ANTIBODIES TO TYPES I, 11, IX, AND XI COLLAGEN IN THE SERUM OF PATIENTS WITH RHEUMATIC DISEASES

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1 325 ANTIBODIES TO TYPES I, 11, IX, AND XI COLLAGEN IN THE SERUM OF PATIENTS WITH RHEUMATIC DISEASES GUY LENE CHARRIERE, DANIEL J. HARTMANN, ERIC VIGNON, MARIE-CLAIRE RONZIERE, DANlEL HERBAGE. and GERARD VlLLE Antibodies to native types I, 11, TX, and XI collagen were measured, using a L51-solid-phase radioimmunoassay, in serum from 104 patients with rheumatic diseases (rheumatoid arthritis, osteoporosis, Paget s disease, or osteoarthritis). In all disease groups, to type 11 collagen occurred with greater frequency than to type I collagen ( versus 5-23%). Antibodies to type XI collagen were the most frequent: They were present in -50% of the patients in the rheumatoid arthritis, Paget s disease, and osteoporosis groups. Antibodies to type IX collagen were found at a high frequency in the rheumatoid arthritis group only (44%). Analysis of the clinical data suggested that the presence of to collagen was associated with disease that was less severe or of shorter duration. A pathogenic role of collagen autoimmunity in rheumatoid arthritis (RA) was first suggested based on the demonstration of collagen in patients serum and synovial fluid (1,2). This hypothesis was later supported by the observation that native type I1 collagen, the major collagen component of cartilage, is arthritogenic when injected into rodents (3). Furthermore, this experimental arthritis is associated with From the Centre de Radioanalyse, lnstitut Pasteur; the Clinique de Rhumatologie, HBpital Edouard Herriot; and the Laboratoire d Histologie experimentale. UniversitC Claude Bernard, Lyon, France. Supported by grants from CNAMTS and CNRS. Guylene Charrikre: Predoctoral Student, Centre de Radioanalyse; Daniel J. Hartmann. PhD: Centre de Radioanalyse; Eric Vignon. MD: Clinique de Rhumatologie: Marie-Claire Ronziere, PhD: Laboratoire d Histologie expkrimentale; Daniel Herbage. PhD: Laboratoire d Histologie experimentale: Gerard Ville. PhD: Centre de Radioanalyse. Address reprint requests to Guylene Charriere, Centre de Radioanalyse, rue Domer, Lyon cedex 07, France. Submitted for publication February 9, 1987; accepted in revised form September both cellular and humoral immunity to type I1 collagen (4) and can be passively transferred with purified anticollagen IgG (5). Subsequently, several reports indicated elevated serum levels of to types I and 11 collagen in a variety of rheumatic diseases, including RA (64, relapsing polychondritis (9, lo), ankylosing spondylitis (9, I I), gout (6), juvenile rheumatoid arthritis (l2,13), systemic lupus erythematosus (14), and Dupuytren s disease (15). Recently, new collagens in cartilage, types IX and XI (la, 2a, and 3a chains) collagen, called minor collagens, have been described (16-18). Type IX collagen was demonstrated to be a potent immunogen in animals (l8,19), and type XI collagen, like type 11 collagen, appeared to be arthritogenic in rodents (20). We therefore investigated whether to these 2 collagens could also be implicated in rheumatic diseases in humans. We previously developed a solidphase radioimmunoassay (RIA) for the detection of serum IgG to various collagen types (21,22); this RIA was used to study patients with RA, osteoarthritis (OA), osteoporosis (OP), and Paget s disease (PD). The results obtained with types 1X and XI collagen were compared with those obtained with the more frequently studied types I and 11 collagen. PATIENTS AND METHODS Patients and controls. The 104 patients included in this study were attending the Clinique de Rhumatologie, HBpital Edouard Herriot, Lyon. Thirt y-one patients had definite or classic RA, according to the American Rheumatism Association criteria (23). There were 22 women and 9 men, with a mean age of 60 years (range 35-82) and a mean disease duration of 12 years (range 1-39). Each patient s clinical and radiologic characteristics were assessed using the Steinbrocker criteria (24), and the results of tests for erythrocyte sedimentation rate (ESR), rheumatoid factor (RF; by latex fixation and Rose-Waaler tests), and antinuclear (ANA) were recorded. Arthritis and Rheumatism, Vol. 31, No. 3 (March 1988)

