Haplotype-Independent Costimulation of IL-10 Secretion by SDF-1/CXCL12 Proceeds via AP-1 Binding to the Human IL-10 Promoter

Abstract

Costimulation by the chemokine, stromal cell-derived factor-1 (SDF-1)/CXCL12, has been shown to increase the amount of IL-10 secreted by TCR-stimulated human T cells; however, the molecular mechanisms of this response are unknown. Knowledge of this signaling pathway may be useful because extensive evidence indicates that deficient IL-10 secretion promotes autoimmunity. The human IL-10 locus is highly polymorphic. We report in this study that SDF-1 costimulates IL-10 secretion from T cells containing all three of the most common human IL-10 promoter haplotypes that are identified by single-nucleotide polymorphisms at −1082, −819, and −592 bp (numbering is relative to the transcription start site). We further show that SDF-1 primarily costimulates IL-10 secretion by a diverse population of CD45RA− (“memory”) phenotype T cells that includes cells expressing the presumed regulatory T cell marker, Foxp3. To address the molecular mechanisms of this response, we showed that SDF-1 costimulates the transcriptional activities in normal human T cells of reporter plasmids containing 1.1 kb of all three of the common IL-10 promoter haplotypes. IL-10 promoter activity was ablated by mutating two nonpolymorphic binding sites for the AP-1 transcription factor, and chromatin immunoprecipitation assays of primary human T cells revealed that SDF-1 costimulation enhances AP-1 binding to both of these sites. Together, these results delineate the molecular mechanisms responsible for SDF-1 costimulation of T cell IL-10 secretion. Because it is preserved among several human haplotypes and in diverse T cell populations including Foxp3+ T cells, this pathway of IL-10 regulation may represent a key mechanism for modulating expression of this important immunoregulatory cytokine.

The Sp1 transcription factor binds to the G-allele of the –1087 IL-10 gene polymorphism and enhances transcriptional activation

“…23 Conversely, other reports suggested that the presence of a G-allele at À1087 was associated with an increase in the IL-10 promoter activity. 24,25,28,29 In earlier studies from our laboratory, 30,31 we demonstrated that severe chronic periodontitis was strongly associated with the GG genotype of the À1087 IL-10 gene polymorphism and that the GG genotype correlated with enhanced fractions of IL-10-positive cells in periodontitis lesions.…”

“…25,28 Transcriptional regulation of the IL-10 gene was studied in several cell types, 23,24,29,33 but the results regarding the influence of the À1087 polymorphism on IL-10 gene expression are conflicting. Rees et al 23 using transient transfections in an Epstein-Barr virustransformed human B-cell line showed that the A-allele was associated with a twofold increase in transcriptional activity as compared to the G-allele.…”

“…[20][21][22] The IL-10 promoter contains several single nucleotide polymorphisms (SNPs) among which the -1087 G/A SNP (sometimes referred to as À1082 G/A) was associated with differences in IL-10 promoter activity and IL-10 production. [23][24][25][26][27][28][29] Results of studies on the influence of the -1087 SNP on the IL-10 production in different cell types, 23,[25][26][27][28][29] however, are conflicting. Thus, the presence of an A-allele was shown to result in an increase in the IL-10 promoter activity, which may lead to enhanced production of the IL-10 protein.…”

Sp1 binds to the G allele of the−1087 polymorphism in the IL-10 promoter and promotes IL-10 mRNA transcription and protein production

“…19,20,26,27,32 The present series of experiments were performed to study the influence of the binding of the Sp1 transcription factor to the À1087 SNP on IL-10 transcription. The binding of the transcription factors PU.1, Spi-B and Sp1 to this site in the IL-10 promoter was earlier analyzed using electromobility shift assay.…”

“…19,[25][26][27]32,40,41 Thus, Turner et al 27 found that subjects who were negative for the A allele exhibited a higher production of IL-10 than subjects who were positive for the A allele. These results are in concordance with data presented in a study on stimulated purified blood lymphocytes.…”