Signal Transduction Mechanisms Regulating the Activation, Adhesion and Migration of Human Eosinophils and T-Lymphocytes in Allergic Inflammation

Transcription

1 Signal Transduction Mechanisms Regulating the Activation, Adhesion and Migration of Human Eosinophils and T-Lymphocytes in Allergic Inflammation IP Wai-Ki M. Phil., CUHK A Thesis Submitted in Partial Fulfilment of the Requirements for the Degree of Doctor of Philosophy in Chemical Pathology The Chinese University of Hong Kong July 2003 The Chinese University of Hong Kong holds the copyright of this thesis. Any person(s) intending to use a part or whole of the materials in the thesis in a proposed publication must seek copyright release from the Dean of the Graduate School.

2 UMI Number: UMI UMI Microform Copyright 2003 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml

3 ACKNOWLEDGEMENTS Acknowledgements I would like to deliver my most sincere gratitude to my supervisor. Professor C.W.K. Lam, the Chairman of the Department of Chemical Pathology, The Chinese University of Hong Kong, for his encouragement and continuous guidance throughout the learning process of these three-year PhD program. I will never forget his kindness, tolerant and enthusiastic working manner. I have greatly benefited from his valuable teaching and guidance. I am grateful to my co-supervisor, Professor C.K. Wong, Associate Professor of the Department of Chemical Pathology, The Chinese University of Hong Kong, for his encouragement, constructive advice, detailed instructions and valuable discussions on all of my research works, presentations, and publications. This thesis could not have been completed without the patience of my supervisor and co-supervisor in reading and checking as well as their helpful criticisms. I would also like to thank Professor T.R Leung, Associate Professor of the Department of Paediatrics, The Chinese University of Hong Kong, for his astute recruitment of asthmatic patients and helpful supply of blood samples throughout the entire clinical study. My gratitude also goes to my colleagues: Dr. RM.K, Pooii, Miss RS. Tse, Miss S.K.W. Lee, Dr. I.H.S, Chan, Miss H.H.W. Cheung, Dr. W.C.B. Wang and Ms. X.Y. Bao for their help and encouragement. I am also grateful to all laboratory technicians and staff of the Department of Chemical Pathology, The Chinese University of Hong

4 Acknowledgements Kong, for their valuable technical assistance and emotional support-1 am indebted to all my friends. Dr. T.C.Y. Ho,Dr. J J.R Zhang, Mr. T.Y. Tsim, Mr. W.K. Ng, Mr. CM. Cho, for their friendship, understanding and constant supports. Lastly, I want to express my deepest thanks to my family for their boundless concern, support and comfort. Their greatest love will always be my motive power. This study was supported by a donation from Zindart (De Zhen) Foundation Ltd., Hong Kong, and Direct Grants for Research ( , , , ) of The Chinese University of Hong Kong. II

5 Abbreviations ABBREVIATIONS AHR AP-1 APC APS ATF-2 BAL CBA CBP CCR3 cdna CS CSAID CTLA-4 CREB dbcamp DEPC D-MEM/F12 DMSO dntp DTT ECL ECM ECP EDN or EPX EGTA ELISA EMSA EPO ERK FCS FEIA fmlp GINA GM-CSF HEPES Airway hypersensitivity response Activator protein-1 Antigen presenting cells Ammonimn persulfate Activating transcription factor-2 Bronchoalveolar lavage Cytometric beads array CREB binding protein CC chemokine receptors Complementary deoxyribonucleic acid Corticosteroid Cytokine suppressant anti-inflammatory drugs Cytotoxic T-lymphocyte-associated molecule-4 Cyclic AMP response element binding protein Dibutyryl cyclic AMP Diethyl pyrocarbonate Dulbecco's Modified Eagle Medium: nutrient mixture F-12 Dimethyl sulfoxide 2 -Deoxyribonucleoside 5 -triphosphate Dithiothreitol Enhanced chemiluminescence Extracellular matrix Eosinophil catioiiic protein Eosinophil-derived neurotoxin Ethylene glycol-bis(p-aminoethyl Etlier)- N,N, N,,N'-tetraacetic acid Enzyme-linked immunosorbent assay Electromobility shift assay Eosinophil peroxidase Extracellular signal-regulated protein kinases Fetal calf serum Fluorescence enzyme immuiioassay Formyl-methionyl-leucyl-phenylalanine Global initiative for asthma Granulocyte-macrophage colony-stimulating factor N-2-hydroxy-ethyl-piperazine-N'-2-ethane-siilfonic acid III

6 ICAM-1 ICE IFN-Y Ig TR IMDM inos IKB JAK JNK LPS LFA-1 LT C4 Mac-1 MACS MAPK MAPKK or MF,K MAPKKK or MEKK MBP MCP MDC MIP NF-KB NSAID PAF PAGE PB PBS PBMC PGE2 PKC PS PTK PYDF RANTES RAST Intercellular cell adhesion molecule-1 Interleukin-1 fj converting enzyme Interferon-"/ Immunoglobulin Interleukin Iscove's Modified Dulbecco's Medium Inducible nitric oxide synthase Inhibitor kappa B J alius kinase c-jun N-temiinal kinases Lipopolysaccharide Leukocyte function-association antigen-1 Leukotriene C4 Macrophage antigen-1 Magnetic cell sorting system Mitogen-activated protein kinase MAPK kinase or MAP/ERK kinase MAPKK kinase or MEK kinase Major basic protein Monocyte chemotactic protein Macrophage-derived chemokine Macrophage inflammatory protein Nuclear factor-icb Non-steroidal anti-inflammatory drug Platelet activation factor Polyacrylamide gel electrophoresis Periphera blood Phosphatc-buffered-saline Peripheral blood mononuclear cells Prostaglandin E2 Protein kinase C Phosphatidylserine Protein tyrosine kinase Polyvinylidene di fluoride Regulated upon activation normal T-cell expressed and secreted Radioallergosorbent test Abbreviations RPMI1640 medium Roswell Park Memorial Insitute tissue culture medium 1640 RT-PCR Reverse transcription-polymerase chain reaction IV

7 Abbreviations SAPK SD SDS SLE STAT TAE TARC TBP TBST TCR TEMED TGF Th TNF UV VCAM-l VLA-4 Stress-activated protein kinase Standard deviation Sodium dodecyl sulfate Systemic lupus erythematosus Signal transducers and activators of transcription Tris-acetate buffer Thymus and activation-regulated chemokine TATA-binding protein Tris-buffered saline Tween 20 T-cell receptor N, N,N,,N'-tetra-methylethylenediamine Transforming growth factor T helper cells Tumor necrosis factor Ultraviolet Vascular-cell adhesion molecule-1 Very late activated antigen-4 V

