Department of Rheumatology and
1 Department of Clinical Immunology, Karolinska Hospital, Stockholm, Sweden
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Abstract |
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Patients and methods. Forty-one consecutive patients with early RA, in whom sulphasalazine was used as the first disease-modifying anti-rheumatic drug in single therapy and was maintained for at least 6 months, were investigated for the occurrence of lupus-related events. Longitudinal analyses of rheumatoid factor (RF), antinuclear antibodies (ANA), anti-double-stranded DNA antibodies and serum IL-10 (ELISA) and the typing of HLA DR and DQ alleles were performed.
Results. Four of the 41 patients developed lupus-like disease. Three of four patients who had lupus-related events vs four of 37 patients without side-effects had an HLA DR 0301 haplotype. The patients developing lupus-related side-effects had increased levels of serum IL-10 and a high frequency of ANA in speckled patterns before the onset of therapy.
Conclusion. The development of SLE-like symptoms and SLE-related autoantibody production was observed more commonly than expected, with an increased risk in patients with SLE-related HLA haplotypes, increased serum IL-10 levels and ANA in speckled patterns. The data suggest that immunomodulation associated with sulphasalazine treatment may contribute to the development of lupus-related reactions in genetically predisposed individuals.
KEY WORDS: Sulphasalazine, SLE, Rheumatoid arthritis, Drug-induced lupus, HLA, IL-10
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Introduction |
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IL-10 is a cytokine with multiple effects on the immune system, such as enhancement of antibody formation, inhibition of cytokine synthesis and inhibitory effects on T-cell and monocyte functions [79]. In rheumatoid arthritis (RA) there is evidence that interleukin-10 (IL-10) may have a dual role in being able to both enhance antibody production and to suppress the proinflammatory events mediated by cytokines such as tumour necrosis factor (TNF-
), IL-1 and interferon
[10, 11]. Administration of IL-10 in vivo has been reported to ameliorate disease both in patients with RA [12] and in experimental collagen-induced arthritis [13]. Additional evidence that IL-10 may exert a physiological immunoregulatory function in RA derives from observations of increased serum levels of IL-10 [14, 15] and increased IL-10 levels in inflamed joints [14].
Conversely, a more clear-cut association of IL-10 with SLE has been deduced from the generally increased IL-10 production by peripheral blood mononuclear cells [15], high serum IL-10 levels [16] and an enhancing effect on anti-double-stranded DNA (dsDNA) antibody formation in vitro [17] in patients with SLE. In experimental models, treatment with anti-IL-10 has been reported to delay the onset of autoimmunity in lupus-prone NZB/W F1 mice and to decrease anti-dsDNA production and the severity of renal disease [17, 18]. That increased production of IL-10 precedes disease development rather than being a result of the disease process per se is suggested from the observation of enhanced IL-10 production in healthy first-degree relatives of patients with SLE [19, 20]. It thus appears that increased production of IL-10 due to genetic and environmental influences may be of significant importance in the development of SLE and SLE-related autoantibody production in an RA population treated with sulphasalazine.
The aim of this study was to define the prevalence of lupus-related symptoms during sulphasalazine treatment in RA patients, and to further document the mechanisms involved in the putative development of SLE-like disease in these patients. Against the background of the possible general role of IL-10 in the triggering of SLE, we put special emphasis on the investigation of the relationship between serum levels of IL-10 and the development of SLE-like symptoms and SLE-related autoantibody production.
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Materials and methods |
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Control population
The control population consisted of 24 healthy volunteers (seven males and 17 females, mean age 48 yr, age range 2762 yr) and 16 untreated SLE patients (two males and 14 females, mean age 45 yr, age range 2076 yr). Sera from these individuals were analysed for IL-10.
Clinical and laboratory evaluations
The patients were evaluated every third month at the out-patient clinic by assessment of the numbers of swollen and tender joints using a 28 joint scale. C-reactive protein (CRP) levels were analysed (nephelometry) at the time of each visit. Sera were obtained before the initiation of sulphasalazine therapy and after 3, 6, 12, 18, 24 months and then yearly, and were stored at -70° before use. All patients had given informed consent to blood sampling for research purposes. The study was approved by the local ethics committee.
