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Jimmunol.1001338.Full.Pdf Published January 14, 2011, doi:10.4049/jimmunol.1001338 The Journal of Immunology An Essential Role of STIM1, Orai1, and S100A8–A9 Proteins for Ca2+ Signaling and FcgR-Mediated Phagosomal Oxidative Activity Natacha Steinckwich,1 Ve´ronique Schenten, Chantal Melchior, Sabrina Bre´chard, and Eric J. Tschirhart 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 Ca2+ concentrations, the mechanisms by which Ca2+ could regulate the different phases of phagocytosis remain unknown. The aim of this study was to investigate the Ca2+ signaling pathways involved in the regulation of FcgRs- 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 Ca2+ by BAPTA or emptying of the in- tracellular Ca2+ store by thapsigargin reduced the efficiency of zymosan internalization. Using an small interfering RNA strategy, our data establish that the observed Ca2+ 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 Ca2+ entry. We dem- onstrate that the observed Ca2+ influx is supported by ORAI calcium release-activated calcium modulator 1 (Orai1) and stromal interaction molecule 1 (STIM1). This result suggests that extracellular Ca2+ entry, which is required for ROS production, is mediated by a store-operated Ca2+ mechanism. Finally, our data identify the complex formed by S100A8 and S100A9 (S100 calcium-binding protein A8 and A9 complex), two Ca2+-binding proteins, as the site of interplay between extracellular Ca2+ entry and intraphagosomal ROS production. Thus, we demonstrate that FcgR-mediated phagocytosis requires intracellular Ca2+ store depletion for the internalization phase. Then phagosomal ROS production requires extracellular Ca2+ entry mediated by Orai1/ STIM1 and relayed by S100A8–A9 as Ca2+ sensor. The Journal of Immunology, 2011, 186: 000–000. hagocytosis is one of the most important innate immune of specific receptors for opsonized particles. Activation of these responses that infected systems use to eliminate invading receptors triggers engulfment of the pathogen and secretion of P pathogenic agents. Phagocytosis is accompanied by the proinflammatory mediators involved in the development of the activation of antimicrobial enzymes, which contribute to the re- adaptive immune response (2). FcgRs constitute an important fam- spiratory burst and allow for the production of reactive oxygen ily of receptors implicated in the recognition of IgG-coated com- species (ROS), which lead to the destruction of ingested micro- plexes. Human neutrophils possess three structurally distinct re- organisms (1). Professional phagocytes, such as polymorphonuclear ceptors for the Fc region of IgGL FcgRI (CD64), FcgRII (CD32), granulocytes, dendritic cells, and macrophages, possess an array and FcgRIII (CD16) (3). Binding of IgG to FcgRs leads to tyrosine phosphorylation of ITAM motifs, within CD32 cytoplasmic tails Life Sciences Research Unit, University of Luxembourg, L-1511 Luxembourg, Lux- or g-chain subunits associated with CD16 and CD64, and subse- embourg quent production of diacylglycerol and inositol 1,4,5-triphosphate 1Current address: Laboratory of Signal Transduction, National Institute of Environ- mental Health Sciences, Department of Health and Human Services, National Insti- (IP3) via phospholipase Cg (PLCg) activation (4, 5). 2+ tutes of Health, Research Triangle Park, NC. IP3, through the activation of its receptor, the Ca channel Received for publication April 23, 2010. Accepted for publication December 10, (inositol 1,4,5-triphosphate receptors [ITPRs]), is a second mes- 2010. 2+ senger. IP3 is known to initiate Ca release from internal stores, This work was supported by a research grant from the University of Luxembourg and which triggers extracellular Ca2+ entry through store-operated a postdoctoral grant from the Fonds National de la Recherche (Luxembourg) (Grant 2+ TR-PDR BFR07-091 to N.S.). Ca channel (SOC) activation. This phenomenon is referred to 2+ Address correspondence and reprint requests to Dr. Eric J. Tschirhart, University of as store-operated Ca entry (SOCE) (6, 7). Alternatively, extracel- 2+ Luxembourg, 162A, Avenue de la Faı¨encerie, L-1511 Luxembourg, Luxembourg. lular Ca entry can also occur through a pathway completely inde- E-mail address: [email protected] 2+ pendent of the initial IP3-mediated Ca store depletion, by a The online version of this article contains supplemental material. non-SOCE mechanism (8). 