Yves T. Falanga

A specific sphingosine kinase 1 inhibitor attenuates airway hyperresponsiveness and inflammation in a mast cell–dependent murine model of allergic asthma

BACKGROUND Sphingosine-1-phosphate (S1P), which is produced by 2 sphingosine kinase (SphK) isoenzymes, SphK1 and SphK2, has been implicated in IgE-mediated mast cell responses. However, studies of allergic inflammation in isotype-specific SphK knockout mice have not clarified their contribution, and the role that S1P plays in vivo in a mast cell- and IgE-dependent murine model of allergic asthma has not yet been examined. OBJECTIVE We used an isoenzyme-specific SphK1 inhibitor, SK1-I, to investigate the contributions of S1P and SphK1 to mast cell-dependent airway hyperresponsiveness (AHR) and airway inflammation in mice. METHODS Allergic airway inflammation and AHR were examined in a mast cell-dependent murine model of ovalbumin (OVA)-induced asthma. C57BL/6 mice received intranasal delivery of SK1-I before sensitization and challenge with OVA or only before challenge. RESULTS SK1-I inhibited antigen-dependent activation of human and murine mast cells and suppressed activation of nuclear factor κB (NF-κB), a master transcription factor that regulates the expression of proinflammatory cytokines. SK1-I treatment of mice sensitized to OVA in the absence of adjuvant, in which mast cell-dependent allergic inflammation develops, significantly reduced OVA-induced AHR to methacholine; decreased numbers of eosinophils and levels of the cytokines IL-4, IL-5, IL-6, IL-13, IFN-γ, and TNF-α and the chemokines eotaxin and CCL2 in bronchoalveolar lavage fluid; and decreased pulmonary inflammation, as well as activation of NF-κB in the lungs. CONCLUSION S1P and SphK1 play important roles in mast cell-dependent, OVA-induced allergic inflammation and AHR, in part by regulating the NF-κB pathway.

Essential roles of sphingosine-1–phosphate receptor 2 in human mast cell activation, anaphylaxis, and pulmonary edema

Allergic disease is endemic in developed nations, and none is more dramatic or deadly than systemic anaphylactic shock (Finkelman, 2007). Unfortunately, this collection of rapid changes to the cutaneous, vasculature, and pulmonary systems may also be the least understood of atopic diseases. A better understanding of the eliciting factors for systemic anaphylaxis is therefore critical for accurate diagnosis and therapeutic intervention. Mast cells, which reside in vascularized tissues and are strategically located at the interfaces of host and environment in skin and mucosal surfaces, are key effectors of IgEmediated allergic disorders, including anaphylaxis, hay fever, eczema, and asthma. Mast cells express FcRI (high-affinity receptors for IgE), which binds antigen (Ag)-specific IgE antibodies, and subsequent exposure to Ag initiates cross-linking and aggregation of FcRI. This cascade of events triggers mast cell activation and secretion of a wide array of inflammatory mediators, such as histamine and other preformed mediators, bioactive lipids, including eicosanoids and platelet-activating factor (PAF), and numerous proinflammatory cytokines and chemokines (Kalesnikoff and Galli, 2008), which are essential to the pathogenesis of allergic diseases and anaphylaxis (Finkelman, 2007). A recent addition to the recognized repertoire of lipid mediators secreted by mast cells is the sphingolipid metabolite sphingosine1–phosphate (S1P; Jolly et al., 2004; Mitra et al., 2006; Olivera et al., 2006), which has been implicated in initiation and maintenance of diverse aspects of immune cell activation and function (Rosen et al., 2008; for review see Schwab and Cyster, 2007; Rivera et al., 2008). Focus on S1P in immune responses has increased after the elucidation of its critical role in lymphocyte trafficking (Rosen et al., 2008; for review see Schwab and Cyster, 2007), and observations of local increases of S1P in inflammatory disorders such as asthma (Ammit et al., 2001) and rheumatoid arthritis (Kitano et al., 2006). S1P is a ligand for five G protein–coupled receptors, designated S1P1-5, through which it exerts many of its actions (Spiegel and Milstien, 2003). CORRESPONDENCE Carole Oskeritzian: coskerit@vcu.edu

IL-4 and TGF-β1 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-β1 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-β1 receptor expression and signaling, and vice versa. In vitro studies demonstrated that IL-4 and TGF-β1 had balancing effects on mast cell survival, migration, and FcεRI 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-β1. These data support the existence of a cytokine network involving the Th2 cytokine IL-4 and the Treg cytokine TGF-β1 that can regulate mast cell homeostasis. Dysregulation of this balance may impact allergic disease and be amenable to targeted therapy.

Lyn but not Fyn kinase controls IgG-Mediated Systemic Anaphylaxis

Anaphylaxis is a rapid, life-threatening hypersensitivity reaction. Until recently, it was mainly attributed to histamine released by mast cells activated by allergen crosslinking (XL) of FcεRI-bound allergen-specific IgE. However, recent reports established that anaphylaxis could also be triggered by basophil, macrophage, and neutrophil secretion of platelet-activating factor subsequent to FcγR stimulation by IgG/Ag complexes. We have investigated the contribution of Fyn and Lyn tyrosine kinases to FcγRIIb and FcγRIII signaling in the context of IgG-mediated passive systemic anaphylaxis (PSA). We found that mast cell IgG XL induced Fyn, Lyn, Akt, Erk, p38, and JNK phosphorylation. Additionally, IgG XL of mast cells, basophils, and macrophages resulted in Fyn- and Lyn-regulated mediator release in vitro. FcγR-mediated activation was enhanced in Lyn-deficient (knockout [KO]) cells, but decreased in Fyn KO cells, compared with wild-type cells. More importantly, Lyn KO mice displayed significantly exacerbated PSA features whereas no change was observed for Fyn KO mice, compared with wild-type littermates. Intriguingly, we establish that mast cells account for most serum histamine in IgG-induced PSA. Taken together, our findings establish pivotal roles for Fyn and Lyn in the regulation of PSA and highlight their unsuspected functions in IgG-mediated pathologies.

