Rheumm!ogY

Rheumatol Int (1987) 7:123-126

Clinical and Experimental Investigations

© Springer-Verlag 1987

Organ-specific autoantibodies in non-organ-specific autoimmune diseases with special reference to rheumatoid arthritis P. Youinou ~, W. Mangold 2, j. Jouquan 3, H. Swirsky z, P. Le Goff ~, and W. A. Scherbaum 2 1 Department of Immunology, University Hospital Medical School, BP 824, F-29285 Brest Cedex, France 2 Department of Internal Medicine I, University Hospital of Ulm, D-7900 Ulm, Federal Republic of Germany 3 Department of Rheumatology, University Hospital Medical School, BP 824, F-29285 Brest Cedex, France Received October 30, 1986 / Accepted April 8, 1987

Summary. Sera from 367 patients with rheumatoid arthritis (RA) and from 102 patients with other non-organspecific (NOS) autoimmune diseases were examined for the presence o f organ-specific (OS) autoantibodies. The incidence of these OS autoantibodies was not increased in patients with NOS autoimmune diseases with the exception of thyroglobulin antibodies, which were significantly more frequent in RA (P < 0.001) and in Sj6gren's syndrome (P < 0.05) patients than in normal controls. Investigation of 169 patients with OS autoimmune diseases did not reveal an increased prevalence o f NOS autoantibodies. In RA patients, OS autoantibodies correlated with N O S autoantibodies (P < 0.04) and with H L A - D R 3 antigen (P < 0.01).

Key words: Rheumatoid arthritis - Thyroglobulin antibodies

Introduction Autoimmune disorders are characterized by clinical and serological overlap. This applies to both non-organ-specific (NOS) diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and primary Sj6gren's syndrome (SS) and to organ-specific (OS) diseases, such as insulin-dependent diabetes mellitus (IDDM), Addison's disease and Hashimoto's thyroiditis. SS is a rather c o m m o n finding in patients with RA [1]. I D D M has been shown to co-exist with Addison's disease [2] and Hashimoto's thyroiditis [3] more often than expected by chance. However, previous studies have failed to show any association between RA and I D D M [4] or RA and Hashimoto's thyroiditis [5], even though a significantly greater proportion of patients with I D D M have positive tests for rheumatoid factor (RF), as compared to patients with osteoarthritis [6]. In addition, the familial aggregation of RA and I D D M has been recognized. The problem therefore still needs to be clarified: is there a serological relationship between OS and NOS autoimmunity? To try to answer this question, a large number of serum samples from patients with RA and

miscellaneous NOS and OS diseases were tested for the presence of NOS and OS autoantibodies.

Material and methods Population studies. A total of 638 disease-associated serum samples were tested. These included specimens from 367 patients with classical or definite RA [8] seen at the Brest University Hospital, France. They were 109 men and 258 women, ranging in age from 18 to 75 years (mean 48 years). The mean duration of disease was 6 years (range: 0.2-34 years). Patients with other diseases were included as controls in the study. The group of NOS autoimmune diseases comprised serum specimens from 62 patients with SLE and specimens from 40 patients with SS. All cases of SLE and SS fulfilled the proposed criteria for the respective diseases [9, 10]. The group of patients with OS autoimmune diseases comprised 34 with Hashimoto's thyroiditis, 50 with Addison's disease, 30 with myasthenia gravis and 55 with recent-onset IDDM. Myasthenia gravis had been diagnosed clinically and serologically at the Department of Neurology, University of Tt~bingen, Federal Republic of Germany. Serum samples from these patients had been included in a previous study [11]. Patients with endocrine diseases had been diagnosed at the Department of Internal Medicine I, University of Ulm, Federal Republic of Germany. Normal controls consisted of healthy hospital staff, medical students, and residents of homes for the elderly. Since we were aware that residents of old people's homes tend to be less healthy that the aged living independently, the subjects were selected after history and physical examination to ascertain that they had been free of symptoms for the preceding month. The sex and age distribution of all normal control groups is given in the footnotes of Tables 1 and 2 in Results. Determination of NOS autoantibodies. All sera used in this study had been stored at -20 °C until used. Non-agglutinating RF (IgM, IgG and IgA) were measured by the indirect immunofluorescence (IFL) technique of Johnson and Holborow [12], using rhesusnegative group 0 human red blood ceils coated with rabbit haemolysin (Institut Pasteur Production, Paris, France) and specific goat anti-IgG, -IgM and -IgA fluorescent labels (Nordic, Tielburg, The Netherlands). A reduction step with 0.01 M dithiothreitol (Sigma, St. Louis, Mo, USA) was carried out to avoid nonspecific interactions with IgM-RF in the IgG-RF test. Sera with titres equal to or greater than 1/100 for IgM and IgA, and 1/10 for IgG, were considered positive. Serum antikeratin antibodies (AKA) were examined using the IFL method of Young et al. [13] on a tissue substrate from the middle third of rat oesophagus. Goat anti-human IgG isotype-

