Background: A single-nucleotide polymorphism (SNP) in the promoter region of the interleukin-6 (IL-6) gene at position −174 (G>C) has been reported to be associated with a variety of major diseases, such as Alzheimer disease, atherosclerosis, and cardiovascular disease, cancer, non-insulin-dependent diabetes mellitus, osteoporosis, sepsis, and systemic-onset juvenile chronic arthritis. However, authors of previous in vitro and in vivo studies have reported conflicting results regarding the functionality of this polymorphism. We therefore aimed to clarify the role of the −174 SNP for the induction of IL-6 in vivo.
Methods: We vaccinated 20 and 18 healthy individuals homozygous for the −174 C and G alleles, respectively, with 1 mL of Salmonella typhii vaccine. IL-1β, IL-6, and tumor necrosis factor-α (TNF-α) were measured in the blood at baseline and up to 24 h after vaccination.
Results: Individuals with the G genotype had significantly higher plasma IL-6 values at 6, 8, and 10 h after vaccination than did individuals with the C genotype (P <0.005). There were no differences between the two genotypes regarding serum concentrations of IL-1β and TNF-α before or after vaccination.
Conclusions: The −174 G>C SNP in the promoter region of the IL-6 gene is functional in vivo with an increased inflammatory response associated with the G allele. Considering the central role of IL-6 in a variety of major diseases, the present finding might be of major relevance.
Interleukin 6 (IL-6)1 is a key proinflammatory cytokine produced by many different cells, including leukocytes, adipocytes, endothelial cells, fibroblasts, and myocytes. IL-6 regulates production of adhesion molecules and induces secretion of monocyte chemotactic protein, an important mediator of release of other cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), that subsequently amplify the inflammatory reaction(1). A G>C single-nucleotide polymorphism (SNP) at position −174 in the promoter region of the IL-6 gene has been identified(2). This polymorphism has been associated with the prevalence, incidence, and/or prognosis of a variety of disease states, such as Alzheimer disease, atherosclerosis, cardiovascular disease, cancer, non-insulin-dependent diabetes mellitus (NIDDM), osteoporosis, sepsis, and systemic-onset juvenile chronic arthritis(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). The G allele of the −174 SNP has been associated with an increased transcriptional response to stimuli such as endotoxin or IL-1β in vitro(2)(20), whereas studies investigating the role of the −174 G>C promoter polymorphism for the plasma IL-6 concentration in vivo have produced conflicting results. In the first report by Fishman et al.(2), unstimulated IL-6 concentrations were associated with the G allele in healthy individuals. Since then, others have reported conflicting results regarding the association between IL-6 and the −174 G>C SNP in healthy and diseased individuals(21)(22)(23). Plasma IL-6 concentrations, stimulated by coronary artery by-pass surgery (CABG), have been associated with either the G allele(21) or the C allele(22), depending of the time of blood sampling after the procedure, whereas administration of endotoxin to healthy male volunteers did not confirm any association between IL-6 and the −174 G>C polymorphism(23). We therefore aimed to clarify the role of the −174 SNP for the induction of plasma concentrations of IL-6 in vivo. For this purpose, healthy individuals homozygous for the respective alleles were exposed to a standardized inflammatory stimulus obtained by vaccination against Salmonella typhii. We chose vaccination as a stimulus because it has been shown to increase plasma IL-6 concentrations and to have a detrimental effect on endothelial function(24). Including only known healthy individuals homozygous for the −174 G>C SNP allowed us to investigate the functionality of the −174 G>C polymorphism in a small number of individuals.
Materials and Methods
Forty healthy individuals were recruited from a cohort of 392 healthy men and women recruited from the general population. These healthy individuals were controls to patients with a first myocardial infarction before the age of 60 years in a study aiming to identify new risk factors for atherosclerosis and coronary heart disease. The first 250 individuals were genotyped for the promoter polymorphisms −174 G>C and −572 G>C of the IL-6 gene. On the basis of homozygosity for the G and C alleles of the −174 SNP and the G allele of the −572 SNP, 70 individuals were invited to participate in the present study, and 40 agreed. Twenty were homozygous for the −174 C allele and 20 for the −174 G allele. Exclusion criteria were treatment for hyperlipidemia, hypertension, ongoing postmenopausal substitution therapy, use of acetyl salicylic acid, and infection at the time of investigation. Smokers were asked to refrain from smoking 24 h before and during the study.
