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The Origin of the Synergistic Effect of Muramyl Dipeptide with Endotoxin and Peptidoglycan

2002; Elsevier BV; Volume: 277; Issue: 42 Linguagem: Inglês

10.1074/jbc.m204885200

1083-351X

Margreet A. Wolfert, Thomas F. Murray, Geert‐Jan Boons, James N. Moore,

Neonatal and Maternal Infections

Although the basis for the high mortality rate for patients with mixed bacterial infections is likely to be multifactorial, there is evidence for a synergistic effect of muramyldipeptide (MDP) with lipopolysaccharide (LPS) on the synthesis of proinflammatory cytokines by mononuclear phagocytes. In this study, co-incubation of human Mono Mac 6 cells with MDP and either LPS or peptidoglycan (PGN) resulted in an apparent synergistic effect on tumor necrosis factor-α (TNF-α) secretion. Although incubation of cells with MDP alone produced minimal TNF-α, it caused significant expression of TNF-α mRNA. These findings suggest that the majority of TNF-α mRNA induced by MDP alone is not translated into protein. Furthermore, simultaneous incubation of cells with MDP and either LPS or PGN resulted in TNF-α mRNA expression that approximated the sum of the amounts expressed in response to MDP, LPS, and PGN individually. These findings indicate that the apparent synergistic effect of MDP on TNF-α production induced by either LPS or PGN is due to removal of a block in translation of the mRNA expressed in response to MDP. In subsequent studies, the effects of MDP alone and its effect on the production of TNF-α by LPS and PGN were determined to be independent of CD14, Toll-like receptor 2, and Toll-like receptor 4. These findings indicate that MDP acts through receptor(s) other than those primarily responsible for transducing the effects of LPS and PGN. Successful treatment of patients having mixed bacterial infections is likely to require interventions that address the mechanisms involved in responses induced by a variety of bacterial cell wall components. Although the basis for the high mortality rate for patients with mixed bacterial infections is likely to be multifactorial, there is evidence for a synergistic effect of muramyldipeptide (MDP) with lipopolysaccharide (LPS) on the synthesis of proinflammatory cytokines by mononuclear phagocytes. In this study, co-incubation of human Mono Mac 6 cells with MDP and either LPS or peptidoglycan (PGN) resulted in an apparent synergistic effect on tumor necrosis factor-α (TNF-α) secretion. Although incubation of cells with MDP alone produced minimal TNF-α, it caused significant expression of TNF-α mRNA. These findings suggest that the majority of TNF-α mRNA induced by MDP alone is not translated into protein. Furthermore, simultaneous incubation of cells with MDP and either LPS or PGN resulted in TNF-α mRNA expression that approximated the sum of the amounts expressed in response to MDP, LPS, and PGN individually. These findings indicate that the apparent synergistic effect of MDP on TNF-α production induced by either LPS or PGN is due to removal of a block in translation of the mRNA expressed in response to MDP. In subsequent studies, the effects of MDP alone and its effect on the production of TNF-α by LPS and PGN were determined to be independent of CD14, Toll-like receptor 2, and Toll-like receptor 4. These findings indicate that MDP acts through receptor(s) other than those primarily responsible for transducing the effects of LPS and PGN. Successful treatment of patients having mixed bacterial infections is likely to require interventions that address the mechanisms involved in responses induced by a variety of bacterial cell wall components. peptidoglycan lipopolysaccharide muramyldipeptide (N-acetylmuramyl-l-alanyl-d-isoglutamine) Toll-like receptor tumor necrosis factor reverse transcription monoclonal antibody interleukin soluble PGN Bacteremia is a critical problem in intensive care units, accounting for high morbidity and mortality rates. The mortality rate associated with bacteremia exceeds 30% (1Roberts F.J. Geere I.W. Coldman A. Rev. Infect. Dis. 1991; 13: 34-46Crossref PubMed Scopus (167) Google Scholar, 2Pittet D. Tarara D. Wenzel R.P. JAMA (J. Am. Med. Assoc.). 