過敏性疾病的患病率一直上升，約30%至40%的世界人口受到一個或多個過敏疾病影響。然而，現時仍然缺乏有效治療過敏性疾病的方法，大多數傳統療法只能改善臨床症狀，卻沒有針對導致過敏性炎症的主要因素。嗜酸性粒細胞浸入和積累於局部炎症部位以及延緩了的細胞凋亡是過敏性炎症的標誌，而嗜酸性粒細胞亦被認為是過敏性炎症的主要效應細胞。因此，針對嗜酸性粒細胞，抑制其活性和衍生產物，是一個有潛力和有效地治療過敏性炎症的策略。發展針對嗜酸性粒細胞的療法需要詳細了解嗜酸性粒細胞介導炎症反應的機制。 / 在第三章中，我們把嗜酸性粒細胞與皮膚成纖維細胞一起培養，建立了一個體外的皮膚炎症模型，用以研究嗜酸性粒細胞在過敏性皮炎發病病理中的作用。我們發現，嗜酸性粒細胞和成纖維細胞的共培養顯著誘導促炎性的細胞因子IL-6和濕疹相關趨化因子CXCL1，CXCL10，CCL2和CCL5的釋放。這些炎症介質的釋放在能導致皮膚瘙癢的細胞因子IL-31和內源性警報因子IL-33的刺激下進一步加強。 IL-31和IL-33可顯著引起嗜酸性粒細胞和成纖維細胞釋放CXCL8，而當兩種細胞共培養後，CXCL8的釋放進一步增強。在共培養系統中，嗜酸性粒細胞是釋放CCL5的主要來源，而成纖維細胞則是釋放IL-6，CXCL1, CXCL8，CXCL10和CCL2的主要來源。嗜酸性粒細胞和成纖維細胞之間的直接相互作用是CXCL1，CXCL10，CXCL8和CCL5的釋放所必需的。在IL-31和IL-33的刺激下，共培養系統中嗜酸性粒細胞和成纖維細胞的細胞表面粘附分子ICAM-1的表達上調。而嗜酸性粒細胞和成纖維細胞間的互作用，以及IL-31和IL-33對兩種細胞的激活，是經由p38、ERK、JNK，NF-κB和PI3K-AK訊息傳遞徑路所調節的。另外，我們亦發現1,25-二羥維生素D3能顯著抑製共培養系統在IL-31或IL-33激活下IL-6，CCL2和CXCL8的釋放。 / 在第四章中，我們探討在嗜酸性粒細胞和支氣管上皮細胞BEAS-2B的共培養系統中，兩種細胞的細菌相關模式識別受體成員NOD1和NOD2激活機制。我們發現NOD配體iE-DAP和NOD2配體MDP能激活嗜酸性粒細胞內的ERK和NF-κB信號傳導通路，而MDP可以激活嗜酸性粒細胞和BEAS-2B細胞的ERK信號傳導通路。當嗜酸性粒細胞和BEAS-2B細胞共培養時，iE-DAP和MDP均可激活嗜酸性粒細胞和BEAS-2B細胞的ERK和NF-κB信號傳導通路，而且與單獨培養的細胞相比，激活的程度更強。此外，我們還探討了iE-DAP和MDP對 BEAS-2B細胞和嗜酸性粒細胞的核轉錄因子的激活作用。我們發現iE-DAP和MDP可以激活嗜酸性粒細胞和BEAS-2B細胞的某些轉錄因子，包括因子NF-κB、STAT-3和NFAT等，而多數被激活的轉錄因子都跟過敏性炎症和哮喘的發病有關。除了體外研究，我們還建立了過敏性肺部炎症的小鼠模型，用以研究iE-DAP和MDP的生理效應。iE-DAP和MDP均可激活第二型輔助性T細胞相關的免疫球蛋白E生產，以及誘導嗜酸性粒細胞趨化因子CCL5。 / 上述的研究結果表明IL-31與IL-33在過敏性皮炎的發病病理中發揮關鍵作用，通過不同的信號傳導機制激活嗜酸性粒細胞與成纖維細胞的相互作用。另外，我們探討了在嗜酸性粒細胞和BEAS-2B細胞中，調節NOD1和NOD2激活的信號傳導機制，加上動物實驗，所得的結果有助了解細菌感染在哮喘發作中所發揮的影響。這些關於細胞之間和細胞內調控機制的研究結果，進一步增強我們對嗜酸性粒細胞介導炎症的理解，並為發展更高專一性、效用和更少副作用的消炎藥提供了新的標靶和方向。 / The prevalence of allergic diseases has been steadily increased, with about 30-40% of the world population being affected by one or more allergic conditions. Effective treatments on allergic diseases are still lacking, as most of the traditional therapies aim at clinical improvement without targeting the factors that primarily promote the allergic inflammation. Infiltration, delayed apoptosis and accumulation of eosinophils at the local inflammatory sites are the hallmarks of allergic inflammation and eosinophils are considered as the principal effector cells in allergic inflammation. Anti-eosinophils therapeutics inhibit eosinophil activity and eosinophil-derived products are thus potential strategies in treating allergic inflammation effectively. Elucidation of the detailed mechanisms of eosinophil-mediated inflammation is therefore necessary for the development of anti-eosinophils therapeutics. / In Chapter 3, we established an in vitro skin inflammation model by co-culturing eosinophils with dermal fibroblasts in order to study the pathophysiology of eosinophils’ involvement in the pathogenesis of atopic dermatitis (AD). We revealed that co-culture of eosinophils and fibroblasts significantly induced release of pro-inflammatory cytokine IL-6 and AD-related chemokines CXCL1, CXCL10, CCL2 and CCL5. Such inductions were further enhanced with pruritogenic cytokine IL-31 and endogenous alarmin IL-33 stimulation. IL-31 and IL-33 could significantly provoke the release of CXCL8 from eosinophils and fibroblasts, respectively, which was further enhanced upon co-culture. In co-culture, eosinophils and fibroblasts were the main source for the release of CCL5, and IL-6, CXCL1, CXCL8, CXCL10 and CCL2, respectively. Direct interaction between eosinophils and fibroblasts was required for CXCL1, CXCL10, CXCL8 and CCL5 release. Cell surface expression of intercellular adhesion molecule-1 on eosinophils and fibroblasts was upregulated in co-culture upon IL-31 and IL-33 stimulation. The interaction between eosinophils and fibroblasts under IL-31 and IL-33 stimulation differentially activated ERK, JNK, p38 MAPK, NF-κB and PI3KAkt pathways. 1,25-dihydroxy vitamin D3 exerted in vitro suppressive effect on the release of IL-6, CCL2 and CXCL8 release from IL-31 or IL-33 activated co-culture of eosinophils and fibroblasts. / In Chapter 4, we revealed the mechanisms underlying bacterial-related pattern recognition receptor members NOD1 and NOD2 activation in eosinophils and bronchial epithelial cells BEAS-2B from the co-culture system and animal model experiment. NOD1 ligand iE-DAP and NOD2 ligand MDP could activate ERK and NF-κB pathways in eosinophils, while MDP stimulation could activate ERK in both eosinophils and BEAS-2B cells. When the eosinophils and BEAS-2B cells were co-cultured together, both iE-DAP and MDP could induce ERK and NF-κB activation in the two cells, and the activation was enhanced when compared with that in cells cultured alone. Besides, we also investigated the transcription factors activation in eosinophils and