* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Sensitizing B Cells for TLR2 Ligands Cell
Survey
Document related concepts
Biochemical switches in the cell cycle wikipedia , lookup
Tissue engineering wikipedia , lookup
Endomembrane system wikipedia , lookup
Extracellular matrix wikipedia , lookup
Signal transduction wikipedia , lookup
Cell encapsulation wikipedia , lookup
Programmed cell death wikipedia , lookup
Cell growth wikipedia , lookup
Cell culture wikipedia , lookup
Cellular differentiation wikipedia , lookup
Cytokinesis wikipedia , lookup
Transcript
Staphylococcus aureus Protein A Triggers T Cell-Independent B Cell Proliferation by Sensitizing B Cells for TLR2 Ligands This information is current as of August 3, 2017. Isabelle Bekeredjian-Ding, Seiichi Inamura, Thomas Giese, Hermann Moll, Stefan Endres, Andreas Sing, Ulrich Zähringer and Gunther Hartmann J Immunol 2007; 178:2803-2812; ; doi: 10.4049/jimmunol.178.5.2803 http://www.jimmunol.org/content/178/5/2803 Subscription Permissions Email Alerts This article cites 86 articles, 45 of which you can access for free at: http://www.jimmunol.org/content/178/5/2803.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017 References The Journal of Immunology Staphylococcus aureus Protein A Triggers T Cell-Independent B Cell Proliferation by Sensitizing B Cells for TLR2 Ligands1 Isabelle Bekeredjian-Ding,2* Seiichi Inamura,† Thomas Giese,‡ Hermann Moll,† Stefan Endres,§ Andreas Sing,¶ Ulrich Zähringer,† and Gunther Hartmann储 C rude preparations of Staphylococcus aureus are frequently used as polyclonal B cell activators to analyze T cell-dependent and -independent B cell responses such as proliferation and Ig production. The comparison of different S. aureus strains generated the hypothesis that the expression of a cell wall protein called surface protein A (SpA),3 an Ig-binding protein, largely accounts for the B cell stimulatory activity (1– 6). Therefore, crude preparations of inactivated S. aureus Cowan strain I (SAC), a highly SpA-expressing strain, are mostly used as polyclonal B cell activators. Moreover, SAC *Department of Microbiology, University of Heidelberg, Germany; †Research Center Borstel, Borstel, Germany; ‡Institute for Immunology, University of Heidelberg, Germany; §Department of Internal Medicine, Division of Clinical Pharmacology, University of Munich, Germany; ¶Max-von-Pettenkofer Institute of Microbiology, University of Munich, Germany; 储Department of Internal Medicine, Division of Clinical Pharmacology, University of Bonn, Germany Received for publication March 6, 2006. Accepted for publication December 11, 2006. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This study was supported by the Deutsche Forschungsgemeinschaft Grant DI898/ 1-1 (to I.B.-D.), the Deutsche Forschungsgomeinschaft Sonderforschungsbereich Grant 576-B11 (to A.S.), and the Deutsche Forschungsgomeinschaft Priority Program “Innate Immunity” Grant SPP 1110 (to S.I. and U.Z.). G.H. is supported by the Bundesministerium für Bildung und Forschung Biofuture 0311896, Deutsche Forschungsgomeinschaft Grants HA 2780/5-1 and Sonderforschungsbereich 571, and the Mildred-Scheel-Stiftung (Deutsche Krebshilfe) Joint Grant 10-2074-Wo 2. 2 Address correspondence and reprint requests to Dr. Isabelle Bekeredjian-Ding, Department of Microbiology, University of Heidelberg, Im Neuenheimer Feld 324, Heidelberg, Germany. E-mail address: [email protected] 3 Abbreviations used in this paper: SpA, surface protein A; BHK, baby hamster kidney; LP, lipopeptide; LTA, lipoteichoic acid; MALP-2, macrophage-activating lipopeptide-2; Nod, nucleotide oligomerization domain; ODN, oligodeoxynucleotide; OG, N-octyl--D-glucopyranoside; SAC, S. aureus Cowan I strain; TA, teichoic acid; WTA, wall teichoic acid; HF, hydrofluoridic acid. Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 www.jimmunol.org stimulation in the presence of T cell help, e.g., in PBMC, has been used for diagnostic procedures involving the analysis of Ig secretion such as the phenotyping of common variable immunodeficiency disorders (7). However, the exact mechanisms of B cell stimulation by S. aureus have not been clarified to date. SpA was first described as a B cell “superantigen” (8 –10) promoting B cell activation. This effect was subsequently shown to be due to the binding and activation of surface Igs belonging to the phylogenetic clone VH3, a subgroup of BCRs that is abundantly expressed among the murine innate B1 and MZ B cell subsets and is also expressed in 10 – 60% of human peripheral blood B lymphocytes (11–16). The early in vitro studies on human B cells proposed that SpA presentation on bacterial cell walls resulted in BCR activation via the cross-linking of surface Igs. In contrast, soluble SpA was shown to promote B cell proliferation only in the presence of T cell costimulation (17). Subsequently, in vivo studies in the mouse demonstrated that the injection of SpA induces the apoptosis of VH3-bearing B cells, mainly of the B1 B cell subset (18, 19). Because the innate B cell repertoire failed to regenerate from the bone marrow, the toxicity of SpA manifested as a longterm defect in innate humoral immunity. Crude extracts of S. aureus contain a variety of molecules derived from both the bacterial cell wall as well as from the cytosol. In addition to SpA, such mixtures represent structurally nondefined pathogen-associated molecular patterns containing CpG DNA, wall teichoic acids (WTA), lipoteichoic acid (LTA), lipopeptides (LP), and peptidoglycan, all known to activate receptors of the innate immune system or the complement cascade (20 –26). Among these molecules, peptidoglycan preparations have been extensively studied for their adjuvant activity in Ab production after the immunization of rabbits (20). Peptidoglycan represents the stabilizing element of the bacterial cell wall, consisting of a murein backbone with cross-linking short peptide chains (20). Cell wall teichoic acids (TA), glycoproteins, and LP are covalently Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017 B cells possess functional characteristics of innate immune cells, as they can present Ag to T cells and can be stimulated with microbial molecules such as TLR ligands. Because crude preparations of Staphylococcus aureus are frequently used as polyclonal B cell activators and contain potent TLR2 activity, the scope of this study was to analyze the impact of S. aureus-derived TLR2-active substances on human B cell activation. Peripheral B cells stimulated with chemically modified S. aureus cell wall preparations proliferated in response to stimulation with crude cell wall preparations but failed to be activated with pure peptidoglycan, indicating that cell wall molecules other than peptidoglycan are responsible for B cell proliferation. Subsequent analysis revealed that surface protein A (SpA), similar to BCR cross-linking with anti-human Ig, sensitizes B cells for the recognition of cell wall-associated TLR2-active lipopeptides (LP). In marked contrast to TLR7- and TLR9-triggered B cell stimulation, stimulation with TLR2-active LP and SpA or with crude cell wall preparations failed to induce IgM secretion, thereby revealing qualitative differences in TLR2 signaling compared with TLR7/9 signaling. Notably, combined stimulation with SpA plus TLR2 ligands induced vigorous proliferation of a defined B cell subset that expressed intracellular IgM in the presence of IL-2. Conclusion: S. aureus triggers B cell activation via SpA-induced sensitization of B cells for TLR2-active LP. Combined SpA and TLR2-mediated B cell activation promotes B cell proliferation but fails to induce polyclonal IgM secretion as seen after TLR7 and TLR9 ligation. The Journal of Immunology, 2007, 178: 2803–2812. 2804 Materials and Methods Human peripheral blood cell isolation PBMC from healthy donors were prepared by gradient centrifugation. Blood draw and cell isolation were approved by our local ethics committees. CD19⫹ B cells were isolated from PBMC by positive selection with anti-CD19-coated microbeads by MACS (Miltenyi Biotec). For the enrichment of memory B cells, CD27⫹ cells were positively selected after the CD3⫹ depletion of T cells. Naive B cells were enriched in CD27⫺ fractions after CD3⫹ and CD27⫹ cell depletion by CD19⫹ positive selection. B cell purity lay between 96 and 99% in all experiments; and the purity of cell fractions yielded 95–99% for memory B cells and 90 –98% for naive B cell fractions. Cells were resuspended in RPMI 1640 (Biochrom) supplemented with 10% (v/v) heat-inactivated FCS (Invitrogen Life Technologies), 3 mM L-glutamine, 0.01 M HEPES, 100 U/ml penicillin, and 100 g/ml streptomycin (all from Sigma- Aldrich) and incubated overnight before stimulation. All reagents were tested in regard to endotoxin contamination. B cell stimulation and assessment of B cell proliferation All stimulatory reagents were optimized for cell stimulation in the settings described. Peptidoglycans from S. aureus (I) and Micrococcus luteus were purchased from Fluka/Sigma-Aldrich, and peptidoglycans from Bacillus subtilis and S. aureus (II) were purchased from InvivoGen. All peptidoglycans were used at 5 g/ml in all experiments