1994; 271: 1598-1601Crossref PubMed Scopus (1279) Google Scholar). In a recent 12-year clinical study, Gram-positive and Gram-negative bacteria accounted for 46.9 and 31.5% of bacteremic episodes in an intensive care unit, respectively, with Gram-positive organisms being cultured from more patients (3Crowe M. Ispahani P. Humphreys H. Kelley T. Winter R. Eur. J. Clin. Microbiol. Infect. Dis. 1998; 17: 377-384PubMed Google Scholar). Further, the incidence of combined infections increased more than 4-fold over the 12-year period and was associated with a mortality rate exceeding 55%. Based on the fact that the majority of the deleterious effects of bacteremia are caused by inflammatory responses to specific bacterial components, these findings suggest that the patient's response to a mixture of Gram-positive and Gram-negative organisms may be heightened to the detriment of the patient. The two most commonly studied components of Gram-positive and Gram-negative bacterial cell walls are peptidoglycan (PGN)1 and lipopolysaccharide (LPS), respectively. Although Gram-negative bacterial cell walls also contain PGN, its concentration is far greater in the walls of Gram-positive bacteria (4Yang S. Tamai R. Akashi S. Takeuchi O. Akira S. Sugawara S. Takada H. Infect. Immun. 2001; 69: 2045-2053Crossref PubMed Scopus (184) Google Scholar). Proinflammatory effects of these bacterial cell wall components occur both in vitro after treatment of mononuclear phagocytes and in in vivo after exposure of whole animals, with cells and animals being more sensitive to LPS than to PGN (5Lichtman S.N. Wang J. Schwab J.H. Lemasters J.J. Hepatology. 1994; 19: 1013-1022Crossref PubMed Scopus (56) Google Scholar). The results of recent experimental studies provide evidence for a synergistic effect of LPS with muramyldipeptide (MDP), the minimal structural subunit of PGN, accounting for some of its immunogenicity (6Baschang G. Tetrahedron. 1989; 45: 6331-6360Crossref Scopus (98) Google Scholar). However, the underlying mechanism of action for MDP has not been fully elucidated. For example, there are discrepancies regarding the involvement of specific receptors, with some investigators indicating that MDP exerts its synergistic effect in human leukocytes in a CD14-dependent manner (7Wang J.E. Jorgenson P.F. Ellingsen E.A. Almlof M. Thiemermann C. Foster S.J. Aasen A.O. Solberg R. Shock. 2001; 16: 178-182Crossref PubMed Scopus (53) Google Scholar). Others indicate that the response is CD14- and Toll-like receptor 4 (TLR4)-independent in human monocytic cell lines and that MDP up-regulates expression of one of the primary components (MyD88 mRNA) in the TLR-mediated response to LPS (4Yang S. Tamai R. Akashi S. Takeuchi O. Akira S. Sugawara S. Takada H. Infect. Immun. 2001; 69: 2045-2053Crossref PubMed Scopus (184) Google Scholar). We report here that MDP not only synergizes with LPS but also acts similarly with PGN to induce the synthesis of tumor necrosis factor (TNF)-α in the human monocytic cell line Mono Mac 6. This synergistic effect of MDP with LPS or PGN was investigated in relation to expression and stability of TNF-α mRNA. Furthermore, the role of receptors (e.g. CD14 and TLR2/4) known to be involved in mediating cellular activation in response to bacterial cell wall components was studied. PGN from Staphylococcus aureus was obtained from BioChemika, MDP (N-acetylmuramyl-l-alanyl-d-isoglutamine) was from Calbiochem, Escherichia coli 055:B5 LPS and3H-labeled E. coli K12 LCD25 LPS were from List Biological Laboratories, polymyxin B was from Bedford Laboratories, and actinomycin D was from Sigma. Affinity-purified anti-CD14 antibodies MEM-18 (IgG1) and MY4 (IgG2b) were purchased from SANBIO b.v. and Coulter, respectively. Functional grade purified anti-human TLR2 (clone TL2.1) and TLR4 (clone HTA125) antibodies (IgG2a) were from Bioscience. Affinity-purified mouse IgG1 (Sigma), IgG2b (Coulter), and IgG2a (Sigma) were used as control antibodies. There were no effects of preincubation with these control antibodies for MEM-18, MY4, or TLRs. PGN was assayed for endotoxin using the Limulus Amoebocyte Lysate assay (BioWhittaker). No significant endotoxin contamination of this preparation was detected (≤1 ng of endotoxin/mg). Mono Mac 6 cells, provided by Dr. H. W. L. Ziegler-Heitbrock (University of Munich, Germany), were cultured in RPMI 1640 