BEAS-2B cells by iE-DAP and MDP. iE-DAP and MDP could also activate a panel of transcription factors in eosinophils and BEAS-2B cells, including NF-κB, STAT-3 and NFAT etc, the nuclear transcription factors commonly involved in allergic inflammation and related to the pathogenesis of asthma. Apart from in vitro study, we also established an in vivo allergic pulmonary inflammation mice model to study the physiological effects of iE-DAP and MDP. Both iE-DAP and MDP could activate the Th2 related IgE production and induce eosinophil chemokine CCL5. / The above findings suggest a crucial immunopathological role of IL-31 and IL-33 in AD through the activation of eosinophils-fibroblasts interaction via differential intracellular signaling mechanisms. The intracellular signaling mechanisms regulating NOD1 and NOD2 activation in eosinophils and bronchial epithelial cells and animal experiment were also revealed and give implications of the role of bacterial infections in the exacerbation of asthma. Such information further enhances our understandings on both the intercellular and intracellular mechanisms of eosinophil-mediated inflammation, and gives implications for new targets for the development of anti-inflammatory drugs with higher specificity, potency, and less side effects. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Leung, Ming Lam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 136-153). / Abstracts also in Chinese. / ACKNOWLEDGEMENTS --- p.I / ABSTRACT --- p.III / 摘要 --- p.VI / PUBLICATIONS --- p.IX / ABBREVIATIONS --- p.XI / TABLE OF CONTENTS --- p.XIV / Chapter Chapter 1 --- : General Introduction / Chapter 1.1 --- Allergy --- p.1 / Chapter 1.1.1 --- Definition and characteristics of allergy --- p.1 / Chapter 1.1.2 --- Allergic diseases and their prevalence --- p.2 / Chapter 1.1.3 --- Allergic inflammation --- p.2 / Chapter 1.2 --- Biology of eosinophils --- p.8 / Chapter 1.2.1 --- Development of eosinophils --- p.8 / Chapter 1.2.2 --- Cellular characteristics of eosinophils --- p.9 / Chapter 1.2.3 --- Eosinophils surface and intracellular markers --- p.10 / Chapter 1.2.4 --- Eosinophil-derived mediators --- p.13 / Chapter 1.2.5 --- Accumulation and activation of eosinophils at inflammatory sites --- p.16 / Chapter 1.3 --- Adhesion molecules in allergic inflammation --- p.19 / Chapter 1.3.1 --- Selectins --- p.19 / Chapter 1.3.2 --- Integrins --- p.20 / Chapter 1.3.3 --- Immunoglobulin gene super family --- p.21 / Chapter 1.4 --- Cytokines in allergic inflammation --- p.23 / Chapter 1.4.1 --- Pro-inflammatory cytokines --- p.23 / Chapter 1.4.2 --- Anti-inflammatory cytokines --- p.25 / Chapter 1.5 --- Chemokines in allergic inflammation --- p.27 / Chapter 1.6 --- Signal transduction in allergic inflammation --- p.29 / Chapter 1.6.1 --- Intracellular signaling mechanisms --- p.29 / Chapter 1.6.2 --- Transcription factors activation --- p.33 / Chapter 1.7 --- Perspective treatments --- p.36 / Chapter 1.7.1 --- Inhibition of pro-inflammatory cytokines --- p.36 / Chapter 1.7.2 --- Inhibition of chemokines --- p.37 / Chapter 1.7.3 --- Small interfering (si)RNA against transcription factor --- p.37 / Chapter 1.7.4 --- Signaling pathway inhibitors --- p.38 / Chapter 1.8 --- Aim of study --- p.39 / Chapter Chapter 2 --- : Materials and Methods / Chapter 2.1 --- Materials --- p.42 / Chapter 2.2 --- Methods --- p.60 / Chapter 2.2.1 --- Purification of human eosinophils --- p.60 / Chapter 2.2.2 --- Cell culture --- p.61 / Chapter 2.2.3 --- Cell surface and intracellular immunofluorescence staining --- p.63 / Chapter 2.2.4 --- Western blot --- p.64 / Chapter 2.2.5 --- Allergic asthmatic mice model --- p.64 / Chapter 2.2.6 --- Statistical analysis --- p.65 / Chapter Chapter 3 --- : Activation of Eosinophils Interacting with Dermal Fibroblasts by Pruritogenic Cytokine IL-31 and Alarmin IL-33: Implications in Atopic Dermatitis / Chapter 3.1 --- Introduction --- p.66 / Chapter 3.1.1 --- Atopic dermatitis --- p.66 / Chapter 3.1.2 --- Eosinophils in AD --- p.68 / Chapter 3.1.3 --- IL-31 --- p.68 / Chapter 3.1.4 --- IL-33 --- p.70 / Chapter 3.1.5 --- Vitamin D --- p.71 / Chapter 3.1.6 --- Hypothesis and aim of study --- p.73 / Chapter 3.2 --- Results --- p.74 / Chapter 3.2.1 --- Surface expression of receptors for IL-31 and IL-33 on human eosinophils and dermal fibroblasts --- p.74 / Chapter 3.2.2 --- Cytokine and chemokine release upon the interaction of eosinophils and dermal fibroblasts activated by IL-31 and IL-33 --- p.77 / Chapter 3.2.3 --- Source of the release of cytokines and chemokines in the co-culture system upon IL-31 