Differential metabolic effects of glucosamine and N-acetylglucosamine in human articular chondrocytes.

OBJECTIVE: Aminosugars are commonly used to treat osteoarthritis; however, molecular mechanisms mediating their anti-arthritic activities are still poorly understood. This study analyzes facilitated transport and metabolic effects of glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) in human articular chondrocytes. METHODS: Human articular chondrocytes were isolated from knee cartilage. Facilitated transport of glucose, GlcN and GlcNAc was measured by uptake of [3H]2-deoxyglucose, [3H]GlcN and [3H]GlcNAc. Glucose transporter (GLUT) expression was analyzed by Western blotting. Production of sulfated glycosaminoglycans (SGAG) was measured using [(35)S]SO4. Hyaluronan was quantified using hyaluronan binding protein. RESULTS: Chondrocytes actively import and metabolize GlcN but not GlcNAc and this represents a cell-type specific phenomenon. Similar to facilitated glucose transport, GlcN transport in chondrocytes is accelerated by cytokines and growth factors. GlcN non-competitively inhibits basal glucose transport, which in part depends on GlcN-mediated depletion of ATP stores. In IL-1beta-stimulated chondrocytes, GlcN inhibits membrane translocation of GLUT1 and 6, but does not affect the expression of GLUT3. In contrast to GlcN, GlcNAc accelerates facilitated glucose transport. In parallel with the opposing actions of these aminosugars on glucose transport, GlcN inhibits hyaluronan and SGAG synthesis while GlcNAc stimulates hyaluronan synthesis. GlcNAc-accelerated hyaluronan synthesis is associated with upregulation of hyaluronan synthase-2. CONCLUSION: Differences in GlcN and GlcNAc uptake, and their subsequent effects on glucose transport, GLUT expression and SGAG and hyaluronan synthesis, indicate that these two aminosugars have distinct molecular mechanisms mediating their differential biological activities in chondrocytes.

Chondroprotective activity of N-acetylglucosamine in rabbits with experimental osteoarthritis.

OBJECTIVE: To examine the therapeutic efficacy of N-acetylglucosamine (GlcNAc) in rabbits with experimental osteoarthritis (OA). METHODS: Experimental OA was induced in rabbits by anterior cruciate ligament transection (ACLT). In the first study, rabbits (six in each group) received intramuscular injections of GlcNAc or normal saline three times a week starting 1 week postoperatively. In the second study, rabbits (eight in each group) were injected intra-articularly with GlcNAc (either once or twice a week) or normal saline. In the third study, rabbits (seven in each group) were injected intra-articularly twice a week with either GlcNAc, hyaluronan, or normal saline. Animals were killed 8 weeks after ACLT for macroscopic and histological assessment of the knee joints. RESULTS: Intramuscular administration of GlcNAc in rabbits with experimental knee OA did not show chondroprotective effects but showed mild anti-inflammatory activity. In contrast, intra-articular administration of GlcNAc twice a week reduced cartilage degradation. Additionally, intra-articular GlcNAc also suppressed synovitis. Once a week intra-articular injections of GlcNAc did not demonstrate therapeutic efficacy. The chondroprotective efficacy of GlcNAc was better than that of viscosupplementation treatment with hyaluronan. CONCLUSION: Intra-articular GlcNAc has chondroprotective and anti-inflammatory activity in experimental OA.

Cytokine regulation of facilitated glucose transport in human articular chondrocytes.

Glucose serves as the major energy substrate and the main precursor for the synthesis of glycosaminoglycans in chondrocytes. Facilitated glucose transport represents the first rate-limiting step in glucose metabolism. This study examines molecular regulation of facilitated glucose transport in normal human articular chondrocytes by proinflammatory cytokines. IL-1beta and TNF-alpha, and to a lesser degree IL-6, accelerate facilitated glucose transport as measured by [(3)H]2-deoxyglucose uptake. IL-1beta induces an increased expression of glucose transporter (GLUT) 1 mRNA and protein, and GLUT9 mRNA. GLUT3 and GLUT8 mRNA are constitutively expressed in chondrocytes and are not regulated by IL-1beta. GLUT2 and GLUT4 mRNA are not detected in chondrocytes. IL-1beta stimulates GLUT1 protein glycosylation and plasma membrane incorporation. IL-1beta regulation of glucose transport in chondrocytes depends on protein kinase C and p38 signal transduction pathways, and does not require phosphoinositide 3-kinase, extracellular signal-related kinase, or c-Jun N-terminal kinase activation. IL-1beta-accelerated glucose transport in chondrocytes is not mediated by endogenous NO or eicosanoids. These results demonstrate that stimulation of glucose transport represents a component of the chondrocyte response to IL-1beta. Two classes of GLUTs are identified in chondrocytes, constitutively expressed GLUT3 and GLUT8, and the inducible GLUT1 and GLUT9.