124 Table 1. Organ-specific autoantibodies in patients with non-organ-specific autoimmune diseases and in normal controls. TAB = thyro-

globulin antibodies; MAb = thyroid microsomal antibodies; PC-Ab = gastric parietal cell antibodies; ICA = pancreatic islet cell antibodies; Adr-Ab= adrenal cortex antibodies; R F = rheumatoid factor; A P F = antiperinuclear factor; AKA= antikeratin antibodies; RA= rheumatoid arthritis; SLE = systemic lupus erythematosus; SS = primary Sjtgren's syndrome Organ-specific autoantibodies a

RA SLE SS Normals b

Non-organ-specific autoantibodies

TAb

MAb

PC-Ab

ICA

Adr-Ab

IgG-RF

IgA-RF

IgM-RF

APF

AKA

43/367* 7/62 6/40 29/528

16/367 8/62 9/40 29/528

1/364 0/62 0/38 14/520

0/364 0/60 0/38 3/525

0/364 0/59 0/39 3/520

103/367 9/62 3/40 3/138

92/367 2/62 11/40 1/145

248/367 11/62 10/40 6/210

257/360 7/60 10/38 30/298

135/367 2/57 2/31 6/247

a No. positive/no, tested b The male/female ratios, mean ages and ranges (in years) for normal controls are: 321 : 207, 42 (18-82); 16 : 204, 42 (18-81); 319 : 206, 42 (18-82); 41 : 97, 37 (18-72); 46 : 99, 38 (18-74); 81 : 129, 46; 134; 131 : 167, 48 (18-84) and 99 : 148, 45 (18-84) for TAb and MAb, PC-Ab and Adr-Ab, ICA, IgG-RF, IgA-RF, IgM-RF, APF and AKA normal controls, respectively * RA, SLE and SS groups compared to normals: P < 0.001, > 0.05 and < 0.05, respectively Table 2. Non-organ-specific and organ-specific autoantibodies in patients with organ-specific diseases. HT=Hashimoto's thyroiditis; AD =Addison's disease; MG=myasthenia gravis; D = diabetes; see Table 1 for other abbreviations

Non-organ-specific autoantibodies

HT AD MG TypelD

Organ-specific autoantibodies

IgG-RF

IgA-RF

IgM-RF

APF

AKA

TAb

MAb

PC-Ab

ICA

Adr-Ab

0/34 0/50 0/30 0/55

1/34 0/50 0/30 0/55

3/34 1/5 3/30 6/55

2/34 4/50 6/30 5/55

0/32 0/47 0/29 0/53

24/34 14/49 8/29 5/54

32/34 28/49 14/29 18/54

17/34 20/50 26/30 32/55

4/32 2/45 1/28 37/55

2/33 15/50 1/30 1/55

specific conjugate was used. A titre equal to or greater than 1/10 was considered as positive. Antiperinuclear factors (APF) were measured by the method of Sondag-Tschroots etal. [14]. Human epithelial cheek cells, obtained by scraping the inside of volunteers' cheeks, served as the antigen substrate. Sera with a titre of at least 1/5 were regarded as positive.

