A comparative study of the immunological properties of peptidoglycans of different origins

The action of peptidoglycans (PG) of different origin has been experimentally studied in vivo. In these experiments PG of bacterial origin, such as blastolysin (BL), and synthetic PG, viz. muramyldipeptide (MDP) and its analog glucosaminylmuramyldipeptide (GMDP) have been used. Their toxicity, allergenic action, their effect on the phagocytic activity of peritoneal exudate macrophages (PEM), the accumulation of antibody-producing cells in the spleen, antibody titer in the blood serum and delayed hypersensitivity to nonbacterial antigens have been determined. As revealed in this study, BL does not differ from MDP in its toxicity and allergenic action. The phagocytic activity of PEM under the influence of BL only insignificantly differs from their activity under the influence of MDP, but is lower than under the influence of GMDP. The adjuvant action of BL is somewhat higher than that of synthetic PG.     

Immunostimulating effects of muramyl dipeptide, glucosaminyl muramyl dipeptide and their synthetic derivatives in vitro

The effect of muramyldipeptide (MDP), glucosaminylmuramyldipeptide (GMDP) and their six synthetic derivatives on production of tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-2 (IL-2) by murine spleen cells in vitro was studied. MDP induced insignificant TNF production and did not stimulate production of IL-1 by the murine splenocytes within a 24-hour cultivation period whereas in combination with lipopolysaccharide (LPS) it induced significant production of both the cytokins. GMDP induced marked production of TNF (54 per cent cytotoxic index) and IL-1 (stimulation index 8). Addition of LPS in an amount of 10 ng/ml increased production of TNF by the murine splenocytes under the effect of GMDP but had no effect on production of IL-1. Neither MDP nor GMDP even in combination with LPS induced production of IL-2 by splenocytes of mice DVA/2 and C57B1/6 at activation for 24 hours. All the synthetic derivatives of MDP and GMDP except the MDP polymer activated TNF production by the murine spleen cells. GMDP lysine had the highest effect: 67 per cent cytotoxic index. In combination with LPS its cytotoxic index amounted to 87 per cent. The TNF activity was always higher when LPS in an amount of 10 ng/ml was added to the glycopeptides.     

Immunostimulating properties of synthetic derivatives of muramyl dipeptide and glucosaminyl muramyl dipeptide in vitro

Production of tumor necrosis factor (TNF) and interleukin-1 (IL-1) by macrophages of the spleen and peritoneal exudate of mice as well as cytotoxic factors (CFs) by murine splenocytes after in vitro activation was estimated. All the derivatives of muramyldipeptide (MDP) and glucosaminylmuramyldipeptide (GMDP) were able to induce production of TNF and CFs. In the presence of lipopolysaccharide (LPS), the effect was always higher. The response of the spleen macrophages to the effect of the preparations was higher than that of the peritoneal ones and ++non-fractionated splenocytes. GMDP and GMDP4 especially in the presence of LPS had the highest effect on induction of IL-1 by the murine peritoneal macrophages. On the contrary, MDP induced higher IL-1 synthesis by the spleen macrophages. The most active substances with respect to production of TNF, CFs and IL-1, i.e. MDP3 and GMDP4, might be recommended for immunotherapy of syngeneic tumors in animals.     

Binding of activated C3b component with complement effector

Various nucleophilic agents (acceptors) react with thiolester group of nascent activated fragment (C3b) of the third complement component. The C3b-acceptors binding prevents transformation of C3 convertase to C5 convertase and results in inhibition of the cell-target lysis. A convenient method of monitoring the EAC142 to EAC1423 transformation was elaborated. Character of the inhibition suggests that the covalent binding follows a stage of the reversible C3b-acceptor complex formation. The method allows to determine the maximum of inhibition of the C5 convertase formation and the dissociation constant of the reversible C3b-acceptor complex, which reflects the C3b affinity to this acceptor.     

