Mast cells are critical for the production of leukotrienes responsible for neutrophil recruitment in immune complex-induced peritonitis in mice

Mast cells are secretory cells strategically located in the vicinity of blood vessels where they can readily initiate and modulate various inflammatory processes, including plasma exudation and leukocyte infiltration. We have previously shown that 50% of the neutrophil influx during immune complex peritonitis in mice is due to mast cells. Eicosanoids are important mediators of various inflammatory processes including neutrophil infiltration. The possibility that mast cells are essential for the production of leukotrienes (LT) involved in the elicitation of neutrophils in immune complex peritonitis was investigated in mast cell-deficient, WBB6F1-W/WV, and normal, WBB6F(1-)+/+, mice. The time course and amounts of immunoreactive PGE2, 6-keto-PGF1 alpha, and TX3B2 released into the peritoneal exudates were similar in both sets of mice. LTB4 and LTC4 levels, however, were twofold higher in +/+ than in W/WV mice 2 h after stimulation. HPLC analysis of the peritoneal exudate confirmed the presence of leukotrienes. The 5-lipoxygenase inhibitor A-63162 blocked leukotriene production in a dose-dependent manner in both sets of mice. However, this compound caused a significant reduction (60%) of neutrophil infiltration only in WBB6F(1-)+/+ but not in the mast cell-deficient mice. Mast cell reconstitution of WBB6F1-W/WV mice restored the effect of A-63162 on PMN recruitment. These data suggest that mast cells in the vicinity of blood vessels are important for the synthesis of leukotrienes responsible for PMN recruitment.     

Time-dependent inhibition of immune complex-induced lung injury by catalase: relationship to alterations in macrophage and neutrophil matrix metalloproteinase elaboration

Rats were subjected to acute lung injury by the intra-alveolar formation of IgG immune complexes of bovine serum albumin (BSA) and anti-BSA. In this model of injury, complement activation occurs and large numbers of neutrophils invade the interstitium and alveolar space. In the present study, animals were treated with intratracheal catalase concomitantly with anti-BSA or after a lag period of 5-120 min. Catalase treatment at time-zero or at 5 min post injury failed to prevent lung injury as indicated by permeability change, histological features, and neutrophil influx. However, treatment after a delay of 15-30 min (but not 120 min) afforded substantial protection. Consistent with past findings [19], lung injury was accompanied by an accumulation of matrix metalloproteinase 9 (MMP-9) in bronchoalveolar lavage (BAL) fluid. There was a strong correlation between inhibition of injury and reduction in MMP-9 levels. In vitro studies conducted in parallel revealed that unstimulated alveolar macrophages did not produce measurable MMP-9, while there was a large induction following exposure to the same immune complexes that initiated injury in vivo. MMP-2 was also slightly upregulated under the same conditions. Concomitant treatment with catalase greatly inhibited MMP-9 production by macrophages in response to immune complexes, but this treatment had little effect on basal production of either MMP-9 or MMP-2 by macrophage. The same concentration of catalase that suppressed MMP-9 elaboration also inhibited the production of tumor necrosis factor alpha. In contrast, when neutrophils were treated with catalase and then exposed to immune complexes, the antioxidant failed to prevent the release of either MMP-2 or MMP-9. Taken together, these findings demonstrate that antioxidant treatment interferes with elaboration of MMPs by alveolar macrophages. Protection against lung injury is correlated with reduction in MMP levels in the BAL fluid.     

The urokinase/urokinase receptor system mediates the IgG immune complex-induced inflammation in lung

Immune complex (IC) deposition induces an acute inflammatory response with tissue injury. IC-induced inflammation is mediated by inflammatory cell infiltration, a process highly regulated by the cell surface-specific receptor (uPAR), a binding partner for the urokinase-type plasminogen activator (uPA). We assessed the role of the uPA/uPAR system in IC-induced inflammation using the pulmonary reverse passive Arthus reaction in mice lacking uPA and uPAR compared with their corresponding wild-type controls. Both uPA-deficient C57BL/6J (uPA(-/-)) and uPAR-deficient mice on a mixed C57BL/6J (75%) x 129 (25%) background (uPAR(-/-)) demonstrated a marked reduction of the inflammatory response due to decreased production of proinflammatory mediators TNF-alpha and Glu-Leu-Arg (ELR)-CXC chemokine MIP-2. In uPAR(-/-) animals, the reduction of inflammatory response was more pronounced because of decreased migratory capacity of polymorphonuclear leukocytes. We show that the uPA/uPAR system is activated in lung of wild-type mice, particularly in resident alveolar macrophages (AM), early in IC-induced alveolitis. This activation is necessary for an adequate C5a anaphylatoxin receptor signaling on AM that, in turn, modulates the functional balance of the activating/inhibitory IgG FcgammaRs responsible for proinflammatory mediator release. These data provide the first evidence that the uPA/uPAR plays an important immunoregulatory role in the initiation of the reverse passive Arthus reaction in the lung by setting the threshold for C5a anaphylatoxin receptor/FcgammaR activation on AM. The findings indicate an important link between the uPA/uPAR system and the two main components involved in the IC inflammation, namely, complement and FcgammaRs.     

