added to each well, and the absorbance was measured at 450 nm. The limit of detection in the assay was 5 pg/ml for eotaxin. Reverse Transcriptase-Polymerase Chain Reaction Gene expression of eotaxin and ß-actin was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) as described [32]. Total RNA was extracted from cells 24 h after the incubation with or without indicated cytokines using Isogen reagent (Nippon Gene, Tokyo, Japan). First-stranded cDNAs were synthesized from isolated RNA templates in the presence of Moloney murine leukemia virus reverse transcriptase, random primers, and reaction buffer at 37 ° C for 1 h (Amersham Pharmacia Biotech, Tokyo, Japan). After cDNAs were denatured at 94 ° C for 5 min, the polymerase chain reaction was performed with 5 pmol each of the forward and reverse primers, 5 Ìl cDNA, 0.6 U Taq polymerase (Roche Diagnostics, Tokyo, Japan) and 2.5 Ìl PCR reaction buffer (Tris-HCl 100 mmol/l, MgCl2 15 mmol/l, KCl 500 mmol/l, Roche Diagnostics); distilled water was added to bring the reaction volume to 25 Ìl. The sequences of the primers for eotaxin were: 5)-CCAACCACCTGCTGCTTTAACCTG-3) (forward) and 5)-GCTTTGGAGTTGGAGATTTTTGG-3) (reverse), and for ß-actin: 5)- GTGGGGCGCCCCAGGCACCA-3) (forward) and 5)- CTCCTTAATGTCACGCACGATTTC-3) (reverse). The amplification reaction was performed for 30 cycles with denaturation at 94 ° C for 45 s, annealing at 57 ° C for 45 s, and extension at 72 ° C for 1 min (Perkin-Elmer Cetus, Norwalk, Conn., USA). After incubation at 72 ° C for 10 min, PCR-amplified products were analyzed with 2% agarose gel electrophoresis and ethidium bromide staining followed by visualization with an ultraviolet transilluminator. Nuclear Extraction Nuclear extracts were based on methods described previously [25]. STAT6-BEAS cells were treated as indicated and then harvested with a scraper after washing twice with PBS. The cells were washed with buffer A (10 mM HEPES, 15 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 10 mg/ml leupeptin and 1 mM Na3VO4; Sigma-RBL). The cell pellets were resuspended in buffer B (buffer A containing 0.2% Igepal CA-630) and incubated for 2 min. Nuclei were pelleted by centrifugation and resuspended in buffer C (buffer A containing 0.25 M sucrose). Nuclei were again pelleted, then resuspended in buffer D (50 mM HEPES, 400 mM KCl, 10% glycerol, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 10 mg/ml leupeptin and 1 mM Na3VO4) and incubated with shaking for 30 min. All procedures were performed on ice. The mixture was centrifuged and the supernatant was stored at –80 ° C. Electrophoretic Mobility Shift Assays DNA-protein binding assays were based on methods described previously [25]. Aliquots of 5 Ìg of nuclear extracts were incubated in 10 Ìl of total reaction volume containing 10 mM Tris-HCl, 1 mM DTT, 1 mM EDTA, 10% glycerol, 0.1% Triton X-100, 50 Ìg/ml poly(dI-dC), 0.1 mg/ml BSA and 50 mM KCl (Invitrogen) with 32P-

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Fig. 1. Schema of eotaxin promoter-luciferase reporter plasmids is depicted. Mutated binding sites in the promoter are underlined.

labeled oligonucleotide probe at room temperature for 20 min with or without unlabeled oligonucleotide probe. In some experiments, antibodies against p50 (NF-ÎB1), p65 (Rel A) and STAT6 (Santa Cruz Biotech, Tokyo, Japan) were incubated with the mixture for 30 min after incubation with labeled probe. The reaction products were analyzed by electrophoresis in a 5% polyacrylamide gel with 0.5 ! TBE buffer. The gels were dried and analyzed by autoradiography. The sequence of the oligonucleotide probe used in EMSA is 5)-GGCTTCCCTGGAATCTCCCACA-3), including the binding sites for STAT6 and NF-ÎB in the eotaxin promoter as reported previously [25]. Construction of Eotaxin Promoter-Luciferase Reporter Plasmids A 1,363-bp fragment of the promoter region of the eotaxin gene (site –1,363 to –1) was amplified by PCR and ligated into Mlu I and Bgl II sites of a firefly-luciferase reporter pGL3-basic vector (Promega, Madison, Wisc., USA) and the construct is referred to as pEotx.1363 as described previously [25]. To investigate the function of the putative site of GRE in eotaxin gene which was indicated by Hein et al. [33] and Garcia-Zepeda et al. [34], the putative GRE site in pEotx.1363 was mutated using the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, Calif., USA). This mutant reporter plasmid was synthesized by temperature cycling using sitemutated primers and PfuTurbo DNA polymerase (Stratagene). Nonmutated template plasmid was digested with Dpn I endonuclease (Stratagene). The mutated plasmid was transformed into XL1-Blue cells (Stratagene) and purified using the QIAGEN plasmid purification kit and referred to as pEotx.mGRE. The sequence of the plasmid was confirmed using a gene analysis system (Takara Syuzou, Shiga, Japan) (see fig. 1). Transient Transfections and Luciferase Assay STAT6-BEAS cells were seeded into 6-well plates and allowed to grow to 50–70% confluence. Cells were transfected with 1 Ìg of reporter plasmids and 10 ng of a control Renilla luciferase vector pRL-TK (Promega, Tokyo, Japan) using 3 Ìl of Fugene 6 transfection reagent (Roche Diagnostics) and incubated for 24 h in 2 ml medium. After incubation with or without fluticasone (10 –7 M ) for 30 min, indicated cytokines were then added and 6 h later cells were washed twice with Ca2+ and Mg2+-free PBS, solubilized by incuba-

Matsukura et al.

tion in 500 Ìl of lysis buffer for 20 min, transferred to microtubes and centrifuged to pellet cellular debris. The supernatants were stored at –80 ° C until luciferase activity was measured using the Dual-Luciferase Assay System (Promega) and a Gene-Light 55 luminometer (Microtech Nichion, Chiba, Japan). The firefly luciferase activity of the eotaxin promoter-reporter plasmid was normalized using Renilla luciferase activity and calculated as fold induction compared with the control values. Statistical Analysis Analysis of data was performed using Stat-View IV (Abacus Concept, Berkeley, Calif., USA). Data are expressed as mean B standard error of mean (SEM). Statistical differences were determined by analysis of variance with Fisher PLSD.

Results

A minimal level of eotaxin protein was detected in the medium of unstimulated STAT6-BEAS cells (fig. 2). TNF-· (100 ng/ml) increased eotaxin production to a lesser degree than stimulation with IL-4 (50 ng/ml) after 24 h with stimulation. The combination of TNF-· and IL-4 synergistically stimulated eotaxin production (*p ! 0.05 compared with unstimulated control; the data are presented as the mean B SEM of a total of four independent experiments). Preincubation with fluticasone propionate (10 –7 M) for 1 h significantly inhibited the induction of eotaxin protein by stimulation with IL-4 alone or with a combination of TNF-· and IL-4 (**p ! 0.05 compared with DMA; the data are presented as the mean B SEM of a total of four independent experiments). While induction with TNF-· alone seemed to be inhibited by fluticasone, there was no statistical significance. DMA, diluent of fluticasone, did not interfere in the production of eotaxin. Preincubation with fluticasone for 24 h also significantly inhibited the production of eotaxin, while its inhibitory effect was not different from preincubation for 1 h (data not shown). Then we performed experiments involving the preincubation of fluticasone for 1 h to observe its effects on the transcription of the eotaxin gene. Subsequently, we confirmed the dose-dependent effect of fluticasone. Preincubation with fluticasone for 1 h decreased the induction of eotaxin protein stimulated with the combination of TNF-· (100 ng/ml) and IL-4 (50 ng/ml) in a dose-dependent manner (fig. 3). The inhibitory effects of fluticasone were statistically significant at a concentration of 10 –10 to 10 –6 M (*p ! 0.05 compared with DMA; the data are presented as the mean B SEM of a total of four independent experiments) and then we used 10 –7 M of fluticasone in additional experiments.

Mechanisms of Inhibition of Eotaxin Expression with Fluticasone

Fig. 2. Effect of fluticasone propionate and its diluent DMA on the

induction of eotaxin protein in the media of STAT6-BEAS cells. The cells were incubated with or without TNF-· (100 ng/ml) and/or IL-4 (50 ng/ml) for 24 h after incubation with or without fluticasone (10 –7 M ) or DMA for 1 h. Concentrations of eotaxin protein in the medium were analyzed by ELISA. The data are presented as the mean B SEM of a total of four independent experiments (*p ! 0.05 compared with unstimulated control; **p ! 0.05 compared with DMA).

We next performed RT-PCR to analyze the expression of eotaxin mRNA in STAT6-BEAS cells. TNF-· (100 ng/ ml) slightly increased the expression of eotaxin mRNA by 24 h after stimulation (fig. 4, representing four independent experiments), while IL-4 (50 ng/ml) increased the expression to a larger degree than TNF-·. The combination of TNF-· and IL-4 synergistically increased eotaxin mRNA. Preincubation with fluticasone for 1 h significantly inhibited the expression of eotaxin mRNA. We next focused on the transcriptional regulation of eotaxin gene by fluticasone in airway epithelial cells. In this report, we first hypothesized that fluticasone would inhibit the translocation of transcription factors into nuclei and their subsequent binding to the promoter region of eotaxin gene. EMSA with nuclear extracts from STAT6-BEAS cells stimulated with TNF-· (100 ng/ml) or

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3

4 Fig. 3. Dose-dependent inhibition of eotaxin protein with flutica-

sone propionate in STAT6-BEAS cells. Cells were preincubated for 1 h with fluticasone (10 –11 to 10 –6 M ) or DMA and then incubated with a combination of TNF-· (100 ng/ml) and IL-4 (50 ng/ml) for 24 h. Concentrations of eotaxin protein in the medium were analyzed by ELISA. The data are presented as the mean B SEM of % induction of a total of four independent experiments (*p ! 0.05 compared with DMA). Fig. 4. Repression of eotaxin mRNA expression with fluticasone propionate in STAT6-BEAS cells. RNA was extracted from the cells which had been preincubated for 1 h with fluticasone (10 –7 M ) or its diluent DMA followed by incubation with or without TNF-· (100 ng/ml) and/or IL-4 (50 ng/ml) for 24 h. Extracted RNA was subjected to RT-PCR; representative of four independent experiments. Fig. 5. Fluticasone does not inhibit formation of binding complex composed of NF-ÎB or STAT6 stimulated with TNF-· or IL-4, respectively, in STAT6-BEAS cells. Nuclear protein was extracted from STAT6-BEAS cells which were preincubated for 1 h with fluticasone (10 –7 M ) or its diluent DMA and then incubated with control medium, TNF-· (100 ng/ml) and/or IL-4 (50 ng/ml) for 30 min. The binding activity was analyzed by EMSA using a radiolabeled oligonucleotide probe containing the eotaxin promoter sequence; representative of four independent experiments.

IL-4 (50 ng/ml) resulted in the formation of binding complexes (fig. 5, representing four independent experiments). These binding complexes were confirmed to be a heterodimer of p50 and p65 NF-ÎB Rel family members or a homodimer of STAT6 as reported earlier (data not shown) [25]. Stimulation with a combination of TNF-· and IL-4 resulted in the formation of both of these two binding complexes, NF-ÎB and STAT6. Unexpectedly, preincubation with fluticasone (10 –7 M) did not inhibit any of these binding complexes (fig. 5).

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5

We next analyzed whether fluticasone would repress the activity of the eotaxin promoter, pEotx.1363, and whether it could repress the eotaxin promoter through putative GRE in the promoter. Similar to previous reports using BEAS-2B cells, TNF-· (100 ng/ml) or IL-4 (50 ng/ml) activated the promoter and combination of these two cytokines synergistically activated the promoter pEotx.1363 (wild type) (fig. 6a) and pEotx.mGRE (the eotaxin promoter in which putative GRE was mutated) (fig. 6b) (the data are presented as the mean B SEM of a

Matsukura et al.

Fig. 6. a Activation of pEotx.1363, an eotaxin promoter-luciferase reporter plasmid, by TNF-· (100 ng/ml) and/or IL-4 (50 ng/ml) in STAT6-BEAS cells. The data are presented as the mean B SEM of a total of four independent experiments (*p ! 0.05 compared with unstimulated condition). b Activation of pEotx.mGRE, an eotaxin promoter-luciferase reporter plasmid in which the putative GRE was mutated, by TNF-· (100 ng/ml) and/or IL-4 (50 ng/ml) in STAT6-BEAS cells. The data are presented as the mean B SEM of a total of four independent experiments (*p ! 0.05 compared with unstimulated condition). c Fluticasone partially inhibits the activity of pEotx.1363. It also inhibits the activity of pEotx.mGRE. The data are presented as the mean B SEM of % induction of a total of four independent experiments (*p ! 0.05 compared with DMA). STAT6-BEAS cells were transfected with indicated reporter plasmid and 48 h later cells were preincubated for 1 h with fluticasone (10 –7 M ) or its vehicle DMA, cultured for 6 h with indicated cytokines and then subjected to dual luciferase assay.

total of four independent experiments, *p ! 0.05 compared with unstimulated control) in STAT6-BEAS cells. Fluticasone partially inhibited the induction of the eotaxin promoter, pEotx.1363, indicating that transcriptional repression of eotaxin gene by fluticasone may be involved in the mechanisms (fig. 6c) (the data are presented as the mean B SEM of a total of four independent experiments, *p ! 0.05 compared with DMA). However, these inhibitory effects of fluticasone were also conserved in pEotx.mGRE, suggesting that this putative GRE site might not be important in the process of transcriptional repression of eotaxin by fluticasone. Finally, we observed the effects of the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), to find possible mechanisms by which fluticasone represses the gene

transcription of eotaxin. TSA seemed to reverse the repression of eotaxin expression by fluticasone (fig. 7, the data are presented as the mean B SEM of a total of four independent experiments, *p ! 0.05 compared with FP + ethanol). When cells were stimulated with a combination of TNF-· and IL-4, a statistical significance between preincubation with fluticasone plus ethanol (diluent of TSA) and fluticasone plus TSA was observed. However, the effect of TSA was modest and it could not totally reverse the inhibitory effect of fluticasone. These data indicate that the theory of inhibition of histone acetylation by GC [35] cannot fully explain the mechanisms of transcriptional repression of the eotaxin gene by fluticasone.

