Adhesion Of Human Mast Cells Research Articles

Hypoxia modulates human mast cell adhesion to hyaluronic acid

Hypoxia is an inherent factor in the inflammatory process and is important in the regulation of some immune cell functions, including the expression of mast cell pro- and anti-inflammatory mediators. Hypoxia also influences cell adhesion to the extracellular matrix (ECM). Hyaluronic acid is one of the major components of the ECM that is involved in inflammatory and tissue regeneration processes in which mast cells play a prominent role. This prompted us to investigate the effects of hypoxia on the expression of hyaluronic acid receptors in mast cells and mast cell adhesion to this ECM component. We found that human LAD2 mast cells spontaneously adhered to hyaluronic acid in a CD44-dependent manner and that reduced oxygen concentrations inhibited or even completely abolished this adhesion process. The mechanism of hypoxia downregulation of mast cell adhesion to hyaluronic acid did not involve a decrease in CD44 expression and hyaluronidase-mediated degradation of adhesion substrates but rather conformational changes in the avidity of CD44 to hyaluronic acid. Hypoxia-mediated regulation of mast cell adhesion to extracellular matrix components might be involved in the pathogenic accumulation of mast cells observed in the course of certain diseases including rheumatoid arthritis and cancer.

Hypoxia regulates human mast cell adhesion to fibronectin via the PI3K/AKT signaling pathway

ABSTRACT A decrease in oxygen concentration is a hallmark of inflammatory reactions resulting from infection or homeostasis disorders. Mast cells interact with extracellular matrix and other cells by adhesion receptors. We investigated the effect of hypoxia on integrin-mediated mast cell adhesion to fibronectin. We found that it was mediated by the α5/β1 receptor and that hypoxia significantly upregulated this process. Hypoxia-mediated increases in mast cell adhesion occurred without increased surface expression of integrins, suggesting regulation by inside-out integrin signaling. Hypoxia also mediated an increase in phosphorylation of Akt, and PI3’kinase inhibitors abolished hypoxia-mediated mast cell adhesion. Hypoxia upregulates the function of integrin receptors by PI3’ kinase-dependent signaling. This process might be important for the location of mast cells at inflammatory sites

The Novel Receptor C5aR2 Is Required for C5a-Mediated Human Mast Cell Adhesion, Migration, and Proinflammatory Mediator Production.

C5a generated during complement activation possesses proinflammatory and immunoregulatory properties critical for the development and modulation of allergic immune responses. In immune cells, C5a mediates its effects through binding to two G protein-coupled receptors, C5aR1 and C5aR2. Mast cells are key effectors in allergic reactions, and decades of research have suggested that the majority of C5a effects on mast cells are mediated through C5aR1, whereas the expression and function of C5aR2 have not been explored. We demonstrated that the human mast cell line Laboratory of Allergic Diseases 2 (LAD2) expresses surface C5aR2 but not C5aR1, whereas CD34(+) cell-derived primary mast cells do not express surface C5aR1 or C5aR2. Stem cell factor and IL-4 upregulated C5aR2 expression on LAD2 cells. Furthermore, C5a caused internalization of LAD2 cell-surface C5aR2. We therefore used LAD2 cells as a model to study C5a/C5aR2-induced biological responses and signaling in human mast cells. We found that whereas C5a was unable to induce degranulation, it stimulated GM-CSF, TNF, CXCL10, and CCL2 production. C5a caused ERK phosphorylation, a signaling molecule important in cytokine and chemokine generation. In addition, C5a stimulated adhesion and chemotaxis of mast cells. Wortmannin, an inhibitor of PI3K, and small interfering RNA against β-arrestin-2 blocked C5a-induced adhesion. Silencing of C5aR2 using lentiviral short hairpin RNA rendered the cells unresponsive to C5a-induced adhesion, chemotaxis, and mediator release, as well as ERK phosphorylation. Overall, this study reveals a novel role for C5aR2 in C5a-mediated activation of mast cells and demonstrates that C5aR2 ligation initiates a β-arrestin-2-, PI3K-, and ERK-dependent signaling pathway in these cells.

Evidence for a novel Kit adhesion domain mediating human mast cell adhesion to structural airway cells.

