Inhibition of IgE-induced activation of human mast cells by IL-10

BACKGROUND: IL-10 exhibits anti-inflammatory effects on activated rodent mast cells (MC) in vitro and inhibits allergen-induced airway inflammation in vivo in murine models. The effects of IL-10 on the allergic activation of human MC are presently unknown. OBJECTIVE: In light of the well-known heterogeneity of mast cell reactivity between animal species, one cannot readily predict the response of human MC to IL-10. Moreover, the impact of IL-10 on MC-derived proinflammatory mediators is still unknown. Thus, the objective of this study was to investigate the effects of IL-10 on the release of inflammatory mediators by IgE/anti-IgE-challenged human cord blood-derived mast cells (CBMC), used as an in vitro model of MC phenotypically similar to human lung MC. MATERIALS AND METHODS: Highly purified human MC were obtained by a first step of long-term culture of cord blood mononuclear cells in the presence of human recombinant stem cell factor (rhSCF) and of human recombinant IL-6 (rhIL-6), followed by a second step of purification by depletion of contaminating cells with an immunomagnetic METHOD: The cells were treated with human IgE, then challenged with anti-human IgE, in the presence or the absence of recombinant rhIL-10 used at various concentrations. Histamine, tumour necrosis factor-alpha (TNF-alpha), IL-5 and IL-8 were measured in the various supernatants collected at different times after the beginning of the challenge. RESULTS: IL-10 inhibited the release of TNF-alpha and of IL-8, but not of IL-5, by activated CBMC. Interestingly, IL-10 also inhibited the release of histamine by activated CBMC, contrasting with data reported for rodent MC. CONCLUSIONS: These findings suggest that IL-10 might have anti-inflammatory effects on IgE/anti-IgE-challenged human MC by inhibiting their release of TNF-alpha, IL-8 and histamine. These data provide new insights into the control of human mast cell activation and might lead to a better knowledge of the cellular mechanisms controlling allergic reactions.     

The role of SHIP in mast cell degranulation and IgE-induced mast cell survival

Atopic disorders are on the increase in the Western world and are due, at least in part, to an overactive mast cell response. A better understanding of the intracellular signalling pathways that regulate both mast cell degranulation and the secretion of arachidonic acid metabolites and inflammatory cytokines could help in the treatment of these disorders. The src homology 2-containing inositol-polyphosphate 5'-phosphatase, SHIP, has been shown to be a key 'gatekeeper' of mast cell degranulation. SHIP prevents degranulation from occuring when IgE alone binds to the high-affinity receptor for IgE (FcvarepsilonR1), SHIP restrains it when IgE-bound FcvarepsilonR1 are engaged by multivalent allergens, and SHIP inhibits it when an IgG against the same allergen co-clusters the inhibitory low-affinity receptor for IgG (FcgammaRIIB) with the IgE receptor. SHIP acts as a negative regulator of degranulation by hydrolyzing phosphatidylinositol-3,4,5-trisphosphate, a second messenger generated in activated cells by phosphatidylinositol 3-kinase. Our finding that binding of only IgE to the FcvarepsilonR1 of SHIP-deficient mast cells results in massive degranulation, led us to investigate the signalling pathways that are triggered in normal murine bone marrow-derived mast cells by monomeric IgE. We report here that monomeric IgE activates signalling pathways resulting in mast cell survival, without stimulating degranulation or proliferation. These studies demonstrate that mast cell sensitization by IgE is an active rather than a passive process.     

TRAIL mediated signaling in human mast cells: the influence of IgE-dependent activation

Background:  Mast cells activation through FcɛRI cross-linking has a pivotal role in the initiation of allergic reactions. The influence of this activation on programmed cell death of human mast cells has not yet been clarified. This study evaluates the influence of IgE-dependent activation alone and in synergy with TRAIL on the expression of molecules involved in the apoptotic signal transduction.
Methods:  Human cord blood derived mast cells (CBMC) were cultured with myeloma IgE followed by activation with anti-human IgE. The expression of proteins involved in apoptotic signal transduction was assessed by immunoblot analysis. To test the effect of activation on a pro-apoptotic stimulus, activated, IgE-treated and resting CBMC were incubated with TRAIL, or in a medium with suboptimal concentrations of stem cell factor (SCF).
Results:  In accordance with a previous study of ours, it was found that IgE-dependent activation increased TRAIL-induced caspase-8 and caspase-3 cleavage. However, it did not have a significant influence on CBMC death induced by SCF withdrawal. IgE-dependent activation increased the expression of FLIP and myeloid cell leukemia 1 (MCL-1) anti-apoptotic molecules as well as the pro-apoptotic one, BIM. In addition, a decrease in BID expression was observed. TRAIL could reverse the increase in FLIP but did not influence the upregulation of MCL-1 and of BIM.
Conclusions:  These findings suggest that IgE-dependent activation of human mast cells induces an increase in both pro-survival and pro-apoptotic molecules. We therefore hypothesized that IgE-dependent activation may regulate human mast cell apoptosis by fine-tuning anti-apoptotic and pro-apoptotic factors.     

