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COLLAGEN ANTIBODIES IN POLYCHONDRITIS - FOIDART ET AL.
supported by the fact that none of the patients in the study had responded to long-term therapy with full doses of topical medications such as nystatin or gentian violet. In our experience, no medication other than intravenous amphotericin B has produced such favorable results. Fifteen of the patients have continued to be observed since the completion of the blind phase of their trial. The follow-up duration is as follows: 10 months, one patient; nine months, nine patients; eight months, three patients; four months, one patient; and three months, one patient. All have received clotrimazole troches, since we were interested in determining (roughly) the minimal effective dose as well as the relative frequencies of drug resistance and relapse. Data accumulated thus far indicate that one to three troches daily are sufficient to prevent relapse. All the nine patients who were previously scored as failures while taking the placebo responded to clotrimazole; thus, none of the 20 patients studied harbored drugresistant organisms. Only one patient, a 44-year-old woman with denture stomatitis, has had a recurrence of noteworthy oral candidiasis. None of the remaining 14 patients have had any adverse effects of prolonged treatment. When treatment is stopped, however, symptomatic infections invariably recur within two to four weeks, presumably because the underlying defect
1203
that predisposes the patients to oral candidiasis has not been corrected. Thus far, we have not seen accompanying clinical responses in other infected sites such as the esophagus, vagina, nails or skin. We are indebted to the staff of the Clinical Center Pharmaceutical Development Services for assistance with the study. These studies were conducted in compliance with a protocol that had been reviewed and approved by the appropriate committees and persons of the National Institutes of Health.
REFERENCES 1. Kirkpatrick CH, Rich RR, Bennett JE: Chronic mucocutaneous candidiasis: model-building in cellular immunity. Ann Intern Med 74:955-978, 1971 2. Kirkpatrick CH, Smith TK: Chronic mucocutaneous candidiasis: immunologic and antibiotic therapy. Ann Intern Med 80:310-320, 1974 3. Bennett JE: Chemotherapy of systemic mycoses. N Engl J Med 290:3032, 320-323, 1974 4. Fredriksson T: Topical treatment with Bay b5097, a new broad spectrum antimycotic agent. Br J Dermatol 86:628-630, 1972 5. Zaias N: Topical treatment of superficial mycoses with two new antifungal agents: clotrimazole (1 per cent) lotion and miconazole (2 per cent) cream, Recent Advances in Dermatopharmacology. Edited by P Frost, EC Gomez, N Zaias. New York, Spectrum Publications, 1978, pp 35-39 6. Siegel S: Nonparametric Statistics for the Behavioral Sciences. New York, McGraw-Hill, 1956, pp 116-126 7. Ipp MM, Boxall L, Gelfand EW: Clotrimazole: intermittent therapy in chronic mucocutaneous candidiasis. Am J Dis Child 131:305-307, 1977 8. Meade RH: Treatment of chronic mucocutaneous candidiasis. Ann Intern Med 86:314-315, 1977 9. Montes LF, Soto TG, Parker JM, et al: Clotrimazole troches: a new therapeutic approach to oral candidiasis. Cutis 17:277-280, 1976
ANTIBODIES TO TYPE II COLLAGEN IN RELAPSING POLYCHONDRITIS JEAN-MICHEL FOIDART, M.D., SHIGETO ABE, M.D., GEORGE R. MARTIN, PH.D., THOMAS M. Zizic, M.D., EUGENE V. BARNETT, M.D., THOMAS J. LAWLEY, M.D., AND STEPHEN I. KATZ, M.D., PH.D. Abstract Relapsing polychondritis is a disorder of unknown cause characterized by the destruction of cartilage. To test the hypothesis that immunologic mechanisms are involved in the pathogenesis of relapsing polychondritis, we analyzed the serum of 15 patients for the presence of antibodies to cartilage. Antibodies to Type 11 (cartilage) collagen were found in the serum of five patients at the time of acute symp-
toms. No antibodies were detected either to cartilage proteoglycan or to other collagen types. The antibodies were detected at the onset of the disease and their titers appeared to correlate with severity of disease. Circulating immune complexes were also detected in the serum of these patients. Our findings support an immunologic involvement in this condition. (N EngI J Med 299:1203-1207, 1978)
RELAPSING polychondritis is a disease manifested by recurring episodes of inflammation in cartilaginous tissue throughout the body. Pearson et al. suggested the name relapsing polychondritis in 1960 to emphasize its episodic nature leading to degeneration and replacement of cartilaginous structures by fibrous tissue.' The coexistence of various rheumatic and autoimmune diseases in 30 per cent of
patients has led to the suggestion that an immunologic dysfunction may be involved in the pathogenesis of relapsing polychondritis.2 Attempts to demonstrate anticartilage antibodies in the serum of these patients have yielded variable, but usually negative, results, and the putative antigen (or antigens) has not been identified.2-9 Chondrocytes produce Type II collagen and cartilage proteoglycan, two tissue-specific macromolecules. The serum of five of 15 patients with relapsing polychondritis described below contains antibodies to cartilage matrix after removal of proteoglycan. These antibodies react with the collagenous component of the matrix, and the highest antibody titers are observed during the most active phase of the disorder. Circulating immune complexes are also detected in the same patients during the acute phases of the disease.
From the Laboratory of Developmental Biology and Anomalies, National Institute of Dental Research, and Dermatology Branch, National Cancer Institute, National Institutes of Health, the Rheumatology Division, Johns Hopkins Hospital, Baltimore, MD, and the Institute of Chronic Disease and Rehabilitation, UCLA Medical School, Los Angeles, CA (address reprint requests to Dr. Foidart at Bldg. 30, Room 429, National Institutes of Health, Bethesda, MD 20014). Dr. Foidart is the recipient of an international research fellowship from the John E. Fogarty International Center for Advanced Study in Health, Bethesda, and a fellow of the FNRS in Belgium.
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THE NEW ENGLAND JOURNAL OF MEDICINE
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MATERIALS
AND
METHODS
Patients In each of the 15 patients (seven women and eight men, 19 to 63 years of age) at least three of the following diagnostic criteria were observed: recurrent chondritis of both auricles; nonerosive polyarthritis; chondritis of nasal cartilage; inflammation of ocular structures; chondritis of tracheal or laryngeal cartilage (or both); and cochlear and vestibular damage.2 Biopsy confirmed the auricular chondritis in all but one patient. In three patients serial serum specimens were obtained. The duration of disease ranged from one month to 10 years (mean of 22 months). Eleven of the 15 patients had arthritis; all were seronegative. Five patients had acute symptoms of chondritis, seven were in remission, and three exhibited only mild inflammatory symptoms while they were receiving corticosteroids. We also analyzed serum specimens from 35 patients with rheumatoid arthritis diagnosed according to the criteria of the American Rheumatism Association, 32 with osteoarthritis, 19 with psoriatic arthritis and six with systemic lupus erythematosus and 75 normal controls.
