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J Biol Chem, Vol. 273, Issue 15, 8680-8690, April 10, 1998
From the The hypothesis that soluble peptidoglycan (sPGN,
a macrophage-activator from Gram-positive bacteria) binds to CD14 (a
lipopolysaccharide (LPS) receptor) was tested. sPGN specifically bound
to CD14 in the following three assays: binding of soluble
32P-CD14 (sCD14) to agarose-immobilized sPGN,
enzyme-linked immunosorbent assay, and photoaffinity cross-linking.
sCD14 also specifically bound to agarose-immobilized muramyl dipeptide
or GlcNAc-muramyl dipeptide but not to PGN pentapeptide. Binding of
sCD14 to both sPGN and ReLPS (where ReLPS is LPS from Salmonella
minnesota Re 595) was competitively inhibited by unlabeled sCD14,
1-152 N-terminal fragment of sCD14, sPGN, smooth LPS, ReLPS, lipid A,
and lipoteichoic acid but not by dextran, dextran sulfate, heparin,
ribitol teichoic acid, or soluble low molecular weight PGN fragments.
Binding of sCD14 to sPGN was slower than to ReLPS but of higher
affinity (KD = 25 nM versus
41 nM). LPS-binding protein (LBP) increased the binding of
sCD14 to sPGN by adding another lower affinity KD
and another higher Bmax, but for ReLPS, LBP increased the affinity of binding by yielding two
KD with significantly higher affinity (7.1 and
27 nM). LBP also enhanced inhibition of sCD14
binding by LPS, ReLPS, and lipid A. Binding of sCD14 to both sPGN and
ReLPS was inhibited by anti-CD14 MEM-18 mAb, but other anti-CD14 mAbs
showed differential inhibition, suggesting conformational binding sites
on CD14 for sPGN and LPS, that are partially identical and partially
different.
Peptidoglycan (PGN)1 is
a polymer of alternating GlcNAc and MurNAc cross-linked by short
peptides, present in the cell walls of all bacteria (1). PGN, similar
to lipopolysaccharide (LPS) from the cell walls of Gram-negative
bacteria, can reproduce most of the clinical manifestations of
bacterial infections, including fever, acute-phase response,
inflammation, septic shock, leukocytosis, sleepiness, malaise, abscess
formation, and arthritis. Most of these effects are due to the release
of cytokines and other mediators from macrophages and other cells
(2-7). To prevent deleterious effects of these bacterial products on
the host, it is essential to understand in detail the mechanism of cell
activation by PGN and LPS. Since the first step in cell activation is
the interaction of the activator with the cell surface receptor, we
decided to identify and characterize the receptors for PGN on
macrophages.
CD14 is a glycosylphosphatidylinositol-linked 55-kDa protein present on
the surface of macrophages and polymorphonuclear leukocytes that
functions as the cell surface receptor for LPS (8-14). We have
recently shown that CD14 also functions as a receptor for PGN, because
PGN-unresponsive CD14-negative cells become responsive to PGN following
transfection of these cells with CD14 and expression of CD14 on their
cell surface (15), and because anti-CD14 mAbs inhibit activation of
CD14-positive cells by PGN (16) and also inhibit binding of PGN to
CD14-positive cells (17). Other studies have also shown that CD14 may
function as a receptor for other bacterial products, such as mannuronic
acid polymers from Pseudomonas (18), insoluble cell wall
fragments from several Gram-positive bacteria (15, 19), mycobacterial
lipoarabinomannan (15, 19, 20), rhamnose-glucose polymer from
Streptococcus (21), and lipoteichoic acid (LTA) (22) or
LTA-like molecule (23) from Gram-positive bacteria.
It was, therefore, proposed that CD14 functions as a "pattern
recognition receptor" (19) that can recognize shared features of
microbial cell surface components and enable the host to respond to
pathogenic bacteria but not to a great variety of other non-microbial polysaccharides. This model, which assumes that CD14 can discriminate between different ligands and can control the specificity of macrophage responses, was recently questioned (24) based on the inability of CD14
to discriminate between agonistic and antagonistic derivatives of LPS
(25). The second alternative model was proposed (24), according to
which CD14 would function as an albumin-like carrier molecule that
binds a large variety of molecules without recognition specificity and
that it would then transfer these molecules to another, as yet
unidentified recognition/cell-activating molecule in the cell membrane.
In a third "combinatorial" model (24), both CD14 and the putative
recognition/cell-activating molecule would contribute to the
specificity of the response. In addition, other possible models can be
proposed, e.g. bacterial cell wall products other than LPS
(such as PGN), could bind to another molecule, which in turn would
activate CD14, or CD14 could be a part of a receptor complex, and this
complex would bind PGN without physical binding of PGN to CD14.
To discriminate between these models and to validate the direct
function of CD14 as the receptor for a variety of bacterial products,
detailed analysis of binding of these bacterial products to CD14 is
needed. Because to date detailed studies of binding to CD14 were only
performed with enterobacterial LPS (10-13, 26-35), the aim of this
study was to determine if PGN directly and specifically binds to CD14.
We show that PGN binds with high affinity to both soluble and membrane
CD14 and that this binding can be competitively inhibited not only by
LPS and sPGN but also by LTA. Because the binding of LPS to CD14 is
facilitated by the LPS-binding protein (LBP) present in plasma (8-13,
26-28, 30, 33), we have also determined the effect of LBP on the
binding of PGN to CD14.
Materials--
Soluble PGN (sPGN) is a polymeric uncross-linked
PGN of approximate average Mr = 125,000, released from Staphylococcus aureus grown in the presence of
Binding of Bacterial Peptidoglycan to CD14*
§, Northwest Center for Medical Education,
Indiana University School of Medicine, Gary, Indiana 46408 and
the ¶ Department of Immunology, The Scripps Research Institute,
La Jolla, California 92037
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-lactam antibiotics (5, 36). sPGN was purified by vancomycin
affinity chromatography from three different S. aureus
strains (Rb, 845, and 3528) (5, 36, 37). Vancomycin specifically binds
to the D-Ala-D-Ala part of PGN, the structure
uniquely found in uncross-linked PGN and biosynthetic PGN precursors
(this accounts for the specificity of vancomycin inhibition of
peptidoglycan synthesis). PGN precursors are of low molecular weight
and were removed by extensive dialysis. By quantitative chemical
analysis, the sPGN was more than 98% pure (5, 36), i.e.
known PGN amino acids and amino sugars accounted for more than 98% of
the mass of the sPGN preparation. sPGN contained <24 pg of
endotoxin/mg determined by the Limulus lysate assay (37).
sPGN from three strains yielded the same results in the binding assays.
Ribitol teichoic acid was obtained from S. aureus cell walls
by trichloroacetic acid extraction as described (5) and after
hydrolysis contained glucosamine, anhydroribitol, and alanine.
1-4-MDP) was from Calbiochem. Heparin (Mr = 6,000-30,000, mean
Mr = 13, 500) from porcine intestinal mucosa was
the same as before (37). Clinical grade dextran from Leuconostoc
mesenteroides (mean Mr = 515,000) and
dextran sulfate (17% sulfur content, mean Mr = 515,000) were from Sigma. No significant endotoxin contamination of
these preparations was detected (
1 ng of endotoxin/mg), determined by
the Limulus assay (37). All other chemicals, unless
otherwise indicated, were obtained from Sigma.
