Biomedical subjects
Publications and source records attributed to C J Elson.
Synovial fluids from patients with rheumatoid arthritis and from patients with osteoarthritis degraded the matrix of living, but not killed, normal human cartilage as judged by loss of glycosaminoglycans. Normal human serum did not degrade living cartilage and neither, unexpectedly, did recombinant human cytokines.
An inhibitor of myeloperoxidase has been identified in the synovial fluids and sera from patients with rheumatoid arthritis and sera from normal subjects. Initially, these fluids were found to inhibit stimulus induced degranulation of polymorphonuclear leucocytes independently of the stimulating agent. Subsequently, the fluids were shown to inhibit the released enzyme rather than the degranulation response of polymorphonuclear leucocytes. Both rheumatoid and normal serum samples contained high concentrations of the inhibitor but the concentrations were lower in rheumatoid synovial fluids. The inhibitory activity seemed to be specific for peroxidase as the fluids did not inhibit beta-glucuronidase activity. A protein of relative molecular mass (Mr) 150 kd was purified from synovial fluid by affinity chromatography on myeloperoxidase-Sepharose. It is concluded that serum and synovial fluid contain a novel myeloperoxidase inhibitor, which acts by binding to myeloperoxidase and thereby prevents myeloperoxidase releasing oxidative products in serum.
Several prominent mycobacterial protein antigens involved in antibody and T cell responses have been identified as members of highly conserved heat shock protein families. In particular, immune responses to the mycobacterial 65 kD heat shock protein (hsp65) have been implicated in the pathogenesis of autoimmune diseases both in experimental animal models and in man. Additionally, hsp65 has been shown to modulate the course of autoimmune disease in such experimental animal systems. In this report, we have examined the synthesis of heat shock proteins by a fast growing mycobacterial strain, M. vaccae, in heat stressed cultures and used the pristane induced arthritis model to investigate the immunoprophylactic and immunotherapeutic potential of heat killed M. vaccae. Heat shock of M. vaccae cultures at 48 degrees C demonstrated a 43-fold increase in hsp65 over that expressed at 37 degrees C. It is therefore suggested that heat killed M. vaccae contains sufficient hsp that can be presented in the context of appropriate adjuvant properties for use as an effective immunomodulatory agent. Immunisation experiments with M. vaccae revealed that protection or exacerbation of pristane induced arthritis was dependent on the dose (given in an oil or aqueous suspension), route and time of immunisation. In addition, it was demonstrated that the development of arthritis correlated with high levels of agalactosyl IgG and that "protected" animals had significantly depressed levels.
We have analysed the cellular and humoral immunity to the mycobacterial 65 kD heat shock protein (hsp65) in groups of DBA/1 mice with arthritis induced by intraperitoneal injection of the mineral oil pristane. Here we confirm that DBA/1 mice are highly susceptible to pristane induced arthritis (PIA) and demonstrate that the incidence of arthritis can be modulated by either pretreatment with low dose irradiation or by preimmunisation with recombinant hsp65. Global cellular responses to antigens such as BSA or type II collagen were not enhanced or impaired within groups of arthritic (A) or non-arthritic (NA) mice. However, the cellular response to hsp65 in arthritic animals preimmunised with the 65 kD antigen was significantly elevated in comparison to hsp65 preimmunised mice that were resistant to the induction of disease. On the contrary, the level of hsp65 specific antibodies was much high in NA animals than in PIA mice. CBA/Igb mice are partially susceptible to the induction of PIA. We have previously reported that arthritic CBA/Igb mice have both elevated cellular and humoral reactivity to hsp65. Although a central pivotal role for hsp65 has been postulated in autoimmune diseases these results indicate that there is no simple relationship between the pathogenesis of PIA and immune responses to hsp65.
The development of arthritis induced in mice by intraperitoneal injection of the non-antigenic mineral oil, 2,6,10,14-tetramethylpentadecane (pristane), was shown to depend on an intact immune response possibly to a heat-shock protein (hsp) in the synovium. Initial experiments suggested that some crucial event in the development of arthritis takes place early after pristane injection. First, irradiated pristane-treated mice failed to develop arthritis unless they were reconstituted with spleen cells from normal donors within 25 days of irradiation. Second, mice irradiated up to 50 days after pristane injection, but not later, did not develop arthritis. Evidence for the involvement of an immune response to heat-shock protein (hsp) comes from the finding that mice injected with mycobacterial 65-kDa hsp prior to pristane challenge had a reduced incidence of arthritis in contrast to animals pre-immunized with the E. coli hsp equivalent GroEL or with bovine serum albumin. Other experiments revealed that T cells from mice with gross morphologically defined arthritis proliferated strongly to hsp65 and to normal joint antigens, whereas T cells from animals treated with pristane which did not develop arthritis gave much smaller responses. Mice which developed arthritis also had elevated levels of anti-hsp65 IgG in comparison with non-arthritic animals. These findings strongly suggest that autoimmune reactions to an antigen which cross-reacts with hsp65 are generated in pristane-induced arthritis. It is considered that the autoimmune response is directed to a synovial antigen and that pre-immunization with hsp65 protects the animals from the development of pristane-induced arthritis by altering the specificity or quality of the immune response to this antigen.
