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R Dziarski

Publications and source records attributed to R Dziarski.

At least 37 records · Page 2Linked to original sources
PubMed1996

CD14 is a cell-activating receptor for bacterial peptidoglycan.

The hypothesis that CD14 (an endotoxin receptor present on macrophages and neutrophils) acts as a cell-activating receptor for bacterial peptidoglycan was tested using mouse 70Z/3 cells transfected with human CD14. 70Z/3 cells transfected with an empty vector were unresponsive to insoluble and soluble peptidoglycan, as well as to low concentrations of endotoxin. 70Z/3-CD14 cells were responsive to both insoluble and soluble peptidoglycan, as well as to low concentrations of endotoxin, as measured by the expression of surface IgM, activation of NF-kappaB, and degradation of IkappaB-alpha. Peptidoglycan also induced activation of NF-kappaB and degradation of IkappaB-alpha in macrophage RAW264.7 cells. These peptidoglycan-induced effects (in contrast to endotoxin-induced effects) were not inhibited by polymyxin B. Both peptidoglycan- and endotoxin-induced activation of NF-kappaB were inhibited by anti-CD14 mAb. The N-terminal 151 amino acids of CD14 were sufficient for acquisition of full responsiveness to both peptidoglycan and endotoxin, but CD14 deletion mutants lacking four small regions within the N-terminal 65 amino acids showed differentially diminished responses to peptidoglycan and endotoxin. These results identify CD14 as the functional receptor for peptidoglycan and demonstrate that similar, but not identical sequences in the N-terminal 65-amino acid region of CD14 are critical for the NF-kappaB and IgM responses to both peptidoglycan and endotoxin.

Amino Acid Sequence↗
PubMed1996

Differential activation of extracellular signal-regulated kinase (ERK) 1, ERK2, p38, and c-Jun NH2-terminal kinase mitogen-activated protein kinases by bacterial peptidoglycan.

Soluble staphylococcal peptidoglycan (sPGN) is an inducer of cytokine secretion and may activate macrophages through the CD14 lipopolysaccharide (LPS) receptor. To elucidate sPGN-activated signal transduction pathways, stimulation of mitogen-activated protein (MAP) kinases by sPGN was studied in mouse RAW264.7 macrophages. sPGN strongly activated extracellular signal-regulated kinase (ERK) 1 and ERK2, moderately activated c-Jun NH2 terminal kinase (JNK), and weakly activated p38 MAP kinase, in contrast to LPS, which strongly activated all of these kinases, and phorbol 12,13-dibutyrate (PDB), which strongly activated ERK1 and ERK2 but did not activate p38 or JNK. sPGN- and LPS-induced activation of ERK1 and ERK2, unlike PDB-induced activation, was sensitive to inhibition by herbimycin A and insensitive to inhibition by increased intracellular cAMP. These results demonstrate differential activation of MAP kinases by sPGN, similar but not identical activation of signal transduction pathways by sPGN and LPS, and different mechanisms of MAP kinase activation by bacterial stimulants and phorbol esters.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Peptidoglycan induces transcription and secretion of TNF-alpha and activation of lyn, extracellular signal-regulated kinase, and rsk signal transduction proteins in mouse macrophages.

In soluble peptidoglycan (PGN) from staphylococcal cell walls as well as soluble PGN (sPGN) secreted by staphylococci in the presence of beta-lactam antibiotics induced TNF-alpha mRNA and secretion of bioactive TNF-alpha in the murine RAW264.7 macrophage cell line, PGN and sPGN also induced rapid and dose-dependent tyrosine phosphorylation of several cellular proteins, including lyn and mitogen-activated protein kinases (extracellular signal-regulated kinases; but not hck, fgr, or vav) and increased the activities of mitogen-activated protein and rsk kinases. These PGN- and sPGN-induced effects were qualitatively similar to the effects induced by ReLPS, but higher concentrations of PGN and sPGN than ReLPS were required. In contrast to the ReLPS-induced effects, the PGN- and sPGN-induced effects were not inhibited by polymyxin B. All PGN-, sPGN-, and ReLPS-induced effects were serum independent, since they were observed both in RAW264.7 cells grown and stimulated in the presence of serum and in the cells adapted to growth and stimulated in a serum- and albumin-free medium. These results indicate that lyn, extracellular signal-regulated kinase, and rsk signal transduction molecules may be involved in macrophage activation by PGN and further support the idea that PGN and LPS may activate the cells through similar mechanisms.

