Degradation of Streptococcal Cell Wall Antigens In Vivo

Specific chemical modification of group A polysaccharide antigen to the A-variant structure was demonstrated in the lymphoid organs of mice by autoradiography by use of radioantibodies specific for these structures. Both antigenic moieties persisted and were still discerned 10 weeks after injection of the group A cell wall. In rabbit skin, the group A specificity was altered after a prolonged period. Unlike the situation for the mouse, polysaccharide A was not converted to A-variant structure, but another specificity common to both polysaccharides persisted at the site of injection. Mucopeptide, separated from the polysaccharide of group A cell walls, was eliminated from the site of injection in rabbit skin between 4 and 8 hr after injection. Group D streptococcal cell walls were also rapidly eliminated from tissue, and were no longer detectable 8 hr after injection into rabbit skin or 24 hr after injection into mice. The rapid degradation of these structures was correlated with their susceptibility to lysozyme in vitro and was in contrast to the prolonged persistence of group A cell walls, which were completely resistant to egg white lysozyme. This persistence in tissue correlated with the capacity of group A cell wall fragments to induce a chronic inflammatory process, whereas the isolated mucopeptide or group D cell walls produced only an acute necrotoxic reaction.     

Prevention of chronic erosive streptococcal cell wall-induced arthritis in rats by treatment with a monoclonal antibody against the T cell antigen receptor alpha beta.

Rats treated with an mAb (R73) against the TCR-alpha beta failed to develop chronic persistent arthritis after injection of streptococcal cell walls. Histologically, R73 mAb-treated rats had mild hyperplasia of synovial lining cells and minimal destruction of cartilage. In contrast, control-treated animals developed marked pannus formation, with pronounced infiltration of mononuclear cells and severe destruction of cartilage and subchondral bone. The preventive effect of R73 mAb on streptococcal cell wall-induced arthritis was associated with the marked depletion of alpha beta + T cells by R73 mAb. These results indicate that T cells play a crucial role in chronic erosive streptococcal cell wall-induced arthritis.     

M protein deficient streptococcal cell walls can induce acute and chronic arthritis rats

Cell walls from M+ and M- protein variants of group A streptococci were examined for their arthritogenicity in female Lewis rats. Intraperitoneal administration of both of these sonicated cell wall preparations caused a severe acute and chronic arthritis in recipient rats. Histological evaluation of the hind paw of these rats indicated synovial lining hyperplasia, cell infiltration in the subsynovial space, pannus formation, and erosions of bone and cartilage. Joint pathology was similar in the hind paws of rats immunized with cell walls prepared from either the M+ or the M- protein variants. Cell-mediated immunity was also similar when lymph nodes were exposed to cell walls derived from these two preparations. A recombinant M6 protein from streptococci did not elicit a proliferative response from lymph nodes prepared from arthritic rats. These observations indicate that the M protein that has previously been implicated in auto-immunity does not have a critical role in the pathogenesis of streptococcal cell wall arthritis in rats.     

Induction of Experimental Chronic Arthritis in Rabbits by Cell-free Fragments of Erysipelothrix

A cell-free crude extract of Erysipelothrix rhusiopathiae injected by high pressure jet into the knee-joint of rabbits stimulated an acute, mild inflammatory reaction. Additional injections at 3-day intervals induced a chronic condition characterized by hyperplasia of the synovial cells and hypertrophy of the villi, due to infiltration by lymphocytes and plasma cells which formed aggregates resembling Allison-Ghormley bodies. There was also extensive proliferation of stroma vasculature and fibrous tissue. A similar jet injection of the diluent produced an early, transient, acute, and mild inflammation. A mechanism is postulated for fixation of one or more of the chemically characterized antigens in or near the synovium as a means of inducing the localized inflammatory response that predisposes the joint to infection.     