2 326 CHARRIERE ET AL There were 31 patients (23 women and 8 men) with OP. Their mean age was 64 years (range 31-83), and mean disease duration was 3.8 years (range 1-19). In all cases, diagnosis had been made after iliac crest bone biopsy (performed by Dr. P. J. Meunier). The presence or absence of OA had been investigated and recorded for each patient. Twenty-four patients (13 women and 11 men) had PD for a mean of 10 years (range 3-33). Their mean age was 66 years (range 49-82). All patients had high levels of serum alkaline phosphatase and urinary hydroxyproline. In this group, as in the OP patients, the presence or absence of OA was investigated and noted. Eighteen patients (12 women and 6 men) had primary OA. Their mean age was 63 years (range 43-82), and their mean disease duration was 6 years (range 1-15). The disease stage was evaluated according to the number of involved joints (1 point for each peripheral joint and 1 point each for the cervical, dorsal, and lumbar spine, with a maximum possible score of 19 points). The radiologic stage was recorded as I if the joint space narrowing was incomplete and 2 if it was complete. Forty-four healthy blood donors (24 women and 20 men), with a mean age of 48 years (range 18-61), were used as normal controls. All sera were aliquoted and frozen immediately after collection and were stored at -20 C. s. Acid-soluble type I collagen (CI) and pepsin-soluble type I1 collagen (CII) were extracted and purified from human skin and from fetal cartilage, respectively, as previously described for bovine tissues (25). Bo- f AMOUNT OF INHIBITOR (pg) 1bo * Figure 1. Specificity of the solid-phase radioimmunoassay for the detection of to collagen. Shown are the results of a typical inhibition experiment using a serum known to be reactive with type 11 collagen (CII) tested after preincubation for 18 hours at 4 C with CII (*), type 1 collagen (O), fibronectin (A), or human serum albumin (*). Table 1. Specificity of the solid-phase radioimmunoassay for the detection of to collagen CI1-positive serum, % binding of 1251-protein A* Assayed After preincubation Tested fraction directly with 25 pg of CII Whole serum 55 Purified IgG 51 IgG-free serum 2.0 * C11 = type I1 collagen. 8. I I vine minor collagens type IX (CIX) and type XI (CXI) were isolated from fetal calf cartilage as previously described (18,26). After salt fractionation and purification by chromatography on a DEAE cellulose column under native conditions, the type IX collagen fraction consisted essentially of the high molecular weight fragments (called XI, X2, and X3 in our laboratory [ 18,261) and type XI collagen consisted of the typical 3 chains (la, 2a, and 3a), in a relative proportion of 1:l:l. These collagens were investigated by amino acid analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (18,26). Solid-phase RIA for to collagens. The RIA was performed as previously described (21,22), with some modifications. Briefly, 5 pg of collagen in O.OSM phosphate buffer, 0.15M NaCI, ph 7.6 (phosphate buffered saline; PBS) was incubated overnight at 4 C in polystyrene microplates with removable wells (M17912B, Removawell; Dynatech, Alexandria, VA). On the next day, after 3 washes in PBS, the residual binding sites on the surface of the wells were saturated by contact with 200 p1 of PBS containing 1% bovine serum albumin (PBS-BSA) for 1 hour at room temperature. After removal of the PBS-BSA, 200 pl of human serum diluted 1 : 100 in PBS-BSA was added to the coated wells and incubated overnight at 4 C. Unbound serum was removed by washing 3 times with 200 pl of PBS, and then 200 pl of rabbit IgG specific for human IgG chains, diluted 1 : 1,000 in PBS-BSA, was added and incubated for 6 hours at 4 C. After 3 more washes in PBS, constant amounts of 125 I-protein A (0.03 pg; 50 x lo3 counts per minute) in 200 pi of PBS-BSA were added and incubated overnight at 4 C. After 3 final washes in PBS, the