After completion of the study, the −174 genotype was checked by sequencing. Two individuals in the IL-6 −174G genotype group were heterozygotes, and their results were therefore excluded.
DNA was extracted from whole blood by use of the Qiagen Blood Cell and Culture Midi Kit. Amplification of the region of interest in the IL-6 promoter was performed by PCR. The −174 G>C genotype was determined by digestion of a 639-bp PCR product (primers 5′-GGGCTGCGATGGAGTCAGAG-3′ and 5′-TCCCTCACACAGGGCTCGAC-3′) by the restriction enzyme NlaIII (New England BioLabs). The −572 G>C genotype was determined by digestion of a 161-bp PCR product (primers 5′-GGAGACGCCTTGAAGTAACTGC-3′ and 5′-GGGCTGACTCCATCGCAG-3′) with the restriction enzyme BsrBI (New England BioLabs). Digested PCR products were visualized by electrophoresis on ethidium bromide-stained agarose gels.
The −174 G>C polymorphism and the AnTn tract at position −373 were genotyped after sequencing in both the forward and reverse directions of PCR products generated by primers flanking the polymorphic sites (5′-GGGCTGCGATGGAGTCAGAG-3′ and 5′-TCCCTCACACAGGGCTCGAC-3′)(7).
Participants arrived at the Clinical Research Unit at Danderyd University Hospital in the morning after 10 h of fasting. Venous blood samples were taken before and 2, 4, 6, 8, 10, and 24 h after vaccination. An intramuscular injection of S. typhii vaccine (Merieux) was administrated after the initial blood sampling. All participants were asked to abstain from physical exercise during the following 24 h.
Plasma IL-6 concentrations were measured in duplicate by a commercial high-sensitivity enzyme immunoassay (R&D Systems). Analysis was performed at one occasion; the interassay CV was 7.8% and the intraassay CV was 5.8%. Serum IL-1β and TNF-α concentrations were measured by a commercial ELISA (Biosource). Leukocyte, neutrophil, and platelet counts were assessed by clinical routine instrumental analyses before vaccination and 6 h after inoculation. Plasma LDL, HDL, and triglyceride concentrations were measured by routine clinical analyses.
Values are presented as the number, mean (SD), median (95% confidence interval or interquartile range), or area under the curve (AUC). Differences between basic characteristics of the two groups were tested by χ2 analysis or by unpaired t-test. Differences between genotypes were tested by repeated-measures ANOVA or by repeated unpaired t-tests. Because IL-6 values were skewed, they were logarithmically transformed before statistical testing.
All participants gave informed consent to participate in the study, which was approved by the Ethics Committee of the Karolinska Hospital.
The characteristics of the study participants are shown in Table 1⇓ . There were no differences between the two genotypes. Leukocyte and neutrophil granulocyte counts increased significantly after vaccination in both groups (data not shown).
