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Abstract Background Bullous pemphigoid (BP) is the most frequent autoimmune blistering disease of the skin affecting the elderly. BP is immunopathologically characterized by autoantibodies against BP180 and BP230. With the growing evidence of cell‐mediated autoimmunity in the pathogenesis of BP, it still remains unclear whether mast cells (MCs) are involved, due to conflicting data obtained from Kit‐dependent MC‐deficient mouse models. Objectives To clarify the role of MCs in experimental BP; the dynamics in cutaneous MC numbers, associated immune cells and the development of disease in Kit‐independent MC‐deficient mouse model. Methods Employing a recently established murine adult passive transfer model of BP induced by the transfer of pathogenic immunoglobulin G (IgG), lesional skin biopsies were investigated histologically and immunohistochemically for the time‐dependent MC accumulation and dermal infiltration. Results The numbers of cutaneous MCs increased following the induction of BP, in part, maintained by MC proliferation. Numbers of T cells, neutrophils and eosinophils in the skin also increased after BP induction, with eosinophils showing a preferential co‐localization with MCs. Furthermore, clinical disease manifestation in MC‐deficient Mcpt5Cre/Dicer fl/fl mice remained unchanged compared to MC‐sufficient Dicer fl/fl mice. The composition of the immune cell infiltration including as T cells, neutrophils and eosinophils was largely unaffected by the absence of MCs. Conclusion MCs do not play a pivotal role in the pathogenesis of passive IgG‐transfer mediated BP model. Their increase in number may be a bystander effect following tissue injury. We therefore suggest caution regarding the selection of MCs as sole targets for the development of novel drugs for BP.

Background: Children with asthma have impaired production of interleukin (IL) 37; in mice, IL-37 reduces hallmarks of experimental allergic asthma (EAA). However, it remains unclear how IL-37 exerts its inhibitory properties in asthma. This study aimed to identify the mechanism(s) by which IL-37 controls allergic inflammation., Methods: IL-37 target cells were identified by single-cell RNA-seq of IL-1R5 and IL-1R8. Airway tissues were isolated by laser-capture microdissection and examined by microarray-based gene expression analysis. Mononuclear cells (MNC) and airway epithelial cells (AECs) were isolated and stimulated with allergen, IL-1β, or IL-33 together with recombinant human (rh) IL-37. Wild-type, IL-1R1- and IL-33-deficient mice with EAA were treated with rhIL-37. IL-1β, IL-33, and IL-37 levels were determined in sputum and nasal secretions from adult asthma patients without glucocorticoid therapy., Results: IL-37 target cells included AECs, T cells, and dendritic cells. In mice with EAA, rhIL-37 led to differential expression of >90 genes induced by IL-1β and IL-33. rhIL-37 reduced production of Th2 cytokines in allergen-activated MNCs from wild-type but not from IL-1R1-deficient mice and inhibited IL-33-induced Th2 cytokine release. Furthermore, rhIL-37 attenuated IL-1β- and IL-33-induced pro-inflammatory mediator expression in murine AEC cultures. In contrast to wild-type mice, hIL-37 had no effect on EAA in IL-1R1- or IL-33-deficient mice. We also observed that expression/production ratios of both IL-1β and IL-33 to IL-37 were dramatically increased in asthma patients compared to healthy controls., Conclusion: IL-37 downregulates allergic airway inflammation by counterbalancing the disease-amplifying effects of IL-1β and IL-33., (© 2021 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Background: Bullous pemphigoid (BP) is the most frequent autoimmune blistering disease of the skin affecting the elderly. BP is immunopathologically characterized by autoantibodies against BP180 and BP230. With the growing evidence of cell-mediated autoimmunity in the pathogenesis of BP, it still remains unclear whether mast cells (MCs) are involved, due to conflicting data obtained from Kit-dependent MC-deficient mouse models., Objectives: To clarify the role of MCs in experimental BP; the dynamics in cutaneous MC numbers, associated immune cells and the development of disease in Kit-independent MC-deficient mouse model., Methods: Employing a recently established murine adult passive transfer model of BP induced by the transfer of pathogenic immunoglobulin G (IgG), lesional skin biopsies were investigated histologically and immunohistochemically for the time-dependent MC accumulation and dermal infiltration., Results: The numbers of cutaneous MCs increased following the induction of BP, in part, maintained by MC proliferation. Numbers of T cells, neutrophils and eosinophils in the skin also increased after BP induction, with eosinophils showing a preferential co-localization with MCs. Furthermore, clinical disease manifestation in MC-deficient Mcpt5Cre/Dicer fl/fl mice remained unchanged compared to MC-sufficient Dicer fl/fl mice. The composition of the immune cell infiltration including as T cells, neutrophils and eosinophils was largely unaffected by the absence of MCs., Conclusion: MCs do not play a pivotal role in the pathogenesis of passive IgG-transfer mediated BP model. Their increase in number may be a bystander effect following tissue injury. We therefore suggest caution regarding the selection of MCs as sole targets for the development of novel drugs for BP., Competing Interests: The authors confirm that no conflict of interest to declare., (© 2021 The Authors. Skin Health and Disease published by John Wiley & Sons Ltd on behalf of British Association of Dermatologists.)

Mast cells (MCs) are long-living immune cells highly specialized in the storage and release of different biologically active compounds and are involved in the regulation of innate and adaptive immunity. MC degranulation and replacement of MC granules are accompanied by active membrane remodelling. Tetraspanins represent an evolutionary conserved family of transmembrane proteins. By interacting with lipids and other membrane and intracellular proteins, they are involved in organisation of membrane protein complexes and act as "molecular facilitators" connecting extracellular and cytoplasmic signaling elements. MCs express different tetraspanins and MC degranulation is accompanied by changes in membrane organisation. Therefore, tetraspanins are very likely involved in the regulation of MC exocytosis and membrane reorganisation after degranulation. Antiviral response and production of exosomes are further aspects of MC function characterized by dynamic changes of membrane organization. In this review, we pay a particular attention to tetraspanin gene expression in different human and murine MC populations, discuss tetraspanin involvement in regulation of key MC signaling complexes, and analyze the potential contribution of tetraspanins to MC antiviral response and exosome production. In-depth knowledge of tetraspanin-mediated molecular mechanisms involved in different aspects of the regulation of MC response will be beneficial for patients with allergies, characterized by overwhelming MC reactions.