Recombinant human Interleukin-1 (rhIL-1) beta was found to enhance stimulus-induced granule exocytosis from human polymorphonuclear leukocytes (PMNs). PMNs were incubated with rhIL-1 beta and then stimulated with either heat-aggregated IgG (Hagg) or N-formyl-methionyl leucylphenylalanine (FMLP). The release of the azurophil enzyme myeloperoxidase (MPO) was measured. Low concentrations of stimuli (10 micrograms/ml Hagg, 2.5 X 10(-9) M FMLP) did not stimulate degranulation in the absence of rhIL-1 beta. However, such concentrations elicited marked degranulation from PMNs preincubated with rhIL-1 beta (0.2-100 ng/ml). The enhancement of degranulation was dependent on the concentration of rhIL-1 beta employed and on the period of incubation. In other experiments, the effect of rhIL-1 beta on the PMN oxidative response was determined. rhIL-1 beta did not directly stimulate the production of superoxide anions or enhance the oxidative response to Hagg or FMLP. It is suggested that in rheumatoid joints, IL-1 beta may potentiate PMN degranulation, but not their oxidative response.
No difference was found between the degranulation responses to FMLP of synovial fluid (SF) polymorphonuclear leucocytes (PMNL), from patients with rheumatoid arthritis (RA), and either paired blood PMNL or blood PMNL from a healthy donor. In contrast, the response of SF PMNL to heat-aggregated IgG was often reduced compared with autologous blood PMNL. Similarly, SF from some (35%) RA patients stimulated degranulation of PMNL but the response of SF-derived PMNL to autologous stimulatory SF was reduced compared with the response of blood PMNL. The stimulatory activity of the SF was removed by sepharose-protein A. These results were taken to suggest that the activity is due to immunoglobulin aggregates and that SF PMNL (from some RA patients) are tachyphylactic to stimulation by immunoglobulin aggregates as measured by degranulation because they have been stimulated by immunoglobulin aggregates in vivo. In other studies the concentration of myeloperoxidase (MPO) was measured enzymically in RA SF and was found to be present in varying amounts. However, only a weak relationship was found between MPO levels and either PMNL numbers or levels of complement-bearing IgG aggregates in SF. It is considered that the relationship between MPO and immunoglobulin aggregates levels is obscured by the presence of a peroxidase inhibitor in the fluids and/or because only aggregates bound to tissue stimulate degranulation in vivo.
The dimethylmethylene blue assay showed higher concentrations of glycosaminoglycans in many synovial fluids from patients with rheumatoid arthritis (RA) than in autologous sera or sera or synovial fluids from normal subjects. These results were taken to suggest that the glycosaminoglycans in RA synovial fluid were abnormally raised and derived from cartilage. To discover what stimulated such glycosaminoglycan release in RA joints relations were sought between synovial fluid concentrations of glycosaminoglycans and immunological and inflammatory mediators. It was shown that RA synovial fluid glycosaminoglycan concentrations correlated with synovial fluid C3d concentrations but not with synovial fluid rheumatoid factor concentrations, polymorphonuclear leucocyte numbers, myeloperoxidase concentrations, or the ability of the synovial fluids to release free radicals from normal polymorphonuclear leucocytes. A correlation was found between synovial fluid C3d and interleukin 1 concentrations as judged by both lymphocyte activating factor activity and immunoassay, but no significant correlation was detected between interleukin 1 and glycosaminoglycan concentrations. It is suggested that in the rheumatoid joint locally produced cytokines, in addition to interleukin 1, together stimulate glycosaminoglycan release from cartilage and render it vulnerable to attack by other processes.
