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Staphylococcus aureus Protein A Triggers T
Cell-Independent B Cell Proliferation by
Sensitizing B Cells for TLR2 Ligands
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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
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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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
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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
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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
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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,
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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
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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
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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
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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
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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