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An expansion of CD21 low B cells has been described in a variety of diseases associated with persistent immune stimulation as in chronic infection, immunodeficiency, or autoimmunity. Different developmental stages of CD21 low B cells have been highlighted in specific diseases; however, a systematic comparison of distribution, phenotype, and signaling capacity of these populations has not yet been performed to delineate the pivotal character of this unusual B cell population. Screening of more than 200 patients with autoimmune disease demonstrated that the prevalence of patients with expanded CD21 low B cells varies between diseases. The expansion was frequent in patients with systemic lupus erythematosus, in which it correlated to relative B cell lymphopenia and duration of disease. Different proportions of distinct developmental stages of CD21 low B cells co-occur in nearly all patients with autoimmune disease. Although in most patients, naive-like and CD27 - switched memory B cells were the most prominent CD21 low subpopulations, there was no detectable association of the pattern with the underlying disease. Despite their distinct developmental stage, all CD21 low B cells share a common core phenotype including the increased expression of inhibitory receptors, associated with an elevated constitutive phosphorylation of proximal signaling molecules downstream of the BCR but impaired Ca 2+ mobilization and NF-κB activation after BCR stimulation. Further, this was accompanied by impaired upregulation of CD69, although CD86 upregulation was preserved. Beyond maturation-associated differences, the common core characteristics of all CD21 low B cell populations suggests either a common ancestry or a shared sustained imprint by the environment they originated in., (Copyright © 2020 by The American Association of Immunologists, Inc.)

The number and activity of T cell subsets in the atherosclerotic plaques are critical for the prognosis of patients with acute coronary syndrome. β 2 Integrin activation is pivotal for T cell recruitment and correlates with future cardiac events. Despite this knowledge, differential regulation of adhesiveness in T cell subsets has not been explored yet. In this study, we show that in human T cells, SDF-1α-mediated β 2 integrin activation is driven by a, so far, not-described reactive oxidative species (ROS)-regulated calcium influx. Furthermore, we show that CD4 + CD28 null T cells represent a highly reactive subset showing 25-fold stronger β 2 integrin activation upon SDF-1α stimulation compared with CD28 + T cells. Interestingly, ROS-dependent Ca release was much more prevalent in the pathogenetically pivotal CD28 null subset compared with the CD28 + T cells, whereas the established mediators of the classical pathways for β 2 integrin activation (PKC, PI3K, and PLC) were similarly activated in both T cell subsets. Thus, interference with the calcium flux attenuates spontaneous adhesion of CD28 null T cells from acute coronary syndrome patients, and calcium ionophores abolished the observed differences in the adhesion properties between CD28 + and CD28 null T cells. Likewise, the adhesion of these T cell subsets was indistinguishable in the presence of exogenous ROS/H 2 O 2 Together, these data provide a molecular explanation of the role of ROS in pathogenesis of plaque destabilization., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Of the thousands of long noncoding RNAs (lncRNA) identified in lymphocytes, very few have defined functions. In this study, we report the discovery and functional elucidation of a human B cell-specific lncRNA with high levels of expression in three types of B cell cancer and normal B cells. The AC099524.1 gene is upstream of the gene encoding the B cell-specific phospholipase C γ 2 ( PLCG2 ), a B cell-specific enzyme that stimulates intracellular Ca 2+ signaling in response to BCR activation. AC099524.1 (B cell-associated lncRNA modulator of BCR-mediated Ca + signaling [BCALM]) transcripts are localized in the cytoplasm and, as expected, CRISPR/Cas9 knockout of AC099524.1 did not affect PLCG2 mRNA or protein expression. lncRNA interactome, RNA immunoprecipitation, and coimmunoprecipitation studies identified BCALM-interacting proteins in B cells, including phospholipase D 1 (PLD1), and kinase adaptor proteins AKAP9 (AKAP450) and AKAP13 (AKAP-Lbc). These two AKAP proteins form signaling complexes containing protein kinases A and C, which phosphorylate and activate PLD1 to produce phosphatidic acid (PA). BCR stimulation of BCALM-deficient B cells resulted in decreased PLD1 phosphorylation and increased intracellular Ca + flux relative to wild-type cells. These results suggest that BCALM promotes negative feedback that downmodulates BCR-mediated Ca + signaling by promoting phosphorylation of PLD1 by AKAP-associated kinases, enhancing production of PA. PA activates SHP-1, which negatively regulates BCR signaling. We propose the name BCALM for B-Cell Associated LncRNA Modulator of BCR-mediated Ca + signaling. Our findings suggest a new, to our knowledge, paradigm for lncRNA-mediated modulation of lymphocyte activation and signaling, with implications for B cell immune response and BCR-dependent cancers., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Rheumatoid arthritis (RA) is closely associated with shared epitope (SE)-coding HLA-DRB1 alleles and circulating anticitrullinated protein Abs (ACPA), but neither the respective pathogenic roles of SE and ACPA in RA nor the mechanisms underlying their coassociation are known. It was recently shown that the SE functions as a signal transduction ligand that activates a cell surface calreticulin-mediated, proarthritogenic, bone erosive pathway in an experimental model of RA. In this study, we demonstrate that stimulation of murine macrophages with LPS or DTT facilitated cell surface translocation of calreticulin, which in turn enabled increased SE-activated calcium signaling and activation of peptidylarginine deiminase with the resultant increased cellular abundance of citrullinated proteins. The i.p. administration of LPS to transgenic mice carrying a human SE-coding HLA-DRB1 allele lead to increased serum levels of TNF-α and anticitrullinated cyclic peptide Abs, along with terminal phalanx bone destruction. These data uncover a previously unknown signal transduction pathway by which the SE facilitates protein citrullination, ACPA production, and bone destruction., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Phospholipase C (PLC) isoforms play central roles in signaling cascades by cleaving PIP 2 into the second messengers IP 3 and DAG. In this study, to our knowledge, we uncover that ORP5L interacts physically with PLCγ1 in T cells, extracts PIP 2 from the plasma membrane via its ORD domain (OSBP-related domain), presents it to PLCγ1 (enabling IP 3 generation), and eventually maintains intracellular Ca 2+ homeostasis. Through this mechanism, ORP5L promotes T cell proliferation in a Ca 2+ -activated NFAT2-dependent manner. To our knowledge, our study uncovers a new key function of ORP5L as a critical cofactor for PLCγ1 catalysis and its crucial role in human T cell proliferation., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Appropriate T cell responses are controlled by strict balance between activatory and inhibitory pathways downstream of TCR. Although mice or humans with impaired TCR signaling develop autoimmunity, the precise molecular mechanisms linking reduced TCR signaling to autoimmunity are not fully understood. Engagement of TCR activates Ca 2+ signaling mainly through store-operated Ca 2+ entry activated by stromal interaction molecule (Stim) 1 and Stim2. Despite defective T cell activation, mice deficient in both Stim1 and Stim2 in T cells (conditional double knockout [cDKO]) developed lymphoproliferative disorders and skin inflammation with a concomitant increase in serum IgG1 and IgE levels. In cDKO mice, follicular helper T (Tfh) cells were dramatically increased in number, and they produced IL-4 spontaneously. These inflammatory symptoms were abolished by the deletion of IL-4 in cDKO mice. Tfh development and inflammatory symptoms in cDKO mice were abrogated by further deletion of NFAT2 in T cells. These findings suggest that Tfh cells spontaneously developed in the absence of Ca 2+ signaling and caused unregulated type 2 responses., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Leukotriene B4 (LTB4) contributes to many inflammatory diseases, including genetic and nongenetic forms of chronic obstructive pulmonary disease. α-1 Antitrypsin (AAT) deficiency (AATD) is characterized by destruction of lung parenchyma and development of emphysema, caused by low AAT levels and a high neutrophil burden in the airways of affected individuals. In this study we assessed whether AATD is an LTB4-related disease and investigated the ability of serum AAT to control LTB4 signaling in neutrophils. In vitro studies demonstrate that neutrophil elastase is a key player in the LTB4 inflammatory cycle in AATD, causing increased LTB4 production, and associated BLT1 membrane receptor expression. AATD patients homozygous for the Z allele were characterized by increased neutrophil adhesion and degranulation responses to LTB4. We demonstrate that AAT can bind LTB4 and that AAT/LTB4 complex formation modulates BLT1 engagement and downstream signaling events, including 1,4,5-triphosphate production and Ca(2+) flux. Additionally, treatment of ZZ-AATD individuals with AAT augmentation therapy decreased plasma LTB4 concentrations and reduced levels of membrane-bound neutrophil elastase. Collectively, these results provide a mechanism by which AAT augmentation therapy impacts on LTB4 signaling in vivo, and not only reinforces the utility of this therapy for resolving inflammation in AATD, but supports useful future clinical applications in treatment of other LTB4-related diseases., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Store-operated Ca(2+) entry (SOCE) through Ca(2+) release-activated Ca(2+) (CRAC) channels is essential for immunity to infection. CRAC channels are formed by ORAI1 proteins in the plasma membrane and activated by stromal interaction molecule (STIM)1 and STIM2 in the endoplasmic reticulum. Mutations in ORAI1 and STIM1 genes that abolish SOCE cause severe immunodeficiency with recurrent infections due to impaired T cell function. SOCE has also been observed in cells of the innate immune system such as macrophages and dendritic cells (DCs) and may provide Ca(2+) signals required for their function. The specific role of SOCE in macrophage and DC function, as well as its contribution to innate immunity, however, is not well defined. We found that nonselective inhibition of Ca(2+) signaling strongly impairs many effector functions of bone marrow-derived macrophages and bone marrow-derived DCs, including phagocytosis, inflammasome activation, and priming of T cells. Surprisingly, however, macrophages and DCs from mice with conditional deletion of Stim1 and Stim2 genes, and therefore complete inhibition of SOCE, showed no major functional defects. Their differentiation, FcR-dependent and -independent phagocytosis, phagolysosome fusion, cytokine production, NLRP3 inflammasome activation, and their ability to present Ags to activate T cells were preserved. Our findings demonstrate that STIM1, STIM2, and SOCE are dispensable for many critical effector functions of macrophages and DCs, which has important implications for CRAC channel inhibition as a therapeutic strategy to suppress pathogenic T cells while not interfering with myeloid cell functions required for innate immunity., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Perturbation of intracellular ion homeostasis is a major cellular stress signal for activation of NLRP3 inflammasome signaling that results in caspase-1-mediated production of IL-1β and pyroptosis. However, the relative contributions of decreased cytosolic K(+) concentration versus increased cytosolic Ca(2+) concentration ([Ca(2+)]) remain disputed and incompletely defined. We investigated roles for elevated cytosolic [Ca(2+)] in NLRP3 activation and downstream inflammasome signaling responses in primary murine dendritic cells and macrophages in response to two canonical NLRP3 agonists (ATP and nigericin) that facilitate primary K(+) efflux by mechanistically distinct pathways or the lysosome-destabilizing agonist Leu-Leu-O-methyl ester. The study provides three major findings relevant to this unresolved area of NLRP3 regulation. First, increased cytosolic [Ca(2+)] was neither a necessary nor sufficient signal for the NLRP3 inflammasome cascade during activation by endogenous ATP-gated P2X7 receptor channels, the exogenous bacterial ionophore nigericin, or the lysosomotropic agent Leu-Leu-O-methyl ester. Second, agonists for three Ca(2+)-mobilizing G protein-coupled receptors (formyl peptide receptor, P2Y2 purinergic receptor, and calcium-sensing receptor) expressed in murine dendritic cells were ineffective as activators of rapidly induced NLRP3 signaling when directly compared with the K(+) efflux agonists. Third, the intracellular Ca(2+) buffer, BAPTA, and the channel blocker, 2-aminoethoxydiphenyl borate, widely used reagents for disruption of Ca(2+)-dependent signaling pathways, strongly suppressed nigericin-induced NLRP3 inflammasome signaling via mechanisms dissociated from their canonical or expected effects on Ca(2+) homeostasis. The results indicate that the ability of K(+) efflux agonists to activate NLRP3 inflammasome signaling can be dissociated from changes in cytosolic [Ca(2+)] as a necessary or sufficient signal., (Copyright © 2015 by The American Association of Immunologists, Inc.)