Mast cells (MCs), which are well known for their effector functions in T H 2-skewed allergic and also autoimmune inflammation, have become increasingly acknowledged for their role in protection of health. It is now clear that they are also key modulators of immune responses at interface organs, such as the skin or gut. MCs can prime tissues for adequate inflammatory responses and cooperate with dendritic cells in T-cell activation. They also regulate harmful immune responses in trauma and help to successfully orchestrate pregnancy. This review focuses on the beneficial effects of MCs on tissue homeostasis and elimination of toxins or venoms. MCs can enhance pathogen clearance in many bacterial, viral, and parasitic infections, such as through Toll-like receptor 2-triggered degranulation, secretion of antimicrobial cathelicidins, neutrophil recruitment, or provision of extracellular DNA traps. The role of MCs in tumors is more ambiguous; however, encouraging new findings show they can change the tumor microenvironment toward antitumor immunity when adequately triggered. Uterine tissue remodeling by α-chymase (mast cell protease [MCP] 5) is crucial for successful embryo implantation. MCP-4 and the tryptase MCP-6 emerge to be protective in central nervous system trauma by reducing inflammatory damage and excessive scar formation, thereby protecting axon growth. Last but not least, proteases, such as carboxypeptidase A, released by FcεRI-activated MCs detoxify an increasing number of venoms and endogenous toxins. A better understanding of the plasticity of MCs will help improve these advantageous effects and hint at ways to cut down detrimental MC actions., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Mast cells (MCs) have long been mainly regarded as effector cells in IgE-associated allergic disorders with potential immunoregulatory roles. Located close to the allergen entry sites in the skin and mucosa, MCs can capture foreign substances such as allergens, toxins, or noxious substances and are exposed to the danger signals produced by epithelial cells. MC reactivity shaped by tissue-specific factors is crucial for allergic responses ranging from local skin reactions to anaphylactic shock. Development of Th2 response leading to allergen-specific IgE production is a prerequisite for MC sensitization and induction of FcεRI-mediated MC degranulation. Up to now, IgE production has been mainly associated with proteins, whereas lipids present in plant pollen grains, mite fecal particles, insect venoms, or food have been largely overlooked regarding their immunostimulatory and immunomodulatory properties. Recent studies, however, have now demonstrated that lipids affect the sensitization process by modulating innate immune responses of epithelial cells, dendritic cells, and NK-T cells and thus crucially contribute to the outcome of sensitization. Whether and how lipids affect also MC effector functions in allergic reactions has not yet been fully clarified. Here, we discuss how lipids can affect MC responses in the context of allergic inflammation. Direct effects of immunomodulatory lipids on MC degranulation, changes in local lipid composition induced by allergens themselves and changes in lipid transport affecting MC reactivity are possible mechanisms by which the function of MC might be modulated.

Plant pollen are an important source of antigens that evoke allergic responses. Protein antigens have been the focus of studies aiming to elucidate the mechanisms responsible for allergic reactions to pollen. However, proteins are not the sole active agent present in pollen. It is known that pollen grains contain lipids essential for its reproduction and bioactive lipid mediators. These small molecular compounds are co-delivered with the allergens and hence have the potential to modulate the immune response of subjects by activating their innate immune cells. Previous reports showed that pollen associated lipid mediators exhibited neutrophil- and eosinophil-chemotactic activity and induced polarization of dendritic cells (DCs) toward a Th2-inducing phenotype. In our study we performed chemical analyses of the pollen associated lipids, that are rapidly released upon hydration. As main components we have identified different types of phytoprostanes (PhytoPs), and for the first time phytofurans (PhytoFs), with predominating 16-F 1t -PhytoPs (PPF 1 -I), 9-F 1t -PhytoPs (PPF 1 -II), 16-E 1t -PhytoPs (PPE 1 -I) and 9-D 1t -PhytoPs (PPE 1 -II), and 16( RS )-9- epi -ST-Δ 14 -10-PhytoFs. Interestingly 16-E 1t -PhytoP and 9-D 1t -PhytoPs were found to be bound to glycerol. Lipid-containing samples (aqueous pollen extract, APE) induced murine mast cell chemotaxis and IL-6 release, and enhanced their IgE-dependent degranulation, demonstrating a role for these lipids in the immediate effector phase of allergic inflammation. Noteworthy, mast cell degranulation seems to be dependent on glycerol-bound, but not free phytoprostanes. On murine dendritic cells, APE selectively induced the upregulation of CD1d, likely preparing lipid-antigen presentation to iNKT cells. Our report contributes to the understanding of the activity of lipid mediators in the immediate effector phase of allergic reactions but identifies a yet undescribed pathway for the recognition of pollen-derived glycolipids by iNKT cells.

Background: Food-induced anaphylaxis is a serious allergic reaction caused by Fcε-receptor activation on mast cells (MCs). The exact mechanisms breaking oral tolerance and the effector pathways driving food allergy remain elusive. As complement is activated in food-induced anaphylaxis, we aimed to assess the role of C5a in disease pathogenesis., Methods: Oral antigen-induced food-induced anaphylaxis was induced in BALB/c wild-type (wt) and C5ar1 -/- mice. Readouts included diarrhea development, changes in rectal temperature, hematocrit, antigen-specific serum IgE, MCPT-1, and intestinal MC numbers, as well as FcεR1-mediated MC functions including C5a receptor 1 (C5aR1) regulation. Further, histamine-mediated hypothermia and regulation of endothelial tight junctions were determined., Results: Repeated oral OVA challenge resulted in diarrhea, hypothermia, increased hematocrit, high OVA-specific serum IgE, and MCPT-1 levels in wt mice. Male C5ar1 -/- mice were completely whereas female C5ar1 -/- mice were partially protected from anaphylaxis development. Serum MCPT-1 levels were reduced gender-independent, whereas IgE levels were reduced in male but not in female C5ar1 -/- mice. Mechanistically, IgE-mediated degranulation and IL-6 production from C5ar1 -/- BMMCs of both sexes were significantly reduced. Importantly, FcεR1 cross-linking strongly upregulated C5aR1 MC expression in vitro and in vivo. Finally, C5ar1 -/- male mice were largely protected from histamine-induced hypovolemic shock, which was associated with protection from histamine-induced barrier dysfunction in vitro following C5aR targeting., Conclusions: Our findings identify C5aR1 activation as an important driver of IgE-mediated food allergy through regulation of allergen-specific IgE production, FcεR1-mediated MC degranulation, and histamine-driven effector functions preferentially in male mice., (© 2018 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

IL-9 is a cytokine with pleiotropic function that mediates allergic inflammation and immunity to intestinal helminth parasites. Accumulating evidence suggests that IL-9 acts via both, initiation and regulation of adaptive immune responses and direct activation of intestinal effector pathways. Here we use IL-9 receptor deficient mice on BALB/c and C57BL/6 genetic background to dissect effector and regulatory functions of IL-9 during infection with the parasitic nematode Strongyloides ratti. IL-9 receptor-deficient mice displayed increased intestinal parasite burden and prolonged infection irrespective of the genetic background of the mice. Increased parasite burden was correlated to a reciprocally reduced early degranulation of mucosal mast cells, reduced intestinal IL-13 expression and caused by IL-9 receptor deficiency on hematopoietic cells. We observed additional significant changes in the adaptive immune response to S. ratti infection in the absence of the IL-9 receptor that depended on the mouse strain. However, the generation of protective memory to a second infection was intact in IL-9 receptor-deficient mice, irrespective of the genetic background. In summary, our results support a central role for IL-9 as an early mast cell activating effector cytokine during intestinal helminth infection while non-redundant functions in the initiation and amplification of adaptive immune responses were not apparent.

