Collagen antibodies in rheumatoid arthritis. Significance of antibodies to denatured collagen and their association with HLA-DR4

The frequency, specificity, and HLA associations of antibodies to collagen were examined in 54 patients with severe rheumatoid arthritis (RA) and in 67 control subjects, using native and denatured bovine type II collagen as reactants in a solid-phase radioimmunoassay. Reactivity to denatured collagen was significantly higher in the RA patients than in the controls (P = 0.004). Reactivity to native collagen was substantially lower than reactivity to denatured collagen and was similar in RA patients and controls. DR4 positive RA patients had significantly greater reactivity to denatured collagen compared with DR4 negative RA patients (P = 0.03), but levels of antibody to native collagen were similar among DR4 positive and DR4 negative patients. These data lend support to the idea that denatured collagen is an important secondary reactant in immune-mediated perpetuation of RA.     

Antibodies to denatured type II collagen in rheumatoid arthritis: negative association with IgM rheumatoid factor

Serum samples from 129 patients with definite or classic rheumatoid arthritis (RA) were assayed by ELISA for antibodies to denatured bovine type II collagen (dII). All patients had active disease at the time of serum sampling. Anti-dII antibodies were found in 18 (14%) of 129 patients (95% confidence intervals: 8-20%). The only clinical or laboratory feature associated with the presence of anti-dII antibodies was seronegativity for IgM rheumatoid factor (IgM RF): 6 (37.5%) of 16 seronegative patients had anti-dII antibodies vs 12 (10.6%) of the 113 seropositive patients (OR = 5, p less than 0.01). There were no associations of anti-dII antibodies with age, sex, race, disease activity, disease duration, functional class, or the presence of HLA-DR1, DR4, or DQw3 in these patients. Antibodies to type II collagen may have a pathophysiologic role in RA, especially in patients seronegative for RF.     

Lymphocyte transformation to denatured type i collagen and b lymphocyte alloantigens in rheumatoid arthritis

Peripheral blood lymphocytes transformed significantly to denatured type I collagen in 22 of 38 patients with rheumatoid arthritis (58%), 1 of 9 patients with Reiter's disease (P < 0.05), and 9 of 32 controls (28%) (P < 0.01). Determination of HLA antigens revealed significant differences between 24 rheumatoid arthritis (RA) patients and a control population for HLA-DRw4 (75% versus 23%). No correlation was observed between lymphocyte transformation to collagen and antibodies to collagen, rheumatoid factor, stage or duration of RA, or HLA antigens.     

Specificity of antibodies to type II collagen in rheumatoid arthritis.

To reassess the role of autoantibodies to type II collagen in the pathogenesis of diseases, we studied antibodies from patients with rheumatoid arthritis (RA) and from patients with relapsing polychondritis for species specificity and collagen type specificity, using an improved enzyme-linked immunosorbent assay. Antibodies were found in the sera of 15% of the RA patients and 50% of the relapsing polychondritis patients, as well as in the cartilage of 69% of the RA patients examined. Reaction with both homologous and heterologous type II collagens was common. Analysis of 19 selected RA sera revealed that autoantibodies were generally associated with specific antibodies to some species of heterologous type II collagen. In contrast, antibodies found in 4% of the non-RA controls were specific for either bovine or chick type II collagen. These findings indicate that autoantibody formation in RA and relapsing polychondritis may occur as a result of an immune response to heterologous type II collagen. However, since RA and relapsing polychondritis patient sera differed in their reactivity with the cyanogen bromide-digested peptides, it is possible that the clinical manifestation of collagen autoimmunity might be influenced by the epitope specificity of the antibodies.     

Associations between HLA and antibodies to collagen in rheumatoid arthritis.

Associations between HLA types and serum antibodies to native and denatured type II collagen were sought in 105 patients with rheumatoid arthritis (RA). Antibodies were measured using a solid phase radioimmunoassay. There were no significant associations between any HLA antigen (A, B, or DR) and a high antibody titre to native collagen. There were significant associations, however, between HLA antigens and high antibody titres to denatured collagen. Although DR4 did not show an association, the phenotype A2+DR4+ did; this was not related to A2 as A2+DR- was not associated with a high antibody titre. No single B locus antigen showed an association, but several B locus antigens, B12, B15, and B40, were included in phenotypes with A2 and DR4 which were associated with a high antibody titre to denatured collagen. These HLA associations with anticollagen type II are best explained by a gene other than DR4 (but in linkage with it) which may regulate the antibody response to denatured collagen. If so, this would represent an HLA gene in addition to DR4 that is active in RA.     

