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Calcium is a key regulator of the T cell immune response. Depending on the spatial properties (nucleus versus cytoplasm) of the calcium signals generated after CD3xCD28 stimulation, primary human T cells either mount a productive immune response or develop tolerance. Nuclear calcium acts as a genomic decision maker: during T cell activation, it drives expression of genes associated with a productive immune response while in its absence, stimulated T cells acquire an anergy-like gene profile. Selective inhibition of nuclear calcium signaling in stimulated T cells blocks the productive immune response and directs the cells towards an anergy-like state. Here we show that the two transcriptional programs that include, respectively, the 'activation gene', interleukin 2 (IL2) and 'anergy-related genes', EGR2, EGR3, and CREM have different requirements for transmembrane calcium flux. By either lowering extracellular calcium concentrations with EGTA or using low concentrations of the ORAI blockers, BTP2 or RO2959, we reduced transmembrane calcium flux in human primary T cells stimulated with CD3xCD28. These 'conditions of limited calcium influx' (CLCI) blocked CD3xCD28-induced IL2 expression but only moderately affected induction of the anergy-related genes EGR2, EGR3, and CREM. We observed no difference in NFAT2 nuclear translocation after CD3xCD28 stimulation between normal conditions and CLCI. These results indicate that CLCI favors expression of anergy-related genes in activated human T cells. CLCI may be used to develop novel means for pro-tolerance immunosuppressive treatments., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Protease activity of allergens has been suggested to be involved in the pathogenesis of allergic diseases. The major allergen Der f 3 from Dermatophagoides farinae harbors serine protease activity, but its immunopathogenesis remains unclear. This study aims to explore the effect of Der f 3 on the airway epithelial barrier and on the molecular pathways by which Der f 3 induces inflammation. RNA-seq was performed to identify differentially expressed genes in bronchial airway epithelial cells (AEC) between native Der f 3 and heat-inactivated (H) Der f 3, coupled with real-time PCR (RT-PCR) and ELISA for validation. Unlike other protease allergens such as that induce Th2-promoting alarmins (IL-25, IL-33, TSLP) in AECs, Der f 3 induced pro-inflammatory cytokines and chemokines including IL-6, IL-8 and GM-CSF, which are known to promote Th17 response. These pro-inflammatory mediators were induced by Der f 3 via the MAPK and NF-κB pathways as well as the store-operated calcium signaling. Gene silencing with small interfering RNA in A549 and BEAS-2B cells indicated that activation of AECs by Der f 3 was mainly dependent on protease-activated receptor 2 (PAR-2), while PAR-1 was also required for the full activation of AECs. Double knock-down of PAR-1 and PAR-2 largely impaired Der f 3-inducecd IL-8 production and subsequent signaling pathways. Our data suggest that Der f 3 induces pro-inflammatory mediators in human epithelial cell lines via the PARs-MAPK-NF-κB axis. Our results provide a molecular mechanism by which Der f 3 may trigger the Th17-skewed allergic response toward house dust mites., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The inducible costimulator (ICOS) is a T cell costimulatory receptor that plays crucial roles in T cell differentiation and function. So far, ICOS has been shown to activate three signaling components: phosphoinositide 3-kinase (PI3K), intracellular calcium mobilization, and TANK binding kinase 1 (TBK1). By generating a knock-in strain of mice in which the ICOS gene is modified such that the ICOS-mediated PI3K pathway is selectively abrogated while the capacity of ICOS to mobilize intracellular calcium remains intact, we have shown that ICOS-mediated PI3K activation is required for some but not all T cell responses. This suggests that the ICOS-calcium signaling axis may explain some of the PI3K-independent ICOS functions. Further, a recent in vivo imaging study indicated that ICOS-dependent intracellular calcium flux facilitates cognate T cell-B cell interactions