Activation of the cannabinoid receptor CB₂ has been shown to induce directed leukocyte migration and inhibit leukocyte chemotaxis towards CC chemokines. However, the role that CB₂ plays in regulating macrophage chemotaxis remains understudied. Using a real-time chemotaxis assay and a panel of chemically diverse CB₂ agonists, I set out to examine whether CB₂ modulates primary macrophage chemotaxis. Of 14 agonists tested, only a subset acted as bona fide macrophage chemoattractants. Surprisingly, despite being pertussis toxin-sensitive, neither pharmacological inhibition nor genetic ablation of CB₂ had any effect on CB₂ agonist-induced macrophage chemotaxis. Furthermore, the activation of CB2 had no effect on CCL2 or CCL5- induced macrophage chemotaxis. Therefore, the activation of CB2 does not inhibit CC chemokine-induced macrophage migration and a non-CB₁/CB₂, G_i/o-coupled GPCR must transduce CB2 agonist-induced macrophage chemotaxis. To identify the GPCR responsible, I examined primary murine macrophage GPCR expression and found that they express 124 non-sensory GPCRs. Functional screening of candidate receptors demonstrated that the putative cannabinoid receptors GPR18 and GPR55 and the lipid binding GPCRs LPAR1&5, CYSLTR1&2 and GPER1, were not responsible for CB₂ agonist-induced macrophage chemotaxis. Alongside, a ligand-directed virtual screen, combined with functional testing, uncovered a novel chemotaxis positive chemical scaffold. Importantly, compounds in this series containing a photoaffinity label retained activity and will aid in the identification of the target(s) responsible for CB₂ agonist-induced macrophage chemotaxis in future photocrosslinking experiments. Finally, I assessed whether CB2 controls innate immune cell recruitment in vivo using the zymosan-induced dorsal air pouch inflammation model and animals genetically deleted for CB₂. I found that CB₂^-/- mice had increased air pouch neutrophil and monocyte numbers, as well as pro-inflammatory mediators, during the acute inflammatory phase. Interestingly, mixed bone marrow chimera experiments demonstrated that lack of CB₂ specifically in the myeloid population is responsible for increased neutrophil trafficking. Therefore these data demonstrate that CB₂ acts to regulate neutrophil recruitment during the acute inflammatory response.

Thesis (M.Sc.)--Victoria University of Wellington, 2009. / "Malaghan Institute of Medical Research." Includes bibliographical references.

No description available.