Mechanisms of Inhibition of Eotaxin Expression with Fluticasone

Int Arch Allergy Immunol 2004;134(suppl 1):12–20

17

Fig. 7. Effects of HDAC inhibitor, TSA, on

the release of eotaxin protein into the medium of STAT6-BEAS cells. Cells were preincubated for 30 min with TSA (10 ng/ ml) or its vehicle ethanol, next incubated for 1 h with fluticasone (10 –7 M ) or its diluent DMA, and cultured for 24 h with or without TNF-· (100 ng/ml) and/or IL-4 (50 ng/ml). Concentrations of eotaxin protein in the medium were analyzed by ELISA. The data are presented as the mean B SEM of a total of four independent experiments (*p ! 0.05 compared with DMA + ethanol; **p ! 0.05 compared with FP + ethanol).

Discussion

Many of the molecular mechanisms by which GC suppress inflammatory genes have been reported. In the steps of the action of GC, free GC diffuse across the plasma membrane and become associated with glucocorticoid receptors (GR). Then GC-GR act in the cytoplasm or translocate into nuclei and bind to a specific GRE in the chromatin [36]. The GC-GR complex is able to interfere in activating transcription factors, including NF-ÎB and AP-1. Then the GC-GR complex can inhibit the translocation of these transcription factors into nuclei and prevent the binding of them to the promoter region of the target gene resulting in the repression of the target gene [37–39]. However, in our experiments, fluticasone inhibited binding of neither NF-ÎB nor STAT6 to the DNA derived from the proximal promoter region of eotaxin. This discrepancy may not be due to the difference of GC because we observed the same results using dexamethasone and budesonide (data not shown). We speculate that the difference of cell types and stimulations may influence the results. Recently, Hart et al. [40] and Newton et al. [41] reported that GC

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Int Arch Allergy Immunol 2004;134(suppl 1):12–20

did not inhibit the DNA binding of NF-ÎB in airway epithelial cells. These reports may support our results. GC is also known to directly repress the target gene. Translocated GC-GR can bind to a ‘negative’ GRE in the promoter of a target gene and suppress the gene transcription [42, 43]. Previous reports have indicated that putative GRE might exist in the eotaxin promoter from sequencing of 5)-flanking region of the eotaxin gene [33, 34]. We mutated this putative GRE site in the eotaxin promoter-luciferase reporter plasmid (pEotx.1363), referred to as pEotx.mGRE, and analyzed the promoter activity. However, the inhibitory effects of fluticasone on the promoter activity were conserved in pEotx.mGRE. These data indicate that the putative GRE site in the eotaxin promoter may not be involved in the repression of the eotaxin gene with fluticasone. Fluticasone inhibited the activity of the eotaxin promoter, pEotx.1363, induced by TNF-· and IL-4. These inhibitory effects on the promoter may reflect the effect on the transcription of the eotaxin gene. But this effect seemed modest compared to the significant inhibitory effect of fluticasone on the expression of eotaxin mRNA. We need further studies to come to a conclusion, but these

Matsukura et al.

data and previous reports indicate that both of transcriptional and posttranscriptional regulation including the inhibition of mRNA stability would be important in the repression of eotaxin expression with GC [5]. Ito et al. [35] recently reported that GC dexamethasone repressed the gene expression of GM-CSF by the mechanisms of inhibition of histone acetylation. They suggested that the GC-GR complex inhibits histone acetylation by recruiting HDAC to the complex composed of NF-kB, CBP and histone acetyltransferase and also inhibits it by inhibiting histone acetyltransferase activity directly. In their reports, TSA, an inhibitor of HDAC, remarkably reversed the inhibitory effect of dexamethasone on GM-CSF expression in airway epithelial cells. We expected that this role might be a breakthrough to resolve our question and examined the effect of TSA in our experiments. However, we could detect a moderate effect of TSA on the action of fluticasone in eotaxin expression, indicating that these discrepancies may also be due to the difference of the target gene. We need to perform much more extensive experiments to come to a conclusion on the role of GC in the histone deacetylation of the eotaxin gene, though we think that the mechanisms of eotaxin gene repression by GC may be complex and cannot be fully explained by the mechanisms already established. GC have numerous effects and these may be specific for target factors. Here we report that fluticasone inhibits eotaxin expression in airway epithelial cells in part through repression of eotaxin transcription. But this mechanism may not de-

pend on the direct inhibition of transcription factors, such as NF-ÎB or STAT6. The existence of ‘negative GRE’ in the eotaxin gene might be unclear in the eotaxin gene. The inhibitor of HDAC may not play a pivotal role in the regulation of eotaxin. Alterations of phosphorylation of transcription factors by GC would also be candidates to explain our results; however, it was confirmed that GC budesonide did not interfere in the phosphorylation of STAT6 induced by IL-4 in BEAS-2B cells [44]. We did not manage to find a new theory as regards the mechanisms of GC action; however, we speculate that GC may interfere in the functional interaction between activating transcription factors, such as NF-ÎB and STAT6, and coactivators, such as CBP/p300, because the interaction among these factors has recently been reported [45, 46]. It might be possible that GC may inhibit the trans-activating sites of NF-ÎB and STAT6 which are not related to the translocation or DNA binding of these factors in airway epithelial cells. We hope that further studies on the molecular mechanisms of GC will contribute to the progress in the treatment of inflammatory diseases including asthma.

Acknowledgments The authors would like to thank Dr. Tsutom Hirano and Dr. Takeshi Kasama for excellent assistance and helpful discussions and Ms. Mieko Mori, Ms. Tomoko Akabane, Ms. Setsuko Sukegawa, Ms. Eri Matsukura, and Ms. Bonnie Hebden for skillful assistance. This work was supported by GlaxoSmithKline.

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34 Garcia-Zepeda EA, Rothenberg ME, Weremowicz S, Sarafi MN, Morton CC, Luster AD: Genomic organization, complete sequence, and chromosomal location of the gene for human eotaxin (SCYA11), an eosinophil-specific CC chemokine. Genomics 1997;41:471–476. 35 Ito K, Barnes PJ, Adcock IM: Glucocorticoid receptor recruitment of histone deacetylase 2 inhibits interleukin-1beta-induced histone H4 acetylation on lysines 8 and 12. Mol Cell Biol 2000;20:6891–6903. 36 Schleimer RP: Glucocorticoids. Allergy Principles Pract 1998;1:638–660. 37 Ray A, Prefontaine KE: Physical association and functional antagonism between the p65 subunit of transcription factor NF-kappa B and the glucocorticoid receptor. Proc Natl Acad Sci USA 1994;91:752–756. 38 Adcock IM, Shirasaki H, Gelder CM, Peters MJ, Brown CR, Barnes PJ: The effects of glucocorticoids on phorbol ester and cytokine stimulated transcription factor activation in human lung. Life Sci 1994;55:1147–1153. 39 Adcock IM, Brown CR, Gelder CM, Shirasaki H, Peters MJ, Barnes PJ: Effects of glucocorticoids on transcription factor activation in human peripheral blood mononuclear cells. Am J Physiol 1995;268:C331–C338. 40 Hart L, Lim S, Adcock I, Barnes PJ, Chung KF: Effects of inhaled corticosteroid therapy on expression and DNA-binding activity of nuclear factor kappaB in asthma. Am J Respir Crit Care Med 2000;161:224–231. 41 Newton R, Hart LA, Stevens DA, Bergmann M, Donnelly LE, Adcock IM, Barnes PJ: Effect of dexamethasone on interleukin-1beta-(IL1beta)-induced nuclear factor-kappaB (NFkappaB) and kappaB-dependent transcription in epithelial cells. Eur J Biochem 1998;254:81– 89. 42 Langer SJ, Ostrowski MC: Negative regulation of transcription in vitro by a glucocorticoid response element is mediated by a trans-acting factor. Mol Cell Biol 1988;8:3872–3881. 43 Diamond MI, Miner JN, Yoshinaga SK, Yamamoto KR: Transcription factor interactions: Selectors of positive or negative regulation from a single DNA element. Science 1990;249: 1266–1272. 44 Heller NM, Matsukura S, Georas SN, Boothby M, Schindler U, Stellato C, Schleimer RP: Determination of the importance of signal transducer and activator of transcription 6 (STAT6) as a target of glucocorticoids in human airway epithelial cells (submitted). 45 Gerritsen ME, Williams AJ, Neish AS, Moore S, Shi Y, Collins T: CREB binding protein/ p300 are transcriptional coactivators of p65. Proc Natl Acad Sci USA 1997;94:2927–2932. 46 Gingras S, Simard J, Groner B, Pfitzner E: p300/CBP is required for transcriptional induction by interleukin-4 and interacts with Stat6. Nucleic Acids Res 1999;27:2722–2729.

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Original Paper Int Arch Allergy Immunol 2004;134(suppl 1):21–24 DOI: 10.1159/000077788

Immunotherapy Attenuates Eosinophil Transendothelial Migration Induced by the Supernatants of Antigen-Stimulated Mononuclear Cells from Atopic Asthmatics Makoto Nagata a Keiko Saito a Izumi Kikuchi a Kazuaki Tabe a Koichi Hagiwara a Minoru Kanazawa a Yoshio Sakamoto b a Department

of Respiratory Medicine, Saitama Medical School and b Department of Internal Medicine, Yugawara Kosei Nenkin Hospital, Iruma-gun, Japan

Key Words Immunotherapy W Mononuclear cells W Eosinophils W Transendothelial migration W Bronchial asthma

Abstract Background: Eosinophil transendothelilal migration across vascular endothelial cells is an initial step of eosinophil accumulation in allergic inflammation. There is increasing evidence that specific immunotherapy (SIT) modulates the production of inflammatory molecules from mononuclear cells. Objective: The present study was undertaken to examine whether SIT modifies eosinophil transendothelial migration induced by the supernatants of antigen-stimulated mononuclear cells from atopic asthmatics. Methods: Dermatophagoides farinae (Df)-sensitive mild persistent asthmatics were divided into a SIT-treated group and a control group. Peripheral blood mononuclear cells (PBMC) were isolated before and after SIT using the rush protocol, and cultured for 96 h at 37 ° C in the presence or absence of Df antigen. Eosinophils were isolated from the blood of healthy subjects, and put on transwell filters coated with pulmonary microvascular endothelial cell monolayers stimulated

ABC

© 2004 S. Karger AG, Basel 1018–2438/04/1345–0021$21.00/0

Fax + 41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/iaa

with IL-4 plus TNF-·. The supernatants of PBMC were applied to the lower compartment and the transmigration of eosinophils was examined. Results: Df stimulation of PBMC resulted in an augmentation of eosinophil transendothelial migration. This enhancement was abrogated following SIT. In the control group, the antigeninduced effect on eosinophil transmigration did not show an interval change. Conclusion: SIT attenuates eosinophil transendothelial migration induced by antigen-stimulated mononuclear cells. Copyright © 2004 S. Karger AG, Basel

Introduction

Eosinophil transendothelilal migration across vascular endothelial cells is an initial step of eosinophil accumulation in allergic inflammation. This process is likely regulated by cytokines/chemokines, produced by immunoregulatory cells including mononuclear cells in response to allergen exposure [1]. Specific immunotherapy (SIT) has been shown to decrease the influx of eosinophils induced by the allergen provocation test or by seasonal exposure to an allergen [2–4]. We have previously observed that SIT

Correspondence to: Dr. Makoto Nagata Department of Respiratory Medicine 38 Morohongou, Moroyama-cho Iruma-gun, Saitama, 350-0495 (Japan) Tel. +81 492 76 1319, Fax +81 492 95 8399, E-Mail [email protected]

Table 1. Characteristics of patients with asthma

Patient

IgE IU/ml

Df-IgE RU/ml

Age years

Gender

SIT(+) 1 2 3 4 5

28 27 21 28 21

F F M F M

190 450 2,980 480 560

90.1 36.1 1100 38.1 88.4

SIT(–) 6 7 8 9 10

48 28 28 26 28

M F M F F

1,490 100 420 120 1,000

1100 29.8 1100 28.9 17.2

throughout the study periods. Informed consent was obtained before the initial blood sampling. Maintenance dose of Df antigen, AU

50 50 50 50 50

RU = Reference units; AU = allergen units.

attenuates the generation of eosinophil adhesive and chemotactic activities from antigen-stimulated mononuclear cells of atopic asthmatics [5, 6]. Here we report that SIT also attenuates eosinophil transendothelial migration induced by the antigen-stimulated mononuclear cells.