T cell activation by Ag is one of the key events in adaptive immunity. It is triggered by interactions of the TCR and coreceptor (CD8 or CD4) with antigenic peptides embedded in MHC (pMHC) molecules expressed on APCs. The mechanism of how signal is initiated remains unclear. In this article, we complement our two-dimensional kinetic analysis of TCR-pMHC-CD8 interaction with concurrent calcium imaging to examine how ligand engagement of TCR with and without the coengagement of CD8 initiates signaling. We found that accumulation of frequently applied forces on the TCR via agonist pMHC triggered calcium, which was further enhanced by CD8 cooperative binding. Prolonging the intermission between sequential force applications impaired calcium signals. Our data support a model where rapid accumulation of serial forces on TCR-pMHC-CD8 bonds triggers calcium in T cells., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Fine-tuning of cytokine-inducing pathways is essential for immune homeostasis. Consistently, a dysregulated increase or decrease in pattern-recognition receptor (PRR)-induced signaling and cytokine secretion can lead to inflammatory bowel disease. Multiple gene loci are associated with inflammatory bowel disease, but their functional effects are largely unknown. One such region in chromosome 2q12 (rs917997), also associated with other immune-mediated diseases, encompasses IL18RAP. We found that human monocyte-derived macrophages (MDMs) from rs917997 AA risk carriers secrete significantly less cytokines than G carriers upon stimulation of multiple PRRs, including nucleotide-binding oligomerization domain 2 (NOD2). We identified that IL-18 signaling through IL-18RAP was critical in amplifying PRR-induced cytokine secretion in MDMs. IL-18RAP responded to NOD2-initiated early, caspase-1-dependent autocrine IL-18, which dramatically enhanced MAPK, NF-κB, PI3K, and calcium signaling. Reconstituting MAPK activation was sufficient to rescue decreased cytokines in NOD2-stimulated IL-18RAP-deficient MDMs. Relative to GG carriers, MDM from rs917997 AA carriers had decreased expression of cell-surface IL-18RAP protein, as well as of IL-18R1 and IL-1R1, genes also located in the IL18RAP region. Accordingly, these risk-carrier MDMs show diminished PRR-, IL-18-, and IL-1-induced MAPK and NF-κB signaling. Taken together, our results demonstrate clear functional consequences of the rs917997 risk polymorphism; this polymorphism leads to a loss-of-function through decreased IL-18RAP, IL-18R1, and IL-1R1 protein expression, which impairs autocrine IL-18 and IL-1 signaling, thereby leading to decreased cytokine secretion in MDMs upon stimulation of a broad range of PRRs., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Dual oxidase 1 (Duox1) is the NADPH oxidase responsible for the H2O2 gradient formed in tissues after injury to trigger the early recruitment of leukocytes. Little is known about the signals that modulate H2O2 release from DUOX1 and whether the H2O2 gradient can orchestrate the inflammatory response in vivo. In this study, we report on a dominant-negative form of zebrafish Duox1 that is able to inhibit endogenous Duox1 activity, H2O2 release and leukocyte recruitment after tissue injury, with none of the side effects associated with morpholino-mediated Duox1 knockdown. Using this specific tool, we found that ATP release following tissue injury activates purinergic P2Y receptors, and modulates Duox1 activity through phospholipase C (PLC) and intracellular calcium signaling in vivo. Furthermore, Duox1-derived H2O2 is able to trigger the NF-κB inflammatory signaling pathway. These data reveal that extracellular ATP acting as an early danger signal is responsible for the activation of Duox1 via a P2YR/PLC/Ca(2+) signaling pathway and the production of H2O2, which, in turn, is able to modulate in vivo not only the early recruitment of leukocytes to the wound but also the inflammatory response through activation of the NF-κB signaling pathway., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Siglec-G is an inhibitory receptor on B1 cells. Siglec-G-deficient mice show a large B1 cell expansion, owing to higher BCR-induced Ca(2+) signaling and enhanced cellular survival. It was unknown why Siglec-G shows a B1 cell-restricted inhibitory function. With a new mAb we could show a comparable Siglec-G expression on B1 cells and conventional B2 cells. However, Siglec-G has a different ligand sialic acid-binding pattern on peritoneal B1 cells than on splenic B cells, and its sialic acid ligands are expressed differentially on these two B cell populations, suggesting that cis-ligand binding plays a crucial role on B1 cells. This observation was further studied by generation of Siglec-G knockin mice with a mutated ligand-binding domain. These mice show increased B1 cell numbers, increased B1 cell Ca(2+) signaling, better B1 cell survival, and changes in the B1 cell Ig repertoire. These phenotypes are very similar to Siglec-G-deficient mice. The mutation of the ligand-binding domain of Siglec-G strongly reduces the Siglec-G-IgM association on the B cell surface. Thus, Siglec-G sialic acid-dependent binding to the BCR is crucial for the B1 cell-restricted inhibitory function of Siglec-G and is regulated in an opposite way to that of the related protein CD22 (Siglec-2) on B cells., (Copyright © 2014 by The American Association of Immunologists, Inc.)

