Eur. J. Immunol. 2015. 45: 2143–2153

DOI: 10.1002/eji.201445337

Leukocyte signaling

Orai1 controls C5a-induced neutrophil recruitment in inflammation Georgios Sogkas1 , Timo V¨ ogtle2 , Eduard Rau1 , Britta Gewecke1 , David Stegner2 , Reinhold E. Schmidt1 , Bernhard Nieswandt2 and J. Engelbert Gessner1 1 2

Clinical Department of Immunology and Rheumatology, Hannover Medical School, Germany Chair of Experimental Biomedicine, University Hospital and Rudolf Virchow Center, DFG Research Center for Experimental Biomedicine, University of W¨ urzburg, Germany

Stromal interaction molecule 1 (STIM1)-dependent store operated calcium-entry (SOCE) through Orai1-mediated calcium (Ca2+ ) influx is considered a major pathway of Ca2+ signaling, serving T-cell, mast cell, and platelet responses. Here, we show that Orai1 is critical for neutrophil function. Orai1-deficient neutrophils present defects in fMLP and complement C5a-induced Ca2+ influx and migration, although they respond normally to another chemoattractant, CXCL2. Up until now, no specific contribution of Orai1 independent from STIM1 or SOCE has been recognized in immune cells. Here, we observe that Orai1-deficient neutrophils exhibit normal STIM1-dependent SOCE and STIM1-deficient neutrophils respond to fMLP and C5a efficiently. Despite substantial cytokine production, Orai1−/− chimeric mice show impaired neutrophil recruitment in LPS-induced peritonitis. Moreover, Orai1 deficiency results in profoundly defective C5a-triggered neutrophil lung recruitment in hypersensitivity pneumonitis. Comparative evaluation of inflammation in Stim1−/− chimeras reveals a distinct pathogenic contribution of STIM1, including its involvement in IgG-induced C5a production. Our data establish Orai1 as key signal mediator of C5aR activation, contributing to inflammation by a STIM1-independent pathway of Ca2+ -influx in neutrophils.

Keywords: Complement. Calcium. Inflammation. Fcγ receptors. Neutrophil



Additional supporting information may be found in the online version of this article at the publisher’s web-site

Introduction Apart from serving host’s defense, neutrophils are of pathogenic relevance in inflammation of distinct origin and at diverse sites, including rheumatoid arthritis, systemic lupus erythematosus, and chronic obstructive pulmonary disease [1]. The complement anaphylatoxin, C5a is a potent activator of polymorphonuclear leukocytes (PMN) in vitro, leading to enhanced chemo-

Correspondence: Prof. J. Engelbert Gessner e-mail: [email protected]  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

taxis, degranulation, and inflammatory mediator release [2]. C5a is frequently detected at inflammatory sites characterized by PMN invasion. C5aR (CD88) is the predominant receptor for C5a, but C5a also interacts with another receptor, C5L2 [3]. It is well established that C5a and C5aR contribute to the pathogenesis of various PMN-dependent diseases in humans, including myocardial ischemia/reperfusion injury and respiratory distress syndrome [4–6]. Moreover, genetic deletion of C5aR is very effective in preventing inflammation in animal models of type II and type III hypersensitivity, such as hemolytic anemia, rheumatoid arthritis, and the neutrophil-dependent antiphospholipid syndrome [7, 8]. C5a can enhance cellular activation www.eji-journal.eu

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downstream of other immune receptors. In case of immune complex (IC)-triggered immunopathology, C5a–C5aR interaction potentiates FcγR-mediated activation and the downstream inflammatory response [9]. The pathogenic contribution of C5aR has been firmly established in models of sepsis [10, 11], where the cross-talk between C5aR and innate immune sensors, such as TLRs could be relevant [12, 13]. Although C5aR-based activation is critical for recruitment of PMN, the contribution of C5a-triggered calcium (Ca2+ ) signaling in inflammation is unclear. C5aR is a G-protein-coupled receptor (GPCR) that functions mainly via heterotrimeric Gi -proteins [14, 15]. Binding of C5a to C5aR activates Gαi subunits to exchange GTP for GDP, resulting in the dissociation of the Gαi subunit from Gβγ heterodimers. The release of Gαi -associated Gβγ is necessary for triggering C5a-induced directional migration of myeloid effector cells, with a more critical requirement of Gαi2 as compared to Gαi3 in macrophages and neutrophils [16, 17]. Gβγ regulates the activation of downstream effector enzymes, including PI3K and phospholipase C (PLC) isoforms leading to production of diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3) followed by influx of extracellular Ca2+ into the cytosol [15]. Blockade of Ca2+ influx abrogates PMN migration elicited by several chemoattractants [18]. However, the molecules and the mechanism accounting for C5a-activated Ca2+ influx remain unknown. Store-operated calcium entry (SOCE) is considered a main mode of Ca2+ influx in nonexcitable cells [19]. It follows depletion of intracellular stores of Ca2+ and in particular the endoplasmic reticulum (ER) store. Stromal interaction molecule 1 (STIM1) has been identified as the Ca2+ sensor in the ER membrane that upon store release activates SOC channels in the plasma membrane. Orai1 has been shown a major SOC channel function in immune cells including T cells, mast cells, and platelets [19–21]. While SOCE is also suggested to contribute to polarization and motility of PMN [22, 23], our understanding of the requirement for SOCE as well as the specific roles of Orai1 and STIM1 in C5aR activation, PMN migration and inflammation remain uncertain.

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In the present work, we analyzed Orai1−/− mice and found defective C5aR-mediated Ca2+ entry and chemotaxis in neutrophils. As a consequence, Orai1−/− mice are partially protected from neutrophilia induced by C5a, LPS, and pathogenic IgG IC in lung and peritoneum. Our study also shows that the absence of Orai1 does not affect SOCE in murine PMN. In contrast, STIM1 appears critical for SOCE but not C5a-induced Ca2+ mobilization and Stim1−/− mice showed enhanced PMN peritoneal recruitment in response to LPS. In the acute model of IC lung injury (hypersensitivity pneumonitis), however, the lack of STIM1 resulted in impaired PMN recruitment through reduced C5a. Since Orai1−/− mice show normal C5a, our data suggest that distinct SOCE and nonSOCE-mediated functions of STIM1 and Orai1 differently control IgG-induced C5a and C5aRmediated PMN recruitment in the context of immune complex inflammation.