shown and tested for reactivity in human PBMC (intracellular TNF-␣ secretion and/or TNF-␣ ELISA; see below) and in Nod-transfected HEK293 cells (see below) to ensure their intact activity. Cells were stimulated with anti-human IgG⫹IgM⫹IgA F(ab⬘)2 from Jackson ImmunoResearch Laboratories as indicated in the figure legends. For the [3H]thymidine proliferation assays in Fig. 6A, 0.1 ml of recombinant SpA (Amersham Biosciences) at 10 g/ml in PBS was coated in a 96-well U-bottom plate for 1 h at 37°C and then at 6°C overnight; residual liquid was removed before the addition of cells. For the experiments in Fig. 6, B and C (mRNA expression studies), IL-6 and Ig analysis soluble SpA was added to the wells. R848 (InvivoGen) was used at 0.25 g/ml and CpG-B oligodeoxynucleotide (ODN) 2006 (5⬘tcgtcgttttgtcgttttgtcgtt-3⬘; small letters indicating a full-length phosphorothioate modification, Coley Pharmaceutical Group) at 2 or 3 g/ml as indicated. Loxoribine (7-allyl-7,8-dihydro-8-oxoguanosine) (0.5 mM) and LPS from Escherichia coli (10 g/ml) were obtained from Sigma-Aldrich. Pam3CSK4 (concentration as indicated in the figure legends) was purchased from InvivoGen and microphage-activating lipopeptide-2 (MALP-2; concentration as indicated in the figure legends) was from Alexis. S. aureus LTA (10 g/ml; 100 g/ml when indicated) was provided by T. Hartung (University of Konstanz, Konstanz, Germany). For CD40 ligation, baby hamster kidney (BHK)-CD40L cells and BHK-pTCF control cells (provided by H. Engelmann, Munich, Germany) were used at a ratio of 1:10 B cells. Recombinant human IFN-␣ (Strathman Biotech) was used at 1000 U/ml. Recombinant human IL-2 (R&D Systems) was used at 10 ng/ml. Cell proliferation was measured by [3H]thymidine or BrdU incorporation. Cells (2.5 ⫻ 104) at 0.2 ml/well (triplicates) were pulsed with [methyl3 H]thymidine (1 Ci/well) (Amersham Biosciences) for 16 h. Alternatively, 5 ⫻ 104 cells/well were pulsed with BrdU (0.5 M) (Roche) for 24 h. Total stimulation time was 72 h for both assays. BrdU incorporation was measured with chemiluminescence (Roche). CFSE staining with 1 M CFSE was used to analyze cell division by CFSE dilution after cell stimulation (2.5 ⫻ 105 per 0.2 ml) for 6 days. S. aureus strains and cell wall preparations For the initial cell wall preparations (cell wall 1 and cell wall 2), a clinically isolated S. aureus strain was used (52). For the second series of experiments, the S. aureus Cowan strain I and the Wood 46 strain were purchased from the Deutsche Sammlung von Mikroorganismen (DSM; catalog nos. 20372 and 20491). Cells were grown in Todd-Hewitt broth (Oxoid) to an OD of ⬃0.2– 0.4. Cells were harvested by centrifugation, washed in PBS, and incubated in acetone at 4°C over night. Cells were centrifuged and resuspended in deionized water, mixed with glass beads (0.5 mm, Biospec Products), and disintegrated in a Biospec bead beater. The residual cell walls were washed in H2O and either treated with chloroform/methanol (1:1) for the removal of phospholipids and H2O/2-propanol (1:1) (cell wall 1, cell wall Wood 46, and cell wall SAC) or with phenol/H2O (1:1) (cell wall 2). The residual insoluble material (sediment) was washed with H2O and defined as “whole cell walls.” Subsequent TCA treatment of cell walls was only performed in the first series of experiments with the clinical isolate (72 h in 10% TCA at 4°C; cell wall 1 and cell wall 2 only, not the SAC and Wood 46 cell walls) (53). Subsequently, WTA was isolated from the supernatant by the addition of diethyl ether (3 volumes) and the removal of the ether phase followed by ethanol precipitation (3 volumes) and the removal of ethanol (53). Sediments (cell walls) were washed in water, cooked in 8% SDS for 40 min, and the SDS was removed by more than six washes in deionized water (26, 28, 29, 53). The enzymes used for subsequent treatments were: amylase (1,4-␣-D-glucan-glucanohydrolase from Bacillus sp. (Sigma-Aldrich)) at 0.125 g/ml in 10 mM Tris; RNase A (Sigma-Aldrich) at ⱖ5 g/ml and DNase I (Roche) at ⱖ15 g/ml in 10 mM Tris and 20 mM MgSO4; trypsin (Roche) at 0.1 g/ml in 10 mM Tris/10 mM CaCl2; proteinase K (Roche) at 75–100 g/ml in 10 mM Tris/100 mM NaCl; and lysostaphin (SigmaAldrich) at 250 –300 g/ml in PBS. All treatments with enzymes were performed for 20 –24 h at 37°C, and enzymes were inactivated by the addition of SDS (0.8%) and subsequent cooking for at least 30 min (SDS was removed by washing with deionized water in a 30 ml volume 5–10 Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017 attached to peptidoglycan. Two well-defined motifs containing typical amino acid residues of the peptidoglycan peptide chains are recognized by the nucleotide oligomerization domain (Nod) receptors meso-D-glutamyl-meso-diaminopimelic acid (Nod1) and muramyl dipeptide (Nod2). These cytoplasmic pattern recognition receptors are involved in the innate immune recognition of bacteria (26 –31). Additionally, most peptidoglycan preparations activate TLR2 (22–24, 32, 33), a receptor belonging to a family of pattern recognition receptors that share the characteristic TIR (Toll/IL-1 receptor) domain and two common signaling pathways via the key signaling molecules MyD88 and/or TRIF (34). The demonstration that peptidoglycan-derived TLR2 activity is not related to the peptidoglycan molecule but rather due to other cell wall molecules bound to the peptidoglycan backbone (35–37) was initially considered as highly controversial. But, very recently, other groups have been able to show that S. aureus cells lacking diacylated and triacylated LP fail to induce a significant immune response (38) and that S. aureus cell wall-derived TLR2 activity is due to LP (35, 39). Moreover, TLR2-deficient mice were found to be hypersusceptible toward S. aureus infection (22, 23), thus indicating that TLR2 engagement is essential for the immune response to S. aureus. Among the known microbial motifs engaging TLRs, the most potent stimulus for B cell activation is unmethylated CpG DNA acting through the engagement of TLR9 (40 – 43). Similarly, human B cells have been demonstrated to respond to stimulation via TLR7 in the presence of type I IFN (40) and to be activated by TLR2 ligands (44 – 46). Furthermore, recent publications have shown that cross-linking of the BCR with anti-human Ig Abs (antiIg) enhances B cell sensitivity toward TLR ligands (47, 48). Although the significance of TLR2 polymorphisms in the severity of S. aureus infections in humans is controversial (49, 50), TLR2 ligand recognition seems to be an essential component of the immune response toward S. aureus. The goal of the present study was to provide a better insight into the mechanisms involved in S. aureus-mediated B cell stimulation by defining the impact of TLR2 activation on the human B cell response to crude S. aureus preparations such as the frequently used and commercially available Pansorbin (Calbiochem), a suspension of heat-killed, formalin-fixed S. aureus cells (51). Our data provide evidence that S. aureus cell wall preparations trigger a T cell-independent human B cell response via combined stimulation with SpA and TLR2 ligands. We further demonstrate that anti-Ig or SpA is a prerequisite for the efficient activation of human B cells via TLR2 and that given these circumstances, TLR2 ligands promote B cell proliferation but, in marked contrast to TLR7 and TLR9 ligation, fail to induce significant Ig secretion. Moreover, combined stimulation with TLR2-active LPs and SpA triggers the vigorous proliferation of a small B cell subset that can be induced to synthesize IgM in the presence of IL-2. S. aureus TRIGGERED B CELL ACTIVATION The Journal of Immunology Quantitation of protein, SpA, Ig, and cytokines Protein content of cell wall fractions was quantified by the Bradford method according to standard protocol (Bio-Rad) to ensure successful protein digestion (data not shown). SpA concentrations were determined by ELISA (Immunsystem). Cell walls and OG fractions were resuspended in water at 1 mg/ml and diluted 1/10, 1/20, and 1/50 for SpA content analysis. IL-6 concentrations were determined by ELISA (BD Biosciences) in cellular supernatants of 5 ⫻ 104 B cells/well after 72 h of stimulation. For the quantification of Igs, cells were stimulated for 13 days and IgM and IgG secretion was quantified with ELISA kits from Bethyl Laboratories. For TNF-␣ analysis in cellular supernatants, PBMC were stimulated for 24 h and TNF-␣ was quantified by ELISA (BD Biosciences). FIGURE 1. B cell response to peptidoglycan (PG) stimulation. A and B, Human naive (CD27⫺) B cells were stimulated with commercially available peptidoglycans from different Gram-positive species (A) or with pure peptidoglycans isolated from S. aureus (SA) cultures (B). C, Anti-Ig (aIg) was combined with pure insoluble peptidoglycan (iPG). All peptidoglycans were used at 5 g/ml. Proliferation rates were assessed by measurement of [3H]thymidine incorporation. The peptidoglycans used in A were S. aureus from Fluka (PG SA (Fl)), S. aureus from InvivoGen (PG SA (Inv)), B. subtilis (PG BS), and M. luteus (PG ML). The peptidoglycans used in B and C were soluble peptidoglycans (sPG) from penicillin-treated