Adipose triglyceride lipase regulates eicosanoid production in activated human mast cells

Human mast cells (MCs) contain TG-rich cytoplasmic lipid droplets (LDs) with high arachidonic acid (AA) content. Here, we investigated the functional role of adipose TG lipase (ATGL) in TG hydrolysis and the ensuing release of AA as substrate for eicosanoid generation by activated human primary MCs in culture. Silencing of ATGL in MCs by siRNAs induced the accumulation of neutral lipids in LDs. IgE-dependent activation of MCs triggered the secretion of the two major eicosanoids, prostaglandin D2 (PGD2) and leukotriene C4 (LTC4). The immediate release of PGD2 from the activated MCs was solely dependent on cyclooxygenase (COX) 1, while during the delayed phase of lipid mediator production, the inducible COX-2 also contributed to its release. Importantly, when ATGL-silenced MCs were activated, the secretion of both PGD2 and LTC4 was significantly reduced. Interestingly, the inhibitory effect on the release of LTC4 was even more pronounced in ATGL-silenced MCs than in cytosolic phospholipase A₂-silenced MCs. These data show that ATGL hydrolyzes AA-containing TGs present in human MC LDs and define ATGL as a novel regulator of the substrate availability of AA for eicosanoid generation upon MC activation.     

IL-16 regulation of human mast cells/basophils and their susceptibility to HIV-1

AIDS patients often contain HIV-1-infected mast cells (MCs)/basophils in their peripheral blood, and in vivo-differentiated MCs/basophils have been isolated from the blood of asthma patients that are HIV-1 susceptible ex vivo due to their surface expression of CD4 and varied chemokine receptors. Because IL-16 is a ligand for CD4 and/or an undefined CD4-associated protein, the ability of this multifunctional cytokine to regulate the development of human MCs/basophils from nongranulated progenitors residing in cord or peripheral blood was evaluated. After 3 wk of culture in the presence of c-kit ligand, IL-16 induced the progenitors residing in the blood of normal individuals to increase their expression of chymase and tryptase about 20-fold. As assessed immunohistochemically, >80% of these tryptase(+) and/or chymase(+) cells expressed CD4. The resulting cells responded to IL-16 in an in vitro chemotaxis assay, and this biologic response could be blocked by anti-IL-16 and anti-CD4 Abs as well as by a competitive peptide inhibitor corresponding to a sequence in the C-terminal domain of IL-16. The additional finding that IL-16 induces calcium mobilization in the HMC-1 cell line indicates that IL-16 acts directly on MCs and their committed progenitors. IL-16-treated MCs/basophils also are less susceptible to infection by an M/R5-tropic strain of HIV-1. Thus, IL-16 regulates MCs/basophils at a number of levels, including their vulnerability to retroviral infection.     

Cutting edge: Interleukin 4-dependent mast cell proliferation requires autocrine/intracrine cysteinyl leukotriene-induced signaling

Reactive mastocytosis (RM) in epithelial surfaces is a consistent Th2-associated feature of allergic disease. RM fails to develop in mice lacking leukotriene (LT) C4 synthase (LTC4S), which is required for cysteinyl leukotriene (cys-LT) production. We now report that IL-4, which induces LTC4S expression by mast cells (MCs), requires cys-LTs, the cys-LT type 1 receptor (CysLT1), and Gi proteins to promote MC proliferation. LTD4 (10-1000 nM) enhanced proliferation of human MCs in a CysLT1-dependent, pertussis toxin-sensitive manner. LTD4-induced phosphorylation of ERK required transactivation of c-kit. IL-4-driven comitogenesis was likewise sensitive to pertussis toxin or a CysLT1-selective antagonist and was attenuated by treatment with leukotriene synthesis inhibitors. Mouse MCs lacking LTC4S or CysLT1 showed substantially diminished IL-4-induced comitogenesis. Thus, IL-4 induces proliferation in part by inducing LTC4S and cys-LT generation, which causes CysLT1 to transactivate c-kit in RM.     

