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• W1980179678 abstract "It is well established that CpG promotes pro-inflammatory cytokine and antibody production by B cells via the Toll-like receptor 9 (TLR9)-dependent pathway. However, scavenger receptors (SRs) are also capable of binding such pathogen-derived molecules, yet their contribution to CpG-induced signaling events has not yet been evaluated. Here we identified a novel TLR9-independent mechanism of CpG-induced signaling and immune function that is mediated by the scavenger B1 receptor (SR-B1). Specifically, we show that CpG/SR-B1 triggers calcium entry into primary B lymphocytes via phospholipase Cγ-1-mediated activation of TRPC3 channels and also B cell adhesion to vascular cell adhesion molecule-1. CpG-induced calcium signals and vascular cell adhesion molecule-1 adhesion are TLR9-independent and are mediated exclusively by SR-B1. Although pro-inflammatory cytokine and Ig production induced by CpG require TLR9 expression, we also found that SR-B1 negatively regulates TLR9-dependent production of interleukin-6, interleukin-10, and IgM. Thus, our results provide a novel perspective on the complexity of CpG signaling within B cells by demonstrating that SR-B1 is an alternative pathway for nucleic acid-induced signaling that provides feedback inhibition on specific TLR9-dependent responses of B cells. Consequently, these results have wide implications for understanding the mechanisms regulating immune tolerance to nucleic acids and pathogen-associated molecules. It is well established that CpG promotes pro-inflammatory cytokine and antibody production by B cells via the Toll-like receptor 9 (TLR9)-dependent pathway. However, scavenger receptors (SRs) are also capable of binding such pathogen-derived molecules, yet their contribution to CpG-induced signaling events has not yet been evaluated. Here we identified a novel TLR9-independent mechanism of CpG-induced signaling and immune function that is mediated by the scavenger B1 receptor (SR-B1). Specifically, we show that CpG/SR-B1 triggers calcium entry into primary B lymphocytes via phospholipase Cγ-1-mediated activation of TRPC3 channels and also B cell adhesion to vascular cell adhesion molecule-1. CpG-induced calcium signals and vascular cell adhesion molecule-1 adhesion are TLR9-independent and are mediated exclusively by SR-B1. Although pro-inflammatory cytokine and Ig production induced by CpG require TLR9 expression, we also found that SR-B1 negatively regulates TLR9-dependent production of interleukin-6, interleukin-10, and IgM. Thus, our results provide a novel perspective on the complexity of CpG signaling within B cells by demonstrating that SR-B1 is an alternative pathway for nucleic acid-induced signaling that provides feedback inhibition on specific TLR9-dependent responses of B cells. Consequently, these results have wide implications for understanding the mechanisms regulating immune tolerance to nucleic acids and pathogen-associated molecules. Stimulus-induced dynamic changes in the concentration of cytoplasmic calcium are primary determinants of the activation, immunological function, and developmental fate of lymphocytes. Calcium signaling through the B cell antigen receptor (BCR) 2The abbreviations used are: BCRB cell receptorNSCCnon-selective cation channelCRACcalcium release-activated calciumTRPtransient receptor potentialSR-B1scavenger receptor B1TLRToll-like receptorPLCphospholipase CVCAM-1vascular cell adhesion molecule-1IP31,4,5-inositol trisphosphateILinterleukinsiRNAsmall interference RNA. 2The abbreviations used are: BCRB cell receptorNSCCnon-selective cation channelCRACcalcium release-activated calciumTRPtransient receptor potentialSR-B1scavenger receptor B1TLRToll-like receptorPLCphospholipase CVCAM-1vascular