The IL-8 (CXCL8) receptors CXCR1 and CXCR2 couple to Gαi to induce leukocyte recruitment and activation at sites of inflammation. We recently showed that CXCR1 couples predominantly to the G protein-coupled receptor kinase (GRK)2, whereas CXCR2 interacts with GRK6 to regulate cellular responses. In addition to G protein-coupled receptors, GRKs displayed a more diverse protein/protein interaction in cells. In this study, we sought to identify GRK6 binding partner(s) that may influence CXCL8 activities, using RBL-2H3 cells stably expressing CXCR1 (RBL-CXCR1) or CXCR2 (RBL-CXCR2), as well as human and murine neutrophils. Our data demonstrated that, upon CXCR2 activation, GRK6 interacts with activator of G protein signaling (AGS)3 and Gαi2 to form a GRK6/AGS3/Gαi2 complex. This complex is time dependent and peaked at 2-3 min postactivation. GTPγS pretreatment blocked GRK6/AGS3/Gαi2 formation, suggesting that this assembly depends on G protein activation. Surprisingly, CXCR2 activation induced AGS3 phosphorylation in a PKC-dependent, but GRK6-independent, fashion. Overexpression of AGS3 in RBL-CXCR2 significantly inhibited CXCL8-induced Ca(2+) mobilization, phosphoinositide hydrolysis, and chemotaxis. In contrast, short hairpin RNA inhibition of AGS3 enhanced CXCL8-induced Ca(2+) mobilization, receptor resistance to desensitization, and recycling to the cell surface, with no effect on receptor internalization. Interestingly, RBL-CXCR2-AGS3(-/-) cells displayed a significant increase in CXCR2 expression on the cell surface but decreased ERK1/2 and P38 MAPK activation. Taken together, these results indicate that GRK6 complexes with AGS3-Gαi2 to regulate CXCR2-mediated leukocyte functions at different levels, including downstream effector activation, receptor trafficking, and expression at the cell membrane.

The Lyn tyrosine kinase regulates inhibitory signaling in B and myeloid cells: loss of Lyn results in a lupus-like autoimmune disease with hyperactive B cells and myeloproliferation. We have characterized the relative contribution of Lyn-regulated signaling pathways in B cells specifically to the development of autoimmunity by crossing the novel lyn(flox/flox) animals with mice carrying the Cre recombinase under the control of the Cd79a promoter, resulting in deletion of Lyn in B cells. The specific deletion of Lyn in B cells is sufficient for the development of immune complex-mediated glomerulonephritis. The B cell-specific Lyn-deficient mice have no defects in early bone marrow B cell development but have reduced numbers of mature B cells with poor germinal centers, as well as increased numbers of plasma and B1a cells, similar to the lyn(-/-) animals. Within 8 mo of life, B cell-specific Lyn mutant mice develop high titers of IgG anti-Smith Ag ribonucleoprotein and anti-dsDNA autoantibodies, which deposit in their kidneys, resulting in glomerulonephritis. B cell-specific Lyn mutant mice also develop myeloproliferation, similar to the lyn(-/-) animals. The additional deletion of MyD88 in B cells, achieved by crossing lyn(flox/flox)Cd79a-cre mice with myd88(flox/flox) animals, reversed the autoimmune phenotype observed in B cell-specific Lyn-deficient mice by blocking production of class-switched pathogenic IgG autoantibodies. Our results demonstrate that B cell-intrinsic Lyn-dependent signaling pathways regulate B cell homeostasis and activation, which in concert with B cell-specific MyD88 signaling pathways can drive the development of autoimmune disease.

Induction of an inflammatory monocyte phenotype by activated platelets is implicated in the pathogenesis of inflammatory diseases, including atherosclerosis. In this study, we investigated the early signaling events associated with this platelet-induced inflammatory phenotype. We report that coculture of human monocytes with activated platelets induces phosphorylation of Akt, together with rapid mobilization of intracellular Ca(2+), and show that these signaling events can be uncoupled from monocyte binding to activated platelets. Specifically, Ab-inhibition studies and incubation of monocytes with activated platelet supernatant highlighted a role for secreted product(s) of activated platelets. We also identified a role for pertussis toxin-sensitive G protein-coupled receptors and excluded key candidates platelet-activating factor receptor and CCR5. Our results suggest that inhibition of monocyte-platelet interactions via PSGL-1 or P-selectin is not sufficient to prevent platelet-mediated monocyte activation in an inflammatory context. These findings have important implications for the development of therapeutics to treat diseases in which platelet-monocyte complexes are implicated in pathogenesis.

T cells become polarized during initial interactions with an APC to form an Ag-independent synapse (AIS) composed of membrane rafts, TCR, and TCR-proximal signaling molecules. AISs occur temporally before TCR triggering, but their role in downstream TCR signaling is not understood. Using both human and murine model systems, we studied the signals that activate AIS formation and the effect of these signals on TCR-dependent responses. We show that CD28 produces AISs detectable by spinning disc confocal microscopy seconds following initial interactions between the T cell and APC. AIS formation by CD28 coincided with costimulatory signaling, evidenced by a cholesterol-sensitive activation of the MAPK ERK that potentiated Ca²⁺ signaling in response to CD3 cross-linking. CD45 also enriched in AISs but to modulate Src kinase activity, because localization of CD45 at the cell interface reduced the activation of proximal Lck. In summary, we show that signaling by CD28 during first encounters between the T cell and APC both sensitizes TCR Ca²⁺ signaling by an Erk-dependent mechanism and drives formation of an AIS that modulates the early signaling until TCR triggering occurs. Thus, early Ag-independent encounters are an important window for optimizing T cell responses to Ag by CD28.

Human β-defensins (hBDs) stimulate degranulation in rat peritoneal mast cells in vitro and cause increased vascular permeability in rats in vivo. In this study, we sought to determine whether hBDs activate murine and human mast cells and to delineate the mechanisms of their regulation. hBD2 and hBD3 did not induce degranulation in murine peritoneal or bone marrow-derived mast cells (BMMC) in vitro and had no effect on vascular permeability in vivo. By contrast, these peptides induced sustained Ca(2+) mobilization and substantial degranulation in human mast cells, with hBD3 being more potent. Pertussis toxin (PTx) had no effect on hBD-induced Ca(2+) mobilization, but La(3+) and 2-aminoethoxydiphenyl borate (a dual inhibitor of inositol 1,4,5-triphosphate receptor and transient receptor potential channels) caused substantial inhibition of this response. Interestingly, degranulation induced by hBDs was substantially inhibited by PTx, La(3+), or 2-aminoethoxydiphenyl borate. Whereas human mast cells endogenously express G protein-coupled receptor, Mas-related gene X2 (MrgX2), rat basophilic leukemia, RBL-2H3 cells, and murine BMMCs do not. Silencing the expression of MrgX2 in human mast cells inhibited hBD-induced degranulation, but had no effect on anaphylatoxin C3a-induced response. Furthermore, ectopic expression of MrgX2 in RBL-2H3 and murine BMMCs rendered these cells responsive to hBDs for degranulation. This study demonstrates that hBDs activate human mast cells via MrgX2, which couples to both PTx-sensitive and insensitive signaling pathways most likely involving Gαq and Gαi to induce degranulation. Furthermore, murine mast cells are resistant to hBDs for degranulation, and this reflects the absence of MrgX2 in these cells.

The suppressor function of regulatory T cells (Tregs) is impaired in multiple sclerosis (MS), but the mechanisms underlying this deficiency are not fully understood. As Tregs counteract the sustained elevation of intracellular calcium, which is indispensable for full activation of conventional T cells (Tcons), we hypothesized that interference with this pathway might prompt MS-related Treg dysfunction. Using single-cell live imaging, we observed that Tregs rapidly reduce Ca(2+) influx and downstream signals in Tcons upon cell contact, yet differ in their potency to efficiently suppress several target cells at the same time. Strikingly, individual Tregs harboring a CD4(+)CD25(+)FOXP3(+)CD45RA(+) naive phenotype suppressed significantly more adjacent Tcons than did CD4(+)CD25(+)FOXP3(+)CD45RA(-) memory Tregs. Some constituents even completely failed to dampen Tcon Ca(2+) influx and were contained exclusively in the memory subset. In accordance with their more powerful suppressive performance, the Ca(2+) signature was considerably enhanced in naive Tregs in response to TCR triggering, compared with the memory counterparts. MS Tregs displayed a significantly diminished suppression of mean Ca(2+) influx in the sum of individual Tcons recorded. This reduced inhibitory activity was closely linked to decreased numbers of individual Tcons becoming suppressed by adjacent Tregs and, in turn, correlated with a marked reduction of naive subtypes and concomitant expansion of nonsuppressive memory phenotypes. We conclude that the superior achievement of naive Tregs is pivotal in maintaining Treg efficiency. As a consequence, MS Tregs become defective because they lack naive subtypes and are disproportionately enriched in memory cells that have lost their inherent downregulatory activity.