Only a minority (7/35, 20%) of synovial fluids from patients with rheumatoid arthritis (RA) and none from patients with other arthritides stimulated the oxidative response of polymorphonuclear leucocytes (PMNs). Superoxide anion generation was measured by superoxide dismutase inhibitable reduction of cytochrome c. The same synovial fluids stimulated superoxide release by PMNs regardless of their source, though they elicited a greater response from RA synovial fluid PMNs than from either RA blood PMNs or blood PMNs from normal subjects. The remaining synovial fluids failed to stimulate any of the PMNs, though some (2/10) stimulated PMNs pretreated with cytochalasin B. The stimulatory activity was removed from RA synovial fluids by protein A-Sepharose and eluted with the void volume on gel chromatography. It is considered that immunoglobulin aggregates in some RA synovial fluids may stimulate the oxidative response of PMNs in the joint space but that most do not because these fluids contain either a specific inhibitor or immunoglobulin aggregates of an inappropriate type, or both.
Synovial fluid (SF) from rheumatoid arthritis (RA) patients were tested for their ability to inhibit the proliferative responses of normal peripheral blood mononuclear cells (PBM) to mitogens and interleukin-2 (IL-2). SF significantly inhibited the responses to concanavalin A (CON A) and phytohaemagglutinin (PHA), but significantly enhanced the responses to IL-2. Similarly, SF mononuclear cells (SFM) were hyporesponsive to CON A and PHA compared with autologous PBM, but hyper-responsive to IL-2. It is concluded that an IL-2 inhibitor in RA SF is unlikely to be the cause of SFM hyporesponsiveness to mitogens.
Mice immunized with intact rat red blood cells (RBC) developed serum auto-antibodies (some of which were mouse specific) to the RBC membrane components spectrin and antigens of 100 and 81 kDa as shown by Western blotting and enzyme-linked immunosorbent assay as well as RBC surface-bound autoantibodies detected by the Coombs' test. In order to discover whether these autoantibodies were induced and controlled in similar or different ways, mice were challenged with a variety of rat and mouse RBC preparations. In addition, the ability of recipients given spleen cells from the above donors to generate autoantibody responses to intact rat RBC was measured. It was found that all the autoantibodies were induced in mice challenged with rat RBC ghosts but none following immunization with butanol-extracted rat RBC ghosts or intact mouse RBC. By contrast, mice injected with mouse RBC ghosts made autoantibodies to spectrin and to the 100-kDa band. Spleen cells from mice primed with intact rat RBC, rat RBC ghosts or butanol-extracted rat RBC ghosts curtailed Coombs' autoantibody production of recipient mice challenged with intact rat RBC. Serum from recipients of spleen cells primed with intact rat RBC or the butanol extract generally failed to react with rat or mouse spectrin or with the 81-kDa band, although antibody was detected to the rat 100-kDa band. Recipients of rat RBC ghost-primed spleen cells produced antibody to rat and mouse spectrin and to rat 100-kDa band but not to mouse 100-kDa or rat or mouse 81-kDa bands. Occasionally, suppression of antibody to the rat-specific 38-kDa band was observed in recipients of intact rat RBC-primed spleen cells. It is therefore suggested that regulation of cross-reactive and mouse-specific autoantibodies as well as rat-specific antibodies occurs in an independent, determinant-specific manner.
Synovial fluid IgG may be altered in rheumatoid arthritis (RA) and promote the formation of immune complexes with rheumatoid factor. To investigate this possibility, monomeric IgG was prepared from synovial fluids from a range of arthritides for use as the antigen in a rheumatoid factor microplate radioimmunoassay. In comparisons with normal serum IgG antigen, increased rheumatoid factor binding was shown to IgG antigens prepared from synovial fluids from patients with RA and osteoarthritis (OA). Increased binding was also shown to RA sera IgG, but not to OA sera IgG. This increased binding was not due to increased IgG antigen binding to the plate or to IgG rheumatoid factor in the antigen preparations. It was considered that cause was a structural alteration of the IgG as a result of inflammation within the rheumatoid and OA joint.
Destructive osteoarthritis is characterised by rapidly progressive joint destruction associated with intra-articular deposition of hydroxyapatite crystals. The possible role of such crystals in the pathogenesis of this condition was investigated by testing the ability of hydroxyapatite crystals to stimulate the production of bone resorbing activity from mouse peritoneal macrophages. Urate crystals were used for comparison. Culture supernatants were tested for bone resorbing activity using the mouse calvarial bone resorption assay, for interleukin 1 using a standard lymphocyte activation assay, and for prostaglandin E2 by radioimmunoassay. Culture supernatants from macrophages incubated with hydroxyapatite crystals contained dialysable bone resorbing activity, high concentrations of prostaglandin E2, but no interleukin 1 like activity. The production of the bone resorbing agent was prevented by culturing macrophages with hydroxyapatite crystals in the presence of indomethacin. By contrast, culture supernatants from macrophages incubated with urate crystals contained bone resorbing activity, which was only partly removed by dialysis, and interleukin 1 like activity. The latter was shown to be increased in culture supernatants from macrophages incubated with urate crystals in the presence of indomethacin, while production of bone resorbing activity was partially inhibited. It is considered that the bone resorbing activity liberated from macrophages stimulated by hydroxyapatite crystals can be explained by the presence of prostaglandin E2 alone, whereas the activity liberated by urate crystals is due to both prostaglandin E2 and interleukin 1.