cell adhesion molecule-1IP31,4,5-inositol trisphosphateILinterleukinsiRNAsmall interference RNA. complex is initiated by the activation of proximal tyrosine kinases Lyn and Syk, which phosphorylate the adaptor BLNK to facilitate its association with and activation of PLCγ-2. PLCγ2 hydrolyzes phosphatidylinositol 4,5-bisphosphate into diacylglycerol and 1,4,5-inositol trisphosphate (IP3) (for review see Ref. 1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6135) Google Scholar), which activates IP3 receptor/channels that mediate Ca2+ release from endoplasmic reticulum into the cytosol (2Sugawara H. Kurosaki M. Takata M. Kurosaki T. EMBO J. 1997; 16: 3078-3088Crossref PubMed Scopus (373) Google Scholar) (for review see Refs. 3Miyakawa T. Maeda A. Yamazawa T. Hirose K. Kurosaki T. Iino M. EMBO J. 1999; 18: 1303-1308Crossref PubMed Scopus (336) Google Scholar, 4Hagar R.E. Burgstahler A.D. Nathanson M.H. Ehrlich B.E. Nature. 1998; 396: 81-84Crossref PubMed Scopus (228) Google Scholar). Ca2+ release from endoplasmic reticulum stores and the resulting depletion of Ca2+ (not an increase in cytoplasmic [Ca2+]) are the central and prerequisite events required to activate plasma membrane “store-operated” calcium release-activated calcium (CRAC) channels. B cell receptor non-selective cation channel calcium release-activated calcium transient receptor potential scavenger receptor B1 Toll-like receptor phospholipase C vascular cell adhesion molecule-1 1,4,5-inositol trisphosphate interleukin small interference RNA. B cell receptor non-selective cation channel calcium release-activated calcium transient receptor potential scavenger receptor B1 Toll-like receptor phospholipase C vascular cell adhesion molecule-1 1,4,5-inositol trisphosphate interleukin small interference RNA. CRAC channels are responsible for antigen receptor-triggered calcium entry; however, a growing body of evidence suggests that CRAC channels do not underlie all the diverse calcium-regulated responses of lymphocytes, particularly those triggered by innate stimuli. For example, we previously identified several calcium-permeant non-selective cation channels (NSCCs) that are uniquely activated by distinct arachidonic acid-derived (eicosanoid) inflammatory mediators and by mechanical stimuli (5Liu Q.H. Liu X. Wen Z. Hondowicz B. King L. Monroe J. Freedman B.D. J. Immunol. 2005; 174: 68-79Crossref PubMed Scopus (29) Google Scholar, 6Zhu P. Liu X. Labelle E.F. Freedman B.D. J. Immunol. 2005; 175: 4981-4989Crossref PubMed Scopus (21) Google Scholar, 7Liu X. Zhu P. Freedman B.D. Am. J. Physiol. Cell Physiol. 2006; 290: C873-C882Crossref PubMed Scopus (18) Google Scholar). Thus, multiple calcium-permeant channels with distinct activation mechanisms may underlie stimulus-specific calcium-dependent B cell functions in vivo. Surprisingly, a number of pathogen-associated Toll-like receptor agonists are known to be strong B cell mitogens, yet the potential for calcium-dependent signaling functions by these polyclonal B cell mitogens has not yet been fully evaluated. Studies detailed in this report focus on the mechanism of calcium signaling elicited by unmethylated CpG DNA in primary B cells. Unmethylated CpG DNA is typically considered a pathogen-derived molecule that triggers polyclonal B cell activation, cytokine production, and immunoglobulin production via Toll-like receptor 9 (TLR9) engagement (8Bauer S. Kirschning C.J. Häcker H. Redecke V. Hausmann S. Akira S. Wagner H. Lipford G.B. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 9237-9242Crossref PubMed Scopus (1242) Google Scholar, 9Hemmi H. Takeuchi O. Kawai T. Kaisho T. Sato S. Sanjo H. Matsumoto M. Hoshino K. Wagner H. Takeda K. Akira S. Nature. 