within the germinal center. However, how ICOS promotes TCR-mediated calcium flux has not been clear. Here we identified a membrane proximal motif in the cytoplasmic tail of ICOS that is essential for ICOS-assisted calcium signaling and demonstrate that ICOS can induce calcium flux independently of other signaling motifs. We also provide evidence that ICOS potentiates phospholipase Cγ1 (PLCγ1) activation to enhance calcium release from the intracellular pool. In parallel, acute ligation of ICOS without TCR co-engagement leads to activation of small GTPases, RhoA and Cdc42, consistent with the capacity of ICOS to induce actin remodeling. Importantly, interruption of actin dynamics during acute TCR or TCR-ICOS co-ligation severely impairs calcium flux in T cells even in the presence of activated PLCγ1. Thus, ICOS potentiates TCR-induced calcium flux by enhancing PLCγ1 activation and actin remodeling in a coordinated manner., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Calcium signals are crucial for diverse cellular functions including adhesion, differentiation, proliferation, effector functions and gene expression. After engagement of the B cell receptor, the intracellular calcium ion (Ca(2+)) concentration is increased promoting the activation of various signaling cascades. While elevated Ca(2+) in the cytosol initially comes from the endoplasmic reticulum (ER), a continuous influx of extracellular Ca(2+) is required to maintain the increased level of cytosolic Ca(2+). Store-operated Ca(2+) entry manages this process, which is regulated by an ER calcium sensor, stromal interaction molecule (STIM). STIM proteins sense changes in the levels of Ca(2+) stored within the ER lumen and regulates the Ca(2+)-release activated Ca(2+) channel in the plasma membrane. This review focuses on the signaling pathways leading to Ca(2+) influx and the role of Ca(2+) signals in B cell functions., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Ca(2+) acts ubiquitously as a second messenger in transmembrane signal transduction. In lymphocytes, calcium mobilization is triggered by antigen and chemokine receptors, among others, and controls cell functions ranging from proliferation to migration. The primary mechanism of extracellular Ca(2+) entry in lymphocytes is the CRAC influx. STIM1 is a crucial component of the CRAC influx mechanism in lymphocytes, acting as a sensor of low Ca(2+) concentration in the ER and an activator of the Ca(2+) selective channel ORAI1 in the plasma membrane. While STIM1 function has been studied extensively, little is known regarding whether it is differentially expressed and thereby affects the magnitude of calcium mobilization responses. We report here that STIM1 expression differs in murine T and B lymphocytes, and in respective subsets. For example, mature T cells express ∼4 times more STIM1 than mature B cells. Furthermore, we show that through the physiologic range of expression, STIM1 levels determine the magnitude of Ca(2+) influx responses that follow BCR-induced intracellular store depletion. Considered in view of previous reports that differences in amplitude of lymphocyte Ca(2+) mobilization determine alternate biological responses, these findings suggest that differential STIM1 expression may be important determinant of biological responses., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The molecular mechanisms underlying the non-opsonic phagocytosis of bacteria by neutrophils are poorly understood. We previously reported the efficient uptake of Streptococcus sanguinis by human neutrophils in the absence of opsonins. To characterize the phagocytosis receptor, protein lysates from neutrophils and HL-60 cells were subjected to affinity chromatography using epoxy beads coated with S. sanguinis. Denaturing electrophoresis of the eluted proteins and subsequent mass spectrometry revealed that one of the proteins eluted from neutrophils was proteinase 3 (PR3). Enzymatic cleavage of the glycosylphosphatidylinositol linker of NB1, a co-receptor for membrane-bound PR3 (mPR3), significantly reduced the phagocytosis of S. sanguinis. In addition, the neutralization of mPR3 with antibody reduced both binding and phagocytosis of S. sanguinis. Treatment