寄生虫IgE依赖组胺释放因子抗体抑制致敏肥大细胞释放组胺作用的研究

目的 观察抗寄生虫IgE依赖组胺释放因子(HRF)抗体对重组大鼠IgE依赖组胺释放因子(rRHRF)诱导致敏吧大细胞释放组胺功能的影响.方法 用纯化的日本血吸虫和华支睾吸虫IgE依赖组胺释放因子重组蛋白rSjHRr和rCsHRF分别免疫大鼠,分离免疫血清并通过亲和层析纯化出总IgG.将rRHRF(终浓度为75 μg/mL)分别与2种纯化抗体(浓度梯度为3/5,2/5,1/5,0)37℃预先反应30 min后,再加入卵清蛋白变应原致敏的大鼠肺肥大细胞,利用荧光分光光度法测定rRHRF诱导吧大细胞组胺释放量.实验中采用未致敏大鼠血清纯化所得总IgG作为对照.结果 得到了纯化的抗rSjHRF IgG和抗rC-sHRF LgG,抗rCsHRF IgG和抗rSjHRF IgG可明显抑制大鼠内源性IgE依赖HRF诱导致敏肥大细胞释放组胺的功能,但抗体抑制作用的强弱与抗体浓度之间的剂量依赖关系还需进一步实验验证.结论 抗寄生虫IgE依赖组胺释放因子抗体具有抑制大鼠IgE依赖组胺释放因子诱导致敏肥大细胞释放组胺的作用,可能是参与寄生虫感染抑制宿主Ⅰ型超敏反应发生的免疫学机制之一,为预防和控制Ⅰ型超敏反应提供了新的思路.     

Macromolecular protein signaling complexes and mast cell responses: a view of the organization of IgE-dependent mast cell signaling

The generation of signals following engagement of cell surface receptors is an ordered process that requires tight regulation as spurious signals could result in unwanted, and possibly deleterious, cellular responses. Like other cell surface receptors, stimulation of a mast cell via the high affinity IgE receptor (FcepsilonRI) causes multiple biochemical events that ultimately result in cell activation and effector responses. Recently, our knowledge of how these events are ordered has increased. We now have identified some of the molecules involved, how they are organized into macromolecular complexes by FcepsilonRI stimulation, and the role of some of the constituents of these macromolecular signaling complexes in mast cell effector responses. In brief, we review the knowledge on macromolecular signaling complexes used by FcepsilonRI in mast cell activation and provide our view on the regulation of signal generation and its effect on mast cell activation.     

Role of phospholipase A2 and G-proteins in the IgE-dependent activation of mast cells and macrophages

The effect of para-bromophenacyl bromide (a selective inhibitor of phospholipase A₂) and pertussis toxin has been investigated on IgE-dependent histamine release and on IgE-dependent macrophage-mediated cytotoxicity. Para-bromophenacyl bromide, inhibited dose-dependently IgE-dependent stimulation of mast cells and macrophages (IC₅₀'s of 5.0×10^–7 M and 2.5×10^–7 M, respectively). In contrast, pertussis toxin only inhibited the IgE-dependent stimulation of macrophages, whereas the IgE-dependent activation of mast cells was not affected. These results suggest that the transducing mechanisms following the activation of the high affinity receptor for IgE (FcRI on mast cells) as well as the low affinity receptor for IgE (FcRII on macrophages) induce the activation of phospholipase A₂. FcRII might be coupled to a pertussis toxin sensitive G-protein.     

