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Allergen immunotherapy for patients with allergies begins with weekly escalating doses of allergen under medical supervision to monitor and treat IgE mast cell-mediated anaphylaxis. There is currently no treatment to safely desensitize mast cells to enable robust allergen immunotherapy with therapeutic levels of allergen. Here, we demonstrated that liposomal nanoparticles bearing an allergen and a high-affinity glycan ligand of the inhibitory receptor CD33 profoundly suppressed IgE-mediated activation of mast cells, prevented anaphylaxis in Tg mice with mast cells expressing human CD33, and desensitized mice to subsequent allergen challenge for several days. We showed that high levels of CD33 were consistently expressed on human skin mast cells and that the antigenic liposomes with CD33 ligand prevented IgE-mediated bronchoconstriction in slices of human lung. The results demonstrated the potential of exploiting CD33 to desensitize mast cells to provide a therapeutic window for administering allergen immunotherapy without triggering anaphylaxis., Introduction Type I hypersensitivity, or allergy, is a major public health concern worldwide (1). In the US, food allergy alone affects 15 million Americans (2, 3), and at least 1.6% [...]

Mast cells (MCs) are immune sentinels, but whether they also function as antigen-presenting cells (APCs) remains elusive. Using mouse models of MC deficiency, we report on MC-dependent recruitment and activation of multiple T cell subsets to the skin and draining lymph nodes (DLNs) during dengue virus (DENV) infection. Newly recruited and locally proliferating [gamma][delta] T cells were the first T cell subset to respond to MC-driven inflammation, and their production of IFN-[gamma] was MC dependent. MC-[gamma][delta] T cell conjugates were observed consistently in infected peripheral tissues, suggesting a new role for MCs as nonconventional APCs for [gamma][delta] T cells. MC-dependent [gamma][delta] T cell activation and proliferation during DENV infection required T cell receptor (TCR) signaling and the nonconventional antigen presentation molecule endothelial cell protein C receptor (EPCR) on MCs. [gamma][delta] T cells, not previously implicated in DENV host defense, killed infected targeted DCs and contributed to the clearance of DENV in vivo. We believe immune synapse formation between MCs and [gamma][delta] T cells is a novel mechanism to induce specific and protective immunity at sites of viral infection., Introduction Mast cells (MCs) are tissue-resident cells that are distributed throughout the dermis, where they have an evolutionarily conserved responsibility to the host for pathogen surveillance (1). Although MCs have [...]

Mast cells are classically thought to play an important role in protection against helminth infections and in the induction of allergic diseases; however, recent studies indicate that these cells also contribute to neovascularization, which is critical for tissue remodeling, chronic inflammation, and carcinogenesis. Here, we demonstrate that mast cells are essential for sprouting angiogenesis in a murine model of oxygen-induced retinopathy (OIR). Although mouse strains lacking mast cells did not exhibit retinal neovascularization following hypoxia, these mice developed OIR following infusion of mast cells or after injection of mast cell tryptase (MCT). Relative hypoxia stimulated mast cell degranulation via transient receptor potential ankyrin 1. Subsequent surges in MCT stimulated retinal endothelial cells to produce monocyte chemotactic protein-1 (MCP1) and angiogenic factors, leading to sprouting angiogenesis. Mast cell stabilizers as well as specific tryptase and MCP1 inhibitors prevented the development of OIR in WT mice. Preterm infants with early retinopathy of prematurity had markedly higher plasma MCT levels than age-matched infants without disease, suggesting mast cells contribute to human disease. Together, these results suggest therapies that suppress mast cell activity should be further explored as a potential option for preventing eye diseases and subsequent blindness induced by neovascularization., Introduction Mast cells are important for innate immune defense against microbes and parasites (1, 2). A number of toxins and chemicals can induce host-protective or pathogenic activation of mast cells [...]

The growth factor receptor Kit is involved in hematopoietic and nonhematopoietic development. Mice bearing Kit defects lack mast cells; however, strains bearing different Kit alleles exhibit diverse phenotypes. Herein, we investigated factors underlying differential sensitivity to IgG-mediated arthritis in 2 mast cell-deficient murine lines: [Kit.sup.Wsh/Wsh], which develops robust arthritis, and [Kit.sup.W/Wv], which does not. Reciprocal bone marrow transplantation between [Kit.sup.W/Wv] and [Kit.sup.Wsh/Wsh] mice revealed that arthritis resistance reflects a hematopoietic defect in addition to mast cell deficiency. In [Kit.sup.W/Wv] mice, restoration of susceptibility to IgG-mediated arthritis was neutrophil independent but required IL-1 and the platelet/ megakaryocyte markers NF-E2 and glycoprotein VI. In [Kit.sup.W/Wv] mice, platelets were present in numbers similar to those in WT animals and functionally intact, and transfer of WT platelets did not restore arthritis susceptibility. These data implicated a platelet-independent role for the megakaryocyte, a Kit-dependent lineage that is selectively deficient in [Kit.sup.W/Wv] mice. Megakaryocytes secreted IL-1 directly and as a component of circulating microparticles, which activated synovial fibroblasts in an IL-1-dependent manner. Transfer of WT but not IL-1-deficient megakaryocytes restored arthritis susceptibility to [Kit.sup.W/Wv] mice. These findings identify functional redundancy among Kit-dependent hematopoietic lineages and establish an unanticipated capacity of megakaryocytes to mediate IL-1-driven systemic inflammatory disease., Introduction Mast cells (MCs) were first described as tissue-resident cells that stain brightly with aniline dyes (1). Their highly granular appearance reflects their capacity to release a wide range of [...]