of neutrophils with a serine proteinase inhibitor indicated that protease activity is required for phagocytosis. Thus, we studied whether protease-activated receptor 2 (PAR2) is involved in signal transmission from mPR3 during this process. Indeed, neutralizing antibodies against PAR2 inhibited phagocytosis and S. sanguinis-induced calcium mobilization desensitized PAR2. Furthermore, the phagocytosis of S. sanguinis and the concomitant activation of Rho family GTPases were inhibited by the intracellular calcium chelator, BAPTA-AM. Collectively, mPR3 acts as a non-opsonic phagocytosis receptor for bacteria probably by activating PAR2 in neutrophils., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

To create antibody diversity, B lymphocyte development is characterized by the ordered rearrangement of first immunoglobulin (Ig) heavy chain gene segments and then Ig light-chain gene segments. Early in B-cell development, expression of a pre-B-cell receptor (pre-BCR) composed of membrane-bound Ig heavy chain protein associated with surrogate light-chain (SLC) proteins serves as a critical checkpoint that monitors for functional heavy chain rearrangement. Signaling from the pre-BCR induces clonal expansion, but it also turns off transcription of the genes for the SLC proteins lambda5 and VpreB, which limits this proliferation. Here we show that signaling from the pre-BCR rapidly down-regulates lambda5 and VpreB and also the co-receptor CD19 in primary pre-B-cells. We show that calcium (Ca(2+)) signaling is essential for this silencing of the SLC and CD19 genes. The SLC genes are activated by the E2A transcription factor, and we show that E2A is required for pre-BCR-mediated regulation of the genes. E2A mutated in its binding site for the Ca(2+) sensor protein calmodulin, and thus with calmodulin-resistant DNA binding, makes lambda5, VpreB and CD19 expression resistant to the inhibition following pre-BCR activation. Thus, Ca(2+) down-regulates SLC and CD19 gene expression upon pre-BCR activation through inhibition of E2A by Ca(2+)/calmodulin., ((c) 2009 Elsevier Ltd. All rights reserved.)

Ca(2+) is a highly versatile intracellular second messenger in many cell types, and regulates many complicated cellular processes, including cell activation, proliferation and apoptosis. Influx of Ca(2+) from the extracellular fluid is required for sustained elevation of the cytosolic Ca(2+) concentration and full activation of Ca(2+)-dependent processes. It is widely accepted that Ca(2+) release-activated Ca(2+) channels are the major routes of Ca(2+) influx in electrically non-excitable cells, including hematopoietic cells, whereas voltage-gated Ca(2+) channels such as L-type Ca(2+) channels (LTCCs) serve as the principal routes of Ca(2+) entry into electrically excitable cells such as neurons and myocytes. However, recent pharmacological and molecular genetic studies have revealed the existence of functional LTCCs and/or LTCC-like channels in a variety of immune cells including mast cells. In this article, we review recent advances in our understanding of Ca(2+) signaling in immune cells with a special interest in mast cells. We highlight roles for LTCCs in antigen receptor-mediated mast cell activation and survival., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The recessive lyp allele, which harbors a defective gimap5 (GTPase of immunity-associated nucleotide binding protein 5) gene, causes spontaneous apoptosis of T lymphocytes in the biobreeding diabetes-prone strain of rats. Mechanisms underlying the pro-survival function of GIMAP5 remain unclear. In this study, we show that gimap5(lyp/lyp) T cells display diminished calcium flux in response to thapsigargin or signaling via the T cell antigen receptor. This defect is manifested in mature single positive thymocytes, where the survival defect first occurs. We also show that GIMAP5 deficiency does not affect the thapsigargin-induced calcium release from the intracellular stores but impairs subsequent calcium entry across the plasma membrane. Our findings suggest that GIMAP5 is an important regulator of calcium response in T lymphocytes and impaired calcium signaling might underlie spontaneous apoptosis of gimap5(lyp/lyp) T cells.

