C. Steffen

Autoimmunity and T-cell subpopulations in old age

To investigate the interrelationship between T-cell-dependent immune functions and autoimmune phenomena in old age we determined T-cell subpopulations in 20 aged healthy individuals (80-96 years old) using monoclonal antibodies. These persons were also investigated as to humoral parameters such as antinuclear antibodies, rheumatoid factors (IgG-, IgA-, IgM-RF), antibodies to collagen types I-IV as well as autoantibodies to organ-specific antigens. In addition, immune complexes were determined. We found that aged individuals have an increased frequency of autoantibodies as compared to a young control population, each aged subject presenting with at least one autoantibody species. Immune complexes, however, were only rarely detected. Three individuals showed a slightly increased T-helper/T-suppressor cell ratio, four had a decreased ratio. An increased number of T-suppressor cells was significantly correlated with a lowered incidence of anticollagen antibodies. Other parameters tested by us: fibronectin, laminin, procollagen type III, C3 and C4 complement components, immunoglobulins and acid alpha 1-glycoprotein. Aged individuals have significantly higher serum levels of fibronectin, while laminin and procollagen concentrations are in the normal range. A large percentage of old individuals had increased serum levels of C3 and/or C4. The acute phase protein orosomucoid, however, was in the normal range.

Change in collagen synthesis of human chondrocyte culture

SummaryA research system constituted entirely of components of human origin was developed to study conversion of collagen synthesis by human chondrocytes. Type specificity of affinity chromatography-purified antibodies to human type II or type I collagen was proven by ELISA inhibition and immunofluorescence analysis. Human chondrocytes were isolated from articular cartilage and kept in monolayer cultures for eight subpassages. Conversion of type II to type I synthesis by chondrocytes was investigated by immunofluorescence. Staining with anti-type II collagen antibodies could be detected during primary cultures and in the first subpassage, whereas staining with anti-type I collagen antibodies occurred beginning from the end of primary cultures and was present up to the eighth subpassage. Results are compared to observations obtained in animal systems and their relevance to conditions in osteoarthritis is discussed.

Human serum inhibitors of collagenase as revealed by preparative isoelectric focusing

Single step separation of pooled normal human serum by means of preparative isoelectric focusing in the range from pH 3.5--9.8 revealed more heterogeneous inhibition of collagenolytic activity than previously reported. Essentially three inhibition zones were resolved. According to their electrophoretic behaviour the respective serum fractions displaying inhibitory activity were designated alpha-, beta- and gamma-collagenase inhibitors. The main component responsible for collagenase inhibition in the alpha-zone was found to be alpha 2-macroglobulin. In the beta-zone inhibitory activity focused around pH 6.3. In the gamma-range a non-dialysable cationic component focusing at pH 9.2 was also able to decrease collagenolytic activity derived from rheumatoid arthritis synovial culture supernatant. These findings were supported by single step separation of serum on DEAE-anion exchange chromatography.

Autoantibodies to Collagen in Patients with Chronic Liver Diseases

Sera of patients with various liver diseases were investigated for the presence of autoantibodies to type I collagen with a sensitive radioimmunoassay employing 3H-labelled collagen type I. A high percentage (69%) of patients with chronic liver diseases, equally distributed between chronic active hepatitis and alcoholic liver disease, was found to possess antibodies to denatured type I collagen, while no antibodies against native collagen were found. These antibodies belonged predominantly to the IgA immunoglobulin class. None of the patients with acute hepatitis or cholestatic liver disease, nor the healthy individual investigated had IgA-anticollagen antibodies. These findings may be useful as a parameter for the detection of chronic liver disease.