8 ABSTRACT Abstract In recent years, allergic diseases such as asthma and allergic rhinitis are prevalent and have been increasing worldwide. In allergic asthma, cosinophilia usually develops with the production of haematopoietic cytokines including interleukin (IL)-3, IL-5 and granulocyte macrophage colony stimulating factor (GM-CSF) from activated T-lyinphocytes, and eosinophil-specific chemoattractant, eotaxin from airway epithelium. Both eosinophils and T-lymphocytes are the principal effector cells which infiltrate and accumulate into sites of inflammation where they produce and secrete a variety of inflammatory mediators. In recent years, synthetic inhibitors for p38 mitogen-activated protein kinase (MAPK) and proteasome inhibitor for nuclear factor-kappab (NF-KB) signaling pathways have been developed as potential therapeutic drugs for inflammatory diseases. p38 MAPK and NF-KB signaling pathways have been demonstrated to be the most important mechanisms regulating the major production of inflammatory cytokines and chemoattractants from different inflammatory cells in allergic inflammation. However, the roles of p38 MAPK and NF-KB in regulating the expression of adhesion molecules, adhesion and chemotaxis of human eosinophils have not yet been fully elucidated. In the present study, we have investigated the intracellular signal transduction mechanisms for tumor necrosis factor (TNF)-a, IL-3, IL-5 and GM-CSF-induced expression of adhesion molecules, adhesion and chemotaxis of human eosinophils. TNF-a was found to up-regulate the gene expression of NF-KB and IKB-OC,induce the phosphorylation of p38 MAPK and IKB-OC, rapid loss of total IKB-CX, and nuclear VI

9 Abstract translocation of NF-KB in an in vitro model of eosinophils, eosinophilic leukemia, EoL-1 cells. TNF-a-induced surface expression of intercellular adhesion molecule (ICAM)-l could be inhibited by a proteasome inhibitor N-cbz-Leu-Leu-leucinal (MG-132) but not a specific p38 MAPK inhibitor (SB ) in both human peripheral blood eosinophils and EoL-1 cells. Therefore, TNF-a-induced ICAM-1 expression was through the activation ofnf-icb pathway which is independent to the activity ofp38 MAPK in eosinophils. Similar to that of TNF-a, haematopoietic cytokines, IL-3, IL-5 and GM-CSF, could stimulate a rapid phosphorylation of p38 MAPK and IKB-OC together with the nuclear translocation of NF-KB in human eosinophils. cdna expression array analysis has shown that these cytokines could differentially up-regulate gene expressions of NF-KB, IKB-OC as well as several adhesion molecules and matrix proteins in eosinophils, such as CD44, ICAM-1, a6, P2 integrins and matrix metalloproteinase (MMP)-9. We found that IL-3, IL-5 and GM-CSF could also induce surface expression of adhesion molecules ICAM-1, CD lib and CD 18 but suppress ICAM-3 expression on eosinophils. The IL-3-, IL-5-,and GM-CSF induced expression of ICAM-1 involved both p38 MAPK and NF-KB activities, whereas the regulation of CD lib, CD 18 and ICAM-3 expressions was mediated through p38 MAPK but not NF-KB signal transduction pathway. p38 MAPK inhibitor could suppress the inducing effect of IL-3, IL-5 and GM-CSF on surface expression of ail the adhesion molecules being tested, while the NF-KB inhibitor could only suppress the surface expression of ICAM-1. Furthermore, IL-3 and IL-5 but not GM-CSF could enhance the adhesion of eosinophils onto a human bronchial epithelial cell line, BEAS-2B cells. All these three cytokines could also induce the chemotaxis of VII

10 Abstract eosinophils which was not mediated through the release of eosinophil specific chemokine, eotaxin, or the expression of its receptor, CCR3. Both cytokine-induced adhesion and cliemotaxis of eosinophils could be inhibited by either p38 MAPK or NF-KB inhibitor. The interaction of eosinophils between bronchial epithelial cells is critical for tissue damage and airway remodeling in allergic asthma. To have a better understanding of this process, cytokines released from the co-culture of eosinophils and bronchial epithelial cells was studied by using an in vitro epithelium model, BEAS-2B cells. We found that the release of proinflammatory cytokines, IL-6 and TNF-a, and chemokines, IL-8 and monocyte chemoattractant protein (MCP)-l were significantly increased in the co-cultures which could be differentially regulated through p38 MAPK and NF-KB signaling pathways. A clinical study on allergic asthmatic children with chronic and acute asthma has demonstrated an elevated plasma level of thymus and activation-regulated chemokine (TARC) and soluble cytotoxic T-lymphocyte-associated molecule (CTLA)-4 in children with both chronic stable and acute asthma. Moreover, the levels of both plasma TARC and sctla-4 were significantly decreased following systemic corticosteroid treatment in acute asthmatic patients. Based on the above findings, we suggested that hematopoietic cytokines and inflammatory cytokines, including ll-3, IL-5, GM-CSF, and TNF-a as well as p38 MAPK and NF-KB signaling pathways play a critical role in regulating the adhesion and migration of eosinophils in inflammatory reactions. TARC is an important activated T-cell chemokine for the recruitment of Th2 lymphocytes into inflammatory sites while sctla-4 is an important regulator for T-lymphocyte activation. These VIII

11 Abstract inflammatory mediators may be useful in monitoring the disease severity and therapeutic efficacy of asthmatic children with acute attacks. In conclusion, the above findings can provide better understanding of the roles of cytokines and chemokines in the pathogenesis of inflammatory diseases and biochemical basis for the future development of more effective drugs or strategies used in the treatment of different inflammatory diseases. IX

12 撮 要 近 年 來, 過 敏 性 疾 病 例 如 喘, 過 敏 性 鼻 炎 等 的 發 病 率 在 全 球 普 遍 上 升 在 過 敏 性 孝 喘 的 病 變 中, 活 性 T 淋 巴 細 胞 釋 出 之 造 血 性 細 胞 因 子, 包 括 白 介 素 -3 (IL-3) 白 介 素 -5 (IL-5) 及 粒 細 胞 和 單 核 細 胞 集 落 刺 激 因 子 (GM-CSF); 和 氣 管 上 皮 細 胞 釋 出 之 嗜 酸 性 粒 細 胞 趨 化 因 子 eotaxin, 都 會 促 使 嗜 酸 性 粒 細 胞 增 多 症 嗜 酸 性 粒 細 胞 及 T 淋 巴 細 胞 都 是 過 敏 性 哮 喘 病 變 中 的 主 要 受 動 細 胞 這 些 細 胞 會 渗 透 及 積 聚 在 有 發 炎 的 組 織, 並 且 釋 放 出 不 同 能 導 致 發 炎 的 媒 介 物 近 年, 人 工 合 成 的 p38ffii togen-activated protein kinase (MAPK) 激 _ 抑 制 劑 和 可 用 於 抑 制 胞 核 因 子 -kappab (NF-KB) 信 號 經 路 的 醇 解 抑 制 因 子 都 正 在 發 展 成 有 潛 力 並 可 用 於 醫 治 炎 症 的 藥 物 在 過 敏 性 炎 症 中,P38MAPK 激 酵 和 NF-KB 的 信 號 經 路 已 被 証 實 為 用 以 調 控 不 同 炎 性 細 胞 製 做 主 要 的 炎 性 細 胞 因 子 及 激 化 物 的 重 要 機 制 然 而, 在 調 控 嗜 酸 性 粒 細 胞 的 粘 付 趙 藥 性 及 黎 附 分 子 的 表 達 中,p38 MAPK 激 _ 和 NF-KB 所 扮 演 的 角 色 還 是 未 能 完 全 被 我 們 闡 明 在 這 次 研 習 中, 我 們 著 重 研 究 了 在 IL-3 IL-5 或 GM-CSF 的 誘 導 下 嗜 酸 彳 生 V 粒 細 胞 的 粘 付 分 子 表 達 粘 附 及 趨 藥 彳 生 我 們 並 且 對 調 控 這 些 過 程 的 細 胞 内 信 號 傳 導 機 制 加 以 研 究 及 分 析 本 研 究 發 現 腫 瘤 壞 死 因 子 -a (TNF-a) 能 誘 導 嗜 酸 性 粒 細 胞 的 體 外 模 型 - 嗜 酸 性 粒 細 胞 白 金 病 細 胞 棟 (EoL-1) 内 的 NF-KB 和 KappaB 抑 制 蛋 白 -a (IKB-a) 的 基 因 表 達 p38mapk 激 酵 與 IKB-OT 的 填 酸 化 總 IKB-a 蛋 白 的 減 少 以 及 N F - K B 的 胞 核 傳 遞 只 有 _ 解 抑 制 因 子 N-cbz-Leu-Leu-1 eucina 1 (MG-132) 而 不 是 特 異 性 p38 MAPK 激 酶 抑 X