Preparation of Antigens Type I and III collagens were prepared from fetal-calf skin.10"11 Type II collagen was prepared from a rat chondrosarcoma.12 Proteoglycan was extracted from the same tumor with 0.5 M guanidine plus 0.05 M Tris (Trishydroxymethylaminomethane) hydrochloride, pH 7.4, and purified by diethylaminoethyl-cellulose chromatography."3 Type IV collagen was purified from a murine sarcoma.'4 Nonhelical peptides were removed from the Type II collagen (5 mg per milliliter) by incubation with pepsin (0.2 mg per milliliter) in 0.5 N acetic acid for six hours at 15°C. Type I and II native collagens were reconstituted into fibrils by dialysis at 4'C against 0.01 M sodium phosphate buffer, pH 7.4. We obtained denatured collagens by heating collagens dissolved in 0.1 per cent acetic acid at 56°C for half an hour. The component al (II) chains were isolated by carboxymethyl-cellulose column chromatography'5 followed by diethylaminoethyl-cellulose chromatography." The identity and purity of each collagen was verified by amino acid analysis, by carboxymethyl-cellulose chromatography,'5 and by polyacrylamide-gel electrophoresis both with and without cyanogen bromide digestion. '7
Preparation of Antibodies to Type 11 Collagen and to Proteoglycan Rabbits were immunized by repeated subcutaneous injections of either 2.5 to 5.0 mg of native Type II collagen or 0.5 to 1.0 mg of proteoglycan. Antibodies to Type II collagen were purified by immunoabsorption to Type II collagen covalently bound to Sepharose 4B followed by elution with 0.5 N acetic acid and 0.5 M sodium chloride.'8 Indirect immunofluorescence demonstrated that antibody binding was limited to the cartilage matrix. No reaction of the antibody was observed with other types of collagen or with proteoglycan with a micropassive hemagglutination assay" or an enzymelinked immunoabsorbent assay.20
Immunofluorescence Indirect immunofluorescent staining was performed on sagittal sections (6 Am thick) of newborn-mouse leg and human chondrocostal and tracheal cartilages with previously described procedures and reagents.2' Unless otherwise stated, all sections were preincubated with testicular hyaluronidase (Type IV, Sigma Chemical Company, St. Louis, Missouri) to remove the masking proteoglycan.22 Some sections were also preincubated at 37°C with 500 IU per milliliter of purified collagenase (Form III, Advance Biofactures, Lynbrook, New York) for two hours.
Serum Absorption Studies Serum in amounts of 0.18 ml was incubated for half an hour at 37'C and then overnight at 40C, with 200 ,g of Type I or 100 ug of Type II collagen fibrils. Insoluble material was removed by centri-
fugation, and the supernatant fluids were tested by indirect immunofluorescence for antibody to cartilage. The fibrillar material removed by centrifugation was exposed to 80 units of purified collagenase for three hours at 37°C. Collagenase was inactivated with 0.075 M ethylenediamine tetra-acetic acid; the specimens were centrifuged, and the supernatant fluids tested for the presence of antibody to cartilage.
Passive Mlcrohemagglutinatlon and Hemagglutinatlon Inhibition Assays Passive microhemagglutination was carried out according to the method of Andriopoulos et al.23 using the antigen concentrations recommended by Trentham et al.24 For hemagglutination inhibition studies, 0.025 ml of serum was incubated overnight with 0.025 ml of collagen solution (1 mg per milliliter in sodium phosphate buffer). The absorbed serums were then assayed for agglutinating activity with erythrocytes coated with native Type II collagen or al (II) chains. Reactivity of the human serum was compared to that of a rabbit antiserum to Type II collagen as well as to its specifically purified antibodies.
Assays for Immune Complexes The percentage of total IgG precipitable by 4 per cent polyethylene glycol 6000 was measured as previously described.25 The 125I. Clq binding assay was performed with minor modifications of the method of Zubler et al.26
RESULTS Indirect Immunofluorescence
Antibodies to cartilage were detected in the serum from five of 15 patients with relapsing polychondritis (Fig. 1) and in the rabbit antiserum to Type II collagen only when tissue sections were preincubated with hyaluronidase. Rabbit anti-proteoglycan antibodies were detected, however, when untreated sections were used (Table 1). Antibody binding was limited to cartilage and was not species specific. Incubation of the test sections with hyaluronidase followed by collagenase removed the reacting material (Table 1). These studies suggested that some of the serum specimens in patients with relapsing polychondritis contained antibody reacting with Type II collagen. The antibodies were of the IgG class in all patients except for Case 1 who had IgG and IgA antibodies. They did not bind complement as determined by immunofluorescence. The antibody-containing serum specimens were from patients in the acute phase of the disease; Cases 2 and 3 were suffering recurrent episodes of nasal, auricular and tracheal chondritis with acute arthritis, and Cases 1, 4 and 5 were having their first attack of relapsing polychondritis with involvement of the nose, ears and trachea. The antibody titers ranged from 10 to 320, and in serum obtained from two patients (Cases 4 and 5) at various times, there was a correlation between the titers and the clinical severity (Table 2). Synovial fluid from Case 4 did not contain antibodies to cartilage. Twelve serum specimens from the 10 other patients lacked antibody to cartilage. These patients were undergoing chronic treatment with prednisone or immunosuppressive agents (or both) and had normal erythrocyte sedimentation rates. The organ involve-
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Vol. 299 No. 22
COLLAGEN ANTIBODIES IN POLYCHONDRITIS
-
FOIDART ET AL.
1 205
~
~
*o
S .v;.sO~n^..= A
40
C
Figure 1. Indirect Immunofluorescence Test on an Unfixed Frozen Section of Newborn-Mouse Leg. The section was pretreated with hyaluronidase. Serum of Case 5 was used at a 1:80 dilution. A shows staining of cartilaginous matrix but no staining of the skin or interstitial connective tissue (x 50). B shows staining of cartilage at cartilage-bonemarrow interface during enchondral ossification of newborn-mouse femur (X 125).
and severity of their disease were not clinically different from those of patients in whom antibodies were detected. Seven of the patients were in remission, and three others exhibited mild arthritic symptoms or moderate auricular chondritis and ocular inflammation. Antibody to hyaluronidase-treated cartilage was detected in serum from one of the 35 patients with rheumatoid arthritis; a titer of 10 was determined in two of five serum specimens obtained over a period of several months. No antibody to cartilage was detectable by immunofluorescence in the serum specimens of 75 normal volunteers or of the 91 patients with various arthritides.
ment
lmmunoabsorptlon Studies The antibody to cartilage was
removed by preincubation of the serum with fibers of Type II but not by twice the amount of Type I collagen. After this incubation, the Type II collagen fibers were treated with collagenase to destroy selectively the antigen responsible for antibody binding. After collagenase inactivation and centrifugation, the supernatant fluid was found to contain antibody to cartilage. These studies indicate that the antibody binds to Type II but not to Type I collagen and can be recovered in active form from Type II collagen fibers treated with collagenase. Similar results were obtained with the rabbit antiserum to Type II collagen used as control.