Non-Hodgkin's lymphoma of the synovium simulating rheumatoid arthritis

Skeletal involvement in patients with non-Hodgkin's lymphoma (NHL) is common, although direct involvement of the joints is unusual. We describe 2 adults who presented with features suggestive of a diagnosis of rheumatoid arthritis, but who were found to have diffuse NHL of the synovium. Results of a review of the literature, and assessment of the few similar cases in which NHL presented in the joint, suggest that the lymphoma may mimic either a monarticular or polyarticular synovitis, without lymphadenopathy or hepatosplenomegaly. Radiographic demonstration of associated bone destruction is the best evidence for non-Hodgkin's lymphomatous arthropathy in patients with rheumatic symptoms.     

Evidence for the local production and utilization of immune reactants in rheumatoid arthritis

Immunoglobulins, including rheumatoid factors, are produced by the rheumatoid synovial membrane. A significant contribution of the synovial membrane to the total IgG and IgM detected in the synovial fluid has been documented. The present study was designed to examine the contribution of the synovial membrane to the rheumatoid factors detected in the synovial fluid. Analysis of the data demonstrated that the synovial membrane was the source of a significant component of the total synovial fluid IgA rheumatoid factor and IgM rheumatoid factor. While some fluids possessed extremely elevated concentrations of the IgG rheumatoid factor, the data suggested that IgG rheumatoid factor was preferentially reduced, relative to total IgG, by the rheumatoid inflammatory process. These observations suggest a potentially important role for IgG rheumatoid factor in rheumatoid synovitis.     

IgG antibodies to type II collagen reflect inflammatory activity in patients with rheumatoid arthritis

OBJECTIVE: To determine the clinical significance of IgG antibodies to type II collagen (CII) and to define any correlation of antibodies to CII with the inflammatory response in patients with rheumatoid arthritis (RA). METHODS: IgG antibodies to native human type II collagen (IgG anti-CII) were measured in sera and synovial fluid (SF) from patients with RA, patients with osteoarthritis (OA), and healthy controls by an improved ELISA. Demographic, clinical, and laboratory data including tumor necrosis factor-alpha (TNF-alpha) and interleukin 6 (IL-6) levels were also obtained at the time of sampling in patients with RA. RESULTS: The median level and positivity for circulating IgG anti-CII were higher in patients with RA (n = 297) than patients with OA (n = 34) and healthy controls (n = 50) (p < 0.001). The titers of IgG anti-CII in SF were also higher in RA (n = 45) than in OA (n = 16) (p < 0.001). In paired samples, the levels of IgG anti-CII were significantly higher in SF compared to the sera in patients with RA (n = 45) (p < 0.001), but levels were not different in patients with OA (n = 16). Circulating IgG anti-CII converted from positive to negative in 13 patients (10.7%) and from negative to positive in 18 patients (14.8%) among 122 patients with RA in whom IgG anti-CII were monitored sequentially at a mean interval of 12.2 months. IgG anti-CII positive patients (n = 98) had shorter disease duration (p = 0.04) and less frequent deformity (p = 0.013), and higher median erythrocyte sedimentation rate (ESR) (p = 0.001) and C-reactive protein (CRP) (p < 0.001) than IgG anti-CII negative patients (n = 120). The levels of IgG anti-CII correlated with CRP (r = 0.270) and ESR (r = 0.253). CRP decreased significantly in patients (n = 13) who converted from IgG anti-CII positive to negative (p = 0.013). IgG anti-CII positive patients (n = 40) had higher levels of TNF-alpha and IL-6 than negative patients (n = 40) (p < 0.001). Levels of IgG anti-CII correlated well with TNF-alpha (r = 0.617) and IL-6 (r = 0.347). CONCLUSION: Increased IgG anti-CII in sera and SF in RA correlated directly with acute phase reactants and the proinflammatory cytokines TNF-alpha and IL-6. Our data suggest that IgG anti-CII could reflect inflammatory activity with a potential to destroy cartilage in the early stages of RA.     

Expression of the apoptosis accelerator Bax in rheumatoid arthritis synovium

OBJECTIVE: The aim of this study was to analyze the expression of apoptosis-related molecules in rheumatoid arthritis (RA) synovium, with special emphasis on the apoptosis accelerator Bax. METHODS: Immunohistochemical analysis of Bax, Bcl-2, and Bcl-x(L) was performed in tissue specimens of patients with RA and compared to normal synovial tissue. Expression of Bax was additionally determined by double labeling with CD68, p53, and Ki-67 (clone MIB-1). Apoptotic cells were further identified by the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) method. RESULTS: In RA, expression of Bax was higher than in healthy controls and occurred in CD68-positive and -negative synoviocytes. Strong Bax staining was also found in chondrocytes at sites of cartilage degradation. Bax-positive synoviocytes could be detected with p53 and also with Ki-67. Bax and Bcl-x(L) were markedly colocalized in synovium. The TUNEL method revealed only few positive synoviocytes. CONCLUSIONS: The marked colocalization of Bax and antiapoptotic Bcl-x(L) as well as the low frequency of TUNEL-positive cells in RA synovium suggest that Bax activity is not sufficient to decrease synovial hyperplasia in RA. Apoptotic mechanisms in RA chondrocytes might also be important for the pathogenesis of joint damage.     