13 制 劑 (SB203580) 能 夠 抑 制 腫 瘤 壞 死 因 子 -a(tnf-a) 誘 導 下 EoL-1 細 月 包 和 人 血 中 的 嗜 酸 ' 生 粒 細 月 包 所 表 達 的 細 胞 間 附 分 子 -1 (I CAM-1) 因 此, 嗜 酸 性 粒 細 胞 在 TNF-a 誘 導 下 的 ICAM-1 的 表 達 是 通 過 NF-KB 經 路 而 NF-KB 的 經 路 是 無 需 透 過 P38 MAPK 激 _ 的 活 性 被 激 活 造 血 性 細 胞 因 子 :IL-3 IL-5 及 GM-CSF 與 TNF-a 的 作 用 相 類 似 他 們 能 夠 迅 速 刺 激 嗜 酸 性 粒 細 胞 内 p38 MAPK 激 酵 IKB-a 及 NF-KB 的 麟 酸 化 互 補 脫 氧 核 糖 核 睃 (cdna) 陣 列 膜 的 結 果 分 析 顯 示, 這 些 細 胞 因 子 能 不 同 程 度 地 增 加 嗜 酸 性 粒 細 胞 内 NF-kB IkB 與 一 些 黎 附 分 子 及 基 質 蛋 白, 例 如 :CD44 ICAM-1 a6 p2 和 基 質 金 屬 蛋 白 _(MMP-9) 等 的 基 因 表 達 我 們 發 現 IL-3 IL-5 及 GM-CSF 亦 能 夠 改 變 嗜 酸 性 粒 細 胞 表 面 的 一 些 黎 附 分 子 的 表 達, 例 如 增 加 ICAM-1 CDllb 和 CD18 ; 以 及 抑 制 ICAM-3 的 表 達 被 IL-3 IL-5 及 GM-CSF 所 誘 導 的 ICAM-1 是 需 要 通 過 p38mapk 與 NF-kB 雙 方 的 活 性 而 表 達 的 但 是, 控 制 CDllb CD18 以 及 ICAM-3 的 表 達 是 通 過 p38 MAPK 的 信 號 傳 遞 而 不 是 通 過 NF-kB 的 信 號 傳 遞 這 是 因 為 p38 MAPK 激 酶 抑 制 劑 能 抑 制 IL-3 IL-5 及 GM-CSF 對 上 述 每 一 個 黎 附 分 子 的 誘 導 作 用 但 是,NF-KB 抑 制 劑 就 只 能 抑 制 ICAM-1 的 表 達 此 外, 只 有 IL-3 和 IL-5 能 增 強 嗜 酸 性 粒 細 胞 黎 附 到 人 類 支 氣 管 上 皮 細 胞 細 胞 棟 (BEAS-2B) 上, 而 GM-CSF 就 沒 有 發 揮 這 種 作 用 我 們 證 實 這 三 種 細 胞 因 子 都 能 夠 誘 導 嗜 酸 性 粒 細 胞 的 趨 藥 性, 而 這 種 趨 藥 性 不 是 通 過 釋 放 出 的 嗜 酸 性 粒 細 胞 趨 化 因 子 eotaxin 或 增 加 其 受 體 -CCR3 的 表 達 而 達 致 的 嗜 酸 性 粒 細 胞 被 細 胞 因 子 誘 導 出 的 黎 附 力 和 趨 藥 性 雙 方 都 可 被 p38 MAPK 激 酵 抑 制 劑 或 NF-KB 抑 制 劑 所 抑 制 在 過 敏 性 蜂 喘 中, 嗜 酸 性 粒 細 胞 與 支 氣 管 上 皮 細 胞 的 互 相 作 用 是 XI

14 導 致 組 織 損 壞 及 支 氣 管 組 織 重 組 的 重 要 一 環 為 更 了 解 這 個 過 程, 我 們 利 用 一 氣 管 上 皮 細 胞 的 體 外 模 型 -BEAS-2B 細 胞 研 究 了 嗜 酸 性 粒 細 胞 與 支 氣 管 上 皮 細 胞 在 共 同 培 養 中 釋 出 的 細 胞 因 子 我 們 發 現 在 嗜 酸 性 粒 細 胞 與 支 氣 管 上 皮 細 胞 的 共 同 培 養 中 所 釋 出 的 炎 性 細 胞 因 子 :IL-5 及 TNF-oc 和 細 胞 趨 化 因 子 :IL-8 及 單 核 細 胞 趙 化 蛋 白 -l(mcp-l) 都 有 重 大 和 顯 著 的 增 加, 而 且 他 們 更 是 通 過 p38 MAPK 和 NF-KB 信 號 經 路 來 調 控 的 在 一 個 對 孝 喘 病 童 所 作 的 臨 床 實. 驗 中, 我 們 證 明 胸 腺 和 活 化 - 調 節 的 趙 化 因 子 (TARC) 及 可 溶 性 細 胞 毒 性 T 淋 巴 細 胞 相 關 分 子 4(sCTLA-4) 在 慢 性 和 急 性 哮 喘 病 童 的 血 黎 中 均 有 顯 著 上 升 此 外, 急 性 译 喘 病 童 服 用 及 注 射 皮 質 類 固 醇 後, 其 血 漿 中 的 TARC 和 sctla-4 水 平 均 顯 著 下 降 跟 據 以 上 的 研 究 結 果, 我 們 提 議 在 炎 性 反 應 中, 造 血 性 和 炎 性 細 胞 因 子, 包 括 :IL-3 IL-5 GM-CSF 和 TNF~a ; 以 及 p38 MAPK 和 NF-kB 信 號 經 路 在 調 控 嗜 酸 性 粒 細 胞 的 粘 付 性 和 趨 藥 性 上 扮 演 著 決 定 性 的 角 色 TARC 是 一 重 要 的 活 化 T 細 胞 的 趨 化 因 子, 並 用 以 激 化 及 募 集 輔 助 性 T 淋 巴 細 胞 1 到 發 炎 的 地 點 sctla-4 也 是 一 重 要 的 T 淋 巴 細 胞 活 性 調 整 劑 這 些 炎 性 的 媒 介 物 或 有 用 於 監 控 孝 喘 病 童 在 急 性 寺 喘 病 發 時 的 病 情 和 療 效 最 後, 我 們 希 望 上 述 的 研 究 結 果, 能 對 細 胞 因 子 和 趨 化 因 子 在 過 敏 炎 性 疾 病 的 病 理 中 所 扮 演 的 角 色 有 更 深 入 的 理 解 另 外 我 們 更 希 望 能 提 供 一 些 有 力 的 生 物 化 學 基 礎, 以 促 進 將 來 開 發 新 一 類 更 有 效 的 藥 物 及 策 略, 用 以 醫 治 不 同 的 炎 性 疾 病 驻 XII

