Genotype-dependent effects of TGF-ß1 on mast cell function: targeting the Stat5 pathway.

We previously demonstrated that TGF-ß1 suppresses IgE-mediated signaling in human and mouse mast cells in vitro, an effect that correlated with decreased expression of the high-affinity IgE receptor, FcεRI. The in vivo effects of TGF-ß1 and the means by which it suppresses mast cells have been less clear. This study shows that TGF-ß1 suppresses FcεRI and c-Kit expression in vivo. By examining changes in cytokine production concurrent with FcεRI expression, we found that TGF-ß1 suppresses TNF production independent of FcεRI levels. Rather, IgE-mediated signaling was altered. TGF-ß1 significantly reduced expression of Fyn and Stat5, proteins critical for cytokine induction. These changes may partly explain the effects of TGF-ß1, because Stat5B overexpression blocked TGF-mediated suppression of IgE-induced cytokine production. We also found that Stat5B is required for mast cell migration toward stem cell factor, and that TGF-ß1 reduced this migration. We found evidence that genetic background may alter TGF responses. TGF-ß1 greatly reduced mast cell numbers in Th1-prone C57BL/6, but not Th2-prone 129/Sv mice. Furthermore, TGF-ß1 did not suppress IgE-induced cytokine release and did increase c-Kit-mediated migration in 129/Sv mast cells. These data correlated with high basal Fyn and Stat5 expression in 129/Sv cells, which was not reduced by TGF-ß1 treatment. Finally, primary human mast cell populations also showed variable sensitivity to TGF-ß1-mediated changes in Stat5 and IgE-mediated IL-6 secretion. We propose that TGF-ß1 regulates mast cell homeostasis, and that this feedback suppression may be dependent on genetic context, predisposing some individuals to atopic disease.

ADAM10 is required for SCF-induced mast cell migration.

A Disintegrin and Metalloproteinase (ADAM)-10 plays critical roles in neuronal migration and distribution. Recently, ADAM10 deletion was shown to disrupt myelopoiesis. We found that inducible deletion of ADAM10 using Mx1-driven Cre recombinase for a period of three weeks resulted in mast cell hyperplasia in the skin, intestine and spleen. Mast cells express surface ADAM10 in vitro and in vivo, at high levels compared to other immune cells tested. ADAM10 is important for mast cell migration, since ADAM10-deficiency reduced c-Kit-mediated migration. As with some mast cell proteases, ADAM10 expression could be altered by the cytokine microenvironment, being inhibited by IL-10 or TGFß1, but not by several other T cell-derived cytokines. Collectively these data show that the ADAM10 protease is an important factor in mast cell migration and tissue distribution, and can be manipulated by environmental cues.

IL-4 and TGF-beta 1 counterbalance one another while regulating mast cell homeostasis.

Mast cell responses can be altered by cytokines, including those secreted by Th2 and regulatory T cells (Treg). Given the important role of mast cells in Th2-mediated inflammation and recent demonstrations of Treg-mast cell interactions, we examined the ability of IL-4 and TGF-beta1 to regulate mast cell homeostasis. Using in vitro and in vivo studies of mouse and human mast cells, we demonstrate that IL-4 suppresses TGF-beta1 receptor expression and signaling, and vice versa. In vitro studies demonstrated that IL-4 and TGF-beta1 had balancing effects on mast cell survival, migration, and FcepsilonRI expression, with each cytokine cancelling the effects of the other. However, in vivo analysis of peritoneal inflammation during Nippostrongylus brasiliensis infection in mice revealed a dominant suppressive function for TGF-beta1. These data support the existence of a cytokine network involving the Th2 cytokine IL-4 and the Treg cytokine TGF-beta1 that can regulate mast cell homeostasis. Dysregulation of this balance may impact allergic disease and be amenable to targeted therapy.

Mast cell modulation of the immune response.

Mast cells are present in nearly all vascularized tissues, but not the blood. They are best known for the prominent role they play in atopic disease. However, our current understanding of their direct and indirect roles in the immune response offers a more nuanced picture of both villain and hero. Although they are implicated in many inflammatory disorders, they also defend us from bacterial pathogens, prevent dangerous overreactions by the immune system, and even protect us from snake venom. Perhaps there is more to these maligned cells than we thought.

Mast cell regulation of the immune response.

Mast cells are well known as principle effector cells of type I hypersensitivity responses. Beyond this role in allergic disease, these cells are now appreciated as playing an important role in many inflammatory conditions. This review summarizes the support for mast cell involvement in resisting bacterial infection, exacerbating autoimmunity and atherosclerosis, and promoting cancer progression. A commonality in these conditions is the ability of mast cells to elicit migration of many cell types, often through the production of inflammatory cytokines such as tumor necrosis factor. However, recent data also demonstrates that mast cells can suppress the immune response through interleukin-10 production. The data encourage those working in this field to expand their view of how mast cells contribute to immune homeostasis.

IgE signaling suppresses FcepsilonRIbeta expression.