Induction of anti-pepsin antibodies after immunization with pepsin-extracted collagen

Immunization with pepsin-extracted human type II collagen purified by different precipitation steps, although not showing any contamination with the enzyme on SDS-polyacrylamide gel electrophoresis, resulted in the generation of antibodies to the enzyme in addition to an immune response to collagen. These antibodies could be removed by immune absorption on a pepsin affinity column, leaving reactivity to type II collagen unaltered. High performance liquid chromatography on hydroxylapatite columns indicated that pepsin remained associated with the collagen molecules even after repeated precipitation and coeluted with a fraction of the collagen preparation. These results demonstrate that pepsin-extracted collagens may contain minimal amounts of the enzyme. On immunization, these impurities may induce the formation of unwanted antibodies, which might simulate a false specificity of the antibody preparation.

Concanavalin A-induced suppressor cell activity and in vitro immunoglobulin secretion in rheumatoid arthritis: correlation with clinical and immunological parameters.

SummaryConcanavalin A-induced suppressor cell activity was found moderately but significantly (P<0.05) decreased in RA patients treated with nonsteroidal antiinflammatory drugs (31±7% suppression) as compared to patients on remission-inducing drugs, such as gold, penicillamine, or chloroquine (51±6%) or to healthy individuals (50±6%). Also, lymphocytes from patients with antibodies to collagen mediated lower suppression (33±7%) than lymphocytes from patients without evidence for these autoantibodies (61±11%). No significant difference between patients and controls or between individual groups of patients were observed in regard to IgM and IgG secretion induced by pokeweed mitogen. Thus, although no indication for a severe derangement of regulatory cells in peripheral blood of RA patients could be observed in this study, a slight deficiency of ConA-inducible suppressor cells that may be reverted by remission-inducing drugs seems to be present in RA.

Experimental Arthritis Induced by Granulocyte Collagenase

Purified human granulocyte collagenase (1 mg %, 10 mg % or 50 mg %) was injected into rabbit knee joints (three groups of 6 animals each) three times within one week. Synovium and synovial fluid were investigated 18 hours, 1 week and 3 weeks after the last injection. After 18 hours, synovial fluids showed distinct cellular exudation, its size depending on the amount of collagenase applied. A smaller number of cells was seen after one week, while normal cell counts were observed 3 weeks after the last injection. Histologically, synovium showed an acute arthritis after 18 hours, whereas after 1 and 3 weeks a chronic proliferative form of arthritis with predominant activation of fibroblasts was diagnosed. As compared with an experimental arthritis induced with rheumatoid synovial collagenase, granulocyte collagenase was less arthritogenic. Neither trypsin nor saline injections induced distinct cellular exudation into synovial fluids nor histologic signs of arthritis.

Determination of collagen in culture supernatants of human chondrocytes.

In order to examine the behaviour of human chondrocytes with regard to the production of collagen more precisely, we have established a human model studying human chondrocytes under monolayer conditions with the use of antihuman collagen antibodies. We were able to demonstrate a switch in collagen synthesis during monolayer culture from type II to type I collagen by immunofluorescence techniques [1]. These investigations have now been extended using Elisa inhibition tests to quantify type-specifically the amount of collagen released by chondrocytes into culture supernatants.

Experimental Pulpal Immunization: I. Route of Application and Demonstration of Immune Response

The experimental exposure of the pulpal room of rabbit teeth made it possible to apply an ovalbumin agar gel deposit into the pulpal room and subsequently investigate the immune response. Diffusion of antigen from the agar into environment was proven. After 3 weekly applications 5 rabbits showed hemagglutinating anti-ovalbumin titers between 1:128 and 1:512, while 7 rabbits receiving 10 weekly applications had titers between 1:8000 and 1:128,000. Control rabbits which received the agar deposit alone showed no antibody response. Control rabbits which obtained 3 identical weekly doses of ovalbumin in PBS by subcutaneous injection showed an identical immune response as animals immunized via the pulpal room. Specificity of antibodies was ascertained by passive hemagglutination with control antigens and hemagglutination inhibition. Intradermal injection of ovalbumin in 3 rabbits which obtained pulpal immunization, induced an Arthus reaction. The precipitating property of ovalbumin antibodies and their identity with defined ovalbumin antisera was proven by gel diffusion. The results obtained show, that antigens present in the pulpal room provide a strong immunogenic stimulus.