Enzyme Digestions and Blots-- Unlabeled sPGN, ReLPS, or S. aureus LTA (at 2 mg/ml) and biotin-labeled sPGN, ReLPS, and LPS (at 0.18 mg/ml) were digested for 48 h at 37 °C with 200 µg/ml lysostaphin (affinity purified from Staphylococcus staphylolyticus, from Sigma), N-acetylmuramidase SG (from Streptomyces globisporus, from Dainippon Pharmaceutical, Seikagaku Kogyo, Tokyo, Japan), lysozyme (grade I from chicken egg, from Sigma), trypsin (type II, from bovine pancreas, from Sigma), or buffer alone (as a control), and before some assays (see "Results") were dialyzed four times against PBS at 4 °C (12,000 cutoff for sPGN and 3,500 cutoff for ReLPS and LTA). 125I-ASD-labeled sPGN and LPS were digested and dialyzed as described (37). Enzyme-digested biotin-labeled preparations (not dialyzed) were subjected to SDS-PAGE on 15 or 11% gels (37) and blotted onto Immobilon (6). The membranes were incubated for 2 h at 22 °C with 1 µg/ml streptavidin-peroxidase polymer (Sigma), and biotin-streptavidin complexes were visualized with the enhanced chemiluminescence system (ECL from Amersham Pharmacia Biotech).
sCD14 and LBP-- Recombinant human full-length (residues 1-323) sCD14, containing at the C terminus five amino acids that constitute protein kinase A phosphorylation site followed by an affinity tag of six histidines, was expressed in a baculovirus system, affinity purified on nickel-agarose, and analyzed as described (40). Recombinant human 1-152 sCD14 fragment, tagged at the C terminus with six histidines, was expressed in a baculovirus system, affinity purified on nickel-agarose, and analyzed as described for the full-length sCD14 (40). Recombinant human LBP was purified by affinity chromatography from supernatants of CHO transfectants as described (41).
Anti-CD14 mAbs-- The following mAbs were purchased as affinity purified immunoglobulins: MEM-18 (Sanbio-Monosan, Uden, Netherlands), MY-4 (Coulter, Hialeah, FL), LeuM3 (Becton Dickinson, San Jose, CA), RPA (Zymed, San Francisco, CA), biG 2, biG 3, biG 4, biG 11, biG 14 (Biometec, Greifswald, Germany), and CRIS-6 (Antigenix America, Franklin Square, NY). Affinity purified 3C10 and 60bca were kindly provided by Dr. Theo N. Kirkland, Department of Veterans Affairs Medical Center, University of California, San Diego. 63D3-producing clone was obtained from ATCC (Rockville, MD), and 63D3 was purified from murine ascites by protein G affinity chromatography and desalted using ImmunoPure (G) IgG purification kit (44441, Pierce). Affinity purified mouse IgG2b (clone MPC-11, Coulter) and IgG1 (clone 107.3, Pharmingen, San Diego) were used as controls.
Cells--
Human monocytic THP-1 cell line, obtained from ATCC
(Rockville, MD), was cultured in 1001 Falcon plates (Falcon Plastics, Oxnard, CA) in RPMI 1640 with 10% defined fetal bovine serum (HyClone, Logan, UT, endotoxin content <0.06 endotoxin units/ml). Before each
experiment, cells were allowed to differentiate for 3 days in the
presence of 100 nM 1
,25-dihydroxyvitamin D3
(Biomol, Plymouth Meeting, PA). RAW264.7 cells were cultured in
Dulbecco's modified Eagle's medium with 10% fetal bovine serum as
before (6).
Binding of 32P-sCD14 to sPGN-Agarose or
ReLPS-Agarose--
Five µg of sCD14 or soluble dephosphorylated
casein from bovine milk as a control (C-4765, Sigma) were labeled with
32P by incubating with 0.25 mCi of
[
-32P]ATP (NEN Life Science Products) and 5 units of
catalytic subunit of cAMP-dependent protein kinase from
bovine heart (P2645, Sigma) in a total volume of 50 µl for 2 h
at 37 °C (40). 100 µg of bovine serum albumin (BSA) was then
added, and the reaction mixture was dialyzed twice at 4 °C against
total 700 ml of PBS, 3.5-kDa cutoff. The specific activities of
32P-labeled sCD14 and casein were 12-36 and 2.7-5.7
µCi/µg, respectively, and their purity was checked by
polyacrylamide gel electrophoresis (PAGE) and autoradiography (see Fig.
5 below).
Binding of Biotinylated-sPGN or ReLPS to Solid-phase
CD14--
sPGN, ReLPS, or LPS were labeled with biotin by incubating
2.4 mg/ml sPGN or 5 mg/ml ReLPS (or smooth LPS) with 2.4 or 5 mg/ml, respectively, of sulfo-NHS-LC-biotin (21335, Pierce) in 0.1 M borate buffer, pH 8.5, at 22 °C for 30 min (according
to the manufacturer's protocol), followed by dialysis seven times at
4 °C against total 2100 ml of PBS, 8- (sPGN) or 3.5-kDa (ReLPS and
LPS) cutoff. Biotinylated sPGN, ReLPS, and LPS were stored at
80 °C.
Cross-linking of 125I-ASD-sPGN or 125I-ASD-LPS to CD14-- 125I-ASD (2-p-azido-salicylamido-1,3'-dithiopropionate)-derivatized sPGN and LPS were the same as before (36, 43) and had specific activities of 6.9-17 and 2.1-3.6 µCi/µg, respectively. The following lines of evidence indicated that the label was indeed bound to sPGN and LPS (and not to some minor otherwise undetectable contaminants or to minor subfractions of PGN and LPS) (36, 37): (i) the patterns of the labeled sPGN and LPS on PAGE were unique for each preparation and corresponded to the patterns of biosynthetically labeled sPGN or silver-stained LPS; and (ii) the label was solubilized (converted to low molecular weight migrating on SDS-PAGE with the buffer front) by digestion of 125I-ASD-sPGN (but not 125I-ASD-LPS) with muramidase SG, lysozyme, or lysostaphin but not with heparinase, RNase, DNase, or trypsin, or removed by treatment of 125I-ASD-LPS (but not 125I-ASD-sPGN) with polymyxin B-agarose or tachyplasin-agarose. Before the cross-linking studies, enzyme-digested 125I-ASD-sPGN and 125I-ASD-LPS were dialyzed as described (36, 37).
For cross-linking to sCD14, 2 µg of sCD14 (or BSA as a control) were incubated with 0.225 µg of 125I-ASD-sPGN or 0.336 µg of 125I-ASD-LPS, with or without 29 µg of sPGN or 34 µg of ReLPS, in 30 µl of Hanks' balanced salt solution with 10 mM HEPES and 0.05% gelatin for 4 h at 37 °C in the dark, followed by 8 min exposure to UV light at 22 °C (36, 44), incubation with 4 µg of anti-CD14 63D3 mAb (or anti-BSA 033 mAb) for 18 h at 4 °C, and incubation with 20 µl of anti-mouse IgG-agarose for 4 h at 22 °C. The agarose was washed five times with PBS with 0.05% Tween 20 and boiled with sample buffer with 2% SDS and 10% 2-mercaptoethanol, and the supernatant was subjected to SDS-PAGE on 11% gels and autoradiography as described (36, 44). For cross-linking to mCD14, differentiated THP-1 cells were washed four times with Hanks' balanced salt solution with 0.05% gelatin, and 240 × 106 cells in 2 ml of Hanks' balanced salt solution with 10 mM HEPES and 0.05% gelatin were incubated with 2.25 µg of 125I-ASD-sPGN for 15 min or 3.36 µg of 125I-ASD-LPS for 5 min at 37 °C in the dark (26), then exposed to UV light for 10 min at 4 °C (35), washed three times by centrifugation, solubilized at 4 °C in 6 ml of solubilizing buffer (6) with 1% Nonidet P-40, and centrifuged at 6000 × g. The supernatant was incubated with 4 µg of anti-CD14 63D3 mAb (or control IgG) for 18 h at 4 °C, followed by incubation with anti-mouse IgG-agarose, washing, denaturing, reduction, PAGE, and autoradiography as described above for sCD14. The reduction after cross-linking cleaves the cross-linker and eliminates the ligand but retains the label cross-linked to the ligand-binding sites.TNF- Assay--
RAW264.7 cells were cultured and stimulated
with the indicated concentrations of sPGN, LTA, or ReLPS, and the
concentrations of TNF- in the culture supernatants were determined
by the bioassay as described (6).