Methods Reagents Percoll was obtained from Pharmacia (Uppsala, Sweden). AntiCD16 antibody-coated magnetic beads were purchased from Miltenyi Biotec (Auburn, Calif., USA). RPMI 1640 medium, newborn calf serum (NCS) and fetal calf serum (FCS) were obtained from Life Technologies (Grand Island, N.Y., USA). Recombinant human IL-4 and TNF-· were purchased from R&D Systems (Minneapolis, Minn., USA). Patients Ten mild persistent asthmatic patients who showed high levels of Dermatophagoides farinae (Df)-specific IgE in sera and a strongly positive skin reaction against Df were divided into the SIT-treated and the control group (table 1). Gender, age, and amounts of total or Df-specific IgE were not different between the two groups. In the SIT group, rush immunotherapy using Df antigen (Hollister, Calif., USA) was performed in July 2000. Patients were hospitalized for 5 days, and three to five subcutaneous shots were given daily at 2-hour intervals, until 50 AU, a maintenance dose, is reached. Following this procedure, maintenance injections were repeated every 2 weeks at the outpatient clinic. All patients were allowed to take inhaled medicines including 400 Ìg/day of beclomethasone dipropionate and/or albuterol, but not oral corticosteroids or leukotriene modifiers

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Peripheral Blood Mononuclear Cells Peripheral blood mononuclear cells (PBMC) were isolated in June and November 2000, using the Ficoll-Hypaque density gradient technique, adjusted to 2 ! 106 cells/ml in an RPMI culture medium (RPMI 1640 supplemented with 10% FCS, 100 U/ml penicillin, 100 Ìg/ml streptomycin, and 2 mM L-glutamine), and cultured for 96 h at 37 ° C in 5% CO2 in the presence or absence of 1 Ìg/ml Df antigen (Torii Pharmaceutical Co., Tokyo, Japan) [5, 6]. The purity of lymphocytes was more than 90% in all experiments. The supernatants were stored at –80 ° C until use. Eosinophil Separation Eosinophils were isolated from peripheral blood of 10 normal volunteers with eosinophils representing less than 5% of the total leukocytes. Subjects ranged in age from 22 to 26 years, and gender distribution was equal. Eosinophil isolation was performed by negative immunomagnetic bead selection as previously described [6]. Briefly, heparinized blood was diluted with HBSS and centrifuged for 20 min at 700 g over 1.090 g/ml Percoll. Plasma and mononuclear cell band were removed, and the red blood cells in the pellet were lysed by hypotonic shock. The resulting granulocytes were washed with HBSS supplemented with 2% NCS, and then incubated with anti-CD16 antibody-coated magnetic beads for 40 min at 4 ° C. The cells were filtered through a steel wool column in a magnetic field (Miltenyi Biotec) to remove neutrophils. CD16-negative eosinophils (1 98% purity and 1 99% viability) were collected, washed and resuspended in RPMI supplemented with 10% FCS (RPMI/FCS). Preparation of Endothelial Cells Human pulmonary microvascular endothelial cells (HPMEC; purchased from Clonetics, San Diego, Calif., USA) were prepared as previously described [7, 8]. Briefly, HPMEC were cultured on type IV collagen-coated tissue culture flasks until confluent and passaged into collagen-coated transwell inserts (6.5-mm diameter polycarbonate membrane with 3-Ìm pores, Costar, Cambridge, Mass., USA). HPMEC were cultured for 24 h in 5% CO2 at 37 ° C in the presence of IL-4 and TNF-· (both at 100 pM ) to augment the expressions of adhesion molecules; following the treatment, approximately 80% of the cells expressed both VCAM-1 and ICAM-1 [7, 8]. The incubated mixture was then aspirated, and HPMEC were washed twice with HBSS before use. Eosinophil Transendothelial Migration Eosinophil transendothelial migration was examined using a method as previously described [7, 8]. Briefly, eosinophils (100 Ìl of 5 ! 105 cells/ml in RPMI/FCS) were added to the upper compartment of transwell inserts and the PBMC culture supernatants (500 Ìl) to the lower compartment. After 3 h incubation in 5% CO2 at 37 ° C, the transwell inserts were removed, and the migrated eosinophils were assessed as the residual eosinophil peroxidase activity [8]. As standards, serially diluted cell suspensions were added to empty wells. Eosinophil peroxidase substrate (1 mM o-phenylenediamine, 1 mM H2O2, and 0.1% Triton X-100 in Tris buffer, pH 8.0) was then added to all the wells. After 30 min incubation at room temperature, 100 Ìl of 4 M H2SO4 was added to stop the reaction, and the absorbance at 490 nm was measured. Percent eosinophil migration was calculated from the log dose-response

Nagata/Saito/Kikuchi/Tabe/Hagiwara/ Kanazawa/Sakamoto

curve. Eosinophil viability after the incubation exceeded 98% by trypan blue dye exclusion. Statistics Data are presented as mean B SEM. For paired comparisons, the Student t test was used. Statistical significance was established at the p ! 0.05 level.

Results

In June 2000, eosinophil transmigration induced by the supernatants of Df-stimulated PBMC was greater than that of unstimulated ones in both the SIT group and the control group [% migration: SIT group, 8.0 B 2.6 by medium vs. 13.2 B 3.5 by Df, p = 0.03 (subjects treated with immunotherapy, IT(+) left); control group, 8.5 B 2.1 by medium vs. 10.9 B 2.3 by Df, p = 0.04 (subjects not treated with immunotherapy, IT(–) left)] (fig. 1). In November, the Df-dependent enhancement was abolished in the SIT group [9.0 B 1.3 by medium vs. 10.0 B 2.4 by Df, p = nonsignificant IT(+) right]; however, the enhancement was clearly observed again in the control group [10.0 B 1.5 by medium vs. 14.2 B 1.7 by Df, p ! 0.01 IT(–) right] (fig. 1).

Discussion

In this investigation, we observed that the antigendependent enhancement in eosinophil transmigration induced by the supernatants of PBMC from atopic asthmatics was attenuated by SIT. The effect is not due to seasonal change or inhaled steroid because the control group did not show such a difference between June and November 2000. These results suggest that SIT inhibits the generation of factor(s), which induce eosinophil transendothelial migration, from mononuclear cells in response to antigen exposure. We have previously observed that the antigen-induced eosinophil migration across HPMEC stimulated with IL-4 and TNF-· was dependent on VCAM-1 since it was blocked by anti-·4 integrin mAb [8]. Furthermore, the eosinophil transmigration enhanced by Df-stimulated PBMC was blocked by anti-CCR3 antibody, suggesting an essential role of the CCR3/CC chemokine pathway in this process [8]. CCR3 is a major chemokine receptor operational on eosinophils [9]. It has been shown that the pretreatment of eosinophils with anti-CCR3 antibody abrogates the chemotactic response to eotaxin, regulated upon activation, normal T cell expressed, and secreted

Fig. 1. Eosinophil transendothelial migration induced by the culture supernatants of PBMC obtained from Df-sensitive atopic asthmatics. IT(+) = Subjects treated with rush immunotherapy (n = 5); IT(–) = subjects not treated with immunotherapy; Df(–) = eosinophil migration in response to the supernatants of PBMC cultured with medium alone; Df(+) = eosinophil migration in response to the supernatants of PBMC cultured with Df antigen. Data are expressed as mean B SEM.

Immunotherapy Attenuates Eosinophil Transendothelial Migration

Int Arch Allergy Immunol 2004;134(suppl 1):21–24

(RANTES), monocyte chemoattractant protein (MCP)-2, MCP-3, and MCP-4 in vitro [10]. Thus, it is conceivable that CC chemokines are contributing factors in the eosinophil transendothelial migration, and their productions were inhibited by SIT. The responsible chemokine(s) was not evaluated in this study; however, it should be clarified in a future study. In conclusion, SIT can modify both the adhesion to and transmigration across endothelial cells via modulation of responses of mononuclear cells to an antigen. These effects may contribute to the benefit of SIT in allergic airway diseases.

23

References 1 Bochner BS, Schleimer RF: The role of adhesion molecules in human eosinophil and basophil recruitment. J Allergy Clin Immunol 1994; 94:427–438. 2 Furin MJ, Norman PS, Creticos PS, Proud D, Kagey-Sobotka A, Lichtenstein LM: Immunotherapy decreases antigen-induced eosinophil cell migration into the nasal cavity. J Allergy Clin Immunol 1991;88:27–32. 3 Rak S, Bjornson A, Hakanson L, Sorenson S, Venge P: The effect of immunotherapy on eosinophil accumulation and production of eosinophil chemotactic activity in the lung of subjects with asthma during natural pollen exposure. J Allergy Clin Immunol 1991;88:878– 888.

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4 Durham SR, Ying S, Varney VA, Jacobson MR, Sudderick RM, Mackay IS, Kay AB, Hamid QA: Grass pollen immunotherapy inhibits allergen-induced infiltration of CD4+ T lymphocytes and eosinophils in the nasal mucosa and increases the number of the cells expressing messenger RNA for interferon-Á. J Allergy Clin Immunol 1996;97:1356–1365. 5 Nagata M, Shibasaki M, Sakamoto Y, Fukuda T, Makino S, Yamamoto K, Dohi Y: Specific immunotherapy reduces the antigen-dependent production of eosinophil chemotactic activity from mononuclear cells in patients with atopic asthma. J Allergy Clin Immunol 1994; 94:160–166. 6 Nagata M, Tabe K, Choo JH, Sakamoto Y, Matsuo H: Effect of immunotherapy on the production of eosinophil adhesion-inducing activity from mononuclear cells in house-dustmite-sensitive bronchial asthma. Int Arch Allergy Immunol 1998;117(suppl 1):20–23.

7 Yamamoto H, Sedgwick JB, Busse WW: Differential regulation of eosinophil adhesion and transmigration by pulmonary microvascular endothelial cells. J Immunol 1998;161:971– 977. 8 Nagata M, Yamamoto H, Tabe K, Sakamoto Y: Eosinophil transmigration across VCAM-1expressing endothelial cells is upregulated by antigen-stimulated mononuclear cells. Int Arch Allergy Immunol 2001;125(suppl 1):7–11. 9 Teran LM: Chemokines and IL-5: Major players of eosinophil recruitment in asthma. Clin Exp Allergy 1999;29:287–290. 10 Heath H, Qin S, Rao P, Wu L, LaRosa G, Kassam N, Ponath PD, Machay CR: Chemokine receptor usage by human eosinophils. The importance of CCR3 demonstrated using an antagonistic monoclonal antibody. J Clin Invest 1997;23:178–184.

Nagata/Saito/Kikuchi/Tabe/Hagiwara/ Kanazawa/Sakamoto

Original Paper Int Arch Allergy Immunol 2004;134(suppl 1):25–29 DOI: 10.1159/000077789

Tyk2 Is Essential for IFN-·-Induced Gene Expression in Mast Cells Yumiko Mori a Koichi Hirose a Kotaro Suzuki a Hiroshi Nakajima a Yohei Seto a Kei Ikeda a Kazuya Shimoda b Kei-ichi Nakayama c Yasushi Saito a Itsuo Iwamoto a a Department

of Internal Medicine II, Chiba University School of Medicine, Chiba, and b Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences and c Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan

Key Words Mast cells W Tyk2 W IFN-· W Stat1 W Immunity, innate

Abstract Mast cells are recognized not only as the major effector cells of type I hypersensitivity reactions but also as an important player of innate immune response against bacterial infection. Type I IFNs are also involved in the response against bacterial infection. However, the role of type I IFNs and their associated Janus kinase Tyk2 in mast cell functions remains to be determined. In this study, we addressed this issue using Tyk2-deficient (Tyk2–/–) bone marrow-derived mast cells (BMMCs). When BMMCs from wild-type (WT) mice were stimulated with IFN-·, they expressed mRNA for IFN-Á-inducible protein 10 (IP-10) and monocyte chemoattractant protein-5 (MCP-5). Interestingly, IFN-·-induced expression of IP-10 and MCP-5 was severely decreased in Tyk2–/– BMMCs. In addition, IFN-·-induced Stat1 phosphorylation was decreased in Tyk2–/– BMMCs. On the other hand, IFN-·-induced Stat1 phosphorylation and IP-10 and MCP-5 expression were normal in Tyk2–/– fibroblasts. These results indicate that IFN-· induces the expression of TNF-· and the chemokines IP-10 and MCP-5 in mast cells and thatTyk2 plays a nonredundant role in IFN-· signaling in mast cells. Copyright © 2004 S. Karger AG, Basel

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Introduction

Mast cells are recognized as the major effector cells of type I hypersensitivity reactions by virtue of possessing the high affinity receptors for IgE and are known to play a critical role in allergic diseases such as atopic rhinitis, asthma, and atopic dermatitis [1, 2]. Recently, a number of studies have revealed that mast cells also play important roles in innate immune responses especially against gram-negative bacteria by recruiting neutrophils and monocytes into the inflammatory site through the production of proinflammatory cytokines such as TNF-· [3, 4]. Some of the bacterial components, including lipopolysaccharide (LPS), directly activate mast cells through their surface receptors [5, 6]. However, the precise mechanisms of mast cell activation in innate immune responses are still largely unknown. Type I IFNs (IFN-·/ß), key immunoregulatory cytokines produced by macrophages and plasmacytoid dendritic cells after the exposure to pathogens, modulate innate and adaptive immune responses [7]. Although the function of type I IFNs is principally associated with the protection against viral infections, recent studies have revealed that type I IFNs are also involved in the immune response against other pathogens [8, 9]. In this regard, it has been shown that preceding IFN-· treatment sensitizes the mice for an enhanced production of TNF-· upon LPS stimulation [10]. It has also been shown that type I IFNs

Correspondence to: Dr. Hiroshi Nakajima Department of Internal Medicine II, Chiba University School of Medicine 1-8-1 Inohana Chiba City, Chiba 260-8670 (Japan) Tel. +81 43 226 2093, Fax +81 43 226 2095, E-Mail [email protected]

are rapidly produced by macrophages upon LPS stimulation [11]. These findings suggest that type I IFNs may participate in immune responses against bacterial infection through the production of TNF-·. Two Janus kinases (JAKs), Tyk2 and Jak1, are associated with IFN-·/ß receptor components IFNAR1 and IFNAR2, respectively [12, 13]. Upon ligand binding, Jak1 and Tyk2 are activated and the activated JAKs phosphorylate Stat1 and Stat2 [12, 13]. Subsequently, these activated STATs associate to form either Stat1 homodimers or Stat1/Stat2 heterodimers, which then translocates to the nucleus to induce gene expression [14, 15]. Recently, the physiological function of Jak1 and Tyk2 in type I IFN signaling has been determined using mice lacking Jak1 or Tyk2 [16–19]. These studies have revealed that, whereas Jak1 is essential for responding to type I IFNs in most cell types [16], the requirement of Tyk2 in type I IFN signaling differs depending on cell types [17–19]. It has been demonstrated that Tyk2 is essential for IFN-· signaling in IL-7-dependent B cells [19] but not in fibroblasts [17, 18]. However, the role of Tyk2 in IFN-· signaling in mast cells is unknown. In this study, we determined whether Tyk2 is essential for IFN-· signaling in mast cells. Our findings have clearly demonstrated that using Tyk2-deficient mice, Tyk2 is required for IFN-·-induced Stat1 phosphorylation and subsequent gene induction in mast cells.

