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The Journal of Immunology, 2004, 173: 3215-3222.
Copyright © 2004 by The American Association of Immunologists

Functional Analysis of –571 IL-10 Promoter Polymorphism Reveals a Repressor Element Controlled by Sp11

John W. Steinke2,*, Elizabeth Barekzi*, James Hagman{dagger} and Larry Borish*

* Asthma and Allergic Diseases Center, Beirne Carter Center for Immunology Research, University of Virginia, Charlottesville, VA 22908; and {dagger} Integrated Department of Immunology, National Jewish Medical and Research Center, Denver, CO 80206, and University of Colorado Health Sciences Center, Denver, CO 80220


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Transcriptional dysregulation of the IL-10 gene may contribute to the development and severity of autoimmune, infectious, neoplastic, and allergic diseases. A C to A base substitution has been identified at –571 bp in the IL-10 promoter and has been linked to immune diseases. The role of this polymorphism in IL-10 promoter function was assessed using luciferase reporter constructs. The presence of an A at –571 (A allele) increases promoter activity compared with that of a promoter with a C at this position (C allele). Binding of nuclear extract proteins from IL-10-producing human cell lines to DNA sequences including this base exchange and flanking sequences was demonstrated using EMSAs. Specific binding of the transcription factors Sp1 and Sp3 was demonstrated to a region immediately upstream of the polymorphism. No differences in the binding affinity of recombinant Sp1 were observed between the two forms of the promoter. Reconstitution of Sp1 expression decreased IL-10 promoter function in an Sp1-deficient cell line, demonstrating that this element functions as a repressor. The C to A base exchange relieves the repression mediated by Sp1. Individuals carrying the A allele of the IL-10 promoter may display increased synthesis of IL-10, resulting in suppressed immune responses and a modulation of their susceptibility to autoimmune, infectious, neoplastic, or atopic disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleukin-10 is a cytokine that displays pleiotropic effects in immunoregulation and inflammation (1). IL-10 inhibits both Th1 production of IFN-{gamma} and IL-2 (2, 3) and IL-4 and IL-5 expression by Th2 lymphocytes (4). Monocytes are the major source for human IL-10 (5, 6). The primary T cell source of IL-10 is the T regulatory (Treg)3 cell, with additional amounts being produced by Th1 and Th2 lymphocytes (4, 7, 8, 9). Other sources of IL-10 include CTLs, B lymphocytes (10, 11), myeloid dendritic cells (12), and mast cells (13). In addition to its effects on Th1 and Th2 cytokine production, IL-10 inhibits the production of IL-1{beta}, IL-6, IL-8, IL-12, and TNF-{alpha} from mononuclear phagocytes (14, 15, 16, 17). IL-10 also inhibits monocyte MHC class II, B7.1/B7.2 (CD80/CD86) expression and accessory cell functions (18). Inhibition of accessory cell function may be the primary means by which IL-10 inhibits cytokine production by Th1 and Th2 lymphocytes. The expression of IL-10 by APCs represents an established pathway for induction of tolerance to allergens (19, 20). Support for a modulating role for IL-10 in human allergic diseases is further derived from observations that IL-10 inhibits eosinophil survival (21), inhibits IgE synthesis (22), and enhances IgG4 synthesis (23). Studies in mice have shown that dendritic cells produce IL-10 after respiratory exposure to Ag, and this stimulates CD4+ Treg-like cells to produce high levels of IL-10 and induce tolerance (24).