Evaluation of lupus-related side-effects
All patient charts were evaluated for side-effects characteristic of SLE. Sulphasalazine-related lupus symptoms were defined as the onset of clinical symptoms consistent with SLE in combination with positive antinuclear antibodies (ANA) and/or the development of anti-dsDNA during sulphasalazine treatment. The clinical features evaluated included the onset of skin manifestations, oral ulcers, photosensitivity, serositis and neurological manifestations. Laboratory analyses for the investigation of possible SLE development included the occurrence of cytopenia, haematuria and/or proteinuria indicating development of nephritis.
Serology
Before the initiation of treatment and after 3, 6, 12, 18 and 24 months of treatment, analysis for IgM rheumatoid factor (RF) (nephelometry, normal value <20 IU/ml) and IgG ANA (immunofluorescence using HEp-2 cells) were performed. Patients with ANA in a speckled pattern, regardless of titre, were investigated for the occurrence of antibodies against Ro/SS-A and La/SS-B by immunodiffusion. Anti-dsDNA (IgG) antibodies were analysed, using immunofluorescence (Crithidia luciliae), if an ANA titre of 1/100 in any pattern had been detected.
Determination of serum IL-10
A highly sensitive ELISA (R&D Systems, Minneapolis, USA) was used for serum IL-10 determination, according to the manufacturer's instructions. The assay was investigated thoroughly and was found to be free of interference by RF. Duplicate determinations were performed for each sample and intra-assay variability of <20% was accepted. The median inter-assay variability, determined from eight samples, was 0.35 pg/ml (range 0.12.3). The normal value of IL-10 (in pg/ml) was determined from the mean value +2 S.D. for 24 healthy controls.
HLA typing
Typing of HLA DRB1 and DQB1 was performed by sequence-based typing using polymerase chain reaction (PCR) amplification of exon 2 with subsequent sequencing reactions [22]. HLA DQA1 typing was performed by PCR amplification with sequence-specific primers [23]. These analyses were made as part of the clinical routine at the Department of Clinical Immunology, Akademiska Hospital, Uppsala, Sweden.
Statistics
The MannWhitney U-test, the Spearman rank correlation, Student's t-test and Fisher's exact test were used. Analysis of variance (ANOVA) was used for repeated measurements of serum IL-10. A P value <0.05 was considered significant.
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Results |
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Lupus-related side-effects according to the definition described above were observed in four patients. Two patients (patients 1 and 4) developed anti-dsDNA antibodies after approximately 12 months of therapy, and one of these (patient 4) had simultaneous onset of a sun rash. One patient (patient 3) developed biopsy-proven membranous nephritis with subepithelial deposits of IgG and C3 compatible with SLE nephritis (WHO grade V) after 26 months of treatment. One patient (patient 2) developed a sun rash and an increasing ANA titre after 6 months of therapy but no anti-dsDNA antibodies were detected. Details of the four patients with lupus-related side-effects are presented in Table 1.
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Clinical and laboratory evaluations
Before onset of treatment, 21 of the 41 patients had an increased CRP level. The normal value of CRP was <5 mg/l. For statistical calculations, a CRP concentration of <5 mg/l was arbitrarily set as 4 mg/l. The mean numbers of swollen and tender joints were 9 and 7 respectively. There was no correlation between CRP level, number of swollen or tender joints and the occurrence of lupus-related side-effects (data not shown) before the initiation of treatment or after 6 months of treatment. The CRP levels and numbers of swollen and tender joints in the total RA group are presented in Table 2.
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Serology
Before the start of sulphasalazine treatment, 26 of the 41 (63.4%) patients were positive for RF ( 20 IU/ml). After 6 months, 13 (31.7%) of the patients were RF-positive. A reduction in RF titre was noted in all patients positive for RF at the first examination. This decrease was highly significant (P = 0.0003, Student's t-test). Higher RF titres were noted before the initiation of sulphasalazine therapy in the patients developing lupus-like side-effects compared with the RA patients without side-effects (P = 0.036, MannWhitney U-test).