radioactivity bound to the wells was measured in a gamma counter (Multigamma 1260; LKB, Bromma, Sweden). All runs included determinations of the total radioactivity and the nonspecific binding of protein A to coated wells containing PBS-BSA instead of serum. This nonspecific binding was -1% of the total radioactivity, and for all experimental tubes, was subtracted before calculations were made. Sera from the 104 patients were tested in duplicate at a 1:IOO dilution. The results are expressed as percentage of total radioactivity after subtracting the mean of the percentages obtained with the 44 control sera, assayed in parallel (B/T). Samples with a B/T more than 2 standard deviations above the mean in control sera (i.e., >8%) were considered positive for to a given collagen type. Three classes, positive, highly positive, and very highly positive, were defined as follows: positive = B/T 9-28%; highly

3 COLLAGEN ANTIBODIES 327 Table 2. Frequencies of collagen in rheumatic disease patient groups* % with Any of Patient group (n) CI CII CIX CXI the 4 RA (31) OP (31) PD (24) OA (18) 5 I I * CI = type I collagen; CII = type I1 collagen; CIX = type 1X collagen; CXI = type XI collagen; RA = rheumatoid arthritis; OP = osteoporosis; PD = Paget s disease; OA = osteoarthritis. positive = B/T 29-48%; and very highly positive = B/T 349%. The intra- and interassay coefficients of variation were <8% and <15%, respectively. Inhibition tests. Aliquots of a serum known to be reactive with CII were incubated overnight with either CII, CI, or unrelated molecules, such as fibronectin or human serum albumin. The first incubated serum was then added to CII-coated wells, and the RIA was continued as described above. Results are expressed as percentage of inhibition, which was calculated as follows: cpm of serum alone - (cpm of serum + inhibitor) cpm of serum alone x 100 Similar experiments were carried out using sera known to be reactive with the 5 other types of collagen. Assay with the total IgG fractions of positive sera. To determine whether positive assay results reflected the inter- ference of nonspecific serum factors, the total IgG fractions from selected test sera were isolated by DEAE chromatography after desalting on Trisacryl GF05 (IBF, Villeneuve La Garenne, France) (27). Purified IgG preparations were reconstituted to approximate the original serum IgG concentration and were then used in the RIA in the same manner as was whole serum. The remaining IgG-free serum fractions were also assayed. Statistical analysis. Analysis of the differences in the biologic and clinical features between the study groups was performed by Student s t-test and chi-square analysis. RESULTS Specificity of the solid-phase RIA for to collagen. Results of inhibition tests. Prior incubation with CII strongly inhibited the binding of a serum that was positive for this type of collagen (Figure 1). In contrast, incubation with another type of collagen or with fibronectin or human serum albumin did not affect the results (Figure 1). Similar results were obtained with sera that were reactive with the 3 other types of collagen (data not shown). Results of assay with the total IgG fractions of positive sera. IgG purified from sera known to be reactive with CI1 was assayed directly or after incubation with 50 pg of collagen. Purified IgG tested directly yielded identical results as whole serum, whereas, with IgG-free serum fractions, almost no binding of protein A was obtained (Table 1). Preincubation of purified IgG with collagen inhibited its binding to the solid phase. m m m Figure 2. Serum levels of IgC antibody to types I, 11, IX, XI collagen (CI, CII, CIX, and CXI, respectively) in patients with rheumatoid arthritis (O), Paget s disease (m), osteoporosis (A), or osteoarthritis (*). Solid horizontal and vertical lines show the mean? 1 SD; dashed line shows the upper limit of normal; values in shaded boxes and open boxes are the number of positive sera and the number of negative sera, respectively, for each collagen type. B/T = percentage of total radioactivity (see Patients and Methods).