Leukotriene B4 receptors BLT1 and BLT2: expression and function in human and murine mast cells

Leukotriene B4 (LTB4) is a potent activator and chemoattractant for leukocytes and is implicated in several inflammatory diseases. The actions of LTB4 are mediated by two cell surface receptors, BLT1, which is predominantly expressed in peripheral blood leukocytes, and BLT2, which is expressed more ubiquitously. Recently, BLT1 expression and LTB4-dependent chemotaxis have been reported in immature mast cells (MCs). We now show the first evidence for BLT2 mRNA expression, in addition to BLT1, in murine bone marrow-derived MCs (mBMMCs) and in a human MC line (HMC-1). Protein expression of BLT1 was confirmed by mAb staining in HMC-1 cells and shown to be predominantly intracellular. Both HMC-1 cells and mBMMCs migrated to LTB4 in a dose-dependent manner in chemotaxis assays. Migration to LTB4 could be inhibited by either a BLT1- or BLT2-selective antagonist. Significant dose-dependent migration of mBMMCs also was observed to 12-(S)-hydroxyeicosotetraenoic acid, a BLT2-selective agonist, demonstrating functional BLT2 activity in these cells. Stimulation of mBMMCs with LTB4 induced transient, dose-dependent, ERK phosphorylation and changes in Akt phosphorylation. Dose-dependent ERK phosphorylation also was observed in response to 12-(S)-hydroxyeicosotetraenoic acid, indicating signaling downstream of BLT2. Pretreatment of mBMMCs with stem cell factor significantly down-regulated expression of BLT1 and BLT2 mRNA and inhibited their migration to LTB4. This study demonstrates expression of functional LTB4 receptors, both BLT1 and BLT2, in murine and human MCs and a regulatory role for stem cell factor in their expression. These receptors may mediate recruitment and accumulation of MCs in response to LTB4 production in areas of inflammation.     

Mast cell protease 5 mediates ischemia-reperfusion injury of mouse skeletal muscle

Ischemia with subsequent reperfusion (IR) injury is a significant clinical problem that occurs after physical and surgical trauma, myocardial infarction, and organ transplantation. IR injury of mouse skeletal muscle depends on the presence of both natural IgM and an intact C pathway. Disruption of the skeletal muscle architecture and permeability also requires mast cell (MC) participation, as revealed by the fact that IR injury is markedly reduced in c-kit defective, MC-deficient mouse strains. In this study, we sought to identify the pathobiologic MC products expressed in IR injury using transgenic mouse strains with normal MC development, except for the lack of a particular MC-derived mediator. Histologic analysis of skeletal muscle from BALB/c and C57BL/6 mice revealed a strong positive correlation (R(2) = 0.85) between the extent of IR injury and the level of MC degranulation. Linkage between C activation and MC degranulation was demonstrated in mice lacking C4, in which only limited MC degranulation and muscle injury were apparent. No reduction in injury was observed in transgenic mice lacking leukotriene C(4) synthase, hemopoietic PGD(2) synthase, N-deacetylase/N-sulfotransferase-2 (enzyme involved in heparin biosynthesis), or mouse MC protease (mMCP) 1. In contrast, muscle injury was significantly attenuated in mMCP-5-null mice. The MCs that reside in skeletal muscle contain abundant amounts of mMCP-5 which is the serine protease that is most similar in sequence to human MC chymase. We now report a cytotoxic activity associated with a MC-specific protease and demonstrate that mMCP-5 is critical for irreversible IR injury of skeletal muscle.     

Lipid body formation during maturation of human mast cells

Lipid droplets, also called lipid bodies (LB) in inflammatory cells, are important cytoplasmic organelles. However, little is known about the molecular characteristics and functions of LBs in human mast cells (MC). Here, we have analyzed the genesis and components of LBs during differentiation of human peripheral blood-derived CD34(+) progenitors into connective tissue-type MCs. In our serum-free culture system, the maturing MCs, derived from 18 different donors, invariably developed triacylglycerol (TG)-rich LBs. Not known heretofore, the MCs transcribe the genes for perilipins (PLIN)1-4, but not PLIN5, and PLIN2 and PLIN3 display different degrees of LB association. Upon MC activation and ensuing degranulation, the LBs were not cosecreted with the cytoplasmic secretory granules. Exogenous arachidonic acid (AA) enhanced LB genesis in Triacsin C-sensitive fashion, and it was found to be preferentially incorporated into the TGs of LBs. The large TG-associated pool of AA in LBs likely is a major precursor for eicosanoid production by MCs. In summary, we demonstrate that cultured human MCs derived from CD34(+) progenitors in peripheral blood provide a new tool to study regulatory mechanisms involving LB functions, with particular emphasis on AA metabolism, eicosanoid biosynthesis, and subsequent release of proinflammatory lipid mediators from these cells.     