and IL-33 stimulation --- p.80 / Chapter 3.2.4 --- Effect of transwell inserts on cytokine and chemokine release in IL-31 and IL-33-treated co-culture --- p.83 / Chapter 3.2.5 --- Effect of IL-31 and IL-33 on adhesion molecule expression on eosinophils and dermal fibroblast in co-culture system --- p.86 / Chapter 3.2.6 --- Intracellular signaling pathways involved in the interaction of eosinophils and dermal fibroblasts under IL-31 and IL-33 stimulation --- p.88 / Chapter 3.2.7 --- Surface expression of vitamin D receptor on human eosinophils and dermal fibroblasts --- p.96 / Chapter 3.2.8 --- Suppressive effects of 1,25-dihydroxy vitamin D₃ (calcitriol) on cytokine and chemokine release from IL-31 and IL-33-treated co-culture --- p.97 / Chapter 3.3 --- Discussion --- p.100 / Chapter Chapter 4 --- :Intracellular Signal Transduction Mechanisms of NLR Activation in Human Eosinophils and Bronchial Epithelial Cells: Implication for Bacterial Infection in Allergic Asthma / Chapter 4.1 --- Introduction --- p.107 / Chapter 4.1.1 --- Allergic asthma --- p.107 / Chapter 4.1.2 --- Eosinophils in allergic asthma --- p.108 / Chapter 4.1.3 --- NOD-like receptors --- p.109 / Chapter 4.1.4 --- Hypothesis and aim of study --- p.110 / Chapter 4.2 --- Results --- p.112 / Chapter 4.2.1 --- Intracellular signaling pathways involved in the interaction of eosinophils and BEAS-2B cells under iE-DAP and MDP stimulation --- p.112 / Chapter 4.2.2 --- Transcription factors activation in eosinophils and BEAS-2B cells under iE-DAP and MDP stimulation --- p.119 / Chapter 4.2.3 --- In vivo activating effect of NOD1,2 ligands on the IgE and chemokine concentration in serum and BALF in allergic asthmatic mice --- p.122 / Chapter 4.3 --- Discussion --- p.125 / Chapter Chapter 5 --- : Concluding Remarks and Future Studies / Chapter 5.1 --- Concluding remarks --- p.130 / Chapter 5.2 --- Future studies --- p.131 / REFERENCES --- p.136

Yip Kwok Ho. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 231-260). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iv / Acknowledgements --- p.vi / Publications --- p.vii / Abbreviations --- p.viii / Contents --- p.xi / Chapter 1. --- Introduction --- p.1 / Chapter 1.1. --- Mast cells --- p.2 / Chapter 1.2. --- "Mast cell origin, growth and development" --- p.2 / Chapter 1.2.1. --- Stem cell factor --- p.4 / Chapter 1.2.2. --- Interleukins --- p.6 / Chapter 1.3. --- Mast ceII heterogeneity --- p.7 / Chapter 1.4. --- Mast ceII mediators --- p.9 / Chapter 1.4.1. --- Pre-Synthesized mediators --- p.9 / Chapter 1.4.1.1. --- Histamine --- p.10 / Chapter 1.4.1.2. --- Protease --- p.11 / Chapter 1.4.2. --- Newly synthesized mediators --- p.13 / Chapter 1.4.2.1. --- Prostanoid --- p.14 / Chapter 1.4.2.2. --- Cysteinyl Leukotriene --- p.15 / Chapter 1.4.3. --- Mast cell-derived cytokines and growth factors --- p.16 / Chapter 1.5. --- Mast cell activation --- p.17 / Chapter 1.5.1. --- FceRI-dependent mast cell activation --- p.18 / Chapter 1.5.1.1. --- FceRI and IgE aggregation --- p.19 / Chapter 1.5.1.2. --- Protein-tyrosine kinase activation --- p.21 / Chapter 1.5.1.3. --- Phospholipase activation and calcium ion mobilization --- p.22 / Chapter 1.5.1.4. --- GTPase and MAPK activation --- p.24 / Chapter 1.5.2. --- Non-immunogical mast cell activation --- p.26 / Chapter 1.6. --- Roles of mast cell in inflammatory disease --- p.27 / Chapter 1.7. --- Nitric oxide --- p.28 / Chapter 1.8. --- Nitric oxide synthase --- p.30 / Chapter 1.9. --- Nitric oxide signaling in cellular level --- p.31 / Chapter 1.9.1. --- Direct effects of NO --- p.32 / Chapter 1.9.2. --- Indirect effects of NO --- p.34 / Chapter 1.10. --- Mast cell and nitric oxide --- p.35 / Chapter 1.11. --- Aim of Study --- p.37 / Chapter 2. --- Materials and Methods --- p.43 / Chapter 2.1. --- Material --- p.44 / Chapter 2.1.1. --- Human buffy coat for mast cell culture --- p.44 / Chapter 2.1.2. --- Materials for cell isolation and cell counting --- p.44 / Chapter 2.1.3. --- Materials for mast cell culture --- p.45 / Chapter 2.1.4. --- Material for buffers --- p.45 / Chapter 2.1.5. --- Materials for cytospin and May-Griinwald-Giemsa staining --- p.46 / Chapter 2.1.6. --- Materials for immunocytochemical staining --- p.46 / Chapter 2.1.7. --- Mast cell secretagogues --- p.47 / Chapter 2.1.8. --- Nitric oxide donors --- p.47 / Chapter 2.1.9. --- Soluble Guanylyl Cyclase activators and cGMP analogues --- p.47 / Chapter 2.1.10. --- Drugs involved in NO-sGC-cGMP pathway --- p.48 / Chapter 2.1.11. --- Materials for histamine assay --- p.48 / Chapter 2.1.12. --- Materials for Enzyme