Before the initiation of sulphasalazine treatment, an ANA titre of 1/50 was detected in 16 (39.0%) patients and a titre of
1/100 in 10. None of the patients had detectable anti-dsDNA antibodies before onset of therapy. After 6 months of therapy, ANA was observed in 13 of the 41 (32%) patients; an ANA titre of 1/50 was observed in four patients and a titre of
1/100 in nine. There was no statistically significant difference in ANA titre between the determinations before the initiation of therapy and at 6 months (P = 0.93; Student's t-test). The patients with lupus-related side-effects did not have a significantly higher ANA titre than the RA group without side-effects before the initiation of treatment (P = 0.06; MannWhitney U-test). The numbers of RF- and ANA-positive patients before the initiation of therapy and at 6 months are shown in Table 2
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At the initiation of sulphasalazine treatment, eight of the 16 ANA-positive patients had a speckled ANA pattern; five had a titre of 1/100, and antibodies against SSA and SSB were detected in one patient. This patient (patient 2) developed a lupus-like side-effect with a pronounced sun rash after 6 months of treatment. A correlation was observed between the occurrence of ANA positivity with a speckled pattern before the initiation of sulphasalazine therapy, regardless of titre, and the development of lupus-like side-effects (P = 0.019, Fisher's exact test). In addition, three of five patients with a speckled ANA pattern at a titre of
1/100 before the initiation of therapy developed lupus-related side-effects. One of these had anti-SSA/SSB antibodies.
HLA typing
DRB1, DQA1 and DQB1 alleles were analysed in all patients. The DRB1 0101 allele was recorded in 10 and 0401 was observed in 13 patients, of whom two were homozygous for 0401. The DRB1 0301 allele associated with SLE was present in seven patients, DQA1 0501 in 15 and DQB1 0201/02 in 10, one of whom was homozygous for 0201/02. Of the four patients who developed SLE-like side-effects, three had the DRB1 0301 allele and three had DQA1 0501. The DQB1 0201/02 allele was found in three of the four patients with lupus-like disease; one of the three was homozygous. Three out of four patients with side-effects had at least one of the DRB1 0301, DQA1 0501 and DQB1 0201/02 alleles. A correlation between DRB1 0301 and the development of SLE-related side-effects was observed (P = 0.012, Fisher's exact test), and there was a weak association between DQB1 0201/02 and side-effects (P = 0.039; Fisher's exact test). The DRB1, DQA1 and DQB1 alleles in the total RA group and in the patients developing SLE-like side-effects are presented in Table 3.
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Determination of serum IL-10 in RA and control groups
The serum IL-10 concentration was significantly higher in the total RA group than in the healthy controls (P < 0.0001, MannWhitney U-test), but significantly lower than in the reference group of SLE patients (P = 0.0028, MannWhitney U-test) (Fig. 1).
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There was a considerable variation in IL-10 levels between the different RA patients; 24 of the 41 had IL-10 levels above the reference value, whereas 15 of 16 SLE patients had levels above the reference range. Interestingly, all four patients who developed SLE-related side-effects had a high IL-10 level at baseline. The difference in IL-10 levels between the side-effect RA group and the non-side-effect RA group was statistically significant (P = 0.0028; MannWhitney U-test).
The patients with side-effects had higher IL-10 levels than the SLE reference patients (P = 0.03, MannWhitney U-test), while the RA group without side-effects had significantly lower IL-10 levels (P = 0.0002; MannWhitney U-test) (Fig. 2).
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IL-10 and clinical and serological parameters
At first examination, there was no correlation between IL-10 level and RF, ANA, number of swollen or tender joints or CRP (Spearman rank correlation; data not shown). However, patients positive for ANA in a speckled pattern were found to have a higher IL-10 level than the RA patients with no detectable ANA or who had ANA in a non-speckled pattern (P = 0.0066, MannWhitney U-test).
Correlations between IL-10 and HLA haplotypes
The level of IL-10 at baseline was significantly lower in patients with a DRB1 0101 and/or 0401 allele (shared epitope) than in RA patients lacking the shared epitope (P = 0.0086, MannWhitney U-test). RA patients carrying the DQB1 0201/02 allele had a significantly higher serum IL-10 level (P = 0.0075, MannWhitney U-test) than patients without this allele. There was no significant difference in IL-10 level between RA patients carrying the SLE-associated DRB1 0301 allele and patients without this allele (P = 0.10, MannWhitney U-test).
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Discussion |
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The patient cohort used in this study was homogeneous regarding disease duration, lack of previous therapy and an ethnic background that could have influenced the clinical and serological features, HLA or levels of cytokines. Only two patients received simultaneous oral corticosteroid therapy, which has been reported not to affect the level of IL-10 in RA [14, 16]. Furthermore, the study cohort was representative of a cross-sectional RA population as no selection had been performed regarding disease activity or severity when choosing anti-rheumatic drug therapy. However, there may have been bias because we did not include patients in whom sulphasalazine was withdrawn early because of gastrointestinal or haematological side-effects, lack of response or other causes.
