Rheumatoid factors in patients with silicosis with round nodular fibrosis of the lung in the absence of rheumatoid arthritis. With a note on the failure to induce such factors in animalsкод для вставкиСкачать
Rheumatoid Factors in Patients with Silicosis with Round Nodular Fibrosis of the Lung in the Absence of Rheumatoid Arthritis With a Note on the Failure to Induce Such Factors in Animals By WALTERSCHROEDER, EDWARD C. FRANKLIN AND CURRIER MCEWEN Sera of 10 patients with silicosis and round nodular fibrosis of the lung in the absence of rheumatoid arthritis had positive rheumatoid factor tests. The proteins responsible for this were identical or at least closely related to those seen in patients with rheumatoid arthritis. It was not possible to induce “rheumatoid factor-like’’ proteins in rats and guinea pigs by prolonged injections of silica. The possible significance of these proteins in the pathogenesis of the disease is discussed, Le sero de 10 patientes con silicosis e fibrosis a nodulos ronde del pulmon in le absentia de arthritis rheumatoide habeva positive tests pro factor rheumatoide. Le proteinas responsabile pro isto esseva identic o a1 minus intimemente relationate con illos vidite in patientes con arthritis rheumatoide. I1 non esseva possibile inducer proteinas de “typo factor rheumatoide” in rattos e porcos de India per prolongate injectiones de silice. Es discutite le signification possibile de iste proteinas in le pathogenese del morbo. P ULMONARY LESIONS have been described in patients with rheumatoid arthritis (R.A.) and have on occasion been the first and even the sole manifestation of rheumatoid disease.1-3Serologic studies of a group of such patients with diffuse interstitial pulmonary fibrosis in the absence cf rheum3toid arthritis have revealed the presence of rheumatoid factor in about 50 per cent.5 In 1953 Caplan described the presence of diffuse round nodular fibrosis of the lung, generally consisting of multiple well defined round opacities 0.55.0 cm. in diameter, in coal miners with R.A.6 Subsequent studies have demonstrated conclusively that this association is not fortuitous and have suggested that this form of silicosis is related to the rheumatoid process.7l4 The frequency of Caplan’s syndrome and of R.A. in coal miners is shown in table 1. Similar pulmonary lesions have been noted also in coal miners in the absence of arthritis,8 or have preceded the onset of arthritis by several years. The relationship of these typical pulmonary infiltrates to rheumatoid disease has been further documented by Miehlke, Dickmans and Fritze15 who found positive tests for rheumatoid factor in approximately 50 per cent of miners with the typical pulmonary lesions but without evidence of arthritis, compared to an incidence of less than 10 per cent in the control population and Frmn the Department of Medicine and the Rheumatic Ciseases Study Group, New York University College of Medicine, New York, N . Y. Supported by Grants #A-2594, A-5064jC3) und A-1 431 ( C 3 ) from the National Institute of Arthritis and Metabolic Diseases, U . S. Pubiic Health Seruice, and by The Arthritis and Rheumatism Foundation. 10 ARTHRITIS kr RHEUMATISM,VOL.5, No. 1 (FEBRUARY),1962 11 RHEUMATOID FACTORS IN PATIENTS WITH SILICOSIS Table 1.-Incidence of Rheumatoid Arthritis in Coal Miners Author Caplan (1952) Miall (1955) Fritze-Dickmans (1959) Number of coal-miners Number of cases with R.A. Number of cases with silicosis and R.A. Number of cases with Caplan’s syndrome 14000 6513 51 (0.4% ) 54 (0.7%) 51 54 13 (2570) 19 (35%) 7323 26 ( 0.37 701 26 8 (30%) in subjects with uncomplicated silicosis. In order to delineate more clearly the relationship of “rheumatoid factors” seen in these subjects to those cncountered in patients with rheumatoid arthritis and to evaluate their possible role in the pathogenesis of the disease, detailed physicochemical studies havc been performed Qn the sera of 10 such selected patients with the typical pulmonary lesions in the absence of arthritis, some of whom have been included previously by Miehlke et a1.I: These studies, reported in the first part of. this paper, demonstrate a close similarity between these factors and those seen in patients with rheumatoid arthritis. Because of the well known ability of quartz to interact with a number of proteinsl’j and to induce the formation of several agglutinating substances,li experiments were designed to study the possible role of quartz in eliciting the appearance of proteins with the properties of rheumatoid factor. In these studies, attempts have been made to induce the appearance of such suhstances in rats and guinea pigs by the injection of quartz-either alone or with a protein antigen. However, neither antibodies