Alexander S. Townes

Humoral and Cellular Sensitivity to Collagen in Type II Collagen-Induced Arthritis in Rats

We have recently described a new animal model of arthritis induced by intradermal injection of a distinct type of collagen found in cartilage (type II collagen). Since immunologic sensitivity to collagen could play a role in the pathogenesis of this type II collagen-induced arthritis in rats, the ability of purified types of native collagens to induce cellular and humoral responses was quantified by antigeninduced tritiated thymidine incorporation into lymphocytes by collagen and passive hemagglutination, respectively. Rats injected intradermally with native heterologous or homologous type II collagens in adjuvant developed type-specific cellular as well as humoral reactivity. Types I and III collagens were less immunogenic than was type II. The latter collagen induced brisk cellular and humoral responses that were equivalent whether complete Freunds adjuvant or incomplete Freunds adjuvant were employed. Both responses could be induced by native type II collagens modified by limited pepsin digestion, indicating that they are not attributable to determinants in the telopeptide regions of the molecule. Thus, these studies demonstrate the unique immunogenic as well as arthritogenic properties of the type II collagen molecule and indicate that both result from a helical conformation of its structurally distinct alpha-chains. Further, they suggest that type II collagen may, by humoral or cellular mechanisms, provoke or perpetuate inflammation in other arthritic diseases.

Nature and specificity of the immune response to collagen in type II collagen-induced arthritis in mice.

To determine the role of collagen-immunity in the development of collagen-induced arthritis, DBA/1 mice were immunized with type II collagen and observed for the development of polyarthritis. 96% of the mice immunized with native type II collagen developed inflammatory arthritis between 4 and 5 wk after primary immunization. Immunization with denatured type II collagen in exactly the same manner was not effective in inducing arthritis. Cell-mediated immunity in arthritic mice was assessed by measuring [3H]thymidine incorporation by mononuclear cells cultured in the presence of collagen. The maximal proliferative response to collagen occurred at 2 wk after immunization. Equally good incorporation of label occurred when cells were cultured with native or denatured type II collagen or type I collagen. The cellular response of nonarthritic mice immunized with denatured collagen was indistinguishable from that seen in arthritic mice. Humoral immunity was assessed by an ELISA assay for antibodies to collagen. The immunoglobulin M (IgM) response peaked at 2 wk and the IgG response at 5 wk after immunization. Antisera from arthritic mice immunized with native type II collagen were relatively specific for conformational determinants on the native type II molecule although some reactivity with denatured collagen was noted. Antisera from nonarthritic mice immunized with denatured collagen primarily recognized covalent structural determinants. It was concluded that native type II collagen was essential for the induction of arthritis and that an antibody response specific for native type II collagen may be important for the development of arthritis.

Cell-mediated immunity to collagen and collagen α chains in rheumatoid arthritis and other rheumatic diseases

Peripheral blood mononuclear cells from patients with rheumatoid arthritis, gout, ankylosing spondylitis and degenerative joint disease were cultured in the presence of native types I, II and III collagens and alpha chains from each of these types of collagen. The culture supernatant fluids were harvested and assayed for lymphocyte-derived chemotatic factor for monocytes. Reactions to one or more of the native collagens was found in 50 per cent (10 of 20) of the patients with rheumatoid arthritis, 20 per cent (two of 10) of the patients with gout and ankylosing spondylitis but in none of the 10 patients with degenerative joint disease or in normal subjects. Reaction to one or more alpha chains was found in 90 per cent (18 of 20) of the patients with rheumatoid arthritis, 60 per cent (six of 10) of the patients with gout, 50 per cent (five of 10) of the patients with ankylosing spondylitis, 30 per cent (three of 10) of the patients with degenerative joint disease and in 10 per cent of the normal subjects (one of 10). All the reactions were quantiatively stronger in patients with rheumatoid arthritis. These results indicate that patients with rheumatoid arthritis have cell-mediated immunity to homologous native and denatured collagens but that the reaction is not specific for rheumatoid arthritis. Some patients with gout, ankylosing spondylitis and degenerative joint disease also have low levels of immunity.

Performance of a rosette assay between lymphocytes and sheep erythrocytes at elevated temperatures to study patients with cancer and other diseases

Abstract When the rosette assay between T lymphocytes and sheep erythrocytes was performed at elevated temperatures, significant differences were observed between the proportion of rosette-forming cells (RFC) of normal adults and those from patients with cancer. At 20°C, eight of ten cancer patients had normal levels of RFC. At 29 and 33°C, nine of those ten patients had rosette levels significantly below the normal mean. In a study of nine patients with systemic lupus erythematosus, discrimination between patients and controls was also greater at higher temperatures. A rosette assay was then performed at 29°C with blood specimens from 50 normal controls, 50 patients with malignant disease, and 100 individuals with a variety of benign diseases. Forty-one of 50 patients with cancer had significantly depressed proportions of rosette-forming cells at 29°C when compared to normal volunteers (mean of 38 ± 10% vs 54 ± 5%). Low percentages of 29°C RFC were also observed with viral infection, alcoholic liver disease, and in patients immediately following myocardial infarction or surgery. In contrast, the percentages of 29°C RFC were normal in 57 of 60 patients with other benign diseases. These studies suggest an alteration of T-cell receptors for sheep red blood cells in cancer and certain other diseases resulting in decreased rosette cohesiveness at elevated temperatures.

Impaired cell-mediated immunity in systemic lupus erythematosus (SLE): A controlled study of 23 untreated patients☆

Cell-mediated immunity was evaluated in 23 patients with systemic lupus erythematosus (SLE) prior to therapy and in 23 control subjects. The patients with SLE who had moderate to severe disease activity had significantly fewer positive delayed skin tests to streptokinase-streptodornase (SK-SD) and Candida than the control subjects, and a higher frequency of anergy than either the control subjects or the patients with mild SLE. Significant impairment of lymphocyte transformation to all common antigens tested was found in patients with SLE as compared to both normal subjects and control subjects with disease. Phytohemagglutinin response was reduced in patients with SLE as compared to normal subjects but not to the control subjects with disease. Lymphocyte transformation responses to SK-SD and Candida were also significantly lower in patients with moderate to severe SLE as compared to patients with mildly active SLE. Primary immune response to keyhole limpet hemocyanin (KLH) was impaired in patients with SLE as measured by lymphocyte transformation and total KLH antibody, but not 2-mercaptoethanol resistant antibody. The data indicate defective T-cell function in SLE, and suggest that the impairment relates in part to disease activity.

Physicochemical and Immunological Studies of the Renatured α1(II) Chains and Isolated Cyanogen Bromide Peptides of Type II Collagen

The alpha 1(II) and cyanogen bromide (CB)1-generated peptides of chick type II collagen were isolated, purified, renatured and examined for their physicochemical and immunological properties. The alpha 1(II) chains and peptides CB-6 through CB-12 (3,000 to 40,000 daltons) formed renatured thermostable products as determined by measurements of reduced viscosity, optical rotation and Stokes radius. Moreover, renatured alpha 1 (II) chains and CB-10 were observed to form segment-long-spacing (SLS) crystallites under appropriate conditions. When examined for immunoreactivity with defined rat polyclonal and mouse monoclonal antibodies to chick type II collagen, conformation-dependent epitopes were detected on renatured alpha 1(II) chains and renatured peptides, CB-8, CB-10 and CB-11. Conformation-independent epitopes were also detected on all CB-peptides in their denatured form. These studies demonstrate that the alpha 1 (II) chains and CB-peptides of chick type II collagen can be efficiently renatured and that the renatured products retain some conformation-dependent epitopes present on the naive molecule.