Suppressor of cytokine signaling 1 (SOCS1) is a negative feedback inhibitor of cytoplasmic Janus kinase and signal transducer and activator of transcription (STAT) signaling. SOCS1 also contains a nuclear localization sequence (NLS), yet, the in vivo importance of nuclear translocation is unknown. We generated transgenic mice containing mutated Socs1ΔNLS that fails to translocate in the cell nucleus ( MGL tg mice). Whereas mice fully deficient for SOCS1 die within the first 3 weeks due to excessive interferon signaling and multiorgan inflammation, mice expressing only non-nuclear Socs1ΔNLS ( Socs1 -/- MGL tg mice) were rescued from early lethality. Canonical interferon gamma signaling was still functional in Socs1 -/- MGL tg mice as shown by unaltered tyrosine phosphorylation of STAT1 and whole genome expression analysis. However, a subset of NFκB inducible genes was dysregulated. Socs1 -/- MGL tg mice spontaneously developed low-grade inflammation in the lung and had elevated Th2-type cytokines. Upon ovalbumin sensitization and challenge, airway eosinophilia was increased in Socs1 -/- MGL tg mice. Decreased transepithelial electrical resistance in trachea epithelial cells from Socs1 -/- MGL tg mice suggests disrupted epithelial cell barrier. The results indicate that nuclear SOCS1 is a regulator of local immunity in the lung and unravel a so far unrecognized function for SOCS1 in the cell nucleus.

The interaction between tissue-resident mast cells (MCs) and recruited immune cells contributes to tissue immunosurveillance. However, the cells, mechanisms, and receptors involved in this crosstalk remain ill defined. Invariant natural killer T (iNKT) cells are CD1d-restricted innate lymphocytes that recognize glycolipid antigens and have emerged as critical players in immunity. Here, we show that primary mouse peritoneal MCs express surface CD1d, which is upregulated in vivo following administration of alpha-galactosylceramide. In contrast, in BM-derived MCs CD1d was found to be stored intracellularly and to relocate at the cell surface upon IgE-mediated degranulation. Activated BM-derived MCs expressing surface CD1d and loaded with alpha-galactosylceramide were found to induce iNKT-cell proliferation and the release of IFN-γ, IL-13, and IL-4 in a CD1d-restricted manner. Moreover, the costimulatory molecules CD48, CD137L, CD252, CD274, and CD275 affected MC-induced IFN-γ release and iNKT-cell proliferation. Interestingly, among the costimulatory molecules, CD48 and CD252 exhibited a distinctly regulatory activity on iNKT-cell release of both IFN-γ and IL-13. In conclusion, we demonstrate that the crosstalk between MCs and iNKT cells may regulate inflammatory immune responses., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The common gamma (γc)-chain cytokine interleukin 15 (IL15) is a multifunctional immune-modulator which impacts the generation, maturation and activity of many cell types of the innate, as well as the adaptive immune system, including natural killer (NK) and CD8(+) T cells. Using a new series of transgenic mice, we analyzed the in vivo potential of IL15 as an immune-regulator when available at different concentrations or delivery modes, i.e. soluble monomer or complexed to its specific receptor α (Rα)-chain. We have identified distinct effects on selected IL15-responsive populations. While CD8(+) T cells required complexed forms of IL15/IL15Rα for full functionality, mature NK populations were rescued in an IL15/IL15Rα-deficient environment by high levels of CD11c-restricted IL15. These IL15-conditions were sufficient to limit tumor formation in a lung metastasis model indicating that the NK cell populations were fully functional. These data underline the potential of "free" IL15 in the absence of Rα-complex as a powerful and specific immuno-modulator, which may be beneficial where selective immune-activation is desired.

Background: Mast cells (MCs) play a central role in allergic and inflammatory disorders by rapid degranulation and release of inflammatory mediators upon antigen-driven engagement of the FcεRI. Receptor-mediated MC responses are controlled by the activation of different isoforms of phosphoinositide-3-kinase (PI3K) and the downstream signaling processes. Recent evidence suggests that miRNAs are important molecular players regulating the PI3K/Akt pathway., Methods: The role of miR-155 in the regulation of MC functions in vivo was studied in the passive cutaneous anaphylaxis (PCA) MC-dependent model. WT and miR-155(-/-) mice were injected intradermally with anti-DNP-IgE and intravenously with the antigen DNP-HSA. Ear swelling was assessed to evaluate the anaphylactic response. All investigations, to characterize miR-155 specific activities in MCs, were conducted comparing WT and miR-155(-/-) bone marrow-derived MCs (BMMCs)., Results: We report that miR-155(-/-) mice display enhanced anaphylaxis reactions. Although miR-155(-/-) BMMCs show normal development, proliferation, and survival, miR-155 deficiency enhances FcεRI-mediated degranulation and release of TNF-α, IL-13, and IL-6. Interestingly, the level of Akt phosphorylation on both of its regulatory residues Thr308 and Ser473 was increased in miR-155(-/-) compared to WT BMMCs. Gene expression profiling showed that miR-155(-/-) BMMCs exhibited significantly increased expression of the adapter PI3Kγ subunits Pik3r5 (p101) and Pik3r6 (p84, p87(PIKAP) ). Furthermore, selective blockade of the PI3Kγ pathway inhibited degranulation in miR-155(-/-) BMMCs., Conclusions: Thus, we suggest that miR-155 plays a critical role in FcεRI-mediated MC responses by modulating components of the PI3Kγ pathway. This newly identified mechanism of miRNA-controlled MC activation may affect the initiation and maintenance of allergic disorders., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Mast cells are important cellular constituents of epithelial-mesenchymal interactions, densely located at sites of microbial entry into the host where they are continuously exposed to products from commensals. In order to avoid excessive activation and the associated pathology, mast cell responses to TLR agonists must be tightly regulated. Here, we show that exposure in vitro to subactivating levels of the epithelial cell product, IL-33, renders mast cells insensitive to bacterial cell wall products. Mast cell responsiveness to Ag, cytoplasmic dsDNA, and TLR7/8 agonists is unaffected or enhanced by IL-33. The IL-33-induced mast cell selective tolerance requires the IL-33 receptor ST2 and peritoneal mast cells from St2(-/-) mice display a constitutively activated phenotype, demonstrated by increased expression of activation markers including CD11b and CD28. IL-33 exposure neither affects the levels of TLR4, MyD88, TIRAP, IL-1R associated kinase 2 (IRAK2), or IRAK4, nor induces persistent A20 or Tollip expression, but potently causes ST2-dependent IRAK1 degradation. We show that while IRAK2 is redundant for TLR4 signaling, IRAK1 is essential for TLR4 signaling in mast cells. We suggest that IL-33 produced during homeostasis retains mast cells in an unresponsive state to bacterial cell wall products via IRAK1 degradation, thus preventing chronic inflammation and tissue destruction., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Syntaxin 11 (STX11) controls vesicular trafficking and is a key player in exocytosis. Since Stx11 mutations are causally associated with a familial hemophagocytic lymphohistio-cytosis, we wanted to clarify whether STX11 is functionally important for key immune cell populations. This was studied in primary cells obtained from newly generated Stx11(-/-) mice. Our data revealed that STX11 is not only widely expressed in different immune cells, but also induced upon LPS or IFN-γ treatment. However, Stx11 deficiency does not affect macrophage phagocytic function and cytokine secretion, mast cell activation, or antigen presentation by DCs. Instead, STX11 selectively controls lymphocyte cytotoxicity in NK and activated CD8(+) T cells and degranulation in neutrophils. Stx11(-/-) NK cells and CTLs show impaired degranulation, despite a comparable activation, maturation and expression of the complex-forming partners MUNC18-2 and VTI1B. In addition, Stx11(-/-) CTLs and NK cells produce abnormal levels of IFN-γ. Since functional reconstitution rescues the defective phenotype of Stx11(-/-) CTLs, we suggest a direct, specific and key role of STX11 in controlling lymphocyte cytotoxicity, cytokine production and secretion. Finally, we show that these mice are a very useful tool for dissecting the role of STX11 in vesicular trafficking and secretion., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Allergen-specific immunoglobulin (Ig) E initiates the effector cascade of allergic asthma and has been identified as a valuable target for therapeutic treatment of this disease. The proteasome inhibitor bortezomib was previously shown to deplete Ig-secreting plasma cells and to efficiently suppress Ig serum titers. The present study aimed at evaluating the therapeutic potential of the proteasome inhibitor bortezomib in allergic bronchial asthma., Methods: To address this question, a chronic experimental asthma mouse model was used in a therapeutic setting. Mice were sensitized to ovalbumin (OVA) and challenged with OVA aerosol for 12 weeks. After 6 weeks of challenge, bortezomib treatment was started and continued for 1 week (short-term) or 6 weeks (long-term) with a dosage of 0.75 mg/kg body weight twice a week. Lung function, lung histology, Ig serum titers and plasma cell numbers were assessed., Results: Whereas short-term treatment lowered bronchoalveolar lavage eosinophils, long-term treatment considerably reduced serum titers of anti-OVA IgE in mice with chronic experimental asthma. However, neither short-term nor long-term treatment significantly reduced plasma cell numbers, anti-OVA IgG1 serum titers or allergic airway inflammation or ablated airway hyperresponsiveness., Conclusion: Our results suggest that bortezomib treatment has only limited value as plasma cell-depleting therapy against allergic bronchial asthma., (Copyright © 2011 S. Karger AG, Basel.)