Surface-dependent modulation by H of C5 cleavage by the cell-bound alternative pathway C5 convertase of human complement

Regulation by H of formation of the C3 and C5 alternative pathway convertases of complement on cells is dependent on such chemical characteristics of the cell surfaces as their membrane content in sialic acid. Properdin-stabilized C5 convertase sites were assembled on the non-activating cells of the alternative pathway, sheep erythrocytes (Es), and on the activating cells, desialated Es and rabbit erythrocytes (Er). C5 hemolytic sites were revealed by incubation of the convertase-bearing cells with limiting C5 and excess C6-C9. H inhibited generation of C5 hemolytic sites in a dose-related fashion on Es, Er, and desialated Es at molar ratios of H/C5 of 0.03 to 0.5. H similarly inhibited C5 utilization by the cell-bound C5 convertase on Es and desialated Es regardless of the cell membrane sialic acid content; however, H was three to five times less effective on Er. Kinetic experiments also suggested that C5 hemolytic sites are generated more rapidly on Er than on Es and desialated Es. The inhibition effect of H was independent of the number of C5 convertase sites per cell on all cell types; two to three times more residual hemolytic sites were found on convertase-bearing Es that had been incubated with C5 and H as compared with cells that had been decayed by H before incubation with C5. Furthermore, H also inhibited C5 interaction with a preformed classical pathway C5 convertase. These results suggest that H interacts with C5 so as to alter C5 binding and/or cleavage by the cell-bound C5 alternative pathway convertase. Sialic acid-independent modulation by H of C5 cleavage by the C5 convertase represents an additional regulatory step in the activation of the human alternative complement pathway.     

Mass-spectrometric determination of the structure of glycopeptides from the bacterial cell wall (illustrated by Lactobacillus bulgaricus)

Mass spectrometry has been applied to the structural analysis of one of the glycopeptides from blastolysin, antitumor bacterial preparation isolated from the Lactobacillus bulgaricus cell wall. The glycopeptide (MW 10,000) was subjected to partial acid hydrolysis (6 N HCl, 100 degrees C) and the resulting products were dansylated or trifluoroacetylated and methylated or deuteromethylated. The mixture of these derivatives was examined by high-performance liquid chromatography or gas chromatography followed by mass spectrometry using electron impact and ammonia chemical ionization techniques.     

Immunomodulators of microbial origin enhance cytotoxicity of human mononuclear leukocytes and reduce metastatic progression of Lewis lung carcinoma in mice

Effect of immunomodulators for microbial origin on innate immunity and antitumor system was continued to study. Immunomodificator Immunovac VP-4, purified staphylococcal toxoid and glucosaminyl muramyl dipeptide (GMDP) equally enhanced cytotoxicity of mononuclear leukocytes of peripheral blood of healthy donors. Index of cytotoxicity was 2.78, 2.77 and 2.70 respectively. Reduced metastatic progression of Lewis lung carcinoma in mice was observed after Immunovac VP-4 and GMDP administration. Effectiveness was seen when preparations administered according to schedules including their administration before implantation of the tumor. If preparations were administered number of metastases reduced in 4.4-5.6 times and size of metastases reduced in 7-10 times. Interplay between antitumor activity of studied immunomodulators and cytotoxic activity of NK-cells, which are base effectors of antitumor immune response, are discussed.     

Activation of the human complement cascade by bacterial cell walls, peptidoglycans, water-soluble peptidoglycan components, and synthetic muramylpeptides--studies on active components and structural requirements

Cell walls isolated from 29 strains of 24 gram-positive bacterial species, whose peptidoglycans belong to the group A type of Schleifer and Kandler's classification, with one exception (Arthrobacter sp.), were shown to activate the complement cascade in pooled fresh human serum mainly through the alternative pathway and partly through the classical one. The complement-activating effect of cell walls (5 species) possessing group B type peptidoglycan, except those of Corynebacterium insidiosum, was weaker than that of the walls with group A type peptidoglycan. Preparations of peptidoglycan isolated from cell walls of Staphylococcus aureus, Streptococcus pyogenes, and Lactobacillus plantarum also activated the alternative pathway of the complement cascade, but less effectively than the respective parent cell walls. A water-soluble "polymer" of peptidoglycan subunits (SEPS), which was prepared from Staphylococcus epidermidis peptidoglycans by treatment with a cross-bridge degrading endopeptidase, retained most of the complement-activating ability of the parent cell walls. A peptidoglycan "monomer," SEPS-M, which was obtained by hydrolysis of the glycan chain of SEPS with endo-N-acetylmuramidase to disaccharide units did not activate complement. In conformity with this finding, neither synthetic N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) nor MDP-L-Lys-D-Ala activated the complement cascade. Among several lipophilic derivatives of MDP, 6-O-(3-hydroxy-3-docosylhexacosanoyl)-MDP-L-Lys-D-Ala (BH48-MDP-L-Lys-D-Ala) and 6-O-(2-tetradecylhexadecanoyl)-MDP (B30-MDP) were shown to activate complement through the alternative as well as the classical pathway and exclusively through the classical pathway, respectively. The finding that a D-isoasparagine analog of B30-MDP caused the same effect as the parent molecule strongly suggests that the activation of complement by B30-MDP is different from that caused by cell wall peptidoglycans and a water-soluble "polymer" of peptidoglycan subunits.     

