Mast cells play a critical role in the development of the allergic response. Upon activation by allergens and IgE via the high affinity receptor for IgE (FcεRI), these cells release histamine and other functional mediators that initiate and propagate immediate hypersensitivity reactions. Mast cells also secrete cytokines that can regulate immune activity. These processes are controlled, in whole or part, by increases in intracellular Ca2+ induced by the FcεRI. We show here that N-(4-(3,5-bis(Trifluoromethyl)-1H-pyrazol-1-yl)phenyl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP2), a pyrazole derivative, inhibits activation-induced Ca2+ influx in the rat basophil cell line RBL-2H3 and in bone marrow-derived mast cells (BMMCs), without affecting global tyrosine phosphorylation of cellular proteins or phosphorylation of the mitogen-activated protein kinases Erk1/2 and p38. BTP2 also inhibits activation-induced degranulation and secretion of Interleukin (IL)-2, IL-3, IL-4, IL-6, IL-13, Tumor Necrosis Factor (TNF)-α, and Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) by BMMCs, which correlates with the inhibition of Nuclear Factor of Activated T cells (NFAT) translocation. In vivo, BTP2 inhibits antigen-induced histamine release. Structure-activity relationship analysis indicates that substitution at the C3 or C5 position of the pyrazole moiety on BTP2 (5-triflouromethyl-3-methyl-pyrazole or 3-triflouromethyl-5-methyl-pyrazole, respectively) affected its activity, with the trifluoromethyl group at the C3 position being critical to its activity. We conclude that BTP2 and related compounds may be potent modulators of mast cell responses and potentially useful for the treatment of symptoms of allergic inflammation.

Tissue-specific innate-like γδ T cells are important components of the immune system critical for the first line of defense. But mechanisms underlying their tissue-specific development are poorly understood. Our study with prototypical skin-specific intraepithelial γδT lymphocytes (sIEL) found that among different thymic γδ T cell subsets, fetal thymic precursors of sIELs specifically acquire a unique skin-homing property after the positive selection, suggesting an important role of the TCR selection signaling in “programming” them for the tissue-specific development. Here we identified IL2-inducible T-cell kinase (ITK) as a critical signal molecule regulating the acquirement of the skin-homing property by the fetal thymic sIEL precursors. In ITK-knockout mice, the sIEL precursors could not undergo the positive selection-associated upregulation of thymus-exiting and skin-homing molecules S1PR1 and CCR10 and accumulated in the thymus. On the other hand, the survival and expansion of sIELs in the skin did not require the ITK-transduced TCR-signaling while its persistent activation impaired the sIEL development by inducing apoptosis. These findings provide insights into molecular mechanisms underlying differential requirements of the TCR signaling in peripheral localization and maintenance of the tissue specific T cells.

Store-operated calcium channels are plasma membrane Ca2+ channels that are activated by depletion of intracellular Ca2+ stores, resulting in an increase in intracellular Ca2+ concentration, which is maintained for prolonged periods in some cell types. Increases in intracellular Ca2+ concentration serve as signals that activate a number of cellular processes, however, little is known about the regulation of these channels. We have characterized the immuno-suppressant compound BTP, which blocks store-operated channel mediated calcium influx into cells. Using an affinity purification scheme to identify potential targets of BTP, we identified the actin reorganizing protein, drebrin, and demonstrated that loss of drebrin protein expression prevents store-operated channel mediated Ca2+ entry, similar to BTP treatment. BTP also blocks actin rearrangements induced by drebrin. While actin cytoskeletal reorganization has been implicated in store-operated calcium channel regulation, little is known about actin binding proteins that are involved in this process, or how actin regulates channel function. The identification of drebrin as a mediator of this process should provide new insight into the interaction between actin rearrangement and tore-operated channel mediated calcium influx.

The eosinophil has been perceived as a terminal effector cell in allergic airway diseases. However, recent work has shown that this multifunctional cell could be more involved in the initial stages of allergic disease development than was previously thought, particularly with regard to the ability of the eosinophil to modulate T-cell responses. In this review, we discuss recent advances that suggest that eosinophils can present antigen to naïve as well as to antigen experienced T cells, induce T helper 2 cell development, cytokine production or both, and affect T-cell migration to sites of inflammation. These findings are changing the way that eosinophil function in disease is perceived, and represent a shift in the dogma of allergic disease development.