Animals↗
PubMed1994

Cell-bound albumin is the 70-kDa peptidoglycan-, lipopolysaccharide-, and lipoteichoic acid-binding protein on lymphocytes and macrophages.

The 70-kDa protein that is present on the surface of lymphocytes and macrophages and that binds peptidoglycan, lipopolysaccharide, lipoteichoic acid, heparin, and sulfated heparinoids was identified as cell-bound albumin. It originated from the tissue culture medium or from the serum in vivo and was not produced by the cells. The following results supported this conclusion: (a) mouse and human cell lines grown in serum-free and albumin-free medium did not have this 70-kDa protein and did not bind peptidoglycan and lipopolysaccharide in photoaffinity cross-linking assay; (b) the appearance of the 70-kDa protein in these cells and the ligand binding were restored by 30-min incubation with serum or purified albumin; (c) soluble albumin bound peptidoglycan and lipopolysaccharide, and this binding was inhibited by the same competitive inhibitors that inhibited the binding to the 70-kDa cellular protein; (d) albumin co-migrated with the 70-kDa cellular protein in two-dimensional polyacrylamide gel electrophoresis; (e) peptide maps of albumin and the 70-kDa cellular protein digested with four proteases were identical; (f) the only protein recognized by anti-albumin monoclonal and polyclonal antibodies on Western immunoblots was the 70-kDa protein that exactly matched with the cellular peptidoglycan/lipopolysaccharide-binding protein; (g) anti-albumin monoclonal antibodies immunoprecipitated the 70-kDa cellular protein; and (h) species-specific anti-bovine, anti-human, and anti-mouse albumin antibodies recognized the 70-kDa protein on mouse and human cells according to the species of albumin that was present in the culture medium or in the serum in vivo, but not according to the species of the cells. The cell-bound albumin was not required for cell activation, because macrophage cell lines that were grown in albumin-free medium and did not have the cell-bound albumin (the 70-kDa protein) fully responded to the stimulation by peptidoglycan and lipopolysaccharide with production of tumor necrosis factor-alpha.

Affinity Labels↗
PubMed1994

Heparin, sulfated heparinoids, and lipoteichoic acids bind to the 70-kDa peptidoglycan/lipopolysaccharide receptor protein on lymphocytes.

The same 70-kDa protein, present on the surface of mouse lymphocytes, served as the predominant binding site for heparin, heparinoids, and bacterial lipoteichoic acids, as well as peptidoglycan and lipopolysaccharides. This conclusion was supported by the following results: (a) all of these compounds photoaffinity cross-linked to one major 70-kDa 6.5-7.0 pI protein that co-migrated on two-dimensional polyacrylamide gel electrophoresis; (b) peptide maps of the 70-kDa proteins digested with chymotrypsin, subtilisin, protease V, or papain yielded the same peptides for heparin-, lipoteichoic acid-, peptidoglycan-, and lipopolysaccharide-binding proteins; (c) cross-linking of peptidoglycan, lipopolysaccharide, lipoteichoic acid, and heparin was competitively inhibited by the same compounds with the same order of potency, i.e. carboxyl-reduced sulfated heparin > peptidoglycan > pentosan polysulfate > heparin > chitin > dextran sulfate > trestatin sulfate > polyanetholesulfonate > fucoidan > beta-cyclodextrin tetradecasulfate > heparan sulfate > carrageenan lambda > lipoteichoic acids > Re-lipopolysaccharide > lipopolysaccharide > lipid A > polygalacturonic acid; and (d) cross-linking of each of these ligands was not inhibited by carboxyl-reduced heparin, dextran, beta-cyclodextrin, trestatin, carrageenan kappa, chondroitin 4-sulfate, chondroitin 6-sulfate, beta-D-glucan, carboxy-methylcellulose, levan, alpha-D-mannan, and glycogen. The minimum size of the molecule that bound was 7-9 glycan residues, whereas, di- and trisaccharides did not bind. There was a logarithmic linear relationship between the strength of the binding and the length of the polymer (up to > 1500 glycan residues), which indicates an avidity effect of the cooperative binding of one polymeric molecule to several receptor molecules on the cell surface. The 70-kDa receptor, therefore, has a broad, but limited specificity of binding for non-charged (peptidoglycan and chitin), highly negatively charged (heparin and heparinoids), and weakly negatively charged (lipoteichoic acids, lipopolysaccharides, and lipid A) ligands.