The effect of muramyl dipeptide analog, GMPD, incorporated into liposomes of various composition on the functional activity of macrophages

Liposomes of different composition and N-acetylglucosaminyl-N-acetylmuramyl-L-alanyl-D-isoglutamine (GMPD) encapsulated in them are studied for their effect on the functional activity of macrophages by means of determining the 5'-nucleotidase and adenosine deaminase activity in the in vivo experiments. It is shown that both liposomes and GMDP encapsulated in them increase the activity of adenoside deaminase and decrease that of 5'-nucleotidase. This evidences for a change in the adenosine metabolism in the alveolar and peritoneal macrophages and an increase in the functional activity of cells which resulted from that rise. The manifestation of the process depends both on the lipid composition of liposomes and their charge. The observed increase in the functional activity of the alveolar macrophages under the effect of liposomes and GMDP encapsulated in them correlates with inhibition of the lung metastases development in mice.     

Stabilization of the amplification convertase of complement by monoclonal antibodies directed against human factor B

Three IgM mouse monoclonal antibodies (MoAb), 5B5-A, 2D2-B, and 5B12-A, were prepared by fusion of spleen cells from mice immunized with human B with the SP 2/0 myeloma cell line. They were assessed for their effect on cell-bound and fluid-phase amplification convertases of complement (C) with purified proteins in vitro. 5B5-A and 2D2-B were similar in their effects on cell-bound preformed C3bBb in that they bound to cell-bound C3bBb, stabilized the C3bBb convertase, and rendered the C3bBb convertase relatively resistant to the plasma protein H. These two MoAb were also able to enhance C3 consumption in vitro in reaction mixtures containing C3b, C3, B, and D. At the same time, they presumably stabilized the C3 convertase and caused relative sparing of B hemolytic activity in the reaction mixtures. In contrast, 5B12-A, which also bound to Bb and C3bBb, did not induce any stabilization, but rather caused accelerated decay of cell-bound C3bBb. These results indicate that MoAb against B can have C3NeF-like activity. On the other hand, not all MoAb against B have stabilizing activity on the C3bBb convertase.     

Characteristics of blastolysin preparations in their ability to inhibit hemagglutination caused by bacteria of the genus Lactobacillus

Anabol and blastolysin preparations obtained from L. bulgaricus may contain surface structural components of the initial strain with adhesion activity; of these, one is similar in specificity to L. casei adhesin and the other, to L. plantarum adhesin. The antigenic activity of anabol and blastolysin, evaluated in the immunodiffusion test, does not correlate with their capacity for binding the receptors of susceptible bacterial cells, determined in the Lactobacillus-induced hemagglutination inhibition test.     

Chemical structure and biological activity relationship of bacterial cell walls and muramyl peptides