Th17 cells play major roles in autoimmunity and bacterial infections, yet how T cell receptor (TCR) signaling affects Th17 differentiation is relatively unknown. We demonstrate that CD4+ T cells deficient in Itk, a tyrosine kinase required for full TCR-induced activation of PLC-γ, exhibit decreased IL-17A expression, yet relatively normal expression of RORγT, RORα and IL-17F. IL-17A expression was rescued by pharmacologically-induced Ca2+ influx or expression of activated NFATc1. Conversely, decreased TCR stimulation or FK506 treatment preferentially reduced expression of IL-17A. The promoter of IL-17A but not IL-17F has conserved NFAT binding sites that bind NFATc1 in WT, but not Itk-deficient cells, even though both promoters exhibit epigenetic modifications consistent with open chromatin. Finally, defective IL-17A expression and differential regulation of IL-17A and IL-17F were observed in vivo in Itk−/− mice in an allergic asthma model. Our results suggest that Itk specifically couples TCR signaling strength to IL-17A expression through NFATc1.

The signals that regulate T cell activation have been studied for some time. We know that upon interaction with antigen/MHC complex, the T cell Receptor (TCR) triggers the activation of a number of kinases, including tyrosine and serine/threonine kinases. The Tec family kinase ITK plays a role in this response, but the signaling pathways it regulates are less well known. Even less well known are its binding partners and substrates. Sacristan and colleagues have now extended the known partners and substrates of ITK by reporting in this issue of the European Journal of Immunology, that ITK interacts with the transcriptional regulator TFII-I. The implications of this finding are discussed.

Friend virus induces an erythroleukemia in susceptible mice that is initiated by the interaction of the Friend virus-encoded glycoprotein gp55 with the erythropoietin (Epo) receptor and the product of the host Fv2 gene, a naturally occurring truncated form of the Stk receptor tyrosine kinase (Sf-Stk). We have previously demonstrated that the activation of Sf-Stk, recruitment of a Grb2/Gab2/Stat3 signaling complex, and induction of Pu.1 expression by Stat3 are required for the development of the early stage of Friend disease both in vitro and in vivo. Here we demonstrate that the interaction of gp55 with Sf-Stk is dependent on cysteine residues in the ecotropic domain of gp55 and the extracellular domain of Sf-Stk. Point mutation of these cysteine residues or deletion of these domains inhibits the ability of gp55 to interact with Sf-Stk, resulting in the inability of these proteins to promote the Epo-independent growth of erythroid progenitor cells. We also demonstrate that the interaction of gp55 with Sf-Stk does not promote dimerization of Sf-Stk but results in enhanced phosphorylation of Sf-Stk and the relocalization of Sf-Stk from the cytosol to the plasma membrane. Finally, we demonstrate that a constitutively active form of Sf-Stk (Sf-StkM330T), as well as its human counterpart, Sf-Ron, promotes Epo-independent colony formation in the absence of gp55 and that this response is also dependent on the cysteines in the extracellular domains of Sf-StkM330T and Sf-Ron. These data suggest that the cysteines in the extracellular domains of Sf-Stk and Sf-Ron may also mediate the interaction of these truncated receptors with other cellular factors that regulate their ability to promote cytokine-independent growth.

Allergic asthma is dependent on chemokine mediated Th2 cell migration and Th2 cytokine secretion into the lungs. The tyrosine kinase Itk regulates the production of Th2 cytokines as well as migration in response to chemokine gradients. Mice lacking Itk are resistant to developing allergic asthma, however, the role of kinase activity of Itk in the development of this disease is unclear. In addition, whether distinct Itk derived signals lead to T cell migration and secretion of Th2 cytokines is also unknown. Using transgenic mice specifically lacking Itk kinase activity, we show that active kinase signaling is required for control of Th2 responses and development of allergic asthma. Moreover, dominant suppression of Itk kinase activity led to normal Th2 responses, but significantly reduced chemokine mediated migration, resulting in prevention of allergic asthma. These observations indicate that signals required for Th2 responses and migration are differentially sensitive to Itk kinase activity. Manipulation of Itk’s kinase activity can thus provide a new strategy to treat allergic asthma by differentially affecting migration of T cells into the lungs, leaving Th2 responses intact.