dicate that these serum specimens contain antibody reacting with helical antigenic determinants in Type II collagen. The serum from Cases 3 and 5 agglutinated erythrocytes coated with both native and denatured forms of Type II collagen (Table 2). Absorption with Type II collagen or with at (II) chains removed specifically the reactivity to each antigen. Pepsin-treated Type II collagen also inhibited the hemagglutination of erythrocytes coated with native collagen. The results suggest that the antibodies present in the serum of these two patients have specificity either for the helical antigenic determinants of the molecule or for the linear amino acid sequences in the al(II) chain. As in the immunofluorescent tests, the serum of only one of 35 patients with rheumatoid arthritis caused appreciable hemagglutination of Type II collagen and al (II) chain coated cells (titers 32 on two serum specimens and 16 on the three others). The serum specimens of all the others either were negative or had titers of 8 or less. Table 1. Indirect Immunofluorescence in Serum of Five Patients with Relapsing PolychondrItis.*
None
Hemagglutinatlon Assays and Characterization tigen
of
the An-
The hemagglutination titers correlated with the positive indirect immunofluorescent tests and in Cases 4 and 5 correlated with the activity of clinical disease (Table 2). Absorption of serum from Cases 1, 2 and 4 with native Type II collagen but not with denatured Type II collagen or with isolated at (II) chains inhibited hemagglutination of Type II coated cells. Limited cleavage of Type II collagen with pepsin did not alter its inhibitory capacity. These studies in-
IIUNOFLUOXESCENCR
Taus TaAnssN
Hyaluronidase30 min Hyaluronidase30min + collagonase 30min Hyaluronidam30min + colaan 120 n
Hyaluronidase30miu + heat-in vted cofena
PATENTS
a/n
R/PGS
_
++ ++
++ ++ ++
-
-
++
++
++
++
++ +
lCartilagno tissu. (newborn-mouse leg A human chondrowostal & trache cartie with variou enzyme. a desibed under lages) were uwd as substrate pAMethods. A positive reatio w obswved with the erum of 5 patients. Treatnt with hyaluronidase for 30 min unmasks the colsgen by removing the proteoglycn.22 Th persisten of a positive reaction with a R/PG & of histologic isg of calage (mtoxylin osin, ponodic acddchiffA alcian blue)bhows that thisrmoval is incomplto. tRabt atibody to Type II aoHagm (50 gg/ed) in sodium phosphate buffer. $Rabbit ntarum to cati proteoglyc (diluted 80 tims).
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NOV. 30, 1978
Table 2. Clinical Status, Anticartilage Antibody Titers and Circulating Immune Complexes in Patients with Relapsing Poly-
chondritis. CAsE No.
DATS
CLINICAL
TuaATMesNT
CONDmON
IF TrrERn
TrYP 11
2 3 4
5
10
40
256
2
20
64
0
Asymptomatic Asymptomatic
None Prednisone Prednisone Prodnisone
10 10
16 16
0
Acute symptoms Acute symptoms Mild symptoms Mild symptoms Asymptomatic Asymptomatic
None Prodnisone Prodnisone Prodnisone Prednisone Prednisone
320
1,024
64
160
256
80 40
64 64 64
32 32
40
32
Acute symptoms Acute symptoms Acute symptoms
None Prodnisone None
8/77 10/25/77 11/17/77 11/24/77
Acute symptoms Mild symptoms
8/22/77 10/31/77 11/14/77 12/13/77 1/78
al(n) CsAINSf
512 128 8
8/77 12/18/74 12/74
9/77
IMUNE CoMsLExBst
MicatovAssIV
HMAOGoLUTiNATION TITErn wriH ERYTHROCYTrS COATED WrrH COLsAGBNt
80
20
40
0
4 32
0
64 64 32
CIQ
PRO
13.511 NDII ND
7.91 5.2
5.2 2.4 2 2 ND 14.91
13.31 171 17.41 12.711
161 8.81 5.51 *5.1
6.511 5.1
*Antibody titers asssed by indirect immunofluoresac (IF). tevels of circulating immune complexes eatimated by a Clq binding asy or by precipitation with polyethylene glycol (PEG) - expressed in % Clq binding or in % of total IgG in PEG precipitated. tldentical results were obtained with intact as well as pepsin-treated Type Ii collagen. Identical results were obtained with aI(11) chains or heat-denatured Type II collagen prepared from intact or pepsin-treated Type 11 Collagen. ¶Significantly different from normal. (The upper 95% confidence limit in the control population is 9.9% for the Clq binding assay and 5 % for the PEG assay.) liNot done.
No hemagglutinating activity was detected in the of 15 patients with relapsing polychondritis or in 35 normal serum specimens on testing with erythrocytes coated with either native or denatured Type I, III or IV collagens, their component chains or cartilage proteoglycan. Serum specimens from patients with the other arthritides were not tested for the denatured collagens. serum
Immune Complexes
Low levels of circulating immune complexes were detected in the serum of three patients with relapsing polychondritis who had demonstrable antibodies to Type II collagen (Table 2) and in two others.
DISCUSSION In this study we have demonstrated that patients with relapsing polychondritis have circulating antibodies to Type II collagen during the acute phase of the disease. The specificity of these antibodies is evidenced by a number of circumstances. First of all, they bind only to cartilaginous matrices. Secondly, their demonstration by indirect immunofluorescence requires prior removal of proteoglycan, which is known to mask collagen.22 Thirdly, collagenase treatment of the substrate prevents binding. Fourthly, the antibodies can be completely absorbed by Type II collagen fibrils. Fifthly, the activity of these immunoabsorbed antibodies is recovered by collagenase treatment of the Type II collagen fibril-antibody complexes. Finally, passive microhemagglutination and inhibition of hemagglutination assays demonstrate the specificity of the antibodies. Antibodies to Type 1, III and IV collagens or their
constituent chains were not detected in the serum of patients with relapsing polychondritis. The antibodies to Type II collagen were found only during the acute phase of the disease, and their titers seemed to correlate with activity of disease. Prednisone therapy was clearly associated with progressive clinical improvement and coincident decreases in titers. The apparent lack of antibody to Type II collagen in the remaining 10 patients may have been the consequence of chronic immunosuppressive therapy. The occurrence of antibodies to Type II collagen appears to be almost entirely restricted to relapsing polychondritis since similar antibodies were found in only one (low titer) of 92 patients with other arthritides and in none of 75 normal volunteers. Patients with rheumatoid arthritis have been reported to have antibodies to Type I, II and III collagens and primarily against their unfolded chains.23 27-"3 This finding has been interpreted as evidence that immunization against collagen occurred after destruction of connective tissue, and that antibody formation was the consequence rather than a cause of the disease." In contrast, in some patients with relapsing polychondritis, antibodies against only native Type II collagen was found, and in the others, in whom antibodies to native and denatured Type II collagens were detected, the titers against the native Type II collagen were substantially higher than those against constituent al (II) chains. This observation suggests that the antibodies were not formed after destruction of cartilage and denaturation of collagen. Thus, the formation of antibody to Type II collagen may not be a consequence of inflammation, but may be a primary event in the pathogenesis of relapsing polychondritis.
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COLLAGEN ANTIBODIES IN POLYCHONDRITIS - FOIDART ET AL.
Cell-mediated immunity to cartilage is also thought to play a part in the pathogenesis of relapsing polychondritis since DNA synthesis by lymphocytes from these patients is stimulated by exposure to cartilage extracts.3'6 However, recent evidence indicates that patients with rheumatoid arthritis also have cell-mediated immunity, although at a low level, to native Type II and III collagens.33 The role of the circulating immune complexes in five of our patients is unclear although vasculitis, neuritis, pericarditis and damage to medium and large vessels have all been documented in cases of relapsing polychondritis. Formation of the immune complexes may be secondary to the release of collagen fragments into the circulation after tissue damage and thus may account for some of the clinical manifestations not involving cartilaginous structures. The demonstration of antibodies to Type II collagen in the serum of patients with relapsing polychondritis raises the question of whether the antibodies are functionally active in vivo or represent an epiphenomenon secondary to cartilage injury with exposure of antigen. The observation of placental transfer of relapsing polychondritis with an affected child and subsequent recovery of the offspring34 suggests an important role for antibody in this disease. Experimental autoimmunity to Type II collagen in rats24'35 results in acute arthritis, suggesting that immune responses to Type II collagen could be a factor by inciting cartilage inflammation. Finally, in vivo deposits of IgG, IgA and C3 in an inflamed cartilage of a patient with relapsing polychondritis36 support the contention that the circulating antibodies to Type II collagen described in this study could be autoantibodies involved in the pathogenesis of relapsing polychondritis. We are indebted to Mr. E. William Bere for technical assistance, to Mr. Harry Schaefer for photographic assistance, to Dr. H. Kleinman for reviewing the manuscript and to Drs. P. Plotz, 0. Lawless and L. Gerber for providing many serum specimens from patients with other arthritides.
