1Tampere University Hospital, Tampere, 2University of Tampere Medical School, Tampere, 3Finnish Red Cross Blood Transfusion Service, Department of Tissue Typing, Helsinki and 4Tampere School of Public Health, University of Tampere, Tampere, Finland
Correspondence to: M. Pertovaara, Department of Internal Medicine, Section of Rheumatology, Tampere University Hospital, P.O. Box 2000, FIN-33521 Tampere, Finland. E-mail: marja.pertovaara{at}pshp.fi
SIR, Tumour necrosis factor- (TNF-
) is a pro-inflammatory cytokine which plays an important role in the pathogenesis of many inflammatory and infectious diseases, including primary Sjögren's syndrome (pSS). TNF-
is expressed in minor salivary gland duct cells in patients with pSS [1]. It promotes the influx of mononuclear cells not only into the salivary glands but also, for example, to the renal epithelia [2], and has been shown to be involved in the pathogenesis of experimental tubulointerstitial nephritis. The roles of TNF-
in the tubulointerstitium are undefined, but may include infiltration of the interstitium by inflammatory cells and alteration in the tubular transport of fluid and electrolytes [3]. A base exchange polymorphism (G to A) at position -308 of the gene exists, designated TNF1 (G) and TNF2 (A) alleles. This polymorphism has an effect on susceptibility to and severity of several autoimmune diseases. The rarer TNF2 allele has been found to be associated with enhanced TNF-
production and higher transcriptional activity compared with TNF1 [4].
Renal manifestations, mostly due to tubulointerstitial inflammation, are among the commonest extraglandular findings in pSS. To investigate the possible role of the TNF- (-308) gene polymorphism in the development of renal manifestations in pSS, we analysed this polymorphism after informed consent in 65 pSS patients (63 female, 2 male, mean age 60 ± 12 yr) selected by modified Californian criteria as described previously [5] and in 66 healthy Finnish Red Cross Blood Transfusion Service blood donors matched for sex, ethnic origin and area of residence. Sixty-two of the patients fulfilled the revised AmericanEuropean consensus group criteria for SS [6]. The presence of the HLA-DR3 allele among the patients was also examined because of strong linkage disequilibrium with the TNF-
(-308) gene in northern European populations. The presence of distal renal tubular acidosis (dRTA) was ascertained by ammonium chloride loading tests in 45 of the patients and urinary total protein excretion was measured in 61 [7]. The study protocol was approved by the Ethical Committee of Tampere University Hospital.
Genomic DNA was isolated from blood samples using the salting-out method [8]. The base exchange polymorphism at position -308 of the TNF- gene was analysed by polymerase chain reaction (PCR)-based genotyping [9]. HLA-DRB1 typing was performed with a commercial kit, LiPA HLA-DRB1 (Innogenetics, Murex Biotech Ltd, UK) or using the PCR method as previously described [10]. TNF-
plasma levels were measured using an enzyme-linked immunosorbent assay (Pelikine Compact human TNF-
ELISA kit, CLB, Amsterdam, The Netherlands), following the manufacturer's instructions. The optical density in individual wells was determined with a Multiscan Bichromatic 348 (Labsystems, Helsinki, Finland) spectrophotometer. The detection limit of the assay was 1.4 pg/ml.
The frequency of the TNF2 allele was higher in pSS patients compared with controls (52 vs 29%, P = 0.006, odds ratio 2.71, 95% confidence interval 1.325.58). A total of 48 and 71% of the patients and controls, respectively, were homozygous for TNF1, 51 and 26% heterozygous and 1.5 and 3% homozygous for the TNF2 genotype (P = 0.013, 2-test for 3 x 2 table). The TNF2 allele was encountered more frequently in pSS patients with proteinuria than in those without (65 vs 40%, P = 0.050, n = 61) and in patients with dRTA compared with those with normal acidification capacity (69 vs 41%, P = 0.079, n = 45). The pSS patients with dRTA had significantly higher plasma TNF-
levels than those with normal renal acidification capacity (14.8 ± 21.7 vs 5.1 ± 10.1 pg/ml, P = 0.003). However, plasma TNF-
levels did not differ significantly between pSS patients with proteinuria and those with normal urinary protein excretion and were unaffected by the presence of TNF2 allele in pSS patients.
The pSS patients with the TNF2 allele yielded several laboratory findings suggestive of a more active form of the disease. Their mean levels of serum IgG (20.2 ± 6.8 vs 16.7 ± 7.1 g/l, P = 0.032) and the titres of anti-SSA or anti-SSB antibodies (84 ± 47 vs 48 ± 51 U/l, P = 0.005 and 98 ± 75 vs 24 ± 48 U/l, P < 0.0001, respectively) were higher than those of patients without this allele. The minor salivary gland histological findings were not related to TNF genotypes. The independent effect of the TNF2 allele on the clinical and immunological characteristics of the patients could, however, not be definitely judged as TNF2 allele and HLA-DR3 were highly significantly associated with each other (P < 0.0001) and there were no patients carrying TNF2 allele who were negative for DR3 (Table 1).
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This study was supported by grants from the Medical Research Fund of Tampere University Hospital, Tampere, Finland and the Finnish Medical Foundation.
The authors have declared no conflicts of interest.
References