medium with l-glutamine (BioWhittaker) supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, 1% OPI supplement (Sigma; containing oxaloacetate, pyruvate, and bovine insulin), and 10% fetal calf serum (HyClone). The cells were maintained in a humid 5% CO2 atmosphere at 37 °C. New batches of frozen cell stock were grown up every 2 months, and growth morphology was evaluated. Before each experiment, Mono Mac 6 cells were allowed to differentiate for 2 days in the presence of 10 ng/ml calcitriol (Sigma). Cells were harvested by centrifugation and gently resuspended (106cells/ml) in prewarmed (37 °C) medium. Cells were then incubated for 6 h with different combinations of stimuli in the presence or absence of polymyxin B or antibodies as described under "Results." At the end of the incubation period, cell supernatants were collected and stored frozen (−80 °C) until assayed for TNF-α protein. In the experiments with antibodies, the cells were incubated with each antibody for 30 min at 4 °C before the stimuli were added. Concentrations of TNF-α in culture supernatants were determined in duplicate by a solid phase sandwich enzyme-linked immunosorbent assay. Briefly, 96-well plates (Nalge Nunc International) were coated with purified mouse anti-human TNF-α monoclonal antibody (mAb) (Pharmingen). TNF-α in standards and samples was allowed to bind to the immobilized mAb for 2 h at room temperature. Biotinylated mouse anti-human TNF-α mAb (Pharmingen) was then added, producing an antibody-antigen-antibody "sandwich." After the addition of avidin-horseradish peroxidase conjugate (Pharmingen) and ABTS peroxidase substrate (Kirkegaard & Perry Laboratories), a green color was produced in direct proportion to the amount of TNF-α present in the sample. The reaction was stopped by adding peroxidase stop solution (Kirkegaard & Perry Laboratories), and the absorbance was measured at 405 nm using a microplate reader (Dynatech Laboratories). All data for TNF-α are presented as the means ± S.D. of duplicate cultures. Each experiment was repeated at least twice. To ensure that any increase in TNF-α production was not caused by LPS contamination of the solutions containing the various stimuli, the experiments were performed in the absence and presence of polymyxin B, an antibiotic that avidly binds to the lipid A region of LPS, thereby preventing LPS-induced monokine production (8Tsubery H. Ofek I. Cohen S. Fridkin M. Biochemistry. 2000; 39: 11837-11844Crossref PubMed Scopus (68) Google Scholar). TNF-α concentrations in supernatants of cells preincubated with polymyxin B (1 μg/ml) for 30 min before incubation with E. coli O55:B5 LPS for 6 h were reduced from 2330 ± 111 pg/ml to 10 ± 7 pg/ml, whereas preincubation with polymyxin B had no effect on TNF-α synthesis by cells incubated with PGN (∼1800 pg/ml) or MDP (∼80 pg/ml). Therefore, LPS contamination of the latter preparations was inconsequential. Cells were harvested by centrifugation and gently suspended (1.25 × 106cells/ml) in prewarmed (37 °C) medium. In appropriate experiments, cells were incubated first with antibody or medium (control) for 30 min at 4 °C. Cells were then incubated with the indicated concentrations of the stimuli for 1.5 h, after which cells were harvested and total RNA was isolated using the StrataPrep Total RNA Miniprep Kit (Stratagene) according to the manufacturer's protocol. TNF-α gene expression was quantified in a two-step reverse transcription-PCR (RT-PCR). In the RT step, cDNA was reverse transcribed from total RNA samples (0.625 μg/50 μl) using random hexamers from the TaqMan RT reagents (Applied Biosystems). In the PCR step, PCR products were synthesized from cDNA (22.5 ng/20 μl) using the TaqMan universal PCR master mix and TaqMan pre-developed assay reagents for human TNF-α (Applied Biosystems). Measurements were done using the ABI Prism 7900 HT sequence detection system (Applied Biosystems), according to the manufacturer's protocols. As an endogenous control for these PCR quantification studies, 18 S ribosomal RNA gene expression was measured using the TaqMan ribosomal RNA control reagents (Applied Biosystems). Results represent means ± S.D. of triplicate measurements. Each experiment was repeated at least twice. Kinetic analysis of the [3H]LPS dissociation rate (off rate) from Mono Mac 6 cells was performed by the method of Kitchens and Munford (9Kitchens R.L. Munford R.S. J. Biol. Chem. 1995; 270: 9904-9910Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Mono Mac 6 cells were washed in ice-cold HNE buffer (20 mm HEPES, pH 7.4, 150 mm NaCl, 1 mm EDTA) and preincubated for 30 min at 37 °C in SEBDAF buffer (20 mm HEPES, pH 7.4, 150 mm NaCl, 1 mm EDTA, 300 μg/ml bovine serum albumin, 10 mm NaN3, 2 mmNaF, 5 mm deoxyglucose) to deplete intracellular ATP and prevent ligand internalization. The cells were then centrifuged and resuspended in RPMI containing 300 μg/ml bovine serum albumin, 10 mm NaN3, 2 mm NaF, and 5 mm deoxyglucose (1 × 106 cells in 500 μl, final volume). [3H]LPS (final concentration 30 ng/ml) was first mixed with fetal calf serum (final concentration 7.5%) as a source for LPS-binding protein. It was added to the cells and incubated with frequent mixing for 1 h at 37 °C to reach equilibrium. Next, unlabeled LPS (final concentration 10 μg/ml) or a mixture of unlabeled LPS and MDP (final concentrations 10 and 100 μg/ml, respectively), mixed first with fetal calf serum (final concentration 7.5%), were added to the cells. The cells were harvested at several time points. To determine nonspecific binding, unlabeled LPS alone or in combination with MDP was added to the cells before the addition of [3H]LPS. The cells were harvested by adding ice-cold HNE buffer (500 μl) and centrifuging the mixtures at 15,000 rpm for 2 min at 4 °C. The supernatant was aspirated, and the cells were washed with 500 μl of ice-cold HNE buffer. The cells were lysed in 5 ml of liquid scintillation mixture (Beckman Instruments Inc.), and the cell-associated 3H was counted. Results represent means ± S.D. of triplicate samples. The experiment was repeated with similar results. LPS and PGN concentration-response data for stimulation of TNF-α production in Mono Mac 6 cells were analyzed using nonlinear least-squares curve fitting in Prism (GraphPad Software, Inc.). These data were fit with the logistic equation, Y=Emax/(1+(EC50/X)Hill slope)Equation 1 where Y represents the TNF-α response, Xis the LPS or PGN concentration, Emax is the maximum response, and EC50 is the concentration of LPS or PGN producing 50% stimulation. [3H]LPS dissociation experiments were analyzed by fitting a monoexponential decay equation to the data, Y=Y0e−k1Equation 2 where Y is the amount of [3H]LPS bound at time t, Y 0 is the amount of [3H]LPS bound at time 0, t is time of dissociation, and k is the dissociation rate constant. Half-lives for [3H]LPS dissociation in the presence and absence of MDP were calculated as follows. t1/2=0.693/kEquation 3 The effect of a 30-min preincubation of Mono Mac 6 cells with MDP on TNF-α secretion induced by a wide concentration range of LPS and PGN was compared with incubation with either LPS or PGN alone (Fig.1). Preincubation of cells with MDP resulted in a LPS concentration-response curve having a higher maximal level (a 2-fold increase), whereas the EC50 and Hill slope values did not differ significantly from those obtained in the absence of MDP. In the absence of MDP, the maximum concentration of produced TNF-α in response to LPS was 1477 pg/ml, with a Hill slope of 2.7 and an LPS EC50 of 13.2 ng/ml. Pretreatment with MDP increased the maximum level of LPS-induced TNF-α production to 3003 pg/ml; the Hill slope and EC50 were 3.1 and 8.6 ng/ml, respectively. Stimulation with MDP alone resulted in a TNF-α concentration of 71 pg/ml. Similar results were obtained when cells were preincubated with MDP followed by PGN (Fig. 1 b). The maximum level of TNF-α after incubation with PGN alone was 3313 pg/ml with a Hill slope of 1.1 and a PGN EC50 of 26.1 μg/ml. Preincubation with MDP yielded a maximum level of 5469 pg/ml, a Hill slope of 1.2, and an EC50 of 22.4 μg/ml. These results, derived from four-parameter logistic fits to the data, demonstrate that the singular observable effect of MDP is to increase the maximum TNF-α responses to LPS or PGN in the absence of an influence on their respective potencies. In addition to the experiments presented above, in which Mono Mac 6 cells were incubated with MDP before the addition of LPS or PGN, experiments also were performed in which MDP was added simultaneously