Thesis (doctoral)--Ume̊a Universitet, 1982. / Extra t.p. with thesis statement inserted. eContent provider-neutral record in process. Description based on print version record. Includes bibliographies.

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Background: Mast cells are known to play a role in the pathophysiology of asthma. Determining their contribution to the development of asthma symptoms has been difficult as they remain tissue-resident and do not usually migrate into the airway lumen for detection using expectorated sputum. Objectives: We investigated the presence and activity of mast cells in the blood and sputum of healthy controls and patients with moderate to severe asthma and the relationship with clinical characteristics of asthma and their associated microenvironment. Methods: Cell-free sputum supernatant was used to detect levels of soluble tryptase and T2 and non-T2 cytokines by ELISA. RNA/cDNA isolated from sputum cells measured expression levels of eosinophil and mast cell-specific genes by digital PCR. Relevant clinical characteristics and measurements of lung function, airway hyperresponsiveness, FeNO, blood eosinophils, IgE and tryptase were collected. Results: Tryptase was detectable in the fluid phase portions of sputum, irrespective of the inflammation based on the differential cell count, and was significantly different than healthy controls. Eosinophil and mast cell-specific genes were detected in sputum cells at levels significantly different than healthy controls. Sputum tryptase was not associated with any phenotype or severity of asthma but identified some associations with clinical characteristics. It is associated with a unique cytokine signature. Conclusion: Differences were seen between eosinophilic and non-eosinophilic phenotypes in sputum supernatant and sputum cells. Eosinophil and mast cell-specific genes were detected in sputum cells but were not associated with asthma severity. Greater levels of cytokines IL-4 and IL-13 in sputum, suggest presence of mast cells in the airway epithelium that contribute to mucus secretion observed in patients with uncontrolled symptoms of asthma. Further investigations hope to identify the relationship of mast cells with the quantification of mucus in these patients to understand and confirm those with a predominant mast cell component. / Thesis / Master of Science (MSc) / In patients with asthma, mast cells are one of the inflammatory cells normally associated with allergies however, they also function by non-allergic mechanisms (bacteria, viruses, fungus, pollutants). Inflammatory cells cause swelling of the bronchial tubes, tightening/spasm of the bronchial muscles, and mucus production. The level of inflammation is measured in lung fluids such as sputum, but the mast cell remains in the lung tissue and therefore difficult to detect. When mast cells are activated they release mediators such as tryptase, that travel through the lung tissue. Tryptase was detected in the sputum of patients with moderate to severe asthma, with levels significantly greater than in healthy people. The level of sputum tryptase was independent of other inflammatory cells such as eosinophils, and was associated with biomarkers related to excess secretion of mucus. Further research hopes to understand if the mucus is related to the activity of the mast cell.

Histamine is present in the central nervous system and is believed to be derived from neurons (50 percent) and mast cells (50 percent). This experiment was designed to analyze histologically the numbers and distribution of brain-associated mast cells in normal (+/+), mast cell deficient (W/W^v) and heterozygote (W/+, W^v/+) mice of the WBB6F_1 /J strain. Significant variations in the number and distribution of mast cells between the various genotypes were found. Based on the results, a hypothesis is proposed to account for the observed genotypical differences in mast cell numbers and distribution. Based on the total number of mast cells and the content of histamine in a typical mast cell, it is apparent that the mast cell is not a major source of brain histamine, suggesting that another non-neuronal pool of histamine must be present in the brain.