Distinct tissue site-specific requirements of mast cells and complement components C3/C5a receptor in IgG immune complex-induced injury of skin and lung.

We induced the passive reverse Arthus reaction to IgG immune complexes (IC) at different tissue sites in mice lacking C3 treated or not with a C5aR-specific antagonist, or in mice lacking mast cells (Kit(W)/Kit(W-v) mice), and compared the inflammatory responses with those in the corresponding wild-type mice. We confirmed that IC inflammation of skin can be mediated largely by mast cells expressing C5aR and FcgammaRIII. In addition, we provided evidence for C3-independent C5aR triggering, which may explain why the cutaneous Arthus reaction develops normally in C3(-/-) mice. Furthermore, some, but not all, of the acute changes associated with the Arthus response in the lung were significantly more intense in normal mice than in C3(-/-) or Kit(W)/Kit(W-v) mice, indicating for C3- and mast cell-dependent and -independent components. Finally, we demonstrated that C3 contributed to the elicitation of neutrophils to alveoli, which corresponded to an increased synthesis of TNF-alpha, macrophage-inflammatory protein-2, and cytokine-induced neutrophil chemoattractant. While mast cells similarly influenced alveolar polymorphonuclear leukocyte influx, the levels of these cytokines remained largely unaffected in mast cell deficiency. Together, the phenotypes of C3(-/-) mice and Kit(W)/Kit(W-v) mice suggest that complement and mast cells have distinct tissue site-specific requirements acting by apparently distinct mechanisms in the initiation of IC inflammation.     

Precursor of mast cells fixed in the skin of mice

The presence and origin of mast-cell precursors fixed in the skin tissue of mice were investigated. Giant granules of beige (C57BL/6-bg^j/bg^j, Chediak-Higashi syndrome) mice were used to distinguish different populations of mast cells. Pieces of the skin were grafted from the intact WBB6F¹ (WB × C57BL/6)F¹?+/+ mice onto the back of the WBB6F¹?+/+ mice which had been irradiated and injected with bone marrow cells of C57BL/6-bg^j/bj^j mice (bg^j/bg^j ?+/+ chimeras). Although the number of mast cells in the skin grafts decreased after the transplantation, the mast-cell precursors circulating in the bloodstream of bg^j/bg^j ?+/+ chimeras (bg^j/bg^j type) did not seem to enter into the skin grafts, because most of mast cells were of +/+ type after the recovery of mast-cell number to pregrafting levels. As a considerable proportion of +/+-type mast cells was labeled with ³H-thymidine, the recovery of mast-cell number in the grafts was attributed to the proliferation and differentiation of +/+-type precursor cells fixed in the skin tissue of the donor. On the other hand, the skin of WBB6F¹-W/W^v mice seemed to be depleted of fixed precursors, because most of mast cells were of bg^j/bg^j type in skin grafted from WBB6F¹-W/W^v mice to bg^j/bg^j?+/+ chimeras. Since the fixed precursor cells which proliferate and differentiate into mast cells after skin grafting may be transferred to WBB6F¹-W/W^v mice by bone marrow transplantation, such precursor cells seem to have been derived from the bone marrow.     

The role of mast cells in the development of skin fibrosis in tight-skin mutant mice