Previous studies have shown that pharmacological inhibition of the phosphoinositol-3 (PI3) kinase disrupts the activation of mast cells. Through phosphoinositide-dependent kinase PDK1, PI3 kinase activates the serum- and glucocorticoid-inducible kinase 3 (SGK3). The present study explored the role of SGK3 in mast cell function. Mast cells were isolated and cultured from bone marrow (BMMCs) of gene-targeted mice lacking SGK3 ([sgk3.sup.-/-]) and their wild-type littermates ([sgk3.sup.+/+]). BMMC numbers in the ear conch were similar in both genotypes. Stimulation with IgE and cognate antigen triggered the release of intracellular [Ca.sup.2+] and entry of extracellular [Ca.sup.2+]. Influx of extracellular [Ca.sup.2+] but not [Ca.sup.2+] release from intracellular stores was significantly blunted in [sgk3.sup.-/-] BMMCs compared with [sgk3.sup.+/+] BMMCs. Antigen stimulation further led to a rapid increase of a [K.sup.+]-selective conductance in [sgk3.sup.+/+] BMMCs, an effect again blunted in [sgk3.sup.-/-] BMMCs. In contrast, the [Ca.sup.2+] ionophore ionomycin activated [K.sup.+] currents to a similar extent in [sgk3.sup.-/-] and in [sgk3.sup.+/+] BMMCs. [beta]-Hexosaminidase release, triggered by antigen stimulation, was also significantly decreased in [sgk3.sup.-/-] BMMCs. IgE-dependent anaphylaxis measured as a sharp decrease in body temperature upon injection of DNP-HSA antigen was again significantly blunted in [sgk3.sup.-/-] compared with [sgk3.sup.+/+] mice. Serum histamine levels measured 30 rain after induction of an anaphylactic reaction were significantly lower in [sgk3.sup.-/-] than in [sgk3.sup.+/+] mice. In conclusion, both in vitro and in vivo function of BMMCs are impaired in gene targeted mice lacking SGK3. Thus SGK3 is critical for proper mast cell function. allergy; anaphylaxis; calcium ion channels; degranulation; potassium ion channels; [K.sub.ca] 3.1; phosphoinositol-3 kinase doi: 10.1152/ajpcell.00539.2009.

Altered intestinal barrier function is postulated to be a central predisposing factor to intestinal diseases, including inflammatory bowel diseases and food allergies. However, the mechanisms involved in maintaining homeostatic intestinal barrier integrity remain undefined. In this study, we demonstrate that mice deficient in mast cells ([Kit.sup.W-sh/W-sh] [Wsh]) or mast cell chymase ([Mcpt4.sup.-/-]) have significantly decreased basal small intestinal permeability compared with wild-type (WT) mice. Altered intestinal barrier function was linked to decreased intestinal epithelial cell migration along the villus/crypt axis, altered intestinal morphology, and dysregulated claudin-3 crypt expression. Remarkably, engraftment of Wsh mice with WT but not [Mcpt4.sup.-/-] mast cells restored intestinal epithelial cell migration, morphology, and intestinal epithelial barrier function. Collectively, these findings identify a mechanism by which mast cells regulate homeostatic intestinal epithelial migration and barrier function. homeostasis | intestinal permeability | mast cell protease-4 doi/10.1073/pnas.0906372106

Mast cells have been shown to play a role in development and persistence of various inflammatory bladder disorders. Mast cell-derived tryptase specifically activates protease-activated receptor-2 (PAR-2), and PAR-2 is known to be involved in inflammation. We investigated whether mast cells participate in increase of cyclooxygenase-2 (COX-2) protein abundance in urothelium/suburothelium of bladders of mice subsequent to cyclophosphamide (CYP)-induced bladder inflammation. We also used primary cultures of human urothelial cells to investigate cellular mechanisms underlying activation of PAR-2 resulting in increased COX-2 expression. We found that treatment of mice with CYP (150 mg/kg ip) increased COX-2 protein abundance in bladder urothelium/suburothelium 3, 6, and 24 h after CYP (P < 0.01), and increased COX-2 protein abundance was prevented by treatment of mice with the mast cell stabilizer sodium cromolyn (I0 mg/kg ip) for 4 consecutive days before CYP treatment. Incubation of freshly isolated mouse urothelium/suburothelium with a selective PAR-2 agonist, 2-furoyl-LIGRLO-amide (3 [micro]M), also increased COX-2 protein abundance (P < 0.05). We further demonstrated that 2-furoyl-LIGRLO-amide (3 [micro]M) increased COX-2 mRNA expression and protein abundance in primary cultures of human urothelial cells (P < 0.01), and the effects of PAR-2 activation were mediated primarily by the ERK1/2 MAP kinase pathway. These data indicate that there are functional interactions among mast cells, PAR-2 activation, and increased expression of COX-2 in bladder inflammation. urothelium; cyclophosphamide; mouse; inflammation; bladder doi: 10.1152/ajpregu.00310.2009

Populations of interstitial cells of Cajal (ICC) are altered in several gastrointestinal neuromuscular disorders. ICC are identified typically by ultrastructure and expression of Kit (CDll7), a protein that is also expressed on mast cells. No other molecular marker currently exists to independently identify ICC. The expression of ANO1 (DOG1, TMEMI6A), a [Ca.sup.2+]-activated [Cl.sup.-] channel, in gastrointestinal stromal tumors suggests it may be useful as an ICC marker. The aims of this study were therefore to determine the distribution of Anol immunoreactivity compared with Kit and to establish whether Anol is a reliable marker for human and mouse ICC. Expression of Anol in human and mouse stomach, small intestine, and colon was investigated by immunofluorescence labeling using antibodies to Anol alone and in combination with antibodies to Kit. Colocalization of immunoreactivity was demonstrated by epifluorescence and confocal microscopy. In the muscularis propria, Anol immunoreactivity was restricted to cells with the morphology and distribution of ICC. All Ano 1-positive cells in the muscularis propria were also Kit positive. Kit-expressing mast cells were not Anol positive. Some non-ICC in the mucosa and submucosa of human tissues were Anol positive but Kit negative. A few (3.2%) Anol-positive cells in the human gastric muscularis propria were labeled weakly for Kit. Anol labels all classes of ICC and represents a highly specific marker for studying the distribution of ICC in mouse and human tissues with an advantage over Kit since it does not label mast cells. Kit; mast cells; chloride channels; gastrointestinal motility; immunofluorescence