Incidence of antibodies to native and denatured cartilage collagens (types II, IX, and XI) and to type I collagen in rheumatoid arthritis.

The frequencies of antibodies to the cartilage type IX and XI collagens and to type I collagen were determined in 188 patients with rheumatoid arthritis, of whom 76 were positive for antibodies to native type II collagen. A higher proportion of patients with antibodies to native type II collagen had antibodies to these other collagens, but about one third of patients without antibodies to native type II collagen had antibodies to one or more denatured collagens. The patterns of antibodies present in individual sera suggested that there was a selective response to the collagens in an individual patient. The incidence of patients having antibodies to these native and denatured collagens in a random group of patients with rheumatoid arthritis was calculated.     

Demonstration of antibodies to collagen and of collagen-anticollagen immune complexes in rheumatoid arthritis synovial fluids

Menzel, J., Steffen, C., Kolarz, G., Eberl, R., Frank, O., and Thumb, N. (1976).Annals of the Rheumatic Diseases, 35, 446-450. Demonstration of antibodies to collagen and of collagen-anticollagen immune complexes in rheumatoid arthritis synovial fluids. Twenty-nine synovial fluids from patients with rheumatoid arthritis (RA) and 10 synovial fluids from patients with other joint diseases were investigated with regard to the presence of antibodies to denatured human collagen and of collagen-anticollagen immune complexes. 12 of the 29 RA synovial fluids showed anticollagen titres from 1: 16 to 1: 512 in passive haemagglutination. Only one patient in the group with no arthritis had a significant anticollagen titre of 1: 32. Digestion of the synovial fluids with bacterial collagenase resulted in an anticollagen titre increase from two to four dilution steps in 9 of the RA fluids, while 6 previously negative RA synovial fluids showed anticollagen titres from 1: 32 to 1: 128 after digestion with collagenase. These results indicate the existence of collagen-anticollagen immune complexes in 15 of the 29 RA synovial fluids investigated.     

Evidence for a functional role of IgE anticitrullinated protein antibodies in rheumatoid arthritis

Rheumatoid arthritis (RA) is a systemic autoimmune disease involving inflammation of the joints. Among the autoantibodies described in RA, anticitrullinated protein antibodies (ACPAs) are highly specific and predictive for RA. In addition, ACPAs have been implicated in the pathogenesis of RA. However, a direct functional response of immune cells from ACPA⁺ RA patients toward citrullinated proteins has not been demonstrated. In this study, we show that exposure to citrullinated antigens leads to activation of basophils from ACPA⁺ RA patients within 20 minutes. This was not observed after exposure of basophils to noncitrullinated control antigens or after stimulation of basophils from ACPA⁻ RA patients and healthy controls. Basophil activation was correlated with the binding of citrullinated proteins to basophils. Furthermore, serum from ACPA⁺ RA patients in contrast to that from ACPA⁻ RA patients could specifically sensitize human FcεRI expressing rat basophil cells (RBL), enabling activation by citrullinated proteins. Mast cell degranulation products such as histamine levels were enhanced in synovial fluid of ACPA⁺ RA patients as compared with ACPA⁻ RA and osteoarthritis patients. In addition, histamine levels in synovial fluid from ACPA⁺ RA patients correlated with IgE levels, suggesting degranulation of mast cells by cross-linking IgE. Immunohistochemistry on synovial biopsies demonstrated an increased number of degranulated CD117⁺ mast cells in ACPA⁺ RA patients; IgE and FcεRI expression in synovial mast cells from ACPA⁺ RA patients was increased. In conclusion, our results show an immunological response of immune cells from ACPA⁺ RA patients in a citrulline-specific manner. Moreover, these data indicate a role for IgE-ACPAs and FcεRI-positive cells in the pathogenesis of RA.     