Acute-Phase Proteins↗
PubMed1994

Soluble peptidoglycan-induced monokine production can be blocked by anti-CD14 monoclonal antibodies and by lipid A partial structures.

We have investigated the interaction of soluble peptidoglycan (sPG), in comparison with lipopolysaccharide (LPS), with human mononuclear cells (MNC) by determining the capacity of sPG to induce interleukin-6 (IL-6) and IL-1 release. In addition, we investigated the modulation of their interaction by anti-CD14 monoclonal antibody and by partial structures of LPS. We found that sPG, like LPS, was able to induce IL-6 and IL-1 production by MNC. However, dose-response experiments revealed that at least 3,000 ng of sPG per ml was necessary for induction, whereas the optimal LPS concentration was 1 ng/ml. Anti-CD14 monoclonal antibody reduced sPG- and LPS-induced IL-6 and IL-1 production. Moreover, partial structures of LPS were able to reduce monokine production induced by sPG and LPS. We conclude that sPG constitutes, like LPS, an inflammatory cytokine inducer and that CD14 is involved in the activation of human monocytes not only by LPS but also by sPG.

Acute-Phase Proteins↗
PubMed1992

Synergistic enhancement of T cell responses and interleukin-1 receptor expression by interleukin-1 and heparin or dextran sulfate.

Heparin markedly enhances generation of cytotoxic T lymphocytes against allogeneic cells and histocompatible tumors. In this study, we demonstrated a marked synergism between heparin and low concentrations of recombinant IL-1-alpha and IL-1-beta in enhancement of cytotoxic T cell responses in mice. Low molecular weight (8000 Da) dextran sulfate also enhanced the T cell responses and synergized with IL-1, whereas, de-N-sulfated heparin was devoid of both of these activities. The synergistic effect was selective for IL-1, because there was no synergism between heparin or dextran sulfate and other cytokines (tumor necrosis factor-alpha, IL-4, and, as shown previously, IL-2). Heparin did not increase the production of IL-1 (and IL-2, as shown before). Heparin did not bind to IL-1, despite significant amino acid homology between IL-1 and heparin-binding endothelial cell growth factors. Heparin enhanced the growth-promoting effect of IL-1 on the IL-1-dependent helper T cell clone, D10.G4.1, and enhanced IL-1 receptor expression on these cells. These data indicate that heparin acts directly on the T cells and enhances their responsiveness to IL-1 by up-regulating IL-1 receptor expression.

Animals↗
PubMed1991

Demonstration of peptidoglycan-binding sites on lymphocytes and macrophages by photoaffinity cross-linking.