4 328 CHARRIERE ET AL c I1 N P HP VHP Figure 3. Correlation between serum levels of antibody to type I collagen (CI) and type II collagen (CII) in patients with rheumatoid arthritis (0). Paget s disease (a), osteoporosis (A), or osteoarthritis (*). N = negative; P = positive; HP = highly positive; VHP = very highly positive.. The mean 2 SD percentages of binding to the various collagens obtained with the 44 normal sera were 6.5 & 4.0 for CI, 7.6? 5.2 for CII, for CIX, and for CXI. These mean percentages were subsequently subtracted from those obtained with the patient sera (see above), in order to make meaningful comparisons between results obtained with the different collagen types. Antibodies to types I and II collagen. The 104 patient sera were investigated for the presence of to types I and 11 collagen. In all 4 disease groups, to type 11 collagen were more frequent than to type 1 collagen: 29% versus 13% in the RA and PD groups and 35% versus 23% in the OP group. The lowest frequencies of to types 1 and 11 collagen, 5% and 11%, respectively, were observed in the OA group (Table 2). The levels of IgG to CI and CII, measured in positive sera, are shown in Figure 2. For to both C1 and CII, the mean binding of protein A (expressed in B/T) was -25%, with a range of 8-58% for CI and 8-63% for C11. The correlation between the levels of CI antibody and CII antibody is shown in Figure 3. Thirteen patients had both kinds of. Sixteen patients had to CII and no to C1; in contrast, only 2 patients had to C1 (slightly positive) and no to CII. Antibodies to minor cartilage collagens. Antibodies to types IX and XI collagen were measured in 81 sera and 89 sera, respectively. As was found with Cl and CIl, to minor collagens were present in only a few sera from OA patients (12% for both CIX and CXl) (Table 2). Antibodies to CIX were found in 44% of the patients with RA, but in only 14% of the patients with PD and 17% of the patients with OP (Table 2). Antibodies to CXI were the most frequent c XI c I X c I1 CI A B Figure 4. Correlation between serum levels of antibody to type 11 collagen (CII) and type XI collagen (CXI) (A) or type 1X collagen (CIX) (B) in patients with rheumatoid arthritis (O), Paget s disease (m), osteoporosis (A), or osteoarthritis (*). N = negative; P = positive; HP = highly positive; VHP = very highly positive.

5 COLLAGEN ANTIBODIES 329 Table 3. Clinical and laboratory features in 31 patients with rheumatoid arthritis, by presence or absence of collagen * n Males/females Age, mean t SD (range) Years of disease, mean? SD (range) Radiologic stage, 1-2/3-41 Clinical grade, 1-2/3-4t ESR. mean f SD (range) RF-positive Rose-Waaler Latex fixation AN A-positive present 17 7/ (35-82) (1-20) 7/10 1 l/6 SO rt 26 (5-100) absent 14 2/ (37-80) IS 2 10 (1-39) 2/ t 35 (5-138) * ESR = erythrocyte sedimentation rate (mdhour); RF = rheumatoid factor; ANA = antinuclear. t By Steinbrocker criteria (24). and were present in nearly I of 2 patients in the above 3 groups. In positive sera, the mean level of to both CIX and CXI, expressed in B/T, was 15%, ranging from 8% to 33% for CIX and from 8% to 45% for CXI (Figure 2). The correlation between the levels of to the major cartilage collagen, type 11, and the minor types, IX and XI, is represented in Figure 4. Seventeen patients had to both CII and CXI. Twenty-one patients had to CXI and no to CII, whereas only 3 had to CII and no to CXI. Only 8 patients had to both CII and CIX, whereas 12 patients (7 of whom had RA) had to CIX alone and 5 patients had to C11 and no to CIX. Clinical correlations. Statistical analysis was performed to examine whether to any of the 4 collagen types were associated with any particular clinical or laboratory features of the diseases. Among Table 4. Clinical features in 31 patients with osteoporosis, by presence or absence of collagen present absent n Maledfemales 5/ Age, mean 2 SD 63? 14 64? 11 (range) (31-83) (40-79) Years of disease, mean f SD t 5.6 (range) (1-6) (1-19) Osteoarthritis present 8 4 Table 5. Clinical and laboratory features in 24 patients with Paget s disease, by presence or absence of collagen present absent n 15 9 Males/females Age, mean 2 SD 67 t II 66? 9 (range) (49-82) (53-76) Years of disease, 8.9 f t 12 mean t SD (range) (3-17) (3-33) Osteoarthritis present 9 4 Urinary hydroxyproline, mg/24 hours, 72 t 23* 109 i 58 mean? SD (range) ( ) (44-174) Alkaline phosphatase, unitsfliter, 240 f t 270 mean t SD (range) (42-550) (98-850) * P < 0.05 versus patients without collagen. RA patients, there was no significant correlation with age, sex, ESR, or presence of RF or ANA (Table 3). However, among patients who had collagen, the mean duration of the disease was generally shorter and the radiologic and clinical findings showed less severity, though these differences did not reach statistical significance. No correlation was found between collagen and clinical features of OP (Table 4). Among PD patients, significantly lower levels of urinary hydroxyproline were correlated with the presence of collagen (Table 5). OA patients with collagen generally had fewer involved joints than those without collagen, but the difference was not statistically significant (Table 6). DISCUSSION There is evidence of the presence of auto to collagen in sera from patients with rheumatoid arthritis. Various immunologic methods have been used to detect these, including passive hemagglutination ( I), immunofluorescence (lo), or im- Table 6. Clinical features in 18 patients with osteoarthritis, by presence or absence of collagen present absent n 5 13 Males/females I Age, mean t SD t 14 (range) (43-75) (44-82) No. of involved joints, mean t SD 1.6 t (range) (1-4) (1-13) Radiologic stage, 1/2* 4/ 1 4/6 * See Patients and Methods.