RT-PCR Analysis for IP-10, MCP-5, IFNAR1, IFNAR2, and Jak1 BMMCs or fibroblasts from WT mice or Tyk2–/– mice were stimulated with IFN-· (1,000 U/ml) at 37 ° C for 3 h. Total cellular RNA was prepared using Isogen solution (Nippon Gene, Tokyo, Japan) according to the manufacturer’s instruction. The first-strand complementary DNA (cDNA) was synthesized from total RNA using Moloney murine leukemia virus reverse transcriptase and oligo(dT) primers (Pharmacia Biotech, Buckinghamshire, UK). cDNAs encoding IFN-Á-inducible protein 10 (IP-10), monocyte chemoattractant protein-5 (MCP-5), IFNAR1, IFNAR2, and Jak1 were amplified by PCR using the following primer pairs: IP-10 5)-GAGATCATTGCCACGATGAA-3) and 5)-CACTGGGTAAAGGGGAGT-3), MCP-5 5)-AATCACAAGCAGCCAGTG-3) and 5)-GGGAACTTCAGGGGGAAATA-3), IFNAR1 5)-CCTGCTGAATAAGACCAGCAACTTC-3) and 5)-GTGCTTTACTTCTACAGCGACCGTG-3), IFNAR2 5)-CAAGCCTCTGCAACAAACCTCTGAC-3) and 5)-GATTTCTCAGATGACCCATCTTCAG-3), Jak1 5)-CTGCTAGCATGATGAGACAGGTTTC-3) and 5)-TTGGAGTCTTCAACACACTCAGGAG-3). ß-Actin was used to normalize the cDNA amount to be used. Immunoblotting Preparation of whole cell extracts and immunoblottings were performed as described previously [20]. The following antisera were used: anti-phospho-Stat1 (New England Biolabs, Beverly, Mass., USA) and anti-Stat1 (Upstate Biotechnology, Lake Placid, N.Y., USA). Data Analysis Data are summarized as mean B SD. The statistical analysis of the results was performed by the unpaired t test. p values ! 0.05 were considered significant.

Methods Mice and Cytokines Tyk2-deficient (Tyk2–/–) mice [17] were backcrossed for more than four generations onto BALB/c mice (Japan SLC, Shizuoka, Japan). The mice were genotyped by PCR as described previously [17] and littermate wild-type (WT) mice were used as controls. Mice were housed in microisolator cages under pathogen-free conditions. All experiments were performed according to the guidelines of the Chiba University. Recombinant murine IFN-· and IL-12 were purchased from R&D systems (Minneapolis, Minn., USA). Culture of Bone Marrow-Derived Mast Cells Primary culture of IL-3-dependent bone marrow-derived mast cells (BMMCs) was prepared from 8- to 12-week-old WT or Tyk2–/– mice and maintained as described previously [20]. BMMCs obtained after 4 weeks of culture were 1 99% mast cells. Culture of Fibroblasts Skin fibroblasts were prepared from WT mice or Tyk2–/– mice and maintained as described previously [21]. Flow-Cytometric Analysis BMMCs were stained and analyzed on FACScalibur (Becton Dickinson, San Jose, Calif., USA) using CELLQuest software. Anti-

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CD117 (c-kit) antibody (2B8) was purchased from BD PharMingen (San Diego, Calif., USA). Prior to staining, Fc receptors were blocked with anti-CD16/32 antibody (2.4G2, BD PharMingen).

Int Arch Allergy Immunol 2004;134(suppl 1):25–29

Results and Discussion

Development of IL-3-Dependent BMMCs Is Normal in Tyk2–/– Mice To determine the role of Tyk2 in mast cell development, bone marrow cells from WT mice or Tyk2–/– mice were cultured in the presence of IL-3 and the number of mast cells were evaluated every 7 days. As shown in figure 1a, the number of mast cells recovered from the culture was indistinguishable between WT and Tyk2–/– mice. Over 99% of cells obtained after 4 weeks of culture were morphologically mast cells in Tyk2–/– mice as well as in WT mice (data not shown). In addition, Tyk2–/– BMMCs expressed comparable levels of c-kit to WT BMMCs (fig. 1b). These results suggest that Tyk2 is not required for the development of IL-3-dependent mast cells.

Mori/Hirose/Suzuki/Nakajima/Seto/Ikeda/ Shimoda/Nakayama/Saito/Iwamoto

Fig. 1. Development of IL-3-dependent BMMCs is normal in Tyk2–/– mice. a Bone

marrow cells from WT mice or Tyk2–/– mice were cultured in the presence of IL-3. Indicated days later, the number of BMMCs was evaluated. Data are means B SD of 5 experiments for each genotype. b BMMCs from WT mice or Tyk2–/– mice were stained with anti-c-kit APC and analyzed on FACS. Representative FACS profiles for c-kit staining from five independent experiments are shown.

IFN-·-Induced Expression of IP-10 and MCP-5 Is Diminished in Tyk2–/– BMMCs Jak1 and Tyk2 are associated with receptors for type I IFNs [12–15]. Using Jak1-deficient mice, it has been shown that Jak1 is essential for biological responses in IFN-·/ß signaling [16]. On the other hand, it has been demonstrated that the requirement of Tyk2 in IFN-·/ß signaling differs depending on cell types [17–19]. To examine the role of Tyk2 in IFN-·-mediated functions in mast cells, BMMCs from WT mice or Tyk2–/– mice were stimulated with IFN-· and the expression of IFN-responsive genes was analyzed at mRNA levels. As shown in figure 2, IFN-·-induced expression of IP-10 and MCP-5, which play important roles in the host defense to pathogens [22, 23], was severely decreased in Tyk2–/– BMMCs as compared with that in WT BMMCs. On the other hand, Tyk2–/– fibroblasts expressed mRNA for IP-10 and MCP-5 at a level comparable to that in WT fibroblasts (fig. 2). These results suggest that Tyk2 is essential for IFN-·-mediated gene expression in mast cells but not in fibroblasts. On the other hand, IL-12, another cytokine that utilizes Tyk2 as a signaling molecule [24] and augments innate immune responses [25], exhibited no significant effects even on WT BMMCs because of the absence of functional IL-12R (data not shown). IFN-·-Induced Phosphorylation of Stat1 Is Diminished in Tyk2–/– BMMCs IFN-·-mediated gene induction was diminished in Tyk2–/– BMMCs (fig. 2). Because most IFN-·-induced responses depend on Stat1 activation [26, 27], we next

Role of Tyk2 in Mast Cells

Fig. 2. IFN-·-induced gene expression is diminished in Tyk2–/–

BMMCs. BMMCs or fibroblasts from WT mice or Tyk2–/– mice were stimulated with IFN-· (1,000 U/ml) for 3 h. The expression of IP-10 and MCP-5 mRNA was determined by RT-PCR. Representative data from 5 independent experiments are shown.

examined IFN-·-induced phosphorylation of Stat1 in Tyk2–/– BMMCs. As shown in figure 3, IFN-·-induced Stat1 phosphorylation was severely decreased in Tyk2–/– BMMCs as compared with that in WT BMMCs (fig. 3). In contrast, consistent with previous reports [17, 18], IFN·-induced Stat1 phosphorylation was similarly observed in Tyk2–/– fibroblasts and WT fibroblasts (fig. 3). These results suggest that Tyk2 is essential for IFN-·-induced Stat1 phosphorylation and then for Stat1-dependent gene expression in mast cells. Because IFN-·-induced Stat1 phosphorylation was diminished in Tyk2–/– BMMCs but not in Tyk2–/– fibro-

Int Arch Allergy Immunol 2004;134(suppl 1):25–29

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Concluding Remarks

Fig. 3. Tyk2 is essential for IFN-·-induced Stat1 phosphorylation in BMMCs but not in fibroblasts. WT BMMCs, Tyk2–/– BMMCs, WT fibroblasts, or Tyk2–/– fibroblasts were stimulated with or without IFN-· (1,000 U/ml) for 30 min. Whole cell extracts were subjected to Western blotting with anti-phospho-Stat1 antibody or anti-Stat1 antibody. Representative data from 5 independent experiments are shown.

Fig. 4. Expression of IFNAR1 is lower in BMMCs than that in fibroblasts. Total cellular RNA was prepared from WT BMMCs, Tyk2–/– BMMCs, WT fibroblasts, or Tyk2–/– fibroblasts. Expression of IFNAR1, IFNAR2, and Jak1 transcripts was examined by RT-PCR analysis. Representative data from 5 independent experiments are shown.

In the present study, we show that IFN-· induces the expression of the chemokines IP-10 and MCP-5 in mast cells and that Tyk2 is essential for IFN-·-induced gene expression in mast cells but not in fibroblasts. We found that IFN-· induced mRNA expression of IP-10 and MCP-5, important chemokines for innate immune responses [22, 23], in WT BMMCs, but the chemokine expression was diminished in Tyk2–/– BMMCs but not in Tyk2–/– fibroblasts. In addition, we found that IFN-·induced Stat1 phosphorylation was decreased in Tyk2–/– BMMCs but not in Tyk2–/– fibroblasts. These results suggest that Tyk2 is required for IFN-·-induced Stat1 phosphorylation and subsequent gene expression in mast cells but not in fibroblasts. Recent studies using Tyk2–/– mice have revealed that Tyk2 regulates both acquired and innate immune responses. It has been shown that Tyk2 is essential for IL12-mediated T cell function, including IFN-Á production and Th1 cell differentiation [17, 18]. Tyk2 is also required for the downregulation of Th2 cell-mediated allergic inflammation in murine models of allergic asthma [28]. In addition, it has been demonstrated that Tyk2 plays an important role in endotoxin shock as a component of type I IFN signaling [29]. These findings suggest that Tyk2 is involved not only in acquired immune responses but also in innate immune responses. Our findings that Tyk2 is required for the IFN-·-induced expression of IP-10 and MCP-5 in mast cells also suggest the important roles of Tyk2 in innate immune responses. We have shown here that Tyk2 is essential for IFN-· signaling in mast cells but not in fibroblasts. Although further studies are required, our data suggest that the expression levels of IFNAR1 may account for the different requirement for Tyk2 in IFN-· signaling between mast cells and fibroblasts.

Acknowledgments

blasts, we next compared the expression of IFN-· receptor components (IFNAR1 and IFNAR2) and Jak1 in BMMCs and fibroblasts. As shown in figure 4, regardless of the presence or absence of Tyk2, the expression of IFNAR1 was significantly lower in BMMCs than that in fibroblasts. On the other hand, the expression of IFNAR2 and Jak1 was comparable between BMMCs and fibroblasts (fig. 4). These results suggest that the limited expression of IFNAR1 in mast cells may account for the requirement of Tyk2 in IFN-· signaling.

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Int Arch Allergy Immunol 2004;134(suppl 1):25–29

We thank Dr. A. Suto and Dr. Y. Maezawa for valuable discussions and useful comments. This work was supported in part by grants from the Ministry of Education, Science and Culture, Japan.

Mori/Hirose/Suzuki/Nakajima/Seto/Ikeda/ Shimoda/Nakayama/Saito/Iwamoto

References 1 Galli SJ, Hammel I: Mast cell and basophil development. Curr Opin Hematol 1994;1:33– 39. 2 Metcalfe DD, Baram D, Mekori YA: Mast cells. Physiol Rev 1997;77:1033–1079. 3 Echtenacher B, Mannel DN, Hultner L: Critical protective role of mast cells in a model of acute septic peritonitis. Nature 1996;381:75– 77. 4 Malaviya R, Ikeda T, Ross E, Abraham SN: Mast cell modulation of neutrophil influx and bacterial clearance at sites of infection through TNF-·. Nature 1996;381:77–80. 5 Malaviya R, Gao Z, Thankavel K, Merwe PA, Abraham SN: The mast cell tumor necrosis factor · response to FimH-expressing Escherichia coli is mediated by the glycosylphosphatidylinositol-anchored molecule CD48. Proc Natl Acad Sci USA 1999;96:8110–8115. 6 McCurdy JD, Lin TJ, Marshall JS: Toll-like receptor 4-mediated activation of murine mast cells. J Leukoc Biol 2001;70:977–984. 7 Pestka S, Langer JA, Zoon KC, Samuel CE: Interferons and their actions. Annu Rev Biochem 1987;56:727–777. 8 Decker T, Stockinger S, Karaghiosoff M, Muller M, Kovarik P: IFNs and STATs in innate immunity to microorganisms. J Clin Invest 2002;109:1271–1277. 9 Bogdan C: The function of type I interferons in antimicrobial immunity. Curr Opin Immunol 2000;12:419–424. 10 Nansen A, Randrup Thomsen A: Viral infection causes rapid sensitization to lipopolysaccharide: Central role of IFN-·/ß. J Immunol 2001;166:982–988. 11 Toshchakov V, Jones BW, Perera PY, Thomas K, Cody MJ, Zhang S, Williams BR, Major J, Hamilton TA, Fenton MJ, Vogel SN: TLR4, but not TLR2, mediates IFN-ß-induced STAT1·/ß-dependent gene expression in macrophages. Nat Immunol 2002;3:392–398. 12 Schindler C, Darnell JE Jr: Transcriptional responses to polypeptide ligands: The JAKSTAT pathway. Annu Rev Biochem 1995;64: 621–651. 13 Leonard WJ, O’Shea JJ: Jaks and STATs: Biological implications. Annu Rev Immunol 1998; 16:293–322.

