TNF(tumor necrosis factor) consists of two distinct peptides with multiple immunological and local as well as systemic inflammatory activities. TNF-α also called cachectin, is produced by activated macrophages and other cells and has a broad spectrum of biological actions on many immune and nonimmune target cells. TNF-β, also called lymphotoxin, is primarily a product of T lymphocytes and shares many of the biologic activities of TNF-α¹⁾. TNF-α and TNF-β bind to the same receptor on target cells and are part of a network of interactive signals that orchestrate inflammatory and immunological events²⁾.

Linkage analysis and pulsed-field gel electrophoresis studies have shown that the gene for TNF-α is linked to the gene for TNF-β and is located between the HLA class III region and the HLA-B locus in mice and humans^3–5). In systemic lupus erythematosus (SLE), an autoimmune disease characterized by abnormal immune response and various clinical manifestations, the association with the major histocompatibility complex (MHC) class II and III alleles was reported with some ethnic differences^6–8). In our previous report⁸⁾, HLA-DRBI^*1501 and C4AQO allele were associated with susceptibility to Korean SLE. Despite this association, the role of the MHC molecules in SLE remains largely unknown. The localization of the TNF genes in the MHC, and the link between TNF and regulation of the immune response have led to the hypothesis that polymorphism of TNF gene could be associated with HLA antigens, and might play an important role in the pathogenesis of various HLA associated autoimmune dieseases³⁾. Jacob et al⁹⁾ reported that TNF-α production levies in human, are associated with MHC class II, and also associated with nephritis in patients with SLE. Two TNFB alleles (TNFB^*1 and TNFB^*2) distinguished by a polymorphic Ncol restriction site in the first intron have been identified¹⁰⁾. Some investigators have analyzed the frequency of the Ncol RELP in various autoimmune diseases including SLE¹³⁾. The small number of the patients studied and the strong association between HLA and TNF allele have made it difficult to understand the role of TNFB gene polymorphism in human SLE.

Therefore, we have investigated the frequency of the TNFB alleles using PCR-RFLP to determine the role of TNFB gene polymorphism in SLE, and also its genetic relationship to the development of nephritis in patients with SLE.

Evidence for participation of genetic factors in the pathogenesis of SLE has been accumulated by many investigators. These include family aggregation of the disease, increased concordance of SLE among monozygotic and dizygoic twins, decreased red cell CRI receptors, the abnormal T cell suppressor function in healthy relatives of SLE patients and association with several MHC loci^18–22). Population studies have shown an in-creased association between SLE and HLA-DR2 and/or DR3 antigen, as well as an association with C2 and C4A deficiency of MHC class III^23–24). The strengths of these associations vary from study to study, and they can vary in ethnically different populations^6,24,25). In Koreans, HLA DRB1^* 1501 (RR=3.3), as a subtype of DR2, and C4AQ0 allele (RR=2.1) without C4 gene deletion were reported as an increased allele in SLE patients, which was different from Causcasian SLE whose predisposing MHC genes were known to be HLA DR2, DR3 and C4A gene deletion⁸⁾. Despite these associations, the role of these molecules in SLE is not clear. Furthermore, the relative risk of SLE in a person positive for any one of these alleles has been reported to be weaker than that of some rheumatic diseases such as rheumatoid arthritis for HLA-DR4 and ankylosing spondylitis for HLA-B27²⁶⁾. Therefore, it is even questionable whether the MHC genes are the predisposing genes to the disease rather than markers for other closely linked gene(s).

Rapid progress has been made in the elucidation of the structure and function of class I and II MHC molecules, and several genes located within the MHC have been identified which are potentially involved in immunlolgic process. Because of the MHC localization of the TNFA (α) and TNFB (β) genes and the biologic activities of the gene products, it has been suggested that TNF gene loci might contribute to the susceptibility of HLA-associated autoimmune diseases^3,5). In humans, the EcoRI RFLP of the TNFB gene is less informative than that of Ncol because of the low frequency (6%) of the less common fragments (2.5kb)²⁷⁾. In Ncol-digested genomic DNA, the TNF-β probe detects both a 5.5kb and a 5 kb, or the 10.5 kb frangment^16,28). Despite only a small number of patients, several groups have studied the association of Ncol TNFB gene RFLP with some autoimmune diseases such as primary biliary cirrgosis (PBC), insulin deqendent diabetes mellitus (IDDM), rheumatoid arthritis (RA), primary Sjogren’s syndrome (pSS) and SLE^12,13,16,29). Among 20 patients with SLE, TNFB^*1 homozygote was increased significantly when compared with the control in Fugger’s¹³⁾ report. There was no difference in the phenotypic frequency for each TNFB allele in SLE, but the extremely low number of TNFB^*1 homozygote in control group (6.1% vs 30%) seems to be responsible for their results. Recently, TNF Ncol-RFLP analysis in a larger group of SLE patients has been reported by some investigators^30,31). Bettinotti et al³⁰⁾ investigated the TNFB gene Ncol RFLP in 173 patients with German SLE and 192 unrelated controls. In that study, the phenotypic frequency of the TNFB^*I allele was significantly increased in patients compared to controls (63.6% vs 47%, RR=1.96, P<0.002). Goldstein³¹⁾ also studied the TNF Ncol-RFLP in Canadian Caucasians with SLE. Their results demonstrated that there were no differences in the frequencies of Ncol-RFLP phenotypes and genotypes between SLE and controls (TNFB^*2 homozygote 38% vs 46%, TNFB^* 1/2 42% vs 43%, TNFB^*1 homozygote 20% vs 11% in SLE and controls, respectively).