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RESULTS |
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Introduction
Procedures
Results
Discussion
References
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Binding of CD14 to PGN-Agarose and Determination of Binding Affinity and the Effect of LBP-- To test the hypothesis that CD14 binds to PGN, we have developed a binding assay in which we used sPGN covalently bound to agarose and recombinant affinity purified human full-length sCD14, containing at the C terminus five amino acids that constitute protein kinase A phosphorylation site followed by an affinity tag of six histidines, labeled with 32P to a very high specific activity. To compare CD14 binding of sPGN and LPS, we developed a similar binding assay with ReLPS covalently bound to agarose. Both sPGN-agarose and ReLPS-agarose bound 32P-sCD14 (Fig. 1). The binding was proportional to the amount of sPGN- or ReLPS-agarose with a very high signal-to-noise ratio (i.e. binding to agarose itself was negligible, Fig. 1A). The binding was dependent on CD14, because similarly labeled 32P-casein showed a very low binding (Fig. 1B).
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Binding of Biotinylated-PGN to Solid-phase CD14-- To confirm the binding of PGN to CD14 by another independent method, we have developed an enzyme-linked immunosorbent assay, in which wells of a microtiter plate were sequentially coated with anti-CD14 63D3 mAb and sCD14 and then incubated with biotin-labeled sPGN (or ReLPS). The binding of sPGN-biotin (or ReLPS-biotin) was detected spectrophotometrically using streptavidin-phosphatase. This assay also demonstrated dose-dependent binding of sPGN to sCD14, but the sPGN binding, in contrast to ReLPS binding, was not enhanced by LBP (Fig. 4A). Similar results were obtained with smooth LPS-biotin, but the absorbance values were 50% lower than with ReLPS-biotin (not shown).
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Cross-linking of 125I-ASD-sPGN to CD14-- To confirm further the binding of PGN to sCD14 by yet another method and to test if PGN also binds to membrane CD14 (mCD14) present on the cell surface, we used a photoaffinity cross-linking procedure. Both 125I-ASD-sPGN and 125I-ASD-LPS cross-linked to sCD14, detected on autoradiograms of PAGE gels as a doublet (26, 30, 40), which co-migrated with the standard 32P-labeled sCD14 (Fig. 5). The photoaffinity labeled sCD14 band was clearly distinct from the 70-kDa photoaffinity labeled albumin band. Cross-linking of both 125I-ASD-sPGN and 125I-ASD-LPS to sCD14 was completely inhibited by an excess of underivatized sPGN and partially inhibited by an excess of underivatized ReLPS (Fig. 5). Since these cross-linking studies were done in the absence of serum or LBP, these competitive inhibition results confirm our data (Fig. 3 and Fig. 6 below) on higher affinity of binding of sPGN than LPS to sCD14 in the absence of LBP.
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Specificity of CD14 Binding-- To determine further the specificity of CD14 binding to sPGN and ReLPS and to define the role of LBP in CD14 binding, we compared the competitive inhibition of binding of 32P-sCD14 to sPGN-agarose or ReLPS-agarose by sPGN, LPS, ReLPS, and lipid A in the presence or absence of LBP (Fig. 6). An excess of sPGN completely or almost completely inhibited the binding of sCD14 to sPGN- or ReLPS-agarose, with an IC50 = approximately 20 µg/ml (160 nM), and this inhibition was not significantly affected by LBP (Fig. 6A). An excess of smooth LPS, ReLPS, and lipid A also inhibited the binding of sCD14 to sPGN- or ReLPS-agarose (Fig. 6, B-D), but complete inhibition was not obtained, most likely due to the aggregation of these preparations at high concentrations and nonspecific binding of these aggregates to the agarose (at 0.1 to 1 mg/ml ReLPS or lipid A, no inhibition or an increase of sCD14 binding was observed, not shown).
LBP greatly enhanced (by 10 to 1000 times) the ability of LPS, ReLPS, and lipid A to inhibit the binding of sCD14 to both sPGN- and ReLPS-agarose (Fig. 6, B-D), and the effect of LBP was the greatest with the most hydrophobic preparations (i.e. ReLPS and lipid A), which tend to aggregate more in solution than smooth LPS. These results are consistent with higher affinity binding of LPS in the presence of LBP and with the function of LBP as a transfer molecule that can disaggregate LPS and transfer single LPS molecules to sCD14 (10, 13, 33). To determine which part of PGN binds to CD14, we studied binding of sCD14 to synthetic PGN fragments coupled to agarose. 32P-sCD14 readily bound to MDP-agarose (Fig. 7) and a disaccharide-dipeptide (GlcNAc-MDP)-agarose (not shown) but did not bind to PGN pentapeptide-agarose (Fig. 7A), indicating that the glycan part of PGN is required for sCD14 binding. Binding of 32P-sCD14 to MDP-agarose (similarly to the binding to sPGN-agarose, Figs. 7 and 8) was competitively inhibited by sPGN, LPS, and LTA but not by monomeric soluble MDP. These data further confirm that sCD14 binds to PGN itself (and not to a minor undetectable contaminant of PGN), since it is highly unlikely that synthetic MDP, synthetic GlcNAc-MDP, and natural PGN would all contain the same undetectable contaminant that binds CD14. The lack of inhibition of sCD14 binding to MDP-agarose by monomeric soluble MDP (Fig. 7B) indicates that polymeric, aggregated, or solid-phase bound PGN or MDP is needed for CD14 binding.
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Regions of CD14 Involved in PGN Binding-- Since we have recently shown that the N-terminal 151 amino acids of mCD14 were sufficient for the cellular responsiveness to PGN (15), we next tested if the similar N-terminal sCD14 fragment binds to sPGN. Indeed, the unlabeled 1-152 N-terminal fragment of sCD14 inhibited binding of full-length 32P-sCD14 to both sPGN and ReLPS as efficiently as the unlabeled full-length sCD14 (Fig. 2). These results indicate that the binding site(s) for both sPGN and ReLPS is located in the N-terminal 152-amino acid region of CD14.