2000; 408: 740-745Crossref PubMed Scopus (5264) Google Scholar). Because CpG induces a subset of the B cell responses normally elicited by cognate antigen binding to the BCR complex, we asked whether CpG stimulation mobilizes calcium. We found that while CpG stimulation and BCR engagement both elicit similar biphasic calcium signals, CpG-mediated calcium entry is regulated by TRPC3, a calcium-permeant NSCC of the canonical transient receptor potential (TRPC) channel family (10Hofmann T. Obukhov A.G. Schaefer M. Harteneck C. Gudermann T. Schultz G. Nature. 1999; 397: 259-263Crossref PubMed Scopus (1229) Google Scholar) and that, unlike the BCR, which couples to calcium entry via PLCγ-2, TRPC3 activation involves an adaptor like function of PLCγ-1. We also report that CpG-mediated calcium signals are initiated by the scavenger receptor B1 (SR-B1) independently of TLR9. To our knowledge, this is the first demonstration of SR-B1 function in B lymphocytes; although scavenger receptors have been implicated in the responses of other immune cells. For example, bacterial pathogens and byproducts of apoptotic cells contribute to the pathogenesis of immune-mediated diseases, including lupus in part via MARCO and CD36 expressed by marginal zone macrophages (11Wermeling F. Chen Y. Pikkarainen T. Scheynius A. Winqvist O. Izui S. Ravetch J.V. Tryggvason K. Karlsson M.C. J. Exp. Med. 2007; 204: 2259-2265Crossref PubMed Scopus (105) Google Scholar). In naïve B cells, CD36 expression is largely restricted to marginal zone cells. Notably, CD36 cooperates with TLR2 to produce antibodies against phosphocholine, which is an endogenous antigen (13Won W.J. Bachmann M.F. Kearney J.F. J. Immunol. 2008; 180: 230-237Crossref PubMed Scopus (31) Google Scholar). Given our finding that CpG elicits calcium signals via SR-B1 on lymphocytes, we asked whether SR-B1 might also act cooperatively, in this case with TLR9, to trigger inflammatory responses of B cells. In fact, our results indicate that SR-B1 negatively regulates CpG/TLR9-mediated production of specific immunoglobulins (IgM) and pro-inflammatory cytokines (IL-6 and IL-10) by B cells. These findings have important implications for understanding how calcium is regulated in B cells, but also point to novel mechanisms by which pathogen-associated molecules regulate B cell activation. B lymphocytes purified from spleens of C57Bl/6 mice by negative immunomagnetic separation (Stemcell Technologies) were loaded with fura-2 AM (3.0 μm, Invitrogen) and intracellular Ca2+ was measured by digital imaging microscopy as previously described (5Liu Q.H. Liu X. Wen Z. Hondowicz B. King L. Monroe J. Freedman B.D. J. Immunol. 2005; 174: 68-79Crossref PubMed Scopus (29) Google Scholar). All results are expressed as the fura-2 fluorescence emission ratio at 510 nm. The BCR was engaged using soluble anti-mouse F(ab′)2 antibody (Jackson ImmunoResearch, West Grove, PA). Phosphorothioester derivatives of stimulatory (CpG, ODN1826: 5′-TCC ATG ACG TTC CTG ACG TT-3′) and a control variant of ODN1826 (GpC, 5′-TCC ATG AGC TTC CTG AGC TT-3′) were synthesized and high-performance liquid chromatography purified by Integrated DNA Technologies (Coralville, IA). CpG was used at a final concentration of 2.5 μm for calcium measurements unless stated otherwise. Total RNA was extracted from purified splenic B cells or bone narrow derived macrophages with an RNeasy kit (Qiagen, CA), and cDNA was synthesized using Moloney murine leukemia virus reverse transcriptase (Invitrogen, CA), and random hexamer primers. cDNA was subject to 40 amplification cycles with SR primers (300 nm) of the following sequences: SRA sense, 5′-AGA ATT TCA GCA TGG CAA CTG; SRA antisense, 5′-ACG GAC TCT GAC ATG CAG TG; MARCO sense, 5′-GCA CTG CTG CTG ATT CAA GTT C-3′; MARCO antisense, 5′-AGT TGC TCC TGG CTG GTA TG-3′; SRB1 sense, 5′-GAC GGG CGT CCA GAA TTT C-3′; SR-B1 antisense, 5′-AGC