Endothelial cells and the pathogenesis of rheumatoid arthritis in humans and streptococcal cell wall arthritis in Lewis rats

Endothelial cells play a fundamental role in the pathogenesis of chronic inflammatory arthritis in humans such as rheumatoid arthritis (RA), as well as experimental animal models such as streptococcal cell wall (SCW) arthritis in Lewis (LEW/N) rats. This review summarizes data in support of this concept. The earliest apparent abnormalities in synovial tissues of patients with RA and Lewis rats with SCW arthritis appear to reflect microvascular endothelial cell activation or injury. At the molecular level, the abnormalities include enhanced expression by endothelial cells of activation markers such as class II major histocompatibility complex antigens, phosphotyrosine, leukocyte adhesion molecules, oncoproteins such as c-Fos and c-Myc, and metalloproteinases such as collagenase and transin/stromelysin. The development of severe, chronic, destructive arthritis is dependent upon thymic-derived lymphocytes and is accompanied by tumorlike proliferation of cells in the synovial connective tissue stroma (blood vessels and fibroblastlike cells), which results in resorptive destruction of bone and cartilage. Multiple criteria support the analogy to a neoplastic process. Paracrine and autocrine factors such as interleukin-1 (IL-1), platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-beta), and heparin-binding fibroblast growth factors (HBGF, FGF) appear to play important roles in the generation of these lesions. Finally, in addition to the autocrine and paracrine regulatory factors, neuroendocrine factors, particularly the hypothalamic-pituitary-adrenal axis, appear to be involved in the counterregulation of the inflammatory process. The counterregulatory effects are mediated, in part, by inhibition of endothelial cell activation by corticosteroids.     

Specific and cross-reactive activation of rabbit peripheral blood lymphocytes by streptococcal vaccines.

The ability of primed rabbit blood lymphocytes to respond in vitro to the homologous streptococcal group antigen depends on its presence in culture in the form of vaccine or cell walls thereof. These lymphocytes can also be stimulated in vitro by streptococcal vaccines with chemically related group antigens. The basis for this cross-stimulation apparently resides in shared rhamnose moieties. Activation of these lymphocytes was not achieved by vaccines from unrelated bacteria. There is also the suggestion that rabbits of different genetic origin differentiate between cross-stimulating antigens, probably at the level of antigenic determinants. The data support the view that recognition and response pattern of the immune system rely heavily on a network of antigens.     

Gamma/delta T cell receptor positive T cells in the inflammatory joint: lack of association with response to soluble antigens

In patients with inflammatory synovitis, the proliferative response by lymphocytes from synovial fluid to soluble mycobacterial antigens is enhanced relative to those from peripheral blood. Earlier studies suggested that gamma/delta T cell receptor positive (TCR+) T lymphocytes may significantly contribute to the mycobacterial-specific synovial fluid response. We therefore examined the relationship of the T cell proliferative response to Mycobacterium tuberculosis antigens and the presence of gamma/delta TCR+ T cells employing several monoclonal antibodies. No consistent increase of gamma/delta TCR+ T cells was noted in inflammatory synovial fluids or tissues. Nonetheless, lymphocytes from the majority of the synovial fluids proliferated vigorously in response to water-soluble M. tuberculosis antigens. There was no relationship between the percentage of gamma/delta TCR+ T lymphocytes and the intensity of the proliferative response. In contrast, stimulation with whole mycobacterial organisms was capable of enriching the gamma/delta TCR+ cell population obtained from the peripheral blood of tuberculosis skin test positive normal controls and from some inflammatory synovial fluids. These observations do not support a role for mycobacteria reactive gamma/delta TCR+ synovial T lymphocytes in response to soluble mycobacterial antigens or in the local pathogenesis of inflammatory synovitis.     

Detection by Hemagglutination of Antibodies to Group A and Group E Streptococci by the Use of O-Stearoyl Derivatives of Their Cell Wall Carbohydrate-grouping Antigens

The streptococcal group A and group E cell wall polysaccharide antigens were extracted with trichloroacetic acid from the cell or cell wall and esterified with stearic acid. The stearoyl derivatives contained 5 to 8% (by weight) of the ester. Sheep or human red blood cells were sensitized with the esterified antigens and were shown to agglutinate in the presence of specific rabbit antisera. Sera from (i) children hospitalized with group A streptococcal respiratory disease and (ii) swine possessing group E streptococcal lymphadenitis were shown to possess antibody titers significantly higher than the controls. The use of the two esterified antigens as controls for each other established the specificity of the reaction in each case. The general shape of the antigen-antibody precipitin curves was not changed when the stearoyl antigens were used; however, the quantitative aspects differed markedly. Oligosaccharides which inhibit the normal antigen-antibody precipitin reaction did not inhibit the hemagglutination reaction. The adsorption of antisera with whole streptococcal cells reduced the hemagglutination titer in relation to the quantity of cells employed. Data are given on the (i) optimal concentration of stearoyl antigen for sensitization, (ii) time of adsorption of antigen to red cells, (iii) use of albumin as diluting fluid, and (iv) condition of red cells. Properties of the esterified antigens and the mechanism of the agglutination reaction are discussed. The results indicate that polysaccharide antigens of other bacteria may be esterified and employed in a similar manner.     