with LPS or PGN. Simultaneous treatment of Mono Mac 6 cells with MDP and LPS or PGN produced the same increase in TNF-α secretion (data not shown), suggesting that pretreatment was not necessary for the effect of MDP. Further, we performed several experiments in which the cells were first exposed to MDP for 10 or 20 min, washed, and then exposed to either LPS or PGN. The effect of exposure to MDP for only 10 min on TNF-α concentration was more than additive although not as dramatic as that occurring with simultaneous and continuous treatment of the cells with MDP and LPS or PGN (data not shown). We also performed experiments to determine whether preincubation with MDP had a synergistic effect with subsequent exposure to a second dose of MDP. In these experiments, the response was simply additive (data not shown). To rule out the possibility that Mono Mac 6 cells incubated with MDP produce TNF-α that remains associated with the cells and was not secreted, TNF-α concentrations in the supernatant were compared with total (cell-associated and secreted) TNF-α concentrations. Incubation of cells with LPS, PGN, MDP, MDP plus LPS, and MDP plus PGN produced only small amounts of TNF-α that remained cell-associated; total TNF-α concentrations were indistinguishable from those in the supernatants (data not shown). TNF-α production in response to LPS is controlled both at the transcriptional and post-transcriptional levels (10Biragyn A. Nedospasov S.A. J. Immunol. 1995; 155: 674-683PubMed Google Scholar, 11Beutler B. Am. J. Med. Sci. 1992; 303: 129-133Crossref PubMed Scopus (45) Google Scholar, 12Kontoyiannis D. Pasparakis M. Pizarro T.T. Cominelli F. Kollias G. Immunity. 1999; 10: 387-398Abstract Full Text Full Text PDF PubMed Scopus (1097) Google Scholar). To study whether the synergistic induction of Mono Mac 6 cells by MDP is controlled at the level of gene transcription, TNF-α mRNA expression was measured after treatment with medium, LPS or PGN alone, or LPS or PGN in combination with MDP. The effects of LPS and PGN each were determined at their lowest concentrations causing maximal TNF-α protein production (i.e. LPS 30 ng/ml; PGN 100 μg/ml). Data presented in Fig. 2 a indicate that incubation with LPS or PGN results in similar levels of TNF-α mRNA. Incubation with MDP (100 μg/ml) resulted in a 10-fold increase in TNF-α gene expression compared with control cells, although this was less than that caused by LPS or PGN. The same concentration of MDP caused very low levels of TNF-α protein secretion compared with LPS or PGN. Simultaneous treatment of cells with MDP and either LPS or PGN resulted in further enhancement of TNF-α mRNA expression, which appears to represent the additive effects of MDP and LPS or PGN individually. In contrast, concentrations of TNF-α protein in the supernatants from the same samples, even after only 90-min exposure, were suggestive of a pronounced synergistic effect of MDP with either LPS or PGN (Fig. 2 b). In addition to the experiment presented above, in which Mono Mac 6 cells were stimulated for 90 min before RNA extraction, the same experiments were also performed in which RNA was extracted after 30, 60, and 120 min of stimulation (Table I). In all cases, only a small amount of mRNA was expressed after 30 min. Stimulation with LPS or PGN produced maximal mRNA expression at 60 min, whereas stimulation with MDP or MDP in combination with LPS or PGN reached maximal values at 90 min. TNF-α mRNA expression was reduced markedly by 120 min. Varying the incubation period did not alter the finding that simultaneous treatment of cells with MDP and either LPS or PGN resulted in an additive effect on TNF-α mRNA expression.Table IRelative TNF-α gene expression by Mono Mac 6 cells after stimulation with different stimuli followed in timeRelative quantity of TNF-α mRNA (95% confidence intervals)30 min60 min90 min120 minUntreated1.0–1.00.8–1.21.0–1.00.9–1.1LPS (30 ng/ml)1.1–1.617.3–18.115.8–19.411.5–12.5PGN (100 μg/ml)1.9–2.229.1–32.117.5–22.513.0–15.4MDP (100 μg/ml)0.9–1.53.2–4.27.2–8.52.4–2.6MDP (100 μg/ml) and LPS (30 ng/ml)1.2–1.421.2–22.526.8–33.68.9–11.7MDP (100 μg/ml) and PGN (100 μg/ml)2.0–2.322.4–30.427.1–31.214.2–18.6 Open table in a new tab After determining that incubation of cells with MDP at 100 μg/ml resulted