15 PUBLICATIONS Publications Refereed Publications: 1. Wong CK, Ip WK, Lam CWK. Interleukin-3, -5, and granulocyte macrophage colony-stimulating factor-induced adhesion molecule expression on eosinophils by p38 mitogen-activated protein kinase and nuclear factor-[kappa] B. Am JRespir Cell Mol Biol 2003; 29: Ip WK, Wong CK, Lam CWK. Tumour necrosis factor-alpha-induced expression of intercellular adhesion molecule-1 on human eosinophilic leukaemia EoL-1 cells is mediated by the activation of nuclear factor-kappab pathway. Clin Exp Allergy. 2003; 33: Leung TF,Wong CK, Lam CWK, Li AM, Ip WK, Wong GW, Fok TF. Plasma TARC concentration may be a useful marker for asthmatic exacerbation in children. Eur Respir J. 2003; 21: Leimg TF, Wong CK, Chan ffl,ip WK, Lam CWK, Wong GW. Plasma concentration of thymus and activation-regulated chemokine is elevated in childhood asthma. J Allergy Clin Immunol. 2002; 110: Wong CK, Zhang JP,Ip WK, Lam CWK. Activation of p38 mi to gen- acti vated protein kinase and nuclear factor-kappab in tumour necrosis factor-induced eotaxin release of human eosinophils. Clin Exp Immunol. 2002; 128: Wong CK, Zhang J,Ip WK, Lam CWK. Intracellular signal transduction in eosinophils and its clinical significance. Immunopharmacol Immimotoxicol. 2002: 24: xm

16 Publications 7. Lung HL, Ip WK, Wong CK, Mak NK, Chen ZY, Leung KN. Anti-proliferative and differentiation-inducing activities of the green tea catechin epigallocatechin-3-gallate (EGCG) on the human eosinophilic leukemia EoL-1 cell line. Life ScL 2002; 72: Abstracts of Conference: 1. Wong CK, Ho CY, Zhang JP, Ip WK and Lam CWK. Cytometric bead array of cytokine assay: a high throughput analysis. (6 仇 Symposium and Annual General Meeting) Hong Kong Society of Flow Cytometry, Ip WK, Wong CK, Zhang JP, Ho CY and Lam CWK. The role ofnf-icb and p38 MAPK for the expression of CD49d and ICAM-l on human blood eosinophils, EoL-1 and BEAS-2B cells. Hong Kong Society for Immunology, Ip WK, Wong CK and Lam CWK. Flow cytometric analysis of apoptosis and intracellular nitric oxide. Annual General Meeting and Scientific Meeting) Hong Kong Society of Flow Cytometry^ (Oral presentation) 4. Ip WK, Wong CK, Wang CB and Lam CWK. Regulation of adhesion molecules on human eosinophils by cytokines IL-3, IL-5 and GM-CSF via p38 Mitogen-activated protein kinase and nuclear factor kappa-b pathways. (8 出.Annual General Meeting and Scientific Meeting) Hong Kong Society of Flow Cytometry, (Poster Prize awarded) 5. Ip WK, Wong CK, Zhang JP, Ho CY and Lam CWK. The role of NF-KB and p38 MAPK for the expression of CD49d and ICAM-l on human blood eosinophils, EoL-1 and BEAS-2B cells. (American College of Allergy, Asthma and Immunology, Hong Kong Institute of Allergy) Hong Kong Allergy XIV

17 Publications Convention, Ip WK, Wong CK, Wang CB and Lam CWK. Regulation of adhesion molecules on human eosinophils by cytokines IL-3, IL-5 and GM-CSF via p38 Mitogen-activated protein kinase and nuclear factor kappa-b pathways, (American College of Allergy, Asthma and Immunology, Hong Kong Institute of Allergy), Hong Kong Allergy Convention, Ip WK, Wong CK and Lam CWK, The roles of p38 Mitogen-activated protein kinase and nuclear factor kappa-b in cytokine-induced chemotaxis of human eosinophils. (American College of Allergy, Asthma and Immunology, Hong Kong Institute of Allergy) Hong Kong Allergy Convention, Ip WK, Wong CK and Lam CWK. Tumour necrosis factor-a-induced intercellular adhesion molecule-1 expression on human eosinophilic leukemia, EoL-1 cells is mediated by the activation of NF-KB pathway. (XXI Congress of European Academy of Allergology and Clinical Immunology), Allergy, 2002; 57(Suppl 73): Ip WK, Wong CK and Lam CWK. The roles of NF-KB and p38 MAPK for ECP release and the expressions of CD49d and ICAM-1 on human blood eosinophils and eosinophilic leukemia EoL-1 cells, (The Annual Meeting of the Japanese Society for Immunology) Proceedings of the Japanese Society for Immunology, 2001 ; 31: Ip WK, Wong CK and Lam CWK. The roles of p38 mitogen-activated protein kinase and nuclear factor kappa-b in cytokine-induced chemotaxis of human eosinophils. (The International Congress of Clinical Chemistry and Laboratory Medicine), Clinical Chemistry and Laboratory Medicine, 2002; 40: S277. (Poster Prize awarded) XV

18 Publications Submitted Manuscripts; 1. Ip WK, Wong CK, Wang CB and Lam CWK. Activation of p38 mitogen-activated protein kinase and nuclear factor-kb in interleukin-3, 5 and granulocyte macrophage colony-stimulating factor-induced adhesion and chemotaxis of human eosinophils. (Submitted to Journal of Immunology) 2. Wong CK, Ip WK, Lam CWK. Intracellular Signal Transduction Pathways in Eosinophils and Their Implications for Pharmacotherapy. (Submitted to Critical Reviews in Clinical and Laboratory Sciences) XVI

19 Table o f Contents TABLE OF CONTENTS ACKNOWLEDGEMENTS ABBREVIATIONS ABSTRACT 撮 要 PUBLICATIONS TABLE OF CONTENTS I HI VI X XIII XVII CHAPTER 1: GENERAL INTRODUCTION 1.1 Allergy and Allergic Diseases Allergy Allergic Diseases and their Prevalence 1.2 Eosinophils in Allergic Inflammation Characteristic of Eosinophils Eosinophil Production, Accumulation, Activation and Fate in Allergic Inflammation Adhesion Molecules in Allergic Inflammation Selectins Integrins Intercellular Adhesion Molecules (ICAM) 1.4 Cytokines and Chemokines in Allergic Inflammation Cytokines in Allergic Inflammation Chemokines in Allergic Inflammation 1.5 Intracellular Signal Transduction Pathways in Eosinophils RAS-RAF-Mitogen-Activated Protein Kinases (MAPK) Pathway Janus kinases (JAK)-Signal Transducers and Activators of Transcription (STAT) Phosphatidylinositol 3-Kinase (PI3K) Pathway Nuclear Factor-kappaB (NF-KB) Pathway Activator Protein (AP)-l Pathway Cyclic AMP Response Element Binding Protein (CREB) Pathway XVII

