British Journal of Dermatology (1979) 100, 127.
The ribonucleic acid (RNA) skin test in systemic lupus erythematosus and other connective tissue diseases E.A.JOHANSSON, K.-M.NIEMI AND H.HALME Department of Dermatology, University Central Hospital, Snellmaninkatu 14, 00170 Helsinki 17, Finland Accepted for publication 6 June 1978
SUMMARY
A series of 65 patients with different autoimmune diseases was examined using different RNAsolutions for intradermal skin tests. Clinically positive results were obtained most often in patients with mixed connective tissue disease but quite often also in patients with systemic lupus erythematosus and progressive systemic sclerosis or with some symptoms of an autoimmune nature. The histological examination of the biopsies from the test sites revealed that there was no correlation between the clinically positive tests and the histological criteria usually used as a sign of a positive test.
In typical cases the differential diagnosis of rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and progressive systemic sclerosis (PSS) does not afford difficulties. Increasing attention has been paid to patients with overlapping features; mixed connective tissue disease (MCTD) has been described as a distinct entity. Serologically a speckled pattern of antinuclear fluorescence and the presence of antibody to the soluble fraction ofthe nuclear antigen (pNA) have been defined as characteristic (Sharp et al, 1972). However, these antibodies also occur in SLE and the methods for their further differentiation are time-consuming and unavailable for routine work (Parker, 1973; Reichlin 1976; Farber & Bole, 1976; Winkelmann, Carapeto & Jordon, 1977). The good results with DNA skin tests in patients with SLE (Johansson, Niemi & Lassus, 1975b) encouraged the undertaking of the present investigation into the value of the ribonucleic acid (RNA) skin test in a series of patients with various autoimmune diseases.
PATIENTS AND METHODS
The patients were hospitalized at the Department of Dermatology, Helsinki University Central Hospital during 1973-1976 because of definite or suspected autoimmune disease. Altogether 65 0007-0963/79/0200-0127S0.200
© 1979 British Association of Dermatologists 127
128
E.A.Johansson, K.-M.Niemi and H.Halme
patients (58 women) and 12 controls were tested. The age of the patients varied from 20 to 71 years with a mean of 417 years. In addition to the laboratory tests required for the diagnosis of SLE (Cohen et al, 1971) the RNA antibodies, cryoglobulins, rheumatoid factor, mitochondrial and smooth muscle antibodies and serum complement levels were repeatedly determined (Schur & Munroe, 1969J Wager et al, 1971) and the diagnosis was based on these findings as well as on clinical features. For skin testing three different RNA preparations were used: RNA from calf liver (Sigma chemicals no. 7250), RNA from baker's yeast (Schwartz/Mann no. 906762) and synthetic RNA Poly (I) Poly (C) (Miles Laboratories no. 11-312). The test solutions were prepared as described previously (Johansson et al, 1975a). o-i ml of the o-i% test solution was injected intradermally into the thigh of each patient. The tests were read at 6, 8, 10 and 24 h and in most cases at 48 h. The reaction was considered positive if the induration was over 10 mm as measured in two perpendicular directions. Erythema was quite distinct in most of the cases during the first few hours and subsided rather slowly. Because it did not correlate with the size of the induration it was not taken into consideration in the final results. Altogether 57 biopsies from the test sites were taken from 51 patients. In all instances the histological evaluation was carried out without knowledge of the size of the induration or the patient's diagnosis. The biopsy specimens were divided and one half was examined with routine methods, while the other half was snap frozen for immunofluorescence studies performed as described previously (Reunala et al, I9ii). Necrosis of the vessel wall and diffuse infiltration of polymorphonuclear leukocytes were taken as the criteria for a positive test reaction. RESULTS
The clinical results of the RNA skin tests are presented in Table i with reference to the diagnoses of the patients. Because there was no marked deviation between test results with the different antigens they were not evaluated separately. Of the 145 tests, 29 gave a clinically positive test result. The frequency of positive tests was highest in the MCTD group, in which half of the patients demonstrated a positive reaction. Of patients with definite or probable SLE about one fifth had a positive RNA skin test. TABLE I. The clinical results of the RNA skin tests in relation to the diagnoses of the patients
Diagnosis Definite SLE Probable SLE
MCTD CBFP reactors*
PSSt NCTDJ Dermatomyositis Controls
No. of patients
No. of tests
Clinically positive tests
i8
31 23 17 13
6 9
II
9 9 8 7 3 12
II
5 2
13
4 3
7
0
30
0
* Patients with chronic biological false positive reactions for syphilis. t Progressive systemic sclerosis. X Nondefinite connective tissue disease.