GAG GAT TCG GGT GTC AT-3′; SRB2 sense, 5′-AAAAGGCATGCATCCCAACA-3′; SRB2 antisense, 5′-GTC CTG ATG TCT CCC GTT TCA-3′; CD36 sense, 5′-GTG ACG TGG CAA AGA ACA G-3′; CD36 antisense, 5′-AAA GGA GGC TGC GTC TGT G-3′; Macrosialin sense, 5′-TCC AAG ATC CTC CAC TGT TG-3′; Macrosialin antisense, 5′-CAT TGT ATT CCA CCG CCA TG-3′; LOX1 sense, 5′-ATG AAT TTG GAA ATG GCT TTT G-3′; LOX1 antisense, 5′-TCA CTG AGT TAG CAA TAA ATT TG-3′; SRECI sense, 5′-CTG GGC CGT CAT GGT AAG AA-3′; SRECI antisense, 5′-GGG CCA TAG GGA CCA TCT CT-3′; SRECII sense, 5′-CAA CGT TTT TGT GGA AGC TTC AG-3′; and SRECII antisense, 5′-GCA GTT GAG TGT GTT GTC TAG GTC AT-3′. For Western analysis, B cells lysates with ∼30 μg of protein were separated by electrophoresis. Proteins transferred to nitrocellulose membrane were detected by enhanced chemiluminescence (ECL). siRNA transfection of primary cells was performed by a modification of the procedure previously described (14Gomez T.S. Kumar K. Medeiros R.B. Shimizu Y. Leibson P.J. Billadeau D.D. Immunity. 2007; 26: 177-190Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Purified splenic B cells were first activated with lipopolysaccharide (0.5 μg/ml) for 24 h, then incubated (5 million B cells) in 500 μl of culture medium (without 2-mercaptoethanol or antibiotics) containing 1000 pmol of TRPC3 or SR-B1 SMARTpool siRNA (Dharmacon) for 10 min at room temperature in an electroporation cuvette (BTX, 4-mm gap). Transfection was performed using a single 10-ms pulse at 295 V with a BTX ECM 830 electroporator. Transfected cells were washed and cultured for an additional 48 h in complete medium, and then used in functional experiments. The efficiency of RNA interference-mediated protein suppression was assessed by immunoblot analysis using antibodies against targeted proteins and typically exceeded 90% suppression. Antibodies against TRPC3 were obtained from Alomone Laboratories (Jerusalem, Israel), SR-B1 were obtained from Novus Biologicals (Littleton, CO), and phosphotyrosine (4G11) from Millipore (Billerica, MA). Single cell suspensions were prepared from dissociated spleens. Purified B cells (2 × 105) were stimulated with CpG or other agonists for 48–72 h in RPMI medium containing 10% fetal bovine serum, 2 mm glutamine, 100 units/ml penicillin G, 100 μg/ml streptomycin sulfate, non-essential amino acids, OPI media supplement (Sigma), and 50 μm 2-mercaptoethanol. Ig levels in culture supernatants at 48 h were measured using a kit (Southern Biotech). Supernatants collected at 72 h were analyzed for IL-6 and IL-10 by enzyme-linked immunosorbent assay (eBiosciences). Samples were processed in duplicate and recombinant cytokines used to establish standard curves (Peprotech, Rocky Hill, NJ) ranging from 8 to 4000 pg/ml. Cell integrin avidity changes were assessed using a VCAM-1 adhesion assay as previously described (5Liu Q.H. Liu X. Wen Z. Hondowicz B. King L. Monroe J. Freedman B.D. J. Immunol. 2005; 174: 68-79Crossref PubMed Scopus (29) Google Scholar). Patch clamp recordings were performed on primary B lymphocytes identified visually by negative immunofluorescence (CD3 negative) in the microscope recording chamber. Patch pipettes were fabricated with a 4- to 6-MΩ (for whole cell recording) or 5- to 10-MΩ (for single channel recording) tip resistance (Sutter Instrument, Novato, CA) from borosilicate glass and were back-filled with appropriate internal solution. Liquid junction potentials were calculated and were corrected manually with the patch clamp amplifier or post analysis. Capacitance and access resistance were monitored continuously. Between 50 and 70% of the series resistance was electronically compensated to minimize voltage errors. Command potentials were generated using an EPC-9 patch clamp amplifier and currents acquired, stored, and