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

No description available.

No description available.

No description available.

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.

Mast cells are granulocytes that require signaling from the receptor protein-tyrosine kinase Kit and its ligand stem cell factor (SCF) for their maturation and function. In addition to providing growth and survival signals, the Kit receptor is involved in crosstalk to β1 integrins leading to mast cell adhesion, spreading, and migration on fibronectin (FN). Previous studies reported the involvement of the non-receptor protein-tyrosine kinases Fps/Fes and Fer in signaling downstream of the high affinity IgE receptor in mast cells and identified cell migration defects in Fer-deficient bone marrow-derived mast cells (BMMCs). Fps/Fes also becomes phosphorylated downstream of the Kit receptor in BMMCs, and this involves the action of the Src family kinase Fyn as an upstream activator of Fps/Fes. In this study, the Fps/Fes SH2 domain was observed to bind the phosphorylated Kit receptor in vitro, suggesting that the SH2 domain plays a role in the activation mechanism of Fps/Fes. To investigate the function of Fps/Fes in Kit signaling, BMMCs were generated from wild-type and Fps/Fes-null mice. Analysis of downstream effectors revealed that Fps/Fes is required for maximal p38 MAPK signaling. Further examination of Fps/Fes-deficient BMMCs revealed increases in adhesion, spreading, and a defect in cell polarization on full-length FN (a ligand for multiple β1 integrins), compared to wild-type BMMCs. Similar phenotypes are observed using an α5β1 integrin-specific FN fragment (9-11) as the matrix. Reduced phosphorylation of the putative Fps/Fes substrate HS1 (a cortactin homologue involved in actin regulation) is observed in Fps/Fes-deficient BMMCs, compared to control cells, and this may contribute to the observed cytoskeletal defects. Restoring Fps/Fes expression in Fps/Fes-deficient BMMCs by retroviral transduction results in a rescue of cell spreading, polarization, and chemotaxis defects to levels similar to those of wild-type cells. This thesis provides novel insights into the potential mode of Fps/Fes activation downstream of the Kit receptor, and a role for Fps/Fes in regulating crosstalk between Kit and α5β1 integrins to promote cytoskeletal reorganization and motility of mast cells. / Thesis (Master, Biochemistry) -- Queen's University, 2009-01-28 15:05:04.056

No description available.

Mast cells (MC) are present in nearly all tissues in the body and participate in many physiological processes including allergy, tissue remodelling, fibrosis, angiogenesis, and autoimmunity. They can be activated by many stimuli, including allergic and innate immune stimulation. When activated, MC release mediators through which they can regulate inflammatory processes. Recently, we have discovered that rat and human MC express the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), the gene responsible for Cystic Fibrosis (CF). We showed that CFTR had functional activity in MC and its expression was differentially regulated by IFNg. In this thesis, we compared CFTR expression between MC and epithelial cells (EC) by Western blot analysis and found that CFTR expression in MC is similar to that in EC, but there are some differences which suggest either glycosylation or post-transcriptional/translational differences between MC and EC. We also explored the role of CFTR in human MC secretion from various cellular compartments, in response to various stimuli. When we blocked CFTR using pharmacological inhibitors, there was an inhibition of cAMP-dependent Cl- flux. Our data also shows that CFTR pharmacological inhibition had no effect on IgE/anti-IgE-mediated b-hexosaminidase or eicosanoid release from MC. When we stimulated MC with either IgE/anti-IgE or the adenosine receptor agonist NECA (3 uM) for 24h in the presence of CFTR inhibitors, secretion of several mediators appeared to be dysregulated including IL-8, MIF, IL-13, IL-16, PAI-1 and CCL1. To add to these findings, we also used short hairpin RNA (shRNA) to reduce CFTR expression in MC. CFTR deficient MC were unresponsive to NECA and showed reduced constitutive IL-6 secretion. Finally, we cultured MC from CF and non-CF donor peripheral blood progenitors and compared several phenotypic and functional aspects of the cells. We saw no difference in growth, protease content and surface marker expression between CF and non-CF MC, but stimulation of the cells with IgE/anti-IgE or Pseudomonas aeruginosa appeared to differentially induce cytokine synthesis and secretion from CF and non-CF MC. These findings suggest that MC function is dysregulated in CF and that CF MC may be involved in the pathophysiology of CF. / Experimental Medicine

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / The action of progesterone and estrogen on uterine mast cells of the ovariectomized rat was studied. A single injection of estradiol-17B (0.25 μg) produced a highly significant (P < O.OO1) reduction in both mesometrial and antimesometrial mast cell populations. The mean ± S.E. number of mesometrial and antimesometrial mast cells in the control animals was 19.2 ± 1.3 and 6.3 ± 0.7 respectively, while in the estrogen treated animals the respective values at 15 hours post treatment were 4.1 ± 0.5 and 2.9 ± 0.4. Estrogen treatment also resulted in considerable degranulation of the mast cells. In comparison, progesterone, when administered as a single injection (5 mg) did not produce a drastic reduction of the mast cells. Fifteen hours after its administration, progesterone had produced only a moderate reduction of the mast cell population; however, the decline was significantly smaller than those values obtained at the same time interval following estrogen treatment. Since it is known that progesterone treatment for at least 48 hours, followed by estrogen constitute the basic hormonal sequence for decidualizationin the rat, experiments were designed to study possible relationship between mast cell population and deciduoma development. Results obtained from these experiments demonstrate quite clearly that maximal decidual response was possible only among animals treated over a 48 hour period with progesterone (5 mg/ 24 hours) followed by a small (0.25 μg) injection of estradiol-17B and then traumatized 15 hours later (at a time when mast cell population was reduced to the lowest level). Thus, the hormonal treatment which resulted in the lowest level of mast cell numbers also permitted the largest deciduoma development. Shelesnyak (1957) proposed that histamine play a vital role in decidualization in the rat. On the other hand, it has been shown that mast cell degranulation with the accompanying loss of metachromasia is related to histamine release (Thon, 1967). In order to evaluate the role of estrogen as opposed to that of histamine in decidualization, animals were treated with estrogen and progesterone in addition to an estrogen antagonist -CN-55,945-27. The data from these experiments indicated only a moderate decidual response among the treated animals. In addition, the mast cell population in the uterine wall of these animals, at the time of traumatization, was considerably reduced and degranulated. Thus, uninterrupted estrogen action seems to be necessary for the establishment of sensitivity for maximal deciduoma development. In another set of investigations, uterine mast cells were depleted by administration of compound 48/80. Animals depleted of their mast cells and then treated with progesterone, estrogen, followed by trauma developed only small to moderate deciduomata. However, when rats were allowed to recover for seven days (at which time over 50% of the mast cells had reappeared) and then given the treatment as the preceding group, massive full length deciduoma were produced. The evidence suggests that maximal uterine sensitivity to decidualization is possible only after adequate hormonal treatment, and only in uteri not depleted of mast cells. / 2031-01-01