Extracellular nucleotides have been recognized as important modulators of inflammation via their action on specific pyrimidine receptors (P2). This regulation coexists with the temporal framework of proinflammatory and proresolution mediators released by the cells involved in the inflammatory response, including macrophages. Under proinflammatory conditions, the expression of cyclooxygenase-2 leads to the release of large amounts of PGs, such as PGE2, that exert their effects through EP receptors and other intracellular targets. The effect of these PGs on P2 receptors expressed in murine and human macrophages was investigated. In thioglycollate-elicited and alternatively activated macrophages, PGE2 selectively impairs P2Y but not P2X7 Ca(2+) mobilization. This effect is absent in LPS-activated cells and is specific for PGE2 because it cannot be reproduced by other PGs with cyclopentenone structure. The inhibition of P2Y responses by PGE2 involves the activation of nPKCs (PKCε) and PKD that can be abrogated by selective inhibitors or by expression of dominant-negative forms of PKD. The inhibition of P2Y signaling by PGE2 has an impact on the cell migration elicited by P2Y agonists in thioglycollate-elicited and alternatively activated macrophages, which provide new clues to understand the resolution phase of inflammation, when accumulation of PGE2, anti-inflammatory and proresolving mediators occurs.

Compared with older children and adults, human neonates have reduced and delayed CD4(+) T cell immunity to certain pathogens, but the mechanisms for these developmental differences in immune function remain poorly understood. We investigated the hypothesis that impaired human neonatal CD4(+) T cell immunity was due to reduced signaling by naive CD4(+) T cells following engagement of the αβ-TCR/CD3 complex and CD28. Surprisingly, calcium flux following engagement of CD3 was significantly higher in neonatal naive CD4(+) T cells from umbilical cord blood (CB) compared with naive CD4(+) T cells from adult peripheral blood. Enhanced calcium flux was also observed in adult CD4(+) recent thymic emigrants. Neonatal naive CD4(+) T cells also had higher activation-induced Erk phosphorylation. The microRNA miR-181a, which enhances activation-induced calcium flux in murine thymocytes, was expressed at significantly higher levels in CB naive CD4(+) T cells compared with adult cells. Overexpression of miR-181a in adult naive CD4(+) T cells increased activation-induced calcium flux, implying that the increased miR-181a levels of CB naive CD4(+) T cells contributed to their enhanced signaling. In contrast, AP-1-dependent transcription, which is downstream of Erk and required for full T cell activation, was decreased in CB naive CD4(+) T cells compared with adult cells. Thus, CB naive CD4(+) T cells have enhanced activation-dependent calcium flux, indicative of the retention of a thymocyte-like phenotype. Enhanced calcium signaling and Erk phosphorylation are decoupled from downstream AP-1-dependent transcription, which is reduced and likely contributes to limitations of human fetal and neonatal CD4(+) T cell immunity.

Neutrophil arrest and migration on inflamed endothelium involves a conformational shift in CD11a/CD18 (leukocyte function antigen-1; LFA-1) to a high-affinity and clustered state that determines the strength and lifetime of bond formation with ICAM-1. Cytoskeletal adapter proteins Kindlin-3 and Talin-1 anchor clustered LFA-1 to the cytoskeleton and facilitate the transition from neutrophil rolling to arrest. We recently reported that tensile force acts on LFA-1 bonds inducing their colocalization with Orai1, the predominant membrane store operated Ca(2+) channel that cooperates with the endoplasmic reticulum to elicit cytosolic flux. Because Kindlin-3 was recently reported to initiate LFA-1 clustering in lymphocytes, we hypothesized that it cooperates with Orai1 and LFA-1 in signaling local Ca(2+) flux necessary for shear-resistant neutrophil arrest. Using microfluidic flow channels combined with total internal reflection fluorescence microscopy, we applied defined shear stress to low- or high-affinity LFA-1 and imaged the spatiotemporal regulation of bond formation with Kindlin-3 recruitment and Ca(2+) influx. Orai1 and Kindlin-3 genes were silenced in neutrophil-like HL-60 cells to assess their respective roles in this process. Kindlin-3 was enriched within focal clusters of high-affinity LFA-1, which promoted physical linkage with Orai1. This macromolecular complex functioned to amplify inside-out Ca(2+) signaling in response to IL-8 stimulation by catalyzing an increased density of Talin-1 and consolidating LFA-1 clusters within sites of contact with ICAM-1. In this manner, neutrophils use focal adhesions as mechanosensors that convert shear stress-mediated tensile force into local bursts of Ca(2+) influx that catalyze cytoskeletal engagement and an adhesion-strengthened migratory phenotype.

IFN regulatory factor 3 (IRF3) regulates early type I IFNs and other genes involved in innate immunity. We have previously shown that cells undergoing an endoplasmic reticulum (ER) stress response called the unfolded protein response produce synergistically augmented IFN-β when stimulated with pattern recognition receptor agonists such as LPS. Concomitant ER stress and LPS stimulation resulted in greater recruitment of the IRF3 transcription factor to ifnb1 gene regulatory elements. In this study, we used murine cells to demonstrate that both oxygen-glucose deprivation and pharmacologic unfolded protein response inducers trigger phosphorylation and nuclear translocation of IRF3, even in the absence of exogenous LPS. Different ER stressors used distinct mechanisms to activate IRF3: IRF3 phosphorylation due to calcium-mobilizing ER stress (thapsigargin treatment, oxygen-glucose deprivation) critically depended upon stimulator of IFN gene, an ER-resident nucleic acid-responsive molecule. However, calcium mobilization alone by ionomycin was insufficient for IRF3 phosphorylation. In contrast, other forms of ER stress (e.g., tunicamycin treatment) promote IRF3 phosphorylation independently of stimulator of IFN gene and TANK-binding kinase 1. Rather, IRF3 activation by tunicamycin and 2-deoxyglucose was inhibited by 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, a serine protease inhibitor that blocks activating transcription factor 6 processing. Interfering with ER stress-induced IRF3 activation abrogated IFN-β synergy. Together, these data suggest ER stress primes cells to respond to innate immune stimuli by activating the IRF3 transcription factor. Our results also suggest certain types of ER stress accomplish IRF3 phosphorylation by co-opting existing innate immune pathogen response pathways. These data have implications for diseases involving ER stress and type I IFN.

IL-33 is an IL-1 family cytokine that displays dual functions: a cytokine via its receptor, T1/ST2, or a chromatin-binding factor within the nucleus. Functionally, it promotes Th2-associated immunity by enhancing the activation and survival of several cell types. However, the pathways regulating IL-33 expression are still unclear. Although several cells display constitutive expression of IL-33, we showed previously that mast cells expressed low levels of IL-33 constitutively but that IL-33 was induced upon IgE-mediated activation. This was mediated via a calcium-dependent mechanism. In this study, we define the pathway through which this inducible IL-33 is regulated. Importantly, this pathway does not alter expression in cells with high constitutive IL-33 expression, such as epithelial cells or fibroblasts. Our data show that, upstream of calcium, inhibition of PI3K and Sphk activity decreases inducible IL-33 expression to IgE/Ag activation. Additionally, expression of Sphk1 short hairpin RNA prevents upregulation of IL-33 expression. Downstream of calcium, NFAT activity is necessary and sufficient for inducible IL-33 expression. We also demonstrate calcium-dependent transcription from two regions of the IL-33 gene that contain putative NFAT-binding sites, one upstream of exon 1 and one upstream of the start site. Interestingly, we show that blocking other calcium pathways, including inositol triphosphate receptor, or NF-κB inhibits IgE-driven IL-1β, another IL-1 family cytokine, but it has no influence on inducible IL-33 expression. In summary, our data demonstrate cell-specific differences in the regulation of IL-33 expression and define a pathway critical for the expression of inducible IL-33 by mast cells upon their activation.

BCR signaling regulates the activities and fates of B cells. BCR signaling encompasses two feedback loops emanating from Lyn and Fyn, which are Src family protein tyrosine kinases (SFKs). Positive feedback arises from SFK-mediated trans phosphorylation of BCR and receptor-bound Lyn and Fyn, which increases the kinase activities of Lyn and Fyn. Negative feedback arises from SFK-mediated cis phosphorylation of the transmembrane adapter protein PAG1, which recruits the cytosolic protein tyrosine kinase Csk to the plasma membrane, where it acts to decrease the kinase activities of Lyn and Fyn. To study the effects of the positive and negative feedback loops on the dynamical stability of BCR signaling and the relative contributions of Lyn and Fyn to BCR signaling, we consider in this study a rule-based model for early events in BCR signaling that encompasses membrane-proximal interactions of six proteins, as follows: BCR, Lyn, Fyn, Csk, PAG1, and Syk, a cytosolic protein tyrosine kinase that is activated as a result of SFK-mediated phosphorylation of BCR. The model is consistent with known effects of Lyn and Fyn deletions. We find that BCR signaling can generate a single pulse or oscillations of Syk activation depending on the strength of Ag signal and the relative levels of Lyn and Fyn. We also show that bistability can arise in Lyn- or Csk-deficient cells.