20 Table o f Contents 1.6 T-lymphocytes in Allergic Asthma The Role of T-lymphocytes in the Pathogenesis of Asthma Regulation of T-lymphocytes by CD28 and CTLA-4 in Allergic Inflammation 1.7 Signal Transduction Pathway Inhibitors as New Therapeutic Drugs of Allergic Diseases NF-KB Signaling Pathway Inhibitor p38 MAPK Signaling Pathway Inhibitor 1.8 Aims and Scopes of This Study CHAPTER 2% MATERIALS AND METHODS 48 1 Materials 48.1 Cell Lines 48.2 Patients, Control Subjects and Blood Samples 49 3 Human Buffy Coat 50 A Cell Culture Media, Buffers and Other Reagents 50.5 Reagents and Buffers for Purification of Human Eosinophils 55.6 Reagents and Buffer Solutions for Flow Cytometry 56.7 Reagents and Buffer Solutions for Total RNA Extraction 60 8 Reagents and Buffers for Protein Extractions Reagents and Buffer Solution for Agarose Gel Electrophoresis 62 Reagents and Buffers for SDS-po 1 yaery 1 amide Gel Electrophoresis 63 Reagents and Buffers for Westrcm Blot Analysis 65 Reagents and Buffers for Gene Expression Analysis 67 Reagents and Buffers for Non-radioactive Electroniobility Shift Assay (EMS A) Other Reagent Kits Methods Cell Cultures Purification of Buffy Coat Eosinophils by MACS and Eosinophil Culture Plasma Collection and Purification of Peripheral Blood Mononuclear Cells (PBMC) from EDTA-Blood Measurement of Total and Allergen-specific Immunoglobulin E (IgE) Concentrations Study of Cell Morphology 77 XVIII

21 Table o f Contents Flow Cytometric Analysis Cell Culture Experiments Gel Electrophoresis Total Cellular RNA Extraction cdna Expression Array Analysis Reverse Transcription Polymerase Chain Reaction (RT-PCR) Western Blot Analysis Non-radioactive Electromobility Shift Assay (EMSA) Gel Shift Assay Enzyme-Linked Immunosorbent Assay (ELISA) Statistical Analysis 87 CHAPTER 3: STUDY OF SIGNAL TRANSDUCTION MECHANISMS REGULATING THE EXPRESSION OF ADHESION MOLECULES ON HUMAN EOSINOPHILIC LEUKEMIC EOL-1 CELLS 3.1 Introduction 3.2 Results Effect ofgm-csf and TNF-a on the Expression ofcd49d, ICAM-1 and ICAM-3 on EoL-1 Cells Effect of Specific p38 MAPK Inhibitor SB and Proteasome Inhibitor MG-132 on TNF-a-induced Surface ICAM-1 Expression on EoL-1 Cells TNF-a-induced Phosphorylation of p38 MAPK and Effect of SB and MG-132 on TNF-a-induced Phosphorylation and Degradation of IkB-oc in EoL-1 Cells Effect of MG-132 and SB on TNF-a-induced NF-KB Activity in EoL-1 Cells Effect of TNF-a on the Expression Profile of NF-KB Pathway-related Genes in EoL-1 Cells 3.3 Discussion XIX

22 Table o f Contents CHAPTER 4: STUDY OF SIGNAL TRANSDUCTION MECHANISMS REGULATING THE ADHESION AND CHEMOTAXIS OF HUMAN EOSINOPHILS The Roles ofp38 MAPK and NF-KB in Regulating the Expression of Adhesion Molecules on Human Eosinophils Introduction 112 Results Morphologies of Purified Human Blood Eosinophils Effect of Proinflammatory Cytokines and Chemoattractants on the Surface Expression of Adhesion Molecules and CCR3 on Human Eosinophils In Vitro Effect of SB and MG-132 on TNF-a -induced Expression of ICAM-1 on Human Eosinophils In Vitro Effects of SB and MG-132 on Haematopoietic Cytokines IL-3-, IL-5-, and GM-CSF-modulated Expression of Surface ICAM-1, ICAM-3, CD lib, CD18 and CD49d on Human Eosinophils The Expression of NF-KB Pathway-related Genes in Haematopoietic Cytokine-stimulated Human Eosinophils Gene Expression of Adhesion Molecules and Extracellular Matrix Proteins in Haematopoietic 4. Cytokine-stimulated Human Eosinophils Activation on Phosphorylation of IicB-a and p38 MAPK in Human Eosinophils treated with IL-3, IL-5 and GM-CSF Effect ofmg-132 and SB on IL-3-, IL-5-, and GM-CSF-induced NF-KB Activity in Human Eosinophils Discussion 148 XX

23 Table of Contents 2 The Roles of p38 and NF-KB Regulating the Adhesion and Chemotaxis of Human Eosinophils Introduction Results Effect of SB and MG-132 on Haematopoietic Cytokine-induced Adhesion of Human, Eosinophils onto Bronchial Epithelail Cell Line BEAS-2B Effect of SB and MG-132 on Haematopoietic Cytokine-induced Shape Change of Human Eosinophils Effect of SB and MG-132 on Eotaxin and Haematopoietic Cytokine-induced Transmigration of Human Eosinophils Effect of Haematopoietic Cytokines, SB and MG-132 on CCR3 Expression in Human Eosinophils Discussion 176 CHAPTER 5: CYTOKINES AND CHEMOKINES RELEASE FROM INTERACTION OF EOSINOPHILS AND BRONCHIAL EPITHELIAL CELLS 5.1 Introduction Results In vitro Effect of SB and MG-132 on the Release of TNF-a and IL-8 in Supematants of IL-3-, IL-5- and GM-CSF-stimulated Eosinophils In vitro Effect of SB and MG-132 on the Release of TNF-a in Co-cultures of IL-3-, IL-5- and GM-CSF-stimulated Eosinophils and Epithelial Cells In vitro Effect of SB and MG-132 on the Release of IL-6 in the Co-cultures of IL-3, IL-5 and GM-CSF-stimulated Eosinophils and Epithelial cells In vitro Effect of SB and MG-132 on the Release of IL-8 in the Co-cultures of IL-3, IL-5 and GM-CSF-stimulated Eosinophils and Epithelial cells XXI