Regulatory CD8(+) T cells are critical for self-tolerance and restricting excessive immune responses. The variety of immune functions they fulfill, the heterogeneity of their phenotype, and the mechanism of action are still poorly understood. Here we describe that regulatory CD8(+) T cells exhibiting immunosuppressive actions in vitro and in vivo are recognized as CD38(high) T cells and present in naive mice. CD38 is a glycosylated membrane protein with ectonucleotidase properties. CD8(+)CD38(high) (CD44(+)CD122(+)CD62L(high)) lymphocytes suppress CD4(+) effector T-cell proliferation in an antigen-non specific manner via IFN-γ. While direct cell-to-cell contact is needed for this suppressor activity, it is independent of membrane-bound TGF-β and granzyme B release. IL-15 potentiates the suppressive activity of CD8(+)CD38(high) T cells and controls their survival and expansion. In humans CD8(+)CD38(high) T cells inhibit CD4(+) effector T cell proliferation. In vivo, CD8(+)CD38(high), but not CD8(+)CD38(-) T cells mitigate murine experimental autoimmune encephalomyelitis (EAE) by reducing the clinical score and delaying disease occurrence. EAE suppression is enhanced by pre-treatment of CD8(+)CD38(high) T cells with IL-15. These findings add evidence that the expression of ectoenzyme receptor family members positively correlates with suppressor functions and identifies CD8(+)CD38(high) T cells as potential inhibitors of excessive immune responses.

Mast cell (MC) activation via aggregation of the high affinity IgE receptor (FcεRI) causes degranulation and release of proinflammatory mediators in a process that involves the reorganization of the actin cytoskeleton. However, the regulatory pathways and the molecular links between cytoskeletal changes and MC function are incompletely understood. In this study, we provide genetic evidence for a critical role of the actin-regulatory proteins Coronin1a (Coro1a) and Coro1b on exocytic pathways in MCs: Coro1a(-/-) bone marrow-derived MCs exhibit increased FcεRI-mediated degranulation of secretory lysosomes but significantly reduced secretion of cytokines. Hyperdegranulation of Coro1a(-/-) MCs is further augmented by the additional loss of Coro1b. In vivo, Coro1a(-/-)Coro1b(-/-) mice displayed enhanced passive cutaneous anaphylaxis. Functional reconstitution assays revealed that the inhibitory effect of Coro1a on MC degranulation strictly correlates with cortical localization of Coro1a, requires its filamentous actin-binding activity, and is regulated by phosphorylation of Ser2 of Coro1a. Thus, coronin proteins, and in turn the actin cytoskeleton, exhibit a functional dichotomy as differential regulators of degranulation versus cytokine secretion in MC biology., (© 2011 Föger et al.)

Since cytokines are potent immunoregulators that can determine the fate of an immune response, their expression is tightly regulated at the transcriptional level. Recent research, however, has also revealed complex post-translational mechanisms through which cytokine secretion, and thereby cytokine activity, is regulated. Here, we review the progress in our understanding of the portfolio of pathways that regulate cytokine intracellular storage, transport, and release. Like other secreted proteins, cytokines utilize canonical and non-canonical secretory pathways for extracellular release. Illustrated by IL-1β, IL-2, TNF-α, IL-12 and IL-15 secretion as selected examples, we discuss common and alternative cytokine secretion pathways and relate them to the consequences these distinct pathways have for cytokine function, mode of action and stability., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Mast cell (MC) differentiation, survival, and activation are controlled by the membrane tyrosine kinase c-Kit upon interaction with stem cell factor (SCF). Here we describe a single point mutation induced by N-ethyl-N-nitrosurea (ENU) mutagenesis in C57BL/6J mice-an A to T transversion at position 2388 (exon 17) of the c-Kit gene, resulting in the isoleucine 787 substitution by phenylalanine (787F), and analyze the consequences of this mutation for ligand binding, signaling, and MC development. The Kit(787F/787F) mice carrying the single amino acid exchange of c-Kit lacks both mucosal and connective tissue-type MCs. In bone marrow-derived mast cells (BMMCs), the 787F mutation does not affect SCF binding and c-Kit receptor shedding, but strongly impairs SCF-induced cytokine production, degranulation enhancement, and apoptosis rescue. Interestingly, c-Kit downstream signaling in 787F BMMCs is normally initiated (Erk1/2 and p38 activation as well as c-Kit autophosphorylation) but fails to be sustained thereafter. In addition, 787F c-Kit does not efficiently mediate Cbl activation, leading to the absence of subsequent receptor ubiquitination and impaired c-Kit internalization. Thus, I787 provides nonredundant signals for c-Kit internalization and functionality.

