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Interindividual variations in constitutive interleukin-10 messenger RNA and protein levels and their association with genetic polymorphisms1

Suárez, Ana2; Castro, Patricia3; Alonso, Rebeca4; Mozo, Lourdes3; Gutiérrez, Carmen2 3 5

Author Information
doi: 10.1097/01.TP.0000055216.19866.9A
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Interleukin (IL)-10 is an important immunosuppressor cytokine, involved in the regulation of many aspects of immune responses. Levels of IL-10 are critical in immune regulation, controlling the balance between inflammatory and humoral responses mainly by inhibiting the production of proinflammatory mediators, such as IL-1β, tumor necrosis factor-α, interferon-γ, granulocyte-macrophage colony-stimulating factor, inducible nitric oxide synthase, and COX-2, in addition to chemokines, particularly IL-8 and regulated on activation, normal T-expressed and secreted (RANTES ) (1–3). Also, by diminishing the expression of MHC II and B7 on monocytes, IL-10 reduces their capacity to activate T cells and generate T helper (Th)1 responses (1–3). Considerable functional levels of IL-10 are constitutively produced, mainly by cells of myeloid origin and less abundantly by T and B lymphocytes (4). Basal levels of IL-10 may modulate the production of other immune mediators, leading to a cytokine-mediated steady state that may cause predisposition to the development of certain diseases. In fact, subtle differences in cytokine composition at the initiation of an immune response may play a key role in the differentiation toward specific Th1 or Th2 effector cells. Accordingly, it has been suggested that the cytokine network may have major effects on the outcome of allogenic responses. Both a prolonged survival (5–8) and a greater risk of organ rejection (9) have been associated with high IL-10 activity. Elevated levels of this cytokine have also been involved in the pathology of systemic lupus erythematosus (10), infectious diseases (11), and tumor-related immunosuppression (12). Other pathologies with a deregulated pattern of cytokines, such as inflammatory bowel disease (13), psoriatic skin lesions (14), and severe asthma (15), correlated with a low capacity of IL-10 production.

Previous studies have shown striking differences among healthy individuals in their ability to produce IL-10 after in vitro mitogen stimulation of human peripheral blood cells (16–19). This interindividual variation seemed to have a genetic origin, because analysis of differences in endotoxin-induced IL-10 production between monozygotic or dizygotic twins and nonrelated individuals estimated the heritability of differences in IL-10 secretion to be 74% (11). According to this genetically conditioned capacity to secrete IL-10, individuals have been tentatively classified as high, intermediate, and low IL-10 producers (18–20). The different amounts of secreted cytokine in response to various stimuli were directly proportional to the rate of gene transcription, whereas the mRNA half-life did not exhibit interindividual variations (17). This indicates that secreted protein levels are regulated by differential gene promoter activity. In fact, the 5′-flanking region of the IL-10 gene has proved to be polymorphic. In addition to two polymorphic microsatellites, there are various single nucleotide polymorphisms that seem to be implicated in the secretion of IL-10 after mitogen stimulation (16–20). Single base pair substitutions at positions −1082 (G/A), −819 (C/T), and −592 (C/A) generate three different haplotypes in the Caucasian populations studied—GCC, ACC, and ATA—which correlated with variability in IL-10 secretion after lipopolysaccharide (LPS) (14), concanavalin A (19,20), phytohemagglutinin (16), or anti-CD3 (18) stimulation of peripheral blood cells. The GCC haplotype was associated with high production, whereas the ATA haplotype was detected in low IL-10 secretors. However, nothing is known about the possible influence of promoter genotypes on the constitutive production of IL-10. To this end we have quantified mRNA and protein IL-10 basal levels in a healthy Spanish population and analyzed the distribution according to genotype promoter at positions −1082, −819, and −592. We have also analyzed the production of this cytokine in response to LPS stimulation in individuals with different IL-10 promoter genotypes. Our results indicate that genetic polymorphisms regulate both constitutive and induced levels of IL-10.