Genetic variations in the nucleotide sequences of promoters can contribute to the altered expression of genes in complex inherited diseases (25, 26, 27). In this regard, a genetically determined reduction in IL-10 production could result in either a failure to maintain a milieu promoting tolerance or a failure of Treg cells to keep Th cells in check. Previously, we described a C to A exchange in the IL-10 promoter located 571 bp upstream from the transcription start site, which is located between putative consensus binding sequences for Sp1 and Ets family proteins (Fig. 1A) (28). This polymorphism is associated with elevated total serum IgE levels in subjects heterozygotic or homozygotic for this base exchange. Recently, in a study of Caucasian families the polymorphism was confirmed as an asthma-associated gene associated with an increase in eosinophil cell counts (29). Moreover, the polymorphism has been linked with increased severity to a number of autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus (SLE), and inflammatory bowel disease (30, 31, 32, 33, 34), and is associated with changes in tumorigenesis and transplantation tolerance (35) and rapid progression to AIDS in individuals infected with HIV (36). These are all immune diseases that predictably could be associated with a molecular genetic mechanism linked to loss of either tolerance or repression of immune responses.



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  		FIGURE 1. A, Diagram depicting the IL-10 –571 polymorphism and the putative Sp1 site upstream and Ets-like site downstream of the polymorphism. B, Schematic representation of luciferase constructs used in transient transfection assays. A single construct was made that is truncated at –560 bp and does not contain the polymorphic residue or putative Sp1 binding site. Two constructs (one with the C allele and one with the A allele) were made that were truncated at position –588 and contained the putative Sp1 site. Two longer constructs (one with the C allele and one with the A allele) were created that extended the IL-10 promoter to –1340 bp. C, Transient transfection assays were performed in the human B cell line Raji. Transcription of each IL-10 promoter template was separately normalized relative to the activity of the –560 construct. Data points represent the average of three independent transfection experiments, with error bars representing the SEM.

 
The linkage studies cannot distinguish direct influences of this polymorphism on gene function from differences mediated by other polymorphisms that may be in linkage disequilibrium. To better understand mechanisms that modulate IL-10 expression, we therefore performed studies to determine the functional significance of the –571 nt exchange in the IL-10 promoter and the basis of its linkage with autoimmune, neoplastic and allergic diseases. We demonstrate that the C to A nucleotide exchange results in increased promoter activity. Specific binding of the transcription factors Sp1 and Sp3 was demonstrated to a region immediately upstream of the polymorphism. The region including the Sp1/Sp3 site and adjoining polymorphism functions as a repressor, and the C to A base exchange relieves the repression mediated by Sp1. This is the first study to demonstrate a functional role for the –571 base exchange in the IL-10 promoter, which has wide-ranging implications for a variety of immunological diseases.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reporter constructs

Reporter constructs were generated by cloning promoter segments of different lengths upstream from the putative translation initiation site of the human IL-10 gene into the luciferase reporter plasmid pGL3-basic (Promega, Madison, WI; see Fig. 1B). Numbering of the constructs is from the transcription start site. C- and A-containing plasmids at position –571 were created for both the –588 and –1340 IL-10 promoter constructs. Promoter DNA was amplified using PCR with genomic DNA (from homozygous donors) and appropriate primers (upstream primer for the –588 constructs, 5'-AAGGTACCTGTGACCCCGCCTGT(C/A)CTGTAGGAAGCCAG-3'; primer for the –560 construct, 5'-TCAAGGTACCAGTCTCTGGAAAGTAAAATGG-3'; downstream primer, 5'-GTGGAAGCTTGCCTTCTTTTGCAAGTCTG-3'). Fragments were digested with KpnI and HindIII restriction enzymes and were ligated into the pGL3-Basic vector digested with the same enzymes. A second set of constructs was generated representing 1340 bases with the CT and AT forms of the IL-10 promoter (–1340 upstream primer, 5'-TCAAGGTACCTAGGTCAGTGTTCCTCCCAGTTAC-3'; downstream primer, 5'-GTGGGCTAGCTGCCTTCTTTTGCAAGCTG-3'). The PCR products were processed as described above, except that KpnI and NheI were used for the restriction digests. The Sp1 mutant constructs were identical with the –588 constructs, except that the upstream primer introduced a 4-bp mutation in the Sp1 site (5'-AAGGTACCTGTGACCTTATCTG(C/A)CTGTAGGAAGCCAG-3') (underline indicates mutated sequence). All constructs were confirmed by sequencing.

