Osteoprotegerin reduces the loss of periarticular bone mass in primary and secondary spongiosa but does not influence inflammation in rat antigen-induced arthritis.

OBJECTIVE: To assess the effect of osteoprotegerin (OPG) on joint swelling, synovial inflammation and cartilage destruction, periarticular and axial bone volume, and bone turnover in rat antigen-induced arthritis (AIA). DESIGN: Rats were treated with OPG (3 mg/kg/day) at regular intervals from day 1 to day 20 of AIA. Disease activity was evaluated by measurement of joint swelling as well as, joint inflammation and destruction by histology. Bone volume and cellular turnover parameters of secondary spongiosa of the right tibia head and the third lumbar vertebra were evaluated by histomorphometry. Periarticular bone volume of the primary spongiosa at the right tibia head was measured by linear scanning. The findings were compared with those of PBS-treated AIA and healthy animals. RESULT: OPG treatment did not reduce joint swelling or histological signs of inflammation. Cartilage destruction was reduced. However, this effect did not reach statistical significance . In the secondary spongiosa OPG treatment reduced the loss of periarticular bone volume. However, the latter did not reach the level of healthy controls. OPG treatment significantly reduced parameters of bone formation and bone resorption. In the primary spongiosa, OPG-treatment led to a higher amount of mineralized tissue and a greater number of trabeculae compared to PBS-treated animals with AIA or healthy controls. In the axial skeleton, OPG treatment reduced bone formation and bone resorption parameters compared to healthy animals. This treatment had no influence on bone volume. CONCLUSIONS: In periarticular bone of AIA rats, OPG treatment reduced the loss of bone volume and decreased the bone turnover, thus preventing periarticular bone destruction. OPG treatment had no influence on inflammatory process or on cartilage destruction.

Amelioration of murine antigen-induced arthritis by dehydroepiandrosterone (DHEA).

OBJECTIVE AND DESIGN: Sex hormones have immunomodulatory properties and may play an important role in the pathogenesis of autoimmune diseases like rheumatoid arthritis (RA). This study sought to examine the effects of the natural weak androgen dehydroepiandrosterone (DHEA) and its metabolite androstenediol (AED) on the development of murine antigen-induced arthritis (AIA). METHODS: DHEA and AED were administered orally, approximately 10 mg/day, from the time of AIA induction (i.e., 3 weeks after start of immunization) in young male as well as young and old female C57BL/6 mice. The effects were assessed in terms of joint swelling, histological changes, and cell-mediated and humoral immunity. RESULTS: Compared to untreated AIA animals, continuous administration of DHEA decreased knee joint swelling during acute and chronic AIA, as well as histological signs of inflammation and joint destruction during chronic AIA. These effects were age- and gender-independent. Delayed-type hypersensitivity (DTH) to the specific antigen methylated bovine serum albumin (mBSA) was significantly reduced, but there were no changes in the balance of the T helper (Th) cell subsets Th1/Th2, as tested by the ratio of IgG isotypes in the sera. Whereas serum levels of IgG antibodies to mBSA were not influenced, the formation of IgG autoantibodies to the matrix constituents collagen type I, collagen type II, and cartilage proteoglycans was significantly inhibited. In all experiments, the effects of AED were not significantly stronger than those of DHEA. CONCLUSIONS: Administration of exogenous DHEA ameliorates the severity of acute and chronic AIA, presumably by suppressing cell-mediated immunity against mBSA (the inducing antigen) and formation of autoantibodies. However, because of the fundamentally different DHEA physiology in rodents, the role of such a replacement therapy in human RA deserves further elucidation.

High-dose clodronate therapy prevents joint destruction in chronic antigen-induced arthritis of the rat but inhibits bone formation at the axial skeleton.