肥大細胞是過敏和炎症的主要效應細胞，其激活機制包括了IgE依賴性和非IgE依賴性的激活。IgE依賴性激活是指抗原與IgE的高親和力受體FcεRI上的IgE結合，促使FcεRI受體交聯而引起變態反應。其它的肥大細胞促分泌素如神經肽P物質，能夠激活百日咳毒素（PTX）敏感性的G蛋白而介導非IgE依賴性的細胞激活。最近的研究指出，肥大細胞表達Toll樣受體家族，提示肥大細胞也積極參與固有免疫反應。本研究主要探討Toll樣受體2激動劑肽聚糖（PGN）和合成激動劑Pam3CSK4對人類肥大細胞的影響，及其對抗原和P物質引起的肥大細胞激活的調控。 / Toll樣受體2激動劑本身不引起人類肥大細胞脫顆粒，但抑制抗原和P物質引起的肥大細胞脫顆粒。鈣動員是引起肥大細胞脫顆粒的關鍵因素。Pam3CSK4通過抑制抗原和P物質鈣動員來抑制肥大細胞脫顆粒。PGN只抑制抗原鈣動員，卻對P物質沒有影響。 / PGN和Pam3CSK4皆刺激人類肥大細胞釋放白細胞介素8(IL-8)和腫瘤壞死因子α（TNF-α）。Pam3CSK4通過激活G₀蛋白，Erk，Ca²⁺/calcineurin/NFAT和TAK信號通路引起肥大細胞釋放IL-8。其間，Go蛋白的激活介導Erk和Ca²⁺/calcineurin/NFAT信號通路的活化。與Pam3CSK4不同，PGN通過激活JNK, Erk, PI3K和TAK信號通路引起肥大細胞釋放IL-8。此外，雖然PTX敏感性G蛋白不影響PGN刺激引起的IL-8釋放，它卻抑制PGN刺激引起的Erk激活。 / Pam3CSK4與抗原協同作用刺激肥大細胞釋放IL-8和TNF-α，PGN與抗原卻並無協同作用。PGN與P物質協同作用刺激肥大細胞釋放IL-8和TNF-α，Pam3CSK4卻幹擾P物質的作用。在Pam3CSK4與抗原的協同作用中，Erk，Ca²⁺/calcineurin/NFAT和TAK信號通路起重要作用。PGN與P物質的協同作用則通過Erk, Ca²⁺/calcineurin/NFAT，NF-κB，PI3K和TAK這五條信號通路。 / 本研究表明，不同的Toll樣受體2激動劑能通過不同的作用機制介導和調控人類肥大細胞的反應。同時，我們首次發現G₀蛋白參與人類肥大細胞Toll樣受體2信號的激活。由於Toll樣受體2與感染和炎症息息相關，繼續研究Toll樣受體2激活對人類肥大細胞的調控機制，有助於促進開發抗感染和炎症藥物，意義深遠。 / Mast cells are activated by IgE-dependent and -independent mechanisms and play a pivotal role in both allergic and inflammatory responses. The classical IgE-dependent mechanism involves the binding of antigens to the receptor-bound IgE and crosslinking of the high-affinity receptor for IgE (FcεRI). For the poly-basic secretagogues, such as the neuropeptide substance P, they can directly stimulate pertussis toxin (PTX)-sensitive G proteins in mast cells in an IgE-independent manner. Recent studies also discover the expression of the Toll-like receptors on mast cells, indicating that mast cells are active players in innate immunity against a wide variety of pathogens. In this study, we investigated the effects of Toll-like receptor 2 (TLR2) ligands peptidoglycan (PGN) and Pam3CSK4 on human mast cell line LAD2 cells activation and the modulatory effects of these TLR2 ligands on LAD2 cells activities in response to anti-IgE and substance P. / TLR2 ligands did not cause significant degranulation on their own, but inhibited anti-IgE and substance P induced degranulation. Pam3CSK4 acted through TLR2, while the inhibitory effect of PGN involved other non-TLR2 related mechanisms. Pretreatment of Pam3CSK4 inhibited calcium mobilization induced by anti-IgE and substance P. However, pretreatment of PGN only inhibited calcium mobilization induced by anti-IgE, but failed to demonstrate similar effect on substance P. / Both TLR2 ligands triggered the release of IL-8 and TNF-α from LAD2 cells in TLR2-dependent manner. G protein, MAPKs, Ca²⁺/calcineurin/NFAT, PI3K/Akt and TAK pathways were differentially activated by PGN and Pam3CSK4. Release of IL-8 induced by Pam3CSK4 required the involvement of G₀ protein, Erk, Ca²⁺/calcineurin/ NFAT and TAK signaling pathways, but not PI3K/Akt and NF-κB. Meanwhile, G₀ protein was required for the upstream regulation of Erk and Ca²⁺/calcineurin/NFAT signaling cascades activated by Pam3CSK4. In contrast to Pam3CSK4, IL-8 release induced by PGN required the activation of JNK, Erk, PI3K and TAK signaling pathways, but not Ca²⁺ /calcineurin/NFAT and NF-κB. PTX-sensitive Gi/o protein was also involved in PGN induced Erk phosphorylation without influencing IL-8 release. / Pam3CSK4 acted in synergy with anti-IgE to augment the release of IL-8 and TNF-α, but PGN failed to demonstrate similar effect. In contrast, PGN acted in synergy with substance P, while co-stimulation of Pam3CSK4 with substance P failed to demonstrate similar synergism. Erk, Ca²⁺/calcineurin/NFAT and TAK signaling pathways were required for the synergistic action of Pam3CSK4 combined with anti-IgE, while synergistic release of IL-8 induced by PGN and substance P required the activation of Ca²⁺/calcineurin/NFAT, Erk, NF-κB, PI3K, and TAK signaling networks and was enhanced by