CD117+ Dendritic and Mast Cells Are Dependent on RasGRP4 to Function as Accessory Cells for Optimal Natural Killer Cell-Mediated Responses to Lipopolysaccharide

Ras guanine nucleotide-releasing protein-4 (RasGRP4) is an evolutionarily conserved calcium-regulated, guanine nucleotide exchange factor and diacylglycerol/phorbol ester receptor. While an important intracellular signaling protein for CD117⁺ mast cells (MCs), its roles in other immune cells is less clear. In this study, we identified a subset of in vivo-differentiated splenic CD117⁺ dendritic cells (DCs) in wild-type (WT) C57BL/6 mice that unexpectedly contained RasGRP4 mRNA and protein. In regard to the biologic significance of these data to innate immunity, LPS-treated splenic CD117⁺ DCs from WT mice induced natural killer (NK) cells to produce much more interferon-γ (IFN-γ) than comparable DCs from RasGRP4-null mice. The ability of LPS-responsive MCs to cause NK cells to increase their expression of IFN-γ was also dependent on this intracellular signaling protein. The discovery that RasGRP4 is required for CD117⁺ MCs and DCs to optimally induce acute NK cell-dependent immune responses to LPS helps explain why this signaling protein has been conserved in evolution.     

High density of tryptase‐positive mast cells in human colorectal cancer: a poor prognostic factor related to protease‐activated receptor 2 expression

Tryptase(+) mast cells (MCs), abundant in the invasive front of tumours, contribute to tissue remodelling. Indeed, protease‐activated receptor‐2 (PAR‐2) activation by MC‐tryptase is considered an oncogenic event in colorectal cancer (CRC). Recently, we have suggested NHERF1 as a potential new marker in CRC. In this study, we aimed to determine the distribution of tryptase(+) MCs and PAR‐2 and to examine the relationship between PAR‐2 and NHERF1, investigating their reputed usefulness as tumour markers. We studied a cohort of 115 CRC specimens including primary cancer (C) and adjacent normal mucosa (NM) by immunohistochemical double staining, analyzing the protein expression of MC‐tryptase, PAR‐2 and cytoplasmic NHERF1. MC density was higher in NM than in C. Tumours with high TNM stage and poor grade showed the highest MC density. A higher PAR‐2 immunoreactivity characterized tumours most infiltrated by MCs compared with samples with low MC density. Furthermore, PAR‐2 overexpression was associated with advanced TNM stage, poor grade and lymphovascular invasion (LVI). A positive correlation existed between tryptase(+) MC density and PAR‐2 expression. Cytoplasmic NHERF1 was higher in C than in NM and overexpressing tumours resulted associated with nodal and distant metastases, poor grade and LVI. PAR‐2 correlated with cytoplasmic NHERF1 and the PAR‐2(+)/cytoplasmic NHERF1(+) expression immunophenotype identified tumours associated with unfavourable prognosis and aggressive clinical parameters. Our data indicate that the high density of tryptase(+) MCs at invasive margins of tumours was associated with advanced stages of CRC and was strongly correlated with PAR‐2 expression.     