Results Dependence of PMN C5a-elicited calcium response and chemotaxis on Ca2+ influx Considering that Ca2+ responses downstream of several chemoattractants do not necessarily correlate with the requirement of Ca2+ for serving efficient cell chemotaxis [24], we tested whether complement C5a can elicit a Ca2+ response in PMN and evaluated its dependence on Ca2+ influx. In the first set of experiments, PMN were loaded with Fura2/AM and stimulated with C5a, triggering a sizeable elevation of cytosolic levels of Ca2+ (Fig. 1A). Complete blockade of this response with EGTA reveals it stemming from the mobilization of Ca2+ from the extracellular compartment (Fig. 1B). Next, PMN chemotactic potential was tested using Transwell migration assays and flow cytometry, after cell exposure to C5a in the presence or absence of EGTA. PMN were unable to migrate in response to C5a in EGTA-supplemented media (Fig. 1C), demonstrating the functional relevance of Figure 1. Ca2+ influx after stimulation of PMN cells with C5a is required for efficient chemotaxis. (A, B) PMN cells from C57Bl/6 mice were loaded with Fura2/AM and stimulated or not (w/o C5a) with either C5a (50 ng/ml) in CaCl2 (1 mM) containing buffer (C5a) or in buffer supplemented with EGTA (0.5 mM) (EGTA + C5a). (A) One representative measurement of n = 4 spectrofluorometric measurements are presented. (B) In the same setting, maximal (࢞[Ca2+ ]i ± SEM) values were calculated of n = 4 spectrofluorometric measurements per group; *P < 0.05, determined by 2-tailed Student´s t test. (C) PMN cells from C57Bl/6 mice were incubated with a Ca2+ -specific chelator, EGTA and assayed for efficient C5a (50 ng/ml)elicited chemotaxis in Transwell migration assays. Results are expressed as mean ± SEM of n = 4 independent experiments, each performed in duplicate; **P < 0.01, determined by 2-tailed Student´s t test.

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Leukocyte signaling

Figure 2. Differential involvement of Orai1 and STIM1 in C5a-induced Ca2+ influx and SOCE in PMN. (A, C) Orai1−/− or Stim1−/− PMN cells loaded with Fura2/AM and stimulated with C5a (50 ng/ml) in CaCl2 (1 mM) containing buffer. One representative spectrofluorometric measurement of n = 4 measurements and maximal (࢞[Ca2+ ]i ± SEM) values are presented (*P < 0.05, determined by 2-tailed Student´s t test). (B, D) Orai1−/− or Stim1−/− PMN cells loaded with Fura2/AM and treated with Tg (2 μM) in EGTA-containing buffer (w/o [Ca2+ ]e ) followed by the addition of CaCl2 (1 mM [Ca2+ ]e ) and monitoring of [Ca2+ ]i . Representative spectrofluorometric measurements and maximal (࢞[Ca2+ ]i ± SEM) values (n = 4 per group, in the presence of 1 mM CaCl2 in measuring buffer are shown) (**P < 0.01, determined by 2-tailed Student´s t test).

extracellular Ca2+ and, as a consequence, of Ca2+ -influx for C5aelicited chemotaxis of PMN.

Orai1 controls C5a-induced Ca2+ influx in PMNs and their migration is independent from STIM1 and SOCE To address the function of Orai1 in Ca2+ -influx and neutrophil migration, we analyzed Orai1−/− mice in comparison with Stim1−/− mice. Due to the early lethality of all these mice, studies were performed with irradiated wild-type mice that received a transplant of Orai1−/− or Stim1−/− or control bone marrow cells. Quantitative real-time PCR analysis confirmed the almost complete absence of Orai1 and STIM1 mRNA in PMN from Orai1−/− and Stim1−/− chimeras, respectively (Supporting Information Fig. 1). Furthermore, the levels of STIM2, Orai2, and Orai3 were unaltered in Orai1−/− and Stim1−/− chimeras. We then tested the role of Orai1 in agonist-induced Ca2+ influx. On stimulation of Orai1−/− PMN with C5a, a substantially reduced Ca2+ response was detected (Fig. 2A). A similar defect was also observed for fMLP-induced Ca2+ influx (Supporting Information Fig. 2). To evaluate the role of Orai1 in SOCE, cells were treated with the sarcoplasmatic / endoplasmatic reticulum Ca2+ ATPase pump inhibitor thapsigargin (Tg). Interestingly, TG-dependent SOC influx was not reduced in Orai1−/− PMN compared with wildtype controls (Fig. 2B). This result is different to that observed with Orai1−/− macrophages where TG-dependent SOCE was strongly reduced (Supporting Information Fig. 3), indicating that Orai1 is a major SOC channel in macrophages but not in neutrophils. The role of STIM1 in neutrophil SOCE and C5a/fMLP-induced changes of [Ca2+ ]i was evaluated under the same experimental conditions. In contrast to Orai1−/− PMN, Stim1−/− PMN exhibited a robust and intact Ca2+ response to C5a (Fig. 2C) as well as fMLP (Supporting Information Fig. 2), whereas TG-induced SOCE was nearly abrogated (Fig. 2D). In summary, these data show that Orai1 - in contrast to its role in macrophage SOCE - is not essentially required for proper SOCE function in PMN, despite the fact that it regulates fMLP and C5a-induced Ca2+ influx in these cells. Since STIM1-deficient neutrophils normally respond to  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

C5a and fMLP, the results further suggest that C5a-induced Ca2+ influx through Orai1 is mainly driven by a nonstore-operated Ca2+ entry (nonSOCE) pathway. The participation of both SOCE and nonSOCE was recently suggested for fMLP-induced activation of human neutrophils [23]. The functional relevance of these findings was then evaluated in the context of Transwell migration assays. In line with their role in C5a-induced Ca2+ influx, Stim1−/− PMN migrated efficiently in response to C5a, whereas Orai1−/− PMN exhibited substantially decreased chemotaxis (Fig. 3A). However, these results again differ from those obtained with Orai1−/− macrophages where C5a (as well as CCL2)-induced chemotactic migration was not affected (Supporting Information Fig. 4A), further indicating a cell typespecific role for Orai1 in C5a-induced cell migration. To test the impact of Orai1-deficiency on the migration behavior of myeloid cells in vivo, we examined neutrophil accumulation into the peritoneum of Orai1−/− chimeric mice 3 hours after i.p. exposure to C5a. In accordance with the in vitro-Transwell migration assay data, Orai1−/− chimeric mice showed reduced neutrophil elicitation in response to C5a (Fig. 3B). It is important to note that this reduced migration of Orai1−/− PMN occurred despite similar C5aR plasma membrane expression levels in both the presence or absence of Orai1 (Fig. 3C). We also observed normal recruitment of Orai1−/− macrophages in thioglycollate (Thg)-induced peritonitis (Supporting Information Fig. 4B).

