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Clinical Chemistry 50: 195-200, 2004; 10.1373/clinchem.2003.022459
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(Clinical Chemistry. 2004;50:195-200.)
© 2004 American Association for Clinical Chemistry, Inc.

Clinical Immunology

The Interleukin-6 G(-174)C Promoter Polymorphism Does Not Determine Plasma Interleukin-6 Concentrations in Experimental Endotoxemia in Humans

Georg Endler2,1, Claudia Marsik1,2,1, Christian Joukhadar1, Rodrig Marculescu2, Florian Mayr1, Christine Mannhalter2, Oswald F. Wagner2 and Bernd Jilma1,a

Departments of1 Clinical Pharmacology and2 Medical and Chemical Laboratory Diagnostics, Vienna University, Vienna, Austria.

aAddress correspondence to this author at: Clinical Pharmacology, Division of Hematology and Immunology, Vienna University, Waehringer Guertel 18-20, A-1090 Wien, Austria. Fax 43-1-40400-2998; e-mail Bernd.Jilma{at}univie.ac.at.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Interleukin 6 (IL-6) is a pleiotropic cytokine that plays an essential role in the pathogenesis of acute and chronic infections. As the role of the IL-6 G(-174)C polymorphism in determining serum concentrations of IL-6 is controversial, we studied the genotype-specific IL-6 response in a well-standardized model of systemic inflammation.

Methods: A total of 76 healthy young males (age range, 19–35 years) received a single bolus of 2 ng/kg endotoxin [lipopolysaccharide (LPS)] intravenously. Plasma IL-6 was measured by enzyme immunoassay at 0, 2, 6, and 24 h after LPS infusion, and the IL-6 promoter genotype was analyzed by a mutagenic separated PCR assay.

Results: IL-6 increased 300-fold 2 h after LPS challenge and returned almost to normal within 24 h. Neither basal IL-6 nor the IL-6 response to LPS was significantly affected by the IL-6 promoter genotype.

Conclusions: The IL-6 G(-174)C promoter polymorphism does not significantly influence basal concentrations of IL-6 or peak IL-6 in human endotoxemia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In spite of advances in modern intensive care medicine, sepsis with consecutive multiple organ failure still represents a major cause of death in the developed world (1). A central event in the pathophysiologic cascade of sepsis is the excessive systemic release of proinflammatory cytokines such as tumor necrosis factor-{alpha}, interleukin-1 (IL-1), 2 and IL-6 in response to bacterial invasion (2).

As a further consequence, these cytokines activate multiple pathways (e.g., coagulation and complement pathways and phagocytosis) to eliminate the invasive microorganisms, but occasionally these pathways convert to a self-destructive systemic inflammatory reaction leading to multiorgan failure (3)(4).

Among these cytokines, IL-6 seems to play a central role in the development of the systemic inflammatory reaction, finally leading to septic shock (5). IL-6 is a pleiotropic cytokine secreted mainly by monocytes and macrophages. In sepsis, IL-6 has multiple effects, among them the stimulation of B and T lymphocytes and the induction of fever and hepatic acute-phase proteins (6).

Several studies have indicated that plasma concentrations of IL-6 are closely related with the severity and outcome of sepsis, suggesting a pathogenetic role of IL-6 (2)(5). IL-6 has a short half-life in vivo (<10 min) (7); therefore, plasma concentrations of IL-6 are regulated mainly at the level of RNA expression. A variety of different transcription factors, including activator protein-1, cAMP-responsive element-binding protein, nuclear factor-IL6, and nuclear factor-{kappa}B, have been described to contribute to the regulation of IL-6 in vivo (8).

Because IL-6 plasma concentrations show considerable interindividual variability in acute and chronic inflammation, it has been speculated that these differences may be determined genetically by variations in the promoter region of the IL-6 gene. Indeed, a frequent G/C polymorphism at nucleotide position (-174) and other, less frequent polymorphic variants in the promoter region have been discussed as possibly influencing serum IL-6 (9)(10)(11).