Inhibition of nasal polyp mast cell and eosinophil activation by desloratadine

Nasal polyp tissue which contains mast cells and eosinophils is similar to the inflamed airway mucosa in cellular composition and mediator content. This investigation assessed the effect of desloratadine (DL), on activation of cells in nasal polyp tissue. Polyps were obtained from 22 patients with chronic rhinosinusitis [nine aspirin acetylosalitic acid (ASA)-sensitive and 13 ASA-tolerant]. Polyp tissue was dispersed by digestion, and preincubated with DL and incubated with anti-immunoglobulin E (IgE) or calcium ionophore. LTC4, eosinophil cationic protein (ECP) and tryptase concentrations in supernatants were measured by immunoassays. Desloratadine (1, 10 and 50 microM) inhibited calcium ionophore-induced LTC4 release by a mean of 29%, 50% and 63% respectively, and anti-IgE-induced LTC4 release by a mean of 27%, 35% and 39% respectively. Calcium ionophore-induced tryptase release was inhibited 60% and 69% by 10 and 50 microM of DL, respectively, and anti-IgE-induced tryptase release was inhibited 33%, 47% and 66% for 1, 10 and 50 microM of DL. Desloratadine 10 microM and 50 microM inhibited ECP release by and 45% and 48% respectively. Polyp tissue from ASA-sensitive patients when compared with ASA-tolerant patients released at baseline significantly more ECP (medians 120.0 microg/ml, range: 69.0-182.0 vs 63.4 microg/ml, range: 3.7-172.0; P <0.05), but similar amounts of tryptase and LTC4. This study demonstrated that DL inhibits activation of both eosinophils and mast cells derived from a site of airway mucosal inflammation.     

Akt cross-links IL-4 priming, stem cell factor signaling, and IgE-dependent activation in mature human mast cells

Challenge of human mast cells with both stem cell factor (SCF) and IL-4 enhances antigen-dependent mediator release raising the assumption of intracellular crosstalk between the IL-4, SCF, and Fc?RI signaling pathways. Here, we analyzed the intracellular crosstalk of IL-4-, SCF-, and IgE-dependent activation pathways in mucosal mast cells isolated from human intestine. The release of β-hexosaminidase, leukotriene C₄, and IL-8, but not IL-6, was strongly enhanced in response to sequential challenge of mast cells with IL-4, SCF and Fc?RI cross-linking compared to stimulation by Fc?RI cross-linking alone. Previous studies revealed that MAPK and other serine/threonine kinases are involved in mast cell activation processes. Here we found that activation of mast cells by Fc?RI cross-linking alone results in phosphorylation of ERK and p38, but not of Akt. Stimulation with SCF alone also induced phosphorylation of ERK and p38, and additionally of Akt. IL-4 priming enhanced activation of ERK, but blocked activation of p38. Activation of p38 was required for IL-6 production explaining the inhibitory effect of IL-4 on IL-6 expression in human mast cells. Moreover, IL-4 priming that anteceded Fc?RI cross-linking induced activation of Akt. The combined challenge of mast cells with IL-4, SCF and Fc?RI cross-linking substantially up-regulated activation of Akt, whereas blocking of Akt inhibited the pronounced production and release of IL-8 in response to the three mast cell agonists. In summary, our data demonstrate that ERK, p38, and especially Akt play an important role in cross-linking IL-4 priming, SCF signaling, and IgE-dependent activation of mature human mast cells.     

Inhibition of antibody production in plasmacytoma cells by antigen.

Tissue culture-adapted MOPC 315 mouse plasmacytoma cells which secrete monoclonal anti-DNP IgA antibody, were cultured with a variety of DNP-carrier conjugates and the production of IgA measured by pulse labeling with [3H]leucine and by plaque-forming cell assays. DNP-coupled bovine, human and rabbit gamma-globulins (containing 9 to 12 DNP groups per 50 000 mol wt of carrier) inhibited the synthesis and secretion of IgA by 40 to 80%. This inhibition was specific, since unconjugated globulins were ineffective, non-IgA proteins and DNA synthesis remained unaffected, and DNP-globulins had no effect on X5563 cells which do not bind DNP. The degree of suppression of antibody production dependen on the concentration and epitone density of the antigen and on the duration of exposure of cells to it, and was no attributable to absorption of secreted antibody by cell-bound antigen. Comparably substituted DNP conjugates of F(ab')2 and intact globulins were equally inhibitory. The inhibition of antibody synthesis by DNP-BGG was reversible following removal of the antigen. This phenomenon is similar in many respects to the antigen-induced blockade of normal antibody-secreting cells, and provides a valuable model system for analyzing the mechanisms of antigen-mediated cellular inactivation.     

The role of CD4+ lymphocytes in the activation of non-specific suppressor cells by antigen.

Tetanus toxoid (TT) and hepatitis B surface antigen (HBsAg) can suppress lectin-induced responses. The suppression induced by TT is dose-dependent and can also down-regulate the induction of a blastogenic response by anti-CD3 and anti-CD4 monoclonals. In addition, TT can dampen the blastogenic response induced by phytohaemagglutinin (PHA) and anti-CD3. The cellular mechanism involved in the turning off of the blastogenic response was investigated by transferring cells treated for 5 days with TT to freshly obtained syngeneic cells and stimulation with PHA. The response of cultures that had received TT-treated cells was significantly lower than of those that had received cells treated with medium only. The removal of CD4+ cells at the induction phase of the suppression reversed the suppression, whereas the elimination of most CD8+ cells had no effect. We propose that CD4+ cells together with monocytes can dampen the specific and non-specific blastogenic responses.     