Determination of OS autoantibodies. Antibodies to thyroglobulin (TAb) and to the thyroid microsomal antigen (MAb) were measured by the passive haemagglufination technique using commercial test kits (Wellcome, London, UK). The basal dilution of sera was 1/10 for thyroglobulin, and 1/100 for thyroid microsomal antibodies. For the detection of autoantibodies to pancreatic islet cells (IcA), adrenal cortex (Adr-Ab), and gastric parietal cells (Pc-Ab), the IFL method was applied using fresh human tissues from donors with blood group 0, as previously described in detail [15]. Tissue typing. HLA-DR typing was performed by the standard microcytotoxicity test [16] on B lymphocytes isolated on nylon wool columns in 203 RA patients. A panel of antisera (anti-DR 1, -DR2, -DR3, -DR4, -DR5 and -DR7) was provided by FranceTransplant. DR3 and DR4 were the only alleles taken into account in this report. Statistical analysis. Statistical analysis was performed by Chisquared testing for unpaired and paired data with Yates' correction when required. Results

As shown in Table 1, OS autoantibodies were not frequently detected in R A patients: 43 (11.7%) and 16 (4.4%)

displayed TAb and MAb, respectively, so that 49 (13.4%) were positive for either one or both o f these autoantibodies. Only one R A serum specimen contained PC-Ab, and all were negative for I C A and Adr-AB. TAb was found in 7 (11.3%) SLE and 6 (15.0%) SS patients, a n d in 29 (5.5%) n o r m a l controls. M A b occurred in 8 (12.9%) SEE and 9 (22.5%) SS patients, a n d in 29 (5.5%) n o r m a l controls. P C - A b was not detected in SLE a n d SS patients and in 14 (2.7%) n o r m a l controls. I C A was found in none of the SLE and SS patients a n d in three (0.6%) n o r m a l controls. A d r - A b occurred in none o f the SLE a n d SS patients and in three (0.6%) n o r m a l controls. It is noteworthy that TAb was over-represented in the R A a n d the SS groups, c o m p a r e d to n o r m a l s ( P < 0 . 0 0 1 a n d <0.05, respectively). As shown in Table 2, N O S a u t o a n t i b o d i e s were detected only in patients with OS a u t o i m m u n e disease a n d in n o r m a l controls. The relationship between OS a n d N O S a u t o a n t i b o d i e s in the R A patients is shown in Table 3. A significant association was observed between the presence of OS a n d N O S autoantibodies ( P < 0 . 0 4 ) . O f the R A patients with N O S autoantibodies, 15% h a d OS autoantibodies. In R A patients without N O S autoantibodies, the prevalence o f OS autoantibodies fell to 2.5%. The d u r a t i o n o f the disease was a p p r o x i m a t e l y the same in the R A patients with OS autoantibodies (mean, 5 years; range, 1-18 years) a n d in those without OS a u t o a n t i b o d i e s (mean, 6 years; range, 1-16 years). T r e a t m e n t regimes, including n o n - s t e r o i d a l

125 Table 3. Correlation between organ-specific and non-organ-specific autoantibodies in 367 patients with rheumatoid arthritis. OS = organ-specific; NOS = non-organ-specific

+* NOS -

NOS

OS+

OS -

49 1

278 39

':~ OS autoantibodies are more common (P<0.04) in NOSpositive (NOS + ) patients than in NOS-negative (NOS-) patients

Table 4. Organ-specific autoantibodies in HLA-DR3-positive or -negative, and in HLA-DR4-positive or -negative rheumatoid arthritis patients. NS = non-significant HLA-DR