OBJECTIVE: To investigate the effects of clodronate on clinical disease activity, inflammatory alterations and cartilage destruction, periarticular and axial bone volume and bone turnover in chronic antigen-induced arthritis (AIA; day 28). METHODS: Rats with AIA were treated with clodronate (5 mg/kg/day continuously; 20 mg/kg/day intermittently or high-dose with 300 mg/kg 3 hours after arthritis induction +20 mg/kg/day continuously, respectively). Joint pathology was examined by histology. Bone volume and cellular turnover parameters of the right tibia head and the third lumbar vertebra were evaluated by histomorphometry. The findings were compared with those of healthy controls, sham-treated AIA and AIA treated continuously with 250 microg/kg of dexamethasone. RESULTS: All three therapy regimens with clodronate resulted in a significant reduction of joint swelling, histopathological inflammatory changes and cartilage destruction in comparison with sham-treated AIA. The antiinflammatory effect of high-dose clodronate was comparable with dexamethasone. The intermittent administration of 20 mg/kg/day of clodronate completely prevented periarticular bone loss by reduction of bone resorption without affecting bone formation at the periarticular and axial bone. Both continuous treatment with 5 mg/kg/day of clodronate and high-dose clodronate therapy partially prevented periarticular bone loss and reduced parameters of bone formation at the axial bone to values below those of healthy controls. CONCLUSION: High-dose clodronate therapy exerts an excellent preventive effect on clinical disease activity and on joint destruction in AIA. However, continuous treatment with high doses of clodronate may result in a low turnover state of bone remodelling.

Periarticular bone alterations in chronic antigen-induced arthritis: free and liposome-encapsulated clodronate prevent loss of bone mass in the secondary spongiosa.

The long-term effects of acutely administered clodronate (free or liposome-encapsulated) on periarticular bone mass and bone turnover were investigated in chronic antigen-induced arthritis (AIA; day 28). Wistar rats were treated intraperitoneally at 3 h and on days 1, 2, and 7 of AIA, with phosphate-buffered saline (PBS; sham), PBS-containing liposomes, free clodronate, or liposome-encapsulated clodronate (cumulative dose, 3.64 mg/animal). In the primary spongiosa (1.25 mm from the growth plate), sham-treated AIA was associated with: (a) a marked significant decrease in trabecular bone volume (-56%); (b) a significant increase of osteoid-covered surface (+135%); and (c) a numerical increase of resorption surface with osteoclasts (+96%). In the secondary spongiosa, free clodronate completely prevented the loss of periarticular bone mass and selectively normalized the parameters of bone formation (i.e., osteoid-covered surface and osteoid-covered surface with osteoblasts). Clodronate liposomes, in addition to these effects, also significantly suppressed bone resorption (i.e., resorption surface covered with osteoclasts). The effects of clodronate liposomes coincided with in vivo targeting of osteoclasts in primary and secondary spongiosa. Thus, low-dose, acutely administered clodronate, both in free and encapsulated forms, exerts an excellent preventive effect on bone loss in the secondary spongiosa of chronic AIA.

Limiting dilution analysis of the frequency of autoreactive lymph node cells isolated from mice with antigen-induced arthritis.

Antigen-induced arthritis (AIA) in mice occurs after the single injection of methylated bovine serum albumin (mBSA) into the knee joint of animals preimmunized with the same antigen in complete Freund's adjuvant. A short acute reaction is followed by a chronic inflammation which shows similar histological features to human rheumatoid arthritis. The mechanisms leading to the chronicity of arthritis are not yet clear. Previous data suggest that autoimmune responses to cartilage components contribute to the persistence of arthritis. In the present study we estimate the frequency of autoreactive cells in the draining lymph nodes of arthritic mice by means of limiting dilution analysis. Graded concentrations of lymph node cells were stimulated for 7 days with type II collagen (CII) or cartilage proteoglycans (PG) in the presence of irradiated syngeneic feeder cells, and the frequency of responding cells was calculated. In the draining lymph nodes of immunized mice without arthritis induction, the frequency of CII reactive lymph node cells ranged from 1/41,182 to 1/57,424 whereas only 1 out of 3 experiments revealed a detectable frequency of PG reactive cells (1/136,128). For comparison, no CII or PG reactive cells were detected in the lymph nodes of normal mice. Intra-articular challenge of immunized mice with mBSA resulted in chronic destructive arthritis and caused a significant increase in the frequencies of CII and PG reactive lymph node cells (1/12,776 to 1/24,611 and 1/79,964 to 1/93,075, respectively). When using incomplete Freund's adjuvant for immunization, a comparable acute arthritis developed after the intra-articular injection of antigen, but the transition into the chronic stage was missing. The frequencies of autoreactive cells in the lymph nodes of these animals were below the level of detection and were judged to be below 1/150,000. The results suggest that autoimmune reactions against cartilage constituents might contribute to the perpetuation of joint inflammation, and that the mycobacteria in the complete Freund's adjuvant play an essential role in this process.