Chronic inflammatory conditions can evolve a fibrotic phenotype often associated with an increase in the number of mast cells (MC) near or within the granulation tissue. Despite the potential of MC to mediate fibrosis, it is unclear whether these cells play a central role in the pathogenesis of ibrosis or whether their presence is simply circumstantial. The tight-skin (Tsk) mouse develops an inherited fibrotic disease (sharing many similarties with the human disease scleroderma, systemic sclerosis) in which the lesions are associated with increased numbers and heightened granule release implicating MC in the pathogenesis of fibrosis. Despite their close association with the skin fibrosis of Tsk mice, the precise role of the MC in the pathogenesis of this inherited disease is unknown. Therefore, to assess directly whether MC are key elements in the pathogenesis of Tsk fibrosis, we generated MC deficient mice carrying the Tsk locus by utilizing selective interbreeding between Tsk and mutant mice deficient in mast cells (W, dominant white-spotting). We found that in the absence of MC, the early natural history of Tsk fibrosis was not altered. Furthermore, in older (5–7 months) Tsk mice, we found that the number of cutaneous MC was correlated with a more pronounced fibrosis. Therefore, we conclude that Tsk skin lesions are a pleiotropic manifestation of the Tsk gene in which MC are ivolved/recruited by an uncharacterized mechanism and that subsequent proliferation and activation of MC leads to augmentation of fibrosis.     

Morphine-modulated mast cell migration and proliferation during early stages of zymosan-induced peritonitis in CBA mice

We have previously shown that supplementation of inflammation-inducing zymosan with a high dose ofmorphine inhibits peritoneal influx ofleukocytes in Swiss, C57C3H, Balb/c, and C57BL/6 strains but not in CBA mice. We have also reported that the different pattern of the response to morphine treatment might be, at least partially, due to the inter-strain differences in the peritoneal mast cell (P-MC) number (high in CBA mice versus other strains) and P-MC specific features (high sensitivity to degranulation upon morphine treatment in CBA mice). The aim of the present study was to investigate the mechanism of morphine action on P-MC in CBA mice. In particular, the effects of morphine on the proliferation and migration of P-MC in CBA mice with ongoing zymosan-induced peritonitis modulated by morphine were studied. Morphine alone acted as a strong chemoattractant for P-MC of CBA mice and this effect was opioid receptor-independent. Moreover, flow cytometric analysis showed that i.p. morphine injection induced significant proliferation of P-MC in CBA mice. Therefore, we conclude that the lack of anti-inflammatory effects of morphine during peritonitis in CBA mice might result not only from a unique sensitivity of CBA mast cells to morphine-induced degranulation but also from the fact that mast cell numbers increase at the inflammatory focus. The latter might be due to morphine-induced mast cell proliferation and/or migration.     

TRPM4 regulates migration of mast cells in mice

We demonstrate here that the transient receptor potential melastatin subfamily channel, TRPM4, controls migration of bone marrow-derived mast cells (BMMCs), triggered by dinitrophenylated human serum albumin (DNP-HSA) or stem cell factor (SCF). Wild-type BMMCs migrate after stimulation with DNP-HSA or SCF whereas both stimuli do not induce migration in BMMCs derived from TRPM4 knockout mice (trpm4^?/?). Mast cell migration is a Ca²⁺-dependent process, and TRPM4 likely controls this process by setting the intracellular Ca²⁺ level upon cell stimulation. Cell migration depends on filamentous actin (F-actin) rearrangement, since pretreatment with cytochalasin B, an inhibitor of F-actin formation, prevented both DNP-HSA- and SCF-induced migration in wild-type BMMC. Immunocytochemical experiments using fluorescence-conjugated phalloidin demonstrate a reduced level of F-actin formation in DNP-HSA-stimulated BMMCs from trpm4^?/? mice. Thus, our results suggest that TRPM4 is critically involved in migration of BMMCs by regulation of Ca²⁺-dependent actin cytoskeleton rearrangements.     

Diminished calcium influx in lectin-stimulated T cells from old mice

T lymphocytes from aged donors function poorly, but the biochemical basis for the defect remains uncertain. We tested the hypothesis that T cells from old mice had a diminished ability to transmit extracellular signals into the cytoplasm, by measuring intracellular free calcium concentrations (Cai) in T cells stimulated by the polyclonal activator concanavalin A (Con A). Using the second-generation fluorochrome indo-1 as a reporter of Cai, we found that the Con A-induced elevation of Cai levels is reduced both in rate and extent in old T cells, as compared to T cells from young mice. Flow cytometric analysis showed that this age-sensitive change represents a decline, with age, in the number of T cells that can respond to Con A by increasing their Cai above resting baseline levels (100-120 nM). These results thus show that defects in activation are manifested by T cells from old donors within the first 5 minutes of the activation process, and suggest that aging may lead to alterations either in the surface molecules that receive extracellular signals, or in the sequence of coupled events by which these extracellular signals bring about alterations in the intracellular ionic milieu.     