2+ 2+ Abbreviations used in this article: [Ca ]i, intracellular Ca concentration; DCFH2, Edberg et al. (9) and Kobayashi et al. (10) demonstrated that dichlorodihydrofluorescein; dHL-60, DMSO-differentiated HL-60; DPI, dibenziodo- 2+ lium chloride; lem, emission wavelength; lex, excitation wavelength; F, fluores- phagocytosis is Ca dependent. In addition, several other studies 2+ cence; IP3, inositol 1,4,5-triphosphate; ITPR, inositol 1,4,5-triphosphate receptor; have suggested that an increase of intracellular Ca concentration NOX2, NADPH oxidase 2; Orai1, ORAI calcium release-activated calcium modula- 2+ ([Ca ]i) is required for phagosome maturation or phagosomal tor 1; PLCg, phospholipase Cg; ROS, reactive oxygen species; S100A8–A9, S100 2+ calcium-binding protein A8 and A9 complex; siRNA, small interfering RNA; SOC, ROS production (9, 11, 12). The source of the Ca has not yet store-operated Ca2+ channel; SOCE, store-operated Ca2+ entry; STIM1, stromal in- been formally established. However, in a previous study, SOCE teraction molecule 1; TG, thapsigargin. did not appear to be involved in these phagocytosis mechanisms Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 (13). But that study was essentially based on the use of BTP2, www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001338 2 Orai1, STIM1, Ca2+, OXIDATIVE ACTIVITY, AND PHAGOCYTOSIS a potential pharmacological inhibitor of SOCE. Thus, further in- siRNA specificity for each ITPR or Orai isoforms was tested at the vestigations are required to establish or disprove this conclusion. mRNA expression level. We did not observe any significant suppression of 2+ mRNA for nontargeted Orai and ITPR isoforms (data not shown). The aim of our work was to determine the source of the [Ca ]i variations and the molecular players involved in the Ca2+ de- Real-time PCR pendence of FcgR-mediated internalization and phagosomal oxi- Total RNA was extracted from the siRNA-transfected dHL-60 cells, and dative activity in granulocytes. Because neutrophils are terminally cDNA was prepared using the Thermoscript RT-PCR system (Invitrogen, differentiated and therefore refractory to genetic manipulation, Merelbeke, Belgium). PCR primers were designed from published se- we used DMSO-differentiated HL-60 (dHL-60) cells, which have quences found in GenBank (Table II). PCR products were cloned and granulocyte-like phenotypes, to investigate the molecular mech- sequenced to control their specificity. Real-time PCR was performed on anisms relating Ca2+ and phagocytosis. Supplementary experi- the iQ5 Real-Time PCR Detection System using the iQSYBR Green Supermix following the manufacturer’s protocol (Bio-Rad, Nazareth Eke, ments, based on a pharmacological approach, were performed in Belgium). The annealing temperature was set at 60˚C, and the reaction was human primary neutrophils to verify our results and conclusions. carried out in a 25 ml volume containing a minimum of 10 ng cDNA. 2 We demonstrate that intracellular Ca2+ store depletion, through Relative mRNA expression was quantified using the 2 DDCt method, as the isoforms 1 and 3 of ITPR, is sufficient to induce IgG- previously described (17). opsonized zymosan phagocytosis. In contrast, we show that Western blotting phagosomal ROS production is dependent on SOCE-induced ex- 3 6 3 tracellular Ca2+ entry. We further identify ORAI calcium release- The dHL-60 cells (2 10 ) were then lysed in a 1 Laemmli buffer (30 mM Tris, pH 6.8, 5% v/v glycerol, 1% w/v SDS, 0.01% w/v bromophenol activated calcium modulator 1 (Orai1) and stromal interaction blue, 2% v/v 2-ME). After resolving on 12.5% SDS-PAGE gels or 3–8% molecule 1 (STIM1) as active players in this mechanism. Finally, Tris Acetate Criterion XT precast gels (Bio-Rad), proteins were trans- we delineate the complex formed by S100 calcium-binding pro- ferred onto HybondTM-ECL (Amersham, GE Healthcare, Belgium) or tein A8 and A9 complex (S100A8 and S100A9), two Ca2+-binding Immobilon-FL (Millipore, Brussel, Belgium) membranes, which were 2+ 2+ saturated with a blocking buffer containing 5% w/v BSA. Immunode- proteins, as a Ca sensor linking Ca influx to phagosomal ROS tection was realized by using appropriate primary and HRP-conjugated production. secondary Abs. Labeled bands were quantified by densitometric scan- ning using the Gel-Pro Analyzer (INTAS, Go¨ttingen, Germany). The in- tegrated intensity of the target protein bands was normalized to the Materials and Methods reference protein (b-actin). The Abs used included: rabbit polyclonal anti- Cell culture STIM1 (Alomone Laboratories, Jerusalem, Israel), rabbit polyclonal anti- Orai1 (Alomone Laboratories),
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