24 Table o f Contents In vitro Effect of SB and MG-132 on the Release ofmcp-1 in the Co-cultures ofil-3, ll-5 and GM-CSF-stimulated Eosinophils and Epithelial cells 5.3 Discussion CHAPTER 6 STUDY ON THE EXPRESSIONS OF CHEMOKINE RECEPTORS, CHEMOKINES, ADHESION AND CTLA-4 IN ALLERGIC ASTHMATIC PATIENTS CYTOKINES, MOLECULES 6.1 Introduction Results Demographic Data and Atopic Status Plasma Level of T-lymphocytes Chemoattractants TARC and MDC, in Asthmatic Children Plasma Level of Proinflammatory Cytokine IL-18, in Asthmatic Children Plasma Level of a soluble T-lymphocyte Negative Regulator, sctla-4, in Asthmatic Children Plasma Level of soluble Adhesion Molecules, se-selectin, in Asthmatic Children Association and Correlation between Atopy and Plasma Concentration of TARC, MDC, IL-18, sctla-4 and se-selectin Gene Expression profiles of Cytokines, Chemokines and Receptors in Peripheral Blood Mononuclear Cells (PBMC) from Acute Asthmatic Children Discussion 231 CHAPTER 7: CONCLUSIONS AND FUTURE PERSPECTIVES 7.1 General Conclusions 7.2 Future Perspectives APPENDIX REFERENCES XXII

25 Chapter 1 General Introduction Chapter 1 General Introduction 1.1 Allergy and Allergic Diseases Allergy The term "allergy" (from the Greek "alios" meaning changed or altered state and "ergon" meaning reaction or reactivity) was introduced in 1906 by von Pirquet of Vienna, Austria, who recognized that in both protective immunity and hypersensitivity reactions,antigens had induced changes in reactivity (Allergy online-vienna Medical School, Austria). Allergies are Type I or immunoglobulin E (IgE)-mediated hypersensitivity reactions of the immune system to specific substances called allergens such as pollens, house-dust mite, cockroach, food, drugs and stings. Atopy is a type of inherited allergic response involving elevated IgE. Sometimes called a reagiii response, it means that you have hay fever, bronchial asthma, or skin problems like urticaria or eczema. It can also be acquired, sometimes following hepatitis or extended contact with solvents or alcohol (Roitt et al 2001). Type I or immediate hypersensitivity is characterized by the production of IgE antibodies against allergens. IgE has the property of binding to high affinity receptors on basophils and mast cells. On encountering allergen, the IgE becomes cross-linked, inducing degranulation and release of vasoactive mediators, chemotactic factors and cytokines that produce allergic reactions (Roitt et al 2001).

26 Chapter 1 General Introduction Allergic Diseases and their Prevalence In recent years,allergic diseases especially asthma, have been increasing worldwide (ISSAC, 1998). Besides, the prevalence of diseases associated with atopy has increased in many parts of the world over the past 20 to 30 years. In the United Kingdom the prevalence of diagnosed asthma and symptoms strongly suggestive of asthma in children has increased at a rate of about 5% a year (Jarvis & Burney, 1998). Increases of similar magnitude have been observed in Sweden, Switzerland, Norway, the United States, Australia, New Zealand, and Taipei (Jarvis & Burney, 1998). In Hong Kong, between 1992 and 1995, the lifetime prevalence of asthma and wheeze in school children increased from 6.6% to 11.2% and from 3.7% to 12.4%, respectively (Leung et al., 1997). A similar increase of 65% in wheeze ever and 57% in rhinitis ever was observed in young adults between 1989 and 1994 (Lai et al., 1996). Up to 5% of the elderly population over the age 70 years are said to suffer from diagnosed asthma (Lai et al, 1995). Asthma is considered to be the most serious of allergic diseases. It can be disabling and even cause death in some cases (Jarvis & Burney, 1998). The increasing incidence and prevalence of allergic diseases especially asthma has already rose awareness of the public on the public health problem, the problem of economic burden and the need for new and efficient treatments of allergic diseases. 1.2 Eosinophils in Allergic Inflammation Eosinophil is thought to make a major contribution to the inflammation underlying the pathogenesis of allergic diseases such as asthma, allergic rhinitis and atopic dermatitis. In allergic asthma, a large number of eosinophils often infiltrate into the bronchial mucosa. The ability of eosinophil to generate an array of 2

27 Chapter27General Introduction proinflammatory mediators, in particular the cytotoxic granule proteins such as eosinophil major basic protein (MBP), has led to the hypothesis that allergic asthma is an on-going mucosal inflammatory disease. Eosinophils are the major effector cells mediating tissue damage by eosinophil-derived mediators. 1,2.1 Characteristic of Eosinophils Eosinophils are actively motile, terminally differentiated leukocytes. They are derived from the bone marrow, and have been identified in many mammalian and non-mammalian species. Human eosinophils are approximately 8 jam in diameter and have a bi-lobed nucleus. They differentiate from stem cell precursors in the bone marrow by the control of T-cell-derived interleukin (IL)-3, ll-5 and granulocyte macrophage colony stimulating factor (GM-CSF) (Giembycz & Lindsay, 1999). Their characteristic membrane-bound specific granules stain reddish brown with basic aniline dyes such as eosin and consist of crystalline core make up of major basic protein (MBP) surrounded by a matrix containing eosinophil cationic protein (ECP), eosinophil peroxidase (EPO) and eosinophil-derived neurotoxin (EDN or EPX) (Figure 1.1) (Weller, 1997). All these basic proteins are cytotoxic and contributing to the tissue damage in allergic inflammation.

28 Chapter28General Introduction Lipid Bodies PAF LTC4 TxBs PGE2 15-oxo-HETE Lipoxins Bilobed Nucleus Cytokines TGF-a, TGF-p L-1a L-3,!L-5, GM-CSF L-2 L-4 L-6 L-10 L-16 TNF-a TNF-p VEGF PDGF-p HB-EGF Chemokines: RANTES MIP-1a IL-8 Eotaxin Figure 1.1 Human eosinophils with bilobed nucleus and eosinophil granules with crystalloids showing eosinophil lipid and protein products (modified from Weller, 1997).

29 Chapter29General Introduction Bone Marrow 介 IL~3 J GM-CSF Growth and Differentiation Matured Eosinophil Endothelium Mast cell Tcell Macrophage IL-3, ll-5 GM-CSF TNF,IL-1 GM-CSF RANTES, Eotaxin,MCP,,MIP,PAF,LTB Chemoattraction IL-3,11^5, GM-CSF Degranulation and Tissue damage cr~s> Figure 1,2 Eosinophil production, activation and accumulation in inflammatory response (modified from Rothenberg, 1998).