Sample DNA

DNA was obtained from the peripheral blood cells of 183 local Caucasoid unrelated healthy blood donors. Blood samples were collected in EDTA tubes and red cells lysed twice in 0.05 mM NH4HCO and 5 mM NH4Cl. After washing, white cells were lysed with 10 mM Tris (pH 8), 10 mM EDTA (pH 8), 50 mM NaCl, and 1% sodium dodecyl sulfate. Protein was digested overnight at 37°C with proteinase K and pelleted with 6 M NaCl after centrifugation at 3000 rpm for 15 min. DNA was precipitated with 2× volume of 96% ethanol.

Analysis of IL-10 Promoter Polymorphisms

Determination of the alleles present at positions −1082 (G/A), −812 (C/T), and −592 (C/A) of the IL-10 gene was assessed by amplification of the region containing each polymorphic site and hybridization with fluorescent-labeled probes. The genotype at each position was determined analyzing the Tm of the probe-target duplex after polymerase chain reaction (PCR) amplification using the LighCycler system with hybridization probes matched with one sequence variant. When a probe hybridizes over the other sequence variant, a mismatch is formed and the duplex is destabilized, lowering the Tm from the completely complementary duplex. For each amplification, 10 ng of DNA were subjected to PCR reactions in a total reaction volume of 12 μL, containing 1.2 μL of 10× PCR buffer (including Taq DNA polymerase, deoxynucleotide triphosphate; Roche Diagnostics, Mannheim, Germany), 4 mM of MgCl2, 0.5 μM of each forward and reverse primer, and 0.2 μM or 0.4 μM of each fluorescein (F) or LC Red450–labeled probes, respectively. The thermocycling conditions were 45 cycles at 95°C for 6 sec, 63°C for 10 sec, and 72°C for 22 sec. Melting curves were generated to obtain melting temperatures. Amplification primers used were as follows: 5′-ATC CAA GAC AAC ACT ACT AAG GC and 5′-ATG GGG TGG AAG AAG TTG AA for −1082; 5′-TCA TTC TAT GTG CTG GAG ATG G and 5′-TGG GGG AAG TGG GTA AGA GT for −819; and 5′-GGT GAG CAC TAC CTG ACT AGC and 5′-GCA GCC CTT CCA TTT TAC TTT C for −592. Hybridization probes (designed by TIB MOLBIOL, Syntheselabor, Berlin, Germany) were as follows: GGA TAG GAG GTC CCT TAC TTT CCT CTT ACC-F and LC Red640-CCC TAC TTC CCC CTCCCA AA for −1082; GGT GAT GTA ACA TCT CTG TGC CTC-F and LC Red640-TTT GCT CAC TAT AAA ATA GAG ACG GTA GGG for −819; and AGC CTG GAA CAC ATC CTG TGA CCC C-F and LC Red640-CCT GTC CTG TAG GAA GCC AGT CTC for −592.

mRNA Isolation and cDNA Synthesis

Peripheral blood was collected from 128 healthy donors in tubes containing EDTA and processed within 1 hr after collection. One and one half milliliter of whole blood was vigorously mixed with 15 mL of RNA/DNA stabilization reagent for blood/bone marrow (Boehringer Mannheim Corp., Indianapolis, IN) producing cell lysis and simultaneous inactivation of nucleases. Samples were then processed immediately for mRNA extraction or stored at −20°C. Control sample mRNA (poli-A+) was isolated from lysed whole blood by magnetic separations using the mRNA isolation kit for blood/bone marrow (Boehringer Mannheim) according to the manufacturer’s instructions. All of the mRNA obtained from the 1.5-mL blood sample was resuspended in 15 μL of RNAse-free distilled water and used for cDNA synthesis.

Reverse transcription of all mRNA isolated from each blood sample was performed in a 30-μL final volume of first-strand buffer (50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2) containing 10 mM dithiothreitol, 0.5 mM of each dNTP (Pharmacia Biotech, Uppsala, Sweden), 2 U/μL of RNasin ribonuclease inhibitor (Clontech, Palo Alto, CA), and 20 U/μL of reverse transcriptase (SuperScript II; GIBCO BRL, Gaithersburg, MD). The mixture was incubated at 42°C for 60 min and at 90°C for 5 min and then placed on ice until required for the PCR reaction or stored at −70°C.