In our study, TNFB gene analysis using Ncol RFLP showed that TNFB^*2 homozygote was significantly increased in SLE compared with the controls (RR=2.36, p=0.011). These results are contradictory to other reported series. But interestingly, no significant difference was observed in the distribution of TNFB alleles between Koreans and Caucasian SLE patients even though this different distribution of TNFB alleles between Korean and Causcasian controls. Because of relatively low frequency of TNFB^*2 homozygote in Korean controls compared with Caucasians, that of TNFB^*2 homozygote in SLE patients seems to be increased over controls in the Korean population.

Despites the efforts of many investigators, little is known about the function of TNFB gene RFLP. Messer and coworkers¹¹⁾ have reported that there is not only a structural but also a functional difference between both TNFB alleles. Upon stimulation with phytohemagglutinin (PHA) of peripheral blood mononuclear cells in vitro, TNFB^*1 homozygotes are higher responders than TNFB^*2 homozygotes. In mouse model systems, autoimmunity has been attributed to a low TNF-α response in NOD-mice with diabetes and mice prone for lupus nephritis (NZWxNZB)^32,33). In humans, Jacob and coworkers reported that DR2, DQwl positive SLE patients showed low levels of TNF-α inducibilityk which was associated with an increased incidence of lupus nephritis. On the other hand, DR3-positive SLE patients are not predisposed to nephritis, and these patients have high TNF-α production. TNF mediates a variety of functions and exerts remarkably diverse effects on the immune system, which includes the regulation of MHC class II molecule dependent on cell differentiation, and the promotion of self tolerance in early induction phase of autoimmune process rather than the late effector phase of the disease³²⁾. Therefore, data from experimental studies, together with the important role of TNF in immune response, could hypothesize that some clinical subset of SLE, such as lupus nephritis, might be attributed to low production of TNF which may be the effect of TNF gene polymorphism.

HLA-DR2 was a major HLA phenotype associated with SLE, which supported the previous report for Korean SLE⁸⁾. We did not find any significant associations between TNFB alleles and HLA-DRB1^*15, which were significant alleles for SLE in this study. Also there was no significant haplotypic association between TNFB alleles and C4AQ0,previously reported as an allele associated with SLE in Koreans⁸⁾ (data not shown). Our data demonstrated that TNFB^*2 allele was more meaningful in DR2 negative patients and DRBI^* 15 affected more significantly in TNFB^*1/2 heterozygote. These findings suggested that TNFB^*2 homozygote appeared to be an additional risk factor for SLE, especially in DR2 negative individuals.

SLE is a heterogeneous autoimmune disorder that involves the skin, joints, serosal surface, kidney, central nervous system and blood elements. Among these various manifestations, nephritis may be the dominant clinical feature and a factor for mortality.

In this study, we categorized the patients into nephritis and non-nephritis groups to evaluate still showed a significantly increased allele over controls, whereas the significance of TNFB^*2 homozygosity conferring risk for SLE was more evident in nephritis group rather than non-nephritis group. The analysis of relative risk for disease susceptibility in nephritis and non-nephritis group revealed that the relative risk of TNFB^*2 homozygote in DR2 negative subjects was more prominently increased in nephritis group than in non-nephritis as a whole (RR=3.06 vs 2.65, data not shown). These observations may have implication that HLA-DR2 may play a role in SLE without nephritis rather than SLE with nephritis.

The disease entity of SLE might be heterogeneous, and the immunogenetic background might also differ among various clinical characteristics. Further study of the larger numbers of patients with different clinical features will be needed to provide more conclusive evidence on the role of TNFB gene in SLE. In addition to TNFB genotyping, we are also testing the association between TNFB genotype and production-rate phenotype in vitro. The results of these studies should also help to elucidate the significance of TNF in the pathogenesis of SLE.