One dominant binding site (70 kDa 6.5 pI protein) for bacterial cell wall peptidoglycan (PGN), a macrophage activator and polyclonal B cell mitogen, was demonstrated on mouse B and T lymphocytes and macrophages by photoaffinity cross-linking and two-dimensional polyacrylamide gel electrophoresis. This binding site was not present on erythrocytes. The binding was specific for polymeric PGN and was competitively inhibited by unlabeled PGN with IC50 = 48 micrograms/ml (0.38 microM). The binding was partially inhibited by O-acetylated PGN monomers (IC50 = 469 micrograms/ml, 521 microM), dextran sulfate (IC50 = 1024 micrograms/ml, 124 microM), and (GlcNAc)3 (IC50 = 6.6 mg/ml, 10 mM), and was not inhibited by non-O-acetylated PGN monomers and dimers, muramyl dipeptide, PGN pentapeptide, GlcNAc, teichoic acid, protein A, and gelatin. The cell surface location of the 70-kDa PGN-binding protein was indicated by the ability of PGN to bind to this protein in intact metabolically inactive cells (at 4 degrees C and in the presence of 0.1% NaN3) and by the ability to extract the 70-kDa PGN-binding protein from viable B lymphocytes by noncytotoxic concentration of n-octyl-beta-D-glucopyranoside.

Affinity Labels↗
PubMed1991

Peptidoglycan and lipopolysaccharide bind to the same binding site on lymphocytes.

Bacterial cell wall peptidoglycan (PGN) and lipopolysaccharide (LPS), which are both macrophage activators and polyclonal B cell mitogens, were shown to bind to the same dominant 70-kDa 6.5 pI protein on the surface of mouse B lymphocytes. This conclusion was supported by the following results: (a) the PGN- and LPS-binding proteins co-migrated following photoaffinity cross-linking and two-dimensional polyacrylamide gel electrophoresis; (b) cross-linking of PGN to this 70-kDa protein was competitively inhibited by LPS (IC50 = 7.3 microM), LPS from a deep rough mutant (IC50 = 6.9 microM), and lipid A (IC50 = 18-72 microM); (c) cross-linking of LPS to this 70-kDa protein was competitively inhibited by polymeric soluble PGN (IC50 = 0.09 microM) and sonicated high Mr PGN (IC50 = 0.6 microM); (d) cross-linking of both PGN and LPS to this 70-kDa protein was also competitively inhibited by dextran sulfate (IC50 = 115-124 microM); (e) cross-linking of both PGN and LPS to this 70-kDa protein was inhibited by a (GlcNAc)2-specific lectin; and (f) peptide maps of the 70-kDa proteins digested with chymotrypsin, subtilisin, staphylococcal protease V, or papain were identical for PGN- and LPS-binding proteins and unique for each enzyme. Based on competitive inhibition experiments, binding of PGN to the 70-kDa protein was 20-1200 times stronger than the binding of LPS or lipid A on a per mol basis. However, when aggregated micellar structures of LPS or lipid A were considered, the avidities of LPS and PGN binding were similar. These results demonstrate binding of PGN and LPS to the same 70-kDa protein on lymphocytes and suggest that the binding is specific for the (GlcNAc-MurNAc)n backbone of PGN and the (GlcNAc)2 part of lipid A.

Affinity Labels↗
PubMed1989

Enhancement of mixed leukocyte reaction and cytotoxic antitumor responses by heparin.

The immunomodulating effects of heparin and natural and synthetic heparinoids (which are now undergoing clinical trials for the treatment of AIDS) on cellular immunity (DNA synthesis and cytotoxic responses of mouse lymphocytes to allogeneic cells and histocompatible tumors) were studied. The results showed that (1) high and low m.w. heparin enhanced mouse antitumor and antiallogeneic cell responses in vitro; (2) other sulfated heparinoids did not have this enhancing activity and some of them (including dextran sulfate) totally suppressed generation of cytotoxic cells; (3) these immunomodulating activities of heparin and heparinoids did not correlate with their anticoagulant effects, degree of sulfation, and mitogenic activity; (4) heparin did not increase the production of IL-2 and did not enhance the action of IL-2 on the cells in MLC, heparin also had no effect on the growth-promoting activity of IL-2 on cloned cytotoxic T cells; (5) heparin had a synergistic enhancing effect with IL-1 on the generation of cytotoxic cells in MLC; and (6) heparin abolished endothelial cell growth factor-induced suppression of cytotoxic response. The latter two effects by themselves, however, could not fully explain the entire immunoenhancing activity of heparin. These results indicate that heparin and heparinoids have multiple effects on the immune system and that some of them can enhance, whereas others can suppress cell-mediated responses.

