Mohit Kashyap

IL-21 signaling is critical for the development of type I diabetes in the NOD mouse

IL-21 is a pleiotropic type I cytokine that shares the common cytokine receptor γ chain and plays important roles for normal Ig production, terminal B cell differentiation to plasma cells, and Th17 differentiation. IL-21 is elevated in several autoimmune diseases, and blocking its action has attenuated disease in MRL/lpr mice and in collagen-induced arthritis. The diabetes-associated Idd3 locus is at the Il2/Il21 locus, and elevated IL-21 was observed in the nonobese diabetic (NOD) mouse and suggested to contribute to diabetes by augmenting T cell homeostatic proliferation. To determine the role of IL-21 in diabetes, Il21r-knockout (KO) mice were backcrossed to NOD mice. These mice were devoid of lymphocytic infiltration into the pancreas, and only 1 of 20 animals had an elevated glucose compared with 60% of NOD mice on a wild-type (WT) background. Although TCR and Treg-related responses were normal, these mice had reduced Th17 cells and significantly higher levels of mRNAs encoding members of the Reg (regenerating) gene family whose transgenic expression protects against diabetes. Our studies establish a critical role for IL-21 in the development of type I diabetes in the NOD mouse, with obvious potential implications for type I diabetes in humans.

Cutting Edge: CD4 T Cell-Mast Cell Interactions Alter IgE Receptor Expression and Signaling

Mast cell activation is associated with atopic and inflammatory diseases, but the natural controls of mast cell homeostasis are poorly understood. We hypothesized that CD4+CD25+ regulatory T cells (Treg) could function in mast cell homeostasis. In this study, we demonstrate that mast cells can recruit both Treg and conventional CD4+ T cells (Tconv). Furthermore, Treg, but not Tconv, suppress mast cell FcεRI expression. Despite the known inhibitory functions of IL-10 and TGFβ1, FcεRI suppression was independent of IL-10 and TGF-β1 and required cell contact. Surprisingly, coculture with either Treg or Tconv cells suppressed IgE-mediated leukotriene C4 production but enhanced cytokine production by mast cells. This was accompanied by a selective increase in FcεRI-mediated Stat5 phosphorylation, which is a critical mediator of IgE-mediated cytokine secretion. These data are the first direct demonstration that mast cells can recruit Treg and illustrate that T cell interactions can alter the mast cell response.

IL-10 Suppresses Mast Cell IgE Receptor Expression and Signaling In Vitro and In Vivo

Mast cells are known for their roles in allergy, asthma, systemic anaphylaxis, and inflammatory disease. IL-10 can regulate inflammatory responses and may serve as a natural regulator of mast cell function. We examined the effects of IL-10 on in vitro-cultured mouse and human mast cells, and evaluated the effects of IL-10 on FcεRI in vivo using mouse models. IgE receptor signaling events were also assessed in the presence or absence of IL-10. IL-10 inhibited mouse mast cell FcεRI expression in vitro through a Stat3-dependent process. This down-regulation was consistent in mice tested in vivo, and also on cultured human mast cells. IL-10 diminished expression of the signaling molecules Syk, Fyn, Akt, and Stat5, which could explain its ability to inhibit IgE-mediated activation. Studies of passive systemic anaphylaxis in IL-10-transgenic mice showed that IL-10 overexpression reduced the IgE-mediated anaphylactic response. These data suggest an important regulatory role for IL-10 in dampening mast cell FcεRI expression and function. IL-10 may hence serve as a mediator of mast cell homeostasis, preventing excessive activation and the development of chronic inflammation.

Interleukin‐10 induces apoptosis in developing mast cells and macrophages

Interleukin (IL)‐10 is a potent immunoregulatory cytokine capable of inhibiting the inflammatory response. As mast cells and macrophages are central effectors of inflammation, we investigated the effects of IL‐10 on mast cell and macrophge development from mouse bone marrow progenitors. Bone marrow cells were cultured in IL‐3 + stem cell factor (SCF), giving rise to mixed populations of mast cells and macrophages. The addition of IL‐10 greatly decreased the expansion of bone marrow progenitor cells through a mechanism requiring signal tranducer and activator of transcription‐3 expression. The inhibitory effects were a result of the induction of apoptosis, which occurred with caspase‐3 activation and reduced mitochondrial membrane potential. Supporting a role for the mitochondrion, bone marrow cells from p53‐deficient or Bcl‐2 transgenic mice were partly resistant to the effects of IL‐10. Further, IL‐10 decreased Kit receptor expression and inhibited survival signaling by SCF or IL‐3. These data indicate that IL‐10 induces an intrinsic, mitochondrial apoptosis cascade in developing mast cells and macrophages through mechanisms involving blockade of growth factor receptor function. The ability of IL‐10 to inhibit survival could support immune homeostasis by dampening inflammatory responses and preventing chronic inflammation.