mRNA Quantification

The LighCycler PCR system (Roche) allows the real-time detection of PCR product formation by monitoring the fluorescence emitted by SYBR Green I dye intercalating in double-strand PCR products. Quantification is based on the principle that the number of original copies of the specific IL-10 cDNA within the reaction is inversely proportional to the number of PCR cycles necessary to generate a signal that rises above background fluorescence. Similar quantification of β2-microglobulin was used as housekeeping gene. By using serial dilutions of IL-10 and β2-microglobulin external standards, a calibration curve was generated that allowed the quantification of cDNA samples, leading to determination of IL-10 mRNA relative units. To exclude fluorescence related to the formation of primer-dimmers, fluorescence was measured at a cytokine-specific temperature, that is, the temperature (as obtained from the melting curves) at which primer-dimmers had already completely melted but at which the specific PCR product was not affected. Hence, during quantitative PCR, the elongation step was followed by incubation at the specific temperature for 3 sec and fluorescence was detected. Reactions without cDNA were always included as a negative control, and cDNA samples were quantified in duplicate.

As external IL-10 standards, we used serial dilutions of the cDNA obtained from peripheral blood mononuclear cells stimulated for 24 hr with LPS (10 ng/mL). For IL-10 amplification, 1 μL of cDNA was subjected to PCR reaction in a total reaction volume of 12 μL, containing 1.2 μL of 10× PCR buffer (including SYBR Green I dye, Taq DNA polymerase, deoxynucleotide triphosphate; Roche), 0.5 μM each forward and reverse primer, and 4 mM MgCl2. PCR consisted of an initial preincubation step of 10 min at 95°C, followed by 30 (β2-microglobulin) or 45 (IL-10) cycles of rapid heating to 95°C for 5 sec, followed by an immediate cooling to the annealing temperature (β2-microglobulin: 53°C; IL-10: 67°C), which was held for 10 sec, an elongation step of 12 sec for β2-microglobulin and 17 sec for IL-10, and a fluorescence detection step (83°C for β2-microglobulin and 87°C for IL-10) for 3 sec. Melting curves were generated to obtain melting temperatures for primer-dimmers and the specific PCR product. The following oligonucleotide primers were used for real-time PCR: IL-10, 5′-AGC TGA GAA CCA AGA CCC AGA and 5′-GGG CTG GGT CAG CTA TCC; and β2-microglobulin, 5′-CCA GCA GAG AAT GGA AAG TC and 5′-GAT GCT GCT TAC ATG TCT CG.

Cell Cultures

For induced IL-10 determination, mononuclear blood cells were obtained from peripheral blood samples by centrifugation over Ficoll-Hypaque gradients (Lymphoprep; Nycomed, Oslo, Norway). Cells were cultured at a concentration of 2×106 cells/mL at 37°C and in 5% CO2 in RPMI 1640 medium containing 2 mM l-glutamine and 25 mM HEPES (BioWhittaker, Verviers, Belgium) and supplemented with 10% heat-inactivated fetal calf serum and antibiotics. Cell activation was set up in 1-mL volume in 24-well culture plates in medium alone or with 10 ng/mL of LPS (Sigma Chemical, St. Louis, MO).

Protein Quantification

Serum IL-10 concentrations were measured using a high-sensitivity sandwich enzyme immunoassay (Quantikine HS; R&D Systems Europe Ltd.). The lower limit of detection was 0.7 pg/mL. IL-10 concentration in culture supernatants was determined by an in-house ELISA test, as follows. Microtiter wells were coated overnight with affinity-purified anti-human IL-10 monoclonal antibody (R&D) and blocked with 1% casein in Tris-buffered saline for 2 hr at 37°C. Samples and IL-10 standards (R&D) were diluted in blocking solution and incubated for 18 hr at 4°C. After washing with Tris-buffered saline/Tween 20 (0.05%), wells were incubated for 2 hr with biotinylated anti-human IL-10 monoclonal antibody (R&D), washed, incubated for 1 hr with streptavidin-alkaline phosphatase conjugate, and revealed using PNPP as substrate. Absorbance was determined at a wavelength of 405 nm. Quantities of IL-10 were calculated according to the standard curves. The lower limit of detection was 7.5 pg/mL.