Multidrug resistance-associated protein 1 (MRP1) is a cysteinyl leukotriene (CysLT) export pump expressed on mast cells. CysLTs are crucial mediators in allergic airway disease. However, biological significance of MRP1 in allergic airway inflammation has not yet been elucidated. In this study, we sensitized wild-type control mice ([mrp1.sup.+/+]) and MRP1-deficient mice ([mrp1.sup.-/-]) to ovalbumin (OVA) and challenged them with OVA by aerosol. Airway inflammation and goblet cell hyperplasia after OVA exposure were reduced in [mrpl.sup.-/-] mice compared with [mrp1.sup+/+] mice. Furthermore, CysLT levels in bronchoalveolar lavage fluid (BALF) from OVA-exposed [mrp1.sup.-/-] mice were significantly lower than those from OVA-exposed [mrp1.sup.+/+] mice. Levels of OVA-specific IgE, IL-4, and IL-13 in BALF were also decreased in OVA-exposed [mrp1.sup.-/-] mice. IgE-mediated release of CysLTs from routine bone marrow-derived mast cells was markedly impaired by MRP1 deficiency. Our results indicate that MRP1 plays an important role in the development of allergic airway inflammation through regulation of IgE-mediated CysLT export from mast cells. cysteinyl leukotrienes; mast cell

Mast cells and other cells such as macrophages have been shown to mediate systemic anaphylaxis. We determined the roles of mast cells and Kupffer ceils in hepatic and systemic anaphylaxis of rats. Roles of mast cells were examined by using the mast cell-deficient white spotting (Ws/Ws) rat; the Ws/Ws and wild type (+/+) rats were sensitized with ovalbumin (1 mg). Roles of Kupffer cells were examined by depleting Kupffer cells using gadolinium chloride or liposome-encapsulated dichloromethylene diphosphonate in the Ws/Ws and Sprague-Dawley rats. An intravenous injection of 0.6 mg ovalbumin caused substantial anaphylactic hypotension in both the Ws/Ws and +/+ rats; however, the occurrence was delayed in the Ws/Ws rats. After antigen, portal venous pressure increased by 13.1 cm[H.sub.2]O in the +/+ rats, while it increased only by 5.7 cm[H.sub.2]O in the Ws/Ws rats. In response to antigen, the isolated perfused liver of the Ws/Ws rats also showed weak venoconstriction, the magnitude of which was one tenth as large as that of the +/+ rats, indicating that hepatic anaphylaxis was primarily due to mast cells. In contrast, Kupffer cell depletion did not attenuate anaphylactic hepatic venoconstriction in isolated perfused livers. In conclusion, mast cells are involved mainly in anaphylactic hepatic presinusoidal portal venoconstriction but only in the early Stage of anaphylactic systemic hypotension in rats. Macrophages, including Kupffer ceils, do not participate in rat hepatic anaphylactic venoconstriction. blood pressure; hemodynamics; macrophages; anaphylactic shock; hepatic circulation

Abdominal aortic aneurysm (AAA), an inflammatory disease, involves leukocyte recruitment, immune responses, inflammatory cytokine production, vascular remodeling, neovascularization, and vascular cell apoptosis, all of which contribute to aortic dilatation. This [...]

A subset of CC chemokines, acting through CC chemokine receptors (CCRs) 1 to 5, is instrumental in shaping inflammatory responses. Recently, we and others have demonstrated that the atypical chemokine [...]

Bacterial infections and other pathologic conditions induce complex dysfunctions of the hypothalamic-pituitary-thyroid axis, collectively known as nonthyroidal illness (NTI). To explore the pathogenesis of bacterial NTI, we injected Mycobacterium tuberculosis extracts or Escherichia coil LPS in mice lacking key components of the Toll-like receptor or crystallizable fragment (Fc) receptor pathways. In wild-type mice, the bacterial components induced a hypothyroidism characterized by elements of both hypothalamic and thyroidal dysfunction. This NTI hypothyroidism did not develop in mice lacking the MyD88 adaptor or in those with a reduced number of mast cells. The hypothyroid responsiveness to LPS, however, was restored upon reconstitution with mast cells derived from the bone marrow of wild-type donors. In addition to bacterial components, whole immunoglobulins induced NTI hypothyroidism in wild-type mice, but not in those lacking activating Fc receptors or mast cells. The study demonstrates a link between Toll-like and Fc receptor signaling and thyroid gland function, uncovering a role of mast cells in murine NTI. innate immunity | euthyroid sick syndrome I mastocyte I hypothyroidism

Neurogenic inflammation is known to induce lowering of interstitial fluid pressure ([P.sub.if]) in mouse skin. This study examined the possible role of mast cell activation secondary to neuropeptide release in lowering of [P.sub.if] by using [Kit.sup.w]/ [Kit.sup.w-v] mice, which are devoid of mast cells, including connective tissue mast cells (CTMCs). Pie was measured in paw skin of anesthetized (fentanylfluanison and midazolam, 1:1) mice with glass capillaries connected to a servo-controlled counterpressure system. In contrast to wild-type mice, intravenous administration of mast cell-activating compound 48/80 induced no lowering of [P.sub.if] in [Kit.sup.w]/[Kit.sup.w-v]`' mice. Intravenous challenge with substance P (SP), calcitonin gene-related peptide (CGRP), or capsaicin induced a significant (P < 0.05) lowering of [P.sub.if] in wild-type mice to -2.16 [+ or -] 0.28, - 1.96 [+ or -] 0.11, and -2.22 [+ or -] 0.19 mmHg, respectively, compared with vehicle (-0.49 [+ or -] 0.11 mmHg). In [Kit.sup.w]/[Kit.sup.w-v] mice the P,- response to SP was completely abolished (-0.53 [+ or -] 0.32 mmHg) while the response to CGRP and capsaicin was attenuated (-1.33 [+ or -] 0.13 and -1.42 [+ or -] 0.13 mmHg, respectively) although significantly (P < 0.05) lowered compared with vehicle. Immunohistochemical analysis revealed no difference in distribution or density of SP- and CGRP-immunoreactive fibers in paws of [Kit.sup.w]/[Kit.sup.w-v] compared with wild-type mice. We conclude that lowering of [P.sub.if] normally depends on mast cells. However, the sensory nerves can also elicit a lowering of [P.sub.if] that is independent of mast cells. [Kit.sup.w]/[Kit.sup.w-v] mice; neuropeptides; micropuncture