Role of Tyk2 in Mast Cells

14 Darnell JE Jr, Kerr IM, Stark GR: Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 1994;264:1415–1421. 15 Ihle JN, Nosaka T, Thierfelder W, Quelle FW, Shimoda K: Jaks and Stats in cytokine signaling. Stem Cells 1997;15:105–111. 16 Rodig SJ, Meraz MA, White JM, Lampe PA, Riley JK, Arthur CD, King KL, Sheehan KC, Yin L, Pennica D, Johnson EM Jr, Schreiber RD: Disruption of the Jak1 gene demonstrates obligatory and nonredundant roles of the Jaks in cytokine-induced biologic responses. Cell 1998;93:373–383. 17 Shimoda K, Kato K, Aoki K, Matsuda T, Miyamoto A, Shibamori M, Yamashita M, Numata A, Takase K, Kobayashi S, Shibata S, Asano Y, Gondo H, Sekiguchi K, Nakayama K, Nakayama T, Okamura T, Okamura S, Niho Y: Tyk2 plays a restricted role in IFN-· signaling, although it is required for IL-12mediated T cell function. Immunity 2000;13: 561–571. 18 Karaghiosoff M, Neubauer H, Lassnig C, Kovarik P, Schindler H, Pircher H, McCoy B, Bogdan C, Decker T, Brem G, Pfeffer K, Muller M: Partial impairment of cytokine responses in Tyk2-deficient mice. Immunity 2000;13:549– 560. 19 Shimoda K, Kamesaki K, Numata A, Aoki K, Matsuda T, Oritani K, Tamiya S, Kato K, Takase K, Imamura R, Yamamoto T, Miyamoto T, Nagafuji K, Gondo H, Nagafuchi S, Nakayama K, Harada M: Tyk2 is required for the induction and nuclear translocation of Daxx which regulates IFN-·-induced suppression of B lymphocyte formation. J Immunol 2002;169: 4707–4711. 20 Suzuki K, Nakajima H, Watanabe N, Kagami S, Suto A, Saito Y, Saito T, Iwamoto I: Role of common cytokine receptor Á chain (Ác)- and Jak3-dependent signaling in the proliferation and survival of murine mast cells. Blood 2000; 96:2172–2180.

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21 Fischer SM, Viaje A, Mills GD, Slaga TJ: Explant methods for epidermal cell culture. Methods Cell Biol 1980;21A:207–227. 22 Khan IA, MacLean JA, Lee FS, Casciotti L, DeHaan E, Schwartzman JD, Luster AD: IP-10 is critical for effector T cell trafficking and host survival in Toxoplasma gondii infection. Immunity 2000;12:483–494. 23 Sarafi MN, Garcia-Zepeda EA, MacLean JA, Charo IF, Luster AD: Murine monocyte chemoattractant protein (MCP)-5: A novel CC chemokine that is a structural and functional homologue of human MCP-1. J Exp Med 1997; 185:99–109. 24 Bacon CM, McVicar DW, Ortaldo JR, Rees RC, O’Shea JJ, Johnston JA: Interleukin 12 (IL-12) induces tyrosine phosphorylation of JAK2 and TYK2: Differential use of Janus family tyrosine kinases by IL-2 and IL-12. J Exp Med 1995;181:399–404. 25 Trinchieri G: Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat Rev Immunol 2003;3:133–146. 26 Meraz MA, White JM, Sheehan KC, Bach EA, Rodig SJ, Dighe AS, Kaplan DH, Riley JK, Greenlund AC, Campbell D, Carver-Moore K, DuBois RN, Clark R, Aguet M, Schreiber RD: Targeted disruption of the Stat1 gene in mice reveals unexpected physiologic specificity in the JAK-STAT signaling pathway. Cell 1996; 84:431–442. 27 Durbin JE, Hackenmiller R, Simon MC, Levy DE: Targeted disruption of the mouse Stat1 gene results in compromised innate immunity to viral disease. Cell 1996;84:443–450. 28 Seto Y, Nakajima H, Suto A, Shimoda K, Saito Y, Nakayama KI, Iwamoto I: Enhanced Th2 cell-mediated allergic inflammation in Tyk2deficient mice. J Immunol 2003;170:1077– 1083. 29 Karaghiosoff M, Steinborn R, Kovarik P, Kriegshauser G, Baccarini M, Donabauer B, Reichart U, Kolbe T, Bogdan C, Leanderson T, Levy D, Decker T, Muller M: Central role for type I interferons and Tyk2 in lipopolysaccharide-induced endotoxin shock. Nat Immunol 2003;4:471–477.

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Review Int Arch Allergy Immunol 2004;134(suppl 1):30–36 DOI: 10.1159/000077790

Peroxisome Proliferator-Activated Receptor Á Regulates Eosinophil Functions: A New Therapeutic Target for Allergic Airway Inflammation Shigeharu Ueki Yoshinori Matsuwaki Hiroyuki Kayaba Hajime Oyamada Akira Kanda Atsuko Usami Norihiro Saito Junichi Chihara Department of Clinical and Laboratory Medicine, Akita University School of Medicine, Akita, Japan

Key Words Eosinophil W Peroxisome proliferator-activated receptor Á W Thiazolidinediones W Chemotaxis W Survival W Asthma

Abstract Peroxisome proliferator-activated receptor Á (PPARÁ) is a nuclear receptor that regulates lipid metabolism and glucose homeostasis. PPARÁ is not only highly expressed in adipose tissue but also in cells involved in the immune system, and it exerts anti-inflammatory activities. We showed that eosinophils, a major inflammatory cell in allergic inflammation, express PPARÁ. PPARÁ negatively modulates eosinophil functions, such as survival, chemotaxis, antibody-dependent cellular cytotoxicity and degranulation. Recently, three independent groups have demonstrated that PPARÁ agonists inhibit airway inflammation in an animal model of asthma. This evidence suggests that PPARÁ agonists may be a new therapeutic modality for the treatment of allergic diseases including asthma.

Introduction

In the 1990s, peroxisome proliferator-activated receptors (PPARs) were identified as a member of the nuclear hormone receptor superfamily of ligand-activated transcription factors including receptors for steroids, thyroid hormone, vitamin D and retinoic acid. PPARs promote the transcription of target genes by forming heterodimers with retinoid X receptor and binding to specific motifs termed PPAR-responsive elements. To date, three mammalian subtypes have been identified, referred to as PPAR·, ß (or ‰), and Á, which are encoded by separate genes. PPARÁ is expressed at a high level in adipose tissue and regulates adipocyte differentiation [1] and glucose homeostasis [see 2 and 3 for reviews]. It has been shown that PPARÁ is present in human hematopoietic cells and involved in immune regulation as well as adipogenesis [4]. The functional role of PPARÁ in allergic conditions, however, is far from clear. In this paper, we focus on the functions of PPARÁ in eosinophils and allergic airway inflammation.

Copyright © 2004 S. Karger AG, Basel

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Correspondence to: Dr. Junichi Chihara Department of Clinical and Laboratory Medicine Akita University School of Medicine 1-1-1, Hondo, Akita 010-8543 (Japan) Tel. +81 18 884 6180, Fax +81 18 836 2624, E-Mail [email protected]

PPARÁ Expression on Human Eosinophils

We investigated the expression of PPARÁ on human eosinophils using RT-PCR and Western blotting. PPARÁ mRNA and protein were detectable in both freshly isolated eosinophils and an eosinophilic cell line, Eol-1 [5]. PPARÁ mRNA and protein were also detected in murine and rat eosinophils [6]. Recently accumulated evidence indicates that the expression of PPARÁ is upregulated in several pathological conditions [7–10]. Compared with PPAR·, PPARÁ expression in eosinophils was present at variable levels in individuals [6]. Using immunofluorescence staining, Benayoun et al. [11] previously reported that the main PPARÁ-expressing cells in bronchial biopsies from asthmatic patients were eosinophils and macrophages. Moreover, the expression of PPARÁ in airway epithelium and smooth muscle of asthmatics was downregulated by corticosteroid therapy. This evidence bolsters the theory that the expression of PPARÁ in eosinophils may reflect an allergic inflammatory response, although PPARÁ protein expression in eosinophils did not change as a result of stimulation with IL-5, a major eosinophil-activating cytokine [5], and showed no correlation with serum eosinophilic cationic protein or CD25 measurements in asthmatic patients [6]. It has been shown that the presence of IL-4, a crucial cytokine for the development of Th2 cells, can induce upregulation of PPARÁ in some cell types, such as lymphocytes and bronchial epithelial cells [12– 14]. PPARÁ-modulating factors in eosinophils are the subject of future investigation.

PPARÁ Agonists Inhibit IL-5-Induced Eosinophil Survival

Activated eosinophils exhibit prolonged survival in proportion to the degree of asthma attacks. Apoptotic eosinophils are recognized by macrophages by a specific process without disgorgement of their histotoxic contents [15]. In this context, inducing apoptosis of inflammatory cells is one way to treat allergic diseases. Several studies have shown that PPARÁ is involved in the regulation of cell survival [7, 12, 16–22]. PPARÁ did not have any agonistic effect on spontaneous apoptosis, which is consistent with results in neutrophils [23]. We found, however, that troglitazone, a synthetic PPARÁ agonist, reduced the prolonged survival of eosinophils stimulated with IL-5 (fig. 1a, b). Since IL-5 is believed to contribute to eosinophil activation during asthma attacks, it is quite

PPARÁ Regulates Eosinophil Functions

interesting that troglitazone induced apoptosis in the IL5-activated eosinophils. Recently, it has been suggested that the Bcl-2 family of proteins is involved in the regulation of eosinophil survival [24], but troglitazone has no effect on the expression of Bcl-2 and Bcl-xl proteins in eosinophils (our unpubl. data). Similar results were observed in cancer cells and vascular smooth muscle cells despite the induction of apoptosis [19–21]. In an eosinophilic cell line, troglitazone suppressed cell proliferation by the induction of the p21WAF/CIP1 cyclin-dependent kinase inhibitor, which regulates cell cycle progression [25].

PPARÁ Agonists Inhibit Various Eosinophil Functions

Next, we examined the effect of troglitazone on eotaxin-directed migration of eosinophils in a chemotaxis assay by using Boyden chambers. Troglitazone significantly inhibited the migration of eosinophils in a concentrationdependent manner (fig. 2). Woerly et al. [6] reported that thiazolidinediones also inhibited IL-5-induced chemotaxis, and that GW9662, a PPARÁ antagonist, led to a virtually complete loss of chemotaxis. Consistent with these results, migration of monocytes was reportedly reduced by synthetic PPARÁ agonists [26]. In the study by Woerly et al. [6], thiazolidinediones did not inhibit MCP-1directed ERK MAP kinase phosphorylation, suggesting that PPARÁ agonists block a target point downstream of MAP kinase or independent of this pathway. The former may be the case in eosinophils, because MAP kinase is critical for eosinophil chemotaxis [27]. In addition, Woerly et al. [6] reported that PPARÁ agonists inhibited eosinophil-mediated antibody-dependent cellular cytotoxicity. We found that thiazolidinediones reduced IL-5-induced eosinophil-derived neurotoxin release in PPARÁ-dependent pathways (unpubl. data). Thus, human eosinophils express functional PPARÁ negatively regulating various eosinophil functions. It has been proposed that PPARÁ regulates inflammation by either downregulating [16, 28–30] or cooperatively enhancing [22, 31] nuclear factor-ÎB-mediated transcription, although the mechanism is unknown in eosinophils.

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Is PPARÁ a Key Molecule in Allergic Airway Inflammation?

Recent findings have led to the working hypothesis that PPARÁ is involved actively in aspects of negative immunoregulation. Expression of adhesion molecules by endothelial cells and adhesion of inflammatory cells are a first essential step in allergic inflammation. PPARÁ activators inhibit cytokine-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule (ICAM-1) in vascular endothelial cells [30, 32]. Furthermore, stimulation of PPARÁ with its agonists reduces cytokine production from monocytes [33], T cells [14, 34], dendritic cells [35, 36], mast cells [37] and bronchial epithelial cells [13]. Recently, Patel et al. [38] reported that airway smooth muscle cells expressed functional PPARÁ. They stated that PPARÁ ligands may prove to be particularly effective in the treatment of steroid-insensitive asthma, because PPARÁ ligands are more potent than steroids in inhibiting cell growth and G-CSF release. Many studies have focused on lymphocytes, but

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Fig. 1. Troglitazone induced apoptosis on the IL-5-activated eosinophil [5]. a Dot plots of FACS-analyzed eosinophils treated with annexin V to stain early-phase apoptotic cells and propidium iodine (PI) to stain the late phase. b The dose-dependent effect of troglitazone on IL-5-induced eosinophil survival (n = 4) is shown. Eosinophils were incubated with and without troglitazone in the presence of 1 ng/ml of IL-5 for 48 h. Data are expressed as mean B SD. *p ! 0.05 vs. without troglitazone.

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Fig. 2. Troglitazone inhibited the eotaxin-

directed eosinophil chemotaxis in a dosedependent manner (n = 4) [5]. Purified eosinophils were preincubated with increasing concentrations of troglitazone for 1 h. Migration assays were performed using Boyden chambers. Chemotactic response to eotaxin alone was considered as 100% and that to buffer was subtracted. Data are expressed as mean B SD. *p ! 0.05 vs. eotaxin alone.

Fig. 3. Possible mechanisms by which PPARÁ regulates eosinophil functions and activation in asthmatic airways.

PPARÁ is expressed by eosinophils and directly inhibits the factor-dependent survival, chemotaxis or degranulation. Furthermore, PPARÁ also has an anti-inflammatory effect in these types of cells including vascular endothelial cells, smooth muscle cells, mast cells, antigen-presenting cells (APCs), lymphocytes and airway epithelial cells. Activation of PPARÁ decreases serum levels of IgE, airway hyperresponsiveness and lung inflammation in animal models of asthma. Thus, administration of PPARÁ agonists can be an alternative approach to the treatment of asthma and related inflammatory airway disorders.

