Systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) are two human autoimmune diseases that are mediated by damaging immune overreactivity to self antigens. Although the pathogenesis of SLE and RA remains unclear, it is possible that dysregulated lymphocyte activation initiates the breakdown of tolerance and predisposes the patient to the development of these autoimmune diseases, because lymphocyte activation appears to be governed by immunostimulatory and immunoinhibitory signals that are delivered through lymphocyte surface receptors. More solid evidence to support this hypothesis comes from the fact that autoimmune diseases can develop in mice that have either an overexpression of a stimulatory receptor or a deficiency of an inhibitory receptor. However, further efforts toward establishing a correlation between each immunoregulatory receptor and human autoimmune diseases are still needed in order to gain more insight into the disease pathogenesis and to develop better therapeutic strategies.
Programmed death 1 (PD-1), which was originally identified in a T cell line undergoing activation-induced cell death, is a CD28 family member that contains a cytoplasmic immunoreceptor tyrosine-based inhibitory motif and is expressed on the surface of activated T cells and B cells (1–5). As an immunoinhibitory receptor, PD-1 has been shown to inhibit lymphocyte activation and cytokine production after interacting with its ligands PDL-1 (B7-H1) and PDL-2 (B7-DC) (6–10). The immunoinhibitory function of PD-1 was further supported by the observation that mice deficient in PD-1 expression developed autoimmune diseases, despite having distinct phenotypes on different genetic backgrounds. C57BL/6 mice with PD-1 deficiency had an increased incidence of progressive glomerulonephritis, and, more impressively, all of these mice exhibited progressive arthritis, with synovial cell proliferation, lymphocyte infiltration, and pannus formation (11). These phenotypes resembled the clinical manifestations of SLE and RA in humans. In contrast, PD-1–deficient BALB/c mice developed autoimmune cardiomyopathy, with IgG deposition in the heart (12). These data, together with the observation of the wide tissue expression of the PD-1 ligands, suggest that interaction between PD-1 and its ligands plays an important role in maintaining peripheral tolerance.
Based on the solid evidence for the immunoinhibitory function of PD-1 in mice, we hypothesized that PD-1 is associated with the development of SLE and RA in humans. We therefore attempted to test this hypothesis by identifying a single-nucleotide polymorphism (SNP) in the human PD-1 gene that we could use for conducting genetic-association studies. Directly validating candidate SNPs, which were predicted by nucleotide mismatches in documented human PD-1 complementary DNA (cDNA) sequences, we discovered a SNP located at nucleotide position +872 (counting from the first base of the PD-1 cDNA sequence) in Chinese subjects (4). This SNP was then used in case–control genetic-association studies to determine whether the PD-1 gene is associated with the development of RA and SLE.
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Lymphocyte activation is strictly regulated by positive and negative signals that are delivered through various immunoregulatory receptors (16). A defect in the negative signals from immunoinhibitory receptors may reduce the threshold of autoreactive lymphocyte activation and lead to the development of autoimmune diseases. This has been evidenced by the expression of the autoimmune phenotype or lymphocyte hyperactivity in genetically manipulated mice with a deficiency in an immunoinhibitory receptor (Fcγ receptor IIB [FcγRIIB], CD22, CTLA-4, or PD-1) and in mice deficient in an inhibitory signaling molecule (Lyn or src homology 2 domain–containing phosphatase 1) (17–21). Thus, negative signals initiated by immunoinhibitory receptors appear to play an important role in the maintenance of peripheral tolerance in mouse.
In humans, the contribution of the immunoinhibitory receptors and their signal molecules to the development of autoimmune diseases remains unclear. Nevertheless, the immunoinhibitory receptors FcγRIIB, CD22, and CTLA-4 have been used as candidate genes in SNP case–control association studies to test their possible association with the human autoimmune diseases SLE and RA. It was found that FcγRIIB and CD22, but not CTLA-4, are associated with SLE susceptibility, whereas neither CD22 nor CTLA-4 appears to be linked to RA susceptibility (22–28).
Our case–control genetic-association study using the C+872T SNP in the PD-1 gene is the first to show evidence of an association of the PD-1 gene with susceptibility to RA, but not to SLE, in the Chinese population. In contrast to our findings, Prokunina and coworkers (15) recently found that the PD-1 gene is associated with SLE susceptibility in European and Mexican multicase families, but not in African American multicase families. This discrepancy may be explained by the influence of the genetic background on susceptibility to autoimmunity, similar to the differential expression of autoimmune phenotypes in different strains of mice deficient in FcγRIIB or PD-1 (11, 12, 17). Yet, further studies of ethnic differences in the expression and function of PD-1 may provide better insight into the role of the PD-1 protein in initiating autoimmune diseases in different human populations.
The molecular mechanisms that account for the association between RA susceptibility and the T allele and the C/T genotype of the C+872T SNP remain to be clarified. One possible mechanism is that the C+872T SNP may be associated with an alteration in the level of expression of the PD-1 gene, possibly as a result of linkage disequilibrium with other PD-1 gene polymorphisms, which differentially control PD-1 gene transcription. This possibility is supported by the identification of several SNPs, including C+872T, in the promoter, introns, exon 5, and 3′-untranslated region of the PD-1 gene in the European population and the finding that a SNP in intron 4 alters the runt-related transcription factor 1 binding site (15). Since there was a significant association between RA and the T allele of the C+872T SNP, we may, by this hypothesis, expect a dose-dependent effect of the T allele on RA susceptibility, but this is inconsistent with the observed lack of a significant association between the T/T genotype and RA susceptibility. However, the absence of an association between RA and the T/T genotype in the present study may be due to the occurrence of a genotype distribution that is skewed toward the C/T genotype in the disease group in a case–control study, because carriage of 1 T allele may be able to dominantly influence susceptibility to RA. An evaluation of the correlation between different genotypes and PD-1 expression levels may help in the assessment of this hypothesis.
Another possible explanation for the significant association of the C+872T SNP with RA susceptibility is the connection between this SNP and the functional change in the PD-1 protein via linkage disequilibrium with other nucleotide polymorphisms that alter the sequence and structure of the PD-1 protein. This possibility may be tested by evaluating the ability of PD-1 protein to transduce inhibitory signals in response to ligand stimulation in individuals with different genotypes. It is possible that finding a correlation between genotypes and modifications in the function of the PD-1 protein may provide an explanation for the significant association between RA susceptibility and the C/T genotype, but not T/T genotype, that was observed in this study. However, we still cannot exclude the possibility that another genuine RA-susceptibility gene is located adjacent to the PD-1 gene in the region of chromosome 2q37.3 and affects the significant association between the PD-1 gene and RA susceptibility, particularly if the expression and function of PD-1 are not correlated with different C+872T SNP genotypes.
In conclusion, we have shown experimental evidence that a genetic locus around an immunoinhibitory receptor gene, PD-1, is linked to RA susceptibility in the Chinese population. These data may provide a starting point for further investigations of whether the defect in PD-1 expression and function plays an important role in the pathogenesis of RA.