Mast cells are immune sentinels that participate in the defense against bacteria and parasites. Resident within the joint, mast cells become activated in human rheumatoid arthritis and are implicated in the pathogenesis of experimental murine synovitis. However, their arthritogenic role remains undefined. Using a model of autoantibody-induced arthritis, we show that mast cells contribute to the initiation of inflammation within the joint by elaboration of IL-1. Mast cells become activated to produce this cytokine via the IgG immune complex receptor Fc[gamma]/RIII. Interestingly, mast cells become dispensable for the perpetuation of arthritis after delivery of IL-1, highlighting the contribution of this lineage to arthritis induction. These findings illuminate a mechanism by which mast cells can participate in the pathogenesis of autoimmune inflammatory arthritis and provide insights of potential relevance to human rheumatoid arthritis. cytokine | mouse model | synovitis

Irritable bowel syndrome and interstitial cystitis frequently overlap. We have shown that acute colitis sensitizes urinary bladder afferents to both mechanical and chemical stimuli and that chronic colitis similarly produces neurogenic cystitis. We hypothesize that chronic irritation of the colon releases neuropeptides from bladder afferents, leading to receptor sensitization and neurogenic inflammation. Female Sprague-Dawley rats received intrarectal trinitrobenzenesulfonic acid (TNBS) or vehicle 3 days following either systemic capsaicin (CP) pretreatment or vehicle. Ten days later, action potentials of single-unit pelvic C-fiber afferents with receptive fields in the bladder were recorded under urethane anesthesia during graded bladder distensions (UBD) or intravesical capsaicin (vCP) administration. In controls, UBD increased bladder afferent firing in proportion to intravesical pressure. At intravesical pressures of 30 mmHg and above, the percent increase in afferent firing was significantly accentuated following TNBS compared with controls (1,222 [+ or -] 176 vs. 624 [+ or -] 54%, P < 0.01). The response to vCP was also enhanced (4,126 [+ or -] 775 vs. 1,979 [+ or -] 438%, P < 0.01). Systemic depletion of neuropeptides from sensory nerves abolished these effects. Histological examination of the bladders revealed an increase in mast cell density in TNBS-treated animals compared with controls (18.02 [+ or -] 1.25 vs. 3.11 [+ or -] 0.27 mast cells/ x 100 field, P < 0.01). This effect was significantly ameliorated with CP (10.25 [+ or -] 0.95, P < 0.5 vs. TNBS-treated animals). In summary, chronic colonic irritation in the rat sensitizes urinary bladder afferents to noxious stimuli and causes mast cell infiltration in the bladder. Depletion of neuropeptides from sensory afferents diminishes these effects, suggesting they play an important role. trinitrobenzenesulfonic acid; interstitial cystitis; irritable bowel syndrome; C fiber; capsaicin; mast cell

Mast cells play a pivotal role in inflammatory and immediate-type allergic reactions by secreting a variety of potent inflammatory mediators, including sphingosine-1-phosphate (S1P). However, it is not known how S1P is released from cells. Here, we report that SIP is exported from mast cells independently of their degranulation and demonstrate that it is mediated by ATP binding cassette (ABC) transporters. Constitutive and antigen-stimulated SIP release was inhibited by MK571, an inhibitor of ABCC1 (MRP1), but not by inhibitors of ABCB1 (MDR-1, P-glycoprotein). Moreover, down-regulation of ABCC1 with small interfering RNA, which decreased its cell surface expression, markedly reduced S1P export from both rat RBL-2H3 and human LAD2 mast ceils. Transport of S1P by ABCC1 influenced migration of mast cells toward antigen but not degranulation. These findings have important implications for S1P functions in mast cell-mediated immune responses. secretion | sphingolipids | multidrug resistance | allergic responses | sphingosine kinase

Mast cells are involved in many disorders where the triggering mechanism that leads to degranulation and/or cytokine secretion has not been defined. Several chronic inflammatory diseases are associated with increased [...]

When exposed to a pathogen, a naive [CD4.sup.+] T cell is forced to make a cell fate decision that leads to a polarized population of Th1 IFN-[gamma]- or Th2 IL-4--producing [...]

We recently reported that RabGEF1 is a negative regulator of high-affinity Fc receptor for IgE (Fc[epsilon]RI)-dependent mast cell activation and that mice lacking RabGEF1 develop severe skin inflammation and increased numbers of dermal mast cells. To better understand how RabGEF1 can regulate signaling events and biological responses in mast cells, we examined the responses of bone marrow-derived cultured mast cells (BMCMCs) from wild-type (+/+) and Rabgef1 knockout (-/-) mice after stimulation with the c-Kit ligand, stem cell factor (SCF), an important regulator of mast cell development, survival, proliferation, and activation. We found that RabGEF1-deficient mast cells exhibited enhanced and prolonged activation of Ras and extracellular regulated kinase, and significantly elevated IL-6 secretion, after stimulation with SCF. SCF-induced activation of c-Jun N-terminal kinase was increased in Rabgef[1.sup.-/-] BMCMCs, but without corresponding significant increases in SCF-induced migration or adhesion. SCF-mediated activation of the survival-enhancing kinase, Akt, also was increased in Rabgef[1.sup.-/-] BMCMCs, and these cells had a survival advantage over their +/+ counterparts in vitro. Despite enhanced Ras activation in the absence of RabGEF1, SCF-induced proliferation was lower in Rabgef[1.sup.-/-] BMCMCs compared with their +/+ counterparts. Finally, we found that c-Kit internalization was delayed in the absence of RabGEF1, probably reflecting a positive role for RabGEF1 in the regulation of endocytic events, and that infection of Rabgef[1.sup.-/-] BMCMCs with a wild-type RabGEF1 lentiviral construct normalized c-Kit internalization to the levels seen in +/+ BMCMCs. Thus, RabGEF1 plays a critical role in the regulation of SCF/c-Kit-mediated signaling events and biological responses in mast cells. endocytosis | proliferation | Rab5 | Rabex-5 | survival