Genotype-Dependent Effects of TGF-β1 on Mast Cell Function: Targeting the Stat5 Pathway

We previously demonstrated that TGF-β1 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-β1 and the means by which it suppresses mast cells have been less clear. This study shows that TGF-β1 suppresses FcεRI and c-Kit expression in vivo. By examining changes in cytokine production concurrent with FcεRI expression, we found that TGF-β1 suppresses TNF production independent of FcεRI levels. Rather, IgE-mediated signaling was altered. TGF-β1 significantly reduced expression of Fyn and Stat5, proteins critical for cytokine induction. These changes may partly explain the effects of TGF-β1, 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-β1 reduced this migration. We found evidence that genetic background may alter TGF responses. TGF-β1 greatly reduced mast cell numbers in Th1-prone C57BL/6, but not Th2-prone 129/Sv mice. Furthermore, TGF-β1 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-β1 treatment. Finally, primary human mast cell populations also showed variable sensitivity to TGF-β1–mediated changes in Stat5 and IgE-mediated IL-6 secretion. We propose that TGF-β1 regulates mast cell homeostasis, and that this feedback suppression may be dependent on genetic context, predisposing some individuals to atopic disease.

Novel Mechanism for FcϵRI-mediated Signal Transducer and Activator of Transcription 5 (STAT5) Tyrosine Phosphorylation and the Selective Influence of STAT5B over Mast Cell Cytokine Production

Background: STAT5 is a transcription factor that is vital for mast cell function. Results: Loss of Fyn kinase prevents, whereas loss of Lyn, Gab2, or SHP-1 enhances, FcϵRI-mediated STAT5 tyrosine phosphorylation. Conclusion: IgE-mediated STAT5 activation in mast cells requires Fyn kinase. Significance: Elucidating the mechanisms of mast cell activity is essential to understanding and treating allergic pathologies. Previous studies indicate that STAT5 expression is required for mast cell development, survival, and IgE-mediated function. STAT5 tyrosine phosphorylation is swiftly and transiently induced by activation of the high affinity IgE receptor, FcϵRI. However, the mechanism for this mode of activation remains unknown. In this study we observed that STAT5 co-localizes with FcϵRI in antigen-stimulated mast cells. This localization was supported by cholesterol depletion of membranes, which ablated STAT5 tyrosine phosphorylation. Through the use of various pharmacological inhibitors and murine knock-out models, we found that IgE-mediated STAT5 activation is dependent upon Fyn kinase, independent of Syk, PI3K, Akt, Brutons tyrosine kinase, and JAK2, and enhanced in the context of Lyn kinase deficiency. STAT5 immunoprecipitation revealed that unphosphorylated protein preassociates with Fyn and that this association diminishes significantly during mast cell activation. SHP-1 tyrosine phosphatase deficiency modestly enhanced STAT5 phosphorylation. This effect was more apparent in the absence of Gab2, a scaffolding protein that docks with multiple negative regulators, including SHP-1, SHP-2, and Lyn. Targeting of STAT5A or B with specific siRNA pools revealed that IgE-mediated mast cell cytokine production is selectively dependent upon the STAT5B isoform. Altogether, these data implicate Fyn as the major positive mediator of STAT5 after FcϵRI engagement and demonstrate importantly distinct roles for STAT5A and STAT5B in mast cell function.

The Fyn-STAT5 pathway: a new Frontier in IgE- and IgG-mediated mast cell signaling

Mast cells are central players in immune surveillance and activation, positioned at the host–environment interface. Understanding the signaling events controlling mast cell function, especially those that maintain host homeostasis, is an important and still less understood area of mast cell-mediated disease. With respect to allergic disease, it is well established that IgE and its high affinity receptor FcεRI are major mediators of mast cell activation. However, IgG-mediated signals can also modulate mast cell activities. Signals elicited by IgG binding to its cognate receptors (FcγR) are the basis for autoimmune disorders such as lupus and rheumatoid arthritis. Using knowledge of IgE-mediated mast cell signaling, recent work has begun to illuminate potential overlap between FcεRI and FcγR signal transduction. Herein we review the importance of Src family kinases in FcεRI and FcγR signaling, the role of the transcription factor STAT5, and impingement of the regulatory cytokines IL-4, IL-10, and TGFβ1 upon this network.

Fyn Kinase Is Required for Optimal Humoral Responses

The generation of antigen-specific antibodies and the development of immunological memory require collaboration between B and T cells. T cell-secreted IL-4 is important for B cell survival, isotype switch to IgG1 and IgE, affinity maturation, and the development of germinal centers (GC). Fyn, a member of the Src family tyrosine kinase, is widely expressed in many cell types, including lymphocytes. This kinase is known to interact with both the B cell and T cell receptor (BCR and TCR, respectively). While Fyn deletion does not impair the development of immature T cells and B cells, TCR signaling is altered in mature T cells. The current study demonstrates that Fyn deficient (KO) B cells have impaired IL-4 signaling. Fyn KO mice displayed low basal levels of IgG1, IgE and IgG2c, and delayed antigen-specific IgG1 and IgG2b production, with a dramatic decrease in antigen-specific IgG2c following immunization with a T-dependent antigen. Defects in antibody production correlated with significantly reduced numbers of GC B cells, follicular T helper cells (TFH), and splenic plasma cells (PC). Taken together, our data demonstrate that Fyn kinase is required for optimal humoral responses.

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.