Cell culture

All cell lines used were purchased from American Type Tissue Culture (Manassas, VA). Raji B cells were grown in suspension to mid-log phase in complete RPMI 1640 medium (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin sulfate (Invitrogen Life Technologies), 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 1.5 g/l sodium bicarbonate, 4.5 g/l glucose, and 10% FBS (HyClone, Logan, UT). Cells were maintained at 37°C in 5% CO2. Schneider’s Drosophila line 2 cells were grown in Schneider’s medium (Invitrogen Life Technologies) supplemented with 10% FBS and were maintained at 24°C without supplemental CO2.

EMSA

Nuclear protein was isolated from human IL-10-expressing Raji B cells as previously described (37). CG and AT oligonucleotides representing bp –568 to –591 of the IL-10 promoter (from the transcription start site) were synthesized: CG, 5'-TCGACATCCTGTGACCCCGCCTGTCCTG-3' and 5'-TCGACAGGACAGGCGGGGTCACAGGATG-3'; and AT, 5'-TCGACATCCTGTGACCCCGCCTGTACTG-3' and 5'-TCGACAGTACAGGCGGGGTCACAGGATG-3' (underline indicates polymorphic residue).

Synthesis of 32P-labeled, double-stranded probe DNA and EMSAs was performed as previously described (37, 38). Labeled probe was electrophoresed on a 6% nondenaturing polyacrylamide gel to separate labeled probe from free nucleotide, single-stranded oligonucleotides, and double-stranded oligonucleotides that were incompletely extended. The probe was eluted from the gel and isolated over a Centrisep separation column (Princeton Separations, Adelphia, NJ), and each probe was counted to determine the radioactivity. From the counts, the femtomoles of each probe to use in a reaction was calculated. Samples were electrophoresed on a 6% nondenaturing polyacrylamide gel. To estimate binding affinity, relative fractions of bound and free probe were quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Specific binding of Sp1 to the IL-10 promoter was evaluated by competition with excess unlabeled double-stranded Sp1 consensus oligonucleotides (5'-ATTCGATCGGGGCGGGGCGAGC-3' and 5'-GCTCGCCCCGCCCCGATCGAAT-3') and mutant consensus probes (5'-ATTCGATCGGTTCGGGGCGAGC-3' and 5'-GCTCGCCCCGAACCGATCGAAT3'; Santa Cruz Biotechnology, Santa Cruz, CA). Supershift assays were performed with anti-Sp1, anti-Sp3, anti-upstream stimulatory factor (USF), anti-STAT6, anti-Ets-1, and anti-Fli1 Abs (Santa Cruz Biotechnology). Binding of Sp1 was also confirmed using recombinant Sp1 protein (Promega).

Transient transfection assays

The activity of the IL-10 promoter was determined by measuring the levels of expression of the luciferase reporter gene. Briefly, plasmids (3 µg of IL-10 promoter-containing plasmid and 2 µg of {beta}-galactosidase expression plasmid) were cotransfected using DEAE-dextran (Sigma-Aldrich, St. Louis, MO) into the human Raji B cell. These experiments were repeated with three independent DNA preparations. DNA was mixed with 500 µg/ml DEAE-dextran in Tris-saline (pH 7.4) to a final volume of 1.5 ml. The DNA mixture was added to 6 x 106 cells and incubated for 1 h at room temperature. Ten milliliters of medium containing 100 µg/ml cholorquine diphosphate was added to the cells and incubated for an additional 1 h at 37°C. The medium was removed, and the cells were washed in PBS. Ten milliliters of complete Raji medium was added, and the cells were plated and incubated for an additional 48 h at 37°C.