Mast cells (MCs) play a critical role in innate and adaptive immunity through the release of cytokines, chemokines, lipid mediators, biogenic amines, and proteases. We recently showed that the activities of MC proteases are transcriptionally regulated by intracellularly retained interleukin-15 (IL-15), and we provided evidence that this cytokine acts as a specific regulator of mouse mast cell protease-2 (mMCP-2). Here, we show that in wild-type bone marrow-derived mast cells (BMMCs) IL-15 inhibits mMCP-2 transcription indirectly by inducing differential expression and mMCP-2 promoter binding of the bifunctional transcription factors C/EBPbeta and YY1. In wild-type BMMCs, C/EBPbeta expression predominates over YY1 expression, and thus C/EBPbeta preferentially binds to the mMCP-2 promoter. In IL-15-deficient BMMCs, the opposite is found: YY1 expression predominates and binds to the mMCP-2 promoter at the expense of C/EBPbeta. Hypertranscription of the mMCP-2 gene in IL-15-deficient BMMCs is associated with histone acetylation and, intriguingly, with methylation of non-CpG dinucleotides within the MCP-2 promoter. This suggests a novel model of cytokine-controlled protease transcription: non-CpG methylation maintains a chromosomal domain in an "open" configuration that is permissive for gene expression.

The characteristics, importance, and molecular requirements for interactions between mast cells (MCs) and CD8(+) T cells have not been elucidated. Here, we demonstrated that MCs induced antigen-specific CD8(+) T cell activation and proliferation. This process required direct cell contact and MHC class I-dependent antigen cross-presentation by MCs and induced the secretion of interleukin-2, interferon-gamma, and macrophage inflammatory protein-1alpha by CD8(+) T cells. MCs regulated antigen-specific CD8(+) T cell cytotoxicity by increasing granzyme B expression and by promoting CD8(+) T cell degranulation. Because MCs also upregulated their expression of costimulatory molecules (4-1BB) and released osteopontin upon direct T cell contact, MC-T cell interactions probably are bidirectional. In vivo, adoptive transfer of antigen-pulsed MCs induced MHC class I-dependent, antigen-specific CD8(+) T cell proliferation, and MCs regulated CD8(+) T cell-specific priming in experimental autoimmune encephalomyelitis. Thus, MCs are important players in antigen-specific regulation of CD8(+) T cells.

The regulation of mast cell activities and survival is a central issue in inflammatory immune responses. Here, we have investigated the role of mouse interleukin-15, a pro-inflammatory and pleiotropic cytokine, in the control of mast cell survival and homeostasis. We report that aged IL-15-/- mice show a reduced number of peritoneal mast cells compared to WT mice. Furthermore, IL-15 deficiency in bone marrow derived mouse mast cells (BMMCs) results in increased susceptibility to apoptosis mediated by growth factor deprivation and A-SMase-treatment. IL-15-/- BMMCs show a constitutive stronger mRNA and protein expression as well as enzymatic activity of the members of the mitochondrial apoptotic pathways including acidic lysosomal aspartate protease cathepsin D (CTSD), endogenous acid sphingomyelinase (A-SMase), caspase-3 and -7 compared to wild type (WT) BMMCs. Furthermore, IL-15-/- BMMCs constitutively generate more A-SMase-derived ceramide than WT controls and display a decreased expression of pro-survival sphingosin-1-phosphate (SPP) both in cytosol and membrane cell fractions. Furthermore, pre-treatment of mast cells with imipramine or pepstatin A, inhibitors of the intracellular acid sphingomyelinase and cathepsin D pathways respectively, increases survival in IL-15-/- BMMCs. These findings suggest that intracellular IL-15 is a key regulator of pathways controlling primary mouse mast cell homeostasis.

Axl, a prototypic member of the transmembrane tyrosine kinase receptor family, is known to regulate innate immunity. In this study, we show that Axl expression is induced by IFN-alpha during human dendritic cell (DC) differentiation from monocytes (IFN/DC) and that constitutively Axl-negative, IL-4-differentiated DC (IL-4/DC) can be induced to up-regulate Axl by IFN-alpha. This effect is inhibited by TLR-dependent maturation stimuli such as LPS, poly(I:C), TLR7/8 ligand, and CD40L. LPS-induced Axl down-regulation on the surface of human IFN-alpha-treated DC correlates with an increased proteolytic cleavage of Axl and with elevated levels of its soluble form. GM6001 and TAPI-1, general inhibitors of MMP and ADAM family proteases, restored Axl expression on the DC surface and diminished Axl shedding. Furthermore, stimulation of Axl by its ligand, Gas6, induced chemotaxis of human DC and rescued them from growth factor deprivation-induced apoptosis. Our study provides the first evidence that Gas6/Axl-mediated signaling regulates human DC activities, and identifies Gas6/Axl as a new DC chemotaxis pathway. This encourages one to explore whether dysregulation of this novel pathway in human DC biology is involved in autoimmunity characterized by high levels of IFN-alpha.

Extracellular ATP mediates a diverse array of biological responses in many cell types and tissues, including immune cells. We have demonstrated that ATP induces purinergic receptor P2X(7) mediated membrane permeabilization, apoptosis, and cytokine expression in murine mast cells (MCs). Here, we report that MCs deficient in the expression of the P2X(7) receptor are resistant to the ATP-induced membrane permeabilization and apoptosis. However, ATP affects the tyrosine phosphorylation pattern of P2X(7)knockout cells, leading to the activation of ERK1/2. Furthermore, ATP induces expression of several cytokines and chemokines in these cells, including IL-4, IL-6, IFN-gamma, TNF-alpha, RANTES, and MIP-2, at the mRNA level. In addition, the release of IL-6 and IL-13 to cell-conditioned medium was confirmed by ELISA. The ligand selectivity and pharmacological profile indicate the involvement of two P2X family receptors, P2X(1) and P2X(3). Thus, depending on genetic background, particular tissue microenvironment, and ATP concentration, MCs can presumably engage different P2X receptor subtypes, which may result in functionally distinct biological responses to extracellular nucleotides. This finding highlights a novel level of complexity in the sophisticated biology of MCs and may facilitate the development of new therapeutic approaches to modulate MC activities.