Regulation and function of the protein inhibitor of nitric oxide synthase (PIN)/dynein light chain 8 (LC8) in a human mast cell line

The protein inhibitor of nitric oxide synthase (PIN) was independently identified as an inhibitor of nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS), and as a member of the cellular dynein light chain family, dynein light chain 8 (LC8), responsible for intracellular protein trafficking. Mast cells (MC) are involved in several homeostatic and pathological processes and can be regulated by NO. This study describes the expression of PIN/LC8 in the human MC line HMC-1. We also studied if PIN/LC8 binds nNOS, and what role this might have in leukotriene (LT) production. We found that PIN/LC8 mRNA and protein was expressed in HMC-1. Using a GST-PIN construct, we showed PIN binds to nNOS, but not endothelial (e)NOS in HMC-1; in our studies HMC-1 did not express inducible (i)NOS. Intracellular delivery of anti-PIN/LC8 antibody enhanced ionophore (A23187)-induced LT production through an unknown mechanism. Thus we established for the first time expression of PIN/LC8 in human MC, its ability to bind nNOS, and the effect that blocking it has on LT production in a human MC lines.     

Transgenic expression of RasGRP1 induces the maturation of double-negative thymocytes and enhances the production of CD8 single-positive thymocytes

RasGRP1 is a guanine nucleotide exchange factor for Ras that is required for the efficient production of both CD4 and CD8 single-positive thymocytes. We found that RasGRP1 expression is rapidly up-regulated in double-negative thymocytes following pre-TCR ligation. Transgenic overexpression of RasGRP1 compensated for deficient pre-TCR signaling in vivo, enabling recombinase-activating gene 2(-/-) double-negative thymocytes to mature to the double-positive stage. RasGRP1 transgenic mice had a 4-fold increase in CD8 single-positive thymocytes, most of which had atypically low levels of CD3. The RasGRP1 transgene lowered the threshold of TCR signaling needed to initiate proliferation of single-positive thymocytes, with this effect being particularly evident among CD8 single-positive cells. In 3-day cultures, TCR stimulation via anti-CD3 caused a 10-fold increase in the ratio of CD8 to CD4 thymocytes among RasGRP1 transgenic vs nontransgenic thymocytes. These results demonstrate that in addition to driving the double-negative to double-positive transition, increased expression of RasGRP1 selectively increases CD8 single-positive thymocyte numbers and enhances their responsiveness to TCR signaling.     

Kit (W-sh) mice develop earlier and more severe experimental autoimmune encephalomyelitis due to absence of immune suppression

Mast cells (MCs) have been thought to play a pathogenic role in the development of autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. However, an immunoregulatory function of these cells has recently been suggested. We investigated the role of MCs in EAE using the W(-sh) mouse strain, which is MC deficient. W(-sh) mice developed earlier and more severe clinical and pathological disease with extensive demyelination and inflammation in the CNS. The inflammatory cells were mainly composed of CD4(+) T cells, monocyte/macrophages, neutrophils, and dendritic cells. Compared with wild-type mice, MC-deficient mice exhibited an increased level of MCP-1/CCR2 and CD44 expression on CD4(+) T cells in addition to decreased production of regulatory T cells, IL-4, IL-5, IL-27, and IL-10. We also found that levels of IL-17, IFN-γ, and GM-CSF were significantly increased in peripheral lymphocytes from immunized W(-sh) mice compared with those in peripheral lymphocytes from wild-type mice. Reconstitution of W(-sh) mice downregulated susceptibility to EAE, which correlated with MC recruitment and regulatory T cell activation in the CNS. These findings indicate that responsiveness is not required in the pathogenesis of inflammatory demyelination in the CNS and that, in the absence of MCs, increased MCP-1, CCR2, IL-17, IFN-γ, CD44, and other inflammatory molecules may be responsible for increased severity of EAE.     

Human intestinal fibroblasts prevent apoptosis in human intestinal mast cells by a mechanism independent of stem cell factor, IL-3, IL-4, and nerve growth factor

In rodents, fibroblasts (FBs) mediate stem cell factor (SCF)-dependent growth of mast cells (MCs). In humans, SCF is mandatory for MC differentiation and survival. Other factors such as IL-3, IL-4, and nerve growth factor (NGF) act in synergism with SCF, thus enhancing proliferation and/or preventing apoptosis in MCs. In this study, we studied in vitro interactions between human MCs and human FBs, both isolated from the intestine and purified to homogeneity. In coculture with FBs, MCs survived for up to 3 wk, whereas purified MCs cultured alone died within a few days. TNF-alpha and IL-1beta, which both did not affect MC survival directly, enhanced FB-dependent MC growth. We provide evidence that FB-derived MC growth factors are soluble, heat-sensitive molecules which down-regulate MC apoptosis without enhancing MC proliferation. However, only low amounts of SCF were measured in FB-conditioned medium (<0.2 ng/ml). Moreover, blocking of SCF/c-kit interaction by anti-SCF or anti-c-kit Abs and neutralization of IL-3, IL-4, and NGF did not affect MC survival in the coculture system. In conclusion, our data indicate that human FBs promote survival of human MCs by mechanisms independent of SCF, IL-3, IL-4, and NGF. Such interactions between MCs and FBs may explain why MCs accumulate at sites of inflammatory bowel disease and intestinal fibrosis.     