analyzed using PulseFit software (Heka Elektronic, Germany). Single channel amplitude frequency analysis was performed using QuB software (15Qin F. Auerbach A. Sachs F. Biophys. J. 1996; 70: 264-280Abstract Full Text PDF PubMed Scopus (368) Google Scholar). Whole cell currents were recorded using the standard whole cell mode of the patch clamp technique. Cells were first held at 0 mV, then stepped to −80 mV and subjected to a linear voltage ramp (100 ms) to 80 mV with a sampling interval of 0.2 ms. This ramp protocol was repeated every 2 s, and the time course of the whole cell currents was obtained by plotting the mean current amplitude at −80 mV for each cycle. Cell membrane capacitance values were used to estimate the cell surface area, and results are expressed as the current amplitude per unit membrane capacitance (pA/pF). All ramp currents were leak-corrected by subtracting ramp currents obtained after establishing a stable whole cell recording from the ramp current obtained after agonist-induced current activation. Measurements of the relative Ca2+ versus Na+ permeability (PCa/PNa) were calculated using the measured reversal potential of CpG-induced currents recorded in a bath solution containing 30 mm Ca2+ and a pipette solution containing 10 mm Na+ using the equation, Erev = RT/2F ln{4PCa2+[Ca2+]o/PNa+[Na+]i}. Single channel recordings were also obtained using the cell-attached configuration of the patch clamp as previously described (7Liu X. Zhu P. Freedman B.D. Am. J. Physiol. Cell Physiol. 2006; 290: C873-C882Crossref PubMed Scopus (18) Google Scholar). Membrane-permeant drugs were typically applied by direct addition to the bath. For some whole cell, patch clamp recording measurements, membrane-impermeant monoclonal antibodies were dialyzed from the recording microelectrode into the cytoplasm of single cells to neutralize the function of PLCγ isoforms as previously demonstrated by us and others (5Liu Q.H. Liu X. Wen Z. Hondowicz B. King L. Monroe J. Freedman B.D. J. Immunol. 2005; 174: 68-79Crossref PubMed Scopus (29) Google Scholar, 16Wang Y.X. Kotlikoff M.I. J. Physiol. 2000; 523: 131-138Crossref PubMed Scopus (44) Google Scholar). Direct visualization of cytoplasmic filling with fluorescein isothiocyanate-conjugated antibodies demonstrated that the time required for complete cytoplasmic dialysis using this approach is <2 min. Given that unmethylated bacterial CpG DNA is a powerful polyclonal B cell mitogen and that calcium regulates lymphocyte activation, proliferation, and effector functions, studies were initiated to examine whether CpG mobilizes calcium in primary B lymphocytes. We found that CpG elicited a large [Ca2+]i increase comparable to that triggered by BCR engagement (Fig. 1A). Notably, this response, which was observed in both murine and human primary B cells (Fig. 1B), did not require TLR9 (Fig. 1C) or MyD88 expression (supplemental Fig. S1), suggesting the presence of a previously unrecognized CpG-induced TLR9-independent pathway. Although CpG-induced calcium mobilization was not observed in a previous studies (17Busconi L. Bauer J.W. Tumang J.R. Laws A. Perkins-Mesires K. Tabor A.S. Lau C. Corley R.B. Rothstein T.L. Lund F.E. Behrens T.W. Marshak-Rothstein A. J. Immunol. 2007; 179: 7397-7405Crossref PubMed Scopus (48) Google Scholar, 18Uccellini M.B. Busconi L. Green N.M. Busto P. Christensen S.R. Shlomchik M.J. Marshak-Rothstein A. Viglianti G.A. J. Immunol. 