In a mouse experimental asthma model, the administration of bacterial lipopolysaccharide (LPS), particularly at low doses, enhances the levels of ovalbumin (OVA)-induced eosinophilic airway inflammation. In an effort to clarify the cellular and molecular basis for the LPS effect, we demonstrate that the OVA-induced eosinophilic inflammation in the lung is dramatically increased by the administration of LPS in wild-type mice, whereas such increase was not observed in mast-cell-deficient mice or Toll-like receptor (TLR)4-deficient mice. Adoptive transfer of bone-marrow-derived mast cells (BMMCs) from wild-type, but not from TLR4-deficient, mice restored the increased eosinophilic inflammation in mast-cell-deficient mice. Wild-type BMMCs pretreated with LPS in vitro also reconstituted the eosinophilic inflammation. Moreover, in vitro analysis revealed that the treatment of BMMCs with LPS resulted in NF-[kappa]B activation, sustained up-regulation of GATA1 and -2 expression, and increased the capability to produce IL-5 and -13. Dramatic increases in the expression of IL-5 and -13 and Eotaxin 2 were detected in LPS-treated BMMCs after costimulation with LPS and IgE/Ag. Overexpression of GATA1, but not GATA2, in MC9 mast cells resulted in increased transcriptional activity of IL-4, -5, and -13. Furthermore, the levels of transcription of Th2 cytokines in BMMCs were decreased by the introduction of small interfering RNA for GATA1. Thus, mast cells appear to control allergic airway inflammation after their activation and modulation through TLR4-mediated induction of GATA1 and subsequent increase in Th2 cytokine production. cytokine | GATA1 | mast cell | lipopolysaccharide | bone-marrow-derived mast cells

Recent studies have shown that accessory proteins that interact with the apical [Na.sup.+]/[H.sup.+] exchanger NHE3 are a vital part of the dynamic nature of the [Na.sup.+]/[H.sup.+] exchanger regulation. We have identified MAST205, a microtubule-associated serine/threonine kinase with a molecular mass of 205 kDa that interacts with NHE3. MAST205 contains a S/T kinase domain and a PDZ domain that mediates interaction with NHE3. Northern blot analysis showed that MAST205 is highly expressed in human and rat kidney. Expression in opossum kidney (OK) cells showed that MAST205 is predominantly expressed in the apical membrane of the cells. Immunohistochemical studies demonstrated the presence of MAST205 at the apical region of the renal proximal tubules. Heterologous expression of MAST205 in OK cells inhibited endogenous NHE3 activity, and this inhibition required the presence of the kinase domain of MAST205, since deletion of the kinase domain or a dominant-negative mutant of MAST205 did not affect the activity of NHE3. Consistent with these results, we found that MAST205 phosphorylated NHE3 under in vitro conditions. However, overexpression of MAST205 did not affect expression of NHE3 proteins, suggesting that the effect of MAST205 was not mediated by a decrease in NHE3 expression. These findings suggest that MAST205 regulates NHE3 activity and, although the precise mechanism is yet to be determined, MAST205 appears to inhibit NHE3 activity through a phosphorylation-dependent mechanism. sodium/hydrogen exchange; kinase

Recently we reported that Toll-like receptor 4 (TLR4)-positive immune cells of unknown identity were responsible for the LPS-induced depression of cardiac myocyte shortening. The aim of this study is to identify the TLR4-positive cell type that is responsible for the LPS-induced cardiac dysfunction. Neither neutrophil depletion alone nor mast cell deficiency had any impact on the impairment of myocyte shortening during LPS treatment. In contrast, LPS-treated, macrophage-deficient mice demonstrated a partial reduction in shortening compared with saline-treated, macrophage-deficient mice. Because the removal of macrophages could only partially restore myocyte shortening, we also investigated the effects of removing both neutrophils and macrophages on myocyte shortening. Interestingly, endotoxemic, neutrophil-depleted, and macrophage-deficient mice had completely restored myocyte shortening. Because both macrophages and neutrophils can produce nitric oxide (NO) and TNF-[alpha], we examined LPS-treated inducible NO synthase knockout (iNOSKO) mice and TNF receptor (TNFR)-deficient mice. Eliminating both TNFR1 and TNFR2 was required to restore myocyte shortening during LPS treatment, whereas iNOS deficiency had no effect. These data suggest that macrophages and to a lesser degree neutrophils cause cardiac impairment, presumably via TNF-[alpha]. neutrophils; mast cells; macrophages; endotoxemia; tumor necrosis factor-[alpha]

Human airway trypsinlike protease (HAT) was isolated from airway secretions and localized to bronchial epithelial cells by immunohistochemistry. In the present study, we examined whether HAT could stimulate DNA synthesis and proliferation of primary human bronchial fibroblasts (HBF). HAT significantly stimulated the proliferation of HBF by 20-55%, a level similar to that of the mitogenic activity of lung mast cell tryptase (MCT). HAT also stimulated the incorporation of [[sup.3]H]thymidine in HBF, and this HAT-induced DNA synthesis was abolished by leupeptin. Protease-activated receptor-2 (PAR-2) mRNA was expressed and localized to the cell surface in HBF. PAR-2 activating peptide (AP) also enhanced DNA synthesis, and both HAT and PAR-2 AP induced receptor internalization, similar to the response to trypsin. Pretreatment of HBF with anti-PAR-2 antibody significantly suppressed both HAT and PAR-2 AP-induced DNA synthesis. In addition, HAT and PAR-2 AP induced intracellular [Ca.sup.2+] mobilization in HBF. The HAT-induced increase in [Ca.sup.2+] was desensitized by pretreatment with trypsin or PAR-2 AP. U0126, a specific MAPK inhibitor, completely inhibited HAT-induced DNA synthesis as well as HAT-induced phosphorylation of MAPK. The effect of HAT and MCT together was additive, whereas the effect of HAT and insulin together on HBF DNA synthesis was synergistic. These results indicate that HAT stimulates fibroblast proliferation in bronchial airways through a PAR-2-dependent MEK-MAPK mediated pathway and that HAT is linked to airway processes involving fibroblasts. mast cell tryptase; mitogen-activated protein kinase