IFN-γ Induces Apoptosis in Developing Mast Cells

Mast cells are critical effectors of allergic disease, and are now implicated in immune responses observed in arthritis, multiple sclerosis, and heart disease. Because of their role in inflammation, understanding how mast cells develop is of clinical importance. In this study we determined the effects of IFN-γ on mast cell survival. Using in vitro culture of bone marrow cells in IL-3 plus stem cell factor, we found that the addition of IFN-γ induced apoptosis, as exhibited by the presence of subdiploid DNA and caspase activation. IFN-γ-mediated apoptosis was Stat1-dependent, and was accompanied by loss of mitochondrial membrane potential. Apoptosis was reduced in cultures of bone marrow cells derived from p53- or Bax-deficient mice, as well as H2K-Bcl-2 transgenic mice. IFN-γ hyperresponsiveness has been shown to result in inflammatory disease and death in mice lacking the regulatory protein suppressor of cytokine signaling (SOCS)-1. Bone marrow cells from SOCS-1 knockout (KO) mice failed to give rise to viable mast cells after culture in IL-3 plus stem cell factor, with profound apoptosis occurring as the cultures matured. However, bone marrow cells lacking both SOCS-1 and IFN-γ survived normally. This in vitro defect in mast cell development was recapitulated in vivo. SOCS-1 KO mice demonstrated a 67% decrease in peritoneal mast cell numbers relative to wild-type mice, a deficiency that was reversed in SOCS-1/IFN-γ KO mice. These data demonstrate the potent regulatory effects of IFN-γ on mast cell survival and show that this cytokine can elicit mast cell death in vitro and in vivo.

IL-3-mediated TNF production is necessary for mast cell development.

Mouse mast cell development and survival are largely controlled by the cytokines IL-3 and stem cell factor (SCF). We have found that IL-3 stimulation of bone marrow cells induces the production of TNF via a PI3K- and MAPK kinase/ERK-dependent pathway. Specifically, Mac-1-positive cells were responsible for TNF production, which peaked on days 7–10 of culture and decreased rapidly thereafter. The importance of IL-3-induced TNF secretion was demonstrated by the failure of TNF-deficient bone marrow cells to survive for >3 wk when cultured in IL-3 and SCF, a defect that was reversed by the addition of soluble TNF. The development of human mast cells from bone marrow progenitors was similarly hampered by the addition of TNF-blocking Abs. Cell death was due to apoptosis, which occurred with changes in mitochondrial membrane potential and caspase activation. Apoptosis appeared to be due to loss of IL-3 signaling, because TNF-deficient cells were less responsive than their wild-type counterparts to IL-3-mediated survival. In vitro cultured mast cells from TNF-deficient mice also demonstrated reduced expression of the high affinity IgE receptor, which was restored to normal levels by the addition of soluble TNF. Finally, TNF-deficient mice demonstrated a 50% reduction in peritoneal mast cell numbers, indicating that TNF is an important mast cell survival factor both in vitro and in vivo.

Stat5: an essential regulator of mast cell biology.

Interleukin-3 (IL-3) and stem cell factor (SCF) are important mast cell growth and differentiation factors. Since both cytokines activate the transcription factor Stat5, a known regulator of proliferation and survival, we investigated the effects of Stat5 deficiency on mast cell development and survival. This article will review data presented at The Fourth International Workshop on Signal Transduction in the Activation and Development of Mast Cells and Basophils. The full set of data is now in preparation for publication. We find that the absence of Stat5 A and B results in a total loss of in vivo mast cell development. Bone marrow-derived mast cell (BMMC) populations can be cultured and maintained from Stat5-deficient mice in IL-3+SCF, but not in either cytokine alone. The absence of Stat5 resulted in aberrant control of Bcl-2, Bcl-x(L) and cyclin A2, with increased apoptosis and delayed cell cycle progression after IL-3 or SCF stimulation. These results indicate that Stat5 A and B are critical regulators of in vitro and in vivo mast cell biology.