to homologous or heterologous gamma-globulins ncr evidence of delayed hypersensitivity to these proteins was obtained in these animals, some of which were injected with quartz for periods as long as 10 months. The results of these studies are briefly discussed in the second part of this report. Part I-Clinical and Serologic Studies of 10 Subjects with Silicosis with Round Nodular Pulmonary Infiltrates in the Absence of Rheumatoid Arthritis MATERIALS AND PV~ETHODS A. Sera Sera* were obtained from 10 coal miners with round pulmonary foci, but without evidence of arthritis, selected from a larger group of patients. Their exposure to dust ranged from 12 to 33 years, and their ages from 48 to 63 years. In all subjects, specimens of sputum, gastric lavage and laryngeal washings were examined on several occasions for tubercle bacilli, but with negative results. B. Serologic Tests: The sera were inactivated at 56 C. for 30 minutes and absorbed repeatedly with packed sheep cells for 60 minutes at 37 C. until they no longer agglutinated unsensitized sheep cells. Englobulin fractions were obtained by dialysis, as described by Ziff and co-workers.18 The agglutination of sensitized sheep cells by tht‘ euglobulin fraction (SCA), and the *The sera used in this study were kindly provided by Dr. H. Dicknians, Med. Klinik, Bergmannsheil, Bochum, Germany. 12 SCHROEDER, FRANKLIN AND MC EWEN inhibition by the euglobulin of agglutination of a positive rheumatoid serum, were carried out according to the method of Ziff and co-workers.l* The latex fixation test was performed following the method of Singer and Plotzlg and the capillary latex test as described by Tanner and Ziff.20 Gamma globulin was determined by using the modification of the zinc turbidity test21 and by paper electrophoresis. C. Ultracentrifugation: Analytical examination of sera containing the highest titers by each of the serologic tests was carried out in a Spinco Model E ultracentrifuge using cells with double sector centerpieces.22 Preparative density gradient ultracentrifugation was performed in a Spinco Model L ultracentrifuge in a SW-39 swinging bucket rotor through a NaCl gradient formed with 21, 14 and 7 per cent saline. The amount of protein in each of the fractions was estimated by the modified Folin technic23 and 7 s and 19s gamma globulins were located with specific antisera to 7s and 19s gamma globulin. The determination of agglutinating activity of the fractions was carried out using the sensitized sheep cell agglutination reaction and the latex fixation test18219 after removal of salt by dialysis. RESULTS In table 2 are listed the results of the serologic tests obtained with the sera from these 10 subjects. Each of them had far advanced round nodular fibrosis of the lung (silicosis stage HI), and none had any clinical evidence of arthritis, other collagen disease or liver disease which might conceivably account for these findings. All 10 sera were positive with the sheep cell inhibition test, and eight were positive by the SCA test. Similarly, when human gamma globulin was used in the latex fixation or capillary latex test, eight of 10 sera were positive. Five of the 10 subjects had elevated levels of gamma globulin. Examination of four of the sera with the highest titers (Gl, Me, Ni, Ho) in the analvtical ultracentrifuge revealed a small amount of a peak sedimenting ahead of the 19s peak in only one. None of the other three contained detectable amounts of a 22s peak or peaks with s rates intermediate between 7s and 19s. This low incidence of detectable 22s material is probably due to tlle lack of sensitivity of the ultracentrifuge as an analytical tool since previous studies have shown that it generally can detect these peaks only in high titer sera.22In order to study further the physicocochemical properties of the agTable B.-Serologic Name HO GR SCHO WA DI NI ME GL SCHI BE Normal Sheep cell agglutination reaction reciprocal titer 112 28 14 56 112 224 224 224 14 28 <14 Findings in Ten Selected Coal Miners with Round Nodular Pulmonary Infiltrates Sheep cell inhibition reciprocal titer Capillary latex fixation test 0 0 0 0 0 0 0 0 0 0 >7 + negative negative negative Latex fixation test reciprocal titer Zinc turbidity 640 320 20 20 80 2560 1280 2560 20 20 21 30 27 20 10 9 22 16 11 20 <20 ________ (U) 13 RHEUMATOID FACTORS I N PATIENTS WITH SILICOSIS Table 3.