Immunosorbent Assay (EIA) --- p.49 / Chapter 2.1.13. --- Pro-inflammatory cytokines --- p.49 / Chapter 2.1.14. --- Materials for RNA extraction and RT-PCR --- p.49 / Chapter 2.1.15. --- Materials for Immunofluorescence staining --- p.50 / Chapter 2.1.16. --- Anti-asthmatic compounds --- p.51 / Chapter 2.1.17. --- Buffer and stock solution --- p.51 / Chapter 2.1.17.1. --- Buffer ingredients --- p.51 / Chapter 2.1.17.2. --- Stock solution --- p.52 / Chapter 2.2. --- Methods --- p.52 / Chapter 2.2.1. --- CD34+ cell isolation from human buffy coat --- p.52 / Chapter 2.2.2. --- CD34+ cell culture --- p.53 / Chapter 2.2.3. --- Human mast cell line (HMC-1 cells) culture --- p.54 / Chapter 2.2.4. --- Mast cell heterogeneity identification --- p.54 / Chapter 2.2.4.1. --- Cell smear preparation --- p.54 / Chapter 2.2.4.2. --- May-Gruwald-Giemsa staining --- p.55 / Chapter 2.2.4.3. --- Immunocytochemical staining --- p.55 / Chapter 2.2.5. --- Histamine release and measurement --- p.56 / Chapter 2.2.5.1. --- Histamine release --- p.56 / Chapter 2.2.5.2. --- Spectroflurometric determination of histamine content --- p.57 / Chapter 2.2.5.3. --- Calculation of histamine level --- p.57 / Chapter 2.2.6. --- Prostaglandin D2 (PGD2) measurement --- p.58 / Chapter 2.2.6.1. --- PGD2 production --- p.58 / Chapter 2.2.6.2. --- EIA methods for PGD2 measurement --- p.58 / Chapter 2.2.6.3. --- Calculation of PGD2 concentration --- p.59 / Chapter 2.2.7. --- Cysteinyl Leukotrienes (Cys-LTs) measurement --- p.59 / Chapter 2.2.7.1. --- Cys-LTs production --- p.59 / Chapter 2.2.7.2. --- EIA methods for Cys-LTs measurement --- p.60 / Chapter 2.2.7.3. --- Calculation of Cys-LTs concentration --- p.60 / Chapter 2.2.8. --- Tumor necrosis factor-alpha (TNF-α) measurement --- p.61 / Chapter 2.2.8.1. --- TNF-α production --- p.61 / Chapter 2.2.8.2. --- EIA methods for TNF-α measurement --- p.61 / Chapter 2.2.8.3. --- Calculation of TNF-α concentration --- p.62 / Chapter 2.2.9. --- Interleukin-8 (IL-8) measurement --- p.62 / Chapter 2.2.9.1. --- IL-8 production --- p.62 / Chapter 2.2.9.2. --- ELISA for IL-8 measurement --- p.62 / Chapter 2.2.9.3. --- Calculation of IL-8 concentration --- p.63 / Chapter 2.2.10. --- Data presentation --- p.63 / Chapter 2.2.11. --- Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) --- p.64 / Chapter 2.2.11.1. --- RNA extraction --- p.64 / Chapter 2.2.11.2. --- Reverse Transcriptase reaction for cDNA synthesis --- p.65 / Chapter 2.2.11.3. --- Polymerase Chain Reaction --- p.66 / Chapter 2.2.11.4. --- Agarose Gel Electrophoresis --- p.67 / Chapter 2.2.11.5. --- Data representation in RT-PCR experiment --- p.67 / Chapter 2.2.12. --- Immunofluorescence staining --- p.67 / Chapter 2.2.12.1. --- Cell smear preparation --- p.68 / Chapter 2.2.12.2. --- Immunofluorescence staining --- p.68 / Chapter 2.3. --- Statistical analysis --- p.69 / Chapter 3. --- Effect of Nitric Oxide Donors on Mast Cell Activation --- p.70 / Chapter 3.1. --- Introduction --- p.71 / Chapter 3.1.1. --- Mechanisms of NO release from NO donors --- p.71 / Chapter 3.1.2. --- Experimental aims --- p.77 / Chapter 3.2. --- Materials and methods --- p.77 / Chapter 3.3. --- Results --- p.78 / Chapter 3.3.1. --- Development of mast cells from buffy coat --- p.78 / Chapter 3.3.2. --- Morphological features of cultured mast cells --- p.78 / Chapter 3.3.3. --- Phenotype of cultured mast cells --- p.79 / Chapter 3.3.4. --- Effects of NO donors on immunologically stimulated mediators release --- p.79 / Chapter 3.3.4.1. --- SIN-1 and NOR-3 --- p.80 / Chapter 3.3.4.2. --- SNP and SNAP --- p.80 / Chapter 3.3.4.3. --- Diazeniumdiolates (NONOates) --- p.80 / Chapter 3.3.5. --- Effects of NO scavenger on NO donors mediated inhibition of immunologically stimulated mediators release --- p.82 / Chapter 3.3.6. --- Discussion --- p.83 / Chapter 4. --- Interaction between NO donors and pharmacological agentsin modulating mast cell activation --- p.123 / Chapter 4.1. --- Introduction --- p.124 / Chapter 4.1.1. --- Modulators of NO-sGC-cGMP pathway --- p.125 / Chapter 4.1.2. --- Anti-asthmatic compounds --- p.128 / Chapter 4.1.3. --- Experimental aims --- p.130 / Chapter 4.2. --- Materials and methods --- p.131 / Chapter 4.3. --- Results --- p.132 / Chapter 4.3.1. --- Effect of sGC activators on immunologically stimulated histamine release and the inhibitory action of DEA/NO --- p.132 / Chapter 4.3.2. --- Effect of cGMP analog on immunologically stimulated histamine release --- p.133 / Chapter 4.3.3. --- "Effects of the sGC inhibitor, ODQ, on DEA/NO induced inhibition on immunologically stimulated