Plasma concentrations of IL-6 and serum concentrations of IL-1β and TNF-α before and after vaccination are shown in the Table 2⇓ . Plasma concentrations of IL-6 increased from 2 h after vaccination and had returned to baseline after 24 h (P <0.001). Overall, individuals with the −174G genotype had significantly higher plasma concentrations of IL-6 than did those with the C genotype (P <0.005; Fig. 1⇓ ). In individuals with the G genotype, plasma IL-6 concentrations reached a peak at 10 h, whereas individuals with the C genotype had no peak but a plateau between 4 and 10 h. The differences in plasma concentrations of IL-6 between the two groups were significant at 6 h (P <0.01), 8 h (P <0.005), and 10 h (P <0.0005). Excluding women from the analysis did not change the results (data not shown). The AUC for IL-6 plasma concentrations differed between the two groups (P <0.0005). Haplotype analysis of the IL-6 promoter polymorphisms [−572(AnTn-373)−174] revealed four common haplotypes: G(A8T12)C, G(A9T11)G, G(A10T10)G and G(A10T11)G. All individuals with the −174C genotype except one [G(A8T12)C/G(A10T10)C] were homozygous for the G(A8T12)C haplotype. Of the individuals with the −174G genotype, four were homozygous for the G(A9T11)G, four were homozygous for the G(A10T10)G, and two were homozygous for the G(A10T11)G haplotype, respectively. The remaining individuals were heterozygous for the G(A9T11)G/G(A10T10)G (n = 4) and G(A9T11)G/G(A10T11)G (n = 4) haplotypes, respectively. Individuals with the G(A9T11)G and G(A10T10)G haplotypes, respectively, had higher IL-6 AUC concentrations than did individuals with the G(A8T12)C haplotype. However, there was no significant difference in IL-6 AUC between the G(A10T11)G and G(A8T12)C haplotypes (Table 3⇓ ).
Serum concentrations of TNF-α decreased (P <0.05), whereas serum concentrations of IL-1β were unchanged after vaccination. There were no differences between the two genotypes regarding serum concentrations of IL-1β and TNF-α.
The main finding of the present study was that healthy individuals homozygous for the G allele of the −174 SNP located in the promoter region of the IL-6 gene had a stronger inflammatory IL-6 response to vaccination against S. typhii compared with individuals homozygous for the C allele. No differences were seen in the IL-6 stimulatory cytokines TNF-α and IL-1β.
Previous in vitro studies(2)(20) have shown that the G allele of the −174 SNP was associated with increased transcription when stimulated by endotoxin and IL-1β. Furthermore, an ex vivo study of whole blood from healthy individuals stimulated by lipopolysaccharide showed that haplotypes of the IL-6 promoter including the G allele were associated with the highest IL-6 concentration(25). Three studies have investigated the role of the IL-6 −174 G>C SNP in the plasma IL-6 response in vivo(21)(22)(23). Brull et al.(22) showed, in contrast to the in vitro and ex vivo studies, that patients homozygous for the C allele had increased plasma IL-6 concentrations 6 h after CABG, whereas in the study by Burzotta et al.(21), CABG patients with the G allele had higher plasma IL-6 values 24 and 48 h after the procedure. The third in vivo study found no significant difference in plasma IL-6 concentrations between genotypes after intravenous endotoxin administration(23).
The results of the present study extend those of previous studies regarding the role of the IL-6 −174 G>C SNP. The results clearly show that the G allele is associated with an increased plasma IL-6 response to a standardized inflammatory stimulus. We could not identify the reason for the discrepancy between the results of the previous studies and our results, but an influence of different stimuli on the IL-6 promoter cannot be excluded. It can be speculated that the effects on the IL-6 promoter of different stimuli are mediated by different sets of transcription factors. The lack of association between the strong inflammatory stimulus of endotoxin administration indicate a lack of functional importance of the −174 G>C SNP. A strong argument against this reasoning is the association between the G allele and survival in sepsis(19). The relatively low frequency and timing of blood sampling in the CABG(21)(22)(26) and endotoxin(23) studies, compared with our study, might have influenced their results. Furthermore, drugs and hemodilution might have obscured the results in the CABG studies.
We used a standardized stimulus to study the in vivo functionality of a promoter polymorphism of a gene. Healthy individuals were investigated according to genotype. This model is suitable for genes with a low basal transcription rate that can be readily increased by a standardized stimulus together with a possibility to measure the phenotype. Examples of such genes are those regulating inflammation and glucose and lipid homeostasis. We measured IL-6 concentrations, which increased significantly after the inflammatory stimulus of vaccination. However, no major TNF-α or IL-1β response was seen. The reason for this is not clear but could be attributable to the nature of the stimulus, vaccination being a mild localized stimulus for cytokine production. Other explanations include short-term peaks that may have been missed because of the interval of blood sampling. Support for the biological relevance of vaccination with S. typhii as a standardized stimulus is that this type of vaccination has been shown to cause endothelial dysfunction(24).