Effect of fish-fat or beef-fat supplemented diet on immune complex-induced entheropathy in the rat

In contrast to animals on a beef fat-supplemented diet (BFD), animals maitained on a fish fat-supplemented diet (FFD) incorporate increased amounts of eicosapentaenoic acid (EPA) into membrane phospholipids. Generation of lipid mediators from such tissues favors the formation of compounds with less pro-inflammatory activity than are derived from tissues poor in EPA. Nevertheless, the FFD has not had a uniformly beneficial effect on animal models of inflammatory diseases. We previously showed that intravenous injection of rat anti-BSA-BSA complexes (IC) prepared in 5x antigen excess rapidly induced a striate pattern of serosal (to mucosal) hemorrhage and vascular congestion throughout the small intestine. In this study, we tested the effect of a BFD and FFD on immune complex-induced enteropathy. After six (Expt. 1) or eight weeks (Expt. 2) on the diet, rats were injected with IC and the severity of serosal hyperemia in the small intestine was scored. In some FFD, no lesions were seen under conditions which elicited moderate to severe lesions in BFD rats. In Expt. 1 involving 22 rats and in Expt. 2 involving 28 rats, those on the FFD had a significantly lower composite lesional score compared to those on the BFD, p < 0.005 and p < 0.005, respectively. These results indicate that the FFD had a beneficial effect on IC-induced enteropathy. It is suggested that this effect of the FFD may be mediated primarily by a reduction in availability of platelet-activating factor.     

Smad3 deficiency in mast cells provides efficient host protection against acute septic peritonitis

Mast cells play an important role in innate immunity as well as in allergic reaction. However, regulatory mechanisms underlying mast cell-mediated innate immune responses remain largely unknown. Here we determined whether Smad3, a major signal transducer of TGF-beta, regulates innate immune response by mast cells against Gram-negative bacteria. Bone marrow-derived mast cells (BMMC) obtained from Smad3 null mutant mice showed augmented capacity to produce proinflammatory cytokines upon stimulation with a Gram-negative bacteria-associated product, LPS. In acute septic peritonitis model induced by cecal ligation and puncture, mast cell-deficient W/W(v) mice reconstituted with Smad3 null BMMC had significantly higher survival rate than W/W(v) mice reconstituted with wild-type BMMC, which was associated with higher production of proinflammatory cytokines in the peritoneal cavity. These in vitro and in vivo results suggest that Smad3 in mast cells functions as inhibitory for mast cell-mediated innate immune response against Gram-negative bacteria. Suppression of Smad3 expression in mast cells may thus have therapeutic potential for Gram-negative bacterial infection such as acute septic peritonitis by augmenting innate immune responses of mast cells.     

Histidine decarboxylase activities in mutant mice deficient in mast cells

The histamine contents were very low in the whole bodies of various types of mutant mice (W^v/W^v, W^v/W, W/W), in which the number of mast cells was decreased, but the L-histidine decarboxylase activities in these mutant mice were not much lower than in control wild type mice. These findings suggest the presence of high histidine decarboxylase activity in cells other than mast cells. Histidine decarboxylase in the whole body of mice was difficult to assay, because the enzyme was rapidly destroyed by proteases, but inclusion of a protease inhibitor, such as Leupeptin, Antipain, Chymostatin, or Pepstatin in the assay mixture permitted the accurate assay of histidine decarboxylase in crude extracts.     

Parasitic infection improves survival from septic peritonitis by enhancing mast cell responses to bacteria in mice

Mammals are serially infected with a variety of microorganisms, including bacteria and parasites. Each infection reprograms the immune system's responses to re-exposure and potentially alters responses to first-time infection by different microorganisms. To examine whether infection with a metazoan parasite modulates host responses to subsequent bacterial infection, mice were infected with the hookworm-like intestinal nematode Nippostrongylus brasiliensis, followed in 2-4 weeks by peritoneal injection of the pathogenic bacterium Klebsiella pneumoniae. Survival from Klebsiella peritonitis two weeks after parasite infection was better in Nippostrongylus-infected animals than in unparasitized mice, with Nippostrongylus-infected mice having fewer peritoneal bacteria, more neutrophils, and higher levels of protective interleukin 6. The improved survival of Nippostrongylus-infected mice depends on IL-4 because the survival benefit is lost in mice lacking IL-4. Because mast cells protect mice from Klebsiella peritonitis, we examined responses in mast cell-deficient Kit(W-sh)/Kit(W-sh) mice, in which parasitosis failed to improve survival from Klebsiella peritonitis. However, adoptive transfer of cultured mast cells to Kit(W-sh)/Kit(W-sh) mice restored survival benefits of parasitosis. These results show that recent infection with Nippostrongylus brasiliensis protects mice from Klebsiella peritonitis by modulating mast cell contributions to host defense, and suggest more generally that parasitosis can yield survival advantages to a bacterially infected host.     