Murine models of allergic asthma have been used to understand the mechanisms of development and pathology in this disease. In addition, knockout mice have contributed significantly to our understanding of the roles of specific molecules and cytokines in these models. However, results can vary significantly depending on the mouse strain used in the model, and in particularly in understanding the effect of specific knockouts. For example, it can be equivocal as to whether specific gene knockouts affect the susceptibility of the mice to developing the disease, or lead to resistance. Here we used a house dust mite model of allergic airway inflammation to examine the response of two strains of mice (C57BL/6 and BALB/c) which differ in their responses in allergic airway inflammation. We demonstrate an algorithm that can facilitate the understanding of the behavior of these models with regards to susceptibility (to allergic airway inflammation) (Saai) or resistance (Raai) in this model. We verify that both C57BL/6 and BALB/c develop disease, but BALB/c mice have higher Saai for development. We then use this approach to show that the absence of the Tec family kinase Itk, which regulates the production of Th2 cytokines, leads to Raai in the C57BL/6 background, but decreases Saai on the BALB/c background. We suggest that the use of such approaches could clarify the behavior of various knockout mice in modeling allergic asthma.

Itk and Txk/Rlk are Tec family kinases expressed in T cells. Itk is expressed in both TH1 and TH2 cells. By contrast, Txk is preferentially expressed in TH1 cells. Although Itk is required for TH2 responses in vivo and Txk is suggested to regulate IFNγ expression and TH1 responses, it remains unclear whether these kinases have distinct roles in TH cell differentiation/function. We demonstrate here that Txk null CD4+ T cells are capable of producing both TH1 and TH2 cytokines similar to those produced by WT CD4+ T cells. To further examine whether Itk and Txk play distinct roles in TH cell differentiation and function we examined Itk-null mice carrying a transgene that expresses Txk at levels similar to the expression of Itk in TH2 cells. Using two TH2 model systems: allergic asthma and Schistosome egg-induced lung granulomas, we found that the Txk transgene rescued TH2 cytokine production and all TH2 symptoms without notable enhancement of IFNγ expression. These results suggest that Txk is not a specific regulator of TH1 responses. Importantly, they suggest that Itk and Txk exert their effects on TH cell differentiation/function at the level of expression.

The Tec family kinase Itk regulates the development of conventional and innate CD8+ T cells, however little is known about the role of Itk in the development of CD4+ T cell lineages, although the role of Itk in the T cell activation and function is well defined. We show here that the Itk null mice have increased percentage of CD62LLoCD44Hi memory phenotype CD4+ T cells compared to WT mice. These cells arise directly in the thymus, express high levels of transcripts for the T-bet and IFNγ and are able to produce IFNγ directly ex vivo in response to stimulation. Itk deficiency greatly decreases the number of CD4+ T cells with CD62LHiCD44Lo naïve phenotype, but has no effect on the number of memory phenotype CD4+ T cells, indicating that the development of memory phenotype CD4+ T cells is Itk independent. We further show that the development of the naïve phenotype CD4+ T cells is dependent on active Itk kinase signals and can be rescued by expression of Itk specifically in T cells. Our data also show that the Itk is required for functional TCR signaling in these cells, but not for the innate function in response to IL-12/IL-18 or L. monocytogenes stimulation. These results indicate that CD62LHiCD44Lo “naïve” and CD62LLoCD44Hi “innate memory phenotype” CD4+ T cells may be independent populations that differ in their requirement for Itk’s signals for development. Our data also suggest that CD4+CD62LLoCD44Hi memory phenotype T cells have innate immune function.

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] and the vitamin D receptor (VDR) are important regulators of autoimmunity. The effect of the VDR on the ability of mice to fight a primary or secondary infection has not been determined. Young and old VDR knockout (KO) mice were able to clear both primary and secondary infections with Listeria monocytogenes. However, the kinetics of clearance was somewhat delayed in the absence of the VDR. Memory T cell development was not different in young VDR KO and wild-type (WT) mice; however, old VDR KO mice had significantly less memory T cells than their WT counterparts but still mounted an adequate immune response as determined by the complete clearance of L. monocytogenes. Although the primary and secondary immune responses were largely intact in the VDR KO mice, the old VDR KO mice had increased cytokines and antibody responses compared with the old WT mice. In particular, old VDR KO mice had elevated antigen non-specific antibodies; however, these magnified immune responses did not correspond to more effective Listeria clearance. The increased antibody and cytokine responses in the old VDR KO mice are consistent with the increased susceptibility of these mice to autoimmunity.