-Fractionation Tube TOP 10 9 8 Anti 7 S gammaglobulin - + ++ +++ +++ +++ ++ 7 6 5 4 Bottom 3 2 1 of 3 Sera from Patients with Advanced Silicosis Anti 19 S gammaglobulin - + +++ +++ ++f 3= i & -+ SCA 0 0 0 0 0 0 0 0 0 7 GL Latex 0 0 0 0 0 0 10 20 10 0 SCA NI Latex SCA ME Latex 0 0 0 0 0 0 0 0 0 0 7 0 0 0 0 0 0 0 20 20 0 0 0 0 0 0 0 0 0 28 14 14 7 0 0 20 20 10 7 0 glutinating factors, five of the sera were separated into fractions with different sedimentation properties by centrifugation in a saline density gradient. The location of the different fractions was determined with specific antisera to albumin, 7s gamma globulin and 19s gamma globulin. After dialysis to remove the excess salt, each of the fractions was tested by the SCA and latex fixation test. In each of the sera examined, serologic activity by both tests was found in the more rapidly sedimenting fractions containing the 19s gamma globulin, and none of the activity remained in the upper part of the tube which contained, however, the bulk of the protein. The results obtained with three of the sera are shown in table 3. Since it is well recognized that this group of proteins is readily dissociated into 7s units which are no longer serologically active by treatment with sulfhydryl r e a g e n t ~ , 2 ~each J ~ of the sera was treated with 0.1 M 2 mercaptoethanol and then tested by the serologic tests. In each of the sera there was complete loss of serologic activitv following reductive cleavage, thus adding further evidence that these pr -teins belonged to the 19s class of gamma globulins and were similar in all respects to rheumatoid factors in patients with rheumatoid arthritis. Part II-Experimental Studies To determine the possible relationship between silicosis and the presence of rheumatoid factor, experiments were performed in rats and guinea pigs in an effort to induce similar factors experimentally by the administration of quartz particles. Previously, many investigatorsz6-36have studied the effects in animals of the injection of quartz and have demonstrated the presence, in sera of some of these, factors that agglutinate quartz particles, zymozanl7 and possibly also factors that react specifically with proteins known to coat quartz If the injection of quartz would cause the production of rheuparticle~.l6*~"-"" matoid factor-like substances in animals, it would be strong evidence for its possible role in the production of such substances in man. Negative resuIts, while not conclusive per se, would favor the concept that rheumatoid factor may have antedated the silicosis and may simply reflect a predisposition of the subject to react to certain exogenous stimuli in an unusual way. 14 SCHROEDER, FRANKLIN AND MC EWEN Table 4.-Course of Injection and Results of Fraction 11 Test and Skin Test in Guinea Pigs Number of guinea pigs Injection of cristobalite i.p. 21 50mg. 14 75 mg. 12 40 20 Total: 107 Table 5.-Course Number of rats 27 10 7 Injection Injection of tridymite of NaCl i.v. after i.p. 27 wks. 10 my. 10 mg. 1.0 cc. 1.0 c c . negative negative neaatioe 1.0 cc. of Skin test for delayed hypersensitivity after 8,16,38 after weeks 7 % weeks Fraction I1 Test ( Heller et al. ) after 6,13,27,38 after weeks R weeks 40 ma. negative negative negative negative negative negative negative Injection and Results of Fraction I1 Test and Skin Test in Rats Injection of cristobalite i.p. i.v. 50mg. 30mg. Injection of NaCl i.v. after 27 wks. Injection of 1 mg. ovalbumin 2 x weekly for 2 months 2nd Injection of cristobalite i.p. after 8 weeks 50 mg. 20mg. 16mg. 16 mg. Skin test for Injection Fraction I1 test delayed hyperof tridymite (Heller et al.) sensitivity i.v. after after 8,14, after 5,12, 27 weeks 27.36 weeks 36 weeks 10 mg. 10 mg. 10 mg. negative negative negative negative negative negative Total: 44 MATERIALS AND METHODS Quartz dnst (cristobalite and tridymite, approximate particle size %I0 p ) were suspended in normal saline to make a concentration of 50 mg./ml. and sterilized by autoclaving." One hundred and seven male guinea pigs weighing between 300-400 Gm. and 44 male rats weighing 200-300 Gm. were used and were bled by cardiac puncture before injection. The course of injection and the results are listed in tables 4 and 5. Twenty-one guinea pigs were injected intraperitoneally with 50 mg. of Cristobalite and 14 guinea pigs intraperitoneally with 75 mg. of cristobalite. Twelve guinea pigs injected with 1.0 cc. of normal saline served as controls. After 27 weeks the same animals were injected intravenously with 10 mg. of Tridymite and the controls with 1.0 cc. of normal saline. The guinea pigs were bled by cardiac puncture 6, 1.3, 27 and 38 weeks after the first injection. Another group of 40 guinea pigs was injected with 40 mg. of tridymite intravenously and 20 controls with 1.0 ce. of normal saline. Their sera were examined after eight weeks. Twenty-seven rats were injected twic.e intraperitoneally with 508 mg. cristobalite during an eight week period. After 27 weeks they received 10 mg. tridymite intravenously. Ten rats were injected with 30 mg. of cristobalite intraperitoneally