REFERENCES 1. Pearson CM, Kline HM, Newcomer VD: Engl J Med 263:51-58, 1960
Relapsing polychondritis. N
2. McAdam LP, O'Hanlan MA, Bluestone R, et al: Relapsing polychondritis: prospective study of 23 patients and a review of the literature. Medicine (Baltimore) 55:193-215, 1976 3. Herman JH, Dennis MV: Immunopathologic studies in relapsing polychondritis. J Clin Invest 52:549-558, 1973 4. Rajapakse DA, Bywaters EGL: Cell-mediated immunity to cartilage proteoglycan in relapsing polychondritis. Clin Exp Immunol 16:497502, 1974 5. Dolan DL, Lemmon GB Jr, Teitelbaum SL: Relapsing polychondritis: analytical literature review and studies on pathogenesis. Am J Med 41:285-299, 1966 6. Hundeiker M, Brehm K, Go M: Infiltrat und Knorpelzerstorung bei polychondritis (Histochemische und immunofluoreszenz-histologische Befunde). Z Haut Geschlechtskr 45:437-443, 1970 7. Menkes C-J, Siaud J-R, Delrieu F, et al: Polychondrite chronique atrophiante: deux cas dont l'un avec cataracte et arterite des membres inf6rieurs. Ann Med Interne (Paris) 121:895-904, 1970 8. Hughes RAC, Berry CL, Seifert M, et al: Relapsing polychondritis: three cases with a clinico-pathological study and literature review. Q J Med 41:363-380, 1972
1207
9. Rogers PH, Boden G, Tourtellotte CD: Relapsing polychondritis with insulin resistance and antibodies to cartilage. Am J Med 55:243-248, 1973 10. Timpl R, Glanville RW, Nowack H, et al: Isolation, chemical and electron microscopical characterization of neutral-salt-soluble type III collagen and procollagen from fetal bovine skin. Hoppe Seylers Z Physiol Chem 356:1783-1792, 1975 11. Lapiere CM, Nusgens B, Pierard GE: Interaction between collagen type I and type III in conditioning bundles organization. Connect Tissue Res 5:21-29, 1977 12. Smith BD, Martin GR, Miller EJ, et al: Nature of the collagen synthesized by a transplanted chondrosarcoma. Arch Biochem Biophys 166:181-186, 1975 13. Miller EJ: Isolation and characterization of a collagen from chick cartilage containing three identical a chains. Biochemistry 10:1652-1659. 1971 14. Orkin RW, Gehron P, McGoodwin EB, et al: A murine tumor producing a matrix of basement membrane. J Exp Med 145:204-220, 1977 15. Piez KA, Eigner EA, Lewis MS: The chromatographic separation and amino acid composition of the subunits of several collagens. Biochemistry 2:58-66, 1963 16. Trelstad RL, Kang AH, Toole BP, et al: Collagen heterogeneity: high resolution separation of native [aI(I)12a2 and [a, (11)13 and their component a chains. J Biol Chem 247:6469-6473, 1972 17. Furthmayr H, Timpl R: Characterization of collagen peptides by sodium dodecylsulfate-polyacrylamide electrophoresis. Anal Biochem 41:510-516, 1971 18. Cuatrecasas P: Protein purification by affinity chromatography: derivations of agarose and polyacrylamide beads. J Biol Chem 245:30593065, 1970 19. Beil W, Furthmayr H, Timpl R: Chicken antibodies to soluble rat collagen. I. Characterization of the immune response by precipitation and agglutination methods. Immunochemistry 9:779-788, 1972 20. Schuurs AHWM, Van Weemen BK: Enzyme-immunoassay. Clin Chim Acta 81:1-40, 1977 21. Katz SI, Hertz KC, Yaoita H: Herpes gestationis: immunopathology and characterization of the HG factor. J Clin Invest 57:1434-1441, 1976 22. von der Mark H, von der Mark K, Gay S: Study of differential collagen
synthesis during development of the chick embryo by immunofluorescence. I. Preparation of collagen type I and type II specific antibodies and their application to early stages of the chick embryo. Dev Biol
48:237-249, 1976 23. Andriopoulos NA, Mestecky J, Wright GP, et al: Characterization of antibodies to the native human collagens and to their component a chains in the sera and the joint fluids of patients with rheumatoid arthritis. Immunochemistry 13:709-712, 1976 24. Trentham DE, Townes AS, Kang AH: Autoimmunity to type II collagen: an experimental model of arthritis. J Exp Med 146:857-868, 1977 25. Barnett EV, Chia D: Quantitation of immunoglobulin G in serum and immune complexes isolated in 4 % polyethylene glycol. Ann Rheum Dis 36:Suppl 1:26, 1977 26. Zubler RH, Lange G, Lambert PH, et al: Detection of immune complexes in unheated sera by a modified '25I-Clq binding test: effect of heating on the binding of C lq by immune complexes and application of the test to systemic lupus erythematosus. J Immunol 116:232-235, 1976 27. Steffen C, Timpl R: Antigenicity of collagen and its application in the serological investigation of rheumatoid arthritis sera. Int Arch Allergy Appl Immunol 22:333-349, 1963 28. Andriopoulos NA, Mestecky J, Miller EJ, et al: Antibodies to native and denatured collagens in sera of patients with rheumatoid arthritis. Arthritis Rheum 19:613-617, 1976 29. Michaeli D, Fudenberg HH: The incidence and antigenic specificity of antibodies against denatured human collagen in rheumatoid arthritis. Clin Immunol Immunopathol 2:153-159, 1974 30. Steffen C, Schuster F, Tausch G, et al: Weitere Untersuchungen ilber Kollagenantikorper bei Patienten mit primar chronischer Polyarthritis. Klin Wochenschr 46:976-981, 1968 31. Cracchiolo A III, Michaeli D, Goldberg LS, et al: The occurrence of antibodies to collagen in synovial fluids. Clin Immunol Immunopathol 3:567-574, 1975 32. Andriopoulos NA, Mestecky J, Miller EJ, et al: Antibodies to human native and denatured collagens in synovial fluids of patients with rheumatoid arthritis. Clin Immunol Immunopathol 6:209-212, 1976 33. Trentham DE, Dynesius RA, Rocklin RE, et al: Cellular sensitivity to collagen in rheumatoid arthritis. N Engl J Med 299:327-332, 1978 34. Arundell FW, Haserick JR: Familial chronic atrophic polychondritis. Arch Dermatol 82:439-441, 1960 35. Trentham DE, Townes AS, Kang AH, et al: Humoral and cellular sensitivity to collagen in type II collagen-induced arthritis in rats. J Clin Invest 61:89-96, 1978 36. Bergfeld WF: Relapsing polychondritis with positive direct immunofluorescence. Arch Dermatol 114:127, 1978
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COLLAGEN ANTIBODIES IN POLYCHONDRITIS - FOIDART ET AL.