CD63 is a member of the tetraspanin superfamily that constitutes a main component of the lysosomal membrane. In mice, two CD63 gene loci are present, with only one of these two being functional. We generated and analyzed mice deficient for active CD63. Disruption of CD63 results in a complete loss of CD63 protein expression. Despite its abundance in late endosomes/lysosomes, the lack of CD63 does not cause obvious endosomal/lysosomal abnormalities. CD63 knockout mice are viable and fertile without gross morphological abnormalities in the majority of tissues. No alterations in the populations of immune cells and only minor differences in platelet function were observed. This suggests that the lack of CD63 could be successfully compensated for, most likely by other tetraspanins. However, CD63 deficiency leads to an altered water balance. CD63 knockout mice show an increased urinary flow, water intake, reduced urine osmolality, and a higher fecal water content. In principle cells of the collecting duct of CD63-deficient mice, abnormal intracellular lamellar inclusions were observed. This indicates that the sorting of apical transport proteins might be impaired in these cells. CD63 knockout mice provide an important tool for analyzing the various postulated functions of CD63 in vivo.

While it is well appreciated that receptors for secreted cytokines transmit ligand-induced signals, little is known about additional roles for cytokine receptor components in the control of ligand transport and secretion. Here, we show that interleukin-15 (IL-15) translocation into the endoplasmic reticulum occurs independently of the presence of IL-15 receptor alpha (IL-15R alpha). Subsequently, however, IL-15 is transported through the Golgi apparatus only in association with IL-15R alpha and then is secreted. This intracellular IL-15/IL-15R alpha complex already is formed in the endoplasmic reticulum and, thus, enables the further trafficking of complexed IL-15 through the secretory pathway. Just transfecting IL-15R alpha in cells, which transcribe but normally do not secrete IL-15, suffices to induce IL-15 secretion. Thus, we provide the first evidence of how a cytokine is chaperoned through the secretory pathway by complexing with its own high-affinity receptor and show that IL-15/IL-15R alpha offers an excellent model system for the further exploration of this novel mechanism for the control of cytokine secretion.

Interleukin-2 (IL-2) treatment is currently used to enhance T cell-mediated immune responses against tumors or in viral infections. At the same time, IL-2 is essential for the peripheral homeostasis of CD4(+)CD25(+)Foxp3(+ )regulatory T cells (Treg). In our study, we show that IL-2 is also an important activator of Treg suppressive activity in vivo. IL-2 treatment induces Treg expansion as well as IL-10 production and increases their suppressive potential in vitro. Importantly, in vivo application of IL-2 via gene-gun vaccination using IL-2 encoding DNA plasmids (pIL-2) inhibited naive antigen-specific T cell proliferation as well as a Th1-induced delayed type hypersensitivity response. The suppressive effect can be transferred onto naive animals by Treg from IL-2-treated mice and the suppression depends on the synergistic action of IL-10 and TGF-beta. These data highlight that during therapeutic treatment with IL-2 the concomitant activation of Treg may indeed counteract the intended activation of cellular immunity.

Sepsis remains a global clinical problem. By using the mouse cecal ligation and puncture model of sepsis, here we identify an important aspect of mast cell (MC)-dependent, innate immune defenses against Gram-negative bacteria by demonstrating that MC protease activity is regulated by interleukin-15 (IL-15). Mouse MCs express both constitutive and lipopolysaccharide-inducible IL-15 and store it intracellularly. Deletion of Il15 in mice markedly increases chymase activities, leading to greater MC bactericidal responses, increased processing and activation of neutrophil-recruiting chemokines, and significantly higher survival rates of mice after septic peritonitis. By showing that intracellular IL-15 acts as a specific negative transcriptional regulator of a mouse MC chymase (mast cell protease-2), we provide evidence that defined MC protease activity is transcriptionally regulated by an intracellularly retained cytokine. Our results identify an unexpected breach in MC-dependent innate immune defenses against sepsis and suggest that inhibiting intracellular IL-15 in MCs may improve survival from sepsis.

Interleukin 15 (IL-15) is a pleiotropic cytokine that is hardly detectable in biological fluids. Here, we show that IL-15 forms functional heterocomplexes with soluble high affinity IL-15 receptor alpha (IL-15Ralpha) chain in mouse serum and cell-conditioned medium, which prevents IL-15 detection by ELISA. We also demonstrate that two soluble IL-15Ralpha (sIL-15Ralpha) sushi domain isoforms are generated through a novel alternative splicing mechanism within the IL-15Ralpha gene. These isoforms potentiate IL-15 action by promoting the IL-15-mediated proliferation of the CTLL cell line and interferon gamma production by murine NK cells, which suggests a role in IL-15 transpresentation. Conversely, a full-length sIL-15Ralpha ectodomain released by tumor necrosis factor-alpha-converting enzyme (TACE)-dependent proteolysis inhibits IL-15 activity. Thus, a dual mechanism of sIL-15Ralpha generation exists in mice, giving rise to polypeptides with distinct properties, which regulate IL-15 function.

The function of mast cells as effector cells in allergy has been extensively studied. However, increasing insight into mast cell physiology has revealed new mast cell functions and has introduced mast cells as key players in the regulation of innate as well as adaptive immunity. For example, mast cells have recently been found to express Toll-like receptors (TLRs), which enable them to participate in the innate immune response against pathogens. Furthermore, mast cells have been reported to interact with B cells, dendritic cells and T cells and thereby modulate the direction of an adaptive immune response. Finally, recent documentation that mast cells express functional MHC class II and costimulatory molecules and release immunologically active exosomes, has raised the possibility that mast cells also engage in (as yet) poorly understood antigen presentation functions. In this review, we explore the hypothesis that mast cells serve as central mediators between innate and adaptive immunity, rather as pure effector cells, during allergic innate responses.

Discrimination between cytokine receptor and receptor tyrosine kinase (RTK) signaling pathways is a central paradigm in signal transduction research. Here, we report a 'promiscuous liaison' between both receptors that enables interleukin (IL)-15 to transactivate the signaling pathway of a tyrosine kinase. IL-15 protects murine L929 fibroblasts from tumor necrosis factor alpha (TNFalpha)-induced cell death, but fails to rescue them upon targeted depletion of the RTK, Axl; however, Axl-overexpressing fibroblasts are TNFalpha-resistant. IL-15Ralpha and Axl colocalize on the cell membrane and co-immunoprecipitate even in the absence of IL-15, whereby the extracellular part of Axl proved to be essential for Axl/IL-15Ralpha interaction. Most strikingly, IL-15 treatment mimics stimulation by the Axl ligand, Gas6, resulting in a rapid tyrosine phosphorylation of both Axl and IL-15Ralpha, and activation of the phosphatidylinositol 3-kinase/Akt pathway. This is also seen in mouse embryonic fibroblasts from wild-type but not Axl-/- or IL-15Ralpha-/- mice. Thus, IL-15-induced protection from TNFalpha-mediated cell death involves a hitherto unknown IL-15 receptor complex, consisting of IL-15Ralpha and Axl RTK, and requires their reciprocal activation initiated by ligand-induced IL-15Ralpha.