Mast cell tryptase is a potent mitogen for many cells in the airways and lung, but the cellular mechanisms for its growth stimulatory effects are poorly understood. Our major goal was to determine whether tryptase activates phosphatidylinositol 3-kinases (PI 3-kinases) in cultured dog tracheal smooth muscle cells to induce its mitogenic effects. After exposure to tryptase, cells were lysed. Immunocomplexes prepared from the lysates using an antibody to the p85 subunit of PI 3-kinase, but not using anti-phosphotyrosine antibodies, possessed increased capacity to phosphorylate inositol on its D3 hydroxyl group. Tryptase also increased phosphorylation of Akt, a downstream target of PI 3-kinases. This effect was abolished by one PI 3-kinase inhibitor, wortmannin, and attenuated by another, LY-294004, which also blocked tryptase's mitogenic effects. Treatment of tryptase with p-amidino phenylmethanesulfonyl fluoride, to abolish its proteolytic activity irreversibly, inhibited its stimulatory effects on Akt phosphorylation. Proteinase-activated receptor-2 (PAR-2)-activating peptides failed to increase Akt phosphorylation in cultured dog tracheal smooth muscle cells, but the PAR-2-activating peptides did induce brisk increases in Akt phosphorylation in Madin-Darby canine kidney cells. We concluded that tryptase activates PI 3-kinases in cultured dog tracheal smooth muscle cells to induce its potent mitogenic effects. These effects of tryptase on PI 3-kinases appear to occur via novel proteolytic mechanisms independent from PAR-2. Also, tryptase, although comparable in mitogenic potency to platelet-derived growth factor (PDGF), induces considerably less tyrosine phosphorylation on proteins than occur in response to PDGF. mast cell; airway smooth muscle hyperplasia; asthma

Histamine, released from mast cells, can modulate the activity of intrinsic neurons in the guinea pig cardiac plexus. The present study examined the ionic mechanisms underlying the histamine-induced responses in these cells. Histamine evokes a small membrane depolarization and an increase in neuronal excitability. Using intracellular voltage recording from individual intracardiac neurons, we were able to demonstrate that removal of extracellular sodium reduced the membrane depolarization, whereas inhibition of [K.sup.+] channels by 1 mM [Ba.sup.2+], 2 mM [Cs.sup.+], or 5 mM tetraethylammonium had no effect. The depolarization was also not inhibited by either 10 [micro]M [Gd.sup.3+] or a reduced [Cl.sup.-] solution. The histamine-induced increase in excitability was unaffected by [K.sup.+] channel inhibitors; however, it was reduced by either blockage of voltage-gated [Ca.sup.2+] channels with 200 [micro]M [Cd.sup.2+] or replacement of extracellular [Ca.sup.2+] with [Mg.sup.2+]. Conversely, alterations in intracellular calcium with thapsigargin or caffeine did not inhibit the histamine-induced effects. However, in cells treated with both thapsigargin and caffeine to deplete internal calcium stores, the histamine-induced increase in excitability was decreased. Treatment with the phospholipase C inhibitor U73122 also prevented both the depolarization and the increase in excitability. From these data, we conclude that histamine, via activation of [H.sub.1] receptors, activates phospholipase C, which results in 1) the opening of a nonspecific cation channel, such as a transient receptor potential channel 4 or 5; and 2) in combination with either the influx of [Ca.sup.2+] through voltage-gated channels or the release of internal calcium stores leads to an increase in excitability. tetraethylammonium; phospholipase C; mast cells; [H.sub.1] receptor

Basophils and mast cells, which are selectively endowed with the high-affinity IgE receptor and mediate a range of adaptive and innate immune responses, have an unknown developmental relationship. Here, by evaluating the expression of the [beta]7 integrin, a molecule that is required for selective homing of mast cell progenitors (MCPs) to the periphery, we identified bipotent progenitors that are capable of differentiating into either cell type in the mouse spleen. These basophil/mast cell progenitors (BMCPs) gave rise to basophils and mast cells at the single-cell level and reconstituted both mucosal and connective tissue mast cells. We also identified the basophil progenitor (BaP) and the MCP in the bone marrow and the gastrointestinal mucosa, respectively. We further show that the granulocyte-related transcription factor CCAAT/ enhancer-binding protein [alpha] (C/EBP[alpha]) plays a primary role in the fate decision of BMCPs, being expressed in BaPs but not in MCPs. Thus, circulating basophils and tissue mast cells share a common developmental stage at which their fate decision might be controlled principally by C/EBP[alpha]. integrin | progenitor | CCAAT/enhancer-binding protein

Mast cells play a key role in allergy and asthma. They reside at the host-environment interface and are among the first cells to make contact with inhaled microorganisms and particulate antigens. Pulmonary surfactant proteins A and D (SP-A and SP-D) function in lung host defense by enhancing microbe phagocytosis and mediating other immune cell functions, but little is known about their effects on mast cells. We hypothesized that SP-A and/or SP-D modulate IgE-dependent mast cell functions. Pollen starch granules (PSG) extracted from Dactylis glomerata and coated with trinitrophenol (TNP) were used as a model of an inhaled organic particulate allergen. Our data revealed that SP-D inhibited by 50% the release of [beta]-hexosaminidase by peritoneal mast cells sensitized with IgE anti-TNP and stimulated with TNP-PSG. In contrast, SP-A had no effect. Furthermore, SP-D aggregated PSG in a dose-dependent manner, and this aggregation was mediated by SP-D's carbohydrate recognition domain. A single arm SP-D mutant (RrSP-Dser 15,20) neither aggregated PSG nor inhibited degranulation, suggesting that multimefization of SP-D is required for maximal PSG aggregation and inhibition of PSG-induced mast cell degranulation. This study is the first to demonstrate that SP-D modulates IgE-mediated mast cell functions, which are important in asthma and allergic inflammation. collectins; allergen; lung; innate immunity; immunoglobulin E