6 330 CHARRIERE ET AL El 2 a H 3: 5 d t 100 w 50 u z W V c: ill a 0 A B t 50 u d z w u a W a AMOUNT OF INHIBITOR (Ug) AMOUNT OF INHIBITOR (Ug) Figure 5. Specificity of to type 11 collagen (Cll) and type XI collagen (CXI). Shown are the results of an inhibition experiment using a CII-reactive serum preincubated with free CII (*) or free CXI ( +) (A) and a CXI-reactive serum preincubated with free CXI (+ ), free C11 (*), or free type V collagen (m) (B). munoenzymatic techniques such as enzyme-linked immunosorbent assay (28) and liquid- (29) or solid-phase (30) radioimmunoassays. These differences in the methodology used have led to great variation in the reported incidence of to collagen. Moreover, some of these techniques could give falsepositive results due to interference of fibronectin or other cryoglobulins (31) or to nonspecific adsorption of IgG in solid-phase assays (8). In this study, we used a solid-phase RIA with 251-protein A, which has been previously developed and validated for measuring IgG to collagen in antisera raised in animals (21,22). This method is not dependent on nonspecific serum factors such as fibronectin, as shown by the fact that the anticollagen reactivity of a positive serum was recovered in the purified IgG fraction and was completely absent in the IgG-depleted one. The inhibition experiments showed that a specific collagen-antibody interaction was measured by this assay. Normal sera yielded a low but significant level of binding, which varied with the type of collagen, as has been previously described (6,8,32). This binding could be attributed to nonspecific factors inherent in the assay system or, more probably, to the existence of natural to collagen that might result from dietary ingestion of collagens and collagen-like molecules, as was recently suggested (33). This level of collagen did not correlate with age, either in the normal subjects or in the patients studied. In this study of the occurrence of to native types I and I1 collagen in 104 patients with rheumatic diseases, we observed a higher frequency of to CII than to CI in all 4 disease groups. Of the 29 sera that were positive for CII, only 13 were simultaneously positive for C1. In contrast, the presence of to CI was almost always associated with the presence of to CII (only 3 of 16 sera were positive for CI alone). These results address the question of whether sera that are positive for both collagen types contain 2 separate antibody populations or whether are crossreactive. Inhibition studies performed on a few sera exhibited type-specific, but further experiments are necessary to investigate whether this is a general finding in all disease states. The increased prevalence of to type 11 collagen as compared with type I collagen in rheumatoid arthritis patients is consistent with the findings of experiments with collagen-induced arthritis which demonstrate that CI1 is arthritogenic in rodents and CI is not (3,34). However, as has been reported in other studies (6,11,35), our data indicate that to type I1 collagen are not confined to patients with RA; we found a comparable frequency in patients with PD and OP. This unexpected occurrence of to cartilage collagen in patients with bone diseases does not seem to be due to accompanying osteoarthritis since, in patients with OA alone, we found the lowest frequency of to all collagen types. This

7 COLLAGEN ANTIBODIES 33 1 finding was also reported by Meyer et al (9). Therefore, the significance and the possible pathogenic role of to CII in PD and OP remain unexplained. There are no previously published reports of investigations of to minor cartilage collagens in humans. In this study, we found that the occurrence of to type XI collagen (la, 2a, and a chains) was frequent in rheumatic disease patients; these were detected in 42% of the patients with RA or OP and 52% of the patients with PD. One of 2 patients with to CXI had no to CII, whereas, the presence of to CII was almost always associated with the presence of to CXI. Although the a,(ii) chain of CII is similar to the 3a chain of CXI (16), the presence of to CII was apparently not due to a potential recognition of epitopes common