as well as 20 mg. cristobalite intravenously at the same time, followed after 27 weeks with 10 nig. tridymite intravenously. Seven rats served as controls and received 1.0 cc. of normal saline instead of quartz. In view of the possible role of quartz as an adjuvant,37 the last group of 10 rats as well as the control rats were given 1.0 nig. ovalbumin intraperitoneally twice weekly for a period of two months. The rats were bled by cardiac puncture after 8, 14, 27 and 36 weeks and their sera tested for ability to react with hoinologous gamma globulin as well as with heterologous human gamma globulin. The test with human globulin was performed with latex particles coated with Cohn Fraction I1 (Squibb), as dericribed by Singer and Plotz.19 Attempts to use guinea pig or rat gamma globulin in the latex fixation test were not successful since these proteins in the native state as well as after heat inactivation at 57 C. for 30' agglutinated the parti "We are indebted to Dr. E. Fritze, Mtd. KIinik, Bergmannsheil, Bochum, Germany, and Dr. B. Pernis, Clinica dei Lavoro, Milan, Italy, for supplying the Cristobalite and Tridynlitc.. RHEUMATOID FACTORS I N PATIENTS WITH SILICOSIS 15 spontaneously over a p H range from 5.1 to 10.0. For this reason tanned sheep cells coated with guinea pig or rat gamma globulin were used. These were prepared according to the method of Heller and co-workers38 and were not agglutinated spontaneously by these proteins. In order to determine the development of delayed hypersensitivity to gamma globulin, all of the animals were tested at three different periods by intradermal injection of 0.1 cc. of a solution of 50 gamma/nil. of homologous gamma globulin, ovalbumin and normal saline. The guinea pigs were tested after 8, 16 and 38 weeks and the rats after 5, 12 and 36 weeks. The reaction was noted 8 hours and 24 hours after each injection. RESULTS All of these studies were entirely negative. None of the animals developed any substance capable of agglutinating homologous or heterologous gamma globulins and none of the animals developed evidence of immediate or delayed hypersensitivity in spite of the presence of silicotic nodules in the lungs, livers, spleens and peritoneum of many. Since completion of this study, essentially similar results have been published by Voisin and c o - w o r k e r ~ . ~ ~ DISCUSSION The finding of pc\sitive rheumatoid factor tests in about one-half of 28 miners with round nodular fibrosis of the lung in the absence of arthritis raises two major questions: first, whether th'e proteins in these subjects are similar to those seen in rhumatoid arthritis and, secondly, the possible role they may play in the pathogenesis of these lesions. It was possible by using a variety of serologic tests as well as analytic and d,ensity gradient ultracentrifugation to demonstrate the presence of a high molecular weight protein which seems to be identical with the rheumatoid factor. A comparison of the results obtained from the 10 sera of coal miners and the serologic reactions and ultracentrifugal patterns from definite rheumatoid sera indicates a close relationship between the rheumatoid factors and the proteins present in the serum of coal miners suffering from round nodular fibrosis of the lung. The positive serologic findings in cases of full blown Caplan's syndrome are due to the rheumatoid arthritis. It is unlikely that the inhalation of quartz dust during a long-stsnding exposure can be responsible for the relatively high incidence of positive serologic tests in cases found with the characteristic Caplan chest x-ray without arthritis, since the number of positive agglutination tests occur no more frequently in coal miners with the common type of pneumoconiosis than in the general population.15 Furthermore, it is worthy of note that none cf the laboratory animals with experimental silicosis studied over a period cf nine and onehalf months gave positive reactions after injection of concentrated and highly active quartz. Many of these animals had gross evidence of silicotic nodules in the lungs. liver, peritoneum and other organs, and previous studi~es29~32,33*35 have demonstrated the high incidence of silicosis in animals treated with a similar schedule of injection. These findings in pati,ents and in the experimental animals therefore offer strong evidence against the hypothesis that silica in the form employed is responsible for the production of positive rheumatoid factor tests. Nevertheless it seems possible that prolonged exposure to silica may play an as yet unde- 16 SCHROEDER, FRANKLIN AND MC EWEN fined role in the d,evelopment of these lesions and circulating factors in view of its ability to serve as an adjuvant,37to induce the formation