Effect of fish-fat or beef-fat supplemented diet on immune complex-induced enteropathy in the rat

In contrast to animals on a beef fat-supplemented diet (BFD), animals maintained on a fish fat-supplemented diet (FFD) incorporate increased amounts of eicosapentaenoic acid (EPA) into membrane phospholipids. Generation of lipid mediators from such tissues favors the formation of compounds with less pro-inflammatory activity than are derived from tissues poor in EPA. Nevertheless, the FFD has not had a uniformly beneficial effect on animal models of inflammatory diseases. We previously showed that intravenous injection of rat anti-BSA-BSA complexes (IC) prepared in 5x antigen excess rapidly induced a striate pattern of serosal (to mucosal) hemorrhage and vascular congestion throughout the small intestine. In this study, we tested the effect of a BFD and FFD on immune complex-induced enteropathy. After six (Expt. 1) or eight weeks (Expt. 2) on the diet, rats were injected with IC and the severity of serosal hyperemia in the small intestine was scored. In some FFD, no lesions were seen under conditions which elicited moderate to severe lesions in BFD rats. In Expt. 1 involving 22 rats and in Expt. 2 involving 28 rats, those on the FFD had a significantly lower composite lesional score compared to those on the BFD, p less than 0.005 and p less than 0.005, respectively. These results indicate that the FFD had a beneficial effect on IC-induced enteropathy. It is suggested that this effect of the FFD may be mediated primarily by a reduction in availability of platelet-activating factor.     

The Src family kinase Fgr is critical for activation of mast cells and IgE-mediated anaphylaxis in mice

Mast cells are critical for various allergic disorders. Mast cells express Src family kinases, which relay positive and negative regulatory signals by Ag. Lyn, for example, initiates activating signaling events, but it also induces inhibitory signals. Fyn and Hck are reported to be positive regulators, but little is known about the roles of other Src kinases, including Fgr, in mast cells. In this study, we define the role of Fgr. Endogenous Fgr associates with FcεRI and promotes phosphorylation of Syk, Syk substrates, which include linkers for activation of T cells, SLP76, and Gab2, and downstream targets such as Akt and the MAPKs in Ag-stimulated mast cells. As a consequence, Fgr positively regulates degranulation, production of eicosanoids, and cytokines. Fgr and Fyn appeared to act in concert, as phosphorylation of Syk and degranulation are enhanced by overexpression of Fgr and further augmented by overexpression of Fyn but are suppressed by overexpression of Lyn. Moreover, knockdown of Fgr by small interfering RNAs (siRNAs) further suppressed degranulation in Fyn-deficient bone marrow-derived mast cells. Overexpression of Fyn or Fgr restored phosphorylation of Syk and partially restored degranulation in Fyn-deficient cells. Additionally, knockdown of Fgr by siRNAs inhibited association of Syk with FcεRIγ as well as the tyrosine phosphorylation of FcεRIγ. Of note, the injection of Fgr siRNAs diminished the protein level of Fgr in mice and simultaneously inhibited IgE-mediated anaphylaxis. In conclusion, Fgr positively regulates mast cell through activation of Syk. These findings help clarify the interplay among Src family kinases and identify Fgr as a potential therapeutic target for allergic diseases.     

Effects of degranulation of mast cells on proliferation of mesenchymal cells in the mesentery of mice

Summary A mast-cell activator, compound 48/80, causes proliferation of mesenchymal cells in the mesentery of rats. Its effect on W/W ^vmice deficient in mast cells was tested to determine whether the proliferation is mediated in the degranulation of mast cells. Incorporation of [³H]thymidine into mesenchymal cells in the mesentery of these mice with or without compound 48/80 was very small compared to their normal litter mates. However, bone marrow transplantation markedly enhanced the effect of compound 48/80, and resulted in an incorporation of [³H]thymidine almost comparable to that observed in normal mice. Our results provide evidence that mesenchymal cell proliferation is caused by a product secreted by mast cells when stimulated by compound 48/80.Supported by a Grant-in-Aid for Scientific Research, No. 366, from the Japanese Ministry of Health and WelfareThe authors are indebted to Drs. Motomu Minamiyama and Yukio Hirata for valuable advices, and to Miss Mitsuko Inoue for technical assistance