Reports of the prevalence of SLE-like side-effects or SLE-related autoantibody production during sulphasalazine treatment, found in approximately 10% of the patients in the present study, have been conflicting. A recently published paper stated that no case of sulphasalazine-induced SLE was observed during long-term follow-up in RA patients treated with sulphasalazine [24], although 10% of the patients in that study were reported to have developed mucocutaneous side-effects. However, no data regarding the occurrence of anti-dsDNA were presented. Conversely, in the study by Chalmers et al. [25], anti-dsDNA antibodies were found in 7% of the patients after 1 yr of sulphasalazine treatment, but no signs of SLE-related symptoms were reported to accompany the serological findings. In a third study, by Mielke et al. [26], only two of 20 patients developing anti-dsDNA antibodies during sulphasalazine therapy showed clinical symptoms of SLE, suggesting that the occurrence of anti-dsDNA antibodies may be transient and not associated with further SLE development. However, prevalence data for sulphasalazine-induced SLE could not be calculated in that study as the size of the original patient cohort was not defined.
No specific diagnostic criteria of drug-induced lupus have been established to date, but diagnosis may be based on the criteria proposed by Cush and Goldings [27], which include lack of previous history of lupus before drug therapy, positivity for ANA and at least one clinical feature in accordance with SLE at presentation of the drug-induced reaction. When these criteria are used, however, patients who develop only autoimmune serology, including anti-dsDNA antibodies, will not be identified.
In the present study, an increased frequency of the SLE-associated alleles DRB1 0301 (DR3) and DQB1 0201/02, in linkage disequilibrium, was demonstrated in the patients who developed sulphasalazine-induced lupus-like side-effects. This finding is in accordance with previous reports on the development of proteinuria during treatment with the anti-rheumatic agents sodium aurothiomalate and D-penicillamine in DR3-positive individuals [28].
The patients who developed lupus-related side-effects had an elevated IL-10 level both at baseline and at the time of onset of the lupus-related event, implying that IL-10 may contribute to the triggering of the sulphasalazine-induced SLE-like reactions. A correlation between HLA haplotype and IL-10 level was found, with a low IL-10 level in patients with the DRB1 0101/0401 alleles (shared epitope), and a high level in patients with haplotypes associated with SLE. On a group basis, however, the findings of increased levels of circulating IL-10 in an RA population compared with controls, as reported previously [14], was confirmed in the present study.
High RF titres before the initiation of sulphasalazine therapy were found at an increased frequency in the group with lupus-related side-effects. Compared with RA patients without side-effects, a trend towards increased ANA levels was noted in the group with side-effects, though this did not reach statistical significance. One possible explanation for the high autoantibody levels in the lupus-susceptible individuals is the stimulatory effect of IL-10 on antibody production in general. It is of interest that the occurrence of ANA in a speckled pattern before the initiation of sulphasalazine was found to be a factor predictive of the later development of lupus-related symptoms. In contrast, no clinical factor, such as the number of swollen or tender joints, the CRP level or the response to therapy (data not shown), was able to predict susceptibility to lupus. It must be emphasized, however, that all patients who developed SLE-like side-effects had erosive arthritis and met the ARA criteria for RA [21]. Furthermore, in no case was there any clinical evidence of SLE before the initiation of sulphasalazine therapy.
Decreasing IL-10 levels were observed in the RA patients after the initiation of sulphasalazine therapy and in the majority of patients who developed SLE-like side-effects or SLE-related autoantibody production. However, in the latter group, recurring high serum IL-10 levels may have contributed to the development of SLE-like disease, as increased levels of IL-10 were observed in conjunction with the onset of SLE-related symptoms or antibody production. Sulphasalazine has also been reported to reduce immunoglobulin levels and the RF titre [29], which may also be explained by a decrease in IL-10 production. Furthermore, sulphasalazine possesses immunomodulatory functions with inhibitory effects on cytokine production, and has been found to reduce the levels of the proinflammatory cytokines IL-1, TNF- and IL-6 [30, 31].
In summary, we confirm the association of HLA with sulphasalazine-induced lupus syndromes that has been suggested previously [6]. Furthermore, apart from the presence of HLA susceptibility genes, we suggest that the occurrence of high levels of IL-10 in conjunction with sulphasalazine treatment may contribute to the development of sulphasalazine-induced SLE. Similar mechanisms might also be operative during the treatment of RA patients with biological agents, such as TNF-blocking substances, in whom SLE-related symptoms and serology have been reported [32].
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Acknowledgments |
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Notes |
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References |
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