Cells suppressing red blood cell (RBC) autoantibody responses were found to exert their effect in Lyt-2+ depleted recipients. Mice injected with monoclonal antibodies to Lyt-2 became deficient in Lyt-2+ cells as judged by indirect immunofluorescence. These mice and normal recipients were given either rat RBC primed spleen cells (suppressor cells) or normal spleen cells and challenged with rat RBC. The autoantibody response of both Lyt-2+ depleted and normal recipients was suppressed as compared with that of mice given normal spleen cells. It is suggested that an Lyt-1+ cell may be involved in the expression of suppression.
The relationship between rat red blood cell (RBC) glycophorins and the antigens recognised by anti-rat RBC antibodies was examined. Initially, murine monoclonal antibodies specific for surface epitopes on whole rat RBCs were tested for their reactivity with RBC membranes on Western blots and two were found which reacted with blotted antigens. These antibodies recognised two bands corresponding to the major PAS-stainable bands of rat RBC membranes (i.e., the glycophorins) and a number of minor bands, thus demonstrating that the bands are antigenically related. This band-pattern was remarkably similar to that obtained with mouse anti-rat RBC serum. Digestion with neuraminidase altered the electrophoretic mobility of most of the bands, providing additional evidence that they are sialoglycoproteins, although sialic acid was shown not to contribute to their antigenicity. The glycophorin nature of the major antigens was verified by reelectrophoresis and blotting of bands excised from SDS gels, which showed that they were interconvertible monomeric and dimeric forms of the same polypeptide chain. It is suggested that rat RBC glycophorins are a related family of sialoglycoproteins with the high molecular weight members being formed by dimerization of five lower molecular weight polypeptide chains in various combinations.
The synovial fluids of patients with a destructive form of osteoarthritis (DOA) were shown to contain high levels of bone resorbing activity as judged by the ability of the fluid to stimulate the release of 45Ca from labelled cultured mouse calvariae. The activity was lost on extended storage of the synovial fluids and was dependent for its effect on cellular activity in bone. Bone resorbing activity was present in most synovial fluids from patients with DOA and rheumatoid arthritis (RA) but occurred at higher levels in the former. In contrast, interleukin 1 (IL1) activity, measured by the mouse thymocytes costimulation assay, was higher in RA than DOA synovial fluids. Little or no bone resorbing or IL1 activity was detected in synovial fluids from patients with pseudogout or non-destructive osteoarthritis. These results suggest that most DOA synovial fluids contain a bone resorbing factor other than IL1. It is considered that the factor may be produced by synovial cells stimulated by hydroxyapatite crystals.
Polymorphonuclear leukocytes (PMN) isolated from the synovial fluid (SF) of patients with rheumatoid arthritis (RA) exhibited an enhanced oxidative response to heat aggregated IgG (Hagg) and N-formyl-methionyl-leucyl-phenylalanine (FMLP) as compared with either autologous or normal blood PMN. Normal blood PMN pretreated with synovial fluid showed a significantly increased response to FMLP which was unaffected by prior dialysis of SF. The degree of enhancement produced varied between SF's and was dependent on the period for which cells were incubated and on the concentration of SF used. In contrast there was no enhancement of the oxidative response when Hagg was used as a stimulus. Indeed, SF produced a dose dependent inhibition of Hagg stimulated superoxide production. These observations suggest that SF's from patients with RA contain factors which both enhance and inhibit the oxidative response of PMN depending on the subsequent stimulus.
An erythrocyte autoantigen has been identified by means of monoclonal autoantibodies raised by immunizing mice with rat red blood cells (RBC). The autoantibodies reacted with intact rat and mouse RBC as judged by a cellular radioimmunoassay, and with a 52K band on western blots of rat and mouse RBC. They did not react with intact sheep RBC or blotted sheep erythrocyte membranes. Although anti-rat erythrocyte antibodies did react with bands in the molecular weight region of 50-55 K (on blots of rat erythrocyte membranes), these bands were susceptible to neuraminidase digestion, thus distinguishing them from the 52 K band recognized by monoclonal autoantibodies. The implications of the above results for the known autoantibody specificity of suppression is discussed, and it is suggested that they favour the existence of autoantigen-specific suppressor cells.