mediators release" --- p.134 / Chapter 4.3.4. --- Effects of anti-oxidants on the actions of NO donors in modulating immunologically stimulated mediators release --- p.134 / Chapter 4.3.5. --- The effects of NO donors on salbutamol mediated inhibition of immunologically stimulated histamine release from human mast cells --- p.135 / Chapter 4.3.6. --- The effects of NO donors on theophylline mediated inhibition of immunologically stimulated histamine release from human mast cells --- p.136 / Chapter 4.3.7. --- The effects of NO donors and DSCG on immunologically stimulated histamine release from human mast cells --- p.137 / Chapter 4.4. --- Discussion --- p.137 / Chapter 4.5. --- Further studies --- p.150 / Chapter 5. --- Human mast cells as a source of nitric oxide --- p.178 / Chapter 5.1. --- Introduction --- p.179 / Chapter 5.1.1. --- Nitric oxide synthases expression in mast cell --- p.180 / Chapter 5.1.2. --- Modulation of NOS expression --- p.182 / Chapter 5.1.3. --- Experimental aims --- p.186 / Chapter 5.2. --- Materials and methods --- p.186 / Chapter 5.3. --- Results --- p.187 / Chapter 5.3.1. --- NOS expression in human mast cell-line HMC-1 --- p.187 / Chapter 5.3.1.1. --- Basal --- p.187 / Chapter 5.3.1.2. --- Effect of cytokines --- p.188 / Chapter 5.3.2. --- NOS expression in cultured CD34+ derived human mast cells --- p.189 / Chapter 5.3.2.1. --- Basal --- p.189 / Chapter 5.3.2.2. --- Effect of cytokines --- p.189 / Chapter 5.3.2.3. --- Effect ofIgE and anti-IgE --- p.190 / Chapter 5.4. --- Discussion --- p.191 / Chapter 5.5. --- Further studies --- p.200 / Chapter 6. --- Conclusion --- p.218 / Chapter 7. --- References --- p.230

Chow, Yin Sau Joyce. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 127-140). / Abstract also in Chinese. / Abstract --- p.I / 摘要 --- p.V / Acknowledgements --- p.VIII / Publications --- p.X / Table of contents --- p.XII / Abbreviations --- p.XV / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter 1.1 --- Allergic diseases and allergic inflammation --- p.1 / Chapter 1.1.1 --- Allergic diseases and their prevalence --- p.1 / Chapter 1.1.2 --- Immunopathology of allergic inflammation --- p.2 / Chapter 1.2 --- Biology of human eosinophils --- p.4 / Chapter 1.2.1 --- Morphology --- p.4 / Chapter 1.2.2 --- Cell surface receptors and mediators --- p.4 / Chapter 1.2.3 --- Origin and development of eosinophils --- p.7 / Chapter 1.3 --- Eosinophils and allergic inflammation --- p.7 / Chapter 1.3.1 --- Adhesion molecules on eosinophils for emigration --- p.8 / Chapter 1.3.2 --- Eosinophil activation and inflammatory mediators --- p.13 / Chapter 1.3.3 --- Survival of eosinophils in allergic inflammation --- p.18 / Chapter 1.3.4 --- Immunopathological roles of eosinophils in allergic inflammation --- p.18 / Chapter 1.4 --- Intracellular signaling mechanisms --- p.20 / Chapter 1.4.1 --- Signal transduction pathways of eosinophils --- p.20 / Chapter 1.4.2 --- Inhibitors of signaling molecules --- p.26 / Chapter 1.5 --- Aim of study --- p.29 / Chapter Chapter 2: --- Materials and Methods --- p.31 / Chapter 2.1 --- Materials --- p.31 / Chapter 2.1.1 --- Human eosinophils --- p.31 / Chapter 2.1.2 --- Cell culture --- p.33 / Chapter 2.1.3 --- Cell surface and intracellular immunofluorescent staining --- p.36 / Chapter 2.1.4 --- Detection of cytokine and chemokine release --- p.39 / Chapter 2.1.5 --- Detection of cell viability and apoptosis --- p.40 / Chapter 2.1.6 --- Protein extraction --- p.40 / Chapter 2.1.7 --- Western blot analysis --- p.40 / Chapter 2.1.8 --- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.42 / Chapter 2.2 --- Methods --- p.45 / Chapter 2.2.1 --- Purification of human eosinophils --- p.45 / Chapter 2.2.2 --- Cell culture --- p.46 / Chapter 2.2.3 --- Cell surface and intracellular immunofluorescent staining --- p.47 / Chapter 2.2.4 --- Detection of cytokine and chemokine release --- p.48 / Chapter 2.2.5 --- Detection of cell viability and apoptoas --- p.49 / Chapter 2.2.6 --- Protein extraction --- p.49 / Chapter 2.2.7 --- Western blot analysis --- p.50 / Chapter 2.2.8 --- SDS-PAGE --- p.50 / Chapter 2.2.9 --- Statistical analysis --- p.51 / Chapter Chapter 3: --- Role of Novel IL-1 Family Cytokine in Allergic Inflammation: IL-33-mediated Eosinophil Activation --- p.52 / Chapter 3.1 --- Introduction --- p.52 / Chapter 3.2 --- Results --- p.54 / Chapter 3.2.1 --- "Total protein expression of IL-33 receptor, ST2, of human eosinophils" --- p.54 / Chapter 3.2.2 --- "Intracellular protein expression of IL-33 receptor, ST2，in human eosinophils" --- p.55 / Chapter 3.2.3 --- "Extracellular protein expression of IL-33 receptor, ST2, on human eosinophils" --- p.56 / Chapter 3.2.4 --- "Effects of IL-1β IL-18, and IL-33 on survival of human eosinophils" --- p.57 / Chapter 3.2.5 --- "Effects of IL-1β, IL-18, and DL-33 on surface adhesion molecule expression on human eosinophils" --- p.60 / Chapter 3.2.6 --- "Effects of IL-1β, IL-18, and IL-33 on chemokine and cytokine release from human eosinophils" --- p.64 / Chapter 3.2.7 --- "Synergistic effects of IL-1β, IH8, and IL-33 on IL-6 release from human eosinophils" --- p.69 / Chapter 3.2.8 --- "Effects of transcription and translation inhibitors on IL- 1β, IL-18, and IL-33-induced chemokine and cytokine release from human eosinophils" --- p.71 / Chapter 3.2.9 --- "Effects of different inhibitors on lL-1β, IL-18, and IL-33-induced survival enhancement of human eosinophils" --- p.75 / Chapter 3.2.10 --- Effects of different inhibitors on IL-1β and IL-33-mediated surface expression of adhesion molecules on human eosinophils --- p.77 / Chapter 3.2.11 --- "Effects of different inhibitors on IL-33, IL-1β，and IL-18-induced release of CCL2，CXCL8，and IL-6 from human eosinophils" --- p.79 / Chapter 3.2.12 --- "Effects of IL-33, IL-1β and IL-18 on activation of ERK, p38 MAPK, and NF-kB pathways in human eosinophils" --- p.83 / Chapter 3.3 --- Discussion --- p.85 / Chapter Chapter 4: --- Co-culture of Eosinophils & Epidermal Keratinocytes Upon IL-33 Stimulation: Implications for Immunopathology of Atopic Dermatitis --- p.95 / Chapter 4.1 --- Introduction --- p.95 / Chapter 4.2 --- Results --- p.98 / Chapter 4.2.1 --- Effect of IL-33 on surface expression of CD18 and ICAM-1 upon the interaction of human eosinophils and epidermal keratinocytes --- p.98 / Chapter 4.2.2 --- Effect of IL-33 on CCL2 release upon the interaction of human eosinophils and epidermal keratinocytes --- p.98 / Chapter 4.2.3 --- Effect of IL-33 on CXCL8 release upon the interaction of human eosinophils and epidermal keratinocytes --- p.101 / Chapter 4.2.4 --- Effect of IL-33 on IL-6 release upon the interaction of human eosinophils and epidermal keratinocytes --- p.101 / Chapter 4.2.5 --- Source(s) of CCL2 in co-culture of human eosinophils and epidermal keratinocytes upon IL-33 stimulation --- p.104 / Chapter 4.2.6 --- Source(s) of CXCL8 in co-culture of human eosinophils and epidermal keratinocytes upon IL-33 stimulation --- p.105 / Chapter 4.2.7 --- Source(s) of IL-6 in co-culture of human eosinophils and epidermal keratinocytes upon BL-33 stimulation --- p.108 / Chapter 4.2.8 --- "Effect of transwell insert on the induction of CCL2，CXCL8, and IL-6 release in co-culture of human eosinophils and epidermal keratinocytes upon IL-33 stimulation" --- p.110 / Chapter 4.3 --- Discussion --- p.113 / Chapter Chapter 5: --- Concluding Remarks and Future Perspectives --- p.120 / Chapter 5.1 --- Concluding Remarks --- p.120 / Chapter 5.2 --- Future Perspectives --- p.123 / Appendix --- p.126 / References --- p.127

Accumulating evidence has indicated that microbial infection could intensify allergic responses. Previous findings demonstrated that eosinophil activation could be elicited by bacterial and viral conserved molecular pattern through TLR. Recently, two cytoplasmic pattern recognition receptors, NLR protein NOD1 and NOD2, have been discovered and the important roles in innate immunity have been elucidated. Eosinophils alone have little responses upon the stimulation with ligands of NOD1 and NOD2. Since airway eosinophils increase in more numbers of asthmatic patients compared to control subjects, we investigated the co-culture system of eosinophils and human bronchial epithelial cells to illustrate the potential immunopathological roles of NOD1 and NOD2 in asthma processes. In the co-culture system, NOD1 ligand gamma-D-Glu-mDAP (iE-DAP) and NOD2 ligand muramyl dipeptide (MDP) could upregulate cell surface expression of CD1 8 and ICAM-1 on eosinophils and ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1) on bronchial epithelial cells, as well as induce chemokines CCL2 and CXCL8 release. These findings therefore imply the direct interaction and activation between the two cells upon NOD1 and NOD2 ligand stimulation. / Allergic diseases are prevalent and their incidences have been increasing worldwide. Eosinophils are the principal effector cells for the late phase response in allergic inflammation. The infiltration of eosinophils together with other inflammatory cells at the local inflammatory sites is the major characteristic in allergic inflammation. However, the detailed innnunopathological responses