Sensitization of mice to chemical allergens modulates the responsiveness of isolated mast cells to IgE-dependent activation.

It is known that the release of granule-associated inflammatory amines by isolated mouse tissue-type mast cells is subject to regulation in vitro by a number of cytokines that are produced during the immune response. In this study we investigated whether mast cell secretory function might also be subject to regulation in vivo during active sensitization. Mice were sensitized with one of three chemical allergens (trimellitic anhydride, TMA; 2,4-dinitrochlorobenzene, DNCB; or oxazolone) all of which induce contact sensitization and one of which (TMA) in addition causes immediate hypersensitivity. Peritoneal mast cells isolated from treated mice and sensitized passively with IgE released a greater proportion of cellular serotonin (5-HT) on stimulation either by anti-IgE or by specific antigen than did cells isolated from vehicle-treated controls. These results show that the function of mast cells is susceptible in vivo to regulatory influences that result from induction of an immune response.     

The role of mast cell activation in cholestatic pruritus

Jaundiced patients experience intense pruritus, the pathophysiology of which is unclear. In this study, blood histamine concentrations, skin mast cell counts and intracellular histamine concentrations in peritoneal mast cells were examined in an experimental model of biliary obstruction. Three weeks after bile duct ligation (BDL), total blood histamine concentrations were significantly elevated compared with those from control animals (p<0.0001). Skin mast cell counts were increased (p<0.05) and peritoneal mast cell histamine content decreased (p<0.05) in jaundiced animals. These results demonstrate that mast cells degranulate in biliary obstruction with consequent release of histamine into the systemic circulation. This may contribute to cholestatic pruritus. These data may have significant pharmacological implications in patients with obstructive jaundice.     

The role of mast cell activation in cholestatic pruritus

Jaundiced patients experience intense pruritus, the pathophysiology of which is unclear. In this study, blood histamine concentrations, skin mast cell counts and intracellular histamine concentrations in peritoneal mast cells were examined in an experimental model of biliary obstruction. Three weeks after bile duct ligation (BDL), total blood histamine concentrations were significantly elevated compared with those from control animals (p<0.0001). skin=" mast=" cell=" counts=" were=" increased=">p<0.05) and=" peritoneal=" mast=" cell=" histamine=" content=" decreased=">p<0.05) in=" jaundiced=" animals.=" these=" results=" demonstrate=" that=" mast=" cells=" degranulate=" in=" biliary=" obstruction=" with=" consequent=" release=" of=" histamine=" into=" the=" systemic=" circulation.=" this=" may=" contribute=" to=" cholestatic=" pruritus.=" these=" data=" may=" have=" significant=" pharmacological=" implications=" in=" patients=" with=" obstructive=">     

Increased IgE-dependent mast cell activation and anaphylactic responses in mice lacking the calcium-activated nonselective cation channel TRPM4

Mast cells are key effector cells in allergic reactions. Aggregation of the receptor FcepsilonRI in mast cells triggers the influx of calcium (Ca(2+)) and the release of inflammatory mediators. Here we show that transient receptor potential TRPM4 proteins acted as calcium-activated nonselective cation channels and critically determined the driving force for Ca(2+) influx in mast cells. Trpm4(-/-) bone marrow-derived mast cells had more Ca(2+) entry than did TRPM4(+/+) cells after FcepsilonRI stimulation. Consequently, Trpm4(-/-) bone marrow-derived mast cells had augmented degranulation and released more histamine, leukotrienes and tumor necrosis factor. Trpm4(-/-) mice had a more severe IgE-mediated acute passive cutaneous anaphylactic response, whereas late-phase passive cutaneous anaphylaxis was not affected. Our results establish the physiological function of TRPM4 channels as critical regulators of Ca(2+) entry in mast cells.     

Inhibition of human basophil and rat mast cell activation by avene spring water

In this study the ability of Avene spring water to inhibit basophil degranulation was confirmed. The active principle is thermo-labile and inactivated by ultraviolet or gamma radiation. Inhibition of basophil degranulation occurred with immunological stimuli (e.g. antigen, anti-IgE) but not with the calcium ionophore A23187 as the secretory stimulus. Freeze drying did not impair the inhibitory properties of the spring water. Freeze dried samples were able to inhibit antigen-induced histamine release from human basophils or rat mast cells. The ability of Avene water to inhibit mast cell/basophil activation may be in part responsible for the anti-inflammatory effect of the spa water observedin vivo.     