The biological activities of the cell walls of bacteria having different types of peptidoglycans, and those of stereoisomers and analogs of muramyl dipeptide (MDP), of N-acetylglucosaminyl-beta(1-4)-N-acetylmuramyl tetrapeptides having different L- and D-amino acids at the COOH-terminus, and of 6-O-acyl-MDPs were examined to elucidate the relationship between structure and activity. Replacement of the L-alanine residue of MDP with glycine and replacement of the D-isoglutamine residue with L-isoglutamine, L-glutamic acid, and D-isoasparagine, but not with D-glutamic acid, caused a marked decrease in the biological activities of the MDP molecule. Test disaccharide tetrapeptides, irrespective of the configuration of COOH-terminal amino acid, showed strong immunoadjuvant activity and stimulation of macrophages, whereas those having COOH-terminal L-amino acids exhibited greater pyrogenicity, induction of acute joint inflammation, and hemorrhagic necrosis at a primed site than those having COOH-terminal D-amino acids. Introduction of an alpha-branched higher fatty acid to the muramic acid residue resulted in the disappearance of pyrogenicity after i.v. injection, an increase of adjuvanticity, and a loss of dependence on administration vehicles. The lack of the immunopotentiating activity (adjuvanticity) in cell walls from group B-type bacterial species was explained by the combined inhibitory effects of the replacement of the L-alanine residue by glycine and involvement of the alpha-carboxyl group of the D-glutamic acid residue in linking with neighboring peptide subunits.     

Quantitative chemical analyses and antigenic properties of peptidoglycans from Clostridium botulinum and other clostridia.

The cell wall peptodoglycans were isolated from Clostridium botulinum and some other species of the genus Clostridium by hot formamide extraction and their quantitative chemical composition and antigenic properties were determined. The petidoglycan of C. botulinum type E was found to be a diaminopimelic acid (DAP)-containing type composed of glucosamine, muramic acid, glutamic acid, alanine and DAP in the molar ratio of 0.76:0.78:1.00:1.88:0.81. All other types of C. botulinum and Clostridium sporogenes also belonged to the same peptidoglycan type. The peptidoglycans of Clostridium bifermentans and Clostridium histoloyticum contained DAP but they differed from those of C. botulinum in the molar ratio of alanine to glutamic acid. The peptidoglycan of Clostridium perfringens was composed of glutamic acid, alanine, DAP and glycine in the molar ratio of 1.00:1.64:0.94:0.90. On the other hand, the peptidoglycan of Clostridium septicum was found to contain lysine instead of DAP and the molar ratio was 1.00:1.41:0.96 for glutamic acid, alanine and lysine. In spite of the difference in amino acid composition of peptidoglycans among the clostridia, the quantitative precipitin test demonstrated that antiserum against C. botulinum type E peptidoglycan cross-reacted with the peptidoglycans from other clostridia as well as various types of C. botulinum.     

Structures and biological activities of peptidoglycans of Listeria monocytogenes and Propionibacterium acnes

The cell-wall skeletons of Listeria monocytogenes strain EGD and Propionibacterium acnes strain C7, which have the ability to induce macrophage activation, were analyzed, and the structures of the peptidoglycans were investigated. The analytical data indicate that both peptidoglycans have glucosamine residues with free amino groups, which are responsible for the resistance to lysozyme. Possible structures of these peptidoglycans were deduced from the composition and the results of determination of N- and C-terminal amino acids, together with the characterization of fragments obtained by enzymatic treatment and partial acid hydrolysis of both peptidoglycans. The results suggested that the peptidoglycan of L. monocytogenes contains a cross-linkage region of peptide chains with meso-diaminopimelic acid and D-alanine, which belongs to the A1 gamma type (Schleifer, K.H. & Kandler, O. (1972) Bacteriol. Rev. 36, 407-477), whereas the peptidoglycan of P. acnes contains a cross-linkage region of peptide chains with L,L-diaminopimelic acid and D-alanine, in which two glycine residues combine with amino and carboxyl groups of two L,L-diaminopimelic acid residues. The latter type should be classified as a new type. These cell-wall skeletons and peptidoglycans were shown to have immunoadjuvant activity on the induction of delayed-type hypersensitivity and suppressive activity on the growth of 3-methylcholanthrene-induced fibrosarcoma in BALB/c mice, and the peptidoglycans were shown to be an immunological-active principle of these cell-wall skeletons.     