PPARÁ Regulates Eosinophil Functions

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the role of PPARÁ in allergic conditions is controversial. Murine Th cell clones and isolated splenocytes express PPARÁ, and thiazolidinediones inhibit the proliferation of these cells [14, 34]. It has been shown that T cells on which PHA has induced PPARÁ expression easily undergo apoptosis when exposed to PPARÁ agonists [39]. It is interesting that Th2 cells expressed greater levels of PPARÁ than Th1 cells [40], because asthma is classically characterized by Th2-dependent immunity. In B cells, PPARÁ activation causes apoptosis [17, 41] and IL-4induced IgE class switching [42]. In contrast, several studies have indicated that the protective effects exhibited by PPARÁ ligands in inflammatory diseases may be due to immune deviation away from Th1 and towards Th2 [37, 43, 44]. Putatively, naturally occurring PPARÁ ligands have been proposed, including oxidized linoleic acid and the final metabolites of prostaglandin (PG) D2 degradation, 15-deoxy-¢12,14-PGJ2 (15d-PGJ2) [2]. Recent findings suggest, however, that 15d-PGJ2 acts through distinct pathways other than PPARÁ [23, 45, 46], and 15d-PGJ2 can be a potent activator of eosinophils by its interaction with the DP2/chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) in a lower concentration than those for the proposed anti-inflammatory effect [47]. PGD2, a bronchoconstrictor, is formed abundantly in bronchoalveolar lavage from asthmatic patients after provocation with the antigen [48]. It undergoes spontaneous dehydration to PGJ2 in vitro, a reaction

enhanced by an albumin-induced catalysis, which yields 15d-PGJ2 [49–51]. Thus, the function of natural PPARÁ agonists in the lung is less well understood.

PPARÁ Agonists as Potent Antiasthma Drugs

A considerable amount of evidence suggests that PPARÁ may be beneficial in the treatment of inflammatory diseases. For example, natural and synthetic PPARÁ ligands have been shown to exert anti-inflammatory effects in models or patients with atherosclerosis [52], inflammatory bowel disease [9, 53], psoriasis [54] and allergic encephalomyelitis [44]. Most recently, Mueller et al. [55] demonstrated that oral administration of thiazolidinedione alleviated lung inflammation and mucous production in a murine model of asthma. In humans, however, the problem is that systemic administration of thiazolidinediones can cause edema or serious hepatic reactions [56]. Interestingly, two independent groups have also shown that local administration of PPARÁ agonists had similar beneficial effects on pathological conditions including serum levels of IgE, airway hyperresponsiveness and lung eosinophilia [6, 57]. These in vivo studies provide evidence for the anti-asthma effects of PPARÁ, which remain a pharmacological target of high potential (fig. 3). We conclude that the inhaled administration of thiazolidinediones may be a new therapeutic modality for the treatment of asthma.

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38 Patel HJ, Belvisi MG, Bishop-Bailey D, Yacoub MH, Mitchell JA: Activation of peroxisome proliferator-activated receptors in human airway smooth muscle cells has a superior antiinflammatory profile to corticosteroids: Relevance for chronic obstructive pulmonary disease therapy. J Immunol 2003;170:2663– 2669. 39 Tautenhahn A, Brune B, von Knethen A: Activation-induced PPARÁ expression sensitizes primary human T cells toward apoptosis. J Leukoc Biol 2003;73:665–672. 40 Chtanova T, Kemp RA, Sutherland AP, Ronchese F, Mackay CR: Gene microarrays reveal extensive differential gene expression in both CD4(+) and CD8(+) type 1 and type 2 T cells. J Immunol 2001;167:3057–3563. 41 Schlezinger JJ, Jensen BA, Mann KK, Ryu HY, Sherr DH: Peroxisome proliferator-activated receptor Á-mediated NF-ÎB activation and apoptosis in pre-B cells. J Immunol 2002;169: 6831–6841. 42 Miyazaki Y, Tachibana H, Yamada K: Inhibitory effect of peroxisome proliferator-activated receptor-Á ligands on the expression of IgE heavy chain germline transcripts in the human B cell line DND39. Biochem Biophys Res Commun 2002;295:547–552. 43 Saubermann LJ, Nakajima A, Wada K, Zhao S, Terauchi Y, Kadowaki T, Aburatani H, Matsuhashi N, Nagai R, Blumberg RS: Peroxisome proliferator-activated receptor Á agonist ligands stimulate a Th2 cytokine response and prevent acute colitis. Inflamm Bowel Dis 2002; 8:330–339. 44 Natarajan C, Bright JJ: Peroxisome proliferator-activated receptor-Á agonists inhibit experimental allergic encephalomyelitis by blocking IL-12 production, IL-12 signaling and Th1 differentiation. Genes Immun 2002;3:59–70. 45 Vaidya S, Somers EP, Wright SD, Detmers PA, Bansal VS: 15-Deoxy-¢12,1412,14-prostaglandin J2 inhibits the ß2 integrin-dependent oxidative burst: Involvement of a mechanism distinct from peroxisome proliferator-activated receptor Á ligation. J Immunol 1999;163:6187– 6192. 46 Harris SG, Smith RS, Phipps RP: 15-Deoxy¢12,14-PGJ2 induces IL-8 production in human T cells by a mitogen-activated protein kinase pathway. J Immunol 2002;168:1372–1379. 47 Monneret G, Li H, Vasilescu J, Rokach J, Powell WS: 15-Deoxy-¢12,1412,14-prostaglandin D2 and J2 are potent activators of human eosinophils. J Immunol 2002;168:3563–3569. 48 Murray JJ, Tonnel AB, Brash AR, Roberts LJ 2nd, Gosset P, Workman R, Capron A, Oates JA: Release of prostaglandin D2 into human airways during acute antigen challenge. N Engl J Med 1986;315:800–804. 49 Kikawa Y, Narumiya S, Fukushima M, Wakatsuka H, Hayaishi O: 9-Deoxy-¢9, ¢12–13,14dihydroprostaglandin D2, a metabolite of prostaglandin D2 formed in human plasma. Proc Natl Acad Sci USA 1984;81:1317–1321.

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Review Int Arch Allergy Immunol 2004;134(suppl 1):37–43 DOI: 10.1159/000077791

Dual Signaling and Effector Pathways Mediate Human Eosinophil Activation by Platelet-Activating Factor Masahiko Kato a Hirohito Kita d, e Atsushi Tachibana b Yasuhide Hayashi a Yoshiaki Tsuchida a Hirokazu Kimura c a Department

of Allergy, Gunma Children’s Medical Center, Hokkitsu, Gunma, and b Department of Pediatrics, Gunma University School of Medicine and c Gunma Prefectural Institute of Public Health and Environmental Sciences, Maebashi, Japan; Departments of d Immunology and e Internal Medicine, Mayo Clinic and Mayo Foundation, Rochester, Minn., USA

Key Words Eosinophils W Platelet-activating factor W Neutrophils W Signal transduction

Abstract Platelet-activating factor (PAF) induces various cellular functions in eosinophils including chemotaxis, adhesion, superoxide anion (O2 – ) production, and degranulation. While PAF shares many biological effects with other chemotactic factors such as N-formyl-methionyl-leucyl-phenylalanine, complement fragments, and lipid mediators, PAF is unique in that its action is relatively resistant to pertussis toxin (PTX), and in activating eosinophils more strongly than neutrophils. In this review we consider how PAF might activate human eosinophils in preference to neutrophils, and discuss possible mechanisms of PAF-induced activation of human eosinophils via two distinct signaling and effector pathways. Recently we analyzed O2 – production by eosinophils using a sensitive, real-time chemiluminescence method. Our results showed that in human eosinophils PAF activates two distinct signaling and effector pathways coupled to the PAF

ABC

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Accessible online at: www.karger.com/iaa

receptor: one linked to PTX-sensitive G protein(s) and another to PTX-resistant G protein(s), phosphatidylinositol 3-kinase, and cellular adhesion. This activation of two different G proteins by the eosinophil PAF receptor may explain the strong and diverse biological responses of human eosinophils to PAF. Copyright © 2004 S. Karger AG, Basel

Introduction

Platelet-activating factor (PAF) has a variety of cellular functions [reviewed in 1, 2] including aggregation and secretion of platelets [2], aggregation and stimulation of neutrophils [3], activation of macrophages [4], an increase in vascular permeability in the skin [5], and contraction of guinea pig ileum and lung parenchymal strips [6, 7]. A specific receptor for PAF has been identified in a number of tissues and cell types including eosinophils [8, 9]. PAF is among the most important inducers of eosinophil function and the action of PAF on eosinophils has been investigated in a number of in vitro studies. For example, PAF is one of the most potent chemoattractants for eosino-

Correspondence to: Dr. Masahiko Kato Department of Allergy Gunma Children’s Medical Center, Hokkitsu Gunma 377-8577 (Japan) Tel. +81 279 52 3551, Fax +81 279 52 2045, E-Mail [email protected]

phils and selectively induces the migration of eosinophils over neutrophils [10, 11]. PAF also has an important role in eosinophil transmigration through basement membrane components [12] or a human epithelial cell monolayer [13]. PAF increases the binding of IgE to human eosinophils and enhances cytotoxicity towards schistosomula of Schistosoma mansoni [14]. Furthermore, PAF promotes actin polymerization [15] and eosinophil adherence to vascular endothelial cells through a ß2 integrin, ·Mß2 (CD11b/CD18, Mac-1) [16] and evokes the release of granule proteins [17, 18], reactive oxygen species [19, 20], leukotriene (LT) C4 [21], and formation of lipid bodies [22]. A recent report suggested that PAF induces the release of neurotrophin nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3 [23], and matrix metalloproteinase-9 [24] in human eosinophils. PAF is also produced by a variety of cells including eosinophils, neutrophils, macrophages, monocytes, basophils, endothelial cells, and platelets [reviewed in 1, 2]. A recent report demonstrated that PAF is generated by human eosinophils themselves, amplifying their effector functions in an autocrine manner [25]. In in vivo studies, the injection of PAF into rabbits causes rapid development of hypotension and an anaphylactoid reaction [26]. In studies of PAF-overexpressing transgenic mice the PAF receptor was also overexpressed, in association with bronchial hyperresponsiveness [27]. PAF-deficient mice, on the other hand, had impaired anaphylactic responses to endotoxin [28]. Although many in vitro and in vivo studies have been reported above, much less is known about the role of PAF in the pathophysiology of eosinophil-associated human diseases. In epidemiologic studies a lack of PAF acetylhydrolase, which catalyzes hydrolysis of PAF, was associated with atopy and severe asthma [29, 30]. While a recent clinical study demonstrated that administration of a new, long-acting PAF receptor antagonist to patients with atopic asthma effectively inhibited eosinophilic inflammation in the airways [31], other clinical studies in patients with asthma found no significant efficacy for PAF receptor antagonists [32]. Thus, further studies are needed to elucidate how PAF contributes to human asthma. In this review, we will consider observations indicating that human eosinophils stimulated with PAF possess a unique signaling and effector pathway differing from that in neutrophils.

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Differences in Cellular Responses to PAF between Human Eosinophils and Neutrophils

Physiologic differences between neutrophils and eosinophils are well established. Neutrophils, the most rapidly recruited leukocytes, are not only essential for phagocytosis and killing of bacteria but can also damage host cells. In contrast, eosinophils are largely localized to mucosal tissues and are recruited more slowly to sites of inflammation; release of their inflammatory mediators is directed toward the targets to which they adhere [1]. Differing responses of neutrophils and eosinophils to chemotactic factors such as PAF may be responsible for differences in functions and effector mechanisms between these cell types in host defenses generally and in specific human diseases. Evidence suggests that PAF shares many activities with other chemotactic factors for granulocytes, such as N-formyl-methionyl-leucyl-phenylalanine (FMLP), complement (C) 5a, and LTB4. In both eosinophils and neutrophils, PAF as well as other chemotactic factors operate via heterotrimeric G proteins as an intermediary, and stimulate an increase in intracellular Ca2+ concentration ([Ca2+]i), or activate protein kinases, and induce actin filament polymerization and chemotaxis [reviewed in 2, 33]. While human eosinophils showed nearly the same chemotactic responses characteristic of human neutrophils [34], several studies have highlighted differences between these two cell types concerning the effects of PAF apart from motility functions. For example, when stimulated with PAF, eosinophils produce at least 3 times more O2 – than neutrophils [20]. Furthermore, PAF alone induced O2 – production from eosinophils but not from neutrophils, while PAF primed both eosinophils and neutrophils for O2 – production stimulated with zymosan or FMLP [35]. We recently demonstrated significantly greater PAFinduced cellular adhesion and O2 – release in human eosinophils than in neutrophils from the same donors upon contact with human serum albumin, a ligand for ß2 integrin on both cells. Morphologically, eosinophils showed marked flattening and deformation following stimulation, while neutrophils showed minimal shape changes, in keeping with the difference observed for cellular adhesion [Fujiu, Kato et al., unpubl. data]. These observations suggested to us that differences in activation profiles between the two cell types were related to their different adhesion properties. Finally, studies of intracellular signaling also showed that PAF activated 42- and 44-kD mitogen-activated protein kinase (MAPK, ERK1 and ERK2) in eosinophils [36], while PAF did not acti-

Kato/Kita/Tachibana/Hayashi/Tsuchida/ Kimura

PAF Activates Two Distinct Effector Pathways in Human Eosinophils

PAF

150 Chemiluminescence intensity, X104 cpm

vate these MAPKs in human neutrophils [37]. These reports suggest quantitative and qualitative differences in cellular responses to PAF between eosinophils and neutrophils. Previous investigations demonstrated similar numbers of PAF receptors in human eosinophils and neutrophils [38]. Furthermore, PAF causes a similar increase in [Ca2+]i in both neutrophils and eosinophils, and the [Ca2+]i increase in neutrophils occurred at a lower PAF concentration than that required in eosinophils [20]. Therefore, receptor densities and the calcium responses cannot account for the distinctive eosinophil properties.