Statistical Analysis

Basal IL-10 expression in different genotype groups was not distributed normally, so nonparametric testing has been used throughout. Basal cytokine production is described by median and interquartile range (IR). To determine whether IL-10 secretion varied with genotype, all genotypes were compared simultaneously by the Kruskal-Wallis test of variance analysis. In some cases, strictly where indicated by the Kruskal-Wallis testing, individual groups were compared by the Mann-Whitney U test. In all cases, P values less than 0.05 were taken as indicating significance. Induced IL-10 expression show Gaussian distribution, and genotypes were compared by two-tailed t test for equality of means. The SPSS 9.0 statistical software package (SPSS Inc.) was used for all calculations.


Constitutive IL-10 mRNA Expression Levels Are Regulated by the Gene Promoter

Based on previous observations on the genetic regulation of mitogen-induced levels of IL-10, we considered the possibility that constitutive levels of IL-10 were also genetically regulated. Thus we extracted DNA from 183 and mRNA from 128 healthy unrelated Caucasian individuals from a Northern Spanish region (Asturias). Polymorphic alleles present at positions −1082, −819, and −592 of the IL-10 gene promoter were determined by testing differential Tm after DNA amplification and hybridization with allele-specific probes using the LighCycler PCR system. Relative IL-10 mRNA levels (IL-10 relative units) were quantified by a highly sensitive real-time reverse transcription (RT)-PCR technique after mRNA (poly-A+) isolation from total whole-blood samples.

Analyzing each individual allele at the three promoter sites, we found only six genotypes, which corresponded to the three haplotypes previously described in other Caucasian populations (GCC, ACC, and ATA). Table 1 shows genotype, haplotype, and allele frequencies of the three single nucleotide IL-10 promoter polymorphisms found in our population. What is remarkable is the low frequency of the GCC haplotype (38.25%) compared with published data from other Northern and Central European populations (50–52%) (21–23).

Table 1:
Table 1. IL-10 promoter polymorphisms

The analysis of the constitutive IL-10 gene expression in our healthy population shows the distribution represented in the histogram of Figure 1A. Using a dichotomy around the median (2.246), we classified individuals as low (<2.25) or intermediate/high (≥2.25) IL-10 producers. Twenty five percent of the population had very low levels of relative IL-10 mRNA (<1), whereas among the intermediate/high producers an elevated interindividual dispersion was observed, with 25% of individuals definitely presenting high IL-10 relative transcript levels (≥11.26). The distribution of relative mRNA IL-10 levels corresponding to each genotype is represented in box plot diagrams (Fig. 1B). To determine whether levels of constitutive mRNA IL-10 production depend on promoter polymorphism, all genotypes were compared simultaneously by the Kruskal-Wallis test of variance analysis, leading to significant differences (P =0.043). In addition, using the Mann-Whitney U test, we observed significant incremented values of mRNA IL-10 levels in individuals carrying the GCC/GCC genotype compared with those carrying the genotypes ATA/ATA (P =0.016), ATA/ACC (P =0.010), and ACC/ACC (P =0.046). What is remarkable is the elevated dispersion observed between individuals carrying the GCC/ATA (IR=17.68), GCC/ACC (IR=9.87), and GCC/GCC (IR=18.87) genotypes compared with that of low IL-10 producers (IR=2.56, 5.56, and 2.61). The contribution of each allele individually and in relation to each other to the control of basal IL-10 mRNA production is represented in Table 2. Alleles at positions −819 and −592 were analyzed together because they exhibit linked transmission. We found significant differences between AA, AG, and GG genotypes at the −1082 position independently of the −819/−592 genotype, indicating that the −1082G* allele is the most important genetic factor in the regulation of high constitutive IL-10 expression.