Axl receptor tyrosine kinase exists as a transmembrane protein and as a soluble molecule. We show that constitutive and phorbol 12-myristate 13-acetate-induced generation of soluble Axl (sAxl) involves the activity of disintegrin-like metalloproteinase 10 (ADAM10). Spontaneous and inducible Axl cleavage was inhibited by the broad-spectrum metalloproteinase inhibitor GM6001 and by hydroxamate GW280264X, which is capable of blocking ADAM10 and ADAM17. Furthermore, murine fibroblasts deficient in ADAM10 expression exhibited a significant reduction in constitutive and inducible Axl shedding, whereas reconstitution of ADAM10 restored sAxl production, suggesting that ADAM10-mediated proteolysis constitutes a major mechanism for sAxl generation in mice. Partially overlapping 14-amino-acid stretch deletions in the membrane-proximal region of Axl dramatically affected sAxl generation, indicating that these regions are involved in regulating the access of the protease to the cleavage site. Importantly, relatively high circulating levels of sAxl are present in mouse sera in a heterocomplex with Axl ligand Gas6. Conversely, two other family members, Tyro3 and Mer, were not detected in mouse sera and conditioned medium. sAxl is constitutively released by murine primary cells such as dendritic and transformed cell lines. Upon immobilization, sAxl promoted cell migration and induced the phosphorylation of Axl and phosphatidylinositol 3-kinase. Thus, ADAM10-mediated generation of sAxl might play an important role in diverse biological processes.

Mast cells play an important role in host defense against various pathogens, but their role in viral infection has not been clarified in detail. dsRNA, synthesized by various types of viruses and mimicked by polyinosinic-polycytidylic acid (poly(I:C)) is recognized by Toll-like receptor 3 (TLR3). In this study, we demonstrate that poly(I:C) injection in vivo potently stimulates peritoneal mast cells to up-regulate a number of different costimulatory molecules. Therefore, we examined the expression and the functional significance of TLR3 activation in mast cells. Mast cells express TLR3 on the cell surface and intracellularly. After stimulation of mast cells with poly(I:C) and Newcastle disease virus (NDV), TLR3 is phosphorylated and the expression of key antiviral response cytokines (interferon beta, ISG15) and chemokines (IP10, RANTES) is upregulated. Interestingly, mast cells activated via TLR3-poly(I:C) potently stimulate CD8+ T-cell recruitment. Indeed, mast-cell-deficient mice (KitW/KitW-v) given an intraperitoneal injection of poly(I:C) show a decreased CD8+ T-cell recruitment, whereas granulocytes normally migrate to the peritoneal cavity. Mast-cell reconstitution of KitW/KitW-v mice normalizes the CD8+ T-cell influx. Thus, mast cells stimulated through engagement of TLR3 are potent regulators of CD8+ T-cell activities in vitro and in vivo.

Extracellular ATP and other nucleotides act through specific cell surface receptors and regulate a wide variety of cellular responses in many cell types and tissues. In this study, we demonstrate that murine mast cells express several P2Y and P2X receptor subtypes including P2X(7), and describe functional responses of these cells to extracellular ATP. Stimulation of bone marrow-derived mast cells (BMMC), as well as MC/9 and P815 mast cell lines with millimolar concentrations of ATP, resulted in Ca(2+) influx across the cellular membrane and cell permeabilization. Moreover, brief exposures to ATP were sufficient to induce apoptosis in BMMCs, MC/9, and P815 cells which involved activation of caspase-3 and -8. However, in the time period between commitment to apoptosis and actual cell death, ATP triggered rapid but transient phosphorylation of multiple signaling molecules in BMMCs and MC/9 cells, including ERK, Jak2, and STAT6. In addition, ATP stimulation enhanced the expression of several proinflammatory cytokines, such as IL-4, IL-6, IL-13, and TNF-alpha. The effects of ATP were mimicked by submillimolar concentrations of 3-O-(4'-benzoyl)-benzoyl-benzoyl-ATP, and were inhibited by pretreatment of mast cells with a selective blocker of human and mouse P2X(7) receptor, 1[N,O-bis(5-isoquinolinesulphonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine, as well as oxidized ATP. The nucleotide selectivity and pharmacological profile data support the role for P2X(7) receptor as the mediator of the ATP-induced responses. Given the importance of mast cells in diverse pathological conditions, the ability of extracellular ATP to induce the P2X(7)-mediated apoptosis in these cells may facilitate the development of new strategies to modulate mast cell activities.

The results from this study implicate membrane-anchored interleukin (IL)-15 constitutively expressed on the cell surface of PC-3 human prostate carcinoma cells and interferon-gamma-activated human monocytes in reverse signaling upon stimulation with soluble IL-15 receptor-alpha or anti-IL-15 antibodies, mediating the outside-to-inside signal transduction that involves the activation of members of the MAPK family (ERK and p38) and focal adhesion kinase. The presence of membrane-bound IL-15 was not dependent on the expression of the trimeric IL-15 receptor complex by these cells and resisted treatment with acidic buffer or trypsin. Reverse signaling through membrane-bound IL-15 considerably increased the production of several pro-inflammatory cytokines by monocytes, such as IL-6, IL-8, and tumor necrosis factor-alpha, thereby indicating the relevance of this process to the complex immunomodulatory function of these cells. Furthermore, stimulation of transmembrane IL-15 also enhanced the transcription of IL-6 and IL-8 in the PC-3 cell line and promoted migration of PC-3 cells as well as LNCaP human prostate carcinoma cells stably expressing IL-15 on the cell surface. Thus, IL-15 can exist as a biologically active transmembrane molecule that possesses dual ligand-receptor qualities with a potential to induce bidirectional signaling. This fact highlights a new level of complexity in the biology of IL-15 and offers novel important insights into our understanding of the cellular responses modulated by this pleiotropic cytokine.

This study shows that the high affinity alpha-chain of the interleukin (IL)-15 receptor exists not only in membrane-anchored but also in soluble form. Soluble IL-15Ralpha (sIL-15Ralpha) can be detected in mouse sera and cell-conditioned media by enzyme-linked immunosorbent assay and by immunoprecipitation and Western blotting. This protein has a molecular mass of about 30 kDa because of the presence of a single N-glycosylation site, which is reduced to 26 kDa after N-glycosidase treatment. Transmembrane IL-15Ralpha is constitutively converted into its soluble form by proteolytic cleavage that involves tumor necrosis factor-alpha-converting enzyme (TACE), and this process is further enhanced by phorbol 12-myristate 13-acetate (PMA) stimulation. The hydroxamate GW280264X, which is capable of blocking TACE and the closely related disintegrin-like metalloproteinase 10 (ADAM10), effectively inhibited both spontaneous and PMA-inducible cleavage of IL-15Ralpha, whereas GI254023X, which preferentially blocks ADAM10, was ineffective. Overexpression of TACE but not ADAM10 in COS-7 cells enhanced the constitutive and PMA-inducible cleavage of IL-15Ralpha. Moreover, murine fibroblasts deficient in TACE but not ADAM10 expression exhibited a significant reduction in the spontaneous and inducible IL-15Ralpha shedding, whereas a reconstitution of TACE in these cells restored the release of sIL-15Ralpha, thereby suggesting that TACE-mediated proteolysis may represent a major mechanism for sIL-15Ralpha generation in mice. The existence of natural sIL-15Ralpha offers novel insights into the complex biology of IL-15 and envisages a new level for therapeutic intervention., (Copyright 2004 American Society for Biochemistry and Molecular Biology, Inc.)