in TNF-α gene expression but minimal protein translation, we wondered whether this effect might be dependent on MDP concentration and whether the degree of mRNA expression in response to MDP might be insufficient to result in protein translation. To address this question, Mono Mac 6 cells were incubated for 90 min with medium alone as control, LPS ranging from 0.01 to 100 ng/ml, or MDP ranging from 0.01 to 300 μg/ml, in an effort to determine whether there were concentrations of LPS causing TNF-α mRNA expression but not translation and to determine the dose dependence for the effects of MDP. Even after only 90 min, there was a clear dose-response for LPS, both for TNF-α protein production and TNF-α mRNA expression (Fig. 3). A plateau in TNF-α protein production was evident at 30 ng/ml LPS, a finding that was in agreement with data presented in Fig. 1, where cells were stimulated for 6 h. However, a plateau was not observed for TNF-α RNA expression. Although incubation with MDP at 100 μg/ml resulted in TNF-α protein production comparable with that caused by 0.01–0.1 ng/ml LPS, TNF-α mRNA expression induced by MDP was comparable with that caused by 1 ng/ml LPS. These results demonstrate that the amount of TNF-α mRNA expressed when cells are incubated with MDP was not a limiting factor for subsequent protein translation. Incubation of the cells with MDP yielded dose-dependent increases in both TNF-α protein production and TNF-α mRNA expression. Although TNF-α protein concentrations barely exceeded background values even at a MDP concentration of 300 μg/ml (Fig.3 a), TNF-α mRNA expression was increased by MDP at 10 μg/ml and reached a maximum at 100 μg/ml (Fig. 3 b). Consequently, we used MDP at 100 μg/ml in the remainder of the study. The above results suggest that the apparent synergistic effects of MDP and either LPS or PGN were caused by different patterns of regulation of mRNA translation. It was reported recently that synergistic production of TNF-α by macrophages exposed to a combination of bacterial DNA and LPS was at least in part caused by prolonged half-life of TNF-α mRNA (13Gao J.J. Xue Q. Papasian C.J. Morrison D.C. J. Immunol. 2001; 166: 6855-6860Crossref PubMed Scopus (79) Google Scholar). To investigate whether this was the case for Mono Mac 6 cells exposed to a combination of MDP and either LPS or PGN, the half-life of TNF-α mRNA was determined in Mono Mac 6 cells exposed to medium, LPS or PGN alone, or LPS or PGN in combination with MDP. After a 90-min exposure, further transcription was inhibited by treating the cells with actinomycin D (10 μg/ml). Total RNA was isolated at various time points after the addition of actinomycin D, and TNF-α mRNA expression was measured by RT-PCR. The half-lives of TNF-α mRNA in Mono Mac 6 cells stimulated with LPS, PGN, MDP, MDP plus LPS, and MDP plus PGN were 14.3, 14.6, 11.5, 14.1, and 13.4 min, respectively (Fig.4). The half-life of TNF-α after stimulation with MDP alone was slightly shorter compared with the other stimuli, but the half-lives of the combinations of MDP and LPS or PGN did not differ significantly from those of LPS or PGN alone. Therefore, the effect of MDP with either LPS or PGN on release of TNF-α cannot be explained by altered stability of TNF-α mRNA. Consequently, other post-transcriptional factors must be responsible for the effect. The fact that maximal TNF-α mRNA expression for cells incubated with LPS or PGN occurred at 60 min whereas maximal mRNA expression for cells incubated with MDP alone or MDP with either LPS or PGN occurred at 90 min suggests that activation of gene expression by MDP might occur through a different route than LPS and PGN. To address this question, we performed experiments to explore the possibility that different receptors are involved in the response of the cells to MDP and either LPS or PGN. Two anti-CD14 mAbs, MY4 and MEM-18, were used to assess the involvement of CD14. It has been reported previously that the binding of LPS and PGN to CD14 involves amino acids 51–64 or 57–64 in the N-terminal region of the receptor (14Dziarski R. Tapping R.I. Tobias P.S. J. Biol. Chem. 1998; 273: 8680-8690Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 15Juan T.S.