to these 2 collagens, since the binding of CII-positive sera to CII-coated wells was inhibited by incubation with free CII but not with CXl (Figure 5A). Moreover, type XI collagen has 2 other chains (la and 2a) which are closely related to the ai and a2 chains of type V collagen (36,37). The positivity observed with type XI collagen was specific, however, since it was inhibited by free type XI collagen but not type I1 or type V collagen (Figure 5B). As was the case for type I1 collagen, to CXI were not specific for RA and were found at a similar percentage in all the disease groups. Antibodies to type 1X collagen differed from to other collagen types in the diseases studied in that they were more frequent in RA patients, 44% of whom had positive results. There was no particular association between the presence of to type IX collagen and the presence of to type 11 collagen. The increase of to ClX in RA patients might reflect a particular role of this collagen type in the pathogenesis of cartilage destruction during the inflammatory process. In fact, it was recently discovered that type IX collagen can stimulate the production of prostaglandin E, and interleukin-1 by monocytes and that it is 2-4-fold more potent than other collagen types (e.g., type I, 11, or XI) (38). Analysis of the patients' clinical features showed few differences between those with and those without elevated serum levels of to collagens. In RA, notably, we failed to demonstrate a correlation between collagen and disease activity as measured by ESR or RF. However, we found that these were generally present in RA of shorter duration or less severity and in OA that was more localized, and they were significantly associated with a slower turnover of collagen in PD. Thus, to collagens seem to be associated with a less severe or an early form of the disease. This is consistent with a recent report by Pereira et a1 (39), who found that collagen present in RA patients whose disease was at a very early stage were often no longer detectable when reassayed at a later date. Further long-term studies on patients, beginning very early in the course of the disease, will be required to assess the clinical significance of these. REFERENCES 1. Andriopoulos NA, Mestecky J, Miller EJ, Bennett JC: Antibodies to human native and denatured collagens in synovial fluids of patients with rheumatoid arthritis. Clin Immunol Immunopathol6: , Menzel J, Steffen C, Kolarz G, Eberl R, Frank 0, Thumb N: Demonstration of to collagen and of collagen-anticollagen immune complexes in rheumatoid arthritis synovial fluids. Ann Rheum Dis 35: , Trentham DE, Townes AS, Kang AH: Autoimmunity to type I1 collagen: an experimental model of arthritis. J Exp Med 146: , Stuart JM, Townes AS, Kang AH: Nature and specificity of the immune response to collagen in type I1 collagen-induced arthritis in mice. J Clin Invest 69: , Kenvar SS, Oronsky AL: Type I1 collagen induced arthritis in rats: clinical and immunological studies. Int J Tissue React 7: , Stuart JM, Huffstutter EH, Townes AS, Kang AH: Incidence and specificity of to types I, 11,111, IV, and V collagen in rheumatoid arthritis and other rheumatic diseases as measured by '*'I-radioimmunoassay. Arthritis Rheum 26: , Gioud M, Monier JC, Bonvoisin B, Meghlaoui A, Venet C, Lejeune E, Bouvier M: Auto-immunite anticollagene humorale et cellulaire en pathologie rhumatismale. Rev Rhum Ma1 Osteoartic , Clague RB, Firth SA, Holt PJL, Skingle J, Greenbury CL, Webley M: Serum to type I1 collagen in rheumatoid arthritis: comparison of 6 immunological methods and clinical features. Ann Rheum Dis , Meyer 0, Semmache M, Cyna J, Mitrovic D, Ryckewaert A: Les anticorps anticollagene: detection au cours de la polyarthrite rhumatoide, de la polychondrite chronique atrophiante et de divers rhumatismes inflammatoires chroniques. Rev Rhum Ma1 Osteoartic 50: , Ebringer R, Rook G, Swana GT, Bottazzo GF, Doniach D: Auto to cartilage and type I1 collagen in relapsing polychondritis and other rheumatic diseases. Ann Rheum Dis 40: , Clague RB, Shaw MJ, Holt PJL: Incidence of serum to native type I and type I1 collagens in

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