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

Ho, Wing-Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 115-131). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abbreviations --- p.iii / Abstract --- p.v / 摘要 --- p.ix / Publications --- p.xii / Table of Contents --- p.xiv / Chapter 1. --- General Introduction / Chapter 1.1. --- Diabetes Mellitus (DM) and Diabetic Nephropathy --- p.1 / Chapter 1.1.1. --- "Prevalence, Diagnosis and Classification of DM" --- p.1 / Chapter 1.1.2. --- Type 2 DM and its Complications: Diabetic Nephropathy --- p.5 / Chapter 1.1.3. --- Diagnosis and Impacts of Diabetic Nephropathy --- p.7 / Chapter 1.1.4. --- Current Treatment of Type 2 DM and Diabetic Nephropathy --- p.8 / Chapter 1.2. --- Cytokines and Chemokines --- p.9 / Chapter 1.2.1. --- Types and Properties --- p.9 / Chapter 1.2.2. --- Cytokines and chemokines in Type 2 DM and Diabetic Nephropathy --- p.13 / Chapter 1.3. --- T Lymphocyte Costimulatory Molecules --- p.15 / Chapter 1.3.1. --- Types and Properties --- p.15 / Chapter 1.3.2. --- T Lymphocyte Costimulatory Molecules in Type 2 DM and Diabetic Nephropathy --- p.16 / Chapter 1.4. --- Adhesion Molecules --- p.18 / Chapter 1.4.1. --- Types and Properties --- p.18 / Chapter 1.4.2. --- Adhesion Molecules in Type 2 DM and Diabetic Nephropathy --- p.20 / Chapter 1.5. --- Intracellular Signaling Pathways --- p.21 / Chapter 1.5.1. --- Types and Properties --- p.21 / Chapter 1.5.2. --- Intracellular Signaling Pathways in Type 2 DM and Diabetic Nephropathy --- p.23 / Chapter 1.6. --- Objectives of Our Study --- p.24 / Chapter 2. --- Materials and Methods / Chapter 2.1. --- Materials --- p.26 / Chapter 2.1.1. --- "Patients, Control Subjects and Blood Samples" --- p.26 / Chapter 2.1.2. --- Cell Line --- p.27 / Chapter 2.1.3. --- "Cell Culture Media, Buffers and Other Reagents" --- p.28 / Chapter 2.1.4. --- "Recombinant Human Cytokines, Inhibitors and Other Stimulators" --- p.30 / Chapter 2.1.5. --- Reagents and Buffers for Flow Cytometric Analysis --- p.31 / Chapter 2.1.5.1. --- Cytometric Bead Array (CBA) of Cytokines and Chemokines --- p.33 / Chapter 2.1.5.2. --- Multiplex Fluorescent Bead Immunoassay (FBI) of Soluble Adhesion Molecules --- p.33 / Chapter 2.1.5.3. --- Phosphorylation State Analysis of Signaling Molecules --- p.34 / Chapter 2.1.5.4. --- Immunofluorescent Staining of Cell Surface Molecules --- p.36 / Chapter 2.1.6. --- Reagents and Buffers for Protein Array Analysis --- p.37 / Chapter 2.1.7. --- "Reagents and Buffers for 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenylytetrazolium Bromide (MTT) Assay" --- p.37 / Chapter 2.1.8. --- Reagents for Human Enzyme-Linked Immunosorbent Assay (ELISA) --- p.37 / Chapter 2.2. --- Methods --- p.38 / Chapter 2.2.1. --- Whole Blood Culture Experiments --- p.38 / Chapter 2.2.2. --- "Collection of Serum and Plasma, and Purification of PBMC from EDTA-Blood" --- p.39 / Chapter 2.2.3. --- HK-2 Cell Cultures --- p.39 / Chapter 2.2.4. --- HK-2 Cell Treatments --- p.40 / Chapter 2.2.5. --- Flow Cytometric Analysis --- p.41 / Chapter 2.2.5.1. --- CBA of Cytokines and Chemokines --- p.41 / Chapter 2.2.5.2. --- Multiplex FBI of Soluble Adhesion Molecules --- p.41 / Chapter 2.2.5.3. --- Phosphorylation State Analysis of Signaling Molecules --- p.42 / Chapter 2.2.5.4. --- Immunofluorescent Staining of Cell Surface Molecules --- p.43 / Chapter 2.2.6. --- Protein Array Analysis --- p.44 / Chapter 2.2.7. --- MTT Assay --- p.44 / Chapter 2.2.8. --- ELISA --- p.45 / Chapter 2.2.9. --- Statistical Analysis --- p.46 / Chapter 3. --- "Clinical Study on the Expressions of Cytokines, Chemokines, Co-stimulatory Molecules, Phosphorylated Signaling Molecules in Patients with Diabetic Nephropathy" / Chapter 3.1. --- Introduction --- p.47 / Chapter 3.2. --- Results --- p.49 / Chapter 3.2.1. --- Demographic Data of Participants --- p.49 / Chapter 3.2.2. --- Expression Profile in Plasma of Patients --- p.49 / Chapter 3.2.2.1. --- Cytokines and Chemokines --- p.49 / Chapter 3.2.2.2. --- Soluble Costimulatory Molecules --- p.55 / Chapter 3.2.2.3. --- Soluble Adhesion Molecules --- p.55 / Chapter 3.2.2.4. --- "Correlations between Plasma Concentrations of Cytokines, Chemokines, soluble Costimulatory Molecules and soluble Adhesion Molecules and UACR in Patients" --- p.60 / Chapter 3.2.3. --- Effects ofTNF-α and IL-18 on the ex vivo Production from Whole Blood of Patients --- p.65 / Chapter 3.2.3.1. --- Ex vivo Production of Cytokines and Chemokines --- p.65 / Chapter 3.2.3.2. --- Ex vivo Production of Soluble Costimulatory Molecules --- p.70 / Chapter 3.2.4. --- "Expression of Phosphorylated p38 MAPK, JNK and ERK in PBMC of Patients" --- p.73 / Chapter 3.3. --- Discussion --- p.77 / Chapter 3.3.1. --- "Cytokines, Chemokines and Diabetic Nephropathy" --- p.77 / Chapter 3.3.2. --- Soluble Costimulatory Molecules and Diabetic Nephropathy --- p.80 / Chapter 3.3.3. --- Soluble Adhesion Molecules and Diabetic Nephropathy --- p.83 / Chapter 3.3.4. --- Intracellular Signaling and Diabetic Nephropathy --- p.87 / Chapter 4. --- In vitro Study on the Signal Transduction Mechanism Regulating the Expression of CCL2 and Cell Surface Adhesion Molecules in Tumour Necrosis Factor (TNF)-α-Stimulated HK-2 Cells / Chapter 4.1. --- Introduction --- p.90 / Chapter 4.2. --- Results --- p.93 / Chapter 4.2.1. --- Expression Profile of Cytokines and Chemokines of TNF-α-activated HK-2 Cells --- p.93 / Chapter 4.2.2. --- "TNF-α Upregulated CCL2, ICAM-1 and VCAM-1 Expression in HK-2 Cells" --- p.95 / Chapter 4.2.3. --- "TNF-α Activated the p38 MAPK, JNK and ERK Signaling Pathways in HK-2 Cells" --- p.96 / Chapter 4.2.4. --- Cytotoxicity of MAPK Inhibitors --- p.96 / Chapter 4.2.5. --- "Effects of p38 MAPK, JNK and ERK Inhibitors on TNF-α-induced Expressions of CCL2, ICAM-1 and VCAM-1" --- p.100 / Chapter 4.3. --- Discussion --- p.102 / Chapter 5. --- Conclusion and Future Prospects / Chapter 5.1. --- Conclusion --- p.107 / Chapter 5.2. --- Future Prospects --- p.111 / References --- p.115