To date, several studies have been published suggesting that the G(-174)C polymorphism is associated with susceptibility and outcome of several acute and chronic inflammatory diseases, such as diabetes mellitus (12)(13), atherosclerosis (14)(15)(16), Alzheimer disease(17), juvenile chronic polyarthritis (9), and the outcome of sepsis (2). For the reader’s convenience we have summarized selected studies analyzing the IL-6 genotype–phenotype association in Table 1 . However, there is disagreement in the data concerning which genotype is associated with higher IL-6 plasma concentrations. In some studies, the homozygous GG genotype has been associated with increased serum IL-6, e.g., in patients with juvenile chronic polyarthritis (9)(18)(19), whereas the authors of other studies have reported that the homozygous CC genotype might be associated with higher systemic IL-6 expression (15)(20).


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  		Table 1. Selected studies that analyzed the genotype–phenotype association of the IL-6 G(-174)C promoter polymorphism.1

On the other hand, large studies have failed to establish any association between the IL-6 genotype and basal serum concentrations (21), although most studies were able to attribute some effect to either the GG or CC genotype. We thus hypothesized that the IL-6 genotype might influence IL-6 expression only after stimulation of an IL-6 response. Experimental endotoxin challenge in healthy individuals has been shown to be an excellent in vivo model for studying the cytokine cascade underlying systemic inflammation and has been applied in numerous studies (22)(23)(24)(25). However, patient samples are very heterogeneous with respect to underlying diseases, and concomitant ailments and different pathogens and their burden [e.g., lipopolysaccharide (LPS) load in sepsis] are very heterogeneous. The LPS model is well standardized and allows study of the IL-6 response to a defined stimulus in healthy volunteers free of confounding diseases.

In our study we aimed to evaluate whether the IL-6 G(-174)C promoter polymorphism contributes to the individual variability in IL-6 responses in healthy volunteers after a single challenge with a bolus of LPS in 76 healthy males.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
study participants
The study protocols were approved by the Ethics Committee of the Vienna University, and all participants gave written informed consent before entering the study.

We obtained complete data and DNA samples from 76 healthy male volunteers who had participated in several clinical trials in which they had received LPS only, including recently published studies (26)(27) and several unpublished trials.

All participants were nonsmokers 19–40 years of age with a body mass index between the 15th and 85th percentiles. Determination of health status included a medical history, physical examination, laboratory values, and virologic and standard drug screening. Exclusion criteria were regular or recent intake of medications, including nonprescription medications, and clinically relevant abnormal findings in the medical history or laboratory values.

study protocol
The experimental procedures of our LPS infusion studies have been described elsewhere (23)(27). Briefly, volunteers were admitted to the study ward at 0800 after an overnight fast. Throughout the entire study period, participants were confined to bed rest and kept fasting for 8.5 h after LPS infusion. Participants received an intravenous bolus containing 2 ng/kg LPS (National Reference Endotoxin, E. coli; USP Convention Inc.).

sampling and analysis
Sampling times were selected based on the kinetics of IL-6 plasma concentrations observed in previous trials (26)(27). Blood samples were collected by venipuncture into Vacutainer Tubes containing EDTA anticoagulant (Becton Dickinson) before LPS infusion and thereafter at the times indicated in the figures. Plasma samples were processed immediately by centrifugation at 2000g at 4 °C for 15 min and stored at -80 °C before analysis. Plasma IL-6 was analyzed in single measurements with a high-sensitivity enzyme immunoassay (R&D-Systems; interassay CV, 7.8%; intraassay CV, 5.8%; detection limit, 0.094 ng/L; range, 0.156–10 ng/L), and samples from individuals were run in the same assay. To overcome the narrow measuring range (0.16–10 ng/L), we analyzed the 2 and 6 h post-LPS samples after adequate dilution (23). C-Reactive protein (CRP) invariably increases 24 h after endotoxin administration and is an integral measure of IL-6 bioactivity (Jilma et al., own unpublished data). Thus, CRP was measured 24 h after LPS infusion by an immunonephelometric test (28).

pcr analysis of the il-6 g(-174)c promoter polymorphism
DNA was isolated from EDTA blood by standard procedures. For detection of the G(-174)C polymorphism, the principle of mutagenic separated PCR was adapted as previously described (29)(30).