Distinct requirement of Orai1 for C5a and CXCL2-induced PMN chemotaxis Having shown that Orai1-mediated Ca2+ influx is of relevance in the context of C5a-induced PMN migration in vivo, we next examined the role of Orai1 for C5aR activation under more complex inflammatory conditions. We employed LPS peritonitis and IC hypersensitivity pneumonitis, two classical models of neutrophilic inflammation previously shown to depend on several PMN recruitment factors, such as CXCL2 and C5a [10, 12, 25]. Biological fluids collected from lung and peritoneum of C56BL/6 mice at 2 to 4 hours after i.n. anti-OVA IgG / i.v. OVA antigen treatment www.eji-journal.eu

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Figure 3. Orai1 is required for efficient C5aelicited chemotaxis of PMN in vitro and peritoneal recruitment in vivo. (A) PMN cells from Stim1−/− or Orai1−/− and matched wildtype (Stim1+/+ and Orai1+/+ ) BM chimeras were assayed for efficient C5a (50 ng/ml)-elicited chemotaxis in Transwell migration assays. Results are expressed as mean ± SEM of n = 4 independent experiments, each performed in duplicate (**P < 0.01, determined by 2-tailed Student´s t test). (B) Orai1−/− or Orai1+/+ BM chimeras were injected with 200μl of C5a (1 μg/ml) or 200 μl of PBS. Two hours later PMN cells infiltrating the peritoneal cavity were counted. Results are expressed as mean ± SEM of n = 4-5 mice per group (*P < 0.05, determined by 2-tailed Student´s t test). Groups of 2– 3 Orai1−/− BM chimeras and two matched wildtype mice were assayed together. (C) Orai1−/− or Orai1+/+ Gr1-positive PMN were characterized for C5aR expression by flow cytometry. Representative histogram (left) and mean fluorescence intensity (MFI) ± SEM of n = 3 measurements (right) are depicted. P: ns = non significant, determined by 2-tailed Student´s t test.

(4h IC-BALF) and i.p. LPS challenge (2h LPS-PLF) contained high levels of bioactive C5a and CXCL2 (Fig. 4A and B). Sensing of the chemotactic potential within LPS-PLF and IC-BALF by PMN partially depends on Ca2+ influx as documented by decreased PMN chemotaxis in the presence of EGTA (Fig. 4C). The EGTA sensitivity of the PLF and BALF fluids, however, appeared lower as compared to C5a alone (see Fig. 1). In these fluids, additionally induced CXCL2 may explain to some extent the difference. We found that PMN chemotaxis induced by CXCL2 was insensitive to EGTA (Fig. 4D) and did not require the presence of Orai1 (Fig. 4E). Moreover, Orai1−/− chimeric mice showed a robust CXCL2-induced PMN recruitment in vivo (Fig. 4F), indicating that Orai1 contributes to C5a- but not CXCL2-induced migratory function of PMN into tissue. Given the apparent differential requirements of Orai1 for C5aR- and CXCR2-mediated PMN migration, we then examined the efficiency of Orai1 in sensing chemotactic activity of LPS and IgG IC primed fluids that both co-contain C5a and CXCL2. Orai1−/− PMN exhibited markedly reduced migration in response to LPS-PLF and IC-BALF (Fig. 5A and B). In contrast, when the function of Stim1−/− PMN was evaluated the same way, cells did show increased migration (Fig. 5C and D). Overall, the data suggest a critical function of Orai1 in controlling PMN chemotaxis induced by C5a and inflammatory fluids and may indicate some negative role of STIM1 for PMN migration that could be of relevance in acute tissue injury.

Orai1 and STIM1 differently control PMN invasion in LPS-induced inflammation We then evaluated the in vivo consequences of Orai1 or STIM1 deficiency in LPS-induced inflammation. Orai1−/− chimeric mice were injected with 10 mg/kg LPS and 2 h later PLF was collected and peritoneal recruitment of PMN was determined (Fig. 6). Despite similar levels of PMN chemoattractant potential  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

in Orai1−/− and Orai1+/+ WT chimeras, including LPS-activated bioactive C5a (Fig. 6A) and CXCL2 production (Fig. 6B), decreased numbers of PMN were obtained from the peritoneal cavity of Orai1−/− mice as compared to control animals (Fig. 6C). Apart from a normal LPS-induced chemotactic activity of PLF, similar levels of proinflammatory cytokines were detected in the serum of LPS-injected mice (Orai1+/+ : TNF-α: 9.16 ±1.29 ng/ml, IL-1β: 365±42.92 pg/ml, IL-6: 80.67±5.34 ng/ml, n = 4 and Orai1−/− : TNF-α: 10.1 ±1.45 ng/ml, IL-1β: 329±43.81 pg/ml, IL6: 70.63±9.48 ng/ml, n = 4, p>0.42), suggesting that decreased PMN invasion in Orai1 deficiency is unlikely to be caused by impaired or reduced sensing of LPS. To substantiate the in vitro finding of enhanced migration of Stim1−/− PMN, we also induced LPS inflammation in Stim1−/− chimeras. Like in Orai1 deficiency, normal production of C5a and CXCL2 was detected in inflamed peritoneum (Fig. 6D and E). In contrast, Stim1−/− mice display substantially higher numbers of PMN as compared with control chimeras (Fig. 6F). Together, these data show that Orai1 in PMN is necessary for neutrophil migration in LPS inflammation, whereas STIM1 is not only dispensable in this process, but may play a negative regulatory role.