CD37 is a leukocyte-specific protein belonging to the tetraspanin superfamily. Previously thought to be predominantly a B cell molecule, CD37 is shown in this study to regulate T cell proliferation. CD37-deficient (CD37(-/-)) T cells were notably hyperproliferative in MLR, in response to Con A, or CD3-TCR engagement particularly in the absence of CD28 costimulation. Hyperproliferation was not due to differences in memory to naive T cell ratios in CD37(-/-) mice, apoptosis, or TCR down-modulation. Division cycle analyses revealed CD37(-/-) T cells to enter first division earlier than wild-type T cells. Importantly, proliferation of CD37(-/-) T cells was preceded by enhanced early IL-2 production. We hypothesized CD37 to be involved in TCR signaling and this was supported by the observation that CD4/CD8-associated p56(Lck) kinase activity was increased in CD37(-/-) T cells. Remarkably, CD37 cross-linking on human T cells transduced signals that led to complete inhibition of CD3-induced proliferation. In the presence of CD28 costimulation, CD37 engagement still significantly reduced proliferation. Taken together, these results demonstrate a regulatory role for CD37 in T cell proliferation by influencing early events of TCR signaling.

Mast cells previously have been reported to be regulated by IL-15 and to express a distinct IL-15R, termed IL-15RX. To further examine IL-15 binding and signaling in mast cells, we have studied the nature of the IL-15R and some of its biological activities in these cells. In this study, we report the existence of three novel isoforms of the IL-15R alpha chain in murine bone marrow-derived mast cells as a result of an alternative exon-splicing mechanism within the IL-15R alpha gene. These correspond to new mRNA transcripts lacking exon 4; exons 3 and 4; or exons 3, 4, and 5 (IL-15R alpha Delta 4, IL-15R alpha Delta 3,4, IL-15R alpha Delta 3,4,5). After transient transfection in COS-7 cells, all IL-15R alpha isoforms associate with the Golgi apparatus, the endoplasmic reticulum, the perinuclear space, and the cell membrane. Analysis of glycosylation pattern demonstrates the usage of a single N-glycosylation site, while no O-glycosylation is observed. Importantly, IL-15 binds with high affinity to, and promotes the survival of, murine BA/F3 cells stably transfected with the IL-15R alpha isoforms. Furthermore, we report that signaling mediated by IL-15 binding to the newly identified IL-15R alpha isoforms involves the phosphorylation of STAT3, STAT5, STAT6, Janus kinase 2, and Syk kinase. Taken together, our data indicate that murine mast cells express novel, fully functional IL-15R alpha isoforms, which can explain the selective regulatory effects of IL-15 on these cells.

ATP-gated ion channel P2X receptors are expressed on the surface of most immune cells and can trigger multiple cellular responses, such as membrane permeabilization, cytokine production, and cell proliferation or apoptosis. Despite broad distribution and pleiotropic activities, signaling pathways downstream of these ionotropic receptors are still poorly understood. Here, we describe intracellular signaling events in Jurkat cells treated with millimolar concentrations of extracellular ATP. Within minutes, ATP treatment resulted in the phosphorylation and activation of p56(lck) kinase, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase but not p38 kinase. These effects were wholly dependent upon the presence of extracellular Ca(2+) ions in the culture medium. Nevertheless, calmodulin antagonist calmidazolium and CaM kinase inhibitor KN-93 both had no effect on the activation of p56(lck) and ERK, whereas a pretreatment of Jurkat cells with MAP kinase kinase inhibitor P098059 was able to abrogate phosphorylation of ERK. Further, expression of c-Jun and c-Fos proteins and activator protein (AP-1) DNA binding activity were enhanced in a time-dependent manner. In contrast, DNA binding activity of NF-kappa B was reduced. ATP failed to stimulate the phosphorylation of ERK and c-Jun N-terminal kinase and activation of AP-1 in the p56(lck)-deficient isogenic T cell line JCaM1, suggesting a critical role for p56(lck) kinase in downstream signaling. Regarding the biological significance of the ATP-induced signaling events we show that although extracellular ATP was able to stimulate proliferation of both Jurkat and JCaM1 cells, an increase in interleukin-2 transcription was observed only in Jurkat cells. The nucleotide selectivity and pharmacological profile data supported the evidence that the ATP-induced effects in Jurkat cells were mediated through the P2X7 receptor. Taken together, these results demonstrate the ability of extracellular ATP to activate multiple downstream signaling events in a human T-lymphoblastoid cell line.

Surface expression of IgM is a characteristic feature of the development of most B cells. Only pre-B cells bearing functional IgM heavy chains mu chains) are selected for clonal expansion and differentiation. Cells lacking mu chains are normally eliminated. muMT mice carrying a deletion of the first exon coding for the transmembrane domain of the immunoglobulin mu chain gene were described as mice deficient for mature B cells, plasma cells and immunoglobulins in serum. In this study, we describe in muMT/BALB/c mice the presence of a novel B cell population, producing IgG, IgA and IgE in the absence of IgM membrane expression. Moreover, this small population of B cells is able to recognize antigens and to differentiate into plasma cells. These "non-conventional" mu(- / -) B cells produce functional immunoglobulins after immunization, undergo germinal center reactions, and maintain B cell memory. Our findings support the concept, that a small percentage of mu -non-expressing pre-B cells can escape elimination, switch to downstream immunoglobulin heavy chains and respond to antigens. It remains an open question how the reactivity of these B cells is regulated and in which extent such B cells play a role in physiological and pathological processes such as autoantibody production and autoimmunity.

CD37 is a membrane protein of the tetraspanin superfamily, which includes CD9, CD53, CD63, CD81, and CD82. Many of these molecules are expressed on leukocytes and have been implicated in signal transduction, cell-cell interactions, and cellular activation and development. We generated and analyzed mice deficient for CD37. Despite the high expression of CD37 on cells of the immune system, no changes in development and cellular composition of lymphoid organs were observed in mice lacking CD37. Analyses of humoral immune responses revealed a reduced level of immunoglobulin G1 (IgG1) in the sera of nonimmunized mice and an alteration of responses to T-cell-dependent antigens. Antibody responses to model antigen administered in the absence of adjuvant and to viral infections were generally poor in CD37-deficient mice. These poor antibody responses could be overcome by the immunization of antigen together with adjuvant. These results suggest a role for CD37 in T-cell-B-cell interactions which manifests itself under suboptimal costimulatory conditions.