supported by the fact that none of the patients in the study had responded to long-term therapy with full doses of topical medications such as nystatin or gentian violet. In our experience, no medication other than intravenous amphotericin B has produced such favorable results. Fifteen of the patients have continued to be observed since the completion of the blind phase of their trial. The follow-up duration is as follows: 10 months, one patient; nine months, nine patients; eight months, three patients; four months, one patient; and three months, one patient. All have received clotrimazole troches, since we were interested in determining (roughly) the minimal effective dose as well as the relative frequencies of drug resistance and relapse. Data accumulated thus far indicate that one to three troches daily are sufficient to prevent relapse. All the nine patients who were previously scored as failures while taking the placebo responded to clotrimazole; thus, none of the 20 patients studied harbored drugresistant organisms. Only one patient, a 44-year-old woman with denture stomatitis, has had a recurrence of noteworthy oral candidiasis. None of the remaining 14 patients have had any adverse effects of prolonged treatment. When treatment is stopped, however, symptomatic infections invariably recur within two to four weeks, presumably because the underlying defect
1203
that predisposes the patients to oral candidiasis has not been corrected. Thus far, we have not seen accompanying clinical responses in other infected sites such as the esophagus, vagina, nails or skin. We are indebted to the staff of the Clinical Center Pharmaceutical Development Services for assistance with the study. These studies were conducted in compliance with a protocol that had been reviewed and approved by the appropriate committees and persons of the National Institutes of Health.
REFERENCES 1. Kirkpatrick CH, Rich RR, Bennett JE: Chronic mucocutaneous candidiasis: model-building in cellular immunity. Ann Intern Med 74:955-978, 1971 2. Kirkpatrick CH, Smith TK: Chronic mucocutaneous candidiasis: immunologic and antibiotic therapy. Ann Intern Med 80:310-320, 1974 3. Bennett JE: Chemotherapy of systemic mycoses. N Engl J Med 290:3032, 320-323, 1974 4. Fredriksson T: Topical treatment with Bay b5097, a new broad spectrum antimycotic agent. Br J Dermatol 86:628-630, 1972 5. Zaias N: Topical treatment of superficial mycoses with two new antifungal agents: clotrimazole (1 per cent) lotion and miconazole (2 per cent) cream, Recent Advances in Dermatopharmacology. Edited by P Frost, EC Gomez, N Zaias. New York, Spectrum Publications, 1978, pp 35-39 6. Siegel S: Nonparametric Statistics for the Behavioral Sciences. New York, McGraw-Hill, 1956, pp 116-126 7. Ipp MM, Boxall L, Gelfand EW: Clotrimazole: intermittent therapy in chronic mucocutaneous candidiasis. Am J Dis Child 131:305-307, 1977 8. Meade RH: Treatment of chronic mucocutaneous candidiasis. Ann Intern Med 86:314-315, 1977 9. Montes LF, Soto TG, Parker JM, et al: Clotrimazole troches: a new therapeutic approach to oral candidiasis. Cutis 17:277-280, 1976
ANTIBODIES TO TYPE II COLLAGEN IN RELAPSING POLYCHONDRITIS JEAN-MICHEL FOIDART, M.D., SHIGETO ABE, M.D., GEORGE R. MARTIN, PH.D., THOMAS M. Zizic, M.D., EUGENE V. BARNETT, M.D., THOMAS J. LAWLEY, M.D., AND STEPHEN I. KATZ, M.D., PH.D. Abstract Relapsing polychondritis is a disorder of unknown cause characterized by the destruction of cartilage. To test the hypothesis that immunologic mechanisms are involved in the pathogenesis of relapsing polychondritis, we analyzed the serum of 15 patients for the presence of antibodies to cartilage. Antibodies to Type 11 (cartilage) collagen were found in the serum of five patients at the time of acute symp-
toms. No antibodies were detected either to cartilage proteoglycan or to other collagen types. The antibodies were detected at the onset of the disease and their titers appeared to correlate with severity of disease. Circulating immune complexes were also detected in the serum of these patients. Our findings support an immunologic involvement in this condition. (N EngI J Med 299:1203-1207, 1978)
RELAPSING polychondritis is a disease manifested by recurring episodes of inflammation in cartilaginous tissue throughout the body. Pearson et al. suggested the name relapsing polychondritis in 1960 to emphasize its episodic nature leading to degeneration and replacement of cartilaginous structures by fibrous tissue.' The coexistence of various rheumatic and autoimmune diseases in 30 per cent of
patients has led to the suggestion that an immunologic dysfunction may be involved in the pathogenesis of relapsing polychondritis.2 Attempts to demonstrate anticartilage antibodies in the serum of these patients have yielded variable, but usually negative, results, and the putative antigen (or antigens) has not been identified.2-9 Chondrocytes produce Type II collagen and cartilage proteoglycan, two tissue-specific macromolecules. The serum of five of 15 patients with relapsing polychondritis described below contains antibodies to cartilage matrix after removal of proteoglycan. These antibodies react with the collagenous component of the matrix, and the highest antibody titers are observed during the most active phase of the disorder. Circulating immune complexes are also detected in the same patients during the acute phases of the disease.
From the Laboratory of Developmental Biology and Anomalies, National Institute of Dental Research, and Dermatology Branch, National Cancer Institute, National Institutes of Health, the Rheumatology Division, Johns Hopkins Hospital, Baltimore, MD, and the Institute of Chronic Disease and Rehabilitation, UCLA Medical School, Los Angeles, CA (address reprint requests to Dr. Foidart at Bldg. 30, Room 429, National Institutes of Health, Bethesda, MD 20014). Dr. Foidart is the recipient of an international research fellowship from the John E. Fogarty International Center for Advanced Study in Health, Bethesda, and a fellow of the FNRS in Belgium.
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THE NEW ENGLAND JOURNAL OF MEDICINE
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MATERIALS
AND
METHODS
Patients In each of the 15 patients (seven women and eight men, 19 to 63 years of age) at least three of the following diagnostic criteria were observed: recurrent chondritis of both auricles; nonerosive polyarthritis; chondritis of nasal cartilage; inflammation of ocular structures; chondritis of tracheal or laryngeal cartilage (or both); and cochlear and vestibular damage.2 Biopsy confirmed the auricular chondritis in all but one patient. In three patients serial serum specimens were obtained. The duration of disease ranged from one month to 10 years (mean of 22 months). Eleven of the 15 patients had arthritis; all were seronegative. Five patients had acute symptoms of chondritis, seven were in remission, and three exhibited only mild inflammatory symptoms while they were receiving corticosteroids. We also analyzed serum specimens from 35 patients with rheumatoid arthritis diagnosed according to the criteria of the American Rheumatism Association, 32 with osteoarthritis, 19 with psoriatic arthritis and six with systemic lupus erythematosus and 75 normal controls.