Purification and characterization of a Streptomyces albus endo-N-acetylmuramidase lytic for group A and other beta haemolytic streptococci.

The purification and characterization of the streptolytic exo-enzyme from the Maxted-McCarty strain of Streptomyces albus is described. This enzyme was shown to be an endo-N-acetylmuramidase with a molecular weight of 10 to 12,000 and optimal activity at pH 8 and 45 degrees C. The enzyme is lytic for streptococci of various groups, Micrococcus lysodeikticus, Staphylococcus aureus, as well as Escherichia coli. It closely resembles the F1 endo-N-acetylmuramidase described by Ghuysen et al. (1966) except for small differences in the products of lysis of streptococcal cell walls and the resistance of Escherichia coli to lysis by the F1 enzyme. Lysates of group A and A variant streptococcal cell walls prepared with purified Streptomyces albus muramidase contained serologically active M protein and C carbohydrate-peptidoglycan complexes. The chemical and immunological characteristics of these enzymmatic products of streptococcal cell walls are reported and their utility as immunologic reagents is described.     

Acrylamide Gel Electrophoresis of Group A Streptococcal Cell Walls

Patterns obtained by acrylamide gel electrophoresis of group A streptococcal cell walls were characteristic for M types 1, 2, 3, 4, and 5, respectively. Best results were obtained with cell walls which had been solubilized with a phenol-acetic acid-water mixture. The method will make it possible to compare group A streptococci of epidemiological and clinical significance and to separate components for more critical analysis.     

Suppression of bacterial cell wall-induced polyarthritis by recombinant gamma interferon

Group A streptococcal cell wall fragments (SCW) induce erosive polyarthritis, characterized by synovial cell hyperplasia and intense mononuclear cell infiltration, in susceptible rats. Because of the known antiproliferative and immunomodulatory effects of interferon (IFN), we evaluated the effect of systemically administered alpha, beta and gamma IFN on the evolution of these destructive lesions. Treatment with gamma IFN not only reduced the acute response, but had an even greater suppressive effect on the chronic mononuclear cell-mediated destructive phase of the disease (articular index 10.2 +/- 1.2 for SCW only versus 3.8 +/- 0.7 for SCW + gamma IFN; p less than 0.01). Treatment with gamma IFN was more effective in the suppression of the arthritis than alpha, beta IFN. Histopathologic evaluation of the joints demonstrated that gamma IFN-treated animals had significantly fewer inflammatory cells, and less synovial hyperplasia and erosions than the SCW controls. gamma IFN suppression of mononuclear cell prostaglandin synthesis and synovial fibroblast proliferation was consistent with its anti-arthritic effects. These data indicate that the pathophysiology of SCW-induced erosive polyarthritis is subject to regulatory control by gamma IFN and that the mechanisms of suppression may be relevant in the treatment of rheumatoid arthritis.     

Echinostoma caproni: intestinal pathology in the golden hamster, a highly compatible host, and the Wistar rat, a less compatible host

The histopathological changes induced by Echinostoma caproni (Trematoda: Echinostomatidae) in a high (golden hamster) and a low compatible host (rat) were compared at 15 and 30 days post-infection. Infection of rats was characterized by a progressive increase in erosion of villi and elevated numbers of goblet cells, which could be related to the early expulsion of the parasite in a host of low compatibility. In contrast to rats, the number of goblet cell in E. caproni-infected hamsters was low, but increased numbers of neutrophils and mesenteric inflammatory cells were observed. This indicated that local inflammatory responses in hamsters were greater than in rats. An immunohistochemical study using polyclonal IgG anti-E. caproni excretory-secretory antigens demonstrated a greater level of passage of E. caproni antigens through the intestinal mucosa in hamsters than in rats, probably in relation to the greater inflammatory response. Our results indicate the fact that early inflammatory responses could be important for the establishment of E. caproni chronic infections in highly compatible hosts.     