To define further the regions of CD14 that bind sPGN and LPS, we studied the inhibition of binding of sCD14 to sPGN or ReLPS by a number of anti-CD14 mAbs with known specificities for different amino acid sequences of CD14 (11, 29, 31, 35). All antibodies were used at 33 µg/ml, which was the highest concentration that did not cause nonspecific inhibition of binding (Fig. 9A). MEM-18, whose epitope is located between amino acids 51 and 64, was the most efficient inhibitor of binding of sCD14 to both sPGN and ReLPS (Fig. 9B). The second most effective antibody was CRIS-6, whose epitope (amino acids 58-62) is located within the MEM-18 epitope. Several other mAbs (such as 3C10, 60bca, RPA, biG 4, biG 14, and MY-4, whose epitopes are located more N-terminal to the MEM-18 epitope) were also quite effective (although not as effective as MEM-18) in inhibiting sCD14 binding to ReLPS. However, most of these antibodies (except MY-4) were less effective, or completely ineffective, in inhibiting sCD14 binding to sPGN. Combining three of these mAbs (3C10, MY-4, and CRIS-6) caused greater inhibition of binding of sCD14 to ReLPS than each of these mAbs alone, but it did not further decrease the binding of sCD14 to sPGN (beyond the inhibition caused by CRIS-6 alone). Only one mAb (Leu-M3) inhibited sCD14 binding to sPGN but did not inhibit sCD14 binding to ReLPS (Fig. 9B).
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DISCUSSION |
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Abstract
Introduction
Procedures
Results
Discussion
References
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Our results demonstrate that PGN directly binds both soluble and membrane CD14 and together with our previous results (15-17) prove that CD14 acts as a true PGN receptor. Both in the absence and presence of LBP, CD14 binds PGN with a slower kinetics than LPS. CD14 binds PGN with high affinity both in the absence and presence of LBP (KD = 24.9 ± 4.7 and 20.4 ± 3.4 nM, respectively), and this affinity is higher than the affinity of binding of CD14 to LPS in the absence of LBP (KD = 41.2 ± 3.2 nM). LBP increases the binding of CD14 to PGN by adding an additional low affinity binding constant (KD = 71.6 ± 12 nM) and by increasing the Bmax. By contrast, LBP increases the affinity of CD14 binding to LPS by yielding two significantly lower dissociation constants (KD1 = 7.1 ± 2.7 nM and KD2 = 27.3 ± 7.2 nM). The second apparent KD for LPS in the presence of LBP detected in our assay is identical to the KD reported by Kirkland et al. (28) (27.4 nM) and Tobias et al. (30) (29 nM), and similar to the KD reported by Stelter et al. (35) (31.9 nM) for the binding of LPS in the presence of LBP to recombinant CD14 expressed on the cell membrane (28, 35) or sCD14 (30).
Our results, therefore, demonstrate different mechanisms of enhancement of CD14 binding by LBP to sPGN and LPS. The increase in the affinity of binding of sCD14 to LPS by LBP is consistent with the previously proposed mechanism (10, 13, 33), in which LBP acts as a transfer molecule, i.e. binds LPS and transfers it to sCD14. This increased affinity of binding of LPS results in the lowering of the cell-activating concentration of LPS in the presence of LBP (8-10, 46, 47). By contrast, LBP does not enhance CD14-dependent cell activation by PGN (15, 16), because it only increases the low affinity binding of PGN to CD14, which apparently does not result in enhanced cell activation.
Our results demonstrate that the glycan part of PGN is required for CD14 binding and that the minimal structure of PGN that binds CD14 is MDP. However, CD14 binds only to MDP immobilized on agarose, which indicates that polymeric PGN or polymeric-like immobilized MDP is required for the binding. This requirement for polymeric PGN is supported by the loss of CD14-binding capacity of PGN following digestion with PGN-lytic enzymes. These results differ from the previous report showing inhibition of fluorescein isothiocyanate-sPGN binding to cellular CD14 by soluble monomeric MDP (17). The reason for this discrepancy is not clear, but the fluorescein isothiocyanate-sPGN binding assay does not stringently show binding to CD14, because the target is the whole cell and it is only presumed that this assay detects binding of fluorescein isothiocyanate-sPGN to CD14 (because this binding is inhibited by anti-CD14 mAbs) (17). Most recent results from Dr. Ulmer's laboratory confirm our current findings and indicate that monomeric soluble MDP does not bind to sCD14 and does not inhibit binding of sPGN or LPS to sCD14 in a gel shift assay.2
Our results of competitive inhibition experiments support the notion (15-24) that CD14 binds other polymeric macrophage activators of microbial origin (besides LPS and PGN), since LTAs from four bacterial species were as active as LPS and sPGN in inhibiting CD14 binding to LPS and PGN. Contrary to the previous report (45), however, other polymers, such as dextran sulfate or heparin and also cell wall ribitol teichoic acid, do not inhibit or only marginally inhibit binding of sCD14 to PGN or LPS.
Since it was recently suggested (48) that a PGN contaminant might have been responsible for PGN-induced CD14-dependent cell activation, we have carefully examined if PGN itself bound to CD14. The following lines of evidence indicate that PGN itself, and not an undetectable minor contaminant, binds to CD14. (i) CD14 binds to natural sPGN and also two synthetic PGN fragments, MDP and a disaccharide GlcNAc-MDP, immobilized to agarose, and this binding is inhibited by PGN, LPS, and LTA. It is highly unlikely that synthetic MDP, synthetic GlcNAc-MDP, and natural PGN would contain the same undetectable contaminant that binds CD14. (ii) Binding of PGN to CD14 and the inhibitory capacity of PGN for CD14 binding to PGN and LPS are lost when the PGN preparations are digested with PGN-lytic enzymes (lysostaphin, muramidase SG, and lysozyme) but are not affected by digestion with other enzymes (heparinase, RNase, DNase, and trypsin). Gel electrophoresis and blotting results also show that the labels (ASD and biotin) are bound to representative molecular species of PGN and that the PGN-lytic enzymes also solubilize ASD- or biotin-labeled PGN. By contrast, the binding of LPS to CD14, the inhibitory capacity of LPS or LTA, and the labeling of LPS with ASD and biotin are not diminished or destroyed by the same treatment with PGN-lytic enzymes. (iii) In a gel-shift assay, which measures binding of unlabeled sPGN to unlabeled sCD14, the stoichiometry of the binding of sPGN to sCD14 was approximately 1:1 (Fig. 9 in Ref. 17). (iv) Binding of sCD14 to immobilized sPGN was inhibited by sPGN with an IC50 = 20 µg/ml (160 nM) (Fig. 6A), which is of similar magnitude as the two KD values that we have obtained for the binding of sCD14 to immobilized PGN (20-72 nM, Fig. 3A). If a minor contaminant, undetectable by chemical analysis, was responsible for CD14 binding, the IC50 of sPGN in this experiment would have been at least 2 orders of magnitude larger. (v) We also previously excluded the possibility that sPGN, LPS, and LTA form hetero-aggregates with each other (Fig. 8 in Ref. 37). Such an aggregation could have been responsible for the inhibition of binding of sPGN by LTA and LPS, and of LPS by sPGN and LTA. Moreover, if a contaminant was responsible for the binding of PGN and LTA to CD14, it would be highly unlikely that the same contaminant was present in insoluble PGN, sPGN, and LTA from several bacterial species, given entirely different methods of purification of LTA, insoluble PGN, and sPGN. (vi) The sPGN preparation used in this study was of the highest purity available (see "Experimental Procedures").