S. aureus cultures and insoluble PG (iPG) from S. aureus cell walls. Diagrams depict the means ⫾ SEM of four (A), three (B), and four (C) individual donors. Flow cytometry Cells were stained in FACS buffer (PBS, 0.5 mM EDTA, and 1% FCS) according to standard procedures. Analysis was performed on a FACSCalibur (BD Biosciences) with CellQuest Software. Unconjugated anti-human CD36 (FA6 –152) was purchased from Immunotech, and secondary anti-mouse IgG FITC was purchased from Sigma-Aldrich. All of other anti-human Abs used were purchased from BD Pharmingen: IgD FITC, CD27 PE, HLA-DR PerCP-Cy5.5, CD20 allophycocyanin, lineage FITC, CD123 PE, CD11c allophycocyanin, CD40 FITC, CD80 PE, CD86 allophycocyanin, CD19 PerCP or allophycocyanin, CD14 FITC, CD36 PE, TNF-␣ PE, and the necessary isotypes. Intracellular TNF-␣ secretion from CD14⫹ monocytes in PBMC was measured after a 4-h stimulation period in the presence of 1 g/ml brefeldin A following a standard protocol for intracellular flow cytometry. Intracellular IgM expression was measured on day 6 poststimulation. B cells were preincubated with unlabelled mouse-anti-human IgM at 10 g/ml (BD Biosciences), fixed, permeabilized, and stained with PElabeled anti-IgM Ab (BD Biosciences). Live cells were gated by forward scatter/side scatter exclusion of dead cells based on annexin V/propidium iodide-established reference standards. microliter/well) were gently mixed, incubated for 20 min at room temperature, and subsequently pipetted into the wells. After 16 –18 h, cells were washed with PBS and resuspended in 0.2 ml of RPMI 1640 medium with 10% FCS plus stimulants (cell walls at 5 g/ml, Pam3CSK4 at 100 ng/ml unless otherwise stated, MALP-2 at 5 ng/ml, and CpG DNA ODN 2006 at 3 g/ml). Stimulation time was 24 h. Supernatants were frozen and analyzed for IL-8 secretion by ELISA (BD Biosciences). For control experiments, Nod1- and Nod2-transfected HEK293 cells were cotransfected with an NF--B reporter construct and luciferase activity was determined after stimulation as described previously (59). The expression plasmids pNod1 and pNod2-HA were provided by Gabriel Nuñez (Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, MI). Statistics Data are depicted as mean ⫾ SEM. Statistical significance of the differences was determined by the paired two-tailed Student t test using Microsoft Excel software. Statistically significant differences are indicated with ⴱ for p ⱕ 0.05 and ⴱⴱ for p ⱕ 0.005. Quantitative real-time PCR After a 3- and 6-h stimulations of cells, the cell pellets were lysed in lysis buffer from the MagnaPure mRNA isolation kit I supplemented with 1% DTT (Roche). The reparation of mRNA was performed with the MagnaPure-LC device using the mRNA-I standard protocol as previously described (56, 57). Measurement of TLR2 and Nod activity HEK293 cells (5 ⫻ 104 per well) were plated in a 96-well flat-bottom plate on day 0 in RPMI 1640 medium with 10% FCS (see above). After 24 h the medium was substituted by 175 microliter of the HEK293 FreeStyle 293 expression medium (Invitrogen Life Technologies). For one well, 100 ng TLR2 plasmid (gift from C. Kirschning, Technical University of Munich, Munich Germany; Refs. 58 and 59), 0.25 l of Lipofectamine 2000 (Invitrogen Life Technologies), and HEK293 medium (to total volume of 25 Results Peptidoglycan from S. aureus stimulates B cell proliferation Previous studies on the adjuvant effects of peptidoglycan have described peptidoglycan preparations from S. aureus as strong adjuvants in comparison to peptidoglycan preparations from other bacterial species (20). We confirmed this finding by comparing commercially available peptidoglycan preparations derived from Gram-positive bacterial species in regard to their B cell activity. As shown in Fig. 1A, we found that the S. aureus peptidoglycan induced marked proliferation of human naive (CD27⫺CD19⫹) B cells, whereas the peptidoglycans from other Gram-positive bacteria (M. luteus and B. subtilis) failed to stimulate significant B cell Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017 times). After enzyme digestion, cell walls were incubated with hydrofluoridic acid (HF) 48% (Merck) for 48 h (26, 28, 29). After dialysis, the removal of residual LTA and WTA was controlled by absence of ribitol using gas chromatography. Lithium chloride was used as an 8 M solution to remove noncovalently bound proteins. As a final step, cell walls were washed in acetone. Cell wall fractions and peptidoglycan were lyophilized and weighed. For stimulation, 5 g/ml cell wall or peptidoglycan turned out to be optimal and were used throughout the experiments unless otherwise stated. Wood 46 cell walls gave better results at higher concentrations (15–50 g/ml) without reaching the potency of SAC cell walls (data not shown) but were also used at 5 g/ml. To ensure that residual SDS was not influencing cell survival, SDS was titrated and it was found that the concentrations of interest had no effect on B cell survival (data not shown). Some cell wall fractions as well as the commercially available peptidoglycans contain traces of endotoxin, most likely due to contamination during the isolation procedure; but in our assays the low amounts should be functionally irrelevant. For LP-enriched fractions, cell walls were digested with RNase and DNase without proteinase K or lysostaphin and subsequently stirred in 10 mM N-octyl--D-glucopyranoside (OG) (Sigma-Aldrich) for 24 h at 6°C (54). OG supernatants were filtrated on and Amicon BioSeparartion Ultrafree-CL, 5,000 nominal molecular weight limit device (Millipore) and washed with H2O, and the collected fractions were lyophilized. For stimulation, OG fractions were also used at 5 g/ml. Soluble peptidoglycan was prepared from penicillin-stimulated S. aureus cells (7.5 ml of a 10 mg/ml solution per 1 liter; Sigma-Aldrich) as described previously (55). 2805 2806 proliferation. Because S. aureus, M. luteus, and B. subtilis peptidoglycan all bear the muramyl dipeptide motif, these results indicated that the mechanism underlying B cell activation by S. aureus could not be explained by muramyl dipeptide recognition via Nod2. This hypothesis was further supported by the finding that human B cells lack Nod2 mRNA expression (data not shown). Commercially available peptidoglycan preparations usually consist of insoluble peptidoglycan preparations and may contain traces of TLR2-activating LP and other contaminating cell wall components (37). To exclude the effects of contaminating cell wall molecules, we decided to isolate peptidoglycan in its pure form (26, 28, 29, 36). To this end we isolated S. aureus soluble peptidoglycan and insoluble peptidoglycan as described in Materials and Methods. As shown in Fig. 1B, both soluble peptidoglycan isolated from the supernatant of S. aureus cultures after penicillinmediated inhibition of peptidoglycan synthesis (55) and insoluble peptidoglycan isolated from S. aureus cell walls failed to induce B cell proliferation. Because BCR stimulation has been shown to sensitize B cells to microbial ligands, the subsequent experiments were performed in the presence of anti-Ig. They showed that BCR stimulation did not rescue B cell activity of pure S. aureus peptidoglycan preparations; no synergy was observed with combined anti-Ig and insoluble peptidoglycan (Fig. 1C, left panel) or soluble peptidoglycan stimulation (data not shown). We concluded that B cell proliferation requires microbial molecules lost during the peptidoglycan isolation procedures. BCR engagement sensitizes B cells for TLR2 ligands Having excluded pure peptidoglycan as an important B cell stimulus, we concluded that LP-derived TLR2 activity may play an FIGURE 3. Stimulatory activity of S. aureus cell walls (CW) before and after chemical elimination of defined microbial substances. S. aureus whole cell walls were washed with either chloroform/methanol and H2O/ propanol (CW1) or with phenol/H2O (CW2). The insoluble fraction was treated with TCA, SDS, amylase (Amy), DNase, RNase, HF, trypsin (Tryps), LiCl and acetone (Acet) as described in Materials and Methods. The chronological order is visualized in the table below the diagram. The end product is purified insoluble peptidoglycan (iPG). Aliquots were preserved after each isolation step and tested for stimulatory activity; the results are shown in the diagrams. A, CD19⫹ B cells were stimulated with untreated and chemically treated cell walls at 5 and 50 g/ml as indicated. CpG DNA ODN 2006 (3 g/ml) was used as a positive control. The diagram shows the means of triplicate values of one representative experiment of four experiments. B, HEK293 cells were transfected with Lipofectamine with or without TLR2-encoding plasmid and stimulated with the cell wall fractions. TLR2-activity was quantified by measuring IL-8 secretion in the supernatants after 24 h. The diagram shows the means ⫾ SEM from exemplary cell wall fractions. The experiment is representative of three experiments. essential role in S. aureus-mediated B cell activation. Therefore, B cells were stimulated with known TLR2 ligands, e.g., diacylated and triacylated LP (MALP-2 and Pam3CSK4) and S. aureus LTA in the presence and absence of anti-Ig. The