2008; 181: 5875-5884Crossref PubMed Scopus (70) Google Scholar), low concentrations (∼100 nm) utilized also did not produce a response in our hands (Fig. 1D, left panel). These findings suggested that different thresholds may exist for unique CpG-induced functional effects. Importantly, the calcium signals we observe are not selective for CpG, as a control oligonucleotide that differed from CpG 1826 only by the inversion of two CG motifs (GpC) elicited a signal with the identical dose sensitivity as CpG 1826 (Fig. 1D, right panel). Thus, together with the data demonstrating CpG-induced calcium mobilization in TLR9-deficient B cells, these data indicate that DNA (both CpG and GpC) elicits calcium signals through a novel TLR9-independent pathway that is less restrictive in its nucleotide recognition. Because the calcium signal triggered by CpG superficially resembles that initiated by BCR engagement, we next asked whether it occurs by a similar mechanism. Antigen receptor-induced calcium entry occurs through CRAC channels, which are activated by intracellular calcium store depletion. In calcium-free medium, we found that CpG produced a transient elevation in cytoplasmic calcium due to release from intracellular stores and subsequent perfusion with calcium replete medium produced a secondary sustained [Ca2+] elevation in primary mouse and human B cells (see Fig. 1, B and C) consistent with entry via plasma membrane calcium-permeant channels. We used the patch clamp to determine whether CpG, like the BCR, activates CRAC channels or if distinct store-operated or store-independent channels are responsible for CpG-induced calcium entry. CpG treatment elicited relatively large amplitude inward currents (−37.4 ± 10.0 picoamps/picofarad, Vm = −80 mV, n = 26) that exhibited a linear current-voltage relationship and a reversal potential near 0 mV in primary mouse (−4.2 ± 1.6 mV; Fig. 2A, left panels) and human B cells (Fig. 2A, right panels). The single channel conductance (Fig. 2B, 18.8 ± 0.2 pS, n = 5) and the relative Ca2+ versus Na+ permeability (2.2 ± 0.3, n = 4) suggest that calcium-permeant NSCCs and not CRAC channels are responsible for CpG-induced calcium entry. We previously identified the expression of eight TRP family NSCCs in murine B cells (5Liu Q.H. Liu X. Wen Z. Hondowicz B. King L. Monroe J. Freedman B.D. J. Immunol. 2005; 174: 68-79Crossref PubMed Scopus (29) Google Scholar), and, of these, TRPC3 has biophysical properties that most closely resemble currents elicited by CpG (Fig. 2C). To determine if TRPC3 contributed functionally to the CpG-induced calcium elevations that we observe, primary B cells were transfected with siRNA, to suppress TRPC3 expression (Fig. 2D). Notably reduction of TRPC3 expression substantially attenuated CpG-induced calcium signals (Fig. 2E) but had no effect on BCR-induced responses. Neither CpG- nor BCR-induced Ca2+ signals were affected by suppression of the SR MARCO (supplemental Fig. S2). Together, these results indicate that CpG-induced calcium signaling is regulated by activation of TRPC3. Native TRPC3 is not store-operated; however, some studies have reported that ectopically expressed TRPC3 channels can operate in a store-dependent manner (19Löf C. Blom T. Törnquist K. J. Cell Physiol. 2008; 216: 245-252Crossref PubMed Scopus (12) Google Scholar). Given that CpG triggers calcium release from stores (see Fig. 1, B and C), we examined whether calcium store depletion activates endogenous TRPC3-like currents in primary B cells. Thapsigargin, which blocks sarco-endoplasmic reticulum ATPase that maintain stores in a filled state, elicited CRAC currents in B cells (Fig. 2F) but did not elicit non-selective cation currents. Together, these data suggest that CpG-induced calcium signals are mediated by TRPC3 activation via a store-independent mechanism. Given that CpG, like BCR engagement, induces calcium release from stores, we next examined whether CpG activates the proximal effector proteins involved in BCR-mediated calcium mobilization. Although CpG increases phosphorylation of dual protein bands with the mobility of Lyn kinase, it does not induce phosphorylation of other critical