100 mIgG1

50

Anti-CD18

0 0

5

10

15

20

25

Time, minute

To obtain greater details concerning the molecular mechanisms of human eosinophil and neutrophil responses to PAF, we measured O2 – production by a chemiluminescence method [39], which produces results that correlate linearly with results from a conventional cytochrome c reduction method [40, 41]. Furthermore, the chemiluminescence method has proven to be approximately 100 times more sensitive than the conventional method, as well as providing a real-time kinetic analysis of O2 – production as opposed to cumulative data [39–41]. Using this approach we found that in human eosinophils PAF induced O2 – production biphasically with a sharp, transient first phase and a larger, sustained second phase; in contrast, PAF induced an essentially monophasic response in human neutrophils. The two phases of eosinophil responses were mediated by different mechanisms since the second phase was dependent on cellular adhesion and ß2 integrins while the first phase was independent of these (fig. 1). Furthermore, the first- and secondphase responses were mediated separately by pertussis toxin (PTX)-sensitive G protein(s) and PTX-resistant G protein(s) coupled to phosphatidylinositol 3-kinase (PI3K), respectively. Interestingly, the second-phase response was observed even in the absence of a first-phase response, and was more resistant to inhibition by a competitive PAF receptor antagonist [42]. These results suggest that PAF activates two distinct effector pathways coupled to PAF receptors in human eosinophils: one linked to PTX-sensitive G protein(s), while the other involves the activation of PTX-resistant G protein(s), PI3K, and cellular adhesion. We summarize these results in table 1. While the many details of the mechanisms representing these two distinct signaling and effector pathways

Dual Signaling Pathways in Human Eosinophils

Fig. 1. Effect of anti-CD18 antibody on PAF-induced bimodal superoxide anion generation in human eosinophils. Isolated eosinophils (2.5 ! 105 cells/sample) were preincubated with anti-CD18 antibody or control antibody, mouse IgG1, for 30 min, 1 ÌM PAF was added as indicated, and then chemiluminescence was measured as described by Kato et al. [42]. Real-time production of superoxide was measured as chemiluminescence intensity. Eosinophils show a bimodal response (a peak at 20–30 s and another sustained peak at 3–10 min). Anti-CD18 antibody almost completely inhibited the second phase of superoxide but did not affect the first phase of superoxide.

Table 1. Summary of the effect of cellular adhesion and pharmacological agents on the first and the second phase of superoxide anion production in human eosinophils

Treatment Stirring Anti-CD18 PTX (IC50) Genistein (IC50) Herbimycin A (IC50) Wortmannin (IC50) CV6209 (IC50)

First phase No effect No effect 20 ng/ml 0.7 Ìg/ml 0.1 Ìg/ml 1100 nM 0.1 nM

Second phase Inhibited Inhibited 1500 ng/ml 0.2 Ìg/l 0.03 Ìg/ml 4 nM 40 nM

Isolated eosinophils (2.5 ! 105 cells/sample) were preincubated with each drug for indicated time or stirring during incubation. Cells were then stimulated with 1 ÌM PAF and chemiluminescence was measured as described [42]. The IC50 values of drugs were calculated from at least four experiments for each drug.

Int Arch Allergy Immunol 2004;134(suppl 1):37–43

39

Fig. 2. A model of PAF-induced signaling and effector pathways in human eosinophils. Stimulation of eosinophils

with PAF induces activation of PTX-sensitive G protein(s) such as G·i2, which leads to the first phase of superoxide response or cell migration from blood vessels to inflammatory sites. Concomitantly, possibly by actions involving PAF receptor dimers, PAF induces activation of PTX-resistant G protein(s) such as G·q/11, leading to PI-3K; this causes the adhesion-dependent, sustained second phase of superoxide response and degranulation that results in epithelial cell damage.

remain unknown, at least two receptor-pathway relationships could be proposed. First, individual PAF receptors might be coupled to at least two different classes of G proteins in human eosinophils. Second, eosinophils might have multiple PAF receptor types, respectively, coupled to PTX-sensitive G proteins or PTX-resistant G proteins. While G proteins involved in chemotactic factor responses often are PTX-sensitive, PAF shows an unusual degree of PTX resistance [43, 44]. In our recent experiments we found that PAF stimulated both PTX-sensitive and PTX-resistant pathways in human eosinophils. When we performed similar analyses with neutrophils, the firstphase chemiluminescence response of neutrophils to PAF was resistant to PTX, consistent with previous observations [45]. Our recent studies also suggest that PAFinduced eosinophil chemotaxis is completely blocked by PTX. In contrast, PAF-induced eosinophil degranulation is only partially inhibited by PTX, suggesting that eosinophil chemotaxis but not degranulation is regulated almost entirely by PTX-sensitive G protein [Motegi and Kato,

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unpubl. data]. Indeed, the expression of G proteins appears to differ between granulocytes; human neutrophils were found to express PTX-sensitive G·i2 and PTX-resistant G·q/11 and G·13, while human eosinophils expressed only G·i2 and G·q/11 [46]. In addition, studies in a rat basophil cell line transfected with human PAF receptor suggested that a single PAF receptor population may interact with multiple PTX-sensitive and PTX-resistant G proteins to mediate biological responses to PAF [44]. These observations suggest that the PAF receptor is coupled to at least two different classes of G proteins in human eosinophils. As for the possibility of involving multiple receptor types, this view is supported by differences in the threshold PAF concentration required to induce the two response phases. However, up to now, only one gene for PAF receptor has been identified in humans. Possibly a single PAF receptor might exist in two biochemically different forms, dependent on the activation state. Alternatively, a single PAF receptor may exist in two different

Kato/Kita/Tachibana/Hayashi/Tsuchida/ Kimura

spatial locations, such as the cell surface membrane and intracellularly [47], resulting in different responses to ligands and signaling molecules. Increasing evidence suggests that the dimerization of the G protein-coupled receptor may play an important role in the regulation of its biological activity and dimerization may also be important in the subsequent internalization of the receptor [48]. Recently, a new exciting insight has emerged from studies of receptors for chemokine [49, 50] and lipid mediators [51, 52]. The simultaneous presence of chemokine receptor (CCR) 2 and CCR5 induced the formation of CCR2-CCR5 heterodimers with unique features, including a reduction in the threshold concentration of the chemokine required to induce PTX-resistant responses. Interestingly, the heterodimeric complex also specifically promoted the recruitment of G·q/11, which distinctly activated PI-3K and preferentially activated cell adhesion. Finally, the heterodimeric complex was not internalized or desensitized. Thus, dimeric chemokine receptors may mediate a set of signals and functions completely distinct from those involving monomeric chemokine receptors [49, 50]. In a similar manner PAF receptors might form homodimers in COS transformants upon ligand stimulation [53] and PAF receptor dimerization potentiates ligand-induced internalization of the receptor in CHO cells [54]. In other recent reports, the human LTB4 receptor, BLT1, was shown to form homodimers according to LTB4 ligand concentration; in its high-affinity state the receptor interacts with a heterotrimeric GDP-loaded G protein, G·i2ß1Á2. Furthermore, a receptor antagonist or overexpression of the cytoplasmic region of transmembrane helix 6 blocked the dimerization of the receptor [51, 52]. This suggests that receptor dimerization may be crucial to the transduction of the LTB4-induced signal. Based on these observations and our results, we propose a model of PAF-induced O2 – production in human eosinophils. Stimulation of eosinophils with PAF induces activation of PTX-sensitive G protein(s), leading to the first phase of superoxide response and chemotaxis; this could involve the PAF receptor monomer. Alternatively, PAF induces activation of PTX-resistant G protein(s) and PI-3K, and causes adhesion-dependent activation of the second phase of superoxide response and degranulation, resulting in bronchial hyperresponsiveness. This pathway involves PAF receptor dimers (fig. 2). Furthermore, a recent report showed that the protein kinase C (PKC) inhibitors staurosporine and GF109203X blocked the desensitization of PAF receptor, suggesting the implication of PKC in the molecular mechanism mediating the PAF

Dual Signaling Pathways in Human Eosinophils

receptor desensitization and internalization triggered by dimerization [54]. Indeed, we found that the PKC inhibitor such as GF109203X (bisindolylmaleimide I, Bis I) augmented cellular adhesion induced by PAF in a dosedependent manner [55]. Moreover, our preliminary study showed that Bis I at a lower concentration enhanced the second phase of superoxide production but inhibited it at a high concentration and that the second phase of superoxide production by PAF was more resistant to inhibition by Bis I than the first phase (data not shown). These results indicated that PKC might also modulate PAF receptor dimerization in human eosinophils.

Conclusion

We demonstrated that PAF activates two distinct signaling and effector pathways in human eosinophils to produce a response distinct from that in human neutrophils. Although more studies are needed to better understand regulatory mechanisms of the PAF receptor, its biochemical characteristics, and coupling to downstream signaling molecules, we propose that dual signaling mechanisms may be involved in a flexible system for the control of the eosinophil response to a single chemotactic factor, PAF. Demonstration of two distinct signaling pathways in eosinophils thus is an important step toward more fully understanding the broad range of biological responses to PAF.

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37 Nick JA, Avdi NJ, Young SK, Knall C, Gerwins P, Johnson GL, Worthen GS: Common and distinct intracellular signaling pathways in human neutrophils utilized by platelet activating factor and FMLP. J Clin Invest 1997;99: 975–986. 38 Korth RM: Specific high affinity binding of platelet activating factor to intact human blood neutrophils and eosinophils. Int Arch Allergy Immunol 1996;110:124–131. 39 Nakano M, Sugioka K, Ushijima Y, Goto T: Chemiluminescence probe with Cypridina luciferin analog, 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one, for estimating the ability of human granulocytes to generate O2 – . Anal Biochem 1986;159:363–369. 40 Nakano M: Determination of superoxide radical and singlet oxygen based on chemiluminescence of luciferin analogs. Methods Enzymol 1990;186:585–591. 41 Kato M, Tokuyama K, Morikawa A, Kuroume T, Barnes PJ: Platelet-activating factor-induced enhancement of superoxide anion generation in guinea-pigs. Eur J Pharmacol 1993; 232:7–12. 42 Kato M, Kimura H, Motegi Y, Tachibana A, Minakami H, Morikawa A, Kita H: Plateletactivating factor activates two distinct effector pathways in human eosinophils. J Immunol 2002;169:5252–5259. 43 Haribabu B, Zhelev DV, Pridgen BC, Richardson RM, Ali H, Snyderman R: Chemoattractant receptors activate distinct pathways for chemotaxis and secretion: Role of G-protein usage. J Biol Chem 1999;274:37087–37092.

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44 Ali H, Richardson RM, Tomhave ED, DuBose RA, Haribabu B, Snyderman RJ: Regulation of stably transfected platelet activating factor receptor in RBL-2H3 cells: Role of multiple G proteins and receptor phosphorylation. J Biol Chem 1994;269:24557–24563. 45 M’Rabet L, Coffer PJ, Wolthuis RM, Zwartkruis F, Koenderman L, Bos JL: Differential fMet-Leu-Phe- and platelet-activating factorinduced signaling toward Ral activation in primary human neutrophils. J Biol Chem 1999; 274:21847–21852. 46 O’Flaherty JT, Taylor JS, Kuroki M: The coupling of 5-oxo-eicosanoid receptors to heterotrimeric G proteins. J Immunol 2000;164: 3345–3352. 47 Nigam S, Eskafi S, Muller S, Zhang H: Evidence for the predominant role of intracellular PAF binding sites in the regulation of phospholipase A2 in human neutrophils. Adv Prostaglandin Thromboxane Leukot Res 1995;23: 471–473. 48 Pin JP, Galvez T, Prezeau L: Evolution, structure, and activation mechanism of family 3/C G-protein-coupled receptors. Pharmacol Ther 2003;98:325–354. 49 Mellado M, Rodriguez-Frade JM, Vila-Coro AJ, Fernandez S, Martin de Ana A, Jones DR, Toran JL, Martinez AC: Chemokine receptor homo- or heterodimerization activates distinct signaling pathways. EMBO J 2001;20:2497– 2507.

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50 Rodriguez-Frade JM, Mellado M, Martinez AC: Chemokine receptor dimerization: Two are better than one. Trends Immunol 2001;22: 612–617. 51 Baneres JL, Martin A, Hullot P, Girard JP, Rossi JC, Parello J: Structure-based analysis of GPCR function: Conformational adaptation of both agonist and receptor upon leukotriene B4 binding to recombinant BLT1. J Mol Biol 2003;329:801–814. 52 Baneres JL, Parello J: Structure-based analysis of GPCR function: Evidence for a novel pentameric assembly between the dimeric leukotriene B(4) receptor BLT1 and the G-protein. J Mol Biol 2003;329:815–829. 53 Ishii I, Saito E, Izumi T, Ui M, Shimizu T: Agonist-induced sequestration, recycling, and resensitization of platelet-activating factor receptor. Role of cytoplasmic tail phosphorylation in each process. J Biol Chem 1998;273:9878– 9885. 54 Perron A, Chen ZG, Gingras D, Dupre DJ, Stankova J, Rola-Pleszczynski M: Agonist-independent desensitization and internalization of the human platelet-activating factor receptor by coumermycin-gyrase B-induced dimerization. J Biol Chem 2003;278:27956–27965. 55 Takizawa T, Kato M, Kimura H, Suzuki M, Tachibana A, Oninata H, Izumi T, Tokuyama K, Morikawa A: Inhibition of protein kinases A and C demonstrates dual modes of response in human eosinophils stimulated with plateletactivating factor. J Allergy Clin Immunol 2002; 110:241–248.