RNA skin test in systemic lupus erythematosus
129
Biopsies from the clinically positive test sites were taken in 23 cases but only 10 tests were also histologically positive. From the clinically negative test sites biopsies were taken in 34 cases, of which 20 were histologically negative and 14 positive. Oedema of the vessel wall or a dilated lumen were occasionally observed but these signs were not considered significant. In some of the reactions necrosis and large numbers of neutrophils were found in the dermal connective tissue, but if the vascular wall was intact this was interpreted as negative. Direct immunofluorescence studies of 47 test site biposies revealed IgG in 3 cases, IgM in 6, IgA in 2, and C3 in 4 cases at the basement membrane; on the dermal capillaries IgM was found in 7 cases, IgA in i case and C3 in 5. In 4 cases epidermal cells showed nuclear epidermalfluorescenceas described by Gilliam & Prystowsky (1977). Three of these patients had MCTD and one SLE. Of the 12 control patients 3 were healthy, 3 had primary syphilis, 3 had mild toxic dermatitis and 3 patients had acne. None had a clinically positive test but 2 of the 5 examined were histologically positive. One ofthe latter had primary syphilis and had previously suffered urticaria; the other was an apparently healthy brother of a MCTD patient. Treatment with steroids or antimalarial drugs did not aflfect test results.
COMMENT
Clinically positive results with RNA skin tests were obtained in both MCTD and SLE patients, just as RNA antibodies in the serum have been found in both disorders (Schur & Monroe, 1969). The correlation between clinically and histologically positive reactions in this study was poor, because ofthe extensive necrosis found in dermal tissue causing difficulties in histological interpretation. The reason for this necrosis is not known and was not seen to the same extent when skin tests with DNAsolutions were made (Johansson, Niemi & Lassus, 1975b). The RNA skin test, at least with the antigens used in this series, can not be considered of diagnostic value in the differentiation of SLE and other connective tissue diseases of autoimmune nature.
REFERENCES COHEN, A.S., REYNOLDS, W.E., FRANKLIN, E . C , KULKA, J.P., ROPES, W.W., SHULMAN, L.E. & WALLACE, S.L.
(1971) Preliminary criteria for the classification of systemic lupus erythematosus. Bulletin on the Rheumatic Diseases, 21, 643. FARBER, S.J. & BOLE, G.G. (1976) Antibodies to components of extractable nuclear antigen. Clinical characteristics of patients. Archives of Internal Medicine, 136, 425. GILLIAM, J.N. & PRYSTOWSKY, D.S. (1977) Mixed connective tissue disease syndrome. Archives of Dermatology, " 3 , 383JOHANSSON, E.A., NIEMI, K . - M . & LASSUS, A. (1975b) The deoxyribonucleic acid (DNA) skin test in systemic lupus erythematosus. 2. Histological findings. British Journal of Dermatology, 93, 451. JOHANSSON, B.A., KUSTALA, U . , NIEMI, K . - M . , HALME, H . & LASSUS, A. (1975a) The deoxyribonucleic acid
(DNA) skin test in systemic lupus erythematosus i. Clinical evaluation. British Journal of Dermatology, 93, 443PARKER, M.D. (1973) Ribonucleoprotein antibodies: frequency and clinical significance in systemic lupus erythematosus, scleroderma and mixed connective tissue disease. Journal of Laboratory and Clinical Medicine, 82, 769. REICHLIN, M . (1976) Problems in differentiating SLE and mixed connective tissue disease. New England Journal of Medicine, 295, 1194. REUNALA, T . , KARVONEN, J., TIILIKAINEN, A. & SALO, O.P. (1977) Herpes gestationis. British Journal of Der-
matology, 96, 563. SCHUR, P.H. & MUNROE, M . (1969) Antibodies to ribonucleic acid in systemic lupus erythematosus. Proceedings of the National Academy of Sciences of the United States of America, 63, 1108. SHARP, G.C, IRVIN, W.S., TAN, E.M., GOULD, R.G. & HOLMAN, H.R. (1972) Mixed connective tissue disease
130
E.A.Johansson, K.-M.Niemi and H.Halme
—an apparently distinct rheumatic disease syndrome associated with a specific antibody to an extractable nuclear antigen (ENA). American Journal of Medicine, 52, 148. WAGER, C , RASANEN, J.A., HALTIA, K . & WASASTJARNA, C. (1971) M-components with antibody activity. Antismooth muscle, anti-thyroglobulin and anti-streptolysin-0 activity in five M-component sera. Annals of Clinical Research, 3 1 , 86.
WINKELMANN, R.K., CARAPETO, F . J . & JORDON, R.E. (1977) Direct immunofluorescence in the diagnosis of
scleroderma syndromes. British Journal of Dermatology, 96, 231.