BackgroundHuman lung mast cells (HLMCs) infiltrate the airway epithelium and airway smooth muscle (ASM) in asthmatic airways. The mechanism of HLMC adhesion to both cell types is only partly defined, and adhesion is not inhibited by function-blocking anti-Kit and anti-stem cell factor (SCF) antibodies. Our aim was to identify adhesion molecules expressed by human mast cells that mediate adhesion to human ASM cells (HASMCs) and human airway epithelial cells.MethodsWe used phage-display to isolate single chain Fv (scFv) antibodies with adhesion-blocking properties from rabbits immunised with HLMC and HMC-1 membrane proteins.ResultsPost-immune rabbit serum labelled HLMCs in flow cytometry and inhibited their adhesion to human BEAS-2B epithelial cells. Mast cell-specific scFvs were identified which labelled mast cells but not Jurkat cells by flow cytometry. Of these, one scFv (A1) consistently inhibited mast cell adhesion to HASMCs and BEAS-2B epithelial cells by about 30 %. A1 immunoprecipitated Kit (CD117) from HMC-1 lysates and bound to a human Kit-expressing mouse mast cell line, but did not interfere with SCF-dependent Kit signalling.ConclusionKit contributes to human mast cell adhesion to human airway epithelial cells and HASMCs, but may utilise a previously unidentified adhesion domain that lies outside the SCF binding site. Targeting this adhesion pathway might offer a novel approach for the inhibition of mast cell interactions with structural airway cells, without detrimental effects on Kit signalling in other tissues.

Differential Regulation of Adaptive and Innate Immunity activated Human Mast Cell by Osteopontin

Osteopontin (OPN), a RGD‐containing protein which has been shown to enhance inflammatory responses by modulating different functions of different cell types such as macrophages, dendritic cells and natural killer cells. However, OPN has also been shown to suppress inflammatory responses and is important in the healing process. To understand more about OPN, anti‐IgE and PGN activated human mast cells developed from human CD34+ stem cells were employed to study the role of this protein in the inflammatory responses. The release of histamine and cytokines (IL‐5, IL‐8 and TNF‐α) releases as well as cell adhesion were studied by spectro‐fluorescence assay, ELISA and CyQUANT assay respectively. OPN induced adhesion of human mast cells after anti‐IgE and PGN stimulation, and the adhesion was able to inhibit anti‐IgE‐induced IL‐5, IL‐8 and TNF‐α release while only PGN‐induced IL‐5 was enhanced by OPN. To investigate the interaction between OPN and human mast cells, cyclic RGD (cRGD) and neutralizing antibodies against integrin αVβ3 and CD44 were employed. cRGD was able to inhibit both anti‐IgE and PGN‐activated adhesion and adhesion‐mediated regulation of cytokines release. While only antibody against Integrin αVβ3 was able to reverse the anti‐IgE‐induced adhesion and the inhibition of anti‐IgE‐mediated cytokines release by OPN. In conclusion, OPN suppresses anti‐IgE‐induced inflammatory response by inhibiting the synthesis of chemokine (IL‐8) and both Th1 (TNF‐α) and Th2 (IL‐5) cytokine and the interaction is through the RGD domain in OPN and integrin αVβ3 expressed on human mast cells. On the other hand, OPN enhances Th2 (IL‐5) synthesis when human mast cells are stimulated by PGN.Research Support: RGC Grant 469310

Activation of toll-like receptor 3 (TLR3) inhibits human mast cell adhesion to extra cellular matrix proteins and inhibits adhesion-dependent degranulation

Activation of toll-like receptor 3 (TLR3) inhibits human mast cell adhesion to extra cellular matrix proteins and inhibits adhesion-dependent degranulation

STI571 inhibits growth and adhesion of human mast cells in culture.

Stem cell factor (SCF)/c-kit system is critical for human mast cell development. We thus examined the effects of STI571, an inhibitor of the c-kit tyrosine kinase receptor, on the proliferation and function of human mast cells. STI571 at concentrations of 10(-6) M or higher almost completely abolished the SCF-dependent progeny generation from cord blood-derived cultured mast cells through an inhibition of the tyrosine phosphorylation of c-kit. The compound also suppressed the early phase of mast cell development. The extinction of mast cell growth induced by STI571 may be due largely to apoptosis according to the flow cytometric analysis and gel electrophoresis. Two-hour exposure to STI571 that failed to influence the total viable cell number suppressed adhesion of the cells to fibronectin in the presence of SCF without altering the expressions of integrin molecules. Our results may provide a fundamental insight for the clinical application of STI571 in allergic disorders.