OS autoantibodies °

Significance

Positive

Negative

DR3 + DR3 -

11 20

24 148

P < 0.01

DR4+ DR4-

18 13

116 56

NS

At least one of the following: thyroglobulin, thyroid microsomal, gastric parietal cell, pancreatic islet-cell and adrenal-cortex antibodies

anti-inffammatory drugs and second-line medications were the same in the two groups of patients. With regard to the HLA type, a significant association (P<0.01) was demonstrated between HLA-DR3 antigen (present in 35 of 203 patients, i.e. 17.2%) and OS autoantibody (detected in 11 of 35 DR3-positive patients, i.e. 31.4%, as opposed to 20 of 168 DR3-negative patients, i.e. 11.9%), whereas there was no association between HLADR4 antigen (present in 134 of 203 patients, i.e. 66.0%) and OS autoantibody (detected in 18 of 134 DR4-positive patients, i.e. 13.4%, as opposed to 13 of 69 DR4-negative patients, i.e. 18.8%). The phenotype D R 3 - D R 4 did not appear to be significantly associated with OS autoantibody production, since DR3-DR4-positive patients were not positive for OS autoantibody more often than DR3-X- and DR4-X-positive patients were. Discussion

The link between NOS and OS autoimmunity has not been unequivocally substantiated to date. Consequently, the present study was carried out in a large series of patients with a variety of immune-mediated diseases, in order to ascertain the prevalence of OS autoantibodies in patients with NOS autoimmune diseases on the one hand, and that of NOS autoantibodies in patients with OS autoimmune diseases on the other. We were able to show a substantial difference in the incidence of TAb between patients with RA, SLE or SS and normal controls, but unable to show any significant difference in the prevalence

of MAb, PC-Ab, ICA and Adr-Ab between the former and the latter groups. The occurrence o f N O S autoantibodies in patients with OS autoimmune diseases was the same as that in normal controls. These findings appear to disagree with some case reports and family studies [7, 11, 18]. However, to explain some positive results, it must be taken into account that, as previously emphasized by Mulhern etal. [19], patients with more than one disease are more likely to present to hospital than patients with one disease. Despite the existence of this bias in our series, we failed to show a consistent relationship between OS and NOS autoantibodies. For many years, circumstantial evidence has been accumulating to suggest that the mechanisms leading to OS autoimmunity are different from those leading to NOS autoimmunity. No increased prevalence of OS autoantibodies has been reported in mouse strains prone to develop RA [20], SLE [21] or SS [22]. Furthermore, diabetic BB rats and thyroiditis-prone obese chickens do not display increased levels of NOS autoantibodies [23, 24]. A number of B-cell clones have been established from RA patients' blood by immortalization with Epstein-Barr virus [25] and from SLE patients' blood using the hybridoma technique [26]. Organ-specific reactivity did not occur in either of the diseases studied. Interestingly, it was shown by Notkins' group [27] that intraperitoneal injection of type-I retrovirus into mice produced a viral infection of the pancreas and anterior pituitary characterized by glucose intolerance and growth retardation. As yet, virus-induced equivalents of RA, SLE and SS have not been convincingly obtained. Genetic markers of the HLA system are different in NOS and in OS autoimmune diseases. There is an increased prevalence of D R 4 in individuals with RA [28], I D D M [29] or autoimmune Addison's disease [30]. The latter two diseases are also associated with DR3, as is SS [31]. SLE, however, is weakly but significant associated with DR2 and possibly D R 7 [32]. Graves' disease (thyrotoxicosis), primary hypothyroidism (myxoedema) and generalized myasthenia gravis of early onset have been found to be associated with DR3 [11, 28]. DR3-positive individuals also seem to display an increased frequency of thyroid antibodies as compared to DR3-negative individuals [33]. This is in accordance with our findings that TAb production tends to be associated with DR3 in RA patients whatever the second D R antigen is. The finding in this study that the prevalence of thyroglobulin and thyroid microsomal antibodies is increased in RA is a notable observation. We have previously reported an even higher frequency of thyroid antibodies in SS [34], but this difference may be due to differing criteria for selection of patients. Together, our data expand the experimentally derived conclusion that non-organ-specific and organ-specific autoimmune diseases differ in pathogenesis although overlap does occur. This may be related to the occurrence of DR antigens shared by patients with different disorders.

126

Acknowledgements. We are grateful to Simone Forest for invaluable help in the preparation of the manuscript.

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