Hexosaminidase inhibitors as new drug candidates for the therapy of osteoarthritis.

BACKGROUND: Articular cartilage from patients with osteoarthritis is characterized by a decreased concentration and reduced size of glycosaminoglycans. Degeneration of the cartilage matrix is a multifactorial process, which is due in part to accelerated glycosaminoglycan catabolism. Recently, we have demonstrated that hexosaminidase represents the dominant glycosaminoglycan-degrading glycosidase released by chondrocytes into the extracellular compartment and is the dominant glycosidase in synovial fluid from patients with osteoarthritis. Inhibition of hexosaminidase activity may represent a novel approach to the prevention of cartilage matrix glycosaminoglycan degradation and a potentially new strategy to treat osteoarthritis. RESULTS: We have synthesized and investigated a series of iminocyclitols designed as transition-state analog inhibitors of human hexosaminidase, and demonstrated that the five-membered iminocyclitol 4 expresses the strongest inhibitory activity with K(i)=24 nM. Inhibition of hexosaminidase activity in human cultured articular chondrocytes and human chondrosarcoma cells with iminocyclitol 4 resulted in accumulation of hyaluronic acid and sulfated glycosaminoglycans in the cell-associated fraction. Similarly, incubation of human cartilage tissue with iminocyclitol 4 resulted in an accumulation of glycosaminoglycans in the pericellular compartment. CONCLUSIONS: Inhibition of hexosaminidase activity represents a new strategy for preventing or even reversing cartilage degradation in patients with osteoarthritis.

N-acetylglucosamine prevents IL-1 beta-mediated activation of human chondrocytes.

Glucosamine represents one of the most commonly used drugs to treat osteoarthritis. However, mechanisms of its antiarthritic activities are still poorly understood. The present study identifies a novel mechanism of glucosamine-mediated anti-inflammatory activity. It is shown that both glucosamine and N-acetylglucosamine inhibit IL-1beta- and TNF-alpha-induced NO production in normal human articular chondrocytes. The effect of the sugars on NO production is specific, since several other monosaccharides, including glucose, glucuronic acid, and N-acetylmannosamine, do not express this activity. Furthermore, N-acetylglucosamine polymers, including the dimer and the trimer, also do not affect NO production. The observed suppression of IL-1beta-induced NO production is associated with inhibition of inducible NO synthase mRNA and protein expression. In addition, N-acetylglucosamine also suppresses the production of IL-1beta-induced cyclooxygenase-2 and IL-6. The constitutively expressed cyclooxygenase-1, however, was not affected by the sugar. N-acetylglucosamine-mediated inhibition of the IL-1beta response of human chondrocytes was not associated with the decreased inhibition of the mitogen-activated protein kinases c-Jun N-terminal kinase, extracellular signal-related kinase, and p38, nor with activation of the transcription factor NF-kappaB. In conclusion, these results demonstrate that N-acetylglucosamine expresses a unique range of activities and identifies a novel mechanism for the inhibition of inflammatory processes.

Profile of glycosaminoglycan-degrading glycosidases and glycoside sulfatases secreted by human articular chondrocytes in homeostasis and inflammation.