Adoptive transfer of susceptibility to antigen-induced arthritis into severe combined immunodeficient (SCID) mice: role of CD4+ and CD8+ T cells.

Antigen-induced arthritis (AIA) in mice occurs after immunization and a subsequent intra-articular injection with methylated bovine serum albumin (mBSA). The role of T lymphocytes in the adoptive transfer of susceptibility to AIA into SCID mice was investigated. Pooled spleen and lymph node cells from immunized syngeneic or allogeneic donor mice, isolated either before or after the induction of arthritis, could transfer the capacity both to develop arthritis and to produce antibodies to mBSA, collagen type II and cartilage proteoglycans into SCID mice. The intra-articular injection of mBSA in responder animals, immediately after the cell transfer, resulted in a chronic arthritis in the induced joint. The histologic examination revealed synovial hyperplasia, mononuclear infiltration of the synovial membrane, exudation of polymorphonuclear leucocytes into the joint space, and chondrocyte death. The depletion of CD4+ T cells before transfer prevented the manifestation of arthritis in SCID mice, with a concomitant decrease in antibody levels to mBSA, collagen type II and cartilage proteoglycans. In contrast, removal of CD8+ T cells did not significantly affect the transfer of arthritis into SCID mice. The results demonstrate an essential role of CD4+ T cells in the pathogenesis of AIA, whereas CD8+ T cells do not seem to be required for the induction and perpetuation of this disease.

Degradation of articular cartilage during the progression of antigen-induced arthritis in mice. A scanning and transmission electron microscopic study.

Ultrastructural changes of knee articular cartilage in C57B1/6 mice were studied in the course of antigen-induced arthritis by means of scanning and transmission electron microscopy. First damages of the cartilage surface were seen one hour after arthritis induction. The earliest signs were the loss of the superficial electron-dense layer as well as a progressive loss of proteoglycans in the cartilaginous matrix on the surface. Breaks of collagen fibres were already detected at the first day of arthritis. The chondrocytes of the superficial cartilage layer showed an increase of the intracellular membraneous system in the early phase of arthritis. Thereafter chondrocytes on the surface became more and more necrotic. On the 7th day of arthritis acute alterations of cartilage had developed completely. Many lacunae of chondrocytes were opened and the cartilage surface showed deep structural defects with adhering cells, probably lymphocytes and macrophages. In the following time these destructive processes were demonstrated along with cellular proliferation as a sign of repair attempts in hyaline cartilage.

Immunomodulation of rat antigen-induced arthritis by leflunomide alone and in combination with cyclosporin A.

The effects of the new immunomodulating isoxazol derivative leflunomide, in comparison with cyclosporin A, on established antigen-induced arthritis in rats as well as serum antibody levels were determined. When treatment with leflunomide, at concentrations from 2.5 to 10 mg/kg/d, was started on day 3 of arthritis, the acute and chronic phases of arthritis were effectively inhibited. This was demonstrated by decreased joint swelling and reduced histopathological arthritis score at the end of experiment (day 26). Furthermore, the treatment resulted in a significantly reduced level of serum antibodies to the matrix components collagen type I, type II and proteoglycans. Neither leflunomide nor cyclosporin A, at doses of 1 mg/kg/d, had an effect on the severity of arthritis and antibody levels. However, when both drugs were used together, at these non-effective doses, the histopathological score of chronic arthritis was significantly reduced. The results of our experiments demonstrate that leflunomide has a strong suppressive effect on both acute and chronic phases of antigen-induced arthritis and formation of autoantibodies in rats. Furthermore, orally administered doses of leflunomide were as effective as doses of cyclosporin A given intraperitoneally. The combination of sub-effective doses of leflunomide and cyclosporin A resulted in significant inhibition of chronic arthritis.