Orai1 and STIM1 act at distinct levels of the inflammatory cascade in hypersensitivity pneumonitis The contribution of Orai1 and STIM1 may differ between inflammatory triggers and cells in tissue injury. As previously suggested for several IgG pathologies, the C5aR is a positive regulator of FcγR function in macrophages, where STIM1 controls IgG-induced C5a synthesis and phagocytosis [9, 21]. Therefore, we further assessed the function of Orai1 and STIM1 in IC-induced hypersensitivity pneumonitis. OVA-specific IgG was applied i.n., which was followed by an i.v. injection of OVA antigen. The acute IC reaction entails the activation of alveolar macrophages and is characterized www.eji-journal.eu

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Figure 4. The requirement of extracellular Ca2+ and Orai1 for efficient PMN chemotaxis activated by LPS and IgG IC primed biological fluids is not related to CXCL2. (A) C57Bl/6 mice were injected with (LPS 10 mg/ kg) i.p. Two hours later mice were sacrificed and PLF samples were evaluated. Measurement of CXCL2 content of PLF by ELISA (left) and PLF bioactive C5a (right). Results are expressed as mean ± SEM of n = 4 independent experiments, each performed in duplicate (***P < 0.001, determined by 2-tailed Student´s t test). (B) C57Bl/6 mice were exposed to IgG IC hypersensitivity alveolitis. Four hours later mice were sacrificed and BALF samples were evaluated. Measurement of CXCL2 content of BALF by ELISA (left) and BALF bioactive C5a (right). Results are expressed as mean ± SEM of n = 4 experiments (***P < 0.001, determined by 2-tailed Student´s t test) (C) PMN cells from C57Bl/6 mice were incubated with EGTA and assayed for efficient PLF- (left) or BALF-elicited (right) chemotaxis in Transwell migration assays. Results are expressed as mean ± SEM of n = 4 independent experiments, each performed in duplicate (*P < 0.05; **P < 0.01, determined by 2tailed Student´s t test). (D) PMN cells from C57Bl/6 mice were incubated with a Ca2+ specific chelator, EGTA and assayed for efficient CXCL2 (25 ng/ml)-elicited chemotaxis in Transwell migration assays. Results are expressed as mean ± SEM of n = 4 independent experiments, each performed in duplicate. (E) PMN cells from Orai1−/− and matched Orai1+/+ BM chimeras were assayed for efficient CXCL2 (25 ng/ml)-elicited chemotaxis in Transwell migration assays. Results are expressed as mean ± SEM of n = 4 independent experiments, each performed in duplicate (ns = nonsignificant, determined by 2-tailed Student´s t test). (F) Orai1−/− or Orai1+/+ BM chimeras were injected with 200μl of CXCL2 (1 μg/ml) or 200 μl of PBS. 2 hours later PMN cells infiltrating the peritoneal cavity were counted. Results are expressed as mean ± SEM of n = 4 mice per group (ns = nonsignificant, determined by 2-tailed Student´s t test).

by PMN influx in lung tissue as well as in the bronchoalveolar compartment [26]. Mice were sacrificed 4 h post i.v. injection of OVA antigen and BALF was analyzed for alveolar PMN recruitment. Moreover, lung interstitial PMN accumulation was evaluated by analyzing myeloperoxidase (MPO) activity. Analysis of Orai1 sufficient and Orai1 deficient mice-derived BALF, each revealed the mounting of a robust chemoattractant potential, characterized by bioactive C5a, CXCL2 production, and TNF-α (Fig. 7A–C). However, IC challenged Orai1−/− chimeric mice exhibited only minor inflammation as exemplified by the maintenance of the alveolar architecture of their lungs and reduced PMN invasion in interstitium and alveoli (Fig. 7D–F), suggesting intrinsically defective migration of Orai1−/− PMN. On the other hand, similarly treated Stim1−/− chimeric mice exhibited an impaired chemoattractant potential in the BALF with markedly decreased levels of IC-induced C5a bioactivity and reduced CXCL2 production (Fig. 7G and H). TNF-α production appeared normal (Fig. 7I). Despite the fact that WT IC-BALF can trigger enhanced migration of STIM1 defi C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

cient PMN in vitro (see Fig. 5D), IC-challenged Stim1−/− chimeric mice showed minor inflammation with reduced PMN numbers in lung tissue and a strongly lowered PMN influx into alveoli (Fig. 7J–L). Similar to previous findings [21], the data confirm the crucial role for STIM1 in the production of chemoattractants (like C5a) by resident cells of the lung to induce PMN recruitment in IC inflammation.

Discussion In the current work, we studied the dependence of PMN activation and migration on Ca2+ influx and the biological relevance of Orai1 in acute inflammation involving C5aR activation. Leukocyte chemotaxis was long ago known to depend on Ca2+ signaling [27, 28]. In particular, for PMN and for several chemoattractants this has been demonstrated to involve Ca2+ influx [18, 24, 28, 29]. As recently reported, TRPC6 channels are essential components www.eji-journal.eu

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Figure 5. Distinct requirements of Orai1 and STIM1 for PMN chemotaxis stimulated by LPS and IgG IC primed biological fluids. (A, B) PMN cells from Orai1−/− or matched Orai1+/+ BM chimeras were exposed to either PLF or BALF derived from C57Bl/6 mice subjected to (A) LPS (10mg/ kg)-induced peritonitis and (B) hypersensitivity pneumonitis, respectively, and assayed for chemotaxis in Transwell migration assays. (C, D) PMN cells from Stim1−/− or matched Stim1+/+ BM chimeras were similarly assayed with C57Bl/6-derived biological fluids from (C) LPS (10mg/ kg)-induced peritonitis and (D) hypersensitivity pneumonitis. Results are expressed as mean ± SEM of n = 5 independent experiments, each performed in duplicate (***P < 0.001, determined by 2-tailed Student´s t test).