and mechanisms of the activation of eosinophils in allergic inflammation are not well defined. In the present study, we investigated and attempted to elucidate the mechanisms of eosinophil activation induced by various stimuli, including thymic stromal lymphopoietin (TSLP), the novel interleukin (IL)-12 family cytokine IL-27, and ligands of nucleotide-binding oligomerization domain (NOD) like receptor (NLR) protein NOD1 and NOD2 upon interaction with bronchial epithelial cells. / In conclusion, the above findings demonstrated that eosinophils could be potently activated by diverse stimuli and regulated by multiple intracellular regulatory mechanisms. The elucidation of eosinophil activation may offer new therapeutic stategies and clues for the treatment of allergic diseases. / Recently, the novel IL-12 family member IL-27 was found to regulate immune responses, exerting either stimulation or suppression effects. We found that eosinophils constitutively expressed IL-27 receptor heterodimer, gp130 and WSX-1. IL-27 could prolong eosinophil survival by reducing apoptosis, modulate the expression of adhesion molecules to facilitate eosinophil adhesion and accumulation, and induce the release of proinflammatory cytokines IL-6, tumor necrosis factor (TNF)-aalpha IL-1beta and chemokines CCL2, CXCL8 and CXCL1. The stimulatory effects of IL-27 on eosinophils could not be abrogated by IL-25, hematopoietic cytokine granulocyte-macrophage colony stimulating factor (GM-CSF) and toll-like receptor (TLR) 4 ligand lipopolysaccharide (LPS). These findings were different from the effects of IL-27 and LPS on monocytes. Intracellular signaling mechanistic studies showed that IL-27-mediated eosinophil activation was differentially regulated by MAPKs and NF-kappaB. Based on the above results, IL-27 could play crucial roles in allergic diseases by the activation of eosinophils via differential intracellular signaling cascades. However, IL-27 has been shown to suppress allergic diseases in mouse models. According to our findings of its activating effects on human eosinophils, IL-27 may play pleiotropic roles in human allergic responses. / TSLP is a novel IL-7-like cytokine highly expressed by bronchial epithelial cells and skin keratinocytes in allergic diseases. TSLP acts as a master switch for allergic inflammation through the activation of dendritic cells and mast cells for initiating inflammatory Th2 responses. To elucidate the immunological cascades of epithelium/keatinocyte-eosinophil mediated allergic inflammation, we examined the modulating effects of TSLP on human eosinophils. We observed that human eosinophils constitutively expressed TSLP receptor complex comprising TSLP-binding chain TSLPR and IL-7Ralpha chain. TSLP could significantly delay eosinophil apoptosis, up-regulate the cell surface expression of adhesion molecule CD18 and intercellular adhesion molecule-1 (ICAM-1) but down-regulate L-selectin, enhance eosinophil adhesion to fibronectin, and induce the release of inflammatory cytokine 1L-6 and chemokines CXCL8, CXCL1 and CCL2. All these effects were concentration-dependent and TSLP-specific. TSLP regulated the above effects through the activation of extracellular signal-regulated protein kinase (ERK), p38 mitogen activated protein kinase (MAPK) and nuclear factor (NF)-kappaB signaling pathway, but not signal transducer and activator of transcription (STAT)-5 and STAT-3 which were usually activated in other effector cells upon TSLP stimulation. Collectively, the above findings elucidated the pro-allergic mechanisms of TSLP via the activation of distinct intracellular signaling pathways in eosinophils. / Hu, Shuiqing. / Adviser: Wong Chin Kwok. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 176-216). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Airway bronchial epithelial cells play important roles in host defense, inflammation and regulation of immune responses. Activated bronchial epithelial cells are potent sources of a wide variety of soluble and cell-surface molecules that can alter the biological functions of inflammatory cells in the airways. Molecular mechanisms regulating the production of inflammatory mediators from bronchial epithelial cells remain to be fully elucidated. / All of the above findings suggest that human bronchial epithelial cells could be activated by a variety of stimuli in airway inflammatory reactions. Besides, different intracellular signaling pathways could regulate the activation of human bronchial epithelial cells in response to different stimuli. Our results therefore provide new insight