Activation of the high-affinity receptor for IgE, FcepsilonRI, is known to elicit its rapid down-regulation through internalization and degradation. In keeping with this, expression of all three FcepsilonRI subunits is decreased at the protein level after cross-linkage of IgE with antigen. However, we find that the FcepsilonRI beta-subunit is also selectively suppressed at the mRNA level, through a pathway primarily involving Fyn, Syk, PI3K, and NF-kappaB. IgG or calcium ionophore, stimuli known to mimic portions of the IgE signaling cascade, similarly suppressed beta-subunit expression. LPS, a NF-kappaB-activating TLR ligand, did not alter beta-subunit expression. As IgE increases FcepsilonRI expression, we examined the coordinated regulation of FcepsilonRI subunits during culture with IgE, followed by cross-linkage with antigen. IgE increased the expression of all three FcepsilonRI subunits and strikingly induced expression of the antagonistic beta(T). The ratio of beta:beta(T) protein expression decreased significantly during culture with IgE and was reset to starting levels by antigen cross-linkage. These changes in protein levels were matched by similar fluctuations in beta and beta(T) mRNAs. FcepsilonRIbeta is a key regulator of IgER expression and function, a gene in which polymorphisms correlate with allergic disease prevalence. The ability of IgE and FcepsilonRI signaling to coordinate expression of the beta and beta(T) subunits may comprise a homeostatic feedback loop-one that could promote chronic inflammation and allergic disease if dysregulated.

Endogenous suppression of mast cell development and survival by IL-4 and IL-10.

Mast cell development is an important component of atopic and chronic inflammatory diseases such as asthma, multiple sclerosis, rheumatoid arthritis, and atherosclerosis. In this study, we found that IL-4 and IL-10 were produced constitutively in cultures of developing mast cells, correlating with mast cell purity. Deletion of either gene increased mast cell numbers and Fc epsilon RI expression during culture in IL-3 + stem cell factor (SCF). By adding exogenous IL-4 and IL-10 to bone marrow (BM) cultures containing IL-3 + SCF, we found that IL-4 + IL-10 suppressed mast cell development through mechanisms not used by either cytokine alone. IL-4 + IL-10 elicited a rapid cell death coincidental with reduced Kit receptor expression and signaling and enhanced mitochondrial damage and caspase activation. IL-4 or IL-10 costimulation, unlike either cytokine alone, altered mast cell ontogeny to yield predominantly macrophages in cultures that typically produce mast cells. This effect was observed consistently with unseparated BM cells, purified mouse BM stem cells, and erythrocyte-depleted human umbilical cord blood cells. These experiments demonstrated a major role for Stat6 and Stat3, but not the Stat3-induced transcriptional repressor Ets variant gene 3. Genetic background was also a critical factor, as BALB/c-derived BM cells were completely resistant to IL-10-mediated killing and expressed lower levels of IL-10R. Collectively, these results support the theory that IL-4 and IL-10 function as endogenous regulators of mast cell progenitor development, consistent with a role in immune homeostasis. Loss of this homeostasis, perhaps via genetic polymorphism, could contribute to the etiology of mast cell-associated disease.

Activation of bile acid biosynthesis by the p38 mitogen-activated protein kinase (MAPK): hepatocyte nuclear factor-4alpha phosphorylation by the p38 MAPK is required for cholesterol 7alpha-hydroxylase expression.

Bile acids are required for intestinal absorption and biliary solubilization of cholesterol and lipids. In addition, bile acids play a crucial role in cholesterol homeostasis. One of the key enzymes in the bile acid biosynthetic pathways is cholesterol 7alpha-hydroxylase/cytochrome P450 7alpha-hydroxylase (7alpha-hydroxylase), which is the rate-limiting and regulatory step of the "classic" pathway. Transcription of the 7alpha-hydroxylase gene is highly regulated. Two nuclear receptors, hepatocyte nuclear factor 4alpha (HNF-4alpha) and alpha(1)-fetoprotein transcription factor, are required for both transcription and regulation by different physiological events. It has been shown that some mitogen-activated protein kinases, such as the c-Jun N-terminal kinase and the ERK, play important roles in the regulation of 7alpha-hydroxylase transcription. In this study, we show evidence that the p38 kinase pathway plays an important role in 7alpha-hydroxylase expression and hence in bile acid synthesis. Inhibition of p38 kinase activity in primary hepatocytes results in approximately 5-10-fold reduction of 7alpha-hydroxylase mRNA. This suppression is mediated, at least in part, through HNF-4alpha. Inhibition of p38 kinase activity diminishes HNF-4alpha nuclear protein levels and its phosphorylation in vivo and in vitro, and it renders a less stable protein. Induction of the p38 kinase pathway by insulin results in an increase in HNF-4alpha protein and a concomitant induction of 7alpha-hydroxylase expression that is blocked by inhibiting the p38 pathway. These studies show a functional link between the p38 signaling pathway, HNF-4alpha, and bile acid synthesis.