Transfection of Schneider’s Drosophila line 2 (which lacks Sp1) was performed using 20 µl of Lipofectamine 2000 (Invitrogen Life Technologies) in serum-free medium. Each reaction contained 3 µg of IL-10 promoter-containing plasmid and 2 µg of {beta}-galactosidase expression plasmid. For the Sp1 expression vector titration, increasing amounts of the pPacSp1 expression vector were cotransfected along with the appropriate concentrations of empty vector pPac0 to give 2 µg of total DNA. After 4 h of incubation of the cells with the Lipofectamine:DNA complexes, the medium was changed to Schneider’s Drosophila medium (Invitrogen Life Technologies) supplemented with 10% FBS, and the cells were maintained at 24°C without supplemental CO2 for 48 h.

Cells were collected by centrifugation and lysed in reporter lysis buffer (Promega). Cellular extracts were assayed for luciferase activity using a MonoLight 2010 (Analytical Luminescence Laboratories, Ann Arbor, MI) by counting and integrating for 30 s. Data were normalized to {beta}-galactosidase expression (Promega), were calculated as stimulation indexes in comparison with resting (luciferase negative) cells, and were subsequently normalized in comparison with the activity of the –560 construct.

Chromatin immunoprecipitation (ChIP) assay

ChIP assays were performed using a ChIP assay kit from Upstate Biotechnology (Lake Placid, NY). Briefly, 1 x 106 cells for each condition were treated with 1% formaldehyde for 10 min at 37°C to cross-link the protein to the DNA. The cells were collected, lysed in SDS lysis buffer, and subjected to eight 30-s cycles of sonication using a Sonic Dismembrator 550 (Fisher Scientific, Pittsburgh, PA). The positive control was phenol/chloroform-extracted and ethanol-precipitated. The remaining samples were diluted in ChIP dilution buffer and precleared with salmon sperm DNA. The samples either received no Ab (negative control) or were given with anti-Sp1, -Sp3 or -Stat3 (Ab control; Santa Cruz Biotechnology) and incubated overnight at 4°C. Abs were precipitated with a salmon sperm/protein A agarose slurry and extensively washed. The protein-DNA cross-links were reversed with 0.2 M NaCl and by heating the samples for 4 h at 65°C. The samples were phenol/chloroform-extracted and ethanol-precipitated before being used in a PCR with primers that amplified 100 bases surrounding the –571 residue. The sequence for the upstream primer was 5'-GCGAGAATCCTAATGAAATCGG-3', and that for the downstream primer was 5'-TATCCTCAAAGTTCCCAACG-3'.

Statistical analysis

Data are expressed as the mean ± SEM. Statistical significance was determined by paired t test. A value of p < 0.05 was considered to indicate statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-10 –571 promoter polymorphism results in altered transcriptional activity

To determine whether the C to A polymorphism at position –571 affects IL-10 promoter function, we measured the transcriptional activity of IL-10 promoter fragments in transient transfection assays (Fig. 1A). Fragments of the IL-10 promoter were cloned into a luciferase reporter plasmid (Fig. 1B), and the activity was measured in the human B cell line Raji (Fig. 1C). Raji was chosen because these cells constitutively produce IL-10 (39). Activity determined in each transfection was normalized to that of the –560 construct, which was assigned a relative transcription index of 1.0. Addition of 28 bases with the C allele at position –571 resulted in a 3.3-fold decrease in transcription, suggesting that the additional IL-10 promoter DNA included a repressor element. Within this segment, changing the sequence of the C to the A allele resulted in a 3.1-fold increase in transcription (p < 0.02), resulting in levels comparable to that observed with the –560 construct. Extending the IL-10 promoter to bp –1340 resulted in an increase in the basal levels of transcription compared with those of the –588 constructs, but the difference between the C- and A-containing promoters was maintained (p < 0.01).