Local production of complement proteins in rheumatoid arthritis synovium

OBJECTIVE: Complement has been repeatedly implicated in the pathogenesis of rheumatoid arthritis (RA) based on studies showing reduced levels of native complement components and increased levels of complement metabolites in plasma, synovial fluid (SF), and synovial tissue (ST) of RA patients. However, there is limited information on local production and activation of key factors of the complement cascade in RA synovium and their potential modulation by novel anticytokine therapies. This study was undertaken to characterize the expression of complement proteins and receptors in RA SF and ST. METHODS: Using in situ hybridization, immunohistochemistry, and Western blot techniques, we assessed the presence of complement proteins C3, factor B (FB), and C5b-9, as well as the expression of complement receptors C3aR and C5aR in rheumatoid synovium. C3 and FB levels in SF were determined by enzyme-linked immunosorbent assay. Functional assessment was performed by examining the effects of soluble tumor necrosis factor receptor (sTNFR) p55 gene transfer in the SCID mouse model of RA. RESULTS: Complement proteins and receptors could be localized in all RA synovial specimens, whereas in osteoarthritis (OA) synovium, only a few, single cells expressed complement proteins and receptors. No differences were noted in the concentration of C3 between RA and OA in SF; however, FB levels were markedly reduced in RA versus OA SF. In RA synovium, in contrast to OA synovium, local expression of complement factor and complement receptor messenger RNA was found throughout the various ST compartments, suggesting that activation of the complement cascade occurs in all parts of the rheumatoid synovium. Moreover, C5aR expression was up-regulated following overexpression of sTNFR p55 by adenovirus-based gene transfer. CONCLUSION: In summary, local complement production and activation may play an important role in RA, and specific modulation and inhibition of local complement production could be an attractive therapeutic target for RA.     

Epitope specificity of antibodies to type II collagen in rheumatoid arthritis and systemic lupus erythematosus

Summary Antibodies to human type II collagen were examined in the sera of 105 patients with rheumatoid arthritis (RA), 44 patients with systemic lupus erythematosus (SLE) and 11 patients who fulfilled the criteria of both diseases (RA-SLE overlap), using a solid-phase radioimmunoassay (RIA). The frequencies of antibodies to native and denatured human type II collagen were 20% and 27% in RA, 14% and 16% in SLE, and 45% and 36% in RA-SLE overlap. The specificity of the antibodies was further examined by inhibition with native and denatured type II collagen, by immunoblotting on native and denatured type II collagen, and by immunoblotting on cyanogen-bromide derived polypeptides of type II collagen. We could not identify any disease-specific patterns of reactivity. Thus, in the three disease groups the antibody response was polyclonal; there were antibody populations that reacted with native and/or denatured collagen, and epitopes could be assigned to at least three CB peptides, CB10.5, CB11 and CB8.     

Histological analysis of synovium by treatment of etanercept for rheumatoid arthritis

Aims: In order to investigate the histological change in effect attenuation cases of etanercept compared with methotrexate (MTX), we performed immunohistochemical examination by seven different molecules. Methods: We histologically assessed synovial tissue from five MTX‐treated rheumatoid arthritis (RA) patients as control and six etanercept and MTX‐treated RA patients after synovectomy by arthroscopy. The synovium of both groups were assessed by hematoxylin and eosin (HE) and we also analysed the expression of tumour necrosis factor‐α (TNF‐α), interleukin‐6 (IL‐6), matrix metalloproteinase‐3 (MMP‐3), B‐cell precursors and mature B‐cell transmembrane protein, CD20, macrophage marker, CD68, bromodeoxyuridine (BrdU) and vascular endothelial growth factor (VEGF) by immunohistochemistry. Results: HE staining showed vascular and cell proliferations of the synovium of the RA patients who received etanercept compared with the control MTX group. TNF‐α and IL‐6 were expressed in both groups.MMP‐3 and CD68 expressed less significantly in the etanercept group compared with the control (P < 0.05). CD20 strongly expressed in the etanercept group significantly (P < 0.05). BrdU expressed in the synovium in the etanercept group significantly (P < 0.05). VEGF was not found overall in both group. Conclusion: Based on the histological change of synovium, treatment by etanercept may be involved in vascular and cell proliferations with inhibition of the expression of CD68 and MMP‐3 in synovium of RA patients. These findings indicate immunohistochemical change of synovium with etanercept is one of the mechanism of efficacy of etanercept.     

Demonstration of antibodies to collagen and of collagen-anticollagen immune complexes in rheumatoid arthritis synovial fluids.

Twenty-nine synovial fluids from patients with rheumatoid arthritis (RA) and 10 synovial fluids from patients with other joint diseases were investigated with regard to the presence of antibodies to denatured human collagen and of collagen-anticollagen immune complexes. 12 of the 29 RA synovial fluids showed anticollagen titres from 1:16 to 1:512 in passive haemagglutination. Only one patient in the group with no arthritis had a significant anticollagen titre of 1:32. Digestion of the synovial fluids with bacterial collagenase resulted in an anticollagen titre increase from two to four dilution steps in 9 of the RA fluids, while 6 previously negative RA synovial fluids showed anticollagen titres from 1:32 to 1:28 after digestion with collagenase. These results indicate the existence of collagen-anticollagen immune complexes in 15 of the 29 RA synovial fluids investigated.