Little is known about the interplay between pathophysiological processes of allergy and infection, particularly with respect to mast cell (MC)-mediated responses. The presence and recognition of pathogen-associated molecular patterns (PAMPs) might have broad impact on the development and severity of diseases. In this study, we assessed the influence of toll-like receptor 2 (TLR 2)-dependent synthetic analogs of bacterial lipopeptides (LPs), Pam(3)CSK(4) and MALP-2, on Ag (DNP-HSA)-triggered responses in bone marrow-derived MCs (BMMCs). Both LPs strongly synergized with sub-optimal amounts of Ag in the stimulation of cytokine release. Intriguingly, Pam(3)CSK(4), but not MALP-2 suppressed Ag-induced degranulation of BMMCs (together with early tyrosine phosphorylation and calcium mobilization) in a TLR2-independent manner. Further analysis revealed that Pam(3)CSK(4), most probably by electrostatic forces, reduced the level of active DNP-HSA and that this, in turn, was responsible for the suppression of Ag-induced degranulation. Thus, our work demonstrates that LPs can synergize with IgE+Ag in stimulating the production of IL-6 by BMMCs. As well, our findings with Pam(3)CSK(4) indicate that one must be cautious when interpretating results obtained with "model" substances and the combination of ligands must be carefully chosen when functional interactions between the high-affinity receptor for IgE (FcepsilonR1) and TLR2 are examined.

Background: Allergic airway diseases are more common in females than in males during early adulthood. A relationship between female hormones and asthma prevalence and severity has been suggested, but the cellular and molecular mechanisms are not understood., Objective: To elucidate the mechanism(s) by which estrogens enhance the synthesis and release of mediators of acute hypersensitivity., Methods: Two mast cell/basophil cell lines (RBL-2H3 and HMC-1) and primary cultures of bone marrow derived mast cells, all of which naturally express estrogen receptor-alpha, were examined. Cells were incubated with physiological concentrations of 17-beta-estradiol with and without IgE and allergens. Intracellular Ca(2+) concentrations and the release of beta-hexosaminidase and leukotriene C(4) were quantified., Results: Estradiol alone induced partial release of the preformed, granular protein beta-hexosaminidase from RBL-2H3, BMMC and HMC-1, but not from BMMC derived from estrogen receptor-alpha knock-out mice. The newly synthesized LTC(4) was also released from RBL-2H3. Estradiol also enhanced IgE-induced degranulation and potentiated LTC(4) production. Intracellular Ca(2+) concentration increased prior to and in parallel with mediator release. Estrogen receptor antagonists or Ca(2+) chelation inhibited these estrogenic effects., Conclusion: Binding of physiological concentrations of estradiol to a membrane estrogen receptor-alpha initiates a rapid onset and progressive influx of extracellular Ca(2+), which supports the synthesis and release of allergic mediators. Estradiol also enhances IgE-dependent mast cell activation, resulting in a shift of the allergen dose response.

Mycobacterium leprae, the causative agent of leprosy, challenges host defense mechanism by impairing the signal transduction of T cells which leads to downregulation of T cell proliferation, mainly as a consequence of interference with IL-2 production. In this study we sought to identify how soluble forms of M. leprae antigen(s) or particulate (liposome) delivery of the same antigens with two immunomodulators Murabutide and T cell peptide of Trat protein influence the transcription of IL-2 gene in anergic T cells of lepromatous patients. It was demonstrated that MLCwA/ManLAM stimulated cells of BL/LL patients showed defects in both jun-NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) activities there by resulting in decreased AP-1 activity. Additionally these cells showed reduced calcium levels, PKC activity and calcineurin (CN) activity. This led to impaired nuclear translocation of NFkappaB and NFAT in these patients. In contrast, when same M. leprae antigen(s) were incorporated with the two immunomodulators in liposomal form, increased transcription of IL-2 gene was observed especially in BL/LL patients which appears to be due to, at least in part, to increased expression of AP-1 Fos and Jun family members, NFkappaB and NFAT1 proteins. The increased expression of these transcription factors correlated with increased ERK/JNK, PKC and CN activities in these patients. Since activation of ERK/JNK/PKC kinases and CN phosphatase are required for stimulation of IL-2 transcription, these data provide a molecular explanation for the block in IL-2 production by M. leprae antigens. Thus the above study revealed suppression of all the three distinct biochemical pathways, viz. Ca-CN-NFAT pathway, PKC-NF-kappaB pathway, and MAPK-AP-1 pathway by M. leprae antigen(s) in anergized T cells of lepromatous patients which were activated by liposomal delivery of M. leprae antigens containing the two immunomodulators leading to optimal induction of IL-2 gene expression, which was required for the activation, and proliferation of T cells in lepromatous patients.