molecules which link the BCR to calcium release from stores, including Syk, Btk, BLNK, or PLCγ-2 (Fig. 3A). We confirmed that Lyn was indeed phosphorylated by CpG by performing a Lyn immunoblot analysis of phosphotyrosine immunoprecipitates (Fig. 3B, top panel). Given that PLCg-2 is not phosphorylated by CpG, we next examined whether CpG activates PLCγ-1. PLCγ-1 is not considered a major isoform in B cell; however, it was detectable in primary B cells (Fig. 3B, bottom panel, right lane). Because neither CpG nor BCR engagement induced a measurable change in PLCγ-2 or PLCγ-1 phosphorylation (Fig. 3B, bottom panel), we used a direct approach to determine whether TRPC3 activation by CpG requires either PLCγ-1 or PLCγ-2. To do this we neutralized these distinct isoforms in situ by dialyzing isoform-specific antibodies into the cytoplasm of single B cells. Antibodies used in this approach are capable of recognizing two-dimensional forms of these proteins and were identical to those we used previously to define the mechanism of mechanical signaling in B cells (see “Experimental Procedures” and Ref. 5Liu Q.H. Liu X. Wen Z. Hondowicz B. King L. Monroe J. Freedman B.D. J. Immunol. 2005; 174: 68-79Crossref PubMed Scopus (29) Google Scholar). As expected, we found that PLCγ-2 neutralization blocked BCR-induced CRAC channel activation and did not affect CpG-induced TRPC3 activation. Surprisingly the converse was observed for CpG responses. PLCγ-1 neutralization in situ efficiently blocked CpG activation of TRPC3 channels, yet it did not disrupt activation of CRAC channels by BCR engagement (Fig. 3C). Together, these results demonstrate that neutralization of these proteins in situ is specific, as the BCR response served as both a positive and negative control for these antibodies, and that although CpG-mediated activation of TRPC3 is store independent, it is regulated by PLCγ-1. Direct interactions between PLCγ-1 and TRPC3 have been identified in cell lines, and it appears that this physical interaction regulates TRPC3 activity (20van Rossum D.B. Patterson R.L. Sharma S. Barrow R.K. Kornberg M. Gill D.L. Snyder S.H. Nature. 2005; 434: 99-104Crossref PubMed Scopus (164) Google Scholar, 21Patterson R.L. van Rossum D.B. Ford D.L. Hurt K.J. Bae S.S. Suh P.G. Kurosaki T. Snyder S.H. Gill D.L. Cell. 2002; 111: 529-541Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). To determine if PLCγ-1 serves a similar adaptor-like role for TRPC3 activation in B cells, we generated a membrane permeant peptide, corresponding to the domain of PLCγ-1 that interacts with TRPC3, to serve as a competitive inhibitor of this interaction (Fig. 3D, 10987). Notably, peptide 10987 completely blocked CpG (Fig. 3E, left panel)-, but not BCR-mediated calcium mobilization (Fig. 3E, right panel). These conditions had no effect on CpG-induced calcium release from stores but strongly suppressed calcium entry. The small degree of residual calcium entry in the presence of the PLCγ-1 peptide was similar to that observed upon solution change and did not reflect cell activation by CpG. Consequently, our inability to elicit CRAC currents in B cells with CpG and the fact that CpG-induced calcium entry is fully suppressed when TRPC3 activation is blocked indicates that TPC3 alone underlies this response. Together, these data suggest that TRPC3 activation is regulated by a physical interaction with PLCγ-1. Given that TLR9 and MyD88 are not required for CpG-induced calcium mobilization, we next sought to identify the responsible receptor. We focused on the scavenger receptors (SRs) as candidates; because, like TLRs, they bind pathogen-associated molecules and have been shown to regulate macrophage and dendritic cell responses to TLR ligands such as lipopolysaccharide and CpG (22Józefowski S. Sulahian T.H. Arredouani