Preferential development of CD4 and CD8 T regulatory cells in RasGRP1-deficient mice

RasGRP1 and Sos are two Ras-guanyl-nucleotide exchange factors that link TCR signal transduction to Ras and MAPK activation. Recent studies demonstrate positive selection of developing thymocytes is crucially dependent on RasGRP1, whereas negative selection of autoreactive thymocytes appears to be RasGRP1 independent. However, the role of RasGRP1 in T regulatory (Treg) cell development and function is unknown. In this study, we characterized the development and function of CD4(+)CD25(+)Foxp3(+) and CD8(+)CD44(high)CD122(+) Treg lineages in RasGRP1(-/-) mice. Despite impaired CD4 Treg cell development in the thymus, the periphery of RasGRP1(-/-) mice contained significantly increased frequencies of CD4(+)Foxp3(+) Treg cells that possessed a more activated cell surface phenotype. Furthermore, on a per cell basis, CD4(+)Foxp3(+) Treg cells from mutant mice are more suppressive than their wild-type counterparts. Our data also suggest that the lymphopenic environment in the mutant mice plays a dominant role of favored peripheral development of CD4 Treg cells. These studies suggest that whereas RasGRP1 is crucial for the intrathymic development of CD4 Treg cells, it is not required for their peripheral expansion and function. By contrast to CD4(+)CD25(+)Foxp3(+) T cells, intrathymic development of CD8(+)CD44(high)CD122(+) Treg cells is unaffected by the RasGRP1(-/-) mutation. Moreover, RasGRP1(-/-) mice contained greater numbers of CD8(+)CD44(high)CD122(+) T cells in the spleen, relative to wild-type mice. Activated CD8 Treg cells from RasGRP1(-/-) mice retained their ability to synthesize IL-10 and suppress the proliferation of wild-type CD8(+)CD122(-) T cells, albeit at a much lower efficiency than wild-type CD8 Treg cells.     

RasGRP1 transmits prodifferentiation TCR signaling that is crucial for CD4 T cell development

TCR signaling plays a governing role in both the survival and differentiation of bipotent double-positive thymocytes into the CD4(+) and CD8(+) single-positive T cell lineages. A central mediator of this developmental program is the small GTPase Ras, emitting cytoplasmic signals through downstream MAPK pathways and eventually affecting gene expression. TCR signal transduction orchestrates the activation of Ras by integrating at least two Ras-guanyl nucleotide exchange factors, RasGRP1 and Sos. In this study, we have characterized the relationship between RasGRP1 function and its potential roles in promoting ERK activity, cell survival, maturation, and lineage commitment. Investigations on RasGRP1(-/-) mice expressing a transgenic (Tg) MHC class II-restricted TCR revealed that the development of CD4 T cells expressing this Tg TCR is completely dependent on RasGRP1. Unexpectedly, a small number of functional CD8 single-positive thymocytes expressing the Tg MHC class II-restricted TCR exists in mutant mice. In addition, RasGRP1(-/-) double-positive thymocytes exhibit marked deficits in TCR-stimulated up-regulation of the positive selection marker CD69 and the antiapoptotic protein Bcl-2, whereas CD5 induction is unaffected. To evaluate the role of RasGRP1 in providing cellular survival signaling, we enforced Bcl-2 expression in RasGRP1(-/-) thymocytes. These studies demonstrate that RasGRP1 function cannot be fully complemented by Tg Bcl-2 expression. Therefore, we propose that RasGRP1 transmits differentiation signaling critically required for CD4 T cell development.