We do not agree with the recent conclusion of Kusunoki and Wright (48)
that a minor undetectable contaminant of insoluble PGN was responsible
for its cell-activating effect for the following reasons. (i) As
indicated in Ref. 48, the activity of PGN was abolished by treatment
with SDS. We have treated the same insoluble PGN preparation with SDS
(5%, 80 °C, 30 min), and after removal of SDS by 10 washes with
PBS, the SDS-treated PGN was as active or more active than the
untreated PGN in induction of TNF- production in RAW264.7
cells.3 Most likely in Ref.
48, SDS was not adequately removed from the insoluble PGN, and since
SDS is toxic to cells, the activity was lost. (ii) As indicated in Ref.
48, the activity of PGN was abolished by hot phenol/water extraction.
In this procedure, after the extraction, the phenol/water/PGN mixture
was centrifuged, and the insoluble PGN, sedimented in the bottom of the
phenol layer, was not active (whereas the "active component" was
supposedly extracted, similar to LPS or LTA, into the water phase).
However, the procedure used in Ref. 48 did not include removal of
phenol (which is toxic to cells) from insoluble PGN sedimented in the phenol phase, and this most likely accounted for the lack of activity of treated PGN. We have repeated this phenol/water extraction (45%
phenol, 60 °C, 60 min), but then we removed residual phenol from the
PGN by ethyl ether extraction, and the recovered PGN had full activity
(for induction of TNF- production in RAW264.7 cells), equal to the
activity of PGN before the extraction. Moreover, the water fraction
obtained from the hot phenol/water extraction of this PGN had no
activity (for induction of TNF- production), indicating that no
active component was removed from PGN by phenol/water extraction.3 In Ref. 48, the authors did not test the
activity of the water fraction, which should have contained the
"active fraction" if it had been removed from the PGN preparation.
(iii) In Ref. 48, when insoluble PGN was incubated with sCD14 and then
spun down, the remaining supernatant containing sCD14 was more active
in activating neutrophils than the sedimented PGN or untreated PGN. The
authors concluded that the putative active contaminant from PGN became
bound to sCD14 and remained in the supernatant in an active sCD14-bound
form. This experiment, however, is inconclusive, because it does not
show what was active in the supernatant, and there are several
alternative explanations of these results, such as release of soluble
PGN fragments aggregated with the insoluble PGN or binding of a large
portion of sCD14 to insoluble PGN, which would make the sCD14 remaining
in the supernatant more active (because high concentrations of sCD14
inhibit cell activation and low concentrations enhance cell
activation). Moreover, sedimented PGN was equally active as the
untreated PGN, which indicates that no significant activity was removed
from the PGN by sCD14. Another unexplained aspect of this experiment is
the requirement for an overnight incubation of PGN with sCD14 at
37 °C, which is inconsistent with much faster binding of sCD14 to
PGN shown here by us. Another possibility includes contamination of the
PGN-sCD14 mix that would produce enough endotoxin during an overnight
incubation to activate cells in conjunction with sCD14. None of these
possibilities were formally excluded (48). (iv) In Ref. 48, only
insoluble PGN (isolated from the cell walls by enzyme digestions and
trichloroacetic acid extraction) was used, and the affinity purified
soluble PGN (used in all experiments in this paper) was not tested
(48). (v) In our experiments, the cell-activating capacity (for
induction of TNF- production in RAW264.7 cells) of sPGN is lost
following digestion with PGN-lytic enzymes and dialysis (6). All these results indicate that PGN itself activates cells.
The difference in the affinity of binding of CD14 to LPS and PGN in the presence of LBP (KD, 7.1 nM versus 20.4 nM) does not fully explain the 4 log difference (in µg/ml, or 200 times difference on per mol basis) between the effective macrophage-activating concentrations of ReLPS and sPGN (Fig. 10). This difference cannot be fully attributed to LBP, because even in the absence of serum (and serum-derived LBP), LPS is a more effective macrophage activator than sPGN (6). Therefore, one possibility is that this difference is related to a much greater ability of CD14 to transfer LPS (than sPGN) to another, as yet unidentified (13, 24), cell-activating molecule. LTA, which has an intermediate ability to activate macrophages (Fig. 10), would then also have an intermediate ability to be transferred from CD14 to the putative cell-activating molecule.
|
Our results demonstrate that less than half of the CD14 molecule (N-terminal 152 amino acids) is sufficient for PGN binding and suggest that the sequences that are most critical for CD14 binding to both PGN and LPS are located within the N-terminal region between amino acids 51-64. This region appears to be necessary for both PGN and LPS binding, because MEM-18, an anti-CD14 mAb specific for this region, inhibited CD14 binding to ReLPS by over 95% and to PGN by over 80%. It is also possible, however, that the effect of MEM-18 could be through a conformational change of CD14, but this would also indicate similar primary binding sites for both PGN and ReLPS (since the same conformational change would abolish binding for both ligands). This region may not be sufficient for binding of LPS or PGN, because mAbs specific to other regions of CD14 also partially inhibited binding of LPS and PGN. Of note, these other sequences that contribute to PGN and LPS binding are different, because there are several mAbs that inhibit LPS binding but not PGN binding (3C10, specific for amino acids 7-14, and 60bca and RPA specific for amino acids 34-38) and, significantly, one mAb that inhibits PGN binding but not LPS binding (Leu-M3, specific for amino acids 135-146). But again, it is also possible that these effects might have been due to conformational changes induced by these mAbs. In summary, it appears that PGN and ReLPS bind to conformational rather than linear CD14 epitopes that are partially identical (amino acids 51-64) and partially different.
This conclusion is consistent with the results of Juan et al. (29) indicating that amino acids 57-64 are the primary LPS-binding site, but it does not fully agree with the conclusion of Stelter et al. (35) that amino acids 39-44 are the main LPS-binding site on CD14 and with the conclusion of Shapiro et al. (34) that Glu-37 was necessary for LPS binding. However, Stelter et al. (35) also noticed full inhibition of LPS binding with MEM-18 mAbs and concluded that the LPS-binding site is a conformational site that includes amino acids 9-13, 39-44, and 51-63.
We have noted some differences in the ability of anti-CD14 mAbs to inhibit binding of sCD14 to ReLPS, compared with the previously reported ability of the same mAbs to inhibit binding of LPS to membrane or soluble CD14. In particular, it was reported that mAbs 3C10 (31), RPA (11), and biG 4 (35) did not inhibit LPS binding to sCD14 (3C10) or mCD14 (RPA, biG 4), whereas we have observed significant inhibition of sCD14 binding to ReLPS by all three mAbs. Most likely these differences are due to the differences in the assay systems, the use of soluble versus membrane CD14, the presence or absence of LBP, or the use of ReLPS versus smooth LPS, which was shown to affect the interaction of LPS with CD14, LBP, and anti-CD14 mAbs (47). Indeed, other studies (26) showed inhibition of LPS cross-linking to mCD14 by 3C10 mAb.
In general, our binding results are consistent with our previous activation results showing that similar but not identical sequences in mCD14 were critical for the responsiveness of CD14-transfected cells to PGN and LPS (15) and that MEM-18 is the mAb that is most active in both inhibiting PGN and LPS binding to CD14 and in inhibiting PGN- and LPS-induced CD14-mediated cell activation (16).3 Detailed comparison of the sequences that are most crucial for the binding and cell activation is difficult at the moment, because the activation analysis was done using CD14 deletion mutants (15) and the current binding studies were done using mAbs, whose epitopes are somewhat different than the epitopes deleted in those mutants.