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of Ca2+ influx and neutrophil chemotaxis triggered by CXCR2 activation [30]. However, TRPC6 channels are not activated by fMLP, suggesting that distinct Ca2+ influx mechanisms are operative downstream of different GPCR. Both fMLP and C5a are classified as end-target chemoattractants. They differ from intermediary chemoattractants, such as CXCL2, in their MAPK and PI3K signaling requirements [31, 32]. Recently, we showed functional coupling of C5aR and CXCR2 to Gi -proteins, Gαi2 , and Gαi3 , in PMN chemotaxis elicited by C5a and CXCL2 [16]. The present results demonstrate that C5aR and CXCR2 activate distinct Ca2+ influx mechanisms in neutrophils. Importantly, C5a (but not CXCL2)induced chemotaxis and peritoneal neutrophilia were specifically dependent on the presence of Orai1 in PMN. STIM isoforms and Orai1 have been revealed to be the major components of the store-operated mechanism of Ca2+ influx, known as SOCE [19, 33]. This applies for several immune cells, including T cells and mast cells [34, 35], where STIM1 and Orai1 activate SOCE by sensing of the ER content of Ca2+ and mediating SOC channel function. It was thus tempting to speculate that STIM1 and Orai1 would play a similar role in PMN, serving SOCE-dependent functions, such as cell migration in response to C5a and fMLP. Here, indeed, STIM1 operates as dominant mediator of SOCE in myeloid cells (Fig. 2D and [21]), whereas Orai1 appears required for SOC channel function in macrophages but not neutrophils. PMN polarization was recently shown to involve high-affinity integrin activation and Orai1 calcium dynamics [22]. Here, we demonstrate that Orai1 regulates both C5a and fMLPinduced Ca2+ entry and PMN chemotaxis through a nonSOCE pathway. PMN from STIM2-deficient mice also showed a normal C5a and fMLP-induced chemotaxis (data not shown). In neutrophils, DAG and some of its metabolites have been shown to induce nonSOCE through TRP channel proteins independent of initial IP3-mediated depletion of ER calcium stores [36, 37]. TRPC6 has been linked to CXCR2 but not fMLP-induced chemotaxis in these cells [32]. Previously, we have shown the

Figure 6. Normal C5a and CXCL2 production, but reduced versus enhanced LPS-induced peritoneal neutrophil accumulation in Orai1−/− and Stim1−/− BM chimeras. Orai1−/− (A-C) or Stim1−/− (D-E) and matched wild-type chimeras were injected with LPS (10 mg/ kg) i.p. 2 hours later, mice were sacrificed and PLF samples were evaluated. (A, D) Functional detection of bioactive C5a in PLF from LPS-challenged mice. Chemotactic activity was determined with Transwell migration assays of neutrophils (PMNs isolated from C5aR+/+ and C5aR–/– mice). (B, E) Measurement of CXCL2 content of PLF by ELISA. (C, F) Evaluation of peritoneal PMN infiltration by measurement of PMN in PLF. Results are expressed as mean ± SEM of n = 5 mice per group (*P < 0.05; **P < 0.01, determined by 2-tailed Student´s t test). Groups of 2–3 Orai1−/− or Stim1−/− BM chimeras and 2–3 matched wild-type mice were assayed together.  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Figure 7. Orai1 and STIM1 act at distinct levels of the inflammatory cascade in IgG IC-induced hypersensitivity pneumonitis. Orai1−/− (A-F) or Stim1−/− (G-L) and matched wild-type chimeras were either exposed to IgG immune complex lung injury. 4 hours later, mice were sacrificed and lung tissue injury and BAL fluid samples were evaluated. (A, G) Functional detection of bioactive C5a in BALF from IC-challenged BM chimeras. Chemotactic activity was determined with Transwell migration assays of neutrophils (PMNs isolated from BM of C57BL/6 and C5aR–/– mice). (B, H) Measurement of CXCL2 content of BALF by ELISA. (C, I) Measurement of TNF-α content of BALF by ELISA. (D, J) Lung H&E section (original magnification 40x). (E, K) Evaluation of lung interstitial PMN infiltration by measurement of MPO activity, normalized to the weight of the lavaged lung. (F, L) Evaluation of PMN accumulation in BALF. Results are expressed as mean ± SEM of n = 5 mice per group (*P < 0.05; **P < 0.01;***P < 0.001, determined by 2-tailed Student´s t test). Groups of 2–3 Orai1−/− or Stim1−/− BM chimeras and 2–3 matched wild-type mice were assayed together.

crosstalk of Orai1 with TRPC6 through Orai1-induced DAG production via phospholipase C and D in platelets [38]. In neutrophils, however, Orai1 (see Fig. 4 D–F) and STIM1 [21] appear not to be involved in CXCR2-induced intermediary chemotaxis regulated by TRPC6. In contrast, a specific role of Orai1 is evident in fMLP and C5a-induced signaling and neutrophil recruitment, independently of STIM-mediated SOCE. Since TRPC6 is not a likely candidate of end-target chemoattractants such as fMLP [30], Orai1 could cooperate with channels of the TRP family other than TRPC6 (i.e. TRPC1, 3, and 4) [39]. Also, C5a-induced Ca2+ entry could cause the generation of specific second messengers, capable of activating Orai1 without affecting the integrity of intracellular stores, proving an example of second messenger-operated Ca2+ influx [40]. Oncogenic activity in breast cancer is linked to constitutive STIM-independent Ca2+ signaling through Orai1 via altered secretory pathway Ca2+ ATPase, SPCA2, in mammary epithelium [41].