-C. Hailman E. Kelley M.J. Busse L.A. Davy E. Empig C.J. Narhi L.O. Wright S.D. Lichenstein H.S. J. Biol. Chem. 1995; 270: 17237-17242Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). MEM-18, with its epitope at residues 57–64, is an anti-CD14 mAb specific for this region. The epitope of MY4 is located closer to the N-terminal end of CD14 at amino acids 34–44. MY4 is quite effective in inhibiting binding of both LPS and sPGN to soluble CD14 (14Dziarski R. Tapping R.I. Tobias P.S. J. Biol. Chem. 1998; 273: 8680-8690Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). In experiments performed in our laboratory, both MEM-18 and MY4 completely neutralized the effect of LPS, whereas the effect of PGN was reduced by 86 and 54% by MEM-18 and MY4, respectively (Fig. 5). We have previously reported that increasing the concentration of these mAbs did not further increase their blocking effect on PGN (16Siriwardena A. Jorgenson M.R. Wolfert M.A. Vandenplas M.L. Moore J.N. Boons G.-J. J. Am. Chem. Soc. 2001; 123: 8145-8146Crossref PubMed Scopus (18) Google Scholar). Even when LPS appeared to be completely blocked by the anti-CD14 mAbs, the presence of MDP resulted in significant amounts of TNF-α secretion (Fig. 5 a). A similar effect of MDP on the response to PGN was observed, even when the effects of PGN alone were partially blocked with the anti-CD14 mAbs (Fig. 5 b). These results suggest that the effect of MDP on the subsequent response to LPS and PGN was CD14-independent. Expression of TNF-α mRNA was determined for cells incubated for 90 min with medium (control), LPS or PGN alone, or LPS or PGN in combination with MDP in the absence or presence of MY4. Whereas LPS-induced expression of TNF-α mRNA was almost completely abolished by MY4, this antibody had minimal effect on the responses to PGN or MDP alone, and the additive effects of MDP on the response to either LPS or PGN persisted in the presence of MY4 (TableII). These results provide further evidence that this effect of MDP was CD14-independent.Table IIEffect of anti-CD14 mAb MY4 on the induction of TNF-α mRNA by Mono Mac 6 cells incubated with different stimuli for 90 minRelative Quantity of TNF-α mRNA (95% confidence intervals)ControlMY4 (20 μg/ml)Untreated0.9–1.21.0–1.1LPS (30 ng/ml)7.7–9.71.0–1.9PGN (100 μg/ml)7.1–8.96.2–8.2MDP (100 μg/ml)4.5–5.54.9–5.7MDP (100 μg/ml) and LPS (30 ng/ml)12.3–15.75.0–6.3MDP (100 μg/ml) and PGN (100 μg/ml)9.2–11.28.1–10.9 Open table in a new tab TLR2 and -4 have been implicated as critical receptors responsible for initiation of signaling events and cellular activation in response to bacterial cell wall components (17Tapping R.I. Akashi S. Miyake K. Godowski P.J. Tobias P.S. J. Immunol. 2000; 165: 5780-5787Crossref PubMed Scopus (301) Google Scholar). For instance, there is compelling information that TLR4 plays a critical role in LPS-induced cell signaling and serves as a cell surface co-receptor for CD14. These two receptors are necessary for LPS-mediated NF-κB activation and subsequent cellular events (18Chow J.C. Young D.W. Golenbock D.T. Christ W.J. Gusovsky F. J. Biol. Chem. 1999; 274: 10689-10692Abstract Full Text Full Text PDF PubMed Scopus (1612) Google Scholar). Although incubation with the mAb directed against TLR4 reduced the effect of LPS by 80% (Fig. 6), it did not affect the synergism between MDP and LPS. MDP increased LPS-induced synthesis of TNF-α by 3.3-fold in the absence of the anti-TLR4 mAb and by 3.7-fold in the presence of the antibody. These findings provide evidence that the synergism of MDP with LPS was TLR4-independent. The results of recent studies suggest that TLR2 is involved in cellular responses to a wide variety of infectious pathogens and their products, including PGN (17Tapping R.I. Akashi S. Miyake K. Godowski P.J. Tobias P.S. J. Immunol. 2000; 165: 5780-5787Crossref PubMed Scopus (301) Google Scholar). Incubation of cells with the mAb against TLR2 reduced the effect of PGN by 51% (Fig.7). In the absence of the anti-TLR2 mAb, MDP increased PGN-induced synthesis of TNF-α protein by 2.4-fold. Similarly, PGN-induced TNF-α production was increased 2.0-fold in cells co-incubated with MDP and the anti-TLR2 mAb. These findings suggest that the synergistic effect of MDP on the cellular response to PGN was also TLR2-independent. Further, TNF-α mRNA expression was determined for cells