experimental results showed that naive (CD27⫺CD19⫹) B cell stimulation with TLR2active LP required simultaneous BCR ligation (Fig. 2A) and significantly enhanced memory B cell proliferation (Fig. 2B). Moreover, S. aureus-derived LTA failed to induce B cell proliferation despite the presence of anti-Ig and the use of high concentrations of LTA (up to 100 g/ml; data not shown). B cell stimulatory activity of S. aureus cell walls is lost after protein digestion Having defined the stimulatory conditions for TLR2-mediated B cell stimulation and knowing that TLR2-active LP would not be sufficient to stimulate B cell proliferation, we decided to isolate the elements responsible for S. aureus-derived B cell stimulatory activity from S. aureus cell walls. To this end, we decided to reduce the cell walls to pure peptidoglycan in a stepwise procedure. Partially purified peptidoglycan (insoluble peptidoglycan) was isolated from S. aureus whole cell walls as described in Materials and Methods and summarized in Fig. 3. Most importantly, Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017 FIGURE 2. TLR2 stimulation of human B cells. Naive (CD27⫺CD19⫹) (A) and memory (CD27⫹CD3⫺) (B) B cells were stimulated with the TLR2 ligands MALP-2 (M) (25 ng/ml) and Pam3CSK4 (P3) (500 ng/ml) in the presence and absence of anti-Ig (aIg) at 10 g/ml. Proliferation was assessed by the quantification of incorporated [3H]thymidine (counts per minute). The diagrams summarize the data (expressed as means ⫾ SEM) from independent experiments data in A, where n ⫽ 5 (TLR2), and B, where n ⫽ 7 (TLR2). In A, ⴱ, p ⫽ 0.03 for anti-Ig with or without MALP-2, and ⴱ, p ⫽ 0.04 for anti-Ig with or without Pam3CSK4; in B, ⴱⴱ, p ⫽ 0.001 for anti-Ig with or without MALP-2, and ⴱⴱ, p ⫽ 0.002 for anti-Ig with or without Pam3CSK4. S. aureus TRIGGERED B CELL ACTIVATION The Journal of Immunology SpA expression correlates with B cell activating capacity Because SpA has been described as a BCR stimulus and proposed to be an important inducer of B cell proliferation (4, 15–17, 51), we decided to test whether SpA could represent the protein component supporting cell wall-induced B cell proliferation. To prove this hypothesis, we compared cell wall preparations from two welldescribed S. aureus strains: S. aureus Cowan I (SAC; DSM 20372), a strain known for high SpA expression, and S. aureus Wood 46 (DSM 20491), a strain known to be deficient for SpA. As expected, the cell walls isolated from the SAC strain induced 10to12-fold higher proliferation rates (Fig. 4A) and ⬃4-fold higher IL-6 secretion rates (data not shown) than those from the Wood 46 strain. Again, digestion of the cell walls of both S. aureus strains with RNase and DNase as well as treatment with SDS and HF did not significantly alter their B cell stimulatory capacity (Fig. 4A), indicating that contaminating nucleic acids and TA were not essential for B cell activity. In contrast, the removal of protein from the cell walls with proteinase K digestion eliminated SpA from SAC cell walls (Fig. 4B) and abolished SAC cell wall B cell proliferation (Fig. 4A) and IL-6 secretion (data not shown) despite preserved TLR2 activity (Fig. 4C). SAC-derived B cell stimulatory activity does not depend on structural integrity of the cell walls Cell-bound SpA has been shown to serve as a more potent B cell stimulus than soluble SpA. This finding has been explained by a more efficient cross-linking of the BCR through its three-dimensional presentation of SpA on the cell wall (17). We argued that the stimulatory differences observed between cell-bound and soluble SpA could rather be based on the additional presence of TLR2 activity in the cell walls. Hence, we decided to use OG to extract FIGURE 4. Comparison of cell wall (CW) activities from the SAC strain and the Wood 46 strain. The activities of cell walls derived from S. aureus Wood 46 strain (■) or Cowan strain I (SAC; open columns) were compared with controls (unstimulated cells, cells stimulated with CpG ODN 2006 (3 g/ml), and HEK293 cells stimulated with TLR2-active LP ( )). As indicated in the diagrams whole cell walls (wCW) were compared with cell walls treated with SDS, RNase, DNase, and HF (CW-A) and additional proteinase K digestion (CW-PK). A, CD27⫺ B cells were stimulated with CpG ODN 2006 or S. aureus cell walls and compared with unstimulated cells. Proliferation was assessed by quantification of BrdU incorporation (RLU, relative light units). The diagram gives the means ⫾ SEM of n ⫽ 3 experiments. ⴱⴱ, p ⫽ 0.004 for CW Wood 46 and CW SAC; ⴱ, p ⫽ 0.007 for CW SAC with or without proteinase K. B, SpA content of the cell wall fractions (Wood 46 (W)) and Cowan I (SAC) was determined by SpA ELISA and is given as nanograms of SpA per milligram of cell wall fraction. The diagrams show representative results from one of two or more measurements. C, TLR2-transfected HEK293 cells were stimulated with S. aureus cell walls, Pam3CSK4 (P3, given in ng/ml), MALP-2 (M) (2.5 ng/ml), or CpG DNA ODN 2006 (3 g/ml). IL-8 secretion was quantified from supernatants after 24 h. The experiment shown is representative of two experiments with triplicates. lipids, LP, and proteins from bacterial cell walls (54), thus conserving the components but abolishing cell wall integrity. OG extraction from cell walls was performed after RNase and DNase treatment without proteinase K digestion. In line with the experiments using whole cell walls, the results showed that the OG fraction of Wood 46 lacked B cell stimulatory activity (Fig. 5A). In contrast, the OG fraction from the SAC strain displayed B cell stimulatory activity that was greatly diminished by proteinase K treatment (Fig. 5A). Accordingly, SpA was only identified in the OG fraction from SAC cell walls without proteinase treatment (data not shown). TLR2 activity was detected in all OG fractions including those from Wood 46 cell walls (Fig. 5B). Because B cell proliferation was observed with solubilized cell wall components, we concluded that S. aureus-derived B cell activity is dependent on the presence of SpA but independent of cell wall integrity. TLR2-active LP stimulate B cells in the presence of recombinant SpA We next sought to test whether recombinant SpA would synergize with synthetic TLR2-activating LPs or other TLR ligands. Indeed, recombinant SpA was found to act synergistically with both TLR ligands and CD40L (Fig. 6). The results shown in Fig. 6A demonstrated that the costimulation of enriched naive (CD27⫺CD19⫹) B cells with SpA enhances CD40L- (left panel), TLR7- (data not shown), and TLR9 (CpG DNA)-mediated (right panel) B cell proliferation and enables naive B cells to respond to the TLR2 stimuli such as Pam3CSK4. Similar results were obtained with memory B Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017 contaminating nucleic acids were removed by DNase and RNase treatment and TA were removed by treatment with TCA and HF which, in addition, destroys any residual contaminating nucleic acids. The absence of nucleic acids was controlled on ethidium bromide-stained agarose gels, and the successful removal of LTA and WTA was monitored by combined gas-liquid chromatography/mass spectrometry (data not shown). Amylase was used to remove hexoses and protein was removed by trypsin digestion and washes in LiCl (controlled by Bradford method). In a final step, acetone was used to remove residual lipids. After every single step, aliquots were preserved and tested for induction of B cell proliferation, which was assessed by nucleotide incorporation in ⱖ4 independent experiments. A representative experiment is shown in Fig. 3A comparing the B cell proliferative activity of chloroform/methanol (cell wall 1)- and phenol/H2O (cell wall 2)-pretreated whole cell walls in two different concentrations (5 and 50 g/ml) (Fig. 3A). Negative results in proliferation studies were defined as counts per minute values ⱕ120% of unstimulated control wells. Quantitative differences in proliferation rates should not be evaluated, because chemical procedures alter the proportions of the residual cell wall components and dry weight was used as a reference parameter in the absence of a better means for standardization. TLR2 activity was measured by quantification of IL-8 concentrations in the supernatants of TLR2-transfected HEK293 cells (Fig. 3B). Most strikingly, neither TCA nor SDS, amylase, RNase, DNase, or HF treatment affected the B cell stimulatory capacity of S. aureus cell walls (Fig. 3A). In contrast, trypsin digestion of the cell walls abolished the proliferative activity, indicating that a protein cell wall component is required for B cell stimulation. Moreover, TLR2 activity was preserved after trypsin digestion but was not sufficient on its own for B cell stimulation. 