of substances agglutinating zymozan and other particles,li and to alter the antigenic prop, ~ ~ , ~the ~ j , injection ~~ into rabbits and guinea pigs erties of p r o t e i n s . ’ ~ , z ~ ’While of denatured autologous and homologous gamma globulins can lead to the development of circulating antibodie~,~~) as well as delayed hyper~ensitivity~l directed against heterologous as well as homologous gamma globulins, silica in the form and amount used does not appear to exert such an effect. An alternate explanation for the high correlation between rheumatoid factor and the characteristic pulmonary lesion seen in these coal miners, as well as in subjects with idiopathic diffuse pulmonary fibrosis, may be that the presence of rheumatoid factor may simply be an index of a predisposition to an abnormal tissue response of the individual. There is abundant evidence from many observers that rheumatoid factors are found in a high per cent of asymptomatic relatives of patients with R.A. and other collagen diseases,42and it seems quite conceivable that this material may therefore be an index of an altered host response, possibly genetically determined. Studies are currently in progress to determine the incidence of rheumatoid factors in relatives of patients with Caplan’s syndrome as well as those with the typical pulmonary picture without arthritis described in this report. SUMMARY Sera of 10 selected coal miners suffering from round nodular fibrosis of the iung in pneumoconiosis without joint disease were chosen for serologic and ultracentrifug,al studies. Various serologic tests used for the diagnosis of rheumatoid arthritis gave positive results in each case. Bv density gradient ultracentrifugation the presence of a high molecular weight protein complex was demonstrated. This was confirmed by a complete loss of serologic activity after dissociation with mercaptoethanol. In one of the cases with the highest titer which was examined in the analytical ultracentrifuge, it was possible to demonstrate a peak with a sedimentation coefficient of 22s. The accumulated evidence suggests that the agglutinating property of the sera examined in the present study is identical or at least closely related to the rheumatoid factors. After intravenous and intraperitoneal injection of fine quartz dust in 107 guinea pigs and 44 rats, 110evidence of delayed hypersensitivity or circulating factors reactive with homologous or heterologous gamma globulin was obtained. Possible relationships between rheumatoid arthritis and pulmonary abnormalities in coal miners with pneumoconiosis are briefly discussed. REFERENCES I . Ellmann, P., and Ball, R. E.: “Rheumatoid disease” with joint and pulmonary manifestations. Brit. M. J. 2:816, 1948. 2. Rubin, E. H.: Pulmonary lesions in “rheumatoid disease” with remarks on diffuse interstitial pulmonary fibrosis. Am. J. Med. 19:569, 1955. RHEUMATOID FACTORS I N PATIENTS WITH SILICOSIS 3. Cruickshank, B.: Interstitial pneumonia and its consequences in rheumatoid disease. Brit. J. Dis. Chest 53:226, 1959. 4. Aronoff, A., Bywaters, E. G. L., and Fearnley, G. R.: Lung lesions in rheumatoid arthritis. Brit. M. J. 2: 228, 1955. 5. Franklin, E. 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Hyg. 145:92, 1958. 36. Powell, D. E. B., and Gough, J . : The effect on experimental silicosis of hypersensitivity induced by horse serum. Brit. J. Exper. Path. 1:40, 1959. 37. Kikuth, W.: Neuere Ergebnisse der Silikoseforschung. Deutsch. med. Wchnschr. 85:917, 1960. 38. Heller, G., Kolodny, M. H., Lepow, I. H., Jacobson, A. S., Rivera, M. E., Marks, G. H.: The Hemagglutination Test for rheumatoid arthritis. J. Immunol. 74:340, 1955. 39. Voisin, G. A., Collet, A., Daniel-Moussard, H., and Toullet, F.: Recherches sur un pouvoir antigene et une capaciti. inductrice d'auto-antigenes de quarz et de I'oxyde de Beryllium. Rev. FranG &Etudes Clin. et Biolog. 6:252, 1961. 40. Milgrom, F., and Witebsky, E . : Studies on the rheumatoid and related serum factors. J. A .M. A. 174:56, 1960. 41. Miller, F., McCluskey, R. T., and Benacerraf, B.: Autosensitization to denatured guinea pig gamma-globulin. Fed. Proc. 20:37, 1961. 42. Ziff, M.: Genetics, hypersensitivity and the connective tissue disease. Am. J. Med. 30:1, 1961. Walter Schroeder, M.D., Fellow, Rheumatic Diseases Study Group, New York University School of Medicine. Present address: Mecl. Klinik, Bergmannsheil, Bochum, Germany. Edward C . Franklin, M.D., Assistant Professor of Medicine, New York University School of Medicine, and Senior Investigator, Art'hhritis and Rheumatism Foundation, New York, N. Y. Currier McEwen, M.D., Professor of Medicine and Chairman, Rheumatic Diseases Study Group, New York University School of Medicine, New York, N . Y .