OBJECTIVE: To determine enzymatic activities of the 8 key glycosaminoglycan-degrading glycosidases and glycoside sulfatases in cultured human articular chondrocytes and in synovial fluid from patients with osteoarthritis. METHODS: The following enzymes were analyzed: hexosaminidase and its isoenzyme A, N-acetyl-alpha-D-glucosaminidase, beta-galactosidase, beta-glucuronidase, alpha-L-iduronidase, aryl sulfatase, and galactose-6-sulfate sulfatase. Activity of the selected enzymes was analyzed by fluorometry with the aid of 4-methylumbelliferryl derivatives of the appropriate monosaccharides. RESULTS: Hexosaminidase was found to be the dominant enzyme released by chondrocytes into the extracellular compartment. Stimulation of chondrocytes with interleukin-1beta resulted in a selective increase of the extracellular hexosaminidase activity and, to a lesser degree, of the extracellular beta-galactosidase activity, without significant changes in the activity of the other studied enzymes. Analysis of the pH dependency of the enzymatic activities revealed that even at neutral pH, hexosaminidase expressed a measurable activity, much higher than the activity of the other studied enzymes. Chondrocyte apoptosis did not result in increased extracellular glycosidase activities, including hexosaminidase activity. The spectrum of glycosidase and glycoside sulfatase activities in the synovial fluid from patients with osteoarthritis was similar to that in cultured human articular chondrocytes. CONCLUSION: These data support the concept that lysosomal glycosidases, in particular hexosaminidase, represent a distinct subset of cartilage matrix-degrading enzymes that are activated by proinflammatory stimuli.

Molecular analysis of polyreactive monoclonal antibodies from rheumatic carditis: human anti-N-acetylglucosamine/anti-myosin antibody V region genes.

Anti-myosin Abs are associated with inflammatory heart diseases such as rheumatic carditis and myocarditis. In this study, human cross-reactive anti-streptococcal/anti-myosin mAbs 1.C8, 1.H9, 5.G3, and 3.B6, produced from peripheral blood lymphocytes of patients with rheumatic carditis, and mAb 10.2.5, produced from a tonsil, were characterized, and the nucleotide sequences of their V(H) and V(H)L genes were analyzed. Human mAbs 1.C8, 1.H9, 10.2.5, and 3.B6 reacted with human cardiac myosin while mAb 5.G3 did not. The mAbs were strongly reactive with N-acetyl-beta-D-glucosamine, the dominant epitope of the group A streptococcal carbohydrate. mAb 1.H9 was moderately cytotoxic to rat heart cells in vitro in the presence of complement. The anti-myosin mAbs from rheumatic carditis were found to react with specific peptides from the light meromyosin region of the human cardiac myosin molecule. Anti-streptococcal/anti-myosin mAbs from normal individuals reacted with distinctly different light meromyosin peptides. The mAbs were encoded by V(H)3 gene segments V3-8, V3-23, and V3-30 and by the V(H)4 gene segment V4-59. The variable region genes encoding the anti-streptococcal/anti-myosin repertoire were heterogeneous and exhibited little evidence of Ag-driven somatic mutation.

Cytokeratin peptide SFGSGFGGGY mimics N-acetyl-beta-D-glucosamine in reaction with antibodies and lectins, and induces in vivo anti-carbohydrate antibody response.

Recently, we found that human anti-GlcNAc mAbs reacted with keratin from human skin and recognized specific peptide epitopes of human cytokeratin 14. Our data demonstrate that anti-keratin Ab responses in mice might be driven by Ags bearing terminal O-linked GlcNAc residues. To determine if mouse anti-GlcNAc mAbs recognized peptide epitopes of keratin, several mouse anti-GlcNAc mAbs were reacted with a panel of overlapping synthetic decapeptides of the entire amino acid sequence of human cytokeratin 14 in the ELISA. Results of the ELISA demonstrated that three mAbs, HGAC 54, HGAC 78, and 101.4.1, expressed maximal binding activity to keratin peptide B1 with the amino acid sequence SFGSGFGGGY. In addition, we found that mouse anti-cytokeratin 14 mAb CKB-1 recognized the same peptide and expressed anti-GlcNAc activity as well. Keratin peptide B1 was shown to react not only with anti-GlcNAc mAbs but also with several lectins such as wheat germ agglutinin, Datura stramonium lectin, Lycopersicon esculentum lectin, Solanum tuberosum lectin, and lectin from Wisteria floribunda. Reaction of the lectins with solid-phase keratin peptide B1 was inhibited by soluble GlcNAc and was not inhibited by glucose demonstrating specificity of binding. Using a panel of 24 synthetic keratin B1 peptides, each with a single amino acid substitution, we found that aromatic-aromatic and hydrophobic interactions were the major driving forces in the stabilization of the peptide-protein complexes. Finally, we demonstrated that immunization of BALB/c mice with peptide B1 conjugated to BSA induced an anti-GlcNAc Ab response. Results of our experiments indicated that certain peptides may express functional activity similar to GlcNAc and induce in vivo anti-carbohydrate Ab responses.