Sp1 and Sp3 bind to a sequence immediately upstream of polymorphic residue

The effects of the base exchange in the IL-10 promoter on factor binding were investigated by EMSA. Preliminary EMSAs were performed using DNA fragments comprising 50 bp of the two forms of the promoter (labeled CG or AT), including the two different bases at position –571. The assay detected slow-migrating electrophoretic complexes using extracts derived from numerous IL-10-producing or nonproducing cell lines (data not shown). The data suggest that a widely expressed transcription factor can regulate IL-10 promoter function through binding to this region.

The –571 base substitution is situated between consensus sequences for an Sp1 site and an Ets-like binding site (Fig. 1A). We hypothesized that complexes detected in our EMSA could include Sp1. To address this hypothesis, EMSAs were repeated with nuclear extract from the IL-10-producing cell line Raji using shorter oligonucleotide probes containing only the putative Sp1 binding site and including each of the polymorphic residues. As demonstrated in Fig. 2, each probe produced similar patterns of shifted complexes, but binding to the CG probe was always more efficient than that to the AT probe. All shifted complexes were specifically competed by excess unlabeled probe DNA. Efficient competition was also detected using excess unlabeled Sp1 consensus oligonucleotides, but not by mutated Sp1 consensus sequences. The presence of Sp1 in the slowest migrating complexes was confirmed by supershifting with anti-Sp1 Abs (Fig. 3). Interestingly, inhibition of DNA binding was also observed with Abs specific for the related family member Sp3, suggesting that both Sp1 and Sp3 recognize the site upstream of the –571 polymorphism. No supershifting was observed with an isotype-matched control (anti-Ets-1, anti-Fli1, anti-STAT6, or anti-USF) Abs.



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  		FIGURE 2. Sp1-specific consensus competition. The human B cell Raji nuclear extract was allowed to interact with 32P-labeled oligomers. The labeled probes represent the CG and AT forms of the IL-10 promoter at base –571. Nonspecific binding was mitigated by addition of poly(dI-dC). FP, free probe without nuclear extract (NE) added. Competition of shifted bands was performed with increasing concentrations (100- to 1000-fold) of unlabeled oligonucleotide. Three major shifted complexes were observed when NE was added to both probes. Unlabeled self-oligonucleotide was able to specifically compete for binding of these shifted complexes. Bands were also eliminated by competition with excess unlabeled Sp1 consensus probe, but not by a mutated form of the Sp1 probe.

 


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  		FIGURE 3. A supershift assay demonstrates that both Sp1 and Sp3 are part of a complex on the IL-10 promoter. Raji nuclear extract was allowed to interact with 32P-labeled oligomers. The labeled probes represent the CG and AT forms of the IL-10 promoter at base –571. Anti-Sp1 supershifts the higher m.w. band, and anti-Sp3 competes with binding of both the lower and higher m.w. bands. Isotype-matched (anti-Ets-1, anti-Fli-1, anti-STAT6, or anti-USF) Abs did not alter any of the shifted complexes.

 
Relative binding of recombinant Sp1 to the two forms of the IL-10 promoter

Specific binding of the site by Sp1 was confirmed by EMSA using recombinant Sp1 and the labeled probes (Fig. 4). Using increasing concentrations of recombinant Sp1 protein, we evaluated the binding affinity of Sp1 to the CG and AT forms of the promoter. Data were evaluated by phosphorimager, and we were able to calculate relative Ka values for each form of the promoter. No differences were observed in the binding affinity of recombinant Sp1 to the C or A form of the IL-10 promoter (data not shown).



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  		FIGURE 4. Binding of recombinant Sp1 to the IL-10 promoter. The labeled probes represent the CG and AT forms of the IL-10 promoter at base –571. One footprinting unit of recombinant Sp1 was added to each reaction. Unlabeled Sp1 consensus probe, but not a mutated form of the Sp1 probe, was able to specifically compete for binding of these shifted complexes.