Preparation of Antigens Type I and III collagens were prepared from fetal-calf skin.10"11 Type II collagen was prepared from a rat chondrosarcoma.12 Proteoglycan was extracted from the same tumor with 0.5 M guanidine plus 0.05 M Tris (Trishydroxymethylaminomethane) hydrochloride, pH 7.4, and purified by diethylaminoethyl-cellulose chromatography."3 Type IV collagen was purified from a murine sarcoma.'4 Nonhelical peptides were removed from the Type II collagen (5 mg per milliliter) by incubation with pepsin (0.2 mg per milliliter) in 0.5 N acetic acid for six hours at 15°C. Type I and II native collagens were reconstituted into fibrils by dialysis at 4'C against 0.01 M sodium phosphate buffer, pH 7.4. We obtained denatured collagens by heating collagens dissolved in 0.1 per cent acetic acid at 56°C for half an hour. The component al (II) chains were isolated by carboxymethyl-cellulose column chromatography'5 followed by diethylaminoethyl-cellulose chromatography." The identity and purity of each collagen was verified by amino acid analysis, by carboxymethyl-cellulose chromatography,'5 and by polyacrylamide-gel electrophoresis both with and without cyanogen bromide digestion. '7
Preparation of Antibodies to Type 11 Collagen and to Proteoglycan Rabbits were immunized by repeated subcutaneous injections of either 2.5 to 5.0 mg of native Type II collagen or 0.5 to 1.0 mg of proteoglycan. Antibodies to Type II collagen were purified by immunoabsorption to Type II collagen covalently bound to Sepharose 4B followed by elution with 0.5 N acetic acid and 0.5 M sodium chloride.'8 Indirect immunofluorescence demonstrated that antibody binding was limited to the cartilage matrix. No reaction of the antibody was observed with other types of collagen or with proteoglycan with a micropassive hemagglutination assay" or an enzymelinked immunoabsorbent assay.20
Immunofluorescence Indirect immunofluorescent staining was performed on sagittal sections (6 Am thick) of newborn-mouse leg and human chondrocostal and tracheal cartilages with previously described procedures and reagents.2' Unless otherwise stated, all sections were preincubated with testicular hyaluronidase (Type IV, Sigma Chemical Company, St. Louis, Missouri) to remove the masking proteoglycan.22 Some sections were also preincubated at 37°C with 500 IU per milliliter of purified collagenase (Form III, Advance Biofactures, Lynbrook, New York) for two hours.
Serum Absorption Studies Serum in amounts of 0.18 ml was incubated for half an hour at 37'C and then overnight at 40C, with 200 ,g of Type I or 100 ug of Type II collagen fibrils. Insoluble material was removed by centri-
fugation, and the supernatant fluids were tested by indirect immunofluorescence for antibody to cartilage. The fibrillar material removed by centrifugation was exposed to 80 units of purified collagenase for three hours at 37°C. Collagenase was inactivated with 0.075 M ethylenediamine tetra-acetic acid; the specimens were centrifuged, and the supernatant fluids tested for the presence of antibody to cartilage.
Passive Mlcrohemagglutinatlon and Hemagglutinatlon Inhibition Assays Passive microhemagglutination was carried out according to the method of Andriopoulos et al.23 using the antigen concentrations recommended by Trentham et al.24 For hemagglutination inhibition studies, 0.025 ml of serum was incubated overnight with 0.025 ml of collagen solution (1 mg per milliliter in sodium phosphate buffer). The absorbed serums were then assayed for agglutinating activity with erythrocytes coated with native Type II collagen or al (II) chains. Reactivity of the human serum was compared to that of a rabbit antiserum to Type II collagen as well as to its specifically purified antibodies.
Assays for Immune Complexes The percentage of total IgG precipitable by 4 per cent polyethylene glycol 6000 was measured as previously described.25 The 125I. Clq binding assay was performed with minor modifications of the method of Zubler et al.26
RESULTS Indirect Immunofluorescence
Antibodies to cartilage were detected in the serum from five of 15 patients with relapsing polychondritis (Fig. 1) and in the rabbit antiserum to Type II collagen only when tissue sections were preincubated with hyaluronidase. Rabbit anti-proteoglycan antibodies were detected, however, when untreated sections were used (Table 1). Antibody binding was limited to cartilage and was not species specific. Incubation of the test sections with hyaluronidase followed by collagenase removed the reacting material (Table 1). These studies suggested that some of the serum specimens in patients with relapsing polychondritis contained antibody reacting with Type II collagen. The antibodies were of the IgG class in all patients except for Case 1 who had IgG and IgA antibodies. They did not bind complement as determined by immunofluorescence. The antibody-containing serum specimens were from patients in the acute phase of the disease; Cases 2 and 3 were suffering recurrent episodes of nasal, auricular and tracheal chondritis with acute arthritis, and Cases 1, 4 and 5 were having their first attack of relapsing polychondritis with involvement of the nose, ears and trachea. The antibody titers ranged from 10 to 320, and in serum obtained from two patients (Cases 4 and 5) at various times, there was a correlation between the titers and the clinical severity (Table 2). Synovial fluid from Case 4 did not contain antibodies to cartilage. Twelve serum specimens from the 10 other patients lacked antibody to cartilage. These patients were undergoing chronic treatment with prednisone or immunosuppressive agents (or both) and had normal erythrocyte sedimentation rates. The organ involve-
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Vol. 299 No. 22
COLLAGEN ANTIBODIES IN POLYCHONDRITIS
-
FOIDART ET AL.
1 205
~
~
*o
S .v;.sO~n^..= A
40
C
Figure 1. Indirect Immunofluorescence Test on an Unfixed Frozen Section of Newborn-Mouse Leg. The section was pretreated with hyaluronidase. Serum of Case 5 was used at a 1:80 dilution. A shows staining of cartilaginous matrix but no staining of the skin or interstitial connective tissue (x 50). B shows staining of cartilage at cartilage-bonemarrow interface during enchondral ossification of newborn-mouse femur (X 125).
and severity of their disease were not clinically different from those of patients in whom antibodies were detected. Seven of the patients were in remission, and three others exhibited mild arthritic symptoms or moderate auricular chondritis and ocular inflammation. Antibody to hyaluronidase-treated cartilage was detected in serum from one of the 35 patients with rheumatoid arthritis; a titer of 10 was determined in two of five serum specimens obtained over a period of several months. No antibody to cartilage was detectable by immunofluorescence in the serum specimens of 75 normal volunteers or of the 91 patients with various arthritides.
ment
lmmunoabsorptlon Studies The antibody to cartilage was
removed by preincubation of the serum with fibers of Type II but not by twice the amount of Type I collagen. After this incubation, the Type II collagen fibers were treated with collagenase to destroy selectively the antigen responsible for antibody binding. After collagenase inactivation and centrifugation, the supernatant fluid was found to contain antibody to cartilage. These studies indicate that the antibody binds to Type II but not to Type I collagen and can be recovered in active form from Type II collagen fibers treated with collagenase. Similar results were obtained with the rabbit antiserum to Type II collagen used as control.