30 Chapter 1 General Introduction Eosinophil Production, Accumulation, Activation and Fate in Allergic Inflammation Eosinophil turnover or eosinopoiesis occurs almost exclusively in the bone marrow. The bone marrow from normal individuals contains about 3% eosinophils, of which 37% are mature, non-dividing granulocytes. The development and differentiation of eosinophils within bone marrow are promoted by IL-3, IL-5 and granulocyte-macrophage colony-stimulating factor. In humans, eosinophils comprise 2 to 10% of the peripheral leukocytes. Eosinophils are predominantly tissue cells. It has been estimated that normal tissues contain 100 to 300 times as many eosinophils as in blood (Parwaresch et al. 1976). IL-5 has the most cell-specific effects on the differentiation and production of eosinophils while eotaxin and IL-5 can promote the release of eosinophil into circulation from bone marrow (Figure 1.2) (Gierabycz & Lindsay, 1999; Rothenberg, 1998). Once in the circulation, eosinophils have a half-life of approximately 18h. However, in the presence of inflammatiory reaction, the half-life of eosinophils can be prolonged under the influence of proinflammatory cytokines or chemokines that enhance the survival and results in eosinophilia (Rothenberg, 1998). Under the interaction of adhesion molecules on the surface of activated eosinophils and endothelial cells, circulating eosinophils will roll along the epithelium and then firmly attached to the epithelial cells and subsequently migrated across the endothelium lining towards the inflammatory sites by differential expression of adhesion molecules under the influence of inflammatory cytokines and chemokines (Rothenberg, 1998). Eosinophils accumulated and further induced to release granular toxic proteins by different proinflammatory cytokines such as TNF-a, IL-1 and GM-CSF at the inflammatory sites where tissue damage occurs (Figure 1.2). 6

31 Chapter31General Introduction Eosinophil Surface Receptors and Proteins Eosinophils are either constitutively or after induction to express a number of receptors or protein ligands on the plasma membrane such as receptors for cytokine: TNF-a, IFN-y, IL-2, IL-4 and chemokine: IL-8,eotaxin. For some of the ligands, such as C5a, the chemokines and certain cytokines, directed migration of eosinophils can be elicited. Some of these ligands directly promote effector responses, including enhanced respiratory burst activity and degranulation. Other ligands, including the eosinophil growth factor cytokines (GM-CSF, IL-3 and IL-5), enhance eosinophil functional responses elicited by other stimuli and prolong the survival (by antagonizing apoptosis) of mature eosinophils (Lacy & Moqbel, 2000). Eosinophils also possess receptors for IgA,IgG and IgE (Dombrowicz et al, 2000). The increased eosinophil number and IgE level are frequently found in allergic diseases. This may imply the important function of IgE receptor in eosinophil activation (Rothenberg, 1998) Eosinophil Derived Mediators Eosinophils contain plenty of specific granules which store lysosomal hydrolases, major basic proteins, as well as most proteins preferentially distinct for eosinophils (Figure 1.1). Apart from the granule-derived basic proteins, eosinophils are capable of producing large quantities of bioactive lipid mediators on stimulation such as platelet activating factor (PAF), leukotriene (LT) C4 and Prostaglandin E2 (PGE2), which are generally proinflammatory, are multiple and include the potent smooth muscle contractile and vasoactive effect (Weller, 1997). In addition, a number of cytokines and chemokines are also produced and stored by the specific granules of eosinophils. These cytokines include the haematopoietic growth factors like IL-3,

32 Chapter32General Introduction I.L-5 and GM-CSF, other growth factors like transforming growth factor (TGF)-a, TGF-pl,vascular endothelial growth factor and platelet derived growth factor-p, and other immunoregulatory cytokines like IL-2, IL-4 and IL-10, and proinflammatory cytokines like IL-1, ll-6, and TNF-a as well as chemokines like IL-8, macrophage inflammatory protein (MlP)-a and regulated upon activation normal T-cell expressed and secreted (RANTES) (Rothenberg, 1998; Tachimoto & Bochner, 2000). 1.3 Adhesion Molecules in Allergic Inilammatioii In allergic disease such as asthma, cell adhesion molecules are involved in all stages of leukocytes infiltration and accumulation at inflammatory sites of the airway. Adhesion molecules involved in leukocyte trafficking are grouped into three families based on structural features: (1) selectins, (2) integrins, (3) immunoglobulin (Ig) gene superfamily. In the development of inflammatory response, for example, eosinophilia, eosinophil recruitment involves transient adhesive interaction between selectins on circulating eosinophils and their glycoprotein ligands which results in cell rolling along the endothelium. Subsequent activation of the rolling eosinophils by inflammatory cytokines or chemoattractants such as eotaxin and IL-5, initiates the differential expression of integrins and Ig superfamily on cell surfaces of eosinophils and endothelial cells which cause the rolling eosinophils to arrest, firmly arrest and flatten. Finally the eosinophils migrate across the endothelium (diapedesis) into the inflamed tissues and move towards the source of stimulus (chemotaxis) (Rothenberg, 1998).

33 Chapter33General Introduction Selectins There are three members of selectin family, E-selectin (CD62E), P-selectin (CD62P) and L-selectin (CD62L), which are glycoproteins sharing an N-terminal lectin domain that is directly responsible for ligand binding. Due to this lectin domain, physiological selectin ligands are a diverse group of glycosylated proteins and glycolipids that can be recognized by the extracellular lectin domain. L-selectin is expressed constitutively on a wide variety of leukocytes (Kansas, 1996). It is expressed on the majority of B cells, thymocytes and native 丁 cells. Exposure of eosinophils to inflammatory mediators causes L-selectin to be rapidly shed from the cell surface facilitating the development of firm adhesion. P-selectin is stored preformed in Weibel-Palade bodies of endothelial cells and granules of platelets. Following activation with inflammatory mediators, it is rapidly mobilized to the cell surface (McEver et al. 1989). Expresion of P-selectin at the surface is transient and decreases substantially within minutes. E-selectin expression is restricted to activated endothelial cells. Expression of E-selectin is induced by LPS and by cytokines such as IL-1, TNF-a and IL-4 (Kansas, 1996) Integrins Integrins are heterodimeric proteins consisting of non-covaleiitly linked a- and P-chains with cytoplasmic tails which associate with the cytoskeleton and provide a connection to the extracellular matrix. Currently, at least 8 different (3 and 17 different a chains have been identified (Gahmberg et al. 1997). These receptors bind to variety of extracellular matrix and cell adhesion molecules. Leukocytes express 13 different integrins that mediate binding to endothelial and epithelial cells and matrix

34 Chapter 1 General Introduction proteins. The most importants for these adhesions are the pi (CD29) and p2 (CD 18) integrins which bind to immimoglobulin gene superfamily receptors. The pi integrins comprise the largest subfamily of integrins and are expressed on a wide variety of cells. The very late activated antigen (VLA)-4 (a4pl; CD49d/CD29) is expressed on all blood leukocytes except neutrophils. It can bind to both fibronectin and vascular cell adhesion molecule-1 (VCAM-1). This interaction mediates leukocyte binding to activated endothelial cells. In eosinophils, it is the only integrin that shares with selectins the ability to mediate rolling adhesion under conditions of shear stress (Sriramarao et ah, 1994), The p2 integrins (CD 18) has been demonstrated to form heterodimers with 4 different a chains, leukocyte function-associated antigen-1 (LFA-1; alj32; CD 11 a/cd 18), CR3 or macrophage-1 antigen (Mac-1; amp2; CDilb/CD18), axp2 (CDllc/CD18), and adp2. (Galimberg et al, 1997). Ligands for p2 integrins include intercellular adhesion molecule (ICAM)-l, ICAM-2, and ICAM-3, as well as fibronectin and the complement fragment C3bi (Larson & Springer, 1990). Levels of Mac-1, ax 32, and adp2 are rapidly up-regulated on the cell surface following activation, presumably the result of mobilization from preformed intracellular stores (Kroegel et al, 1994). In vitro studies have demonstrated that interactions through p2 integrins and their ligands are important for degranulation, leukocytes-endothelial adhesion, and transendothelial migration (Horie and Kita, 1994; Kato et al, 1998), Interceiliilar Adhesion Molecules (ICAM) The Ig superfamily is composed of cell surface molecules that contain immunoglobulin-like domains. There are over 100 members of this superfamily and 10