into the molecular mechanisms involved in airway inflammatory diseases and may have important therapeutic implications. / Basophils are the accessory cell type required for T helper (Th)2 induction and initiators in IgE-mediated chronic allergic inflammation in response to allergens. Number of basophils and Th17 cells increases at the sites of allergic inflammation in the airways of allergic asthmatic patients. To elucidate the interaction among the activation of human bronchial epithelial cells, Th17 cells, and basophils, we investigated the activation effects of Th17 hallmark cytokine IL-17A on the human primary bronchial epithelial cells/BEAS-2B bronchial epithelial cells and human primary basophils/ KU812 basophilic cells. Human bronchial epithelial cells and basophils were cultured either together or separately in the presence or absence of IL-17A stimulation. Co-culture of human bronchial epithelial cells and basophils could significantly increase the release of inflammatory cytokine IL-6 and mononuclear chemoattractant protein-1 (MCP-1/CCL2), a chemokine for basophils, eosinophils and monocytes, while human bronchial epithelial cells were the main source for releasing IL-6 and CCL2. Such induction was synergistically enhanced upon the activation of IL-17A. The use of transwell inserts in the co-culture system demonstrated that the direct interaction between these two cell types was necessary for IL-6 and CCL2 release induced by IL-17A. Surface expression of intercellular adhesion molecule-1 (ICAM-1) on the human bronchial epithelial cells was also up-regulated upon their interaction. The interaction of human bronchial epithelial cells and basophils under IL-17A stimulation was differentially regulated by extracellular signal-regulated kinase (ERK), c-Jun N-terminal protein kinase (JNK), p38 mitogen activated protein kinase (MAPK) and nuclear factor (NF)-kappaB pathways. Our findings therefore suggest a novel immunopathological role of human Th17 cells and basophils in allergic asthma through the activation of granulocytes-mediated inflammation initiated by the direct interaction between human basophils and bronchial epithelial cells. / IL-27 is a novel member of the IL-6/IL-12 family cytokines that are produced early by antigen-presenting cells (APCs) during immune responses. IL-27 can drive the commitment of naive T cells to a Th1 phenotype and inhibit inflammation in later phases of infection. Recent evidence has suggested that human bronchial epithelial cells with the expression of IL-27 receptor complex are potential target cells of IL-27. Here we investigated the in vitro effects of IL-27, alone or in combination with inflammatory cytokine TNF-alpha on the pro-inflammatory activation of human bronchial epithelial cells, and the underlying intracellular signaling molecules were also studied. IL-27 was found to up-regulate ICAM-1 expression on the surface of human bronchial epithelial cells, and a synergistic effect was observed in the combined treatment of IL-27 and TNF-alpha on the surface expression of ICAM-1. Although IL-27 did not alter the basal IL-6 secretion from human bronchial epithelial cells, it could significantly enhance TNF-alpha-induced IL-6 production. The synergistic effects on the induction of ICAM-1 and IL-6 were partially due to the up-regulated expression of TNF-alpha receptor (p55TNFR) on the surface of human bronchial epithelial cells induced by IL-27. Further investigations showed that the enhanced production of ICAM-1 and IL-6 in human bronchial epithelial cells activated by IL-27 and TNF-alpha was differentially regulated by phosphatidylinositol 3-OH kinase (PI3K)-Akt, p38 MAPK and NF-kappaB pathways. Our study therefore suggests a potential role of IL-27 and TNF-alpha in the pathogenesis of airway infection or inflammatory diseases. / In the present study, we investigated the mechanisms of the activation of human bronchial epithelial cells induced by various stimuli including interleukin (IL)-17A, IL-27, tumor necrosis factor (TNF)-alpha and human basophils. The activation of human bronchial epithelial cells was studied in terms of the expression of cytokines, chemokines and adhesion molecules. Using intracellular staining with flow cytometry and selective pharmacological inhibitors, we further investigated the underlying intracellular signaling mechanisms regulating the activation of human bronchial epithelial cells. / Cao, Ju. / Advisers: Chun K. Wong; Christopher W. K. Lam. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 175-202). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.