Therapeutic effects of antibodies against adhesion molecules in murine collagen type II-induced arthritis.

Adhesion molecules play important roles in immune reactions and inflammatory processes and may constitute attractive targets for immunomodulatory approaches. In this study, blocking mAbs against a series of adhesion molecules were tested for their therapeutic effect on developing arthritis in a mouse model. MAbs were given for a period of 4 weeks at the time of exspected incidence of visible disease symptoms, i.e. 4 weeks after priming with collagen type II. A significant reduction of incidence down to values of 13% and 29% of the controls was obtained with mAbs against CD44 and alpha 4-integrin, respectively, during an observation time of 13 weeks. MAbs against CD4 and LFA-1 resulted only in weaker, non-significant effects or a delay in the incidence. MAbs against other molecules including L-selectin, ICAM-1 or VCAM-1 were not effective. The development of antibodies against collagen type II, collagen type I, proteoglycans and the immunogen, bovine collagen type II was affected by mAb treatment to a different extent. In this case, the anti CD4 mAb was the most effective, followed by the anti alpha 4-antibodies in most cases, whereas anti CD44 showed less clear effects on the development of humoral responses. In a skin delayed type hypersensitivity model analyzed for comparison, mAbs against LFA-1/ICAM-1 and alpha 4-integrin showed the largest effects on ear swelling. These data show that mAbs against several adhesion molecules are able to block selectively distinct aspects of immune reactions, and that CD44 and alpha 4-integrins could be promising targets for an immunotherapy of rheumatoid arthritis with receptor-interfering agents.

Different immunological mechanisms contribute to cartilage destruction in antigen-induced arthritis.

Antigen-induced arthritis in guinea pigs was used as a model to investigate the pathogenic mechanisms responsible for cartilage destruction in chronic joint inflammation. The activation of macrophages, their effects on cartilage metabolism, and the development of autoimmunity to cartilage constituents were studied during the progression of arthritis. The results show that in arthritic animals the macrophages are systemically activated, with a peak in the early phase of inflammation. Interleukin 1, produced by the activated cells, suppresses the proteoglycan synthesis in cartilage explants and cultured chondrocytes and increases the proliferation of the cells in vitro. During the progression of arthritis humoral and cell-mediated immune responses to collagen type II and cartilage proteoglycans occur correlating with the severity of arthritis. It is concluded that different immunological mechanisms may be involved in cartilage destruction during antigen-induced arthritis. Mediator-induced metabolic reactions dominate in the early phase, whereas autoimmunity to cartilage might play an essential role in later phases of arthritis.

Influence of cyclosporin A on cytokine levels in synovial fluid and serum of rats with antigen-induced arthritis.

The effects of the T-cell-directed immunosuppressant cyclosporin A (CsA) on the development of antigen-induced arthritis (AIA) in rats as well as on cytokine levels in synovial fluid and serum were determined. The treatment with CsA effectively inhibited the chronic phase of arthritis as demonstrated by decreased joint swelling and reduced histological arthritic score. In animals with AIA the level of IL-6 in the synovial fluid and serum is increased, showing good correlation with the severity of the disease. The CsA treatment reduced IL-6 levels to normal. IL-1, IL-2 and TNF-alpha do not seem to be directly involved in the pathogenic mechanisms of chronic arthritis.

The effects of immunomodulatory thymic and splenic peptides and cyclosporin A on antigen-induced arthritis in the rat.