Adjuvants, Immunologic↗
PubMed1989

Correlation between ribosylation of pertussis toxin substrates and inhibition of peptidoglycan-, muramyl dipeptide- and lipopolysaccharide-induced mitogenic stimulation in B lymphocytes.

Selective inhibition by pertussis toxin (PT) of mitogenic activation of mouse B lymphocytes by bacterial mitogens (peptidoglycan and lipopolysaccharide) and muramyl dipeptide (a synthetic analog of peptidoglycan fragment) was demonstrated. Mitogenic activation of B cells by protein kinase C activators and ionomycin was insensitive to PT. Also PT did not inhibit peptidoglycan- and lipopolysaccharide-induced differentiation of B cells into Ig-secreting cells, when it was added to the cultures after the proliferative stage of the response. B lymphocyte membranes contained two major PT substrates (40 and 41 kDa). The extent of PT-mediated ADP ribosylation of these substrates correlated with the degree of PT-mediated inhibition of mitogenic stimulation of B cells. B cell stimulation by all mitogens tested was not inhibited by cholera toxin at nontoxic concentrations that are known to cause maximal increase in cAMP in B cells. Since the only known substrates for PT-mediated ADP ribosylation in mammalian cells are the alpha subunits of some G proteins, our data suggest that G proteins are present in B cell membranes and that they are involved in B cell activation induced by bacterial mitogens.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1988

Enhancement of B-cell stimulation by muramyl dipeptide through a mechanism not involving interleukin 1 or increased Ca2+ mobilization or protein kinase C activation.

Muramyl dipeptide (MDP) enhanced mitogenic stimulation of mouse lymphocytes by polyclonal B cell activators (peptidoglycan, lipopolysaccharide, Staphylococcus aureus Cowan I cells, and pokeweed mitogen), but not by T-cell mitogens (phytohemagglutinin and concanavalin A). Only adjuvant-active MDP analogs were effective, whereas adjuvant-inactive MDP analogs, muramic acid, peptidoglycan pentapeptide, and low Mr digests of peptidoglycan were not. The half-maximal enhancement was seen at 5-10 microM MDP and occurred at both optimal and suboptimal concentrations of B cell mitogens. The enhancing effect of MDP was exerted on the B cells, since it was T cell- and macrophage-independent and was not mediated by IL-1. MDP was effective during the first 12 hrs of culture, and most strongly enhanced the mitogen-induced DNA synthesis, although significant enhancement of RNA synthesis and B cell differentiation into antibody-secreting cells was also observed. The enhancement of mitogenic response was not due to changed requirements for extracellular or intracellular Ca2+ or to increased activation of protein kinase C. These results demonstrate a novel immunoenhancing effect of MDP that should be useful in the studies on the mechanism of B cell activation.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1987

Binding sites for peptidoglycan on mouse lymphocytes.