T cell development is critically dependent on both the environment and signals delivered by the T cell Receptor (TCR). The Tec family kinase Itk has been suggested to be an amplifier of signals emanating from the TCR and the loss of Itk partially affects most stages of thymopoiesis. Loss of Itk also differentially affects the development of conventional vs. non-conventional or innate memory phenotype T cells. Here, we examine whether these lineage choices are affected by a combination of TCR affinity and Itk by analyzing mice lacking Itk and carrying two TCR transgenes with differing affinities, OT-II and DO11.10. Our results show that developing thymocytes receive a gradient of signals, DO11.10>OT-II>DO11.10/Itk−/−>OT-II/Itk−/−. We also show that the development of CD4+ T cells is controlled by TCR signaling via Itk, which regulates the expression of the transcription factor, Th-POK, an enforcement factor for CD4 commitment. This results in a reduction in CD4+ T cell development, and an increase in the development of MHC class II restricted TCR transgenic CD8+ T cells that resemble non-conventional or innate memory phenotype CD8 T cells. This alteration accompanies increased expression of Runx3 and its target genes Eomesodermin, Granzyme B and Perforin in Itk null OT-II CD4+ thymocytes. All together, these data suggest that Itk plays an important role in CD4/CD8 commitment by regulating signal thresholds for the lineage commitment. Our data also suggest that the lower level of TCR signaling that occurs with a low affinity TCR in the absence of Itk can redirect some MHC class II restricted CD4+ T cell to class II-restricted CD8+ innate memory phenotype T cells.

Interleukin-2-inducible T cell kinase (ITK) is a non-receptor tyrosine kinase expressed in T cells, NKT cells and mast cells which plays a crucial role in regulating the T cell receptor (TCR), CD28, CD2, chemokine receptor CXCR4 and FcεR mediated signaling pathways. In T cells, ITK is an important mediator for actin reorganization, activation of PLCγ, mobilization of calcium, and activation of the NFAT transcription factor. ITK plays an important role in the secretion of IL-2, but more critically, also has a pivotal role in the secretion of Th2 cytokines, IL-4, IL-5 and IL-13. As such, ITK has been shown to regulate the development of effective Th2 response during allergic asthma as well as infections against parasitic worms. This ability of ITK to regulate Th2 responses, along with it's pattern of expression, has led to the proposal that it would represent an excellent target for Th2 mediated inflammation. We discuss here the possibilities and pitfalls of targeting ITK for inflammatory disorders.

Summary

T cells with a memory like phenotype and possessing innate immune function have been previously identified as CD8+CD44hi cells. These cells rapidly secrete IFNγ upon stimulation with IL-12/IL-18 and are involved in innate responses to infection with Listeria monocytogenes. The signals regulating these cells are unclear. The Tec kinase Itk regulates T cell activation and we report here that a majority of the CD8+ T cells in Itk null mice have a phenotype of CD44hi similar to memory like innate T cells. These cells are observed in mice carrying an Itk mutant lacking the kinase domain, indicating that active Tec kinase signaling suppresses their presence. These cells carry preformed message for and are able to rapidly produce IFNγ upon stimulation in vitro with IL-12/IL-18, and endow Itk null mice the ability to effectively respond to infection with L. monocytogenes or exposure to LPS by secretion of IFNγ. Transfer of these cells rescues the ability of IFNγ null mice to reduce bacterial burden following L. monocytogenes infection indicating that these cells are functional CD8+CD44hi T cells previously detected in vivo. These results indicate that active signals from Tec kinases regulate the development of memory like CD8+ T cells with innate function.