Long-term treatment with natural and synthetic thymic and splenic peptides as well as cyclosporin A inhibited the development of antigen-induced arthritis in rats. This was demonstrated by decreased joint swelling and reduced degree of macroscopically and histologically evaluated severity of synovitis. The drug treatment also decreased serum levels of antibodies against the specific antigen methylated bovine serum albumin (mBSA) and against cartilage proteoglycans and collagens type I and II. The conclusion from these studies is that the treatment with immunomodulatory thymic and splenic peptides and with the T-cell-directed immunosuppressive drug cyclosporin A inhibits the specific immune response against mBSA and/or the development of autoimmunity against cartilage constituents. The decreased immune reactivity in the joint may reduces the severity of chronic joint inflammation.

Humoral and cell-mediated sensitivity to cartilage constituents in mice with antigen-induced arthritis.

Serum antibodies against native collagen type II and cartilage proteoglycans as well as proliferative responses of spleen lymphocytes to these antigens were evidenced during the progression of antigen-induced arthritis in mice. Because there were some correlations to the severity of arthritis, it is suggested that autoimmunity to cartilage constituents play a role for the persistence of the joint inflammation and cartilage destruction in this experimental model of chronic arthritis.

Effects of the immunomodulator diacetyl-splenopentin on antigen-induced arthritis in rabbits.

Long-term treatment with the immunomodulator diacetyl-splenopentin reduces the severity of chronic joint inflammation and cartilage destruction in rabbits with antigen-induced arthritis. The level of specific antibodies as well as specific and non-specific cell-mediated immune reactivities including the proliferative response of spleen lymphocytes to cartilage proteoglycans in treated animals are lower than in untreated arthritic rabbits. Moreover, suppressor cell activity, which normally decreases during the early phase of inflammation, is enhanced and hyperreactive helper cell potential is reduced. These findings suggest that treatment with diacetyl-splenopentin normalizes the immune regulation, which is disturbed in the early phase of inflammation. This might result in a depression of the hyperreactive immune system including the autoimmunity developed against cartilage. Lowered immune reactivity in the joint in turn reduces the severity of chronic joint inflammation.

Beta-2-microglobulin-derived amyloidosis: onset, distribution and clinical features in 13 hemodialysed patients.

Postmortem examinations were carried out in 13 patients (6 males, 7 females, age 58 +/- 9 years) who had been on regular hemodialysis treatment for 10-90 months using disposable regenerated cellulose membrane dialyzers. The prevalence of beta 2-microglobulin (beta 2m)-derived AB-amyloid deposits in different sites was determined. At autopsy, specimens were obtained from different joints, paravertebral tissue, intervertebral discs and from visceral organs. During life, routine laboratory parameters and radiographic studies had been carried out at 6-month intervals. Serum levels of beta 2m were elevated in all patients (57.5 +/- 13.4 mg/l). Synovial AB-amyloid deposits were shown in different joints of 4 patients, aged between 59 and 73 years, and dialysed for 10-90 months, respectively. All had been unremarkable by X-ray and asymptomatic. No amyloid could be detected in the intervertebral discs of 2 further patients suffering from destructive spondylarthropathy. In 11 of the 13 patients, extracellular beta 2m deposits were observed by immunohistochemistry in different tissues. The results document that (a) AB-amyloidosis may occur in elderly patients even when dialysed for less than 5 years; (b) most cases are completely asymptomatic; the appearance of symptoms must be dependent on additional factors, e.g., site of AB-amyloid deposition and intensity of inflammatory reaction, and (c) AB-amyloid is not the exclusive cause of destructive spondylarthropathy, as 2 typical cases were observed who were devoid of amyloid.

Beta 2-microglobulin serum concentration and associated amyloidosis in dialysis patients.

Using radioimmunological estimation of beta 2-microglobulin (beta 2M), significantly greater serum values were found in 36 dialysis patients (44.4 +/- 20.3 mg/l) in comparison to healthy probands (1.5 +/- 0.2 mg/l). A significant relation to the duration of dialysis, diuresis and serum aluminium and ferritin was found. The used dialysers MLW 1.3/1.8 m2 (regenerated cellulose membrane) did not eliminate beta 2M from the blood. Significantly greater beta 2M concentrations were observed in patients suffering from arthralgia and bone pain, but not in radiologically verified arthropathy and destructive spondylarthropathy. Post-mortem examinations of 13 patients on haemodialysis treatment for between 10 and 90 months revealed synovial beta 2M-derived (AB-)amyloid deposits in four patients at different joints, but not in radiologically suspect areas. The results suggest that independent of serum beta 2M, beta 2M-derived amyloidosis may occur in elderly patients on dialysis for less than 5 years. Several cases were completely asymptomatic.

