Ligation of the T cell receptor with peptide bound on the major histocompatibility complex provides the first signal for T cell activation. However, activation does not occur until a second signal is provided by costimulatory molecules. Expressed on T cells, positive costimulators such as CD28 enhance activation, while CTLA-4 (cytotoxic T lymphocyte–associated antigen 4) down-regulates T cell activation (1, 2). CTLA-4 ligation leads to T cell quiescence or anergy, terminating T cell activation (3). CTLA-4 represents an essential checkpoint for immune function in vivo. In mice, CTLA-4 deficiency leads to death due to lymphoproliferative disease (4–6).
The gene encoding human CTLA-4 (Ctla4) contains multiple polymorphisms (7). Certain of them affect the expression and function of the protein. To date, 3 Ctla4 polymorphisms have been studied extensively (8–10) (Figure 1). A single-nucleotide polymorphism (SNP) at position −318 (relative to the transcriptional start site) in the promoter region has been shown to influence gene expression in in vitro gene reporter assays (11). The higher-secretor allele T of this SNP at −318 is protective against autoimmunity and inflammatory responses, but occurs with low frequency in Caucasian populations (11, 12). Carriers of allele G in another SNP coding sequence 1 (CDS1) at position +49 have reduced levels of messenger RNA (mRNA) and T cell surface protein expression of CTLA-4. Allele G is associated with increased susceptibility to autoimmune/inflammatory diseases (13, 14). There is strong linkage disequilibrium between the +49 SNP and a dinucleotide repeat polymorphism, (AT)n, located at position +642 of the 3′-untranslated region (3′-UTR) (10, 13, 15). It is already recognized that allele A in CDS1 is strongly linked to the shortest allele, 86, of the (AT)n polymorphism (7, 14, 16), while allele G is linked to longer alleles (alleles that have polymerase chain reaction [PCR] products longer than 86 bp) (17).
Figure 1. Common polymorphisms in the human gene encoding CTLA-4. Multiple CTLA-4 polymorphisms have been found; shown are the 3 that have been reported early and studied extensively. The genetic variations and locations of the polymorphisms are indicated by arrows.
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We have previously shown that individuals with long alleles of Ctla4 (AT)n have hyperreactive T cells and that the length of the AT repeat in Ctla4 has a significant linear correlation with levels of soluble interleukin-2 (IL-2) receptor in the circulation (18). Heterozygotes have less CTLA-4 mRNA from the longer allele than from the shorter allele in vivo, in part because the longer allele, which contains a higher number of AT repeats, is responsible for reduced mRNA stability, thus resulting in less protein synthesis (19). Microsatellite (AT)n in the 3′-UTR might be the major, primary genetic mutation that affects CTLA-4 gene expression. While haplotypes, rather than single mutations, may account for the altered gene expression (20), haplotypes of CTLA-4 have not yet been studied in healthy individuals or patients with Wegener's granulomatosis (WG). Nevertheless, the shortest allele of microsatellite (AT)n in Ctla4 serves as a marker for high-level CTLA-4 expression.
WG is a systemic disorder that most often affects the upper and lower airways and the kidneys. However, any organ in the body may be involved. The classic pathologic triad includes necrotizing inflammation with granulomas and vasculitis. Without treatment, patients with generalized WG have a mean survival of only 5 months (21). The etiology of WG is largely unknown. Evidence suggests that the disease is multifactorial (22), with both environmental and genetic factors contributing to its pathogenesis. Previous studies revealed a genetic association with certain Ctla4 polymorphisms in a Swedish population (16, 23). In view of the vital function of CTLA-4 in immune regulation and the ample evidence of immune dysregulation in WG, we hypothesized that variation in Ctla4 might play a role in the pathogenesis of WG. We therefore conducted this genetic association study of CTLA-4 and candidate proinflammatory cytokine genes in American patients with WG.