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Future studies on the expression and function of SPCA and TRPCs in neutrophils will lead to further insights into the mechanisms that determine the neutrophil-specific and STIM1-independent effects of Orai1 in C5aR activation. Orai1 regulates adhesion in tumor cells where they promote tumor cell migration, suggesting a role for Orai1 in integrinmediated signaling that guide PMN migration in response to inflammation. Shear force acting on high-affinity LFA-1 in the presence of fMLP can trigger PMN adhesion and Ca2+ influx to promote migration. Orai1-defective PMNs are unable to induce LFA-1-mediated outside-in Ca2+ flux, indicating a possible role for SOCE in integrin-dependent PMN arrest [22]. However, a contribution of STIM1 was not specified by these studies. Here, we established distinct roles of STIM1 and Orai1 for PMN migration in acute inflammation. LPS-induced peritonitis exemplifies a model of inflammation, where complement cascade and C5aR pathogenic

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involvement have been demonstrated [12]. Our results with biological fluids from LPS-challenged mice show highly induced peritoneal levels of bioactive C5a that, however, are not influenced by the absence of either Orai1 or STIM1 in PMN. Stim1−/− PMN show normal, not increased, C5a-induced chemotaxis and a mildly more responsive LPS-PLF-stimulated migratory phenotype. Of note, PMN lacking STIM1 also show an increased capacity to migrate in response to inflammatory fluids from IgG IC-challenged mice. The data suggest that STIM1, while dispensable for C5a-mediated PMN migration, may mediate an inhibitory function in neutrophils during inflammation. In accordance, Stim1−/− chimeric mice display enhanced PMN invasion at the site of LPS challenge. In contrast, Orai1−/− animals exhibit defective neutrophilia in this model. These observations, together with the findings that Orai1 is required for C5aR activation and is critical for inflammatory fluid-mediated PMN chemotaxis in vitro, suggest that reduced recruitment of Orai1−/− PMN into tissue likely stems from the intrinsic chemotactic defect of these cells to respond to C5a, which is present at the site of inflammation. Hypersensitivity pneumonitis (or passive reverse Arthus reaction of the lung) exemplifies IgG IC-triggered acute inflammation, dually affected by C5aR and FcγR [reviewed in 42]. Several lines of evidence also reveal the pathogenic role of C5aR in other animal models of type II and III hypersensitivity [43–47]. The balance between opposing signaling of FcγR sets the threshold of cellular activation by IgG antibodies. In macrophages, C5a production and C5aR-mediated stimulation interferes with FcγR expression, altering the ratio activating to inhibitory FcγR expression [9, 45, 48]. This appears crucial for local production of C5a and the mounting of a robust PMN chemotactic activity by FcγR through a mechanism that involves LAT adaptor, PI3 kinase δ, and STIM1 signaling molecules [21, 47, 49]. Comparing here the role of STIM1 and Orai1 upstream and downstream of PMN activation, our data confirm the importance of STIM1 for productive formation of an IgG IC-induced C5a and chemokine milieu, whereas Orai1 is specifically required for efficient neutrophil C5aR activation. In both cases of STIM1 and Orai1 deficiency, impaired PMN influx into the bronchoalveolar compartment was observed. These results are consistent with a linked cascade of events in which STIM1 triggers FcγR-mediated C5a release that in turn activates C5aR in PMN through Orai1-mediated Ca2+ signals for their IC-induced migration into inflamed lung. Moreover, the use of distinct models of inflammation suggested a dual role of STIM1. STIM1-negative PMNs show enhanced potentials to migrate towards soluble factors derived from LPS- and IgG IC-challenged mice. As a consequence, a stronger PMN invasion is seen in LPS injury in Stim1−/− chimeric mice, whereas this is masked and inversed in lung pathology because of the requirement of STIM1 for FcγR-mediated C5a that together with other chemokines mostly account for PMN attraction in IC inflammation. Overall, in the present study, we have provided a first example of immune cell, where SOC channel function and thus, SOCE are not strictly dependent on Orai1 expression. Despite that, in PMN, C5a-elicited Ca2+ influx and C5a-triggered chemotaxis, both, depend on Orai1. The critical role of Orai1 channel in medi C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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ating C5a- (but not CXCL2-) induced responses is of biological relevance as demonstrated by defective PMN recruitment in case of LPS- or IgG IC-triggered inflammation in the absence of Orai1. The fact that Orai1 and STIM1 are able to function both dependently [19] and independently (this study) from each other in different cell types suggests that these two Ca2+ regulators mediate discrete receptor- and cell-specific functions, extending the current model of STIM1 and Orai1 in SOCE and underscores the importance of Orai1 for SOCE-independent functions, such as C5aR activation in neutrophils (a summarized model of the roles of STIM1 and Orai1 in IC inflammation and of SOCE versus nonSOCE in PMN activation are presented in Fig. 8). Since C5aR is considered a potential target in therapy of inflammatory diseases in humans, the understanding of both the complex signaling mechanisms of C5aR and the pathways that contribute to C5a production is likely to lead to novel therapeutics.

Materials and methods Mice The generation of Orai1−/− and Stim1−/− mice has been described previously [21, 50]. For the generation of bone marrow (BM) chimeras, 5- to 6-week-old C57Bl/6 female mice were lethally irradiated with a single dose of 10 Gy and transplanted with Stim1−/− , Orai1−/− or wild type mice-derived BM (4 × 106 cells/animal). Recipient mice received acidified water containing 2 g/liter neomycin sulfate for 2 weeks after transplantation. PCRbased genotyping of Orai1−/− mice-derived neutrophils was performed using published primers [50]. To verify efficient chimerism of Stim1−/− chimeras, STIM1 immunoblot analysis was performed in the BM of Stim1−/− transplanted or wild-type transplanted animals as described previously [21]. Western blot detection of Orai1 was not possible, as no antibodies are available that recognize murine Orai1. The expression status of Orai1, Orai2, Orai3, Stim1, and Stim2 mRNA was assessed in sorted PMN from Orai1−/− and Stim1−/− chimeras. Primer sequences used are described in Supporting Information Fig. 1. All mice were assayed at 12- to 14-week of age, 6- to 8-weeks post transplantation. C57BL/6 (purchased from Charles River) and C5aR−/− mice were assayed at 10- to 14-week of age. Animal studies were conducted in accordance with current laws and approved by the local animal care and use committees.