Our conclusion that PGN and LPS bind to conformational sites can
accommodate both the similarities (1-4- or 1-6-linked
N-acetylated glucosamine residues with closely located
carbonyl groups) and differences (presence of fatty acids and phosphate
groups in ReLPS and presence of amino acids and a long glycan chain in
PGN) in the structures of PGN and ReLPS (37). This conclusion is also consistent with the hypothesis that CD14 binds to the glycan part of
LPS and other ligands (49). Our data, showing that agarose-immobilized MDP (but not PGN derived pentapeptide) binds CD14, further support this
hypothesis.
In summary, our results support the hypothesis that CD14 does have a defined specificity and that it is not merely a nonspecific transfer molecule. This specificity of CD14 is different from the specificity of other molecules that bind multiple ligands, such as albumin or the scavenger receptor (37, 44). These results also support the hypothesis that CD14 functions as a "pattern recognition receptor" and suggest that recognition of different patterns is encoded in somewhat different regions of CD14. However, our results also do not exclude the possibility that additional specificity in cell activation may be determined by other events occurring after the CD14 binding step.
| |
ACKNOWLEDGEMENTS |
|---|
We are grateful to Dr. Theo N. Kirkland for providing 3C10 and 60bca mAbs and for advice on developing CD14 binding assays, to Dr. Harry S. Courtney for providing S. pyogenes LTA, to Dr. Raoul S. Rosenthal for providing muramidase SG, and to Dr. Dipika Gupta for reviewing the manuscript.
| |
FOOTNOTES |
|---|
* This work was supported by the National Institutes of Health Grants AI28797 (to R. D.), AI32021, and HL23584 (to P. S. T.), and a Human Frontiers Science Program Organization fellowship (to R. I. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Northwest Center for Medical Education, Indiana University School of Medicine, 3400 Broadway, Gary, IN 46408. Tel.: 219-980-6535; Fax: 219-980-6566, E-mail: rdziar{at}iunhaw1.iun.indiana.edu.
1
The abbreviations used are: PGN, peptidoglycan;
LBP, lipopolysaccharide-binding protein; LPS, lipopolysaccharide;
mCD14, membrane CD14; ReLPS, LPS from Salmonella minnesota
Re 595; LTA, lipoteichoic acid; MDP, muramyl dipeptide
(MurNAc-L-Ala-D-isoglutamine); sCD14, soluble CD14; sPGN, soluble PGN; TNF-, tumor necrosis factor-; ELISA, enzyme-linked immunosorbent assay; PAGE, polyacrylamide gel electrophoresis; mAb, monoclonal antibody; PBS,
phosphate-buffered saline; TBS, Tris-buffered saline; ASD,
(2-p-azido-salicylamido-1,3'-dithiopropionate; BSA, bovine
serum albumin.
2 A. J. Ulmer, unpublished observations.
3 R. Dziarski, unpublished observations.
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REFERENCES |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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J. Branger, S. Knapp, S. Weijer, J. C. Leemans, J. M. Pater, P. Speelman, S. Florquin, and T. van der Poll Role of Toll-Like Receptor 4 in Gram-Positive and Gram-Negative Pneumonia in Mice Infect. Immun., February 1, 2004; 72(2): 788 - 794. [Abstract] [Full Text] [PDF]
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Z.-M. Wang, X. Li, R. R. Cocklin, M. Wang, M. Wang, K. Fukase, S. Inamura, S. Kusumoto, D. Gupta, and R. Dziarski Human Peptidoglycan Recognition Protein-L Is an N-Acetylmuramoyl-L-alanine Amidase J. Biol. Chem., December 5, 2003; 278(49): 49044 - 49052. [Abstract] [Full Text] [PDF]
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 F. C. Neuhaus and J. Baddiley A Continuum of Anionic Charge: Structures and Functions of D-Alanyl-Teichoic Acids in Gram-Positive Bacteria Microbiol. Mol. Biol. Rev., December 1, 2003; 67(4): 686 - 723. [Abstract] [Full Text] [PDF]
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M. Hashimoto, Y. Asai, and T. Ogawa Treponemal Phospholipids Inhibit Innate Immune Responses Induced by Pathogen-associated Molecular Patterns J. Biol. Chem., November 7, 2003; 278(45): 44205 - 44213. [Abstract] [Full Text] [PDF]
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R. Dziarski, K. A. Platt, E. Gelius, H. Steiner, and D. Gupta Defect in neutrophil killing and increased susceptibility to infection with nonpathogenic gram-positive bacteria in peptidoglycan recognition protein-S (PGRP-S)-deficient mice Blood, July 15, 2003; 102(2): 689 - 697. [Abstract] [Full Text] [PDF]
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P. A. Majcherczyk, E. Rubli, D. Heumann, M. P. Glauser, and P. Moreillon Teichoic Acids Are Not Required for Streptococcus pneumoniae and Staphylococcus aureus Cell Walls To Trigger the Release of Tumor Necrosis Factor by Peripheral Blood Monocytes Infect. Immun., July 1, 2003; 71(7): 3707 - 3713. [Abstract] [Full Text] [PDF]
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U. Koedel, B. Angele, T. Rupprecht, H. Wagner, A. Roggenkamp, H.-W. Pfister, and C. J. Kirschning Toll-Like Receptor 2 Participates in Mediation of Immune Response in Experimental Pneumococcal Meningitis J. Immunol., January 1, 2003; 170(1): 438 - 444. [Abstract] [Full Text] [PDF]
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M. A. Wolfert, T. F. Murray, G.-J. Boons, and J. N. Moore The Origin of the Synergistic Effect of Muramyl Dipeptide with Endotoxin and Peptidoglycan J. Biol. Chem., October 11, 2002; 277(42): 39179 - 39186. [Abstract] [Full Text] [PDF]
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 C. Stamme, M. Muller, L. Hamann, T. Gutsmann, and U. Seydel Surfactant Protein A Inhibits Lipopolysaccharide-Induced Immune Cell Activation by Preventing the Interaction of Lipopolysaccharide with Lipopolysaccharide-Binding Protein Am. J. Respir. Cell Mol. Biol., September 1, 2002; 27(3): 353 - 360. [Abstract] [Full Text] [PDF]
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 V. J. McCracken, T. Chun, M. E. Baldeon, S. Ahrne, G. Molin, R. I. Mackie, and H. R. Gaskins TNF-{alpha} Sensitizes HT-29 Colonic Epithelial Cells to Intestinal Lactobacilli Experimental Biology and Medicine, September 1, 2002; 227(8): 665 - 670. [Abstract] [Full Text] [PDF]
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D. Iwaki, H. Mitsuzawa, S. Murakami, H. Sano, M. Konishi, T. Akino, and Y. Kuroki The Extracellular Toll-like Receptor 2 Domain Directly Binds Peptidoglycan Derived from Staphylococcus aureus J. Biol. Chem., June 28, 2002; 277(27): 24315 - 24320. [Abstract] [Full Text] [PDF]
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 F.-G. Zhu, C. F. Reich, and D. S. Pisetsky Inhibition of murine macrophage nitric oxide production by synthetic oligonucleotides J. Leukoc. Biol., April 1, 2002; 71(4): 686 - 694. [Abstract] [Full Text] [PDF]