Formation of high affinity C5 convertase of the classical pathway of complement

C3/C5 convertase is a serine protease that cleaves C3 and C5. In the present study we examined the C5 cleaving properties of classical pathway C3/C5 convertase either bound to the surface of sheep erythrocytes or in its free soluble form. Kinetic parameters revealed that the soluble form of the enzyme (C4b,C2a) cleaved C5 at a catalytic rate similar to that of the surface-bound form (EAC1,C4b,C2a). However, both forms of the enzyme exhibited a poor affinity for the substrate, C5, as indicated by a high Km (6-9 microM). Increasing the density of C4b on the cell surface from 8,000 to 172,000 C4b/cell did not influence the Km. Very high affinity C5 convertases were generated only when the low affinity C3/C5 convertases (EAC1,C4b,C2a) were allowed to deposit C3b by cleaving native C3. These C3b-containing C3/C5 convertases exhibited Km (0.0051 microM) well below the normal concentration of C5 in blood (0.37 microM). The data suggest that C3/C5 convertase assembled with either monomeric C4b or C4b-C4b complexes are inefficient in capturing C5 but cleave C3 opsonizing the cell surface with C3b for phagocytosis. Deposition of C3b converts the enzymes to high affinity C5 convertases, which cleave C5 in blood at catalytic rates approaching Vmax, thereby switching from C3 to C5 cleavage. Comparison of the kinetic parameters with those of the alternative pathway convertase indicates that the 6-9-fold greater catalytic rate of the classical pathway C5 convertase may compensate for the fewer numbers of C5 convertase sites generated upon activation of this pathway.     

Multiligand specificity of pathogen-associated molecular pattern-binding site in peptidoglycan recognition protein

The peptidoglycan recognition protein PGRP-S is an innate immunity molecule that specifically interacts with microbial peptidoglycans and other pathogen-associated molecular patterns. We report here two structures of the unique tetrameric camel PGRP-S (CPGRP-S) complexed with (i) muramyl dipeptide (MDP) at 2.5 Å resolution and (ii) GlcNAc and β-maltose at 1.7Å resolution. The binding studies carried out using surface plasmon resonance indicated that CPGRP-S binds to MDP with a dissociation constant of 10⁻⁷ m, whereas the binding affinities for GlcNAc and β-maltose separately are in the range of 10⁻⁴ m to 10⁻⁵ m, whereas the dissociation constant for the mixture of GlcNAc and maltose was estimated to be 10⁻⁶ m. The data from bacterial suspension culture experiments showed a significant inhibition of the growth of Staphylococcus aureus cells when CPGRP-S was added to culture medium. The ELISA experiment showed that the amount of MDP-induced production of TNF-α and IL-6 decreased considerably after the introduction of CPGRP-S. The crystal structure determinations of (i) a binary complex with MDP and (ii) a ternary complex with GlcNAc and β-maltose revealed that MDP, GlcNAc, and β-maltose bound to CPGRP-S in the ligand binding cleft, which is situated at the interface of molecules C and D of the homotetramer formed by four protein molecules A, B, C, and D. In the binary complex, the muramyl moiety of MDP is observed at the C-D interface, whereas the peptide chain protrudes into the center of tetramer. In the ternary complex, GlcNAc and β-maltose occupy distinct non-overlapping positions belonging to different subsites.     

Reconstitution of C5 convertase of the alternative complement pathway with isolated C3b dimer and factors B and D

C5 convertase of the alternative complement pathway is a trimolecular complex consisting of two molecules of C3b and one molecule of Bb. We previously proposed a model of the alternative pathway C5 convertase in which the second C3b molecule binds covalently to the first C3b molecule bearing Bb, and the C5 molecule binds to each C3b molecule of the covalently linked C3b dimer, resulting in its appropriate presentation to the catalytic site on Bb. In the present study, we purified the covalently linked C3b dimer and reconstituted the C5 convertase with the C3b dimer and factors B and D to obtain evidence in support of this model. An insoluble glucan, OMZ-176, was incubated with human serum to activate the alternative pathway and to allow formation of the alternative C5 convertase on the surface of the glucan, and the glucan bearing the C5 convertase was then solubilized by incubation with glucosidases. In this way, the covalently linked C3b dimer was obtained in solution without using a detergent. The C3b dimer was then separated from enzymes, C3b monomer, C3b oligomer, and other materials by chromatographies. SDS-PAGE analysis demonstrated that the purified C3b dimer had intact alpha'-chains. Alternative pathway C5 convertase was reconstituted when the isolated C3b dimer was incubated with factors B and D. The presence of P enhanced C5 convertase formation threefold. These results support the notions that the formation of the covalently linked C3b dimer is a general phenomenon associated with activation of the alternative pathway and that the C3b dimer acts as a part of the C5 convertase.     