Interleukin-6 Enhances Whereas Tumor Necrosis Factor α and Interferons Inhibit Integrin Expression and Adhesion of Human Mast Cells to Extracellular Matrix Proteins

Integrins are expressed on mast cells and constitute an essential prerequisite for the accumulation of the cells at sites of inflammation. In order to clarify a potential contribution of inflammatory cytokines to this process, we have studied the modulation of integrin expression and adhesion of immature human mast cells (HMC-1) to extracellular matrix proteins by interleukin-6, tumor necrosis factor alpha, interferon-alpha and interferon-gamma. Corticosteroids were used for comparison. On fluorescence-activated cell sorter analysis, preincubation of cells for 48 h with different concentrations of interleukin-6 induced a significant, up to 40%, increase of alpha v alpha 5, CD49b (alpha 2), CD49e (alpha 5), CD49f (alpha 6), and CD51 (alpha v). In contrast, different concentrations of tumor necrosis factor alpha, interferon-alpha, interferon-gamma, and dexamethasone (10-8-10-10 M) inhibited expression of adhesion receptors by up to 60%, reaching significance for some but not all integrins. On semiquantitative polymerase chain reaction analysis, interleukin-6, the other cytokines, and corticosteroids significantly modulated expression of alpha1, alpha v and alpha 5 integrin chains at mRNA level. Functional significance of these findings was proven in adhesion assays using fibronectin, laminin, and vitronectin, with interleukin-6 causing significant enhancement of adhesion in all cases, tumor necrosis factor alpha and dexamethasone inducing significant reduction of adhesion to fibronectin and laminin, and interferon-gamma significantly inhibiting adhesion to fibronectin only. Specificity of interleukin-6-induced changes was demonstrated using antibodies against alpha1 and alpha 5 integrins in unstimulated and interleukin-6-prestimulated cells. These data show that interleukin-6 stimulates mast cell adhesion to extracellular matrix and thus allows for the accumulation of the cells at tissue sites by enhancing integrin expression, whereas tumor necrosis factor alpha, interferon-alpha, interferon-gamma, and dexamethasone downmodulate this process.

Inhibition of calpain is a component of nitric oxide-induced down-regulation of human mast cell adhesion.

Nitric oxide is an important messenger that regulates mast cell activity by modifications to gene expression and intracellular pathways associated with exocytosis and adhesion. Integrin interactions with extracellular matrix components modulate an array of cell activities, including mediator production and secretion. To investigate the molecular mechanisms underlying NO regulation of mast cell function, we studied its effects on adhesion of a human mast cell line (HMC-1) to fibronectin (FN). The NO donors S-nitrosoglutathione and S-nitroso-N-acetylpenicillamine strongly down-regulated the adhesion of HMC-1 to FN. Inhibitors of soluble guanylate cyclase and protein kinase G did not alter the response of cells to NO. A peroxynitrite scavenger did not affect modulation of adhesion by NO, nor could the effect of NO be mimicked by the peroxynitrite-producing compound 3-morpholinosydnonimine. NO donors inhibited the cysteine protease, calpain, while calpain inhibitors mimicked the effect of NO and led to a decrease in the ability of HMC-1 cells to adhere to FN. Thus, NO is an effective down-regulator of human mast cell adhesion. The mechanism for this action does not involve peroxynitrite or activation of soluble guanylate cyclase. Instead, a portion of NO-induced down-regulation of adhesion may be attributed to inhibition of the cysteine protease, calpain, an enzyme that has been associated with control of integrin activation in other cell types. The inhibition of calpain is most likely mediated via nitrosylation of its active site thiol group. Calpain may represent a novel therapeutic target for the regulation of mast cell activity in inflammatory disorders.