2807 2808 cell fractions (data not shown). The results also visualize the extent of the donor variability. In another set of experiments, recombinant SpA was titrated to test whether B cell proliferation can be stimulated by SpA in higher concentrations. In contrast to anti-Ig, which induces B cell proliferation in a concentration-dependent manner (Fig. 6B, right panel), recombinant SpA alone did not induce significant changes in B cell proliferation rates (Fig. 6B, left panel). Both SpA and anti-Ig synergized with Pam3CSK4, resulting in enhanced proliferation rates (Fig. 6B). Moreover, CD19⫹ B cell proliferation was monitored with CFSE dilution. In contrast to TLR9-mediated B cell activation, TLR2-triggered stimulation of B cell proliferation required a costimulus via anti-Ig or SpA. Notably, combined stimulation with SpA and TLR2-LP induced the vigorous proliferation of a small subfraction of B cells (Fig. 6C, arrow) not observed in combination with anti-Ig stimulation or with CpG DNA. Interestingly enough, this B cell subset was also observed when CD19⫹ B cells were stimulated with Wood 46 whole cell walls combined with recombinant SpA (Fig. 6D). B cell activation with SpA and TLR2 ligands is not sufficient to induce Ig synthesis B cell proliferation and cytokine secretion precedes plasma cell differentiation and Ig secretion. We therefore wanted to analyze Ab secretion from CD19⫹ B cells stimulated with S. aureus whole cell walls or TLR ligands in the presence or absence of recombinant SpA. We found that only CpG DNA ODN 2006 and R848, a TLR7 ligand, induced significant Ig production, which occurred FIGURE 6. SpA-mediated B cell activation in the presence of costimulation through TLRs. A, Human CD27⫺ B cells were stimulated with TLR ligands in the presence and absence of recombinant SpA (coated; 10 g/ ml). Cells were stimulated with BHK-CD40L or BHK-pTCF control cells, Pam3CSK4 (500 ng/ml), or CpG DNA ODN 2006 (2 g/ml). The data shown in the diagrams show the absolute counts per minute values for five independent donors. ⴱ, p ⫽ 0.02 for BHK-CD40L with or without SpA; ⴱ, p ⫽ 0.05 for Pam3CSK4 with or without SpA; ⴱ, p ⫽ 0.03 for CpG DNA ODN 2006 with or without SpA. B, Total CD19⫹ B cells were stimulated with anti-Ig (left panel) or recombinant SpA (right panel) in the presence (䊐) and absence (■) of Pam3CSK4 (P3; 1 g/ml). The anti-Ig and SpA concentrations used (given in g/ml) are indicated below the columns in the diagram. Proliferation was assessed by [3H]thymidine incorporation. The diagram gives the mean counts per minute values ⫾ SEM of four independent experiments. C, CFSE-stained total (CD19⫹) B cells were stimulated with the TLR ligands MALP-2 (1 g/ml), Pam3CSK4 (1 g/ ml), or CpG DNA ODN 2006 (2 g/ml) in the presence or absence of anti-Ig (5 g/ml) or SpA (5 g/ml). CFSE dilution was used to assess proliferation in comparison to unstimulated cells. The data shown are representative of six or more experiments. The arrow marks a population characterized by high CFSE dilution, e.g., vigorous proliferation. D, CFSEstained CD19⫹ B cells were stimulated with Wood 46 whole cell walls (5 g/ml) in the presence and absence of recombinant SpA (5 g/ml). Proliferation was assessed by CFSE dilution. The data shown are representative of four experiments. The arrow marks a strongly proliferating B cell population. independently of the addition of SpA. IgM secretion was not seen with either cell walls or TLR2 ligands despite the presence or absence of SpA (Fig. 7A). We therefore concluded that the induction of Ig synthesis may be a unique feature of TLR7- and TLR9mediated B cell activation not shared by other TLRs. Because previous reports had claimed that SpA and SAC could only activate B cells in the presence of T cell help, e.g., either T cell-derived cytokines (IL-2) or CD40 activation (60, 61), we costimulated SpA and Pam3CSK4-stimulated B cells with IL-2. The Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017 FIGURE 5. Protein and LP extraction from S. aureus cell walls. Whole cell walls from S. aureus were treated with RNase and DNase with or without proteinase K (PK). Afterward, OG was used for the extraction of LP and proteins from whole cell walls. After dialysis, the OG fractions were used for the stimulation of CD27⫺ B cells. Extracts from S. aureus Wood 46 strain (left, ■) are compared with extracts from Cowan strain I (right, 䊐). A, CD27⫺ B cells were stimulated with the protein/LP-containing OG-fractions (5 g/ml) and compared with unstimulated cells. Proliferation was assessed by measurement of BrdU incorporation (relative light units (RLU)). The diagram gives the means ⫾ SEM of n ⫽ 3 experiments. ⴱ, p ⫽ 0.04 for OG Wood 46 and OG SAC; ⴱ, p ⫽ 0.04 for OG SAC with or without protein kinase. B, TLR2-transfected HEK293 cells were stimulated for 24 h with OG fractions (■, Wood 46; 䊐, Cowan I) or TLR2 ligands ( ) as indicated (Pam3CSK4 (P3) at 200 ng/ml; MALP-2 (M) at 2.5 ng/ml). IL-8 concentrations in the supernatants were used to quantify TLR2-activity. S. aureus TRIGGERED B CELL ACTIVATION The Journal of Immunology 2809 results obtained showed that costimulation with IL-2 enhances CpG DNA-induced intracellular IgM expression (Fig. 7, B (left panel) and C) and induces intracellular IgM expression in a small B cell population (2– 4.5% of live cells) stimulated with TLR2active LP and SpA or anti-Ig (Fig. 7, B (right panel) and C). Thus, TLR9 activation may represent a polyclonal B cell activating principle triggering the expansion and Ig synthesis of a high percentage of human peripheral blood B cells. In contrast, TLR2-mediated B cell activation in the presence of SpA or a BCR stimulus may trigger T cell-independent B cell proliferation of a limited number of B cells, and only a restricted subset of B cells will subsequently synthesize Ig in the additional presence of T cell-derived cytokines such as IL-2. Discussion S. aureus lysates and cell wall preparations are frequently used as B cell mitogens. The present study introduces a new point of view on S. aureus-mediated B cell activation by providing evidence that SpA sensitizes human B cells for cell wall-derived TLR2-ligands. The experimental results show that a TLR2-active LP induce polyclonal B cell proliferation in the presence of anti-Ig or SpA stimulation but fail to induce significant Ig production. This stands in contrast to B cell activation via TLR7 and TLR9 ligands that both induce Ig secretion. Furthermore, our data attract attention to a small B cell subset proliferating vigorously in response to TLR2active LP and SpA. This B cell subset can be induced to express intracellular IgM in the presence of rhIL-2. Innate immune recognition of S. aureus and other Gram-positive bacteria has mainly been attributed to the presence of TLR2-active substances in the Gram-positive cell wall. For several years peptidoglycan was generally accepted as a TLR2 ligand (32). But recently Travassos et al. (37) provided evidence that peptidoglycan is only recognized by the Nod receptors and that highly purified peptidoglycan does not bind to TLR2. In our hands TLR2 activity is detectable in most crude peptidoglycan preparations but we have recently succeeded in separating the TLR2 activity from peptidoglycan, indicating that peptidoglycan is not the TLR2 ligand under investigation (62). This finding was later confirmed by Suda and coworkers (35), who found that LP rather than peptidoglycan possesses TLR2 activity, and by Hashimoto et al. (35, 39, 56), who demonstrated that S. aureus-derived TLR2 activity is mediated via LP. Additionally, Stoll et al. (38) created a mutant S. aureus strain deficient in lipoprotein diacylglyceryl transferase (lgt) that completely lacked acylated lipoproteins and produced only prelipoproteins. The lgt deletion mutant and its crude cell lysates were only very weak inducers of proinflammatory cytokines when compared with the wild-type strain. Based on the strong evidence available, we assume that the substances responsible for TLR2 activity in our cell wall preparations are LP. This is further supported by the observations that TLR2 activity in the solubilized OG fractions could be separated from insoluble peptidoglycan and that B cell activity of pure peptidoglycan preparations could not be rescued by the addition of anti-Ig (Fig. 1C), thus excluding the peptidoglycan molecule as a major B cell stimulus. Furthermore, TLR2 activity in the cell wall preparations proved to be heat and acid stable (e.g., not eliminated by boiling in SDS or treatment with TCA) (Fig. 3) but sensitive to Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017 FIGURE 7. IgM secretion after stimulation with S. aureus cell walls or TLR2 ligands. A, IgM secretion in CD19⫹ B cells stimulated with Wood 46 (W46) and Cowan I (SAC) whole cell walls or TLR ligands (Pam3CSK4 (500 ng/ml) (P3), MALP-2 (25 ng/ml) (M), R848 (0.25 g/ml), and CpG DNA ODN 2006 (3 g/ ml)) with (■) and without ( ) recombinant SpA (10 g/ml). The diagram depicts the mean IgM concentrations ⫾ SEM of three independent experiments. B and C, CD19⫹ B cells were stimulated with CpG ODN 2006 (1 g/ml) or Pam3CSK4 (P3) (1 g/ml) in combination with SpA (5 g/ml) or anti-Ig (aIg) (5 g/ml) in the presence or absence of rhIL-2 (10 ng/ml). Intracellular IgM expression was measured on day 6. B, The diagrams show the means ⫾ SEM of the percentage of intracellular IgM⫹ B cells of gated live cells for n ⫽ 4 experiments. ⴱ, p ⫽ 0.05 for Pam3CSK4 with SpA and with or without IL-2 C, Contour blots of three donors are depicted. The percentages of cells positive for intracellular IgM are indicated. 