The Tec-family protein tyrosine kinase IL-2–inducible T cell kinase (ITK) mediates T cell activation, as does the adaptor protein SLP-76 (SH2-domain–containing leukocyte protein of 76 kD), which forms a complex with ITK and other intracellular signaling enzymes. One of these enzymes is phospholipase C–γ1 (PLC-γ1), which mediates T cell receptor (TCR)–stimulated intracellular calcium mobilization leading to the activation of transcription factors such as nuclear factor of activated T cells. The Src-family tyrosine kinase Lck and the Syk-family tyrosine kinase ζ chain–associated protein kinase of 70 kD (ZAP-70), together with ITK, are necessary for the phosphorylation of PLC-γ1 in response to TCR stimulation. ITK is thought to phosphorylate a specific tyrosine residue of PLC-γ1 that is required for its activation. The mechanism of activation of ITK appears to involve the interaction between SLP-76 and ITK, which not only initiates ITK activity but is also important to maintain the kinase activity of ITK. This suggests that SLP-76 acts as more than a neutral adaptor in mediating T cell activation; SLP-76 also directly influences the kinase activity of ITK, allowing ITK to phosphorylate PLC-γ1.

Elevated expression of p130Cas/BCAR1 (breast cancer anti estrogen resistance 1) in human breast tumors is a marker of poor prognosis and poor overall survival. Specifically, p130Cas signaling has been associated with antiestrogen resistance, for which the mechanism is currently unknown. TAM-R cells, which were established by long-term exposure of estrogen (E2)-dependent MCF-7 cells to tamoxifen, displayed elevated levels of total and activated p130Cas. Here we have investigated the effects of p130Cas inhibition on growth factor signaling in tamoxifen resistance. To inhibit p130Cas, a phosphorylated substrate domain of p130Cas, that acts as a dominant-negative (DN) p130Cas molecule by blocking signal transduction downstream of the p130Cas substrate domain, as well as knockdown by siRNA was employed. Interference with p130Cas signaling/expression induced morphological changes, which were consistent with a more epithelial-like phenotype. The phenotypic reversion was accompanied by reduced migration, attenuation of the ERK and phosphatidylinositol 3-kinase/Akt pathways, and induction of apoptosis. Apoptosis was accompanied by downregulation of the expression of the anti-apoptotic protein Bcl-2. Importantly, these changes re-sensitized TAM-R cells to tamoxifen treatment by inducing cell death. Therefore, our findings suggest that targeting the product of the BCAR1 gene by a peptide which mimics the phosphorylated substrate domain may provide a new molecular avenue for treatment of antiestrogen resistant breast cancers.

HIV-1 assembly and egress are driven by the viral protein Gag and occur at the plasma membrane in T cells. Recent evidence indicates that secretory vesicles and machinery are essential components of virus packaging in both T cells and macrophages. However, the pathways and cellular mediators of Gag targeting to the plasma membrane are not well characterized. Lck, a lymphoid specific Src kinase critical for T cell activation, is found in the plasma membrane as well as various intracellular compartments and it has been suggested to influence HIV-1 replication. To investigate Lck as a potential regulator of Gag targeting, we assessed HIV-1 replication and Gag induced virus-like particle release in the presence and absence of Lck. Release of HIV-1 and virus like particles was reduced in the absence of Lck. This decrease in replication was not due to altered HIV-1 infection, transcription or protein translation. However, in T cells lacking Lck, HIV-1 accumulated intracellularly. In addition, expressing Lck in HeLa cells promoted HIV-1 Gag plasma membrane localization. Palmitoylation of the Lck unique domain, which is essential for directing Lck to the plasma membrane, was critical for its effect on HIV-1 replication. Furthermore, HIV-1 Gag directly interacted with the Lck unique domain in the context of infected cells. These results indicate that Lck plays a key role in targeting HIV-1 Gag to the plasma membrane in T cells.