Transwell migration assay of neutrophils BM from the indicated mice was resuspended at 0.75×106 cells in 100 μl RPMI 1640 medium, 0.5% fatty acid free BSA, placed into the insert of a 3-μm pore diameter Transwell chemotaxis chamber (Greiner, Frickenhausen, Germany) and incubated for 2 hours with RPMI/0.5% BSA supplemented with either single chemoattractants (C5a or CXCL2) or complex biological fluids www.eji-journal.eu

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Figure 8. A summarized model of the distinct roles of STIM1 and Orai1 in IC inflammation and of SOCE vs. nonSOCE in the activation of neutrophils. (A), 1: Initial contact between IC and resident effector cells induces C5a and CXCL2 production that is STIM1-dependent [21]; 2: PMN migration induced by C5a and CXCL2 involves the activation of distinct calcium channels, Orai1 and TRPC6 [30]; 3: TNF-α-mediated regulation of endothelial ICAM-1 promotes PMN adhesion through LFA-1 and Orai1 calcium dynamics [22]; all these steps contribute to the recruitment of PMN to sites of inflammation, but CXCL2 appears not sufficient to overcome C5a dysfunction in the absence of Orai1 (indicated by the dashed arrow). (B) Binding of C5a to the Gαi -coupled C5aR activates Orai1 at the plasma membrane independent from STIM1-mediated SOCE, implicating the existence of a nonSOCE pathway for increased Ca2+ in the regulation of C5aR-mediated PMN migration. The minor role of (IP3 -IP3 R) Ca2+ store depletion and SOCE pathway for C5a-induced neutrophil chemotaxis is indicated by the dashed arrows.

from LPS and IgG IC-challenged mice in the presence or absence of EGTA. The neutrophils that transmigrated into the lower chamber were vigorously suspended and counted with a FACSCalibur for 1 minute at 240 events/sec. Migration of PMN from the insert to the bottom well was quantitated as described [9]. PMN were determined by their typical appearance in the forward/sideward scatter and Gr-1 expression. C5aR on Gr1-positive PMN was analyzed with Alexa 647-conjugated anti-C5aR mAb [51] by flow cytometry, using a FACScalibur flow cytometer (Becton Dickinson, Heidelberg, Germany).

SOCE and chemokine-induced Ca2+ response recording Gr1 positive PMN (106 /ml) were sorted out of BM suspension, prepared from the each time indicated mice with an XDP, BeckmanCoulter cell sorter (Fullerton, California) at low voltage. Sorted cells were washed twice in RPMI 1640 medium, resuspended at 107 /ml in HBSS buffer (120 mM NaCl, 5 mM KCl, 1 mM MgCl2 , 20 mM Hepes pH 7.4, 1 mM CaCl2 , 10 mM Glucose, 0,5% BSA) and loaded with Fura-2/AM (5 μM; Calbiochem, Darmstadt, Germany) in the presence of pluronic F-127 (0,2 μg/ml; SigmaAldrich, Munich, Germany) for 30 min at 37ο C. After labeling, cells were washed twice and treated with 2 μM Thapsigargin (Tg; Invitrogen, Darmstadt, Germany) or stimulated with recombinant mouse C5a (50 ng/ml) (R&D Systems, Wiesbaden, Germany) in buffer supplemented or not with 0.5 mM EGTA as indicated. Fluorescence was measured with an LS 50 spectrofluorimeter (PerkinElmer, Waltham, USA). Excitation was alternated at 340 and 380 nm, and emission was measured at 509 nm. Each  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

measurement was calibrated with Triton X-100 and EGTA and intracellular concentration of Ca2+ was calculated.

Agonist-induced peritonitis PMN recruitment was induced by i.p. injection of 200 ng of either recombinant mouse C5a or CXCL2 (R&D Systems, Wiesbaden, Germany). Three hours post injection, mice were sacrificed and the peritoneal cavity was rinsed two times with 1 ml ice cold PBS / 2 mM EDTA. Total cell count of the collected peritoneal lavage fluid (PLF) was assessed with a hemocytometer (Neubauer Z¨ ahlkammer, Gehrden, Germany). For quantification of neutrophil influx, differential cell counts were performed on cytospins (10 min at 55 x g) stained with May-Gr¨ unwald-Giemsa as described [16].

LPS-induced peritonitis Mice were injected i.p. with a dose of 10 mg/kg LPS / 5% FBS (Sigma-Aldrich, Munich, Germany). Two hours post injection, mice were sacrificed and PLF was prepared by a single wash of the peritoneal cavity with 5 ml PBS at 4ο C. Peritoneal PMN were quantified as described [16]. The concentration of CXCL2 in PLF was quantified by ELISA. The presence of bioactive C5a in PLF was determined by Transwell migration assays, using C5aR−/− and C5aR+/+ PMN [16, 21]. Blood was collected from the retroorbital plexus. Serum was prepared after blood was left at RT for 45 min and spun at 3000 rpm for 10 min. Serum cytokine levels were assayed by TNF-α, IL-6- ,and IL-1β-specific ELISA kits. www.eji-journal.eu

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2 Guo, R. F. and Ward, P. A., Role of C5a in inflammatory responses. Annu.

IC-triggered hypersensitivity pneumonitis was performed as described [21]. Briefly, ketamine and xylazine anesthetized mice received an intranasal (i.n.) dose of 150 μg of protein G purified anti-ovalbumin (OVA), which was immediately followed by an intravenous (i.v.) injection of 20 mg/kg OVA antigen. 4 hours later mice were sacrificed. For bronchoalveolar lavage fluid (BALF) preparation, mouse trachea was cannulated and the bronchoalveolar tree was washed three times, each with 1 mL PBS at 4ο C. For quantitation of alveolar PMN accumulation, differential cell counts were performed on cytospins (10 min at 55g) stained with May-Gr¨ unwald-Giemsa using 300 μl BALF. The concentrations of CXCL2 and TNF-α were assayed with ELISA kits. Overall chemoatractant potential and bioactive C5a in BALF were determined by Transwell chemotaxis assays. The difference in the percentage migration of C5aR+/+ and C5aR−/− PMN was quantitated [16, 21]. ELISA detection of C5a in BALF was not possible, because of the low sensitivity of the assay (detection limit >10 ng/ml). Myeloperoxidase (MPO) activity of lavaged lung tissue was assayed as described [9]. To process lung tissues for histological examination after lavage, they were fixated in 4% buffered paraformaldehyde, embedded in paraffin, and stained with hematoxylin and eosin according to conventional procedures.