Immunological mimicry between N-acetyl-beta-D-glucosamine and cytokeratin peptides. Evidence for a microbially driven anti-keratin antibody response.

We discovered recently that a subset of mouse anti-streptococcal mAbs cross-reacted with N-acetyl-beta-D-glucosamine (GlcNAc) and certain cytoskeletal proteins, and recognized both carbohydrate and peptide antigenic determinants. To further study the nature and biologic significance of immunologic mimicry between carbohydrate and peptide Ags, eight human hybridomas secreting anti-GlcNAc mAbs were produced by in vitro stimulation of PBL with streptococcal peptidoglycan-polysaccharide complexes and pokeweed mitogen. All human anti-GlcNAc mAbs described in this study were shown to express marked cross-reactivity with keratin from human skin in the ELISA and Western immunoblot. Mapping of the mAbs with overlapping synthetic decapeptides of the entire amino acid sequence of human cytokeratin 14 revealed that human anti-GlcNAc mAbs recognized specific cytokeratin decapeptides. Four human anti-GlcNAc mAbs recognized a single cytokeratin decapeptide whereas two mAbs reacted with several individual peptide epitopes in different fragments of cytokeratin 14. In addition, two mAbs, 1.C8 and 9.B12, reacted with multiple cytokeratin decapeptides, predominantly in the head domain of the molecule, and their reactivity correlated with positive binding of the mAbs to cytokeratin 14 in the Western immunoblot and with positive staining of human epidermis in the indirect immunofluorescent assay. Finally, we demonstrated that Abs to keratin and synthetic keratin decapeptides were induced in BALB/c mice immunized with GlcNAc-BSA but not with BSA, suggesting that the anti-keratin Ab response in vivo may be driven by nonkeratin Ags containing terminal O-linked GlcNAc.

A subset of mouse monoclonal antibodies cross-reactive with cytoskeletal proteins and group A streptococcal M proteins recognizes N-acetyl-beta-D-glucosamine.

It is well known that antibodies to N-acetyl-beta-D-glucosamine (GlcNAc) cross-react with cardiac valves, skin, and other host tissues. However, molecular targets of these antibodies have not been identified. For this reason, anti-streptococcal mAb cross-reactive with group A streptococci and heart proteins were studied for their reactivity with GlcNAc, the immunodominant epitope of group A streptococcal carbohydrate. Characterization of the mAb that recognized GlcNAc revealed that each mAb had its own unique antigen-binding profile and pattern of immunofluorescence on rat heart cells. In the ELISA and Western blot these mAb reacted with cytoskeletal and heart proteins such as actin, keratin, myosin, and vimentin, as well as with streptococcal recombinant M5 and M6 proteins. Binding of the mAb to cytoskeletal proteins was inhibited by GlcNAc conjugated with BSA in a dose-dependent manner, and the mAb preferentially reacted with high-density GlcNAc-BSA conjugates. Antigenic determinants on the proteins recognized by the mAb were resistant to sodium periodate and N-acetylglucosaminidase treatment, suggesting reactivity with peptide and not carbohydrate structures. On reaction of the mAb with a panel of synthetic streptococcal, viral, and myosin peptides, one of the mAb, 49.8.9, was found to react most strongly with a synthetic peptide sequence synthesized from the coxsackievirus B3 capsid protein VP1, which shows homology with and cross-reacts with sequences in the streptococcal M6 protein and human cardiac myosin. This most interesting mAb, previously shown to neutralize coxsackie viruses, recognized the amino acid sequence RRKLEFF, which may mimic the GlcNAc epitope. The data collected show that we have identified a new group of multireactive autoantibodies that recognize GlcNAc and cytoskeletal proteins, as well as defined peptide epitopes.

Role of heavy chain constant domains in antibody-antigen interaction. Apparent specificity differences among streptococcal IgG antibodies expressing identical variable domains.