dicate that these serum specimens contain antibody reacting with helical antigenic determinants in Type II collagen. The serum from Cases 3 and 5 agglutinated erythrocytes coated with both native and denatured forms of Type II collagen (Table 2). Absorption with Type II collagen or with at (II) chains removed specifically the reactivity to each antigen. Pepsin-treated Type II collagen also inhibited the hemagglutination of erythrocytes coated with native collagen. The results suggest that the antibodies present in the serum of these two patients have specificity either for the helical antigenic determinants of the molecule or for the linear amino acid sequences in the al(II) chain. As in the immunofluorescent tests, the serum of only one of 35 patients with rheumatoid arthritis caused appreciable hemagglutination of Type II collagen and al (II) chain coated cells (titers 32 on two serum specimens and 16 on the three others). The serum specimens of all the others either were negative or had titers of 8 or less. Table 1. Indirect Immunofluorescence in Serum of Five Patients with Relapsing PolychondrItis.*
None
Hemagglutinatlon Assays and Characterization tigen
of
the An-
The hemagglutination titers correlated with the positive indirect immunofluorescent tests and in Cases 4 and 5 correlated with the activity of clinical disease (Table 2). Absorption of serum from Cases 1, 2 and 4 with native Type II collagen but not with denatured Type II collagen or with isolated at (II) chains inhibited hemagglutination of Type II coated cells. Limited cleavage of Type II collagen with pepsin did not alter its inhibitory capacity. These studies in-
IIUNOFLUOXESCENCR
Taus TaAnssN
Hyaluronidase30 min Hyaluronidase30min + collagonase 30min Hyaluronidam30min + colaan 120 n
Hyaluronidase30miu + heat-in vted cofena
PATENTS
a/n
R/PGS
_
++ ++
++ ++ ++
-
-
++
++
++
++
++ +
lCartilagno tissu. (newborn-mouse leg A human chondrowostal & trache cartie with variou enzyme. a desibed under lages) were uwd as substrate pAMethods. A positive reatio w obswved with the erum of 5 patients. Treatnt with hyaluronidase for 30 min unmasks the colsgen by removing the proteoglycn.22 Th persisten of a positive reaction with a R/PG & of histologic isg of calage (mtoxylin osin, ponodic acddchiffA alcian blue)bhows that thisrmoval is incomplto. tRabt atibody to Type II aoHagm (50 gg/ed) in sodium phosphate buffer. $Rabbit ntarum to cati proteoglyc (diluted 80 tims).
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NOV. 30, 1978
Table 2. Clinical Status, Anticartilage Antibody Titers and Circulating Immune Complexes in Patients with Relapsing Poly-
chondritis. CAsE No.
DATS
CLINICAL
TuaATMesNT
CONDmON
IF TrrERn
TrYP 11
2 3 4
5
10
40
256
2
20
64
0
Asymptomatic Asymptomatic
None Prednisone Prednisone Prodnisone
10 10
16 16
0
Acute symptoms Acute symptoms Mild symptoms Mild symptoms Asymptomatic Asymptomatic
None Prodnisone Prodnisone Prodnisone Prednisone Prednisone
320
1,024
64
160
256
80 40
64 64 64
32 32
40
32
Acute symptoms Acute symptoms Acute symptoms
None Prodnisone None
8/77 10/25/77 11/17/77 11/24/77
Acute symptoms Mild symptoms
8/22/77 10/31/77 11/14/77 12/13/77 1/78
al(n) CsAINSf
512 128 8
8/77 12/18/74 12/74
9/77
IMUNE CoMsLExBst
MicatovAssIV
HMAOGoLUTiNATION TITErn wriH ERYTHROCYTrS COATED WrrH COLsAGBNt
80
20
40
0
4 32
0
64 64 32
CIQ
PRO
13.511 NDII ND
7.91 5.2
5.2 2.4 2 2 ND 14.91
13.31 171 17.41 12.711
161 8.81 5.51 *5.1
6.511 5.1
*Antibody titers asssed by indirect immunofluoresac (IF). tevels of circulating immune complexes eatimated by a Clq binding asy or by precipitation with polyethylene glycol (PEG) - expressed in % Clq binding or in % of total IgG in PEG precipitated. tldentical results were obtained with intact as well as pepsin-treated Type Ii collagen. Identical results were obtained with aI(11) chains or heat-denatured Type II collagen prepared from intact or pepsin-treated Type 11 Collagen. ¶Significantly different from normal. (The upper 95% confidence limit in the control population is 9.9% for the Clq binding assay and 5 % for the PEG assay.) liNot done.
No hemagglutinating activity was detected in the of 15 patients with relapsing polychondritis or in 35 normal serum specimens on testing with erythrocytes coated with either native or denatured Type I, III or IV collagens, their component chains or cartilage proteoglycan. Serum specimens from patients with the other arthritides were not tested for the denatured collagens. serum
Immune Complexes
Low levels of circulating immune complexes were detected in the serum of three patients with relapsing polychondritis who had demonstrable antibodies to Type II collagen (Table 2) and in two others.
DISCUSSION In this study we have demonstrated that patients with relapsing polychondritis have circulating antibodies to Type II collagen during the acute phase of the disease. The specificity of these antibodies is evidenced by a number of circumstances. First of all, they bind only to cartilaginous matrices. Secondly, their demonstration by indirect immunofluorescence requires prior removal of proteoglycan, which is known to mask collagen.22 Thirdly, collagenase treatment of the substrate prevents binding. Fourthly, the antibodies can be completely absorbed by Type II collagen fibrils. Fifthly, the activity of these immunoabsorbed antibodies is recovered by collagenase treatment of the Type II collagen fibril-antibody complexes. Finally, passive microhemagglutination and inhibition of hemagglutination assays demonstrate the specificity of the antibodies. Antibodies to Type 1, III and IV collagens or their
constituent chains were not detected in the serum of patients with relapsing polychondritis. The antibodies to Type II collagen were found only during the acute phase of the disease, and their titers seemed to correlate with activity of disease. Prednisone therapy was clearly associated with progressive clinical improvement and coincident decreases in titers. The apparent lack of antibody to Type II collagen in the remaining 10 patients may have been the consequence of chronic immunosuppressive therapy. The occurrence of antibodies to Type II collagen appears to be almost entirely restricted to relapsing polychondritis since similar antibodies were found in only one (low titer) of 92 patients with other arthritides and in none of 75 normal volunteers. Patients with rheumatoid arthritis have been reported to have antibodies to Type I, II and III collagens and primarily against their unfolded chains.23 27-"3 This finding has been interpreted as evidence that immunization against collagen occurred after destruction of connective tissue, and that antibody formation was the consequence rather than a cause of the disease." In contrast, in some patients with relapsing polychondritis, antibodies against only native Type II collagen was found, and in the others, in whom antibodies to native and denatured Type II collagens were detected, the titers against the native Type II collagen were substantially higher than those against constituent al (II) chains. This observation suggests that the antibodies were not formed after destruction of cartilage and denaturation of collagen. Thus, the formation of antibody to Type II collagen may not be a consequence of inflammation, but may be a primary event in the pathogenesis of relapsing polychondritis.
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COLLAGEN ANTIBODIES IN POLYCHONDRITIS - FOIDART ET AL.
Cell-mediated immunity to cartilage is also thought to play a part in the pathogenesis of relapsing polychondritis since DNA synthesis by lymphocytes from these patients is stimulated by exposure to cartilage extracts.3'6 However, recent evidence indicates that patients with rheumatoid arthritis also have cell-mediated immunity, although at a low level, to native Type II and III collagens.33 The role of the circulating immune complexes in five of our patients is unclear although vasculitis, neuritis, pericarditis and damage to medium and large vessels have all been documented in cases of relapsing polychondritis. Formation of the immune complexes may be secondary to the release of collagen fragments into the circulation after tissue damage and thus may account for some of the clinical manifestations not involving cartilaginous structures. The demonstration of antibodies to Type II collagen in the serum of patients with relapsing polychondritis raises the question of whether the antibodies are functionally active in vivo or represent an epiphenomenon secondary to cartilage injury with exposure of antigen. The observation of placental transfer of relapsing polychondritis with an affected child and subsequent recovery of the offspring34 suggests an important role for antibody in this disease. Experimental autoimmunity to Type II collagen in rats24'35 results in acute arthritis, suggesting that immune responses to Type II collagen could be a factor by inciting cartilage inflammation. Finally, in vivo deposits of IgG, IgA and C3 in an inflamed cartilage of a patient with relapsing polychondritis36 support the contention that the circulating antibodies to Type II collagen described in this study could be autoantibodies involved in the pathogenesis of relapsing polychondritis. We are indebted to Mr. E. William Bere for technical assistance, to Mr. Harry Schaefer for photographic assistance, to Dr. H. Kleinman for reviewing the manuscript and to Drs. P. Plotz, 0. Lawless and L. Gerber for providing many serum specimens from patients with other arthritides.