3 Klos, A., Wende, E., Wareham, K. J. and Monk, P.N., International Union

Rev. Immunol. 2005. 23: 821–852.

of Pharmacology. LXXXVII. Complement peptide C5a, C4a, and C3a receptors. Pharmacol. Rev. 2013. 65: 500–543. 4 Peng, Q., Li, K., Smyth, L. A., Xing, G., Wang, N., Meader, L., Lu, B. et al., C3a and C5a promote renal ischemia-reperfusion injury. J. Am. Soc. Nephrol. 2012. 23: 1474–1485. 5 Riedemann, N. C., Neff, T. A., Guo, R. F., Bernacki, K. D., Laudes, I. J., Sarma, J. V., Lambris, J. D. and Ward, P. A., Protective effects of IL-6 blockade in sepsis are linked to reduced C5a receptor expression. J. Immunol. 2003. 170: 503–507. 6 Huber-Lang, M., Younkin, E. M. Sarma, J. V., Riedemann, N., McGuire, S. R., Lu, K. T., Kunkel, R. et al., Generation of C5a by phagocytic cells. Am. J. Pathol. 2002. 161: 1849–1859. 7 Karsten, C. M. and Kohl, ¨ J., The immunoglobulin, IgG Fc receptor and complement triangle in autoimmune diseases. Immunobiology 2012. 217: 1067–1079. 8 Redecha, P., Tilley, R., Tencati, M., Salmon, J. E., Kirchhofer, D., Mackman, N. and Girardi, G., Tissue factor: a link between C5a and neutrophil activation in antiphospholipid antibody induced fetal injury. Blood 2007. 110: 2423–2431. 9 Shushakova, N., Skokowa, J., Schulman, J., Baumann, U., Zwirner, J., Schmidt, R. E. and Gessner, J. E., C5a anaphylatoxin is a major regulator of activating versus inhibitory FcγRs in immune complex induced lung disease. J. Clin. Invest. 2002. 110: 1823–1830. 10 Guo, R. F., Riedemann, N. C. and Ward, P. A., Role of C5a-C5aR interaction in sepsis. Shock 2004. 21: 1–7.

Statistical analysis

11 Rittirsch, D., Flierl, M. A., Nadeau, B. A., Day, D. E., Huber-Lang, M., Mackay, C. R., Zetoune, F. S. et al., Functional roles for C5a receptors in

Statistical analysis was performed using Prism 5 statistical software package (GraphPad Software). Comparisons between groups were analyzed with 2-tailed Student´s t test.

sepsis. Nat. Med. 2008. 14: 551–557. 12 Zhang, X., Kimura, Y., Fang, C., Zhou, L., Sfyroera, G., Lambris, J. D., Wetsel, R. A. et al., Regulation of Toll-like receptor-mediated inflammatory response by complement in vivo. Blood 2007. 110: 228–236. 13 Bosmann, M., Haggadone, M. D., Hemmila, M. R., Zetoune, F. S., Sarma, J. V. and Ward, P. A., Complement activation product C5a is a selective suppressor of TLR4-induced, but not TLR3-induced, production of IL-27(p28) from macrophages. J. Immunol. 2012. 188: 5086–5093.

Acknowledgements: This work was supported by Deutsche Forschungsgemeinschaft (DFG) SFB 587, GE 892/ 11-1 (to J.E.G.) and Ni 556/10-1 (to B.N.). G.S. received a fellowship from the international PhD program of Hannover Medical School. We would like to thank A. Klos for helpful advice on calcium measurements. We also like to thank J. Zwirner for providing anti-C5aR antibody.

14 Monk, P. N., Scola, A. M., Madala, P. and Fairlie, D. P., Function, structure and therapeutic potential of complement C5a receptors. Br. J. Pharmacol. 2007. 152: 429–448. 15 Gerard, C. and Gerard, N. P., C5A anaphylatoxin and its seven transmembrane-segment receptor. Annu. Rev. Immunol. 1994. 12: 775–808. 16 Wiege, K., Ali, S. R., Gewecke, B., Novakovic, A., Konrad, F. M., Pexa, K., Beer-Hammer, S. et al., Gαi2 is the essential Gαi protein in immune complex-induced lung disease. J. Immunol. 2013. 190: 324–333. 17 Wiege, K., Le, D. D., Syed, S. N., Ali, S. R., Novakovic, A., Beer-Hammer, S., Piekorz, R. P. et al., Defective macrophage migration in Gαi2- but not

Conflict of interest: The authors declare no commercial or financial conflict of interest.

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Abbreviations:

BALF:

bronchoalveolar

lavage

fluid

·

BM:

bone

marrow · C5aR: C5a anaphylatoxin receptor · FcγR: Fc gamma receptors · ER: endoplasmic reticulum · Fura-2/AM: Fura-2 acetoxymethyl ester · GPCR: G protein-coupled receptor · IC: immune complex · PLF: peritoneal lavage fluid · PMN: polymorphonuclear leukocytes · SOC: storeoperated Ca2+ · SOCE: SOC entry · STIM: stromal interaction molecule · Tg: thapsigargin · Thg: thioglycollate

37 DeHaven, W. I., Jones, B. F., Petranka, J. G., Smyth, J. T., Tomita, T., Bird, G. S. and Putney, Jr., J. W., TRPC channels function independently of STIM1 and Orai1. J. Physiol. 2009. 587: 2275–2298. 38 Chen, W., Thielmann, I., Gupta, S., Subramanian, H., Stegner, D., vanKruchten, R., Dietrich, A. et al., Orai1-induced store-operated calcium entry enhances phospholipase activity and modulates TRPC6 function in murine platelets. J. Thromb. Haemost. 2014. 4:528-539.

¨ Molekulare Full correspondence: Prof. J. Engelbert Gessner, Labor fur ¨ Immunologie und Rheumatologie, Immunologie, Klinik fur Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany fax: +49-511-532-5648 e-mail: [email protected].

39 Cheng, K. T., Liu, X., Ong, H. L., Swaim, W. and Ambudkar, I. S., Local Ca2+ entry via Orai1 regulates plasma membrane recruitment of TRPC1 and controls cytosolic Ca2+ signals required for specific cell functions. PLoS Biol. 2011. 9: e1001025

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Received: 14/11/2014 Revised: 5/3/2015 Accepted: 25/4/2015

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Orai1 controls C5a-induced neutrophil recruitment in inflammation.

Stromal interaction molecule 1 (STIM1)-dependent store operated calcium-entry (SOCE) through Orai1-mediated calcium (Ca(2+) ) influx is considered a m...
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