 
In vivo detection of Sp1 and Sp3 binding to the IL-10 promoter

The EMSA experiments indicated that Sp1 and Sp3 could bind to the IL-10 promoter using in vitro binding assays, but these assays may not be indicative of in vivo binding of these proteins. To determine whether the Sp1 and Sp3 proteins bind to the IL-10 promoter within a cell, we used the ChIP assay. THP-1 monocytes, Raji B cells, and Jurkat T cells were examined because all three cell types support transcription of the IL-10 gene. As shown in Fig. 5, Sp3 binding to the –571 region of the IL-10 promoter was detected in all three cell types. A complex of Sp1 with the IL-10 promoter was only detected in the THP-1 and Raji cell lines. To confirm the specificity of Ab immunoprecipitation, an anti-Stat3 Ab was used as a control. This protein does not interact with this region of the IL-10 promoter as determined by EMSA, and the Ab was not able to immunoprecipitate a DNA fragment containing the –571 residue.



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  		FIGURE 5. Chromatin immunoprecipitation assay performed on THP-1 monocytes, Raji B cells, and Jurkat T cells to measure in vivo Sp1 and Sp3 binding. Proteins were cross-linked to the DNA with formaldehyde, and Abs directed against Sp1, Sp3, or Stat3 (Ab control) were added to precipitate any protein-DNA complexes. The – control lanes were processed according to the protocol, but did not have any Ab added to the samples. The + control lane was DNA that went through sonication and reversal of the protein-DNA contacts followed by ethanol precipitation. PCRs were performed on isolated DNA using primers that span the –571 site in the IL-10 promoter and were analyzed by agarose gel electrophoresis.

 
Sp1 inhibits transcription of –571 C containing IL-10 promoter in Drosophila SL2 cells

To directly test the role of Sp family in mediating transcriptional activity through the –571 polymorphic residue, we performed transient transfection assays in a cell line deficient of Sp transcription factor family members. Increasing amounts of a Sp1 expression vector were transfected with the –588CG, –588AT, or –560 IL-10 constructs, and promoter activity was measured. Transcription from the –588AT and –560 IL-10 constructs was stimulated as the concentration of the Sp1 expression vector was increased (Fig. 6). This increase in transcription is probably due in part to Sp1 binding to a putative site downstream of the –571 residue (40, 41). In contrast, no stimulation of transcriptional activity was observed for the –588CG promoter at any concentration of Sp1 expression vector, indicating that Sp1 can inhibit transcription through the –571 residue. To confirm the inhibitory role for the Sp1 site in the –588CG construct, the Sp1 site was mutated in the context of the C allele-containing promoter. Transcription of the IL-10 gene was increased from the Sp1 mutant compared with that of the –588CG construct when transfected into Raji cells (data not shown).



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  		FIGURE 6. Effect of Sp1 on the IL-10 promoter in Drosophila SL2 cells that lack endogenous Sp family transcription factors. SL2 cells were transfected with 3 µg of each IL-10 promoter construct with the indicated amount of pPacSp1. The total amount of the expression plasmid was maintained at 2 µg with pPac0. Transcription of each IL-10 promoter template was separately normalized relative to the luciferase activity of the construct without addition of the Sp1 expression vector. Data points represent the average of at least three independent transfection experiments, with error bars representing the SEM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The concept that promoter polymorphisms may influence transcription levels by altering promoter strength and thereby produce phenotypic differences has been shown for polymorphisms in the human MHC class II locus (42, 43), CD14 gene (44), CCL5 (RANTES) promoter (45, 46), and many others. Polymorphisms in the MHC class II locus are associated with allele- and tissue-specific differences in expression of these genes. The CD14 and CCL5 promoter polymorphisms have been linked to the complex inheritance of serum IgE and atopic dermatitis, respectively.