The Tec family tyrosine kinase Itk is expressed in T cells and mast cells. Mice lacking Itk exhibit impaired TH2 cytokine secretion; however, they have increased circulating serum IgE but exhibit few immunological symptoms of allergic airway responses. We have examined the role of Itk in mast cell function and FcεRI signaling. We report here that Itk null mice have reduced allergen/IgE induced histamine release as well as early airway hyperresponsiveness in vivo. This is due to the increased levels of IgE in the serum of these mice, since the transfer of Itk null BMMC into mast cell deficient W/Wv animals is able to fully rescue histamine release in the W/Wv mice. Further analysis of Itk null BMMC in vitro revealed that while they have normal degranulation responses, they secrete elevated levels of cytokines, including IL-13 and TNF-α, particularly in response to unliganded IgE. Analysis of biochemical events downstream of the FcεRI revealed little difference in overall tyrosine phosphorylation of specific substrates or calcium responses, however, these cells express elevated levels of NFAT which was largely nuclear. Our results suggest that the reduced mast cell response in vivo in Itk null mice is due to elevated levels of IgE in these mice. Our results also suggest that Itk differentially modulates mast cell degranulation and cytokine production in part by regulating expression and activation of NFAT proteins in these cells.

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Eosinophils have been implicated as playing a major role in allergic airway responses. However, the importance of these cells to the development of this disease has remained ambiguous despite many studies, partly because of lack of appropriate model systems. In this study, using transgenic murine models, we more clearly delineate a role for eosinophils in asthma. We report that, in contrast to results obtained on a BALB/c background, eosinophil-deficient C57BL/6 ΔdblGATA mice (eosinophil-null mice via the ΔDblGATA1 mutation) have reduced airway hyperresponsiveness, and cytokine production of interleukin (IL)-4, -5, and -13 in ovalbumin-induced allergic airway inflammation. This was caused by reduced T cell recruitment into the lung, as these mouse lungs had reduced expression of CCL7/MCP-3, CC11/eotaxin-1, and CCL24/eotaxin-2. Transferring eosinophils into these eosinophil-deficient mice and, more importantly, delivery of CCL11/eotaxin-1 into the lung during the development of this disease rescued lung T cell infiltration and airway inflammation when delivered together with allergen. These studies indicate that on the C57BL/6 background, eosinophils are integral to the development of airway allergic responses by modulating chemokine and/or cytokine production in the lung, leading to T cell recruitment.

Extracellular domains of internalizing cell surface receptors are often targeted to enable drug delivery through the mechanism of receptor-mediated endocytosis. To circumvent natural receptors required for endocytic drug delivery, we constructed a small artificial cell surface receptor comprising the membrane anchor N-alkyl-3β-cholesterylamine linked to a D-Phe-D-Ala motif that binds the glycopeptide antibiotic vancomycin. By mimicking membrane association and trafficking properties of cholesterol, this cholesterol-derived synthetic receptor functions as a prosthetic molecule, inserting into plasma membranes of mammalian cells, and rapidly cycling between the cell surface and intracellular endosomes. Human HeLa cells treated with this receptor gained the ability to internalize vancomycin by endocytosis, enabling this cell-impermeable antibiotic to eradicate the lethal intracellular pathogen Listeria monocytogenes, and preserving the viability of the host cells in vitro. Moreover, when combined with the synthetic receptor, vancomycin penetrated the blood brain barrier, accumulating in the brain and other tissues of mice in vivo. Because of their ability to define new pathways across biological membrane barriers, small synthetic cell surface receptors represent promising tools for drug delivery.

The order Mononegavirales (comprised of nonsegmented negative-stranded RNA viruses or NNSVs) contains many important pathogens. Parainfluenza virus 5 (PIV5), formerly known as simian virus 5, is a prototypical paramyxovirus and encodes a V protein, which has a cysteine-rich C terminus that is conserved among all paramyxoviruses. The V protein of PIV5, like that of many other paramyxoviruses, plays an important role in regulating viral RNA synthesis. In this work, we show that V interacts with Akt, a serine/threonine kinase, also known as protein kinase B. Both pharmacological inhibitors and small interfering RNA against Akt1 reduced PIV5 replication, indicating that Akt plays a critical role in PIV5 replication. Furthermore, treatment with Akt inhibitors also reduced the replication of several other paramyxoviruses, as well as vesicular stomatitis virus, the prototypical rhabdovirus, indicating that Akt may play a more universal role in NNSV replication. The phosphoproteins (P proteins) of NNSVs are essential cofactors for the viral RNA polymerase complex and require heavy phosphorylation for their activity. Inhibition of Akt activity reduced the level of P phosphorylation, suggesting that Akt is involved in regulating viral RNA synthesis. In addition, Akt1 phosphorylated a recombinant P protein of PIV5 purified from bacteria. The finding that Akt plays a critical role in replication of NNSV will lead to a better understanding of how these viruses replicate, as well as novel strategies to treat infectious diseases caused by NNSVs.