Briefly, allele-specific primers that differed in length by 9 bp with single base mismatches at defined positions were used to minimize cross-reactions of the PCR products. In every sample, one or two different products were generated, depending on the genotype. PCR amplification was carried out in 50-µL volumes containing 1.5 U of AmpliTaq Gold (Perkin-Elmer Cetus), 1.5 mM MgCl2, 200 µM each deoxynucleotide triphosphate (Amersham Pharmacia Biotech), 8 pmol of IL-6 -174G forward primer (5'-TCC CCC TAG TTG TGT CTC GCG-3'), 12 pmol of IL-6 -174C forward primer (5'-CTG CAC TTT ATC CCC TAG TTG TGT CAT GCC-3'), 10 pmol of IL-6 reverse primer (5'-TGA GGG TGG GGC CAG AGC-3'; all from MWG Biotech; GenBank accession no. Y00081), and ~50 ng of DNA. Amplifications were performed in an Eppendorf Thermo Cycler (Eppendorf Inc.). A 10-min denaturation at 95 °C was followed by 37 cycles at 95 °C for 1 min, 58 °C for 2 min, and 72 °C for 1 min. A final extension step of 10 min at 72 °C completed the reaction. The PCR generated a 94-bp product for the IL-6 -174G allele and a 103-bp product for the IL-6 -174C allele, which were separated on 6% precast Tris-borate-EDTA polyacrylamide gels (Novex) by electrophoresis for 50 min at 160 V. After staining with SYBR Green (Molecular Probes) for 20 min, bands were visualized on an ultraviolet transilluminator at 306 nm and photographed with a Polaroid land camera. In each experiment, an individual known to be heterozygous for IL-6 G(-174)C was included as a positive control to ensure amplification of both alleles. A reagent control without DNA served as a negative control.

data analysis
Data are expressed as the median and the interquartile range for description in the text unless otherwise stated. Nonparametric statistics were applied. All statistical comparisons between groups were done with the Kruskal–Wallis test and the Mann–Whitney U-test. All statistical calculations were performed with commercially available statistical software (SPSS 10.0; SPSS Inc.). The areas under the time–IL-6 curves (AUCs) were calculated by the trapezoid rule (31). An estimation of the statistical power of the study was performed as described by Stolley and Strom (32).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Of the 76 healthy volunteers, 26 (34%) had the (-174)GG genotype, 40 (53%) were heterozygous (-174)GC, and 10 (13%) were homozygous (-174)CC. Genotype frequencies were in Hardy–Weinberg equilibrium.

Median (interquartile range) IL-6 plasma concentrations increased from baseline before LPS infusion [2.3 (0.8–2.4) ng/L] to a peak at 2 h after LPS [427 (227–754) ng/L] and then decreased continually to 12.8 (7.2–20.6) ng/L after 6 h and returned to baseline values after 24 h [1.2 (0.8–1.7) ng/L]. IL-6 concentrations showed considerable interindividual variability, as indicated by the range of values in Table 2 .


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  		Table 2. IL-6 G(-174)C genotype-dependent IL-6 concentrations.

We observed no significant differences in IL-6 response to LPS (Table 2Up and Fig. 1 ) after stratification of the data according to IL-6 genotype. Although peak IL-6 values were somewhat lower in patients homozygous for the C allele [377 (120–713) ng/L in CC homozygotes compared with 437 (67–3473) ng/L in GC heterozygotes and 423 (278–804) ng/L in GG homozygotes], these effects did not reach statistical significance. Additionally, at 6 h a trend in the opposite direction occurred, i.e., IL-6 concentrations were highest in individuals carrying the CC genotype (Table 2Up ), which makes a consistent genotype–phenotype interaction unlikely. In parallel, the AUC0–24 h values for IL-6 revealed no genotype-specific differences (Table 2Up ).



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  		Figure 1. Genotype–phenotype association of IL-6.

IL-6 concentrations are similar in all three IL-6 G(-174)C genotypes. Data are means (SE; error bars).

Consistently, the IL-6 genotypes were not associated with differences in serum CRP 24 h after LPS challenge, and we found no significant correlation between body mass index and either IL-6 basal or peak concentrations (data not shown).

Analysis of the study’s power for comparing IL-6 (-174)CC and IL-6 (-174)GG individuals revealed that the current sample size should be sufficient to detect a twofold increase or decrease in either IL-6 basal or peak concentrations.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In our study, genotype frequencies in the healthy volunteers were comparable to those reported in other studies (21)(33).