The current studies were performed to examine the effects of base exchanges in the IL-10 promoter on the regulation of IL-10 transcription. EMSAs were used to characterize specific binding of nuclear protein to promoter sequences flanking these base substitutions. This protein was a ubiquitous factor, as it was found in both IL-10-producing and nonproducing cell lines. We demonstrated specific binding of the transcription factors Sp1 and Sp3 to the IL-10 promoter to a site immediately upstream of the –571 residue (Figs. 2 and 3). No difference in the affinity of binding for recombinant Sp1 was observed on the C- or A-containing probes. When crude nuclear extract was used, multimeric complexes were observed. This most likely reflects multimer formation of Sp1 with other proteins or post-translational modifications of Sp1 that were not present in the recombinant protein. These multimeric complexes contain at least Sp1 and Sp3, as can be seen in the supershift (Fig. 3) where anti-Sp3 eliminates binding of the Sp1-containing band. Support for the concept that the interactions of Sp1 with gene promoters involves ternary complexes comes from studies with the CD14 promoter where Sp2 was demonstrated to form a complex with Sp1 (44), and regulation of the parathyroid hormone-related protein gene involves interaction of Sp1 with Ets1 on the promoter (47). The combination of Sp1 with other transcription factors may lead to the opposite functions of activation and repression observed for this protein depending on the expression levels and functions of these factors in various cell lines. The binding of both Sp1 and Sp3 was confirmed in vivo using the ChIP assay (Fig. 5). Three different cell lines that express IL-10 were chosen: a monocyte cell line, THP-1; a B cell line, Raji; and a T cell line, Jurkat. Binding of Sp1 and Sp3 to the IL-10 promoter was observed in the THP-1 and Raji cell lines, whereas only Sp3 was found to be bound to the promoter in the Jurkat cell line. This may be a reflection of lineage-specific differences in IL-10 promoter occupancy by these proteins. An alternative explanation may be that differences in promoter occupancy are due to genotype, because the THP-1 cell line is homozygous for the A allele, Jurkat is homozygous for the C allele, and Raji is heterozygous for the two alleles. Additional experiments are needed to clarify these possibilities.

An influence on gene expression was confirmed with functional assays by comparing the expression of reporter genes linked to different forms of the IL-10 promoter (Fig. 1C). The polymorphism is present in the promoter in a region with negative enhancer activity and is associated with loss of this activity. When the Sp1 site was mutated in the context of the C allele-containing promoter, transcription of the IL-10 gene was increased further, supporting its role as a repressor element (data not shown). This base exchange occurs between what we confirmed as being an Sp family member binding site and a sequence with similarity to that recognized by members of the Ets family (AGGAA; Fig. 1A). We have not yet identified the other transcription factor(s) that specifically binds to this region of the IL-10 promoter; however, the binding affinity of this factor was influenced by the base exchange (our unpublished observations). Sp1 is a ubiquitous factor that has been shown to be important in both transcriptional activation and repression. The interaction of Sp1/Sp3 to this region of the IL-10 promoter was associated with decreased promoter function. It is possible that Sp1 may have direct inhibitory influences on the IL-10 promoter. Alternatively, Sp1 may displace an important stimulating transcription factor from the IL-10 promoter and thereby interrupt the architecture of the functional IL-10 promoter. Transfection of Drosophila SL2 cells with the IL-10 promoter constructs along with a Sp1 expression vector supports a direct role of Sp1 acting as a repressor (Fig. 6). Transcription from the –588 promoter construct was inhibited if the promoter contained a C at position –571. When the promoter contained the A allele or the site upstream of bp –571 was mutated, Sp1 was able to activate transcription of the IL-10 promoter. An Sp1 site that is important for unstimulated and LPS-stimulated transcription has been described for the murine IL-10 promoter located between nucleotides –89 to –78 (40, 41). This Sp1 site differs from the typical GC box consensus normally described, because it has the sequence CCTCCT. On the human IL-10 promoter, a site containing a one-base mismatch to this sequence is located 12 bp upstream of the TATA box. Activity from this potential Sp1 site has not been assessed, but is indirectly supported by our data.