Mice lacking the vitamin D receptor (VDR) are resistant to airway inflammation. Pathogenic immune cells capable of transferring experimental airway inflammation to wildtype (WT) mice are present and primed in the VDR KO mice. Furthermore, the VDR KO immune cells homed to the WT lung in sufficient numbers to induce symptoms of asthma. Conversely, WT splenocytes, Th2 cells and hematopoetic cells induced some symptoms of experimental asthma when transferred to VDR KO mice, but the severity was less than that seen in the WT controls. Interestingly, experimentally induced vitamin D deficiency failed to mirror the VDR KO phenotype suggesting there might be a difference between absence of the ligand and VDR deficiency. Lipopolysaccharide (LPS) induced inflammation in the lungs of VDR KO mice was also less than in WT mice. Together the data suggest that vitamin D and the VDR are important regulators of inflammation in the lung and that in the absence of the VDR the lung environment, independent of immune cells, is less responsive to environmental challenges.

NKT cells are a heterogeneous population characterized by the ability to rapidly produce cytokines, such as interleukin 2 (IL-2), IL-4, and gamma interferon (IFN-γ) in response to infections by viruses, bacteria, and parasites. The bacterial superantigen staphylococcal enterotoxin B (SEB) interacts with T cells bearing the Vβ3, -7, or -8 T-cell receptors, inducing their expansion and cytokine secretion, leading to death in some cases due to cytokine poisoning. The majority of NKT cells bear the Vβ7 or -8 T-cell receptor, suggesting that they may play a role in regulating this response. Using mice lacking NKT cells (CD1d−/− and Jα18−/− mice), we set out to identify the role of these cells in T-cell expansion, cytokine secretion, and toxicity induced by exposure to SEB. We find that Vβ8+ CD4+ T-cell populations similarly expand in wild-type (WT) and NKT cell-null mice and that NKT cells did not regulate the secretion of IL-2. By contrast, these cells positively regulated the secretion of IL-4 and IFN-γ production and negatively regulated the secretion of tumor necrosis factor alpha (TNF-α). However, this negative regulation of TNF-α secretion by NKT cells provides only a minor protective effect on SEB-mediated shock in WT mice compared to mice lacking NKT cells. These data suggest that NKT cells may regulate the nature of the cytokine response to exposure to the superantigen SEB and may act as regulatory T cells during exposure to this superantigen.

Background

Exposure to Staphylococcal Enterotoxin B (SEB), a bacterial superantigen secreted by the Gram-positive bacteria Staphyloccocus aureus, results in the expansion and eventual clonal deletion and anergy of Vβ8+ T cells, as well as massive cytokine release, including Interleukin-2 (IL-2). This IL-2 is rapidly secreted following exposure to SEB and may contribute to the symptoms seen following exposure to this bacterial toxin. The Tec family kinase ITK has been shown to be important for the production of IL-2 by T cells stimulated in vitro and may represent a good target for blocking the production of this cytokine in vivo. In order to determine if ITK represents such a target, mice lacking ITK were analyzed for their response to SEB exposure.

Results

It was found that T cells from mice lacking ITK exhibited significantly reduced proliferative responses to SEB exposure in vitro, as well as in vivo. Examination of IL-2 production revealed that ITK null mice produced reduced levels of this cytokine in vitro, and more dramatically, in vivo. In vivo analysis of c-jun phosphorylation, previously shown to be critical for regulating IL-2 production, revealed that this pathway was specifically activated in SEB reactive Vβ8+ (but not non-reactive Vβ6+) T cells from WT mice, but not in Vβ8+ T cells from ITK null mice. However, toxicity analysis indicated that both WT and ITK null animals were similarly affected by SEB exposure.

Conclusion

These data show that ITK is required for IL-2 production induced by SEB in vivo, and may regulate signals leading IL-2 production, in part by regulating phosphorylation of c-jun. The data also suggest that perturbing T cell activation pathways leading to IL-2 does not necessarily lead to improved responses to SEB toxicity.