It is well known that mast cells are derived from hematopoietic stem cells. However, in adult hematopoiesis, a committed mast cell progenitor has not yet been identified in any species, nor is it clear at what point during adult hematopoiesis commitment to the mast cell lineage occurs. We identified a cell population in adult mouse bone marrow, characterized as [Lin.sup.-]c-[Kit.sup.+] [Sca-1.sup.-][Ly6c.sup.-][Fc[epsilon]Rl[[alpha].sup.-][CD27.sup. -][[beta]7.sup.+]T1/[ST2.sup.+], that gives rise only to mast cells in culture and that can reconstitute the mast cell compartment when transferred into c-kit mutant mast cell-deficient mice. In addition, our experiments strongly suggest that these adult mast cell progenitors are derived directly from multipotential progenitors instead of, as previously proposed, common myeloid progenitors or granulocyte/macrophage progenitors. hematopoietic stem cell | multipotential progenitor

Mast cells are not only important effector cells in immediate hypersensitivity reactions and immune responses to pathogens but also can contribute to T cell-mediated disorders. However, the mechanisms by which mast cells might influence T cells in such settings are not fully understood. We find that mast cells can enhance proliferation and cytokine production in multiple T cell subsets. Mast cell-dependent enhancement of T cell activation can be promoted by Fc[epsilon]RI-dependent mast cell activation, TNF production by both mast cells and T cells, and mast cell-T cell contact. However, at high concentrations of cells, mast cells can promote T cell activation independent of IgE or TNF. Finally, mast cells also can promote T cell activation by means of soluble factors. These findings identify multiple mechanisms by which mast cells can influence T cell proliferation and cytokine production. allergy | asthma | autoimmunity | cytokines | immune response

Recent reports including those from our laboratories indicate that hyperhomocysteinemia (Hhe) is an independent risk factor for cardiac dysfunction and clinical heart failure. Mast cell accumulation is a prominent feature in our model of Hhe-induced cardiac dysfunction. Because mast cell-derived mediators can potentially attenuate cardiac remodeling, we investigated the possible protective role of mast cells in Hhe-induced cardiac remodeling using a mast cell-deficient rat model that in our recent report did not demonstrate any adverse cardiac function at younger age (6 mo) than mast cell-competent control animals. Mast cell-deficient (Ws/Ws) rats and mast cell-competent (+/+) littermate control animals (3 mo of age) were treated with a Hhe-inducing diet for 10 wk. Cardiac remodeling was assessed structurally utilizing histomorphometric methods and functionally using an isolated Langendorff-perfused heart preparation. The Hhe-inducing diet caused similar elevations of homocysteine levels in the two groups. Compared with Hhe +/+ rats, the Hhe Ws/Ws rats demonstrated strikingly exacerbated adverse cardiac remodeling and myocardial fibrosis. Cardiac function measurement showed worsened diastolic function in Hhe Ws/Ws rats compared with Hhe +/+ rats. The absence of mast cells strikingly exacerbates Hhe-induced adverse cardiac remodeling and diastolic dysfunction. These findings indicate a potential dual rather than sole deleterious role for mast cells in cardiac injury. myocardial fibrosis; diastolic dysfunction; collagen

Upregulation of CGRP-immunoreactive (IR) primary afferent nerve fibers accompanied by mastocytosis is characteristic for the Schistosoma mansoni-infected murine ileum. These mucosal mast cells (MMC) and CGRP-IR fibers, which originate from dorsal root (DRG) and nodose ganglia, are found in close apposition. We examined interactions between primary cultured MMC and CGRP-IR DRG neurons in vitro by confocal recording of intracellular [Ca.sup.2+] concentration ([[Ca.sup.2+].sub.i]). The degranulatory E[C.sub.50] for the mast cell secretagogue compound 48/80 (C48/80; 10 [micro]g/ml) and the neuropeptides CGRP (2.[10.sup.-8] M) and substance P (SP; 3.[10.sup.-8] M) were determined by measurement of extracellular release of the granule chymase, mouse mast cell protease-1. Application of C48/80 (10 [micro]g/ml) and CGRP and SP (both [10.sup.-7] M) to Fluo-4-loaded MMC induced a transient rise in [[Ca.sup.2+].sub.i]] after a lag time, indicative of mast cell degranulation and/or secretion. The CGRP response could be completely blocked by pertussis toxin (2 [micro]g/ml), indicating involvement of [G.sub.i] proteins. Application of MMC juice, obtained by C48/80 degranulation of MMC, to Fluo-4-loaded DRG neurons induced in all neurons a rise in [[Ca.sup.2+].sub.i], indicative of activation. Degranulation of MMC by C48/80 in culture dishes containing Fluo-4-loaded DRG neurons also caused activation of the DRG neurons. In conclusion, these results demonstrate a bidirectional cross-talk between cultured MMC and CGRP-IR DRG neurons in vitro. This indicates that such a communication may be the functional relevance for the close apposition between MMC and CGRP-IR nerve fibers in vivo. calcium imaging; electrophysiology; cultured mucosal mast cells; cultured dorsal root ganglia neurons: mast cell juice