ORAI1 is a pore subunit of Ca(2+) release-activated Ca(2+) channels that mediate TCR stimulation-induced Ca(2+) entry. A point mutation in ORAI1 (ORAI1(R91W)) causes SCID in human patients that is recapitulated in Orai1(-/-) mice, emphasizing its important role in the immune cells. In this study, we have characterized a novel function of ORAI1 in T cell death. CD4(+) T cells from Orai1(-/-) mice showed robust proliferation with repetitive stimulations and strong resistance to stimulation-induced cell death due to reduced mitochondrial Ca(2+) uptake and altered gene expression of proapoptotic and antiapoptotic molecules (e.g., Fas ligand, Noxa, and Mcl-1). Nuclear accumulation of NFAT was severely reduced in ORAI1-deficient T cells, and expression of ORAI1 and a constitutively active mutant of NFAT recovered cell death. These results indicate NFAT-mediated cell death pathway as one of the major downstream targets of ORAI1-induced Ca(2+) entry. By expressing various mutants of ORAI1 in wild-type and Orai1(-/-) T cells to generate different levels of intracellular Ca(2+), we have shown that activation-induced cell death is directly proportional to the intracellular Ca(2+) concentration levels. Consistent with the in vitro results, Orai1(-/-) mice showed strong resistance to T cell depletion induced by injection of anti-CD3 Ab. Furthermore, ORAI1-deficient T cells showed enhanced survival after adoptive transfer into immunocompromised hosts. Thus, our results demonstrate a crucial role of the ORAI1-NFAT pathway in T cell death and highlight the important role of ORAI1 as a major route of Ca(2+) entry during activated T cell death.

The CXCR4 chemokine receptor is a G protein-coupled receptor that signals in T lymphocytes by forming a heterodimer with the TCR. CXCR4 and TCR functions are consequently highly cross regulated, affecting T cell immune activation, cytokine secretion, and T cell migration. The CXCR4-TCR heterodimer stimulates T cell migration and activation of the ERK MAPK and downstream AP-1-dependent cytokine transcription in response to stromal cell-derived factor-1 (SDF-1), the sole chemokine ligand of CXCR4. These responses require Gi-type G proteins as well as TCR ITAM domains and the ZAP70 tyrosine kinase, thus indicating that the CXCR4-TCR heterodimer signals to integrate G protein-coupled receptor-associated and TCR-associated signaling molecules in response to SDF-1. Yet, the phospholipase C (PLC) isozymes responsible for coupling the CXCR4-TCR heterodimer to distinct downstream cellular responses are incompletely characterized. In this study, we demonstrate that PLC activity is required for SDF-1 to induce ERK activation, migration, and CXCR4 endocytosis in human T cells. SDF-1 signaling via the CXCR4-TCR heterodimer uses PLC-β3 to activate the Ras-ERK pathway and increase intracellular calcium ion concentrations, whereas PLC-γ1 is dispensable for these outcomes. In contrast, PLC-γ1, but not PLC-β3, is required for SDF-1-mediated migration via a mechanism independent of LAT. These results increase understanding of the signaling mechanisms employed by the CXCR4-TCR heterodimer, characterize new roles for PLC-β3 and PLC-γ1 in T cells, and suggest that multiple PLCs may also be activated downstream of other chemokine receptors to distinctly regulate migration versus other signaling functions.

Acute inflammation triggers the innate immune response of neutrophils that efficiently traffic from the bloodstream to concentrate at high numbers at the site of tissue infection or wounding. A gatekeeper in this process is activation of β(2) integrins, which form bond clusters with ICAM-1 on the endothelial surface. These bond clusters serve dual functions of providing adhesive strength to anchor neutrophils under the shear forces of blood flow and directional guidance for cell polarization and subsequent transmigration on inflamed endothelium. We hypothesized that shear forces transmitted through high-affinity LFA-1 facilitates the cooperation with the calcium release-activated channel Orai1 in directing localized cytoskeletal activation and directed migration. By using vascular mimetic microfluidic channels, we observed neutrophil arrest on a substrate of either ICAM-1 or allosteric Abs that stabilize a high- or low-affinity conformation of LFA-1. Neutrophils captured via low-affinity LFA-1 did not exhibit intracellular calcium flux, F-actin polymerization, cell polarization, or directional migration under shear flow. In contrast, high-affinity LFA-1 provided orientation along a uropod-pseudopod axis that required calcium flux through Orai1. We demonstrate how the shear stress of blood flow can transduce distinct outside-in signals at focal sites of high-affinity LFA-1 that provide contact-mediated guidance for neutrophil emigration.

Coronins are WD repeat-containing proteins highly conserved in the eukaryotic kingdom implicated in the regulation of F-actin. Mammalian coronin 1, one of the most conserved isoforms expressed in leukocytes, regulates survival of T cells, which has been suggested to be due to its role in preventing F-actin-induced apoptosis. In this study, we come to a different conclusion. We show that coronin 1 does not modulate F-actin and that induction of F-actin failed to induce apoptosis. Instead, coronin 1 was required for providing prosurvival signals, in the absence of which T cells rapidly underwent apoptosis. These results argue against a role for coronin 1 in F-actin-mediated T cell apoptosis and establish coronin 1 as an essential regulator of the balance between prosurvival and proapoptotic signals in naive T cells.

Cytotoxic lymphocytes kill target cells through polarized release of the content of lytic granules at the immunological synapse. In human NK cells, signals for granule polarization and for degranulation can be uncoupled: Binding of β(2) integrin LFA-1 to ICAM is sufficient to induce polarization but not degranulation, whereas CD16 binding to IgG triggers unpolarized degranulation. In this study, we investigated the basis for this difference. IL-2-expanded human NK cells were stimulated by incubation with plate-bound ligands of LFA-1 (ICAM-1) and CD16 (human IgG). Surprisingly, LFA-1 elicited signals similar to those induced by CD16, including tyrosine phosphorylation of the TCR ζ-chain, tyrosine kinase Syk, and phospholipase C-γ. Whereas CD16 activated Ca(2+) mobilization and LAT phosphorylation, LFA-1 did not, but induced strong Pyk2 and paxillin phosphorylation. LFA-1-dependent granule polarization was blocked by inhibition of Syk, phospholipase C-γ, and protein kinase C, as well as by paxillin knockdown. Therefore, common signals triggered by CD16 and LFA-1 bifurcate to provide independent control of Ca(2+)-dependent degranulation and paxillin-dependent granule polarization.

Selenoprotein K (Sel K) is a selenium-containing protein for which no function has been identified. We found that Sel K is an endoplasmic reticulum transmembrane protein expressed at relatively high levels in immune cells and is regulated by dietary selenium. Sel K(-/-) mice were generated and found to be similar to wild-type controls regarding growth and fertility. Immune system development was not affected by Sel K deletion, but specific immune cell defects were found in Sel K(-/-) mice. Receptor-mediated Ca(2+) flux was decreased in T cells, neutrophils, and macrophages from Sel K(-/-) mice compared with controls. Ca(2+)-dependent functions including T cell proliferation, T cell and neutrophil migration, and Fcγ receptor-mediated oxidative burst in macrophages were decreased in cells from Sel K(-/-) mice compared with that in cells from controls. West Nile virus infections were performed, and Sel K(-/-) mice exhibited decreased viral clearance in the periphery and increased viral titers in brain. Furthermore, West Nile virus-infected Sel K(-/-) mice demonstrated significantly lower survival (2 of 23; 8.7%) compared with that of wild-type controls (10 of 26; 38.5%). These results establish Sel K as an endoplasmic reticulum-membrane protein important for promoting effective Ca(2+) flux during immune cell activation and provide insight into molecular mechanisms by which dietary selenium enhances immune responses.