35 Chapter 1 General Introduction a number of them play an important role in cell adhesion in inflammatory responses. They include ICAM-1, TCAM-2, ICAM-3 and VCAM-1. ICAM-1 is a single transmembrane protein containing five Ig-like domains. It is constitutively expressed at low levels on endothelial cells but is up-regulated by various cytokines such as IL-1, IFN-y and TNF-a, and lipopolysaccharide (LPS) (Dustin & Springer, 1991). ICAM-1 can bind to LFA-1, Mac-1 and fibronectin. ICAM-2 has two Ig-like domains. It is constitutively expressed on endothelial cells at higher levels than ICAM-1 but low levels on surface of resting lymphocytes and monocytes. It is being recognized by both LFA-1 and Mac-1. ICAM-2 is not up-regulated by inflammatory cytokines and the significance of this is not clear (de Fougerolles et al., 1991). Similar to ICAM-1, ICAM-3 also contains five Ig domains. It is not normally found on endothelial cells but is constitutively expressed at high levels on leukocytes. It is important in mediating interactions between leukocytes. It is being recognized by LFA-1 but not Mac-l (Fawcett et al 1992). VCAM-1 has seven Ig domains which can be induced to express on endothelial cells by cytokines like IL-1, ll-4 and TNF-a (Bevilacqua, 1993). 1.4 Cytokines and Chemokines in Allergic Inflammation Cytokines in allergic inflammation Cytokines are usually glycosylated extracellular signaling proteins, which usually have less than 80 kda in size. They are produced from many different cell types and exert effects by paracrine and autocrine manner, through the interaction with specific receptors on the surface of adjacent target cells and cells producing themselves respectively. They act on target cells to cause a wide array of cellular 11

36 Chapter 1 General Introduction functions including activation, proliferation, chemotaxis, immunomodulation, release of other cytokines or mediators, growth and cell differentiation, and apoptosis. It is generally accepted that type 2 T helper (Th2) cytokines such as interleukin (XL)-4, IL-3, IL-5, IL-10 and IL-13 play an essential role in the development of asthmatic response (Wong & Lam, 2003; Chung & Barnes, 1999). Th2 cells represent the only cells in the immune system that can both directly recognize the allergen peptides through the T cell receptor (TCR) and, at the same time, release Th2 cytokines that account for the joint involvement of IgE antibody-producing B cells (IL-4, IL-13), mast cells (IL-4, IL-10), and eosinophils (IL-5) in allergic inflammation (Romagnani, 2000). Moreover, other proinflammatory cytokines such as GM-CSF, IL-6, IL-18 and TNF-a also associated with the inflammatory response characterizing human allergic asthma (Chung and Barnes, 1999) Interleukin (IL)-3, IL-5 and Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) IL-3, IL-5 and GM-CSF are pluripotent haematopoietic growth factor involved in differentiation and activation of cells in the myeloid compartment (de Groot et ah, 1998). The activities of these cytokines are restricted to cell types expressing appropriate cytokine specific cell surface receptors. In human these three cytokines exhibit overlapping biological activities on cells expressing their receptors by sharing a single signaling receptor subunit, the common p-subunit (pc), with the three specific 1L-3/IL-5/GM-CSF binding a-subunits (Scott & Begley, 1999). Cytokines binding to their a-siibunits with high affinity when interacting with the common P-subunit results in receptor dimerization. The receptor complex triggers rapid 12

37 Chapter 1 General Introduction activation of receptor-associated JAK2 and tyrosine, serine/threonine phosphorylation of pc receptor. Consequently, activated receptor then triggers intracellular signaling including JAK/STAT and ras-raf-mapk kinase pathways (Figure 1.3) (Guthridge et ah, 1998). IL-3 and IL-5 are mainly produced from activated T-helper cells (Chung & Bames, 1999) whilst eosinophils can also express both of them in vitro (Weller, 1997; Dubucquoi et al, 1994). Moreover, GM-CSF is produced by a variety of cells including macrophages, eosinophils, T-cells,fibroblasts,endothelial cells, airway smooth muscle cells and epithelial cells (Chung & Bames, 1999). These three cytokines appear to be the major cytokines involved in the development of eosinophilia and tissue remodeling in allergic asthma (Rothenberg, 1998). They are central in determining the number of eosinophils in the circulation and in tissues by promoting production, proliferation, maturation and survival by inhibition of apoptosis. These three cytokines are also implicated in the priming of mature eosinophils to a range of stimuli that activate eosinophil chemotaxis, degranulation, adhesion and activation of superoxide production (Giembycz & Lindsay, 1999) IL-6 IL-6 is produced by variety of cells. The major sources are macrophages, fibroblasts and endothelial and epithelial cells. It can also be synthesized by different immune cells including T-cells, B-cells, macrophages, mast cells and eosinophils (Chung & Barnes, 1999; Akira et al, 1993). IL-6 is a pleiotropic cytokine involved in the regulation of immune responses, the acute phase response and hematopoiesis (Akira et al 1993). Although the role of IL-6 in asthma is still uncertain, there is increased basal release of IL-6 from asthmatic patient compared with normal subjects 13

38 Chapter 1 General Introduction (Gosset et a/.,1991) and IL-6 release is increased from alveolar macrophages of asthmatic patients after allergen challenge (Broide et al 1992). IL-6 is involved in T-cell activation, growth and differentiation. It is also a terminal differentiation factor for B cell and it is an important co-factor for IL-4 dependent IgE synthesis from B-cells (Akira et al 1993). In eosinophils, IL-6 nirna is constitulively expressed and IFN-y can increase IL-6 release from eosinophils in vitro (Hamid et al, 1992). IL-6 has dual functions of both inflammatory and anti-inflammatory effects. It has been demonstrated to inhibit the expression and release of IL-1 and TNF from macrophages in vitro. It can also inhibit endotoxin-induced TNF production and neutrophil influx in the airways in vivo (Ulich et al 1991) IL-18 IL-18 is a novel Thl-inducing, proinflammatory cytokine and is formerly identified as an rfn-y-inducing factor. It is produced not only by various types of immunocompetent cells but also by non-immune cells including Kupffer cells, macrophages, keratinocytes, intestinal epithelial cells, osteoblasts and adrenal cortex cells (Dinarello, 1999; Nakanishi et al, 2001). IL-18 is crucial in promoting type 1 cytokine responses, primarily by its ability to induce IFN-y production in T cells, B-cells and natural killer (NK) cells particularly in the presence of IL-12. IL-18 is related to the IL-1 family. Similar to IL-1 (3, IL-18 is synthesized as a 24-kDa precursor protein (pro-ll-18) and becomes active after cleavage by IL-1 p-converting enzyme (ICE) that is also called caspase-1 (Dinarello, 1998). Similar to the receptor system of IL-1, IL-18 receptor is composed of a ligand-binding component, IL-lSRa and the signaling component, IL-18Rp. Freshly 14