In concordance with previous results (23)(34), IL-6 plasma concentrations showed a peak increase 2 h after a single bolus of LPS and then rapidly decreased and almost normalized after 24 h. Although identical doses were administered (2 ng/kg endotoxin intravenously) to young healthy males under controlled study conditions, we observed the expected considerable interindividual variability in both basal and peak IL-6 concentrations, which differed as much as 10-fold.

Our data indicate that the IL-6 G(-174)C promoter polymorphism does not seem to be a major determinant of the systemic IL-6 response in human endotoxemia. These findings are in accordance with a study on patients with sepsis (2), which also reported no association between IL-6 genotype and systemic IL-6 concentrations in vivo (compare the findings in Table 1Up ). Possibly other polymorphisms (35)(36)(37) may be overruling the putative effect of the IL-6 G(-174)C promoter polymorphism on the release of IL-6 into the circulation, such as the recently described polymorphisms in the Toll-like receptor 4 (38)(39) or in the CD14 gene (40).

In vitro, the IL-6 response of monocytes to endotoxin has been reported to follow a U-shaped genotype–phenotype association, with both the homozygous CC and the homozygous GG genotypes having higher IL-6 release than heterozygous C/G individuals (41). Although the molecular mechanisms are unclear, this reflects some of the divergent results reported for IL-6 genotypes in the literature.

Confusingly, in chronic inflammatory diseases, either of the two alleles has been associated with higher systemic IL-6 concentrations (9)(10)(14)(20)(42)(43). In contrast, in other studies, no effect has been observed (21)(44)(45). Possibly, these findings could be explained by localized tissue-specific IL-6 production modulated by the IL-6 G(-174)C polymorphism (11), whereas systemic IL-6 concentrations remain unaffected.

Our study had several limitations. In our trial we studied genotype-dependent IL-6 expression in healthy young Caucasian males. Age, menopausal state, ethnic background, and body mass index as well as smoking habits have been reported as influencing the genotype-dependent individual expression of IL-6 (21)(42)(43)(46). Because our study population comprised a relatively homogeneous group of healthy, young, nonsmoking, lean males, these effects could not be studied in our study. Another limitation of our study undoubtedly is the sample size. We thus cannot exclude small effects of the IL-6 genotype on systemic IL-6 concentrations in vivo. The authors of most published studies reported genotype-specific changes in IL-6 of <30% (Table 1Up ). However, these small differences, which might not be detected in our study, are likely to be of limited biological relevance in endotoxin-triggered inflammation. In light of the substantial intersubject variability (10-fold) in our study, its calculated power to detect twofold changes of IL-6 seems sufficient to detect biologically relevant differences. We also cannot exclude a clinically relevant genotype-specific IL-6 response to stimuli different from LPS, which have been reported in noninfectious diseases such as diabetes (12)(13) and juvenile polyarthritis (9).

Additionally, several other polymorphisms within the IL-6 promoter, such as the (-572)GC promoter (10) polymorphism or complex haplotypes of other promoter polymorphisms (11), have been discussed as influencing IL-6 concentrations. However, these genetic variants are considerably less frequent than the G(-174)C polymorphism and thus would have required a large sample size, which is not feasible in this setting. Because most of the polymorphisms are in linkage with the IL-6 G(-174)C polymorphism, we thus chose to restrict our study to one frequently occurring polymorphism.

In conclusion, the IL-6 G(-174)C genotype does not contribute significantly to the individual variability of systemic IL-6 plasma concentrations after endotoxin challenge. Thus, if any, the impact of the IL-6 G(-174)C polymorphism on systemic endotoxin-triggered IL-6 concentrations seems to be limited, whereas other, in part unknown factors might exert a major influence on individual IL-6 responses to LPS.


   Acknowledgments
 
The nucleic acid analyses [DNA isolation and genotyping of the IL-6 G(-174)C polymorphism] were supported by the Jubiläumsfonds der Oesterreichischen Nationalbank (Project 9558).


   Footnotes
 
1 These authors contributed equally to the manuscript.

Previously published online at DOI: 10.1373/clinchem.2003.022459

2 Nonstandard abbreviations: IL, interleukin; LPS, lipopolysaccharide; CRP, C-reactive protein; and AUC, area under the curve.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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