Quantitative Chemical Analyses and Antigenic Properties of Peptidoglycans from Clostridium botulinum and Other Clostridia

The cell wall peptidoglycans were isolated from Clostridium botulinum and some other species of the genus Clostridium by hot formamide extraction and their quantitative chemical composition and antigenic properties were determined. The peptidoglycan of C. botulinum type E was found to be a diaminopimelic acid (DAP)-containing type composed of glucosamine, muramic acid, glutamic acid, alanine and DAP in the molar ratio of 0.76:0.78:1.00:1.88:0.81. All other types of C. botulinum and Clostridium sporogenes also belonged to the same peptidoglycan type. The peptidoglycans of Clostridium bifermentans and Clostridium histolyticum contained DAP but they differed from those of C. botulinum in the molar ratio of alanine to glutamic acid. The peptidoglycan of Clostridium perfringens was composed of glutamic acid, alanine, DAP and glycine in the molar ratio of 1.00:1.64:0.94:0.90. On the other hand, the peptidoglycan of Clostridium septicum was found to contain lysine instead of DAP and the molar ratio was 1.00:1.41:0.96 for glutamic acid, alanine and lysine. In spite of the difference in amino acid composition of peptidoglycans among the Clostridia, the quantitative precipitin test demonstrated that antiserum against C. botulinum type E peptidoglycan cross-reacted with the peptidoglycans from other Clostridia as well as various types of C. botulinum.     

Identification of bacterial muramyl dipeptide as activator of the NALP3/cryopyrin inflammasome

Activation of caspase-1 and subsequent processing and secretion of the pro-inflammatory cytokine IL-1beta is triggered upon assembly of the inflammasome complex. It is generally believed that bacterial lipopolysaccharides (LPS) are activators of the inflammasome through stimulation of Toll-like receptor 4 (TLR4). Like TLRs, NALP3/Cryopyrin, which is a key component of the inflammasome, contains Leucine-Rich-Repeats (LRRs). LRRs are frequently used to sense bacterial components, thus raising the possibility that bacteria directly activate the inflammasome. Here, we show that bacterial peptidoglycans (PGN), but surprisingly not LPS, induce NALP3-mediated activation of caspase-1 and maturation of proIL-1beta. Activation is independent of TLRs because the PGN degradation product muramyl dipeptide (MDP), which is not sensed by TLRs, is the minimal-activating structure. Macrophages from a patient with Muckle-Wells syndrome, an autoinflammatory disease associated with mutations in the NALP3/Cryopyrin gene, show increased IL-1beta secretion in the presence of MDP. The activation of the NALP3-inflammasome by MDP may be the basis of the potent adjuvant activity of MDP.     

The low C5 convertase activity of the C4A6 allotype of human complement component C4.

We have compared the C5-convertase-forming ability of different C4 allotypes, including the C4A6 allotype, which has low haemolytic activity and which has previously been shown to be defective in C5-convertase formation. Recent studies suggest that C4 plays two roles in the formation of the C5 convertase from the C3 convertase. Firstly, C4b acts as the binding site for C3 which, upon cleavage by C2, forms a covalent linkage with the C4b. Secondly, C4b with covalently attached C3b serves to form a high-affinity binding site for C5. Purified allotypes C4A3, C4B1 and C4A6 were used to compare these two activities of C4. Covalently linked C4b-C3b complexes were formed on sheep erythrocytes with similar efficiency by using C4A3 and C4B1, indicating that the two isotypes behave similarly as acceptors for covalent attachment of C3b. C4A6 showed normal efficiency in this function. However, cells bearing C4b-C3b complexes made from C4A6 contained only a small number of high-affinity binding sites for C5. Therefore a lack of binding of C5 to the C4b C3b complexes is the reason for the inefficient formation of C5 convertase by C4A6. The small number of high-affinity binding sites created, when C4A6 was used, were tested for inhibition by anti-C3 and anti-C4. Anti-C4 did not inhibit C5 binding, whereas anti-C3 did. This suggests that the sites created when C4A6 is used to make C3 convertase may be C3b-C3b dimers, and hence the low haemolytic activity of C4A6 results from the creation of low numbers of alternative-pathway C5-convertase sites.