2810 Because SpA has recently been shown to bind and activate TNFR1 in human respiratory epithelial cells (68), we stimulated human B cells with TLR ligands (Pam3CSK4 and R848) in combination with TNF (10 –1000 ng/ml) and did not observe synergistic effects in terms of B cell proliferation (I. Bekeredjian-Ding, unpublished observation). These findings indicate that recombinant TNF cannot substitute for SpA in sensitizing B cells for TLR ligands, but they do not exclude the possibility that SpA may bind to receptors other than the VH3-BCR on human B cells. Other groups have convincingly demonstrated that SpA specifically triggers the apoptosis of VH3-BCR⫹ B cells (18, 69, 70). In line with these studies, we observe that SpA alone fails to induce B cell proliferation (Fig. 6) and that only synthetic BCR stimulation with anti-Ig results in proliferative activity (Fig. 6B). However, the present study proposes a different in vivo scenario: based on our data we postulate that under physiological conditions the body will encounter SpA in the presence of TLR2-active substances and that costimulation via TLR2 will counteract SpA-induced apoptosis and induce the expansion of SpA- and TLR2reactive B cells (Fig. 6). Moreover, the vigorously proliferating population shown in Fig. 6, C and D, may comprise VH3-BCR⫹ B cells, and it is tempting to speculate that, along with their B1 and MZ B cell murine counterparts, they may be prone to respond to bacterial stimulation. Our work further implies that S. aureus cell walls and peptidoglycan preparations represent very potent B cell activators by providing at least two signals (SpA and TLR2-active LP) that synergistically induce T cell-independent B cell activation and subsequent expansion. In contrast, we could not detect significant production of IgM after stimulation of B cells with S. aureus cell walls or SpA and TLR2-active LP (Fig. 7). Because TLR7 and TLR9 ligation stimulated IgM production, this finding suggested that different TLR stimuli provide qualitatively different signals and differ in their Ig induction potential. It therefore seems reasonable that Ig induction by commercially available S. aureus preparations such as Pansorbin may be related to the microbial DNA and RNA content of these preparations (71, 72). In the context of an infection, the presence of bacterial nucleic acids may serve as an indicator for the disintegration of a pathogen and will be associated with strong proliferation of bacteria and more severe types of infection. It seems ingenious that human B cells respond more strongly to microbial nucleic acids than to the surface molecules always present on endogenous microbial flora. Because previous studies using SAC lysates for Ig induction used IL-2-containing medium or worked with whole PBMC (7, 17, 60, 61, 73, 74) instead of purified B cells, we were interested in whether the addition of IL-2 would enable SpA plus TLR2-LPstimulated B cells to secrete IgM. Indeed, the addition of IL-2 promoted intracellular IgM expression in a small B cell subset stimulated with SpA and TLR2-LP. Because the arising IgM⫹ B cell population comprised only a very small B cell subset (2– 4.5% of gated live cells), we deducted that this B cell subset may correspond to the vigorously proliferating B cell subset observed with CFSE staining (Fig. 6, C and D). Due to the paucity of cells, we currently lack information on the nature of the Igs secreted and we can only speculate that these Igs represent unspecific IgM Abs directed at bacterial cell wall molecules as have been described in murine B1 cells. Anti-staphylococcal Abs have been shown to be protective against S. aureus infection in both human and mouse, and insufficient humoral responses have been associated with severe infections and relapse (75– 82). Our data suggest that S. aureus induces Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017 alkaline treatment (I. Bekeredjian-Ding, unpublished observation), characteristic features of LP. Although the LTA isolated from the S. aureus lgt mutant deficient in acylated LP was devoid of TLR2 activity (39), S. aureus LTA has repeatedly been shown to activate the innate immune response and therefore remained a possible candidate molecule involved in B cell activation by S. aureus (63, 64), albeit in a TLR2-independent fashion. To clarify the role of LTA or WTA in S. aureus-triggered B cell activation, we stimulated B cells with LTA and WTA with and without anti-Ig and observed no B cell activity (data not shown). This finding was supported by the fact that despite the removal of TA from S. aureus cell walls, both the intrinsic B cell activity and the TLR2 activity were preserved. Moreover, LTA activity has been shown to depend on its interaction with CD36 (65, 66), a surface receptor that was not detectable on stimulated and unstimulated B cells (data not shown). We therefore concluded that TA are not essential for S. aureus-mediated B cell activation. However, the potency of B cell stimulation by S. aureus cell wall preparations has so far been attributed to SpA (4, 15–17), and our results demonstrate that B cell activity is abolished by protein digestion despite preserved TLR2 activity (Figs. 4 and 5). Moreover, our study clearly demonstrates an essential role for SpA in B cell expansion: the SpA-deficient Wood 46 S. aureus strain displayed greatly diminished B cell activity despite conserved TLR2 activity. In contrast soluble recombinant SpA failed to induce B cell proliferation in the absence of a costimulus even when coated to the culture plate (Fig. 6A). Because Romagnani et al. (17) had published that soluble and cell-bound SpA differ in their B cellactivating potential, it was crucial to demonstrate that the B cell activating capacity of S. aureus does not depend on the integrity of the cell walls or SpA presentation on the cell walls. This was achieved by showing that the solubilized cell wall components in the OG fractions display potent B cell activity (Fig. 5). Furthermore, we were able to show that soluble SpA gains B cellactivating capacity in the presence of CD40L or TLR costimulation (Fig. 6). Although it is well-recognized that SpA activates VH3-BCR⫹ B cells (5, 13, 14, 16), its mechanism of action used for sensitization of B cells toward TLR2 ligands remains unclear. The present data only provide evidence that SpA sensitizes human B cells for TLR ligands as seen with anti-Ig stimulation. Because recent reports claimed that BCR engagement induces de novo synthesis of TLRs and thereby enhances the sensitivity of B cells toward TLR stimuli (46, 47), we compared anti-Ig with SpA stimulation in regard to TLR mRNA induction, but TLR and Nod mRNA levels remained unchanged in both conditions (I. Bekeredjian-Ding and T. Giese, unpublished results). We can therefore only conclude that both stimuli most likely sensitize B cells for TLR activation via other more complex mechanisms that may involve subcellular redistribution of TLRs or their adapter proteins. Furthermore, anti-Ig and SpA may act via distinct pathways. Similar to anti-Ig and SpA, CD40 stimulation has been shown to synergize with TLR ligands (46, 67). Because we were able to reproduce this finding with TLR2 ligands (I. Bekeredjian-Ding, unpublished data), we postulate that the requirement for a costimulatory signal in TLR2-mediated B cell activation may follow a general principle and may be found with many other costimuli. In addition, SpA does not stimulate TLR2-induced IL-8 secretion in TLR2-transfected HEK293 cells (I. Bekeredjian-Ding, unpublished data). Taking this into consideration and presupposing that B cell sensitization for TLR ligands require simultaneous stimulation of two distinct signaling pathways, we suggest that SpA does not act via direct TLR2 or most likely other TLR activation. S. aureus TRIGGERED B CELL ACTIVATION The Journal of Immunology B cell proliferation by rendering B cells sensitive to cell wallassociated TLR2-LP with SpA. We therefore speculate that S. aureus-induced T cell-independent B cell expansion may serve the pathogen in circumventing Ag-specific B cell responses. This evasion strategy may, in turn, be used by other bacteria and parasites bearing Ig-binding proteins such as protein G-expressing Streptococcus species, protein L-expressing Peptostreptococcus magnus, and Plasmodium falciparum (83– 85). In addition, any microbial molecule triggering T cell-independent B cell expansion, such as the HIV-derived protein Nef that acts via C-type lectin receptors, may circumvent Ag-specific B cell responses (85, 86). Acknowledgments We thank Thomas Hartung (University of Konstanz, Konstanz, Germany) for providing purified S. aureus LTA, Hartmut Engelmann (University of Munich, Munich, Germany) for providing the CD40L-transfected BHK cells, C. Kirschning (Technical University of Munich, Munich, Germany) for providing the TLR2 and CD14 plasmids and Gabriel Nuñez (University of Michigan Medical School, Ann Arbor, Michigan) for pNod1 and pNod2-HA expression plasmids. The authors have no financial conflict of interest. References 1. Forsgren, A., and J. Sjoquist. 1966. “Protein A” from S. aureus, I: pseudo-immune reaction with human gamma-globulin. J. Immunol. 97: 822– 827. 