M. Kobzik L. J. Leukoc. Biol. 2006; 80: 870-879Crossref PubMed Scopus (55) Google Scholar, 23Gordon S. Cell. 2002; 111: 927-930Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar). To determine if SRs were capable of mediating CpG-induced calcium signals in B cells, we first evaluated the effect of several broadly specific SR antagonists (poly-inosine, fucoidan, and low density lipoprotein). Notably each of the SR antagonists tested blocked CpG-induced calcium responses in primary B cells (Fig. 4A), implicating SRs in this response to CpG. Reverse transcription-PCR analysis of SR expression identified MARCO, CD36, SR-B1, and its splice variant SR-B2 in primary mouse B cells (Fig. 4B). Given that B cells express multiple SR and the broad specificity of antagonists tested, we next evaluated the effect of individual anti-SR antibodies on CpG-induced calcium signaling. Although neither anti-MARCO nor -CD36 antibodies affected CpG-induced calcium signals (supplemental Fig. S2), the anti-SR-B1/2 antibody was clearly inhibitory (Fig. 4C). To directly verify the requirement for SR-B1 in CpG-induced calcium signaling, we examined the response of SR-B1-deficient B cells (24Rigotti A. Trigatti B.L. Penman M. Rayburn H. Herz J. Krieger M. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 12610-12615Crossref PubMed Scopus (746) Google Scholar) to this ligand. In the absence of SR-B1 expression, CpG failed to mobilize calcium (Fig. 4D) or elicit a cation current (Fig. 4E) in primary B cells. This failure of CpG-induced signaling does not reflect a general calcium defect; however, because BCR-induced calcium elevations and CRAC channel activation were unaffected by SR-B1 deficiency (Fig. 4E). Taken together, these data indicate that SR-B1 mediates CpG-induced calcium mobilization in B lymphocytes. Although little is known about the role of SRs in B cells, it is noteworthy that Toll-like receptors and scavenger receptors expressed by B cells bind a similar range of cellular and pathogen-associated molecules (23Gordon S. Cell. 2002; 111: 927-930Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar, 25Gough P.J. Gordon S. Microbes Infect. 2000; 2: 305-311Crossref PubMed Scopus (145) Google Scholar, 26Pearson A.M. Curr. Opin. Immunol. 1996; 8: 20-28Crossref PubMed Scopus (248) Google Scholar, 27Mukhopadhyay S. Gordon S. Immunobiology. 2004; 209: 39-49Crossref PubMed Scopus (140) Google Scholar). Moreover, TLR2 co-engagement with the scavenger receptor CD36 positively regulates B cell activation (13Won W.J. Bachmann M.F. Kearney J.F. J. Immunol. 2008; 180: 230-237Crossref PubMed Scopus (31) Google Scholar). Given our findings that CpG activates the CD36-related class B family member SR-B1, we asked whether SR-B1 serves a similar co-stimulatory role in TLR9-mediated activation of B cells. We found that neither SR-B1 nor TRPC3 activation was required for NFκB activation (IκBα degradation and p65 activation) or B cell proliferation (supplemental Fig. S3). However, in addition to calcium mobilization we found that VLA-4 integrin activation (VCAM-1 adhesion) by CpG was independent of TLR9 (Fig. 5A) and fully dependent upon SR-B1 expression (Fig. 5B). CpG did not increase B cell adhesion to the LFA-1 ligand ICAM-1 (supplemental" @default.
• W1980179678 created "2016-06-24" @default.
• W1980179678 creator A5002551693 @default.
• W1980179678 creator A5010956002 @default.
• W1980179678 creator A5019155182 @default.
• W1980179678 creator A5034908399 @default.
• W1980179678 creator A5087129282 @default.
• W1980179678 date "2009-08-01" @default.
• W1980179678 modified "2023-10-16" @default.
• W1980179678 title "Mechanism and Regulatory Function of CpG Signaling via Scavenger Receptor B1 in Primary B Cells" @default.
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