Phagocytosis is a process of innate immunity that allows for the enclosure of pathogens within the phagosome and their subsequent destruction through the production of reactive oxygen species (ROS). Although these processes have been associated with increases of intracellular Ca(2+) concentrations, the mechanisms by which Ca(2+) could regulate the different phases of phagocytosis remain unknown. The aim of this study was to investigate the Ca(2+) signaling pathways involved in the regulation of FcγRs-induced phagocytosis. Our work focuses on IgG-opsonized zymosan internalization and phagosomal ROS production in DMSO-differentiated HL-60 cells and neutrophils. We found that chelation of intracellular Ca(2+) by BAPTA or emptying of the intracellular Ca(2+) store by thapsigargin reduced the efficiency of zymosan internalization. Using an small interfering RNA strategy, our data establish that the observed Ca(2+) release occurs through two isoforms of inositol 1,4,5-triphosphate receptors, ITPR1 and ITPR3. In addition, we provide evidence that phagosomal ROS production is dependent on extracellular Ca(2+) entry. We demonstrate that the observed Ca(2+) influx is supported by ORAI calcium release-activated calcium modulator 1 (Orai1) and stromal interaction molecule 1 (STIM1). This result suggests that extracellular Ca(2+) entry, which is required for ROS production, is mediated by a store-operated Ca(2+) mechanism. Finally, our data identify the complex formed by S100A8 and S100A9 (S100 calcium-binding protein A8 and A9 complex), two Ca(2+)-binding proteins, as the site of interplay between extracellular Ca(2+) entry and intraphagosomal ROS production. Thus, we demonstrate that FcγR-mediated phagocytosis requires intracellular Ca(2+) store depletion for the internalization phase. Then phagosomal ROS production requires extracellular Ca(2+) entry mediated by Orai1/STIM1 and relayed by S100A8-A9 as Ca(2+) sensor.

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved intracellular bulk degradation pathway that plays critical roles in eliminating intracellular pathogens, presenting endogenous Ags, and regulating T lymphocyte survival and proliferation. In this study, we have investigated the role of autophagy in regulating the endoplasmic reticulum (ER) compartment in T lymphocytes. We found that ER content is expanded in mature autophagy-related protein (Atg) 7-deficient T lymphocytes. Atg7-deficient T cells stimulated through the TCR display impaired influx, but not efflux, of calcium, and ER calcium stores are increased in Atg7-deficient T cells. Treatment with the ER sarco/ER Ca(2+)-ATPase pump inhibitor thapsigargin rescues the calcium influx defect in Atg7-deficient T lymphocytes, suggesting that this impairment is caused by an intrinsic defect in ER. Furthermore, we found that the stimulation-induced redistribution of stromal interaction molecule-1, a critical event for the store-operated Ca(2+) release-activated Ca(2+) channel opening, is impaired in Atg7-deficient T cells. Together, these findings indicate that the expanded ER compartment in Atg7-deficient T cells contains increased calcium stores, and the inability of these stores to be depleted causes defective calcium influx in these cells. Our results demonstrate that autophagy plays an important role in maintaining ER and calcium homeostasis in T lymphocytes.

DRAK2 is a serine/threonine kinase highly enriched in lymphocytes that raises the threshold for T cell activation and maintains T cell survival following productive activation. T cells lacking DRAK2 are prone to activation under suboptimal conditions and exhibit enhanced calcium responses to AgR stimulation. Despite this, mice lacking DRAK2 are resistant to organ-specific autoimmune diseases due to defective autoreactive T cell survival. DRAK2 kinase activity is induced by AgR signaling, and in this study we show that the induction of DRAK2 activity requires Ca(2+) influx through the Ca(2+) release-activated Ca(2+) channel formed from Orai1 subunits. Blockade of DRAK2 activity with the protein kinase D (PKD) inhibitor Gö6976 or expression of a kinase-dead PKD mutant prevented activation of DRAK2, whereas a constitutively active PKD mutant promoted DRAK2 function. Knockdown of PKD in T cells strongly blocked endogenous DRAK2 activation following TCR ligation, implicating PKD as an essential intermediate in the activation of DRAK2 by Ca(2+) influx. Furthermore, we identify DRAK2 as a novel substrate of PKD, and demonstrate that DRAK2 and PKD physically interact under conditions that activate PKD. Mitochondrial generation of reactive oxygen intermediates was necessary and sufficient for DRAK2 activation in response to Ca(2+) influx. Taken together, DRAK2 and PKD form a novel signaling module that controls calcium homeostasis following T cell activation.

Activation of mast cells by aggregation of the high-affinity IgE receptors (FcεRI) initiates signaling events leading to the release of inflammatory and allergic mediators stored in cytoplasmic granules. A key role in this process play changes in concentrations of intracellular Ca(2+) controlled by store-operated Ca(2+) entry (SOCE). Although microtubules are also involved in the process leading to degranulation, the molecular mechanisms that control microtubule rearrangement during activation are largely unknown. In this study, we report that activation of bone marrow-derived mast cells (BMMCs) induced by FcεRI aggregation or treatment with pervanadate or thapsigargin results in generation of protrusions containing microtubules (microtubule protrusions). Formation of these protrusions depended on the influx of extracellular Ca(2+). Changes in cytosolic Ca(2+)concentration also affected microtubule plus-end dynamics detected by microtubule plus-end tracking protein EB1. Experiments with knockdown or reexpression of STIM1, the key regulator of SOCE, confirmed the important role of STIM1 in the formation of microtubule protrusions. Although STIM1 in activated cells formed puncta associated with microtubules in protrusions, relocation of STIM1 to a close proximity of cell membrane was independent of growing microtubules. In accordance with the inhibition of Ag-induced Ca(2+) response and decreased formation of microtubule protrusions in BMMCs with reduced STIM1, the cells also exhibited impaired chemotactic response to Ag. We propose that rearrangement of microtubules in activated mast cells depends on STIM1-induced SOCE, and that Ca(2+) plays an important role in the formation of microtubule protrusions in BMMCs.

Eotaxin-3/CCL26 is a functional ligand for CCR3 and abundantly produced by IL-4-/IL-13-stimulated vascular endothelial cells. CCL26 also functions as a natural antagonist for CCR1, CCR2, and CCR5. In this study, we report that CCL26 is yet a functional ligand for CX3CR1, the receptor for fractalkine/CX3CL1, which is expressed by CD16(+) NK cells, cytotoxic effector CD8(+) T cells, and CD14(low)CD16(high) monocytes. Albeit at relatively high concentrations, CCL26 induced calcium flux and chemotaxis in mouse L1.2 cells expressing human CX3CR1 but not mouse CX3CR1 and competed with CX3CL1 for binding to CX3CR1. In chemotaxis assays using human PBMCs, CCL26 attracted not only eosinophils but also CD16(+) NK cells, CD45RA(+)CD27(-)CD8(+) T cells, and CD14(low)CD16(high) monocytes. Intraperitoneal injection of CCL26 into mice rapidly recruited mouse eosinophils and intravenously transferred human CD16(+) NK cells into the peritoneal cavity. IL-4-stimulated HUVECs produced CCL26 and efficiently induced adhesion of cells expressing CX3CR1. Real-time PCR showed that skin lesions of psoriasis consistently contained CX3CL1 mRNA but not CCL26 mRNA, whereas those of atopic dermatitis contained CCL26 mRNA in all samples but CX3CL1 mRNA in only about half of the samples. Nevertheless, the skin lesions from both diseases consistently contained CX3CR1 mRNA at high levels. Thus, CCL26 may be partly responsible for the recruitment of cells expressing CX3CR1 in atopic dermatitis particularly when the expression of CX3CL1 is low. Collectively, CCL26 is another agonist for CX3CR1 and may play a dual role in allergic diseases by attracting eosinophils via CCR3 and killer lymphocytes and resident monocytes via CX3CR1.

ORAI1 is the pore-forming subunit of the Ca(2+) release-activated Ca(2+) (CRAC) channel, which is responsible for store-operated Ca(2+) entry in lymphocytes. A role for ORAI1 in T cell function in vivo has been inferred from in vitro studies of T cells from human immunodeficient patients with mutations in ORAI1 and Orai1(-/-) mice, but a detailed analysis of T cell-mediated immune responses in vivo in mice lacking functional ORAI1 has been missing. We therefore generated Orai1 knock-in mice (Orai1(KI/KI)) expressing a nonfunctional ORAI1-R93W protein. Homozygosity for the equivalent ORAI1-R91W mutation abolishes CRAC channel function in human T cells resulting in severe immunodeficiency. Homozygous Orai1(KI/KI) mice die neonatally, but Orai1(KI/KI) fetal liver chimeric mice are viable and show normal lymphocyte development. T and B cells from Orai1(KI/KI) mice display severely impaired store-operated Ca(2+) entry and CRAC channel function resulting in a strongly reduced expression of several key cytokines including IL-2, IL-4, IL-17, IFN-γ, and TNF-α in CD4(+) and CD8(+) T cells. Cell-mediated immune responses in vivo that depend on Th1, Th2, and Th17 cell function were severely attenuated in ORAI1-deficient mice. Orai1(KI/KI) mice lacked detectable contact hypersensitivity responses and tolerated skin allografts significantly longer than wild-type mice. In addition, T cells from Orai1(KI/KI) mice failed to induce colitis in an adoptive transfer model of inflammatory bowel disease. These findings reaffirm the critical role of ORAI1 for T cell function and provide important insights into the in vivo functions of CRAC channels for T cell-mediated immunity.