Ca²⁺/calcineurin/NFAT and NF-κB signaling cascades in LAD2 cells, although NF-κB was not required for IL-8 release induced by PGN or substance P. / These ndings suggest that activation of human mast cells LAD2 can be differentially modified by different TLR2 ligands via distinct signaling pathways. We identify for the first time the involvement of G₀ protein in TLR2 signaling transduction in human mast cells. Further studies of the regulation of mast cells by Toll-like receptors will provide important opportunities for the therapeutic manipulation of infection and allergic diseases. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Yu, Yangyang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 205-233). / Abstract also in Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iv / Acknowledgements --- p.vi / Publication --- p.vii / Abbreviations --- p.viii / Contents --- p.x / Chapter 1 --- Introduction --- p.1 / Origin of mast cells --- p.1 / Cytokines and growth factors required for mast cells development --- p.3 / Mediators release from mast cell --- p.7 / Mast cells activation by classical IgE-dependent pathway --- p.13 / Substance P and mast cells --- p.20 / Mast cells in host defense --- p.23 / Toll-like receptors and mast cells --- p.25 / Aims --- p.31 / Chapter 2 --- Materials and Methods --- p.33 / Materials --- p.33 / Methods --- p.42 / LAD2 mast cells culture --- p.42 / Degranulation assay --- p.43 / IL-8 and TNF-α measurement --- p.44 / Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) --- p.44 / Western Blotting --- p.46 / Calcium mobilization assay --- p.47 / Flow cytometry assay --- p.48 / siRNA Transfection --- p.48 / Statistical analysis --- p.49 / Chapter 3 --- Functional Studies of Toll-Like Receptor 2 on Human Mast Cells Activation --- p.51 / Experimental conditions --- p.56 / Results --- p.57 / Discussions --- p.62 / Chapter 4 --- Modulatory Effects of Toll-Like Receptor 2 on Human Mast Cells in Response to Anti-IgE and the Signaling Pathways Involved in the Events --- p.80 / Experimental conditions --- p.92 / Results --- p.93 / Discussions --- p.102 / Chapter 5 --- Modulatory Effects of Toll-Like Receptor 2 on Human Mast Cells Activation in Response to Substance P and Signaling Pathways Involved in the Event --- p.136 / Experimental conditions --- p.140 / Results --- p.141 / Discussions --- p.152 / Chapter 6 --- General Discussion --- p.188 / Chapter 7 --- References --- p.205

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The chemokine receptor CXCR3, which has three known variants (CXCR3-A, CXCR3-B and CXCR3-Alt), has been implicated in the recruitment of mast cells to tissues in many different chronic diseases with its agonists found in elevated levels in many pulmonary diseases. All three variants of CXCR3 were detected in cord blood-derived mast cells at the mRNA level. Using an antibody that is unable to distinguish individual CXCR3 isoforms, we detected a marked down-regulation of intracellular protein during maturation from progenitor cells, with no concomitant changes in the modest surface expression of CXCR3. The known CXCR3 agonists CXCL9, CXCL10 and CXCL11 as well as the reported CXCR3-B agonist CXCL4, were able to induce Akt and ERK1/2 phosphorylation, as well as partial degranulation. Responses to all agonists were inhibited by pre-treatment with selective CXCR3 antagonists and pertussis toxin. Use of novel isoform-selective inhibitors indicates that the p110 isoform of PI3K is required for degranulation and signalling responses to CXCR3 agonists. Unexpectedly, dual (but not individual) isoform inhibition of the class I  and  isoforms substantially inhibited signalling and degranulation responses, indicating a hitherto unrecognised synergy between these isoforms, which provide a conduit for CXCR3 signalling in mast cells.