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R. N. Fichorova, A. O. Cronin, E. Lien, D. J. Anderson, and R. R. Ingalls Response to Neisseria gonorrhoeae by Cervicovaginal Epithelial Cells Occurs in the Absence of Toll-Like Receptor 4-Mediated Signaling J. Immunol., March 1, 2002; 168(5): 2424 - 2432. [Abstract] [Full Text] [PDF]
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R. Nau and H. Eiffert Modulation of Release of Proinflammatory Bacterial Compounds by Antibacterials: Potential Impact on Course of Inflammation and Outcome in Sepsis and Meningitis Clin. Microbiol. Rev., January 1, 2002; 15(1): 95 - 110. [Abstract] [Full Text] [PDF]
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Z. Xu, R. Dziarski, Q. Wang, K. Swartz, K. M. Sakamoto, and D. Gupta Bacterial Peptidoglycan-Induced tnf-{alpha} Transcription Is Mediated Through the Transcription Factors Egr-1, Elk-1, and NF-{kappa}B J. Immunol., December 15, 2001; 167(12): 6975 - 6982. [Abstract] [Full Text] [PDF]
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X. Zhang, M. Rimpilainen, and P. Toivanen Enzyme Degradation and Proinflammatory Activity in Arthritogenic and Nonarthritogenic Eubacterium aerofaciens Cell Walls Infect. Immun., December 1, 2001; 69(12): 7277 - 7284. [Abstract] [Full Text] [PDF]
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Y. Asai, Y. Ohyama, K. Gen, and T. Ogawa Bacterial Fimbriae and Their Peptides Activate Human Gingival Epithelial Cells through Toll-Like Receptor 2 Infect. Immun., December 1, 2001; 69(12): 7387 - 7395. [Abstract] [Full Text] [PDF]
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M. Bilej, P. De Baetselier, E. Van Dijck, B. Stijlemans, A. Colige, and A. Beschin Distinct Carbohydrate Recognition Domains of an Invertebrate Defense Molecule Recognize Gram-negative and Gram-positive Bacteria J. Biol. Chem., November 30, 2001; 276(49): 45840 - 45847. [Abstract] [Full Text] [PDF]
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X. Li, B. U. Bradford, M. D. Wheeler, S. A. Stimpson, H. M. Pink, T. A. Brodie, J. H. Schwab, and R. G. Thurman Dietary Glycine Prevents Peptidoglycan Polysaccharide-Induced Reactive Arthritis in the Rat: Role for Glycine-Gated Chloride Channel Infect. Immun., September 1, 2001; 69(9): 5883 - 5891. [Abstract] [Full Text] [PDF]
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R. R. Ingalls, E. Lien, and D. T. Golenbock Membrane-Associated Proteins of a Lipopolysaccharide-Deficient Mutant of Neisseria meningitidis Activate the Inflammatory Response through Toll-Like Receptor 2 Infect. Immun., April 1, 2001; 69(4): 2230 - 2236. [Abstract] [Full Text] [PDF]
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Q. Wang, R. Dziarski, C. J. Kirschning, M. Muzio, and D. Gupta Micrococci and Peptidoglycan Activate TLR2{right-arrow}MyD88{right-arrow}IRAK{right-arrow}TRAF{right-arrow}NIK{right-arrow}IKK{right-arrow}NF-{kappa}B Signal Transduction Pathway That Induces Transcription of Interleukin-8 Infect. Immun., April 1, 2001; 69(4): 2270 - 2276. [Abstract] [Full Text] [PDF]
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A. Verbon, P. E. P. Dekkers, T. ten Hove, C. E. Hack, J. P. Pribble, T. Turner, S. Souza, T. Axtelle, F. J. Hoek, S. J. H. van Deventer, et al. IC14, an Anti-CD14 Antibody, Inhibits Endotoxin-Mediated Symptoms and Inflammatory Responses in Humans J. Immunol., March 1, 2001; 166(5): 3599 - 3605. [Abstract] [Full Text] [PDF]
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R. Dziarski, Q. Wang, K. Miyake, C. J. Kirschning, and D. Gupta MD-2 Enables Toll-Like Receptor 2 (TLR2)-Mediated Responses to Lipopolysaccharide and Enhances TLR2-Mediated Responses to Gram-Positive and Gram-Negative Bacteria and Their Cell Wall Components J. Immunol., February 1, 2001; 166(3): 1938 - 1944. [Abstract] [Full Text] [PDF]
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D. H. Wyllie, E. Kiss-Toth, A. Visintin, S. C. Smith, S. Boussouf, D. M. Segal, G. W. Duff, and S. K. Dower Evidence for an Accessory Protein Function for Toll-Like Receptor 1 in Anti-Bacterial Responses J. Immunol., December 15, 2000; 165(12): 7125 - 7132. [Abstract] [Full Text] [PDF]
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S. Baveye, E. Elass, D. G. Fernig, C. Blanquart, J. Mazurier, and D. Legrand Human Lactoferrin Interacts with Soluble CD14 and Inhibits Expression of Endothelial Adhesion Molecules, E-Selectin and ICAM-1, Induced by the CD14-Lipopolysaccharide Complex Infect. Immun., December 1, 2000; 68(12): 6519 - 6525. [Abstract] [Full Text] [PDF]
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P. T. Lee, P. G. Holt, and A. S. McWilliam Role of Alveolar Macrophages in Innate Immunity in Neonates . Evidence for Selective Lipopolysaccharide Binding Protein Production by Rat Neonatal Alveolar Macrophages Am. J. Respir. Cell Mol. Biol., November 1, 2000; 23(5): 652 - 661. [Abstract] [Full Text]
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R. Dziarski and D. Gupta Role of MD-2 in TLR2- and TLR4-mediated recognition of Gram-negative and Gram-positive bacteria and activation of chemokine genes Innate Immunity, October 1, 2000; 6(5): 401 - 405. [Abstract] [PDF]
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A. Yoshimura, H. Takada, T. Kaneko, I. Kato, D. Golenbock, and Y. Hara Structural requirements of muramylpeptides for induction of Toll-like receptor 2-mediated NF-{kappa}B activation in CHO cells Innate Immunity, October 1, 2000; 6(5): 407 - 410. [Abstract] [PDF]
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R. R. Ingalls, E. Lien, and D. T. Golenbock Differential roles of TLR2 and TLR4 in the host response to Gram-negative bacteria: lessons from a lipopolysaccharide-deficient mutant of Neisseria meningitidis Innate Immunity, October 1, 2000; 6(5): 411 - 415. [Abstract] [PDF]
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R. Dziarski, S. Viriyakosol, T. N. Kirkland, and D. Gupta Soluble CD14 Enhances Membrane CD14-Mediated Responses to Peptidoglycan: Structural Requirements Differ from Those for Responses to Lipopolysaccharide Infect. Immun., September 1, 2000; 68(9): 5254 - 5260. [Abstract] [Full Text] [PDF]
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 E. C. A. Darcissac, M.-J. Truong, J. Dewulf, Y. Mouton, A. Capron, and G. M. Bahr The Synthetic Immunomodulator Murabutide Controls Human Immunodeficiency Virus Type 1 Replication at Multiple Levels in Macrophages and Dendritic Cells J. Virol., September 1, 2000; 74(17): 7794 - 7802. [Abstract] [Full Text]
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R. E. W. Hancock and M. G. Scott The role of antimicrobial peptides in animal defenses PNAS, August 1, 2000; 97(16): 8856 - 8861. [Abstract] [Full Text] [PDF]