Th cell subsets have unique calcium (Ca(2+)) signals when activated with identical stimuli. The regulation of these Ca(2+) signals and their correlation to the biological function of each T cell subset remains unclear. Trpm4 is a Ca(2+)-activated cation channel that we found is expressed at higher levels in Th2 cells compared with Th1 cells. Inhibition of Trpm4 expression increased Ca(2+) influx and oscillatory levels in Th2 cells and decreased influx and oscillations in Th1 cells. This inhibition of Trpm4 expression also significantly altered T cell cytokine production and motility. Our experiments revealed that decreasing Trpm4 levels divergently regulates nuclear localization of NFATc1. Consistent with this, gene profiling did not show Trpm4-dependent transcriptional regulation, and T-bet and GATA-3 levels remain identical. Thus, Trpm4 is expressed at different levels in Th cells and plays a distinctive role in T cell function by differentially regulating Ca(2+) signaling and NFATc1 localization.

Methamphetamine (METH) abuse is known to be associated with an inordinate rate of infections. Although many studies have described the association of METH exposure and immunosuppression, so far the underlying mechanism still remains elusive. In this study, we present evidence that METH exposure resulted in mitochondrial oxidative damage and caused dysfunction of primary human T cells. METH treatment of T lymphocytes led to a rise in intracellular calcium levels that enhanced the generation of reactive oxygen species. TCR-CD28 linked calcium mobilization and subsequent uptake by mitochondria in METH-treated T cells correlated with an increase in mitochondrion-derived superoxide. Exposure to METH-induced mitochondrial dysfunction in the form of marked decrease in mitochondrial membrane potential, increased mitochondrial mass, enhanced protein nitrosylation and diminished protein levels of complexes I, III, and IV of the electron transport chain. These changes paralleled reduced IL-2 secretion and T cell proliferative responses after TCR-CD28 stimulation indicating impaired T cell function. Furthermore, antioxidants attenuated METH-induced mitochondrial damage by preserving the protein levels of mitochondrial complexes I, III, and IV. Altogether, our data indicate that METH can cause T cell dysfunction via induction of oxidative stress and mitochondrial injury as underlying mechanism of immune impairment secondary to METH abuse.

Sh2d3c is an adaptor protein that has been implicated in T cell activation and shown to associate with different components of the integrin signaling pathway ex vivo. However, the in vivo significance of Sh2d3c expression in the regulation of the immune response and/or hematopoietic cell lineage development is not known. In this study, we show that expression of Sh2d3c is more critical for development and function of marginal zone B (MZB) cells than for T cell maturation. Mice deficient in Sh2d3c expression (Sh2d3c(-/-)) had a reduced number of MZB cells, and the residual MZB cells failed to properly capture polysaccharide Ags. Activation-induced proliferation, cytokine production, and migration of Sh2d3c(-)(/)(-) splenic B cells were also significantly reduced in vitro compared with wild-type (Sh2d3c(+/+)) cells. In contrast, T cell development and function were largely normal in Sh2d3c(-/-) mice. The thymi of Sh2d3c(-/-) mice showed no maturational abnormalities, the number of splenic T cells was only modestly reduced, and the T cells responded normally to in vitro polyclonal activation. The observed B cell deficiency in the Sh2d3c(-/-) mice led to diminished humoral immune response against thymus-independent type 2, but not thymus-dependent Ags, which highlights the primary in vivo role of Sh2d3c in regulating B cell development and function.

Human Vgamma9Vdelta2 T cells, a major innate-like peripheral T cell subset, are thought to play in vivo an important role in innate and adaptive immune responses to infection agents and tumors. However, the mechanisms regulating their broad effector functions, such as cytotoxicity and cytokine responses, remain poorly understood. In this study, we used single-cell calcium video imaging to analyze the early intracellular events associated with TCR-induced Vgamma9Vdelta2 T cell functional responses. When compared with other human T cell subsets, including NKT and Vdelta2(neg) gammadelta T cells, TCR/CD3-activated Vgamma9Vdelta2 T cells displayed an unusually delayed and sustained intracellular calcium mobilization, which was dramatically quickened and shortened on costimulation by NKG2D, a main activating NKR regulating gammadelta T cell tumor cytolysis. Importantly, the protein kinase C transduction pathway was identified as a main regulator of the NKG2D-mediated costimulation of antitumor Vgamma9Vdelta2 cytolytic responses. Therefore, this study identifies a new mechanism regulating Vgamma9Vdelta2 T cell functional plasticity through fine-tuning of early signal transduction events.

Several lines of evidence have demonstrated B cell intrinsic activation defects in patients with common variable immunodeficiency (CVID). The rapid increase of intracellular free calcium concentrations after engagement of the BCR represents one crucial element in this activation process. The analysis of 53 patients with CVID for BCR-induced calcium flux identified a subgroup of patients with significantly reduced Ca2+ signals in primary B cells. This subgroup strongly corresponded to the class Ia of the Freiburg classification. Comparison at the level of defined B cell subpopulations revealed reduced Ca2+ signals in all mature B cell populations of patients with CVID class Ia when compared with healthy individuals and other groups of patients with CVID but not in circulating transitional B cells. BCR-induced Ca2+ responses were the lowest in CD21low B cells in patients as well as healthy donors, indicating an additional cell-specific mechanism inhibiting the Ca2+ flux. Although proximal BCR signaling events are unperturbed in patients' B cells, including normal phospholipase Cgamma2 phosphorylation and Ca2+ release from intracellular stores, Ca2+ influx from the extracellular space is significantly impaired. CD22, a negative regulator of calcium signals in B cells, is highly expressed on CD21low B cells from patients with CVID Ia and might be involved in the attenuated Ca2+ response of this B cell subpopulation. These data from patients with CVID suggest that a defect leading to impaired BCR-induced calcium signaling is associated with the expansion of CD21low B cells, hypogammaglobulinemia, autoimmune dysregulation, and lymphadenopathy.

Recent studies indicate that the process of Ag presentation induces cytoskeleton-dependent mitochondrial redistribution to the immediate vicinity of the immunologic synapse (IS). This redistribution of mitochondria to the IS in T cells is necessary to maintain Ca(2+) influx and Th cell activation. Recently, we demonstrated that n-3 polyunsaturated fatty acids (PUFAs) suppress the localization and activation of signaling proteins at the IS. Therefore, we hypothesized that n-3 PUFAs suppress CD4(+) T cell mitochondrial translocation during the early stages of IS formation and downmodulate Ca(2+)-dependent Th cell activation. CD4(+) cells derived from fat-1 mice, a transgenic model that synthesizes n-3 PUFA from n-6 PUFA, were cocultured with anti-CD3-expressing hybridoma cells (145-2C11) for 15 min at 37 degrees C, and mitochondrial translocation to the IS was assessed by confocal microscopy. Fat-1 mice exhibited a significantly (p < 0.05) reduced percentage of T cells with mitochondria which translocated to the IS; fat-1 (30%) versus wild type control (82%). Regarding the effect on the mitochondrial-to-cytosolic Ca(2+) ratio, wild type cells showed significant increases at the IS (71%) and total cell (60%) within 30 min of IS formation. In contrast, fat-1 CD4(+) T cells remained at basal levels following the IS formation. A similar blunting of the mitochondrial-to-cytosolic Ca(2+) ratio was observed in wild type cells that were coincubated with inhibitors of the mitochondrial uniporter, RU360 or calcium release-activated Ca(2+) (CRAC) channels, BTP2. These observations provide evidence that n-3 PUFAs modulate Th cell activation by limiting mitochondrial translocation to the IS and reducing Ca(2+) entry.

Outside of the TLR paradigm, there is little understanding of how pathogen recognition at the cell surface is linked to functional responses in cells of the innate immune system. Recent work in this area demonstrates that the yeast particle zymosan, by binding to the beta-glucan receptor Dectin-1, activates an ITAM-Syk-dependent pathway in dendritic cells, which is required for optimal cytokine production and generation of an oxidative burst. It remains unclear how activation of Syk is coupled to effector mechanisms. In human macrophages, zymosan rapidly activated a calcium-dependent pathway downstream of Dectin-1 and Syk that led to activation of calmodulin-dependent kinase II and Pyk2. Calmodulin-dependent kinase and Pyk2 transduced calcium signals into activation of the ERK-MAPK pathway, CREB, and generation of an oxidative burst, leading to downstream production of IL-10. These observations identify a new calcium-mediated signaling pathway activated by zymosan and link this pathway to both inflammatory and anti-inflammatory responses in macrophages.