In this report, we examine the influence of CH domains on antibody specificity, in the context of variable epitope density on bacteria and synthetic glycoconjugates. Hybridomas secreting IgG1 and IgG2b mAb, specific for the N-acetyl-glucosamine (GlcNAc) residues of streptococcal group A carbohydrate, were previously generated from a hybridoma secreting a mouse IgG3 mAb. We show that these three mAb have identical H and L chain V domains, as determined by 1) cDNA sequencing, 2) binding to soluble Ag, and 3) binding to nine monoclonal anti-idiotopes. Nevertheless, the IgG3 mAb binds more effectively than the V region-identical IgG1 or IgG2b mAb to each of three strains of group A streptococci that display different amounts of terminal GlcNAc residues on their cell walls. The magnitude of the subclass-associated differential in binding varies with the target strain, and, whereas the IgG3 mAb binds best to the strain expressing an intermediate amount of GlcNAc, the IgG1 and IgG2b mAb and IgG3-derived F(ab')2 fragments bind best to the strain expressing the highest amount of GlcNAc. The IgG3 mAb also binds better than the IgG1 and IgG2b mAb to solid-phase GlcNAc50-BSA, but the IgG2b mAb binds best to otherwise identical conjugates with lower ratios of GlcNAc to BSA (20:1, 10:1, 5:1, and 1:1). These results suggest that epitope density can significantly influence the magnitude of IgG subclass-associated binding differences, and that structural differences in the CH regions, particularly the CH2 and CH3 domains, can influence the apparent specificities of IgG molecules for multivalent Ag.

[Biochemical and immunochemical analyses of the protein components of the cell wall in group-A streptococci isolated by a sparing chemical method]. / Biokhimicheskii i immunokhimicheskii analizy belkovykh komponentov kletichnoi stenki streptokokkov gruppy A, vydelennykh shchadiashchim khimicheskim metodom.

To study the protein components of the cell wall of group A streptococci, type M 29, a special preparative method was developed (extraction with 1 M hydroxylamine solution, pH 6.0, and subsequent purification). Altogether six protein fractions were obtained. The isolated proteins were found to be a heterogeneous group of molecules, consisting of 25-40 individual proteins with molecular weights ranging between 13 and 94 kD. The study of the protein fractions thus obtained in the immunodiffusion test with rabbit antiserum to the initial protein preparation revealed that these proteins contained type-specific components, 3-6 type-nonspecific protein antigens common with protein antigens of M 1 and M 12, as well as one protein antigen common with type M 1. Fc receptor was shown to be absent. The detected type-nonspecific protein antigens were partially separated by ion-exchange chromatography and some of them could be purified from the admixtures of nucleic acids and group-specific polysaccharide.

[Immunological study of lysates from the cell wall of group A Streptococcus]. / Immunologicheskoe izuchenie lizatov kletochnoi stenki streptokokka gruppy A.

The chemical composition and presence of immunogenic components in the lysates of the cell walls of group A Streptococcus, type M29, were studied. The lysates were prepared with the use of muramidase. Fc-Receptors were detected in the lysates. Within the first 30 minutes of cell wall lysis by muramidase, 4 times higher amounts of the protein reacting with fibrinogen excreted than in the subsequent 4 hours. The lysates contained immunogenic proteins. Fraction III isolated by chromatography of the 30-minute lysate on DEAE-trisacryl formed a single precipitation band with lysate antiserum. The lysate Fraction IV forming three precipitation bands contained a protein not specific of the type. The protein was identical to the protein antigen from Triton X-100 extracts of group A Streptococcus, types M1, M12 and M29. The group-specific polysaccharide was detected in the lysate Fraction I and Fraction II of the 4-hour lysate.

[The modification of the cell wall proteins in group A streptococci type M 29 under the influence of spermidine contained in the culture medium]. / Modifikatsiia belkov kletochnykh stenok streptokokkov gruppy A tipa M 29 pod deistviem spermidina, soderzhashchegosia v srede kul'tivirovaniia.