An association between the acute phase response and patterns of antigen induced T cell proliferation in juvenile idiopathic arthritis

The aim of this research was to determine whether all memory T cells have the same propensity to migrate to the joint in patients with juvenile idiopathic arthritis. Paired synovial fluid and peripheral blood mononuclear cell proliferative responses to a panel of antigens were measured and the results correlated with a detailed set of laboratory and clinical data from 39 patients with juvenile idiopathic arthritis. Two distinct patterns of proliferative response were found in the majority of patients: a diverse pattern, in which synovial fluid responses were greater than peripheral blood responses for all antigens tested; and a restricted pattern, in which peripheral blood responses to some antigens were more vigorous than those in the synovial fluid compartment. The diverse pattern was generally found in patients with a high acute phase response, whereas patients without elevated acute phase proteins were more likely to demonstrate a restricted pattern. We propose that an association between the synovial fluid T cell repertoire and the acute phase response suggests that proinflammatory cytokines may influence recruitment of memory T cells to an inflammatory site, independent of their antigen specificity. Additionally, increased responses to enteric bacteria and the presence of αEβ7 T cells in synovial fluid may reflect accumulation of gut associated T cells in the synovial compartment, even in the absence of an elevated acute phase response. This is the first report of an association between the acute phase response and the T cell population recruited to an inflammatory site.     

T cell activation by mycobacterial antigens in inflammatory synovitis

To define which mycobacterial antigens were responsible for the activation of synovial fluid T lymphocytes, acetone-precipitated Mycobacterium tuberculosis (AP-MT) antigens were separated into five fractions following polyacrylamide gel electrophoresis and added to the mononuclear cell cultures of patients with inflammatory synovitis. Fractions 2 (50 to 70 kDa) and 5 (less than 28 kDa) resulted in significantly more proliferation than that of fractions 1, 3, and 4. The response to a purified mycobacterial 65-kDa heat shock protein (hsp), which migrated in fraction 2, was highly correlated (r = 0.89, P less than 0.001) with the response to the crude AP-MT. The proliferative response to a different hsp. the Escherichia coli DnaK, by synovial fluid lymphocytes was marginal. Analysis of the synovial fluid T cell response to mycobacterial culture filtrates by T cell Western blotting revealed dominant responses to antigen(s) in the range of 31 to 21 kDa in each responding patient, although no other consistent pattern of T cell activation was noted. Three lines of evidence suggested that the response to the low molecular weight fractions was directed against degradation fragments of the 65-kDa protein. These observations suggest that the activation of T lymphocytes obtained from inflammatory synovial fluids by crude mycobacterial antigens was due in large part to recognition of the 65-kDa mycobacterial hsp.     

The physicochemical and biochemical characteristics of the cell walls in M+ and M- variants of Streptococcus group A type 29

The amino acid composition of cell walls and surface proteins, isolated from virulent (M+) and avirulent (M-) streptococcal strains (group A, type 29) has been determined by the method of E. H. Beachey et al. The kinetics of the lysis and proteolysis of streptococcal cell walls with muramidase and protease obtained from Actinomyces levoris and streptolysin has been studied. The constants describing the progress rates of these processes has been determined; their values in case of both lysis and proteolysis are higher in virulent strains than in avirulent ones.     

Inhibitors of reactive oxygen intermediates suppress bacterial cell wall-induced arthritis.

Peritoneal and peripheral blood monocyte-macrophages from inbred Lewis (LEW) rats generate higher levels of reactive oxygen intermediates (ROI) in response to group A streptococcal cell walls (SCW) than do similar populations of cells from histocompatible Fischer rats. This differential sensitivity of the phagocytes to SCW is reflected in differences in susceptibility of the two strains to the development of arthritis in response to SCW. After systemic administration of the SCW, LEW rats develop acute and chronic erosive polyarthritis, whereas the Fischer rats are arthritis resistant. Inasmuch as these data suggested that the SCW-induced release of inflammatory cell products such as ROI might be an important contributory factor in the pathogenesis of arthritis in the LEW rats, the animals were injected with SCW and treated with ROI inhibitors. A single intraarticular injection of superoxide dismutase or catalase significantly reduced the SCW-induced inflammatory response and evolution of erosive arthritis in the treated animals (articular index 3.6 +/- 0.36 for SCW only vs 1.4 +/- 0.3 for SCW + SOD; p less than 0.001; n = 6). These data indicate that ROI play a pivotal role in synovitis and, furthermore, that suppression of these inflammatory mediators modulates both acute and chronic SCW-induced inflammation of the joint.     

