Thymic stromal lymphopoietin is produced by dendritic cells.

Thymic stromal lymphopoietin (TSLP) is a type 1 cytokine that contributes to lymphopoiesis and the development of asthma and atopic dermatitis. TSLP acts on multiple lineages, including dendritic cells (DCs), T cells, NKT cells, eosinophils, and mast cells, mediating proliferation and survival and linking innate and adaptive immune responses. TSLP is produced by a range of cells, including epithelial cells, fibroblasts, stromal cells, and keratinocytes. DCs are important primary targets of TSLP, and we unexpectedly demonstrated that DCs also produce TSLP in response to TLR stimulation and that this is augmented by IL-4. Moreover, we demonstrated that when mice were challenged with house dust mite extract, lung CD11c(+) DCs expressed TSLP mRNA at an even higher level than did epithelial cells. These data suggested that DCs not only respond to TSLP but also are a source of TSLP during pathogen and/or allergen encounter.

Thymic stromal lymphopoietin-mediated STAT5 phosphorylation via kinases JAK1 and JAK2 reveals a key difference from IL-7-induced signaling.

Thymic stromal lymphopoietin (TSLP) is a type I cytokine that plays essential roles in allergic/inflammatory skin and airway disorders, in helminth infections, and in regulating intestinal immunity. TSLP signals via IL-7Rα and a specific TSLPR subunit that is highly related to the common cytokine receptor γ chain, γ(c). Although TSLP has effects on a broad range of hematopoetic cells and can induce STAT5 phosphorylation, TSLP was reported to not signal via JAK kinases, and the mechanism by which TSLP regulates STAT5 phosphorylation has been unclear. We now demonstrate the role of JAK1 and JAK2 in TSLP-mediated STAT5 phosphorylation in mouse and human primary CD4(+) T cells, in contrast to the known activation of JAK1 and JAK3 by the related cytokine, IL-7. We also show that just as JAK1 interacts with IL-7Rα, JAK2 is associated with TSLPR protein. Moreover, we demonstrate the importance of STAT5 activation for TSLP-mediated survival and proliferation of CD4(+) T cells. These findings clarify the basis for TSLP-mediated signaling and provide an example wherein a cytokine uses JAK1 and JAK2 to mediate the activation of STAT5.

TSLP: A Key Regulator of Asthma Pathogenesis.

Asthma is a complex disorder of the airways that is characterized by T helper type 2 (Th2) inflammation. The pleiotrophic cytokine TSLP has emerged as an important player involved in orchestrating the inflammation seen in asthma and other atopic diseases. Early research elucidated the role of TSLP on CD4+ T cells, and recent work has revealed the impact of TSLP on multiple cell types. Furthermore, TSLP plays an important role in the sequential progression of atopic dermatitis to asthma, clarifying the key role of TSLP in the pathogenesis of asthma, a finding with therapeutic implications.

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 gamma 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.

Mast cell homeostasis: a fundamental aspect of allergic disease.

Mast cells are well known for their role in allergic disease and have recently been implicated in inflammatory disorders, including autoimmune arthritis, multiple sclerosis, and atherosclerosis. Although aberrant mast cell activation is the focus of many studies, much less is known about normal mast cell homeostasis. Because loss of the normal constraints on mast cell activation, proliferation, and survival may be central to disease etiology, understanding these issues warrants attention. This review summarizes the knowledge of mast cell homeostasis controlled by IgE and the regulatory cytokines IL-4, IL-10, and TGF-beta1. Because each of these proteins plays an important role in immune responses tied to mast cell-associated disease, this group represents a potential set of factors altered in atopic or autoimmune patients. It is interesting to note, for example, that polymorphisms within each of these factors or their receptors are linked to allergic disease. By first understanding how cytokines and IgE regulate mast cell function and survival, we may then predict how these factors may function in disease onset and progression.

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 macrophage 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.

TGFbeta1 induces mast cell apoptosis.

Mast cells are potent effectors of the inflammatory response, playing an important role in atopy, bacterial immunity, and animal models of arthritis, multiple sclerosis, and heart disease. Hence controlling mast cell numbers and responsiveness is essential for preventing inflammatory disease. We demonstrate that the cytokine transforming growth factor (TGF) beta1 is a potent inducer of mast cell apoptosis, a finding that was consistent in cultured mouse bone marrow-derived mast cells, peritoneal mast cells, and human mast cells. Cell death appeared to be caused by TGF-mediated repression of interleukin-3 (IL-3) receptor expression and function, leading to mitochondrial damage and activation of an apoptotic cascade acting via p53 and caspases. Although IL-3 receptor expression was reduced within 1 day of TGFbeta1 stimulation, apoptosis required at least 3 days to occur. This delay in onset is postulated to allow protective mast cell effector functions, protecting the host from infection while preventing the establishment of chronic inflammation. Our data support the theory that TGFbeta1 is an inhibitor of mast cell survival. The widespread expression of TGFbeta1 offers this cytokine as an ideal candidate for control of mast cell homeostasis.

TGF-beta1 inhibits late-stage mast cell maturation.