The ribonucleic acid (RNA) skin test in systemic lupus erythematosus and other connective tissue diseases E.A.JOHANSSON, K.-M.NIEMI AND H.HALME Department of Dermatology, University Central Hospital, Snellmaninkatu 14, 00170 Helsinki 17, Finland Accepted for publication 6 June 1978
SUMMARY
A series of 65 patients with different autoimmune diseases was examined using different RNAsolutions for intradermal skin tests. Clinically positive results were obtained most often in patients with mixed connective tissue disease but quite often also in patients with systemic lupus erythematosus and progressive systemic sclerosis or with some symptoms of an autoimmune nature. The histological examination of the biopsies from the test sites revealed that there was no correlation between the clinically positive tests and the histological criteria usually used as a sign of a positive test.
In typical cases the differential diagnosis of rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and progressive systemic sclerosis (PSS) does not afford difficulties. Increasing attention has been paid to patients with overlapping features; mixed connective tissue disease (MCTD) has been described as a distinct entity. Serologically a speckled pattern of antinuclear fluorescence and the presence of antibody to the soluble fraction ofthe nuclear antigen (pNA) have been defined as characteristic (Sharp et al, 1972). However, these antibodies also occur in SLE and the methods for their further differentiation are time-consuming and unavailable for routine work (Parker, 1973; Reichlin 1976; Farber & Bole, 1976; Winkelmann, Carapeto & Jordon, 1977). The good results with DNA skin tests in patients with SLE (Johansson, Niemi & Lassus, 1975b) encouraged the undertaking of the present investigation into the value of the ribonucleic acid (RNA) skin test in a series of patients with various autoimmune diseases.
PATIENTS AND METHODS
The patients were hospitalized at the Department of Dermatology, Helsinki University Central Hospital during 1973-1976 because of definite or suspected autoimmune disease. Altogether 65 0007-0963/79/0200-0127S0.200
© 1979 British Association of Dermatologists 127
128
E.A.Johansson, K.-M.Niemi and H.Halme
patients (58 women) and 12 controls were tested. The age of the patients varied from 20 to 71 years with a mean of 417 years. In addition to the laboratory tests required for the diagnosis of SLE (Cohen et al, 1971) the RNA antibodies, cryoglobulins, rheumatoid factor, mitochondrial and smooth muscle antibodies and serum complement levels were repeatedly determined (Schur & Munroe, 1969J Wager et al, 1971) and the diagnosis was based on these findings as well as on clinical features. For skin testing three different RNA preparations were used: RNA from calf liver (Sigma chemicals no. 7250), RNA from baker's yeast (Schwartz/Mann no. 906762) and synthetic RNA Poly (I) Poly (C) (Miles Laboratories no. 11-312). The test solutions were prepared as described previously (Johansson et al, 1975a). o-i ml of the o-i% test solution was injected intradermally into the thigh of each patient. The tests were read at 6, 8, 10 and 24 h and in most cases at 48 h. The reaction was considered positive if the induration was over 10 mm as measured in two perpendicular directions. Erythema was quite distinct in most of the cases during the first few hours and subsided rather slowly. Because it did not correlate with the size of the induration it was not taken into consideration in the final results. Altogether 57 biopsies from the test sites were taken from 51 patients. In all instances the histological evaluation was carried out without knowledge of the size of the induration or the patient's diagnosis. The biopsy specimens were divided and one half was examined with routine methods, while the other half was snap frozen for immunofluorescence studies performed as described previously (Reunala et al, I9ii). Necrosis of the vessel wall and diffuse infiltration of polymorphonuclear leukocytes were taken as the criteria for a positive test reaction. RESULTS
The clinical results of the RNA skin tests are presented in Table i with reference to the diagnoses of the patients. Because there was no marked deviation between test results with the different antigens they were not evaluated separately. Of the 145 tests, 29 gave a clinically positive test result. The frequency of positive tests was highest in the MCTD group, in which half of the patients demonstrated a positive reaction. Of patients with definite or probable SLE about one fifth had a positive RNA skin test. TABLE I. The clinical results of the RNA skin tests in relation to the diagnoses of the patients
Diagnosis Definite SLE Probable SLE
MCTD CBFP reactors*
PSSt NCTDJ Dermatomyositis Controls
No. of patients
No. of tests
Clinically positive tests
i8
31 23 17 13
6 9
II
9 9 8 7 3 12
II
5 2
13
4 3
7
0
30
0
* Patients with chronic biological false positive reactions for syphilis. t Progressive systemic sclerosis. X Nondefinite connective tissue disease.