REFERENCES 1. Pearson CM, Kline HM, Newcomer VD: Engl J Med 263:51-58, 1960
Relapsing polychondritis. N
2. McAdam LP, O'Hanlan MA, Bluestone R, et al: Relapsing polychondritis: prospective study of 23 patients and a review of the literature. Medicine (Baltimore) 55:193-215, 1976 3. Herman JH, Dennis MV: Immunopathologic studies in relapsing polychondritis. J Clin Invest 52:549-558, 1973 4. Rajapakse DA, Bywaters EGL: Cell-mediated immunity to cartilage proteoglycan in relapsing polychondritis. Clin Exp Immunol 16:497502, 1974 5. Dolan DL, Lemmon GB Jr, Teitelbaum SL: Relapsing polychondritis: analytical literature review and studies on pathogenesis. Am J Med 41:285-299, 1966 6. Hundeiker M, Brehm K, Go M: Infiltrat und Knorpelzerstorung bei polychondritis (Histochemische und immunofluoreszenz-histologische Befunde). Z Haut Geschlechtskr 45:437-443, 1970 7. Menkes C-J, Siaud J-R, Delrieu F, et al: Polychondrite chronique atrophiante: deux cas dont l'un avec cataracte et arterite des membres inf6rieurs. Ann Med Interne (Paris) 121:895-904, 1970 8. Hughes RAC, Berry CL, Seifert M, et al: Relapsing polychondritis: three cases with a clinico-pathological study and literature review. Q J Med 41:363-380, 1972
1207
9. Rogers PH, Boden G, Tourtellotte CD: Relapsing polychondritis with insulin resistance and antibodies to cartilage. Am J Med 55:243-248, 1973 10. Timpl R, Glanville RW, Nowack H, et al: Isolation, chemical and electron microscopical characterization of neutral-salt-soluble type III collagen and procollagen from fetal bovine skin. Hoppe Seylers Z Physiol Chem 356:1783-1792, 1975 11. Lapiere CM, Nusgens B, Pierard GE: Interaction between collagen type I and type III in conditioning bundles organization. Connect Tissue Res 5:21-29, 1977 12. Smith BD, Martin GR, Miller EJ, et al: Nature of the collagen synthesized by a transplanted chondrosarcoma. Arch Biochem Biophys 166:181-186, 1975 13. Miller EJ: Isolation and characterization of a collagen from chick cartilage containing three identical a chains. Biochemistry 10:1652-1659. 1971 14. Orkin RW, Gehron P, McGoodwin EB, et al: A murine tumor producing a matrix of basement membrane. J Exp Med 145:204-220, 1977 15. Piez KA, Eigner EA, Lewis MS: The chromatographic separation and amino acid composition of the subunits of several collagens. Biochemistry 2:58-66, 1963 16. Trelstad RL, Kang AH, Toole BP, et al: Collagen heterogeneity: high resolution separation of native [aI(I)12a2 and [a, (11)13 and their component a chains. J Biol Chem 247:6469-6473, 1972 17. Furthmayr H, Timpl R: Characterization of collagen peptides by sodium dodecylsulfate-polyacrylamide electrophoresis. Anal Biochem 41:510-516, 1971 18. Cuatrecasas P: Protein purification by affinity chromatography: derivations of agarose and polyacrylamide beads. J Biol Chem 245:30593065, 1970 19. Beil W, Furthmayr H, Timpl R: Chicken antibodies to soluble rat collagen. I. Characterization of the immune response by precipitation and agglutination methods. Immunochemistry 9:779-788, 1972 20. Schuurs AHWM, Van Weemen BK: Enzyme-immunoassay. Clin Chim Acta 81:1-40, 1977 21. Katz SI, Hertz KC, Yaoita H: Herpes gestationis: immunopathology and characterization of the HG factor. J Clin Invest 57:1434-1441, 1976 22. von der Mark H, von der Mark K, Gay S: Study of differential collagen
synthesis during development of the chick embryo by immunofluorescence. I. Preparation of collagen type I and type II specific antibodies and their application to early stages of the chick embryo. Dev Biol
48:237-249, 1976 23. Andriopoulos NA, Mestecky J, Wright GP, et al: Characterization of antibodies to the native human collagens and to their component a chains in the sera and the joint fluids of patients with rheumatoid arthritis. Immunochemistry 13:709-712, 1976 24. Trentham DE, Townes AS, Kang AH: Autoimmunity to type II collagen: an experimental model of arthritis. J Exp Med 146:857-868, 1977 25. Barnett EV, Chia D: Quantitation of immunoglobulin G in serum and immune complexes isolated in 4 % polyethylene glycol. Ann Rheum Dis 36:Suppl 1:26, 1977 26. Zubler RH, Lange G, Lambert PH, et al: Detection of immune complexes in unheated sera by a modified '25I-Clq binding test: effect of heating on the binding of C lq by immune complexes and application of the test to systemic lupus erythematosus. J Immunol 116:232-235, 1976 27. Steffen C, Timpl R: Antigenicity of collagen and its application in the serological investigation of rheumatoid arthritis sera. Int Arch Allergy Appl Immunol 22:333-349, 1963 28. Andriopoulos NA, Mestecky J, Miller EJ, et al: Antibodies to native and denatured collagens in sera of patients with rheumatoid arthritis. Arthritis Rheum 19:613-617, 1976 29. Michaeli D, Fudenberg HH: The incidence and antigenic specificity of antibodies against denatured human collagen in rheumatoid arthritis. Clin Immunol Immunopathol 2:153-159, 1974 30. Steffen C, Schuster F, Tausch G, et al: Weitere Untersuchungen ilber Kollagenantikorper bei Patienten mit primar chronischer Polyarthritis. Klin Wochenschr 46:976-981, 1968 31. Cracchiolo A III, Michaeli D, Goldberg LS, et al: The occurrence of antibodies to collagen in synovial fluids. Clin Immunol Immunopathol 3:567-574, 1975 32. Andriopoulos NA, Mestecky J, Miller EJ, et al: Antibodies to human native and denatured collagens in synovial fluids of patients with rheumatoid arthritis. Clin Immunol Immunopathol 6:209-212, 1976 33. Trentham DE, Dynesius RA, Rocklin RE, et al: Cellular sensitivity to collagen in rheumatoid arthritis. N Engl J Med 299:327-332, 1978 34. Arundell FW, Haserick JR: Familial chronic atrophic polychondritis. Arch Dermatol 82:439-441, 1960 35. Trentham DE, Townes AS, Kang AH, et al: Humoral and cellular sensitivity to collagen in type II collagen-induced arthritis in rats. J Clin Invest 61:89-96, 1978 36. Bergfeld WF: Relapsing polychondritis with positive direct immunofluorescence. Arch Dermatol 114:127, 1978
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