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 V. Terpstra, E. S. van Amersfoort, A. G. van Velzen, J. Kuiper, and T. J. C. van Berkel Hepatic and Extrahepatic Scavenger Receptors : Function in Relation to Disease Arterioscler. Thromb. Vasc. Biol., August 1, 2000; 20(8): 1860 - 1872. [Full Text] [PDF]
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J. E. Wang, P. F. Jorgensen, M. Almlof, C. Thiemermann, S. J. Foster, A. O. Aasen, and R. Solberg Peptidoglycan and Lipoteichoic Acid from Staphylococcus aureus Induce Tumor Necrosis Factor Alpha, Interleukin 6 (IL-6), and IL-10 Production in Both T Cells and Monocytes in a Human Whole Blood Model Infect. Immun., July 1, 2000; 68(7): 3965 - 3970. [Abstract] [Full Text] [PDF]
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K. Imai, A. Takeshita, and S. Hanazawa Transforming Growth Factor-beta Inhibits Lipopolysaccharide-Stimulated Expression of Inflammatory Cytokines in Mouse Macrophages through Downregulation of Activation Protein 1 and CD14 Receptor Expression Infect. Immun., May 1, 2000; 68(5): 2418 - 2423. [Abstract] [Full Text] [PDF]
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M. D. Cunningham, R. A. Shapiro, C. Seachord, K. Ratcliffe, L. Cassiano, and R. P. Darveau CD14 Employs Hydrophilic Regions to ""Capture"" Lipopolysaccharides J. Immunol., March 15, 2000; 164(6): 3255 - 3263. [Abstract] [Full Text] [PDF]
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T. H. Flo, O. Halaas, E. Lien, L. Ryan, G. Teti, D. T. Golenbock, A. Sundan, and T. Espevik Human Toll-Like Receptor 2 Mediates Monocyte Activation by Listeria monocytogenes, But Not by Group B Streptococci or Lipopolysaccharide J. Immunol., February 15, 2000; 164(4): 2064 - 2069. [Abstract] [Full Text] [PDF]
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S. Viriyakosol, J. C. Mathison, P. S. Tobias, and T. N. Kirkland Structure-Function Analysis of CD14 as a Soluble Receptor for Lipopolysaccharide J. Biol. Chem., February 4, 2000; 275(5): 3144 - 3149. [Abstract] [Full Text] [PDF]
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M. G. Scott, M. R. Gold, and R. E. W. Hancock Interaction of Cationic Peptides with Lipoteichoic Acid and Gram-Positive Bacteria Infect. Immun., December 1, 1999; 67(12): 6445 - 6453. [Abstract] [Full Text] [PDF]
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 A. Sediva, K. Smetana Jr., J. Stejskal, J. Bartunkova, F.-T. Liu, N. V. Bovin, and H.-J. Gabius Binding sites for carrier-immobilized carbohydrates in the kidney: implication for the pathogenesis of Henoch-Schonlein purpura and/or IgA nephropathy Nephrol. Dial. Transplant., December 1, 1999; 14(12): 2885 - 2891. [Abstract] [Full Text] [PDF]
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A. Yoshimura, E. Lien, R. R. Ingalls, E. Tuomanen, R. Dziarski, and D. Golenbock Cutting Edge: Recognition of Gram-Positive Bacterial Cell Wall Components by the Innate Immune System Occurs Via Toll-Like Receptor 2 J. Immunol., July 1, 1999; 163(1): 1 - 5. [Abstract] [Full Text] [PDF]
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R. Schwandner, R. Dziarski, H. Wesche, M. Rothe, and C. J. Kirschning Peptidoglycan- and Lipoteichoic Acid-induced Cell Activation Is Mediated by Toll-like Receptor 2 J. Biol. Chem., June 18, 1999; 274(25): 17406 - 17409. [Abstract] [Full Text] [PDF]
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O. D. Moffatt, A. Devitt, E. D. Bell, D. L. Simmons, and C. D. Gregory Macrophage Recognition of ICAM-3 on Apoptotic Leukocytes J. Immunol., June 1, 1999; 162(11): 6800 - 6810. [Abstract] [Full Text] [PDF]
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D. Gupta, Q. Wang, C. Vinson, and R. Dziarski Bacterial Peptidoglycan Induces CD14-dependent Activation of Transcription Factors CREB/ATF and AP-1 J. Biol. Chem., May 14, 1999; 274(20): 14012 - 14020. [Abstract] [Full Text] [PDF]
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P. A. Majcherczyk, H. Langen, D. Heumann, M. Fountoulakis, M. P. Glauser, and P. Moreillon Digestion of Streptococcus pneumoniae Cell Walls with Its Major Peptidoglycan Hydrolase Releases Branched Stem Peptides Carrying Proinflammatory Activity J. Biol. Chem., April 30, 1999; 274(18): 12537 - 12543. [Abstract] [Full Text] [PDF]
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M. Ochiai and M. Ashida A Pattern Recognition Protein for Peptidoglycan. CLONING THE cDNA AND THE GENE OF THE SILKWORM, BOMBYX MORI J. Biol. Chem., April 23, 1999; 274(17): 11854 - 11858. [Abstract] [Full Text] [PDF]
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A. Cauwels, K. Frei, S. Sansano, C. Fearns, R. Ulevitch, W. Zimmerli, and R. Landmann The Origin and Function of Soluble CD14 in Experimental Bacterial Meningitis J. Immunol., April 15, 1999; 162(8): 4762 - 4772. [Abstract] [Full Text] [PDF]
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R. Dziarski and D. Gupta Function of CD14 as a peptidoglycan receptor: differences and similarities with LPS Innate Immunity, February 1, 1999; 5(1-2): 56 - 61. [Abstract] [PDF]
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P.-y. Wang, R. L. Kitchens, and R. S. Munford Phosphatidylinositides Bind to Plasma Membrane CD14 and Can Prevent Monocyte Activation by Bacterial Lipopolysaccharide J. Biol. Chem., September 18, 1998; 273(38): 24309 - 24313. [Abstract] [Full Text] [PDF]
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C. Liu, E. Gelius, G. Liu, H. Steiner, and R. Dziarski Mammalian Peptidoglycan Recognition Protein Binds Peptidoglycan with High Affinity, Is Expressed in Neutrophils, and Inhibits Bacterial Growth J. Biol. Chem., August 4, 2000; 275(32): 24490 - 24499. [Abstract] [Full Text] [PDF]
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H. Mitsuzawa, I. Wada, H. Sano, D. Iwaki, S. Murakami, T. Himi, N. Matsushima, and Y. Kuroki Extracellular Toll-Like Receptor 2 Region Containing Ser40-Ile64 but Not Cys30-Ser39 Is Critical for the Recognition of Staphylococcus aureus Peptidoglycan J. Biol. Chem., October 26, 2001; 276(44): 41350 - 41356. [Abstract] [Full Text] [PDF]
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C. Liu, Z. Xu, D. Gupta, and R. Dziarski Peptidoglycan Recognition Proteins. A NOVEL FAMILY OF FOUR HUMAN INNATE IMMUNITY PATTERN RECOGNITION MOLECULES J. Biol. Chem., September 7, 2001; 276(37): 34686 - 34694. [Abstract] [Full Text] [PDF]
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Z.-M. Wang, C. Liu, and R. Dziarski Chemokines Are the Main Proinflammatory Mediators in Human Monocytes Activated by Staphylococcus aureus, Peptidoglycan, and Endotoxin J. Biol. Chem., June 30, 2000; 275(27): 20260 - 20267. [Abstract] [Full Text] [PDF]
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