Engagement of FcepsilonRI causes its phosphorylation by Lyn kinase. Two alternatively spliced variants, Lyn A and B, are expressed in mast cells, and both isoforms interact with FcepsilonRI. Unlike Lyn A, Lyn B lacks a 21-aa region in the N-terminal unique domain. In this study, we investigated the role of Lyn A and B isoforms in mast cell signaling and responses. Lyn B was found to be a poor inducer of mast cell degranulation and was less potent in both inositol 1,4,5-triphosphate production and calcium responses. Expression of Lyn B alone showed reduced phosphorylation of both phospholipase Cgamma-1 and -2 and decreased interaction of phospholipase Cgamma-1 with the phosphorylated linker for activation of T cells. Lyn B also showed increased binding of tyrosine-phosphorylated proteins, which included the negative regulatory lipid phosphatase SHIP-1. In contrast, both Lyn A and B caused similar total cellular tyrosine phosphorylation and FcepsilonRI phosphorylation and neither Lyn A nor Lyn B alone could completely restore mast cell degranulation or dampen the excessive cytokine production seen in the absence of Lyn. However, expression of both isoforms showed complementation and normalized responses. These findings demonstrate that Lyn B differs from Lyn A in its association with SHIP-1 and in the regulation of calcium responses. However, complementation of both isoforms is required in mast cell activation.

CD22 and Siglec-G are inhibitory coreceptors for BCR-mediated signaling. Although CD22-deficient mice show increased calcium signaling in their conventional B2 cells and a quite normal B cell maturation, Siglec-G-deficient mice have increased calcium mobilization just in B1 cells and show a large expansion of the B1 cell population. Neither CD22-deficient, nor Siglec-G-deficient mice on a pure C57BL/6 or BALB/c background, respectively, develop autoimmunity. Using Siglec-G x CD22 double-deficient mice, we addressed whether Siglec-G and CD22 have redundant functions. Siglec-G x CD22 double-deficient mice show elevated calcium responses in both B1 cells and B2 cells, increased serum IgM levels and an enlarged population of B1 cells. The enlargement of B1 cell numbers is even higher than in Siglecg(-/-) mice. This expansion seems to happen at the expense of B2 cells, which are reduced in absolute cell numbers, but show an activated phenotype. Furthermore, Siglec-G x CD22 double-deficient mice show a diminished immune response to both thymus-dependent and thymus-independent type II Ags. In contrast, B cells from Siglec-G x CD22 double-deficient mice exhibit a hyperproliferative response to stimulation with several TLR ligands. Aged Siglec-G x CD22 double-deficient mice spontaneously develop anti-DNA and antinuclear autoantibodies. These resulted in a moderate form of immune complex glomerulonephritis. These results show that Siglec-G and CD22 have partly compensatory functions and together are crucial in maintaining the B cell tolerance.

The use of cysteinyl leukotriene receptor antagonists (LTRAs) for asthma therapy has been associated with a significant degree of interpatient variability in response to treatment. Some of that variability may be attributable to noncysteinyl leukotriene type 1 receptor (CysLT(1))-mediated inhibitory mechanisms that have been demonstrated for this group of drugs. We used a model of CysLT(1) signaling in human monocytes to characterize CysLT(1)-dependent and -independent anti-inflammatory activity of two chemically different, clinically relevant LTRAs (montelukast and zafirlukast). Using receptor-desensitization experiments in monocytes and CysLT(1)-transfected HEK293 cells and IL-10- and CysLT(1) small interfering RNA-induced downregulation of CysLT(1) expression, we showed that reported CysLT(1) agonists leukotriene D(4) and UDP signal through calcium mobilization, acting on separate receptors, and that both pathways were inhibited by montelukast and zafirlukast. However, 3-log greater concentrations of LTRAs were required for the inhibition of UDP-induced signaling. In monocytes, UDP, but not leukotriene D(4), induced IL-8 production that was significantly inhibited by both drugs at micromolar concentrations. At low micromolar concentrations, both LTRAs also inhibited calcium ionophore-induced leukotriene (leukotriene B(4) and leukotriene C(4)) production, indicating 5-lipoxygenase inhibitory activities. We report herein that montelukast and zafirlukast, acting in a concentration-dependent manner, can inhibit non-CysLT(1)-mediated proinflammatory reactions, suggesting activities potentially relevant for interpatient variability in response to treatment. Higher doses of currently known LTRAs or new compounds derived from this class of drugs may represent a new strategy for finding more efficient therapy for bronchial asthma.

Calcium (Ca(2+)) signaling in T lymphocytes is essential for a variety of functions, including the regulation of differentiation, gene transcription, and effector functions. A major Ca(2+) entry pathway in nonexcitable cells, including T cells, is store-operated Ca(2+) entry (SOCE), wherein depletion of intracellular Ca(2+) stores upon receptor stimulation causes subsequent influx of extracellular Ca(2+) across the plasma membrane. Stromal interaction molecule (STIM) 1 is the Ca(2+) sensor in the endoplasmic reticulum, which controls this process, whereas the other STIM isoform, STIM2, coregulates SOCE. Although the contribution of STIM molecules and SOCE to T lymphocyte function is well studied in vitro, their significance for immune processes in vivo has remained largely elusive. In this study, we studied T cell function in mice lacking STIM1 or STIM2 in a model of myelin-oligodendrocyte glycoprotein (MOG(35-55))-induced experimental autoimmune encephalomyelitis (EAE). We found that STIM1 deficiency significantly impaired the generation of neuroantigen-specific T cell responses in vivo with reduced Th1/Th17 responses, resulting in complete protection from EAE. Mice lacking STIM2 developed EAE, but the disease course was ameliorated. This was associated with a reduced clinical peak of disease. Deficiency of STIM2 was associated with an overall reduced proliferative capacity of lymphocytes and a reduction of IFN-gamma/IL-17 production by neuroantigen-specific T cells. Neither STIM1 nor STIM2 deficiency altered the phenotype or function of APCs. These findings reveal a crucial role of STIM-dependent pathways for T cell function and activation under autoimmune inflammatory conditions, establishing them as attractive new molecular therapeutic targets for the treatment of inflammatory and autoimmune disorders.

Microglia are intrinsic immune cells that release factors, including proinflammatory cytokines, NO, and neurotrophins, following activation after disturbance in the brain. Elevation of intracellular Ca(2+) concentration ([Ca(2+)]i) is important for microglial functions, such as the release of cytokines and NO from activated microglia. There is increasing evidence suggesting that pathophysiology of neuropsychiatric disorders is related to the inflammatory responses mediated by microglia. Brain-derived neurotrophic factor (BDNF) is a neurotrophin well known for its roles in the activation of microglia as well as in pathophysiology and/or treatment of neuropsychiatric disorders. In this study, we observed that BDNF induced a sustained increase in [Ca(2+)]i through binding with the truncated tropomyosin-related kinase B receptor, resulting in activation of the PLC pathway and store-operated calcium entry in rodent microglial cells. RT-PCR and immunocytochemical techniques revealed that truncated tropomyosin-related kinase B-T1 receptors were highly expressed in rodent microglial cells. Sustained activation of store-operated calcium entry occurred after brief BDNF application and contributed to the maintenance of sustained [Ca(2+)]i elevation. Pretreatment with BDNF significantly suppressed the release of NO from activated microglia. Additionally, pretreatment of BDNF suppressed the IFN-gamma-induced increase in [Ca(2+)]i, along with a rise in basal levels of [Ca(2+)]i in rodent microglial cells. We show direct evidence that rodent microglial cells are able to respond to BDNF, which may be important for the regulation of inflammatory responses, and may also be involved in the pathophysiology and/or the treatment of neuropsychiatric disorders.

Foam cell formation is the most important process in atherosclerosis, and low density lipoprotein oxidation by reactive oxygen species (ROS) is the key step in the conversion of macrophages to foam cells. This study reveals the control mechanism of the gene for NADPH oxidase 1 (Nox1), which produces ROS in the formation of foam cells by stimulating TLR4. Treatment of macrophages by the TLR4 agonist LPS stimulated ROS production and ROS-mediated macrophage to foam cell conversion. This LPS-induced ROS production and foam cell formation could be abrogated by pretreatment of macrophages with N-acetyl cysteine or apocynin. LPS increased Nox1 promoter activity, and resultant expression of mRNA and protein. Small interfering RNA mediated inhibition of Nox1 expression decreased LPS-induced ROS production and foam cell formation. LPS-mediated Nox1 expression and the responses occurred in a calcium-independent phospholipase A(2) (iPLA(2))-dependent manner. The iPLA(2)beta-specific inhibitor S-BEL or iPLA(2)beta small interfering RNA attenuated LPS-induced Nox1 expression, ROS production, and foam cell formation. In addition, activation of iPLA(2)beta by LPS caused Akt phosphorylation and was followed by increased Nox1 expression. These results suggest that the binding of LPS and TLR4 increases Nox1 expression through the iPLA(2)beta-Akt signaling pathway, and control ROS production and foam cell formation.