Study of the composition of cell wall of group A Streptococcus after hydrolysis using muramidase from Streptomyces levoris

The aim of the experiment was to study the lysis products of cell walls of group A streptococci resulting from exposure to N-acetylmuramidase. It was shown that for isolating surface proteins free of polysaccharide and peptidoglycan fragments it was necessary to treat the streptococcal cell walls with endo-beta-N-acetylmuramidase for no more than 30 minutes. Prolonged hydrolysis with muramidase led to the presence of polysaccharide and the peptidoglycan fragments in the protein fractions, intracellular wall proteins covalently bound to the peptidoglycan fragments and polysaccharide being also released.     

Monoclonal antibodies cross-reacting with fibroblasts of interstitial connective tissue of the myocardium and cell wall protein antigens of group A Streptococcus

Monoclonal antibodies (MCA) B6/5 and C5/3 were obtained after immunization of BALB/c mice with the protein non-type-specific antigens (NTSA) of streptococcal group A cell wall. MCA B6/5 in the indirect immunofluorescence react with human and animal interstitial connective tissue (ICT) of the myocardium and human fibroblast culture cells. MCA C5/3 react with the bands of muscle fibers of the myocardium. MCA B6/5 and C5/3 are autoantibodies. It was revealed that these MCA are directed to two streptococcal cross-reacting antigens (CRA). Production of B6/5 and C5/3, apparently, does not depend on the possibility of some streptococcal antigens to bind fibrinogen. Bound immunoglobulins were not revealed in the ICT and in the muscle fibres by the cultivation of the C5/3 monoclone. Firstly it was stated that, MCA B6/5, reacting with fibroblasts and with streptococcal CRA, are capable to fix in the ICT of myocardium, what is typical for the phenomenon described in rheumatic fever.     

Fc gamma R expression on macrophages is related to severity and chronicity of synovial inflammation and cartilage destruction during experimental immune-complex-mediated arthritis (ICA)

STATEMENT OF FINDINGS: We investigated the role of Fc gamma receptors (Fc gamma Rs) on synovial macrophages in immune-complex-mediated arthritis (ICA). ICA elicited in knee joints of C57BL/6 mice caused a short-lasting, florid inflammation and reversible loss of proteoglycans (PGs), moderate chondrocyte death, and minor erosion of the cartilage. In contrast, when ICA was induced in knee joints of Fc receptor (FcR) gamma-chain(-/-) C57BL/6 mice, which lack functional Fc gamma RI and RIII, inflammation and cartilage destruction were prevented. When ICA was elicited in DBA/1 mice, a very severe, chronic inflammation was observed, and significantly more chondrocyte death and cartilage erosion than in arthritic C57BL/6 mice. The synovial lining and peritoneal macrophages of na?ve DBA/1 mice expressed a significantly higher level of Fc gamma Rs than was seen in C57BL/6 mice. Moreover, elevated and prolonged expression of IL-1 was found after stimulation of these cells with immune complexes. Zymosan or streptococcal cell walls caused comparable inflammation and only mild cartilage destruction in all strains. We conclude that Fc gamma R expression on synovial macrophages may be related to the severity of synovial inflammation and cartilage destruction during ICA.     

Lysis of the cellular walls of Streptococcus group A by enzymes produced by actinomycetes]

More than 80 cultures of actinomycetes belonging to different taxanomic groups were studied with a purpose of screening actinomycetes actively producing enzymes lyzing the cell walls of group A streptococci. 31 strains of the actinomycetes producing enzymes which lyzed the cell walls by 20-50 and 60-80 per cent within 1 and 4 hours respectively were selected. The proteolytic activity of the enzymes produced by these strains was also studied. It was shown that 4 cultures, i.e. Actinomyces albus, strains 6 and 9, Actinomyces levoris, strain 29 and Actinomyces gibsonii, strain 42 were of interest as organisms producing enzymes which lyzed the streptococcal cell wall without impairing its antigenic components.