Binding of peptidoglycan (PG), a B-cell mitogen and polyclonal activator, to mouse lymphocytes was studied using rosetting with PG-sensitized erythrocytes and a direct binding assay with 125I-labeled PG. Thirty-four percent of splenic lymphocytes formed PG rosettes, 62% of which were inhibited by preincubation of lymphocytes with free PG. Less than 1 or 3% of spleen cells formed rosettes with uncoated or albumin-coated red cells. The formation of rosettes was not inhibited by 0.1% azide and was not dependent on the presence of complement or immunoglobulins. The 125I-PG bound both specifically and nonspecifically to the lymphocytes. The binding was completed within 15-20 min, was proportional to the cell concentration, and was not inhibited by 0.1% azide or treatment of lymphocytes with formalin. The cells had one set of specific binding sites of low affinity (KD = 1.2-4.6 X 10(-7) M +/- 9% SE, based on competitive) experiments. The binding, however, was complex, probably involving interaction of multiple binding sites on PG with the cell surface. The EC50 (920 micrograms/ml) was similar to the optimal lymphocyte-activating concentration of PG (400-1000 micrograms/ml). The binding correlated with the ability of different PG preparations to stimulate lymphocytes, since only high Mr PG (not low Mr PG preparations, muramyl dipeptide (MDP), or PG pentapeptide) had the ability to specifically bind to lymphocytes, to compete with PG binding, and to stimulate lymphocytes. Also, low Mr PG preparations, MDP, or PG pentapeptide did not inhibit the mitogenic stimulation of lymphocytes by high Mr PG. These results indicate the presence of specific binding sites for PG on the surface of murine lymphocytes and suggest that the binding of PG to these binding sites is involved in lymphocyte activation by PG.

Animals↗
PubMed1987

Modulation of polyclonal activation by plasma fibronectin and fibronectin fragments.

Modulation of activation of polyclonal IgM, IgG and IgM anti-DNA antibodies by plasma fibronectin (Fn) was studied because in some autoimmune diseases there appears to be a correlation between the increased level of Fn in the affected tissues and increased polyclonal B-cell activation. Fn caused a dose-dependent polyclonal activation of IgM, IgG and IgM anti-DNA antibody-secreting cells in cultures of mouse splenocytes. Fn significantly inhibited the generation of polyclonal antibodies by Fn-binding stimulants and did not significantly change the generation of polyclonal antibodies by the stimulants that do not bind Fn. Plasmin or trypsin digestion of Fn abolished both the polyclonal activating properties of Fn and the inhibitory effects of Fn that were selective for the Fn-binding polyclonal activators. Digestion of Fn with trypsin also generated immunosuppressive Fn fragments that inhibited polyclonal activation by both Fn-binding and non-binding bacteria. Under our culture conditions Fn or Fn digests were not mitogenic and had no effect on the mitogenicity of Fn-binding and non-binding stimulants. These results indicate that Fn can act as a polyclonal activator and that it can also modulate lymphocyte activation induced by other activators.

Animals↗
PubMed1985

Ontogenic development of proliferative and polyclonal antibody and autoantibody responses to staphylococcal peptidoglycan, protein A and cell walls in mice.

In the spleens of newborn mice, polyclonal IgM, IgG and IgA responses were low to protein A, cell walls (CW) and PWM, intermediate to peptidoglycan (PG), and high to LPS. Small, not significant increases in Ig responses to LPS (which occurred during the first 2 wks) were observed, whereas, the responses to PG, protein A, CW and PWM continued to increase for 8 wks, and the increases were high and significant. In most cases, there was no change in the dose response and kinetics patterns (characteristic for each stimulant) during ontogeny. Ontogenic development of autoantibody-secreting cells was different from the development of cells secreting all Ig and anti-SRBC antibodies. The increases in the numbers of cells secreting IgM anti-DNA and IgM anti-bromelin-treated mouse RBC antibodies in response to LPS and PG during postnatal development were larger than the increases of all IgM-secreting cells. In contrast, the increases of IgM anti-DNA secreting cells in response to protein A and PWM were smaller than the increases of all IgM-secreting cells. The frequencies of cells secreting anti-DNA antibodies in LPS- and PG-stimulated cultures were low in newborns and continued to increase until 8 wks of age, but they were high and did not change throughout ontogeny in protein A-stimulated cultures. Changes in the frequencies of anti-RBC antibody-secreting cells were less distinct and mostly insignificant. Postnatal changes in mitogenic responses were smaller and not correlated with the development of polyclonal Ig responses. Our data indicate different modes of ontogenic development of the ability of cells to produce polyclonal Ig, autoantibodies and heteroantibodies, and to synthesize DNA, in response to different stimulants.

Animals↗