Changes of immunoregulatory properties and induction of autoimmune reactivity to cartilage in rabbits with antigen-induced arthritis.

The chronicity of the antigen-induced arthritis is characterized as dependent on the development of cell-mediated immunity to the antigen, but the exact mechanisms underlying are unclear. We have evidenced decreased suppressor and increased helper cell potential in the early phase of arthritis as result of the immunization procedure. In the late phase of arthritis proliferative responses of spleen lymphocytes to cartilage proteoglycans were revealed which were neither present in immunized animals without arthritis induction nor in the early phase of arthritis. The changes of the regulatory properties on the T-cell level are probably responsible for the transition of acute arthritis into the chronic stage. The deficiency of an effective suppression and/or the increased helper cell potential results in the activation of B- and T-lymphocytes with increased cell-mediated and humoral immune responsiveness to the antigen maintaining the inflammatory process for a long time. In this situation the release of cartilage proteoglycans during the acute joint reaction induces autoimmune responses against cartilage which could contribute to the chronification of inflammation and to cartilage degradation.

Significance of cell-mediated and humoral immunity in the acute and chronic phase of antigen-induced arthritis in rabbits.

In the course of antigen-induced arthritis of rabbit cell-mediated and humoral immune responses were repeatedly tested in order to prove their significance for the acute and chronic phase of inflammation. The arthritis was monitored during the progression of the inflammation by means of the joint swelling and at the end of experiments by histological evaluation of synovitis and cartilage degradation. Following the arthritis induction a strong increase of specific antibodies and of circulating immune complexes was evident. The correlations between antibody levels and joint swellings confirmed that the local formation of immune complexes is responsible for the initiation and perpetuation of arthritis. In the early phase after immunization the responsiveness of lymphocytes to antigenic and mitogenic stimulation was increased, in the late chronic phase of arthritis proliferative responses of lymphocytes to cartilage matrix components were revealed. No direct correlations could be demonstrated between any cell-mediated immune response and the severity of arthritis. The hyperreactivity of cell-mediated immunity is suggested to be responsible for the transition of the acute arthritis into the chronic stage. The deficiency of an effective suppression results in the activation of B-lymphocytes with increased production of antibodies, maintaining the inflammatory process for a long time. Under these conditions the release of cartilage matrix components during the acute joint reaction induces autoimmune responses against cartilage, which could contribute to the chronification of arthritis and to cartilage degradation.

Myocardial bioptic findings in correlation with TL-201 imaging and hemodynamic parameters in patients with latent cardiomyopathies suspected of having myocarditis.

Sixty-eight patients (24 males, 44 females, mean age 37.2 years) belonging to NYHA classes I and II were investigated. All patients had a nondilated ventricle as well as hemodynamic criteria of "latent cardiomyopathy." In 78% of the patients, a high susceptibility to infection, frequent angina, or possible myocarditis were found. In 75.5%, the mean pulmonary artery pressure was abnormal. T1-201 scintigraphy revealed perfusion defects in 78%. Dyskinesia or hypokinesia were found in 52%. The left ventricular ejection fraction was normal in 74%. On biopsy, pathologic findings were detected in 60.9% of patients with a high frequency of hypertrophy and fibrosis (50%). In two patients, lymphocytic infiltrates were found. Immunohistologically, deposits of complement, IgG, IgM, and gammaglobulin, mainly in the sarcolemma, could be identified in 19.1% of cases. The myocardium was normal in 39.1%; insufficient material was found in 5.9% of patients. Morphologic, scintigraphic, and hemodynamic findings could not be correlated but the combination of two pathologic parameters permitted diagnosis. Biopsy was the most effective diagnostic method in myocarditis.