Faculty Bibliography

Lymphocyte function is regulated by a network of ion channels and transporters in the plasma membrane of B and T cells. These proteins modulate the cytoplasmic concentrations of diverse cations, such as calcium, magnesium and zinc ions, which function as second messengers to regulate crucial lymphocyte effector functions, including cytokine production, differentiation and cytotoxicity. The repertoire of ion-conducting proteins includes calcium release-activated calcium (CRAC) channels, P2X receptors, transient receptor potential (TRP) channels, potassium channels, chloride channels and magnesium and zinc transporters. This Review discusses the roles of ion conduction pathways in lymphocyte function and immunity.

Stromal interaction molecule 1 (STIM1) deficiency is a rare genetic disorder of store-operated calcium entry, associated with a complex syndrome including immunodeficiency and immune dysregulation. The link from the molecular defect to these clinical manifestations is incompletely understood. We report two patients with a homozygous R429C point mutation in STIM1 completely abolishing store-operated calcium entry in T cells. Immunological analysis of one patient revealed that despite the expected defect of T cell proliferation and cytokine production in vitro, significant antiviral T cell populations were generated in vivo. These T cells proliferated in response to viral Ags and showed normal antiviral cytotoxicity. However, antiviral immunity was insufficient to prevent chronic CMV and EBV infections with a possible contribution of impaired NK cell function and a lack of NKT cells. Furthermore, autoimmune cytopenia, eczema, and intermittent diarrhea suggested impaired immune regulation. FOXP3-positive regulatory T (Treg) cells were present but showed an abnormal phenotype. The suppressive function of STIM1-deficient Treg cells in vitro, however, was normal. Given these partial defects in cytotoxic and Treg cell function, impairment of other immune cell populations probably contributes more to the pathogenesis of immunodeficiency and autoimmunity in STIM1 deficiency than previously appreciated.

Calcium signals play a critical role in many cell-type specific effector functions during innate and adaptive immune responses. The predominant mechanism to raise intracellular (Ca(2)) used by most immune cells is store-operated Ca(2) entry (SOCE), whereby the depletion of endoplasmic reticulum (ER) Ca(2) stores triggers the influx of extracellular Ca(2). SOCE in immune cells is mediated by the highly Ca(2) selective Ca(2)-release-activated Ca(2) (CRAC) channel, encoded by ORAI1, ORAI2 and ORAI3 genes. ORAI proteins are activated by stromal interaction molecules (STIM) 1 and 2, which act as sensors of ER Ca(2) store depletion. The importance of SOCE mediated by STIM and ORAI proteins for immune function is evident from the immunodeficiency and autoimmunity in patients with mutations in STIM1 and ORAI1 genes. These patients and studies in gene-targeted mice have revealed an essential role for ORAI/STIM proteins in the function of several immune cells. This review focuses on recent advances made towards understanding the role of SOCE in immune cells with an emphasis on the immune dysregulation that results from defects in SOCE in human patients and transgenic mice.

Mutations in genes encoding the calcium-release activated calcium (CRAC) channel abolish calcium influx in cells of the immune system and cause severe congenital immunodeficiency. Patients with autosomal recessive mutations in the CRAC channel gene ORAI1, its activator stromal interaction molecule 1 (STIM1), and mice with targeted deletion of Orai1, Stim1, and Stim2 genes reveal important roles for CRAC channels in adaptive and innate immune responses to infection and in autoimmunity. Because CRAC channels have important functions outside the immune system, deficiency of either ORAI1 or STIM1 is associated with a unique clinical phenotype. This review will give an overview of CRAC channel function in the immune system, examine the consequences of CRAC channel deficiency for immunity in human patients and mice, and discuss genetic defects in immunoreceptor-associated signaling molecules that compromise calcium influx and cause immunodeficiency