Blood substitutes, such as diaspirin cross-linked Hb (DBBF-Hb), have been considered for use during blood transfusions. Unfortunately, bolus injection of modified Hb has been shown to rapidly increase the leakage of microvessels to plasma albumin. This effect may result from production of excess reactive oxygen species (ROS) and could be linked to the observed increase in degranulated mast cells (DMC). Disodium cromoglycate (cromolyn) stabilizes mast cells and therefore might minimize the venular permeability in the rat mesentery. In 10 anesthetized Sprague-Dawley rats, the mesenteric preparation was continuously suffused with cromolyn while the microvasculature was filled with DBBF-Hb solution (10 mg/ml) for 10 min. Six animals received cromolyn pretreatment [two intravascular injections over 30 min (experiment A)] and four animals received pretreatment with 2% HEPES-buffered saline (HBS)-BSA (experiment B). Two more animals were pretreated with HBS-BSA without DBBF-Hb infusion but with cromolyn suffusion (experiment C). Another set of experiments was performed on five animals without cromolyn suffusion or any pretreatment but with DBBF-Hb infusion (experiment D). All groups then received a l-min perfusion of FITC-albumin, fixation for 60 min, and microscopic examination. Experiments A and B demonstrated a significant reduction in the number of venular leaks and DMC compared with experiment D, but not in the area of venular leaks. These results suggest mast cell degranulation is not a major contributor to microvascular leakage induced by DBBF-Hb. blood substitutes; cromolyn; venular leaks; diaspirin cross-linked hemoglobin

Mast cell activation, or neurogenic inflammation, is known to induce lowering of interstitial fluid pressure ([P.sub.if]) and plasma protein extravasation (PPE) in several tissues from both rats and mice. To examine a possible role of connective tissue mast cells (CTMCs) in these inflammatory responses, we used mice with dysfunctional CTMCs due to lack of the N-deacetylase/N-sulfotransferase-2 enzyme (NDST-[2.sup.-/-]). Pit and PPE were measured after challenge with compound 48/80 (C48/80), and [P.sub.if] alone was measured after treatment either with capsaicin, substance P (SP), or calcitonin gene-related peptide (CGRP). Measurements of [P.sub.if] in anesthetized (fentanyl/fluanison and midazolam, 1:1) mice were performed in paw skin with glass capillaries connected to a servo-controlled counterpressure system. PPE was measured with microdialysis by using hollow plasmapheresis fibers (cutoff at 3,000 kDa) placed subcutaneously on the back. Intravenous administration of C48/80 lowered [P.sub.if] significantly (P < 0.05) in NDST-[2.sub.-/-] mice (-1.67 [+ or -] 0.42 mmHg) compared with vehicle (0.57 [+ or -] 0.17 mmHg) but the lowering was significantly (P < 0.05) less compared with that of the NDST-[2.sup.+/+] mice (-2.31 [+ or -] 0.47 mmHg). PPE was increased 300% after treatment with C48/80 in NDST-[2.sup.+/+] mice, whereas there was no increase in PPE in NDST-[2.sup.-/-] mice. Capsaicin, SP, and CGRP lowered Pif significantly (P < 0.05) compared with vehicle and to the same extent in both NDST-[2.sup.+/+] and NDST-[2.sup.-/-] mice. We can conclude that although NDST-[2.sup.-/-] mice demonstrate an altered response in [P.sub.if] after mast cell activation, there was no similar alteration after neurogenic inflammation. Therefore, we suggest that neurogenic inflammation in mouse skin is not exclusively dependent on intact CTMCs. N-deacetylase/N-sulfotransferase-2; mast cell: capsaicin; peptides; micropuncture

Byline: Valeria Solari (1,2), Kei Unemoto (1,2), Anna Piaseczna Piotrowska (1,2), Prem Puri (1,2) Keywords: Mast cells; Reflux nephropathy; Renal scarring Abstract: End-stage renal disease is characterized by interstitial infiltrate of inflammatory cells in association with tubular atrophy and interstitial fibrosis. Mast cells (MCs) secrete a large number of fibrogenic factors and have been implicated in chronic inflammatory processes with fibrous tissue deposition. The aim of this study was to investigate the distribution of MCs in kidneys with reflux nephropathy (RN) and to determine the relationship between MCs and the interstitial fibrotic process in RN. Kidney specimens from 12 patients (aged 2--13 years) with severe RN secondary to primary high-grade vesicoureteral reflux, obtained at the time of nephrectomy, and 5 controls were examined. Sections were investigated histochemically by acid toluidine blue (TB) and immunohistochemically with antibodies for anti MC-tryptase, MC-chymase, c-kit (CD117), and fibronectin. Double staining for fibronectin and MC-tryptase was performed and examined using confocal scanning microscopy. TB histochemistry showed a marked increase of MCs in RN specimens compared with controls. MC-tryptase, chymase, and c-kit immunopositive MC infiltration was significantly higher in RN samples (14.2+-9.6) than controls (1.3+-0.8), (P&lt 0.05). In all the sections there were more MC-tryptase-positive cells than MC-chymase-positive MCs. Double staining showed increased immunoreactivity of MCs and fibrosis in the renal interstitium of kidneys with RN. The number of infiltrating tryptase-positive MCs was correlated with the degree of interstitial renal scarring. This study demonstrates for the first time the increased expression of MCs in RN, suggesting that MCs may be involved in the development of scarring in RN. Author Affiliation: (1) Children&apos;s Research Centre, Our Lady&apos;s Hospital for Sick Children, Crumlin, Dublin 12, Ireland (2) University College, Dublin, Ireland Article History: Registration Date: 04/09/2003 Received Date: 11/06/2003 Accepted Date: 18/08/2003 Online Date: 16/12/2003

We have investigated the influence of mast cells on the barrier function of intestinal epithelium during nematode infection. Trichinella spiralis infection induces a strong type 2 cytokine-mediated inflammation, resulting in a critical mucosal mastocytosis that is known to mediate expulsion of the parasites from the intestine. The host response to infection is also characterized by an increase in mucosal leakiness. We show here that intestinal epithelial permeability is markedly elevated during infection, with kinetics that mirror the adaptive immune response to primary and secondary infection. Furthermore, we have identified degradation of the tight junction protein, occludin, thereby providing a mechanism for increased paracellular permeability during helminth infection. We further demonstrate by using anti-c-kit antibody and IL-9 transgenic mice that mast cells are directly responsible for increasing epithelial paracellular permeability and that mice deficient in a mast cell-specific protease fail to increase intestinal permeability and fail to expel their parasite burden. These results provide the mechanism whereby mucosal mast cells mediate parasite expulsion from the intestine.