Regulatory effects of stem cell factor and interleukin-4 on adhesion of human mast cells to extracellular matrix proteins

AbstractMast cells are inflammatory and immunoregulatory cells resident in tissues. They develop from bone marrow-derived progenitor cells that enter the tissue through the blood circulation. The specific localization and migration of mast cells in tissues is dependent on their interaction with extracellular matrix (ECM) proteins. Adhesion of human mast cells isolated from intestinal mucosa and cultured in the presence of stem cell factor (SCF) to ECM proteins is analyzed. It was observed that SCF is a unique cytokine enhancing mast cell adhesion to all tested ECM proteins (fibronectin, laminin, collagen I, III, IV, VI, XIV) up to 5-fold, particularly to fibronectin (54% ± 12% of mast cells) and to denatured collagens (40% ± 12% on cyanogen bromide-cleaved peptides of collagen I). Most noteworthy, preculture of mast cells with interleukin-4 (IL-4), in addition to SCF, reduced their potency to adhere to ECM proteins to one third compared to mast cells cultured with SCF alone. Mast cell adhesion was preferentially mediated by β1 integrins, and most cells expressed the ECM-binding integrins α2β1, α3β1, α4β1, α5β1, and αVβ3. SCF-induced mast cell adhesion was totally blocked by wortmannin and apigenin, indicating an involvement of phosphatidylinositol 3-kinase and mitogen-activated protein kinase, and it was related to an up-regulation of the HUTS-21 β1 epitope, which is associated with an activated conformation of β1. In conclusion, these data indicate that SCF induces the adhesion of cultured mast cells to ECM proteins, whereas IL-4 may promote detachment from the ECM.

Nitric oxide down-regulates human mast cell adhesion to extracellular matrix proteins

Nitric oxide down-regulates human mast cell adhesion to extracellular matrix proteins

Adhesion of human mast cells to extracellular matrix provides a co-stimulatory signal for cytokine production.

Engagement of integrin receptors during cell adhesion leads to changes in the morphology and the state of activation of cells. We therefore examined whether mast cell adhesion to extracellular matrix proteins affects the synthesis and release of various proinflammatory cytokines. Cells of the human mast cell line HMC-1 were added to fibronectin (FN)-, vitronectin (VN)- or, as a control, bovine serum albumin (BSA)-coated wells and were stimulated with phorbol 12-myristate 13-acetate (PMA) and/or calcium ionophore A23187 (ionophore). Cytokine production was evaluated using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of cell extracts and enzyme-linked immunosorbent assay (ELISA) analysis of cell supernatants. After a 4-hr incubation, mRNA expression of interleukin (IL)-8 (and weakly of IL-6) was up-regulated in matrix-adherent cells, with further increase in the presence of PMA and/or ionophore, compared with unstimulated cells. High-level de novo expression of IL-3 and of granulocyte-macrophage colony-stimulating factor (GM-CSF) was observed mainly in matrix-adherent cells. These changes were paralleled by the secretory pattern of HMC-1 cells after a 24-hr stimulation. Unstimulated cells adherent to FN or VN had already released small amounts of IL-8, and both VN- and FN-adherent cells produced, almost invariably, a higher level of cytokines than BSA-exposed cells after additional stimulation. These results show that mast cell adhesion to matrix proteins by itself has only selected and minor effects, but additional activation of mast cells by secretory stimuli causes significantly enhanced cytokine gene expression and secretion, suggesting that mast cells are far more active in their natural tissue environment than hitherto suggested from data in suspension cultures.

Heterotypic cell-cell adhesion of human mast cells to fibroblasts.

Skin mast cells are typically located in the perivascular or perineural connective tissue. We observed that HMC-1 mast cells growing in suspension adhered efficiently to (> 90% of cells) and spread on top of fibroblast monolayers and to a lesser degree on purified extracellular matrix proteins. Since adhesive interactions determine cell migration and tissue localization we studied the mechanism. It was found that HMC-1 cells attach to collagen I and fibronectin, laminin, collagen IV and vitronectin, but not to collagens III and VI or hyaluronic acid. Adhesion to fibronectin, collagen I and laminin was completely inhibited by mAbs blocking beta 1-integrins, whereas adhesion of HMC-1 cells to vitronectin was inhibited by anti-alpha v-chain mAbs. However, attachment of HMC-1 cells to fibroblasts was not influenced by mAbs blocking beta 1- or alpha v-chain function, by RGD peptides or by mAbs interfering with other receptors, most notably c-kit. Identical results were obtained with normal mast cells isolated from human foreskin. These results indicate that human mast cells attach to fibroblasts independently of beta 1- or alpha v-integrins as well as of c-kit receptor-mediated mechanisms. The functional characteristics observed (i.e. only partial sensitivity to trypsin and EDTA, no increase in trypsin sensitivity by pretreatment with EDTA) suggest that cadherin receptors were not involved, and it is likely that the adhesion process observed involved not-yet-defined heterotypic cell-cell adhesion receptors.