The −174 G>C SNP in the promoter region of the IL-6 gene has gained considerable interest because it has been associated with a variety of disease states, such as Alzheimer disease, atherosclerosis, cardiovascular disease, cancer, NIDDM, osteoporosis, sepsis, and systemic-onset juvenile chronic arthritis(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). The results of the present study, which demonstrate the functionality of this SNP, highlight the results of previous association studies of major disease states. The G allele has been shown to be associated with improved survival in septic patients but not with the incidence of sepsis, indicating that the increased IL-6 response associated with the G allele is beneficial when humans are challenged by an acute infection(19). In contrast, the increased IL-6 response associated with the G allele might be harmful in the development of chronic diseases, such as NIDDM and atherosclerosis, by increasing the inflammatory stress when challenged repeatedly by minor stimuli. For example, insulin resistance has been associated with the G allele(14), and the G allele has been reported more frequently among Pima Indians and Caucasians with NIDDM(13). However, polymorphisms in other genes also need to be taken into account because another study has suggested that the C allele, when interacting with a TNF-α SNP, was associated with the development of NIDDM(12). In several studies, IL-6 has been proposed to play a key role in the development of atherosclerosis. Studies of carotid intima-media thickness and peripheral artery disease have, with one exception(6), suggested that the G allele was associated with atherosclerosis(5)(7)(9). Furthermore, several studies have suggested an important role for IL-6 in the regulation of bone mass in pre- and postmenopausal women as well as in healthy men(15)(17). Studies that examined the association between IL-6 genotype and bone mineral density showed that individuals homozygous for the C allele have higher bone mineral density than do individuals with the G allele. One explanation can be that repeatedly higher IL-6 concentrations during infections and other inflammatory states may lead to a loss of bone mass(16)(18).
It appears that not only the −174 G>C SNP influences IL-6 expression and production in response to a stimulus. In vitro and in vivo studies have indicated that haplotypes including the −174 G>C, −373 AnTn, −572 G>C, and −597 G>A polymorphisms influence IL-6 concentrations(20). Haplotype analysis of the present sample of individuals showed that at least two of three common haplotypes, including −174G, were associated with increased IL-6 concentrations compared with the only common haplotype including −174 C. Interestingly, keeping in mind the low number of participants in our study, the individuals with the G(A10T11)G haplotype had lower plasma IL-6 concentrations, in agreement with the results of the study by Kelberman et al(26), which indicates that the AnTn tract might modulate the influence of the IL-6 response of the −174 G>C SNP. However, our study was not designed primarily to study the effects of haplotypes and cannot therefore refute or confirm such effects.
A limitation of the present study is the lack of genotyping data of the −597G>A SNP. However, previous studies have shown that the −597 A allele is in a strong linkage dysequilibrium with the −174 C allele(20). Another limitation is the lack of data regarding the source of IL-6. There is a clear need for future in vitro and in vivo studies of representative cells and tissues to gain expanded knowledge about IL-6 expression and production.
In conclusion, the results of the present study clearly show that the −174 G>C SNP of the promoter region of the IL-6 gene is functional in vivo, with an increased inflammatory response associated with the G allele. Taking into account the central role of IL-6 in a variety of major diseases, the present finding might be of major relevance.
This work was supported by grants from The Swedish Heart-Lung Foundation and Astra Zeneca unrestricted. We thank Camilla Andersson, RN, at the Heart Laboratory of Danderyds University Hospital for skillful care of the study participants.
1 All values except gender and smoking are the mean (SD).
2 P <0.001 between 0 and 6 h.
1 NS, not significant.
↵1 Nonstandard abbreviations: IL, interleukin; TNF, tumor necrosis factor; SNP, single-nucleotide polymorphism; NIDDM, non-insulin-dependent diabetes mellitus; CABG, coronary by-pass grafting; and AUC, area under the curve.
- © 2004 The American Association for Clinical Chemistry