OBJECTIVE: The objective of this study was to determine the effects of transforming growth factor (TGF)-beta1 on mast cell development. MATERIALS AND METHODS: Mast cells were cultured from mouse bone marrow in interleukin (IL)-3 + stem cell factor, in the presence or absence of TGF-beta1. We assessed mast cell development by measuring the expression of kit, T1/ST2, FcvarepsilonRI, and Fcgamma receptors. Cell morphology was determined by histochemical staining. Alterations in FcvarepsilonRI subunit expression were measured by Western blot analysis. Adoptive transfer of cultured mast cells into mast cell-deficient W/W(v) mice was used to determine if the in vivo environment could reverse the inhibitory effects of TGF-beta1. RESULTS: TGF-beta1 decreased FcvarepsilonRI, c-kit, T1/ST2, and FcgammaR expression, and inhibited granule formation in developing mast cells. Accessory cells were not required for this inhibition. Smad3 deficiency did not alter the response of bone marrow cells to TGF-beta1. TGF-beta1 inhibited expression of the FcvarepsilonRI alpha subunit protein, without decreasing beta or gamma proteins. Mast cells derived in the presence of TGF-beta1 were functionally impaired, as IgE-mediated cytokine secretion was greatly reduced. The changes in granule formation and surface antigen expression were long-standing, as they were not reversed by transfer to W/W(v) mice. CONCLUSIONS: TGF-beta1 may contribute to mast cell homeostasis by inhibiting maturation from bone marrow precursors. The effects of TGF-beta1 result in greatly diminished expression of cell surface markers, reduced granulation, and lack of responsiveness to IgE-mediated activation. Thus TGF-beta1 can serve as a potent and multifunctional regulator of mast cell maturation.

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.

Interleukin-4 elicits apoptosis of developing mast cells via a Stat6-dependent mitochondrial pathway.

OBJECTIVE: The aim of this study was to assess the effects of interleukin-4 and signal transducer and activator of transcription (Stat)-6 on IL-3+SCF-induced mast cell development. PATIENTS AND METHODS: Unseparated mouse bone marrow cells were cultured in IL-3+SCF, giving rise to mast cells and monocytes/macrophages. The addition of IL-4, the use of Stat6-deficient bone marrow cells, and expression of a constitutively active Stat6 mutant were employed to assess the effects of IL-4 and Stat6 on cell viability, proliferation, and differentiation. Bax-deficient and bcl-2 transgenic bone marrow cells were used to assess the importance of the mitochondria in IL-4-mediated effects. RESULTS: IL-4 elicited apoptosis and limited the cell cycle progression of developing bone marrow cells, without affecting cell differentiation. Apoptosis required that IL-4 be present during the first 8 days of the 21-day culture period. Cell death correlated with loss of mitochondrial membrane potential. Accordingly, IL-4-mediated apoptosis was inhibited by Bax deletion or bcl-2 overexpression. Lastly, Stat6 activation was both necessary and sufficient to inhibit cell survival. CONCLUSION: IL-4 exerts potent apoptotic effects on developing mast cells and monocyte/macrophages through mitochondrial damage and Stat6 activation.

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 FcepsilonRI expression. Despite the known inhibitory functions of IL-10 and TGFbeta1, FcepsilonRI suppression was independent of IL-10 and TGF-beta1 and required cell contact. Surprisingly, coculture with either Treg or Tconv cells suppressed IgE-mediated leukotriene C(4) production but enhanced cytokine production by mast cells. This was accompanied by a selective increase in FcepsilonRI-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 FcepsilonRI 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 FcepsilonRI 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 FcepsilonRI expression and function. IL-10 may hence serve as a mediator of mast cell homeostasis, preventing excessive activation and the development of chronic inflammation.

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.

IFN-gamma 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-gamma 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-gamma induced apoptosis, as exhibited by the presence of subdiploid DNA and caspase activation. IFN-gamma-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-gamma 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-gamma 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-gamma KO mice. These data demonstrate the potent regulatory effects of IFN-gamma on mast cell survival and show that this cytokine can elicit mast cell death in vitro and in vivo.

TGF-beta 1 inhibits mast cell Fc epsilon RI expression.

Mast cell activation through the high affinity IgE receptor (FcepsilonRI) is a critical component of atopic inflammation. The cytokine TGF-beta1 has been shown to inhibit IgE-dependent mast cell activation, possibly serving to dampen mast cell-mediated inflammatory responses. We present proof that TGF-beta1 inhibits mast cell FcepsilonRI expression through a reversible pathway that diminishes protein, but not mRNA, expression of the FcepsilonRI subunit proteins alpha, beta, and gamma. The stability of the expressed proteins and the assembled cell surface complex was unaltered by TGF-beta1 treatment. However, TGF-beta1 decreased the rate of FcepsilonRI beta-chain synthesis, arguing that this inhibitory cytokine exerts its effects at the level of mRNA translation. TGF-beta1 consistently diminished FcepsilonRI expression on cultured human or mouse mast cells as well as freshly isolated peritoneal mast cells. The related cytokines, TGF-beta2 and TGF-beta3, had similar effects. We propose that TGF-beta1 acts as a negative regulator of mast cell function, in part by decreasing FcepsilonRI expression.

Stat5 expression is critical for mast cell development and survival.

Interleukin-3 (IL-3) and stem cell factor (SCF) are important mast cell growth and differentiation factors. Since both cytokines activate the transcription factor signal transducer and activator of transcription 5 (Stat5), a known regulator of proliferation and survival, we investigated the effects of Stat5 deficiency on mast cell development and survival. Bone marrow-derived mast cell (BMMC) populations cultured from Stat5A/B-deficient mice survived in IL-3 + SCF, but not in either cytokine alone. These cells demonstrated reduced expression of Bcl-2, Bcl-x(L), cyclin A2, and cyclin B1, with increased apoptosis and delayed cell cycle progression during IL-3 or SCF culture. Finally, the absence of Stat5 resulted in loss of in vivo mast cell development, as judged by assessments of Stat5-deficient mice and transplantation of Stat5-deficient bone marrow cells to mast cell-deficient recipient mice. These results indicate that Stat5A and Stat5B are critical regulators of in vitro and in vivo mast cell development and survival.