BACKGROUND: Stromal interaction molecule 1 (STIM1) was recently identified as a critical component of store-operated calcium entry (SOCE) in platelets. We previously reported the Ca(2+) -sensing guanine nucleotide exchange factor CalDAG-GEFI as a critical molecule in Ca(2+) signaling in platelets. OBJECTIVE: To evaluate the contribution of STIM1/SOCE to Ca(2+) -dependent platelet activation and thrombosis, we here compared the activation responses of platelets lacking STIM1 and platelets lacking CalDAG-GEFI. METHODS: The murine Stim1 gene was conditionally deleted in the megakaryocyte/platelet lineage. CalDAG-GEFI(-/-) and Stim1(fl/fl) PF4-Cre mice, along with littermate control mice, were used for in vitro and in vivo experiments under flow as well as static conditions. RESULTS: Integrin alpha(IIb) beta(3) -mediated aggregation was markedly impaired in CalDAG-GEFI-deficient but not STIM1-deficient platelets, under both static and flow conditions. In contrast, deficiency in either STIM1 or CalDAG-GEFI significantly impaired the ability of platelets to express phosphatidylserine on the cell surface. When subjected to a laser injury thrombosis model, mice lacking STIM1 in platelets were characterized by the formation of unstable platelet-rich thrombi and delayed and reduced fibrin generation in injured arterioles. In CalDAG-GEFI(-/-) mice, fibrin generation was also delayed and reduced, but platelet accumulation was almost abolished. CONCLUSIONS: Our studies suggest that: (i) STIM1/SOCE is critical for the procoagulant activity but not the proadhesive function of platelets; and (ii) at the site of vascular injury, STIM1 and CalDAG-GEFI are critical for the first wave of thrombin generation mediated by procoagulant platelets.

Orai1 proteins have been recently identified as subunits of SOCE (store-operated Ca(2)(+) entry) channels. In primary isolated PACs (pancreatic acinar cells), Orai1 showed remarkable co-localization and co-immunoprecipitation with all three subtypes of IP(3)Rs (InsP(3) receptors). The co-localization between Orai1 and IP(3)Rs was restricted to the apical part of PACs. Neither co-localization nor co-immunoprecipitation was affected by Ca(2)(+) store depletion. Importantly we also characterized Orai1 in basal and lateral membranes of PACs. The basal and lateral membranes of PACs have been shown previously to accumulate STIM1 (stromal interaction molecule 1) puncta as a result of Ca(2)(+) store depletion. We therefore conclude that these polarized secretory cells contain two pools of Orai1: an apical pool that interacts with IP(3)Rs and a basolateral pool that interacts with STIM1 following the Ca(2)(+) store depletion. Experiments on IP(3)R knockout animals demonstrated that the apical Orai1 localization does not require IP(3)Rs and that IP(3)Rs are not necessary for the activation of SOCE. However, the InsP(3)-releasing secretagogue ACh (acetylcholine) produced a negative modulatory effect on SOCE, suggesting that activated IP(3)Rs could have an inhibitory effect on this Ca(2)(+) entry mechanism.

Lymphocytes mediate cytotoxicity by polarized release of the contents of cytotoxic granules toward their target cells. Here, we have studied the role of the calcium release-activated calcium channel ORAI1 in human lymphocyte cytotoxicity. Natural killer (NK) cells obtained from an ORAI1-deficient patient displayed defective store-operated Ca(2+) entry (SOCE) and severely defective cytotoxic granule exocytosis leading to impaired target cell lysis. Similar findings were obtained using NK cells from a stromal interaction molecule 1-deficient patient. The defect occurred at a late stage of the signaling process, because activation of leukocyte functional antigen (LFA)-1 and cytotoxic granule polarization were not impaired. Moreover, pharmacological inhibition of SOCE interfered with degranulation and target cell lysis by freshly isolated NK cells and CD8(+) effector T cells from healthy donors. In addition to effects on lymphocyte cytotoxicity, synthesis of the chemokine macrophage inflammatory protein-1beta and the cytokines TNF-alpha and IFN-gamma on target cell recognition was impaired in ORAI1-deficient NK cells, as previously described for T cells. By contrast, NK cell cytokine production induced by combinations of IL-12, IL-15, and IL-18 was not impaired by ORAI1 deficiency. Taken together, these results identify a critical role for ORAI1-mediated Ca(2+) influx in granule exocytosis for lymphocyte cytotoxicity as well as for cytokine production induced by target cell recognition