IL-10 underproduction has been linked to autoimmune diseases, including human inflammatory bowel disease (48, 49) and more virulent forms of experimental allergic encephalitis (11, 50). In contrast, IL-10 overproduction has also been associated with autoimmune diseases, including insulitis, as demonstrated in an experimental model of diabetes-prone rodents (51); SLE (52); and neoplasia (53). For example, in non-Hodgkin’s lymphoma (NHL), both IL-10 message and protein are found in B cells, and increased levels of IL-10 correlate with severity of disease (54, 55). A more recent study has demonstrated that the A nucleotide at position –571 of the IL-10 promoter is associated with more aggressive forms of NHL (56). Our demonstration that this allele results in higher levels of IL-10 production, which could thereby act to suppress the antineoplastic immune response, supports the negative impact of this polymorphism in NHL. Serum levels and ex vivo studies of monocytes and B cells from patients with SLE and rheumatoid arthritis have shown increased levels of IL-10 production (57, 58). Several genetic studies have confirmed that the –571 polymorphism is overexpressed in populations of patients with SLE (52, 59). IL-10 overproduction may contribute to SLE through its ability to promote humoral immune responses. IL-10 has also been demonstrated to be important for the allergic diseases. Populations of Treg cells have been identified, including CD4+CD25+ T cells (Treg) that can suppress the expansion and cytokine secretion of other peripheral CD4+ T cells. The regulatory effects of these cells may be mediated in part by high expression of IL-10 and TGF-{beta} (60). The production of IL-10 may also mediate differentiation of regulatory T cells. In nonallergic subjects, Treg cells prevent a robust immune response when Ag is encountered in the lung. However, this response is absent or diminished in allergic subjects, allowing Th2-like lymphocytes to proliferate and generate a nonproductive T cell response. In humans, allergen challenges, more severe asthma, and the skin of atopic dermatitis subjects are all associated with the expression of IL-10 mRNA (61, 62, 63). Thus, IL-10 and mutations influencing IL-10 production may have different effects depending on the timing and source. If present early in the immune response, IL-10 should induce nonresponsiveness to allergens and prevent the IgE isotype switch by inhibiting IL-4 and IL-13 production, whereas when present late in the allergic response, it could enhance IgE secretion by B lymphocytes that have undergone the {epsilon} isotype switch.

Our indirect data support the existence of both pro- and anti-inflammatory influences of IL-10 in allergic inflammation via the observation that the A form of the promoter is associated with both enhanced functional activity when linked to a luciferase reporter gene, and subjects having this allele demonstrate increased total and specific IgE (28) and an increase in eosinophil numbers (64). However, a direct linkage of the polymorphism to increased IL-10 production in inflammation cannot be absolutely established without performing experiments in vivo. Consistent with these current results, our studies examining IL-10 production from PBMC indicate that in the basal state, individuals homozygous for the A allele express higher levels of IL-10 than those homozygous for the C allele (our unpublished observations). The established role of IL-10 in infectious, autoimmune, neoplastic, and demyelinating diseases make these important processes in which the linkage of this polymorphism to disease susceptibility provides a rich area for future research.


   Acknowledgments
 
We thank Dr. Robert Tjian for providing the pPac0 and pPacSp1 expression vectors, and Dr. John D Noti for providing the pPacSp3 expression vector.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by American Lung Association Grants AL440-ALA (to J.W.S.) and AI01793 (to L.B.). Back

2 Address correspondence and reprint requests to Dr. John W. Steinke, Asthma and Allergic Diseases Center, University of Virginia, P.O. Box 801355, Charlottesville, VA 22908-1355. E-mail address: js3ch{at}virginia.edu Back

3 Abbreviations used in this paper: Treg, T regulatory cell; ChIP, chromatin immunoprecipitation; NHL, non-Hodgkin’s lymphoma; SLE, systemic lupus erythematosus; USF, upstream stimulatory factor. Back

Received for publication August 21, 2003. Accepted for publication June 16, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Borish, L. C., J. W. Steinke. 2003. Cytokines and chemokines. J. Allergy Clin. Immunol. 111:S460.[Medline]
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