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- PATIENTS AND METHODS
Compared with analyses that utilize genetic markers of the entire genome, candidate gene approaches, as applied in this study, select polymorphisms situated close to or within genes coding proteins that have functional significance in the pathogenesis of a disease. Candidate gene studies have the advantages of being guided by established knowledge of pathogenesis and having lower cost and greater efficiency. Therefore, despite the introduction of genetic map markers many years ago and the disadvantages of possible investigator bias and incomplete evaluation of what might be important unexplored genetic factors, the candidate gene approach remains a powerful, widely used strategy for the study of disease associations and pathogenesis.
The results of the current studies provide evidence of a genetic association between Ctla4 polymorphisms and WG in an American population. This work extends our previous findings of a positive association between longer alleles of (AT)n in the 3′-UTR of Ctla4 and WG (23). These results demonstrate that genetic variations in this T cell–related gene confer susceptibility to WG and that such influence is not limited to our previously reported WG patients from Sweden (23).
Since the discovery of Ctla4, numerous experiments from different groups using both murine and human systems have demonstrated a critical role of CTLA-4 in down-regulating T cell activation, which has a profound impact on inflammation and autoimmunity (1). There are multiple sites of immune dysfunction in WG, including excessive activation of macrophages, T cells, and B cells. CTLA-4 appears to play an important role in down-regulating immune responses. The underrepresentation of the shortest allele in Ctla4 in both Swedish and American patients with WG indicates that the overrepresented longer alleles of Ctla4 (AT)n might contribute to perpetuation of immune dysfunction, especially T cell activation, in WG (34–36).
TNFα is capable of priming neutrophils, leading to a preactivated state, oxidative burst, and neutrophil degranulation. Levels of TNFα in serum and expression and secretion of TNFα by peripheral blood mononuclear cells and T cells are increased in patients with active WG (35, 37, 38). TNFα concentrations are also increased in affected renal tissue (38). The polymorphism located at nucleotide position −308 of the promoter region has been studied extensively (39), and the majority of the published data from independent groups supports the notion that TNFα −308 allele 2 or its haplotype plays a direct role in the increased production of TNFα (20, 39–42).
Our previous preliminary results in Swedish patients did not support the idea that TNF2 plays a major role in the pathogenesis of WG (23). However, that study was limited by its small patient cohort. Moreover, disease associations with specific genes may vary in different ethnic populations. For example, different TNFα gene polymorphisms are associated with differences in susceptibility to severe malaria in East and West Africa (42), and interethnic differences in severity of disease are well recognized in systemic lupus erythematosus (43). In order to study the role of genetic determinants of TNFα in the pathogenesis of WG, we included analysis of the TNFα −308 RFLP in the current study. Results from both studies suggest that enhanced production of TNFα in WG does not derive from this genetic variation. It is likely that increased circulating and tissue levels of TNFα might be determined by posttranscriptional events in WG. Similar conclusions might be drawn with regard to IL-1β and IL-6 in WG (Table 2).
TNF exerts its wide spectrum of proinflammatory effects via 2 receptors, TNFRI and TNFRIII (TNFR p55 and TNFR p75). Both receptors can produce a soluble form of TNFR that binds to TNFα in vivo, limiting its proinflammatory functions. Improvement in scores on the WG modification of the Birmingham Vasculitis Activity Score (44) after 6 months of treatment in a clinical trial of etanercept, a fusion protein consisting of 2 molecules of TNFRII conjugated with the Fc portion of human IgG (45), underscores the significance of TNFα and TNF receptors in the disease. However, no association of polymorphisms in TNF receptor genes was found in the present study. Taken together, these findings indicate that in genetically predisposed patients with WG, e.g., those who are positive for Ctla4 (AT)n longer alleles, certain triggering factor(s), such as yet-unknown microorganisms, might elicit heightened T cell responses, while proinflammatory cytokines, such as TNFα and IL-1β, augment autoimmune/inflammatory injury pathways.