Figure 1:
Constitutive mRNA IL-10 expression in normal healthy individuals. Whole-blood samples were collected from 128 healthy individuals, and mRNA (poly-A+) was extracted. After cDNA synthesis, IL-10 expression was quantified by real time RT-PCR and relativized to β2-microglobulin gene. IL-10 promoter genotype was determined after PCR amplification and hybridization with allele-specific probes. (A) Distribution of relative IL-10 mRNA expression. (B) Distribution of relative IL-10 mRNA levels corresponding to each genotype.
Table 2:
Table 2. Partial contribution of each polymorphism to mRNA basal IL-10 levels

Influence of Genotype on Circulating IL-10 Protein Levels

Serum levels of IL-10 were measured in 171 healthy individuals by a highly sensitive ELISA test and analyzed in comparison with genotypic variation in the IL-10 gene promoter. Figure 2A shows the frequencies and percentiles of IL-10 serum level distribution. Considerable circulating amounts of IL-10 (≥2 pg/mL) were detected in 21.6% of the population, whereas most people had an undetectable (<0.7 pg/mL, 29%) or low concentration (0.7–1.1 pg/mL, 21%) of serum IL-10. These interindividual differences suggest that serum levels of IL-10 are genetically regulated. The contribution of the genotype to basal protein IL-10 expression is shown in boxplot diagrams (Fig. 2B). The highest IL-10 serum levels were found in individuals with the −1082GG genotype, although differences with regard to the other groups were not statistically significant (P =0.087). However, among individuals with high IL-10 serum levels (≥2 pg/mL), we found a significant overrepresentation (chi-square test, P =0.034) of the high producer genotype −1082GG (Table 3). These and the above results indicate that the GCC/GCC genotype is clearly associated with high IL-10 mRNA and protein basal expression.

Figure 2:
Serum IL-10 levels in normal healthy individuals. Serum samples were collected from 171 healthy individuals and IL-10 levels measured by a highly sensitive ELISA technique. IL-10 promoter genotype was determined after PCR amplification and hybridization with allele-specific probes. (A) Distribution of basal IL-10 concentration. (B) Distribution of serum IL-10 levels corresponding to each −1082 genotype.
Table 3:
Table 3. Healthy individuals with IL-10 serum levels ≥2 pg/mL

IL-10 Production After LPS Stimulation

Finally, we wanted to determine whether, in our population (and similar to previous published data), the production of IL-10 after in vitro mitogen stimulation correlated with the IL-10 promoter genotype. Peripheral blood mononuclear cells from 29 healthy individuals with different genotype at the −1082 position were cultured in medium alone or with LPS (10 ng/mL). After 48 hr, culture supernatants were collected and IL-10 concentration was measured by ELISA techniques. The amount of secreted IL-10 corresponding to each genotype was compared by two-tailed t test for equality of means, leading to significant differences (P =0.008). As expected, the major production of IL-10 after LPS stimulation corresponded to the −1082 GG genotype, similar to other previously studied populations (Fig. 3).

Figure 3:
IL-10 production after LPS stimulation. Peripheral blood mononuclear cells from 29 healthy individuals with different genotypes at the −1082 position were cultured in medium alone or with LPS (10 ng/mL). After 48 h, culture supernatants were collected and IL-10 concentration was measured by ELISA techniques. Bars represent IL-10 production after LPS stimulation relative to secretion in medium alone. Mean levels were compared by two-tailed t test.


Basal levels of IL-10 may influence the outcome of acute rejection after organ transplantation. Constitutive and induced levels of IL-10 may be differentially regulated by the activation of different metabolic pathways and, therefore, should be submitted to different genetic control. We considered it important to know whether the three single nucleotide polymorphisms previously described in the gene promoter, which address the levels of in vitro–induced production of IL-10 (−1082 G/A, −819 C/T, and −592 C/A), also regulate the in vivo constitutive production of this cytokine. In the present work we have analyzed, for the first time, the distribution of the three biallelic polymorphisms in a population from the North of Spain and its correlation with mRNA IL-10 constitutive levels, tested by a sensitive real-time RT-PCR using whole-blood samples. In our population, inheritance of these biallelic variants was similar to other Caucasian populations, detecting exclusively the three haplotypes and the six genotypes previously published (21–23). However, allele, haplotype, and genotype distribution in our population differed from Northern and Central European groups (Finland, United Kingdom, Poland), mainly because of an overrepresentation in the Spanish group of the −1082A* allele (0.62 in the local group versus 0.47–0.52 in the others), associated in previous studies with low production of IL-10 after mitogen stimulation, and a subsequent lower frequency of the haplotype GCC, associated with high induced IL-10 secretion (21–23). In contrast, reported data from Italian groups (24–25) are comparable to those of the present study, suggesting a close genetic proximity among Mediterranean populations. More European populations should be tested to confirm this observation.