2. Jansson, B., M. Uhlen, and P. A. Nygren. 1998. All individual domains of staphylococcal protein A show Fab binding. FEMS Immunol. Med. Microbiol. 20: 69 –78. 3. Moks, T., L. Abrahmsen, B. Nilsson, U. Hellman, J. Sjoquist, and M. Uhlen. 1986. Staphylococcal protein A consists of five IgG-binding domains. Eur. J. Biochem. 156: 637– 643. 4. Palmqvist, N., G. J. Silverman, E. Josefsson, and A. Tarkowski. 2005. Bacterial cell wall-expressed protein A triggers supraclonal B-cell responses upon in vivo infection with Staphylococcus aureus. Microbes Infect. 7: 1501–1511. 5. Sasso, E. H., G. J. Silverman, and M. Mannik. 1989. Human IgM molecules that bind staphylococcal protein A contain VHIII H chains. J. Immunol. 142: 2778 –2783. 6. Uhlen, M., B. Guss, B. Nilsson, S. Gatenbeck, L. Philipson, and M. Lindberg. 1984. Complete sequence of the staphylococcal gene encoding protein A. A gene evolved through multiple duplications. J. Biol. Chem. 259: 1695–1702. 7. Ferry, B. L., J. Jones, E. A. Bateman, N. Woodham, K. Warnatz, M. Schlesier, S. A. Misbah, H. H. Peter, and H. M. Chapel. 2005. Measurement of peripheral B cell subpopulations in common variable immunodeficiency (CVID) using a whole blood method. Clin. Exp. Immunol. 140: 532–539. 8. Silverman, G. J. 1998. B cell superantigens: possible roles in immunodeficiency and autoimmunity. Semin. Immunol. 10: 43–55. 9. Silverman, G. J., and C. S. Goodyear. 2002. A model B-cell superantigen and the immunobiology of B lymphocytes. Clin. Immunol. 102: 117–134. 10. Silverman, G. J., J. V. Nayak, K. Warnatz, F. F. Hajjar, S. Cary, H. Tighe, and V. E. Curtiss. 1998. The dual phases of the response to neonatal exposure to a VH family-restricted staphylococcal B cell superantigen. J. Immunol. 161: 5720 –5732. 11. Cary, S., M. Krishnan, T. N. Marion, and G. J. Silverman. 1999. The murine clan V(H) III related 7183, J606 and S107 and DNA4 families commonly encode for binding to a bacterial B cell superantigen. Mol. Immunol. 36: 769 –776. 12. Graille, M., E. A. Stura, A. L. Corper, B. J. Sutton, M. J. Taussig, J. B. Charbonnier, and G. J. Silverman. 2000. Crystal structure of a Staphylococcus aureus protein A domain complexed with the Fab fragment of a human IgM antibody: structural basis for recognition of B-cell receptors and superantigen activity. Proc. Natl. Acad. Sci. USA 97: 5399 –5404. 13. Potter, K. N., Y. Li, and J. D. Capra. 1996. Staphylococcal protein A simultaneously interacts with framework region 1, complementarity-determining region 2, and framework region 3 on human VH3-encoded Igs. J. Immunol. 157: 2982–2988. 14. Potter, K. N., Y. Li, V. Pascual, and J. D. Capra. 1997. Staphylococcal protein A binding to VH3 encoded immunoglobulins. Int. Rev. Immunol. 14: 291–308. 15. Romagnani, S., G. M. Giudizi, F. Almerigogna, R. Biagiotti, G. Bellesi, F. Bernardi, and M. Ricci. 1981. Protein A reactivity of IgM- and IgD-bearing lymphocytes from some patients with chronic lymphocytic leukemia. Clin. Immunol. Immunopathol. 19: 139 –148. 16. Romagnani, S., M. G. Giudizi, R. Biagiotti, F. Almerigogna, E. Maggi, G. Del Prete, and M. Ricci. 1981. Surface immunoglobulins are involved in the interaction of protein A with human B cells and in the triggering of B cell proliferation induced by protein A-containing Staphylococcus aureus. J. Immunol. 127: 1307–1313. 17. Romagnani, S., A. Amadori, M. G. Giudizi, R. Biagiotti, E. Maggi, and M. Ricci. 1978. Different mitogenic activity of soluble and insoluble staphylococcal protein A (SPA). Immunology 35: 471– 478. 18. Goodyear, C. S., and G. J. Silverman. 2004. Staphylococcal toxin induced preferential and prolonged in vivo deletion of innate-like B lymphocytes. Proc. Natl. Acad. Sci. USA 101: 11392–11397. 19. Silverman, G. J. 2001. Adoptive transfer of a superantigen-induced hole in the repertoire of natural IgM-secreting cells. Cell. Immunol. 209: 76 – 80. 20. Stewart-Tull, D. E. 1980. The immunological activities of bacterial peptidoglycans. Annu. Rev. Microbiol. 34: 311–340. 21. Dziarski, R., and D. Gupta. 2005. Peptidoglycan recognition in innate immunity. J. Endotoxin Res. 11: 304 –310. 22. Takeuchi, O., K. Hoshino, T. Kawai, H. Sanjo, H. Takada, T. Ogawa, K. Takeda, and S. Akira. 1999. Differential roles of TLR2 and TLR4 in recognition of gramnegative and gram-positive bacterial cell wall components. Immunity 11: 443– 451. 23. Takeuchi, O., K. Takeda, K. Hoshino, O. Adachi, T. Ogawa, and S. Akira. 2000. Cellular responses to bacterial cell wall components are mediated through MyD88-dependent signaling cascades. Int. Immunol. 12: 113–117. 24. Underhill, D. M., A. Ozinsky, K. D. Smith, and A. Aderem. 1999. Toll-like receptor-2 mediates mycobacteria-induced proinflammatory signaling in macrophages. Proc. Natl. Acad. Sci. USA 96: 14459 –14463. 25. Schwandner, R., R. Dziarski, H. Wesche, M. Rothe, and C. J. Kirschning. 1999. Peptidoglycan- and lipoteichoic acid-induced cell activation is mediated by tolllike receptor 2. J. Biol. Chem. 274: 17406 –17409. 26. Girardin, S. E., L. H. Travassos, M. Herve, D. Blanot, I. G. Boneca, D. J. Philpott, P. J. Sansonetti, and D. Mengin-Lecreulx. 2003. Peptidoglycan molecular requirements allowing detection by Nod1 and Nod2. J. Biol. Chem. 278: 41702– 41708. 27. Fritz, J. H., S. E. Girardin, C. Fitting, C. Werts, D. Mengin-Lecreulx, M. Caroff, J. M. Cavaillon, D. J. Philpott, and M. Adib-Conquy. 2005. Synergistic stimulation of human monocytes and dendritic cells by Toll-like receptor 4 and NOD1and NOD2-activating agonists. Eur. J. Immunol. 35: 2459 –2470. 28. Girardin, S. E., I. G. Boneca, L. A. Carneiro, A. Antignac, M. Jehanno, J. Viala, K. Tedin, M. K. Taha, A. Labigne, U. Zahringer, et al. 2003. Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan. Science 300: 1584 –1587. 29. Girardin, S. E., I. G. Boneca, J. Viala, M. Chamaillard, A. Labigne, G. Thomas, D. J. Philpott, and P. J. Sansonetti. 2003. Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J. Biol. Chem. 278: 8869 – 8872. 30. Watanabe, T., A. Kitani, P. J. Murray, and W. Strober. 2004. NOD2 is a negative regulator of Toll-like receptor 2-mediated T helper type 1 responses. Nat. Immunol. 5: 800 – 808. 31. Watanabe, T., A. Kitani, and W. Strober. 2005. NOD2 regulation of Toll-like receptor responses and the pathogenesis of Crohn’s disease. Gut 54: 1515–1518. 32. Dziarski, R., and D. Gupta. 2005. Staphylococcus aureus peptidoglycan is a toll-like receptor 2 activator: a reevaluation. Infect. Immun. 73: 5212–5216. 33. Yoshimura, A., E. Lien, R. R. Ingalls, E. Tuomanen, R. Dziarski, and D. Golenbock. 1999. Cutting edge: recognition of Gram-positive bacterial cell wall components by the innate immune system occurs via Toll-like receptor 2. J. Immunol. 163: 1–5. 34. Takeda, K., T. Kaisho, and S. Akira. 2003. Toll-like receptors. Annu. Rev. Immunol. 21: 335–376. 35. Hashimoto, M., K. Tawaratsumida, H. Kariya, K. Aoyama, T. Tamura, and Y. Suda. 2006. Lipoprotein is a predominant Toll-like receptor 2 ligand in Staphylococcus aureus cell wall components. Int. Immunol. 18: 355–362. 36. Inamura, S., Y. Fujimoto, A. Kawasaki, Z. Shiokawa, E. Woelk, H. Heine, B. Lindner, N. Inohara, S. Kusumoto, and K. Fukase. 2006. Synthesis of peptidoglycan fragments and evaluation of their biological activity. Org. Biomol. Chem. 4: 232–242. 37. Travassos, L. H., S. E. Girardin, D. J. Philpott, D. Blanot, M. A. Nahori, C. Werts, and I. G. Boneca. 2004. Toll-like receptor 2-dependent bacterial sensing does not occur via peptidoglycan recognition. EMBO Rep. 5: 1000 –1006. 38. Stoll, H., J. Dengjel, C. Nerz, and F. Gotz. 2005. Staphylococcus aureus deficient in lipidation of prelipoproteins is attenuated in growth and immune activation. Infect. Immun. 73: 2411–2423. 39. Hashimoto, M., K. Tawaratsumida, H. Kariya, A. Kiyohara, Y. Suda, F. Krikae, T. Kirikae, and F. Gotz. 2006. Not lipoteichoic acid but lipoproteins appear to be the dominant immunobiologically active compounds in Staphylococcus aureus. J. Immunol. 177: 3162–3169. 40. Bekeredjian-Ding, I. B., M. Wagner, V. Hornung, T. Giese, M. Schnurr, S. Endres, and G. Hartmann. 2005. Plasmacytoid dendritic cells control TLR7 sensitivity of naive B cells via type I IFN. J. Immunol. 174: 4043– 4050. 41. Hartmann, G., and A. M. Krieg. 2000. Mechanism and function of a newly identified CpG DNA motif in human primary B cells. J. Immunol. 164: 944 –953. 42. He, B., X. Qiao, and A. Cerutti. 2004. CpG DNA induces IgG class switch DNA recombination by activating human B cells through an innate pathway that requires TLR9 and cooperates with IL-10. J. Immunol. 173: 4479 – 4491. 43. Lin, L., A. J. Gerth, and S. L. Peng. 2004. CpG DNA redirects class-switching towards “Th1-like” Ig isotype production via TLR9 and MyD88. Eur. J. Immunol. 34: 1483–1487. 44. Borsutzky, S., K. Kretschmer, P. D. Becker, P. F. Muhlradt, C. J. Kirschning, S. Weiss, and C. A. Guzman. 2005. The mucosal adjuvant macrophage-activating lipopeptide-2 directly stimulates B lymphocytes via the TLR2 without the need of accessory cells. J. Immunol. 174: 6308 – 6313. 45. Mansson, A., M. Adner, U. Hockerfelt, and L. O. Cardell. 2006. A distinct Tolllike receptor repertoire in human tonsillar B cells, directly activated by PamCSK, R-837 and CpG-2006 stimulation. Immunology 118: 539 –548. Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017 Disclosures 2811 2812 66. Stuart, L. M., J. Deng, J. M. Silver, K. Takahashi, A. A. Tseng, E. J. Hennessy, R. A. Ezekowitz, and K. J. Moore. 2005. Response to Staphylococcus aureus requires CD36-mediated phagocytosis triggered by the COOH-terminal cytoplasmic domain. J. Cell Biol. 170: 477– 485. 67. Wagner, M., H. Poeck, B. Jahrsdoerfer, S. Rothenfusser, D. Prell, B. Bohle, E. Tuma, T. Giese, J. W. Ellwart, S. Endres, and G. Hartmann. 2004. IL-12p70dependent Th1 induction by human B cells requires combined activation with CD40 ligand and CpG DNA. J. Immunol. 172: 954 –963. 68. Gomez, M. I., A. Lee, B. Reddy, A. Muir, G. Soong, A. Pitt, A. Cheung, and A. Prince. 2004. Staphylococcus aureus protein A induces airway epithelial inflammatory responses by activating TNFR1. Nat. Med. 10: 842– 848. 69. Goodyear, C. S., and G. J. Silverman. 2003. Death by a B cell superantigen: in vivo VH-targeted apoptotic supraclonal B cell deletion by a staphylococcal toxin. J. Exp. Med. 197: 1125–1139. 70. Viau, M., N. S. Longo, P. E. Lipsky, and M. Zouali. 2005. Staphylococcal protein a deletes B-1a and marginal zone B lymphocytes expressing human immunoglobulins: an immune evasion mechanism. J. Immunol. 175: 7719 –7727. 71. Wagner, H. 1999. Bacterial CpG DNA activates immune cells to signal infectious danger. Adv. Immunol. 73: 329 –368. 72. Wagner, H. 2001. Toll meets bacterial CpG-DNA. Immunity 14: 499 –502. 73. Crotty, S., R. D. Aubert, J. Glidewell, and R. Ahmed. 2004. Tracking human antigen-specific memory B cells: a sensitive and generalized ELISPOT system. J. Immunol. Methods 286: 111–122. 74. Devos, R., B. Jayaram, P. Vandenabeele, and W. Fiers. 1985. Recombinant interleukin 2 induces immunoglobulin secretion in Staphylococcus aureus Cowan strain I activated human B-cells. Immunol. Lett. 11: 101–105. 75. Nilsson, I. M., J. M. Patti, T. Bremell, M. Hook, and A. Tarkowski. 1998. Vaccination with a recombinant fragment of collagen adhesin provides protection against Staphylococcus aureus-mediated septic death. J. Clin. Invest. 101: 2640 –2649. 76. Dryla, A., S. Prustomersky, D. Gelbmann, M. Hanner, E. Bettinger, B. Kocsis, T. Kustos, T. Henics, A. Meinke, and E. Nagy. 2005. Comparison of antibody repertoires against Staphylococcus aureus in healthy individuals and in acutely infected patients. Clin. Diagn. Lab. Immunol. 12: 387–398. 77. Monteil, M., J. Hobbs, and K. Citron. 1987. Selective immunodeficiency affecting staphylococcal response. Lancet 2: 880 – 883. 78. Fattom, A. I., J. Sarwar, L. Basham, S. Ennifar, and R. Naso. 1998. Antigenic determinants of Staphylococcus aureus type 5 and type 8 capsular polysaccharide vaccines. Infect. Immun. 66: 4588 – 4592. 79. Fattom, A. I., J. Sarwar, A. Ortiz, and R. Naso. 1996. A Staphylococcus aureus capsular polysaccharide (CP) vaccine and CP-specific antibodies protect mice against bacterial challenge. Infect. Immun. 64: 1659 –1665. 80. Hall, A. E., P. J. Domanski, P. R. Patel, J. H. Vernachio, P. J. Syribeys, E. L. Gorovits, M. A. Johnson, J. M. Ross, J. T. Hutchins, and J. M. Patti. 2003. Characterization of a protective monoclonal antibody recognizing Staphylococcus aureus MSCRAMM protein clumping factor A. Infect. Immun. 71: 6864 – 6870. 81. Domanski, P. J., P. R. Patel, A. S. Bayer, L. Zhang, A. E. Hall, P. J. Syribeys, E. L. Gorovits, D. Bryant, J. H. Vernachio, J. T. Hutchins, and J. M. Patti. 2005. Characterization of a humanized monoclonal antibody recognizing clumping factor A expressed by Staphylococcus aureus. Infect. Immun. 73: 5229 –5232. 82. Josefsson, E., O. Hartford, L. O’Brien, J. M. Patti, and T. Foster. 2001. Protection against experimental Staphylococcus aureus arthritis by vaccination with clumping factor A, a novel virulence determinant. J. Infect. Dis. 184: 1572–1580. 83. Donati, D., L. P. Zhang, A. Chene, Q. Chen, K. Flick, M. Nystrom, M. Wahlgren, and M. T. Bejarano. 2004. Identification of a polyclonal B-cell activator in Plasmodium falciparum. Infect. Immun. 72: 5412–5418. 84. Goodyear, C. S., M. Narita, and G. J. Silverman. 2004. In vivo VL-targeted activation-induced apoptotic supraclonal deletion by a microbial B cell toxin. J. Immunol. 172: 2870 –2877. 85. Silverman, G. J., and C. S. Goodyear. 2006. Confounding B-cell defences: lessons from a staphylococcal superantigen. Nat. Rev. Immunol. 6: 465– 475. 86. He, B., X. Qiao, P. J. Klasse, A. Chiu, A. Chadburn, D. M. Knowles, J. P. Moore, and A. Cerutti. 2006. HIV-1 envelope triggers polyclonal Ig class switch recombination through a CD40-independent mechanism involving BAFF and C-type lectin receptors. J. Immunol. 176: 3931–3941. Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017 46. Ruprecht, C. R., and A. Lanzavecchia. 2006. Toll-like receptor stimulation as a third signal required for activation of human naive B cells. Eur. J. Immunol. 36: 810 – 816. 47. Bernasconi, N. L., N. Onai, and A. Lanzavecchia. 2003. A role for Toll-like receptors in acquired immunity: up-regulation of TLR9 by BCR triggering in naive B cells and constitutive expression in memory B cells. Blood 101: 4500 – 4504. 48. Bourke, E., D. Bosisio, J. Golay, N. Polentarutti, and A. Mantovani. 2003. The toll-like receptor repertoire of human B lymphocytes: inducible and selective expression of TLR9 and TLR10 in normal and transformed cells. Blood 102: 956 –963. 49. Moore, C. E., S. Segal, A. R. Berendt, A. V. Hill, and N. P. Day. 2004. Lack of association between Toll-like receptor 2 polymorphisms and susceptibility to severe disease caused by Staphylococcus aureus. Clin. Diagn. Lab. Immunol. 11: 1194 –1197. 50. Texereau, J., J. D. Chiche, W. Taylor, G. Choukroun, B. Comba, and J. P. Mira. 2005. The importance of Toll-like receptor 2 polymorphisms in severe infections. Clin. Infect. Dis. 41(Suppl. 7): S408 –S415. 51. Kessler, S. W. 1975. Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J. Immunol. 115: 1617–1624. 52. Dziarski, R. 1982. Studies on the mechanism of peptidoglycan- and lipopolysaccharide-induced polyclonal activation. Infect. Immun. 35: 507–514. 53. Rasanen, L., and H. Arvilommi. 1981. Cell walls, peptidoglycans, and teichoic acids of gram-positive bacteria as polyclonal inducers and immunomodulators of proliferative and lymphokine responses of human B and T lymphocytes. Infect. Immun. 34: 712–717. 54. Muhlradt, P. F., M. Kiess, H. Meyer, R. Sussmuth, and G. Jung. 1997. Isolation, structure elucidation, and synthesis of a macrophage stimulatory lipopeptide from Mycoplasma fermentans acting at picomolar concentration. J. Exp. Med. 185: 1951–1958. 55. Rosenthal, R. S., and R. Dziarski. 1994. Isolation of peptidoglycan and soluble peptidoglycan fragments. Methods Enzymol. 235: 253–285. 56. Bekeredjian-Ding, I., S. I. Roth, S. Gilles, T. Giese, A. Ablasser, V. Hornung, S. Endres, and G. Hartmann. 2006. T cell-independent, TLR-induced IL-12p70 production in primary human monocytes. J. Immunol. 176: 7438 –7446. 57. Hornung, V., S. Rothenfusser, S. Britsch, A. Krug, B. Jahrsdorfer, T. Giese, S. Endres, and G. Hartmann. 2002. Quantitative expression of toll-like receptor 1–10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides. J. Immunol. 168: 4531– 4537. 58. Kirschning, C. J., H. Wesche, T. Merrill Ayres, and M. Rothe. 1998. Human toll-like receptor 2 confers responsiveness to bacterial lipopolysaccharide. J. Exp. Med. 188: 2091–2097. 59. Sing, A., D. Rost, N. Tvardovskaia, A. Roggenkamp, A. Wiedemann, C. J. Kirschning, M. Aepfelbacher, and J. Heesemann. 2002. Yersinia V-antigen exploits toll-like receptor 2 and CD14 for interleukin 10-mediated immunosuppression. J. Exp. Med. 196: 1017–1024. 60. Romagnani, S., G. Del Prete, M. G. Giudizi, R. Biagiotti, F. Almerigogna, A. Tiri, A. Alessi, M. Mazzetti, and M. Ricci. 1986. Direct induction of human B-cell differentiation by recombinant interleukin-2. Immunology 58: 31–35. 61. Romagnani, S., G. M. Giudizi, F. Almerigogna, R. Biagiotti, A. Alessi, C. Mingari, C. M. Liang, L. Moretta, and M. Ricci. 1986. Analysis of the role of interferon-␥, interleukin 2 and a third factor distinct from interferon-␥ and interleukin 2 in human B cell proliferation: evidence that they act at different times after B cell activation. Eur. J. Immunol. 16: 623– 629. 62. Inamura, S., E. Woelk, H. Heine, and U. Zähringer. 2004. In search of the TLR2activity in peptidoglycan from Staphylococcus aureus. J. Endotox. Res. 351: 10. 63. Schroder, N. W., S. Morath, C. Alexander, L. Hamann, T. Hartung, U. Zahringer, U. B. Gobel, J. R. Weber, and R. R. Schumann. 2003. Lipoteichoic acid (LTA) of Streptococcus pneumoniae and Staphylococcus aureus activates immune cells via Toll-like receptor (TLR)-2, lipopolysaccharide-binding protein (LBP), and CD14, whereas TLR4 and MD-2 are not involved. J. Biol. Chem. 278: 15587–15594. 64. Ginsburg, I. 2002. Role of lipoteichoic acid in infection and inflammation. Lancet Infect. Dis. 2: 171–179. 65. Hoebe, K., P. Georgel, S. Rutschmann, X. Du, S. Mudd, K. Crozat, S. Sovath, L. Shamel, T. Hartung, U. Zahringer, and B. Beutler. 2005. CD36 is a sensor of diacylglycerides. Nature 433: 523–527. S. aureus TRIGGERED B CELL ACTIVATION