The addition of spermidine into growth medium used for the cultivation of group A streptococci, type M 29, leads to changes in the amino acid composition of cell walls and surface proteins isolated by the method of E. H. Beachey et al. The separation of surface proteins into fibrinogen-binding proteins and fibrinogen receptors by affinity chromatography techniques on cellulose with covalently bound fibrinogen indicates that the proportion of these proteins in pepsin extracts obtained from different strains varies. Both spermidine and avirulent strains have similar content of fibrinogen-binding proteins, although these proteins are absent in virulent strains. Different amounts of fibrinogen receptors are extracted from all strains. As shown in the enzyme immunoassay, fibrinogen receptors contain no group-specific polysaccharide A, Fc-receptors and interact with total antiserum to group A streptococci, type M 29 [correction of 28]. Fibrinogen receptors isolated from the strains under study have been found to have similar amino acid composition. On the basis of these results we believe that neither receptor capacity to fibrinogen nor amino acid composition is indicative of the protective properties of protein M.

[The low-molecular protein of the cell wall in streptococci group A devoid of serological type specificity]. / Nizkomolekuliarnyi belok kletochnoi stenki streptokokkov gruppy A, lishennyi tipovoi serologicheskoi spetsifichnosti.

The scheme for the isolation and purification of low-molecular cell-wall protein without type specificity, including the extraction of the cell walls of group A streptococci, type M 29, with 1% solution of Triton X-100, the separation of the extract by ion-exchange chromatography in DEAE-trisacryl M with the subsequent two-stage gel filtration in superfine Sephadex G-50, is described. The isolated protein had a molecular weight of 4,000 daltons and contained no admixtures of group-specific polysaccharide A, phosphorus, nucleic acids and Fc receptors and interacted with antisera to group A streptococcal cells of heterologous type M in the enzyme immunoassay (EIA). Purified protein was characterized by a high content of glycine. The antigenic determinants of immobilized protein, recognized by antibodies in EIA, were sensitive to the action of trypsin and resistant to the action of pepsin, papain, pronase E and sodium periodate.

[The pharyngeal beta-hemolytic streptococcal carrier state among blood donors]. / Glotochnoe nositel'stvo beta-gemoliticheskikh streptokokkov sredi donorov krovi.

459 blood donors aged 18-50 years were examined in 1987-1988 in Moscow. Among them, carrier state with respect to beta-hemolytic streptococci was detected in 107 donors (23.3%). The number of carriers gradually decreased with the increase of age of the examined donors. Group C streptococci occurred least of all (6.9%). Group A beta-hemolytic streptococci were isolated in 16.7% of the carriers. The isolation rate of streptococci from blood achieved its maximum in autumn and winter months and did not depend on preceding diseases, unhealthy working conditions, the rhesus factor and, with the exception of group A streptococci, the blood group. Among tonsillectomized donors carrier state with respect to beta-hemolytic streptococci occurred 2.2 times less frequently than among donors who had not undergone tonsillectomy. Carrier state with respect to beta-hemolytic streptococci was accompanied by higher levels of salivary sIgA antibodies to polysaccharide A, serum antibodies to polysaccharide A and circulating polysaccharide A. All beta-hemolytic streptococci were sensitive to erythromycin. All groups of streptococci showed the highest percentage of cultures resistant to gentamicin and tetracycline. In 100% of cases group A streptococci were sensitive to benzylpenicillin, methicillin, ampicillin, erythromycin and lincomycin.

[The level of IgM, IgG and IgA antibodies and total antibodies to the low-molecular, cell-wall protein of Streptococcus group A without type specificity in the serum of patients with a streptococcal infection]. / Izuchenie urovnia IgM-, IgG-, IgA-antitel i summarnykh antitel k nizkomolekuliarnomu belku kletochnoi stenki streptokokkov gruppy A, lishennomu tipovoi spetsifichnosti, v syvorotke bol'nykh streptokokkovoi infektsiei.

The levels of IgM, IgG and IgA antibodies, as well as total antibodies, to group A streptococcal low-molecular cell-wall protein without type specificity were studied in the sera of patients with primary erysipelas, rheumatism in the active and inactive phases, seronegative rheumatoid arthritis, as well as in the sera of healthy donors. The average level of antibodies to low-molecular protein in the sera of all groups of patients was significantly higher than the sera of healthy donors. The analysis of the distribution of antibodies in accordance with their isotypes revealed the specific features of response, characteristic of each group of patients. For rheumatism patients, the positive correlation between response to low-molecular protein and response to group-specific polysaccharide A was established. This correlation was most pronounced in patients with rheumatism in the inactive phase.