When analyzing constitutive IL-10 gene expression in the local group, striking individual differences were found. Fifty percent of the population was considered to be low IL-10 producers (<2.25 mRNA relative units), with a 26.56% (34 of 128) presenting very low mRNA IL-10 relative levels (<1). However, a high data dispersion was found among those intermediate-high producers, with 25% yielding a quantification of mRNA greater than 11 relative units. These differences among healthy people suggested that constitutive IL-10 gene expression could be genetically regulated. When transcript levels of IL-10 were compared between groups with different genotypes, statistical data showed significantly higher amounts of IL-10 transcripts among those with the GCC/GCC genotype (median values 7.03) compared with those with the genotypes ATA/ATA, ATA/ACC, and ACC/ACC (median values 1.38, 1.43, and 1.93, respectively). By analyzing the contribution of each allele to sustain transcript levels, we concluded that allelic variants at position −1082 were the most important genetic factor in the regulation of the constitutive mRNA IL-10 levels, supporting the wide use of this polymorphism as a genetic marker for IL-10 production (6,7,15,20,23,24). These findings are in agreement with those previously reported, which demonstrate that the ATA and the GCC haplotypes are, respectively, associated with low and high IL-10 production after in vitro stimulation of peripheral blood mononuclear cells; these finding also indicate that basal and induced levels of IL-10 are regulated by the same polymorphic genetic regions. However, the considerable dispersion of IL-10 transcript concentration among individuals carrying the −1082 GG and GA genotypes (IR=13.85 and 18.87) suggests that other genetic or environmental factors affect IL-10 expression.

Circulating IL-10 protein concentration was also analyzed in the same population and evaluated in relation to IL-10 promoter genotypes. An elevated percentage of individuals had undetectable or very low concentration of IL-10 in their serum samples. Although median protein values were higher in subjects with the biallelism −1082 GG, data were not statistically significant compared with the other two genotypes (−1082 AA and −1082 AG). The lack of a significant correlation was probably because a large number of healthy individuals had levels of systemic IL-10 too low to be detected by currently used immunoassays. However, a significantly higher percentage of individuals with considerable concentration of circulating IL-10 (≥ 2 pg/mL) was found among those with the −1082 GG genotype compared with the other two variants (AA and AG), again supporting the fact that the −1082 GG allele is a determining factor in the regulation of basal IL-10 secretion. Interestingly, we observed a significant association of the high producer polymorphic variants in the IL-10 promoter with low levels of circulating IL-8, probably because of an indirect down-regulation of this chemokine by an overproduction of IL-10 (data not shown). This indicates the physiologic relevance of basal levels of IL-10 in maintaining the steady-state cytokine network. On the other hand, and in accordance with previous reports (13), our results also show that normal subjects with the high IL-10 producer genotype (−1082 GG) secreted elevated amounts of this molecule after LPS stimulation of peripheral blood mononuclear cells.


We have shown that the wide interindividual differences found in constitutive IL-10 levels are genetically regulated by polymorphic regions in the gene promoter. Genetic variants in this region have been previously analyzed in several experimental and clinical studies in relation to organ allograft survival. Although some authors found no association between cytokine gene polymorphisms and renal allograft rejection (26), other reports indicated a major role of genetic IL-10 variants on graft acceptance (6,7). These correlations may be caused by different genetically regulated basal amounts of IL-10, which in turn regulate the expression levels of proinflammatory mediators secreted in response to alloantigen stimulation (1–3). In fact, elevated serum and urine IL-8 levels have been associated with early graft failure (27,28). It has been suggested that immunosuppressive therapy could be individualized on the basis of recipient or donor cytokine genotype, particularly IL-10, interferon-γ, and tumor necrosis factor-α (6,29,30). Although our data support this view, we nevertheless consider it necessary to determine IL-10 basal levels by sensitive techniques before organ transplantation, because we found considerable interindividual differences in IL-10 constitutive levels among carriers of the same promoter genotype. The role of cytokine genetic polymorphisms on the control of protein levels and the regulation of allogenic responses is a subject of great interest, which needs further investigation.


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