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Abstracts Int Arch Allergy Immunol 2004;134(suppl 1):44–45 DOI: 10.1159/000077792

The Functional Roles of the Soluble Form of Adhesion Molecules in Survival and Chemotaxis in Eosinophils Kazutoshi Yamaguchi, Yoshiyuki Yamada, Shigeharu Ueki, Tetsuya Adachi, Hajime Oyamada, Kazuyuki Hamada, Akira Kanda, Hiroyuki Kayaba, Junichi Chihara Department of Clinical and Laboratory Medicine, Akita University School of Medicine, Hondo, Akita, Japan Background: Eosinophils play a pivotal role in the mechanism of allergic diseases including asthma. Adhesion molecules, especially intercellular adhesion molecule-1 (ICAM-1), may play an important role in the accumulation of eosinophils in this process. These molecules may be shed in vivo during an inflammatory process. In this study, the functional roles of the soluble form of adhesion molecules in survival and chemotaxis in eosinophils were examined. Methods: Blood eosinophils were purified by using Percoll and anti-CD16 antibody-coated magnetic beads. Eosinophil apoptosis was analyzed using an FACScan cytometer with annexin V and propidium iodide, gating on the live cell population. The chemotaxis assay was performed in a Boyden microchamber. Results: The soluble form of ICAM-1 and fibronectin (FN), but not that of the vascular cell adhesion molecule-1 (VCAM-1), prolonged the eosinophil survival in a concentration-dependent manner. Moreover, the eosinophil chemotaxis to the soluble form of ICAM-1 and that of VCAM-1, but not to FN, was observed in a dose-dependent fashion. Conclusion: These results suggest the importance of the soluble form of adhesion molecules as well as the expression of these molecules in eosinophilmediated allergic inflammation.

Autocrine Activation of Eosinophil Transmigration by Cysteinyl Leukotrienes Yoshiko Kato, Takao Fujisawa, Hitoshi Kamiya, Osamu Yoshie Clinical Research Institute, National Mie Hospital, Tsu, and Department of Microbiology, Kinki University, Osaka, Japan Rationale: We have previously reported that airway epithelial cells promote eosinophil transmigration by utilizing a novel transmigration system and that among the epithelial cell-derived factors CCR3 ligands and extracellular matrix proteins support migration. To further analyze the interaction of eosinophils and airway epithelial cells, we investigated a possible involvement of cysteinyl leukotrienes (CysLT) in this system. Methods: A three-dimensional eosinophil transmigration system consisted of cultured airway epithelial cells and type I collagen matrix [Kato Y, et al.: Clin Exp Allergy 2002;32:889] was used. Eosinophils were preincubated with a CysLT1 receptor antagonist, montelukast, before being subjected to

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transmigration experiments and eosinophil migration was evaluated. Epithelial supernatants from the system were then added to eosinophils and gene expression of LTC4 synthase and CysLT production from the eosinophils were measured with quantitative RT-PCR and ELISA, respectively. Cytokines in the epithelial supernatants were measured with a bead microarray system and gene expression from the cells were quantified with real-time PCR. Results: Montelukast inhibited epithelial cell-promoted eosinophil transmigration in a concentration-dependent manner. The epithelial supernatants induced LTC4 synthase gene expression and CysLT release from eosinophils. To identify factors that induce CysLT production, a panel of cytokines in the supernatants were quantified with a bead microarray and real-time PCR. GM-CSF was found to be a major cytokine and anti-GM-CSF blocked epithelial supernatant-induced eosinophil CysLT production. Conclusions: Airway epithelial cells induce CysLT production from eosinophils by producing GM-CSF, and consequently enhance eosinophil migration through an autocrine activation mechanism. CysLT1 receptor antagonists may interfere with these interactions.

Anti-Inflammatory Effects of Fenoterol on Human Eosinophils and Neutrophils: A Possible Mechanism of ß2-Adrenoceptor-Independent Effects Atsushi Tachibana a, Hirokazu Kimura b, Kunihisa Kozawa b, Akihiro Morikawa a, Masahiko Kato c a Department

of Pediatrics, Gunma University School of Medicine and b Gunma Prefectural Institute of Public Health and Environmental Sciences, Maebashi, and c Children’s Medical Center, Hokkitsu, Gunma, Japan Background: Agonists at ß2-adrenoceptors are widely used as bronchodilators in treating bronchial asthma. These agents may also have important anti-inflammatory effects on eosinophils and neutrophils in asthma. We examined whether widely prescribed ß2-adrenoceptor agonists differ in their ability to suppress stimulus-induced effector functions of eosinophils and neutrophils such as superoxide anion (O2–) generation. Methods: We investigated the effects of these drugs on O2– generation by stimulated human eosinophils and neutrophils in vitro using a highly sensitive and specific chemiluminescence method for O2–. Results: Fenoterol inhibited platelet-activating factor-induced O2– generation by eosinophils significantly more than salbutamol or procaterol. Fenoterol inhibited phorbol myristate acetate (PMA)-induced O2– generation by eosinophils, while salbutamol or procaterol did not. The inhibition of PMAinduced O2– generation by fenoterol was not reversed by ICI-118551, a selective ß2-adrenoceptor antagonist. Fenoterol significantly inhibited both N-formyl-methionyl-leucyl-phenylalanine (FMLP)- and

PMA-induced O2– generation by neutrophils. In contrast, salbutamol or procaterol partially inhibited O2– generation with FMLP but not with PMA. The effect of fenoterol against O2– generation with PMA was not reversed. None of the drugs scavenged superoxide at the highest concentration used (10 –5 M ). Conclusions: Fenoterol generally inhibited O2– generation more effectively than salbutamol or procaterol. Inhibition might also include a component not mediated via ß2-adrenoceptors. Direct inhibition at or downstream from protein kinase C may be involved. This pharmacologic property of fenoterol could contribute to the mortality associated with the overuse of fenoterol in asthmatic attacks.

Expression of Interleukin-5 and RANTES in the Heart in Murine Ovalbumin-Challenged Myocarditis Masao Hirasawa, Hirofumi Deguchi, Yasushi Kitaura Third Department of Internal Medicine, Osaka Medical College, Osaka, Japan Background: Some types of myocarditis, especially eosinophilic myocarditis, are often recognized as a result of a hypersensitive status (i.e., side effects of drugs or parasite infection). Some of these forms can be dealt with by proper treatment, but the hypereosinophilic syndrome still has a poor prognosis. These heart diseases are reported to be usually associated with increased CD4+ T cells, and the related cytokines, but the pathogenesis remains poorly understood. Unfortunately, the heart does not easily represent an immunomodulative condition under experimental settings. Therefore, an ovalbumin (OVA) challenge was performed to study the cytokine levels in aller-

Abstracts

gic heart disease in DBA/2 mice that are susceptible to myocardial disease. Methods: DBA/2 mice were sensitized to OVA at 3 and 5 weeks of age, and were administered a high-concentration OVA solution at 6 weeks of age. Localization of eosinophils and CD4+ T cells were initially screened at 23, 71, 95, 143 h after OVA challenge. Thereafter, interleukin-5 (IL-5) and RANTES were examined by immunohistochemistry and compared quantitatively throughout the time period. The detected parameters include the IL-5-positive area (Dk), the whole heart area (Sk), the ratio of these parameters (Dk/ Sk %), and the calculated densities (intensity) of both IL-5 and RANTES. Lastly, serum RANTES levels were measured in parallel with the above time points with the enzyme-linked immunosorbent assay (ELISA). Results: Scattered macrophages, eosinophils, granulomas, and some CD4+ T cells were observed within the myocardial interstitium and pericardium. Myocardial cells exhibited partial degenerative changes. Some mononuclear cells were also evident around the small blood vessels. IL-5 was broadly expressed in the heart tissue and was most demonstrable at the pericardium. The Dk/ Sk of IL-5 increased to a maximum of 49.9 B 5.1, 95 h after OVA challenge. However, the intensity of IL-5 exhibited a maximum of 0.74 B 0.15 (p ! 0.05) 71 h postchallenge. The intensity of RANTES, which was also most demonstrable at the pericardium, remained elevated until 95 h and decreased to 0.09 B 0.01 by 143 h (p ! 0.05). In contrast, serum RANTES reached a peak of 60.1 B 6.3 pg/ml at the earliest 23 h postchallenge (p ! 0.05). Conclusion: OVA challenge in mice causes a rapid elevation of systemic RANTES as well as local induction of IL-5 or RANTES in the heart tissue at the site of eosinophil infiltration. These key cytokines and CC chemokine may initially accumulate in the pericardium and spread following myocardiocyte injury. These data suggest the possibility that a similar pathophysiology may occur in human hypersensitive or eosinophilic myocarditis.

Int Arch Allergy Immunol 2004;134(suppl 1):44–45

45

Int Arch Allergy Immunol 2004;134(suppl 1):46 DOI: 10.1159/000077793

List of Lectures by Speakers Who Have Not Submitted Their Manuscripts

P-Selectin Expression in Human Dermal Microvascular Endothelial Cells Yasuhiro Miyazaki et al. Department of Dermatology and Immunodermatology, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan

Augmentation of Allergic Inflammation by Formaldehyde Exposure – Analysis Using an Antigen-Specific Murine Asthmatic Model Hirofumi Ishida et al. Department of Internal Medicine, Teikyo University, School of Medicine, Tokyo, Japan

LTD4 Induces Eosinophil Degranulation and Superoxide Production via ß2 Integrin Keiko Saito et al. Department of Respiratory Medicine, Saitama Medical School, Saitama, Japan

Effects of CpG ODN on Human Eosinophils Kenji Matsumoto et al. Department of Allergy and Immunology, National Center for Child Health and Development Research Institute, Tokyo, Japan

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Expression and Regulation of Toll-Like Receptor 2 in Cord Blood-Derived and Adult Peripheral Blood-Derived Cultured Human Mast Cells Naoko Inomata et al. Department of Dermatology, Yokohama City University of Medicine, Yokohama, Japan

Expression and Function of Toll-Like Receptors in Eosinophils – Activation by Toll-Like Receptor 7 Ligand Hiroyuki Nagase et al. Departments of Respiratory Medicine, University of Tokyo Graduate School of Medicine, Tokyo, Japan

Characteristics of Cells Gathered in Effusion of the Middle Ear in a Patient with Eosinophilic Otitis Media Akiko Tokumaru et al. Department of Otolaryngology and Head and Neck Surgery, Koshigaya Hospital, Dokkyo University School of Medicine, Saitama, Japan

Author Index Vol. 134, Suppl. 1, 2004 (A) = Abstract

Adachi, M. 12 Adachi, T. 44 (A) Akasawa, A. 2 Akiyama, K. 7 Chihara, J. 30, 44 (A)

Matsumoto, K. 2, 46 (A) Matsuwaki, Y. 30 Miyagawa, M. 2 Miyazaki, Y. 46 (A) Mori, A. 1, 7 Mori, Y. 25 Morikawa, A. 44 (A)

Deguchi, H. 45 (A) Fujisawa, T. 44 (A) Hagiwara, K. 21 Hamada, K. 44 (A) Hashida, R. 2 Hashimoto, T. 7 Hayashi, Y. 37 Hirasawa, M. 45 (A) Hirose, K. 25 Ieki, K. 12 Ikeda, K. 25 Inomata, N. 46 (A) Ishida, H. 46 (A) Itoh, M. 2 Iwamoto, I. 25 Kamiya, H. 44 (A) Kanazawa, M. 21 Kanda, A. 30, 44 (A) Kato, M. 37, 44 (A) Kato, Y. 44 (A) Katsunuma, T. 2 Kawaguchi, H. 7 Kawaguchi, M. 12 Kayaba, H. 30, 44 (A) Kikuchi, I. 21 Kimura, H. 37, 44 (A) Kita, H. 37 Kitaura, Y. 45 (A) Kobayashi, N. 7 Kokubu, F. 12 Kozawa, K. 44 (A) Kuga, H. 12 Kurokawa, M. 12 Maeda, Y. 7 Matsukura, S. 12

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Nagase, H. 46 (A) Nagasu, T. 2 Nagata, M. 21 Nakajima, H. 25 Nakayama, K. 25 Odaka, M. 12 Ogawa, K. 2 Oyamada, H. 30, 44 (A) Saito, H. 2 Saito, K. 21, 46 (A) Saito, N. 30 Saito, Y. 25 Sakamoto, Y. 21 Schindler, U. 12 Schleimer, R.P. 12 Seto, Y. 25 Shimoda, K. 25 Sugita, Y. 2 Suzuki, K. 25 Suzuki, S. 12 Tabe, K. 21 Tachibana, A. 37, 44 (A) Takeuchi, H. 12 Taniguchi, M. 7 Tokumaru, A. 46 (A) Tsuchida, Y. 37 Tsujimoto, G. 2 Ueki, S. 30, 44 (A) Usami, A. 30 Watanabe, S. 12 Yamada, Y. 44 (A) Yamaguchi, K. 44 (A) Yoshie, O. 44 (A)

47

Subject Index Vol. 134, Suppl. 1, 2004

Airway epithelial cells 12 Apoptosis 2 Atopic dermatitis 2, 7 Bronchial asthma 7, 12, 21, 30 Cell cycle 2 Chemotaxis 30 Differential display 2 Eosinophil(s) 2, 21, 30, 37 – function regulation 30 Eotaxin 12 Fluticasone propionate 12 Gene expression, peripheral blood eosinophils 2

Mast cells 25 Mononuclear cells 21 Neutrophils 37 Nuclear factor kappa B 12 Peripheral blood T cells, asthma patients 7 Peroxisome proliferator-activated receptor gamma 30 Platelet-activating factor 37 Real-time RT-PCR 2 Signal transduction 37 STAT1 25 STAT6 12 Thiazolidinediones 30 Transendothelial migration, eosinophils 21 Tyk2 25

HSOCP-1 2 Yeast two-hybrid screening 2 Immunity, innate 25 Immunotherapy, bronchial asthma 21 Interferon alpha induced gene expression 25 Interleukin-5 7 Interleukin-13 7

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© 2004 S. Karger AG, Basel

Accessible online at: www.karger.com/iaa