Alternative complement pathway activation by CD4+ T cells of HIV infected individuals: a possible role in AIDS pathogenesis

The mechanisms by which CD4⁺ T cells are eliminated during HIVinfection are poorly understood. We have previously shown thatHIV infected cell lines activate and fix C3 via the alternativecomplement pathway (ACP). In the present study we examined theability of blood lymphocytes from 40 HIV⁺ individuals to fixC3. A large fraction of the CD4⁺ T cells reacted with anti-gp120antibodies. These cells also carried C3 fragments in vivo andcould further fix C3 if exposed to human serum In vitro. C3activation occurred via the ACP. In some cases exposure of thelymphocytes to human serum under conditions allowing ACP activationresulted in partial elimination of CD4⁺ T cells. The resultssuggest that complement activation and fixation by CD4⁺ T cellsopsonized with HIV particles or gp120 may contribute to theirselective destruction.     

Products from human mast cell line cells enhance the production of interferon-gamma by CD8+ and CD4+ T cells

In patients with allergic asthma, T-cell cytokines are implicated in the regulation of the local inflammation in the airways. The ability of sensitized mast cells to release mediators and cytokines early upon allergen stimulation makes them important candidates for local immunoregulation. We have studied the effects of human mast cells on T cells with the use of the human mast cell line HMC-1. We showed that activated human mast cells or their soluble products induced and enhanced the interferon-gamma (IFN-gamma) production by T cells up to about 60-fold. The production of interleukin (IL)-4 was hardly affected and that of IL-5 was slightly enhanced. The enhancement of IFN-gamma production was induced both in polyclonal CD4+ and CD8+ T cells and in CD4+ and CD8+ T-cell clones. Further characterization of the factors involved demonstrated a molecular mass above 30 000. Our results implicate that by this mechanism mast cells may account for a negative feedback system locally down-regulating allergen-induced T helper 2 responses via IFN-gamma production by the T cells.     

An IgE-dependent secretory response of mast cells can be induced by a glycosphingolipid-specific monoclonal antibody

The signal transduction pathway of the type 1 Fcepsilon receptor (FcepsilonRI) has been proposed to be spatially constrained to plasma membrane microdomains enriched in glycosphingolipids and cholesterol. These domains are proposed to serve as platforms that enhance the efficiency of the antigen-receptor stimulus-response coupling process. Here we describe a monoclonal antibody (mAb) designated 2B5, raised by immunizing mice with rat mucosal-type mast cell (line RBL-2H3) membranes, which binds to glycosphingolipids and causes a dose-dependent secretory response of these cells. This secretory response to mAb 2B5 requires binding of IgE to the FcepsilonRI on these cells, although direct interactions between IgE and mAb 2B5 are excluded. The bound IgE- or FcepsilonRI-specific mAb did not affect binding of mAb 2B5 or its Fab fragments to the RBL-2H3 cells and only a limited interference with the binding of IgE to the FcepsilonRI by mAb 2B5 was observed. Binding of mAb 2B5 to the RBL-2H3 cells induced a distribution of fluorescently labeled IgE similar to that produced by antigen-induced aggregation of the IgE-FcepsilonRI. Thus we suggest that mAb 2B5 binds to cell surface glycosphingolipids that are probably associated with the FcepsilonRI-IgE complexes and causes their aggregation, thereby initiating the cascade leading to the cell's secretory response.     

The role of mast cells in inflammatory processes: evidence for nerve/mast cell interactions

In the rat, there is considerable evidence of mast cell/nerve interaction both in the normal and infected intestine. Between 67 and 87% of all mast cells in the intestinal lamina propria of rats infected 22-35 days earlier with Nippostrongylus brasiliensis were touching nerves. These membrane contacts were between subepithelial mast cells and nonmyelinated nerves containing substance P, calcitonin gene-related peptide and neurone specific enolase. 2.5S nerve growth factor (NGF) has a significant enhancement effect on antigen-induced histamine release without addition of phosphatidylserine, and the in vivo administration of NGF to rats causes both connective tissue and mucosal mast cells to dramatically increase in number. All of these effects are both dose dependent and NGF specific, as evidenced by inhibition with anti-NGF. 2.5S NGF also causes in vitro increase of colonies in methylcellulose cultures of human peripheral blood. The effects of NGF in this system are synergistic with other T cell-derived growth factors and relatively specific for metachromatic cell growth. These observations support the conclusions that nerves and mast cells may constantly communicate and provide a structural and conceptual framework whereby the central nervous system may communicate with inflammatory events.