RNA skin test in systemic lupus erythematosus
129
Biopsies from the clinically positive test sites were taken in 23 cases but only 10 tests were also histologically positive. From the clinically negative test sites biopsies were taken in 34 cases, of which 20 were histologically negative and 14 positive. Oedema of the vessel wall or a dilated lumen were occasionally observed but these signs were not considered significant. In some of the reactions necrosis and large numbers of neutrophils were found in the dermal connective tissue, but if the vascular wall was intact this was interpreted as negative. Direct immunofluorescence studies of 47 test site biposies revealed IgG in 3 cases, IgM in 6, IgA in 2, and C3 in 4 cases at the basement membrane; on the dermal capillaries IgM was found in 7 cases, IgA in i case and C3 in 5. In 4 cases epidermal cells showed nuclear epidermalfluorescenceas described by Gilliam & Prystowsky (1977). Three of these patients had MCTD and one SLE. Of the 12 control patients 3 were healthy, 3 had primary syphilis, 3 had mild toxic dermatitis and 3 patients had acne. None had a clinically positive test but 2 of the 5 examined were histologically positive. One ofthe latter had primary syphilis and had previously suffered urticaria; the other was an apparently healthy brother of a MCTD patient. Treatment with steroids or antimalarial drugs did not aflfect test results.
COMMENT
Clinically positive results with RNA skin tests were obtained in both MCTD and SLE patients, just as RNA antibodies in the serum have been found in both disorders (Schur & Monroe, 1969). The correlation between clinically and histologically positive reactions in this study was poor, because ofthe extensive necrosis found in dermal tissue causing difficulties in histological interpretation. The reason for this necrosis is not known and was not seen to the same extent when skin tests with DNAsolutions were made (Johansson, Niemi & Lassus, 1975b). The RNA skin test, at least with the antigens used in this series, can not be considered of diagnostic value in the differentiation of SLE and other connective tissue diseases of autoimmune nature.
REFERENCES COHEN, A.S., REYNOLDS, W.E., FRANKLIN, E . C , KULKA, J.P., ROPES, W.W., SHULMAN, L.E. & WALLACE, S.L.
(1971) Preliminary criteria for the classification of systemic lupus erythematosus. Bulletin on the Rheumatic Diseases, 21, 643. FARBER, S.J. & BOLE, G.G. (1976) Antibodies to components of extractable nuclear antigen. Clinical characteristics of patients. Archives of Internal Medicine, 136, 425. GILLIAM, J.N. & PRYSTOWSKY, D.S. (1977) Mixed connective tissue disease syndrome. Archives of Dermatology, " 3 , 383JOHANSSON, E.A., NIEMI, K . - M . & LASSUS, A. (1975b) The deoxyribonucleic acid (DNA) skin test in systemic lupus erythematosus. 2. Histological findings. British Journal of Dermatology, 93, 451. JOHANSSON, B.A., KUSTALA, U . , NIEMI, K . - M . , HALME, H . & LASSUS, A. (1975a) The deoxyribonucleic acid
(DNA) skin test in systemic lupus erythematosus i. Clinical evaluation. British Journal of Dermatology, 93, 443PARKER, M.D. (1973) Ribonucleoprotein antibodies: frequency and clinical significance in systemic lupus erythematosus, scleroderma and mixed connective tissue disease. Journal of Laboratory and Clinical Medicine, 82, 769. REICHLIN, M . (1976) Problems in differentiating SLE and mixed connective tissue disease. New England Journal of Medicine, 295, 1194. REUNALA, T . , KARVONEN, J., TIILIKAINEN, A. & SALO, O.P. (1977) Herpes gestationis. British Journal of Der-
matology, 96, 563. SCHUR, P.H. & MUNROE, M . (1969) Antibodies to ribonucleic acid in systemic lupus erythematosus. Proceedings of the National Academy of Sciences of the United States of America, 63, 1108. SHARP, G.C, IRVIN, W.S., TAN, E.M., GOULD, R.G. & HOLMAN, H.R. (1972) Mixed connective tissue disease
130
E.A.Johansson, K.-M.Niemi and H.Halme
—an apparently distinct rheumatic disease syndrome associated with a specific antibody to an extractable nuclear antigen (ENA). American Journal of Medicine, 52, 148. WAGER, C , RASANEN, J.A., HALTIA, K . & WASASTJARNA, C. (1971) M-components with antibody activity. Antismooth muscle, anti-thyroglobulin and anti-streptolysin-0 activity in five M-component sera. Annals of Clinical Research, 3 1 , 86.
WINKELMANN, R.K., CARAPETO, F . J . & JORDON, R.E. (1977) Direct immunofluorescence in the diagnosis of
scleroderma syndromes. British Journal of Dermatology, 96, 231.
