HASH(0xbeb934c)

ARTICLE

INTRODUCTION

A major trigger for the release of cytokines is the binding of lipopolysaccharide (LPS) to the monocyte surface receptor CD14. Consequently, monocytes produce and release cytokines such as tumor necrosis factor-alpha (TNF-alpha) and IL-1beta. Recent research on the interindividual variance in cytokine release elucidated the role of genetic polymorphisms [1]. The genes for CD14 [2] and TNF-alpha [3] carry polymorphisms within the promoter region regulating gene transcription.

The TNF-alpha polymorphism is based on a G/A exchange at position -308 of the promoter region [3]. Two in vitro studies demonstrated that the A allele is associated with higher TNF-alpha production [3, 4], a result contrasted by the findings of two other groups [5, 6]. In their report on healthy human blood donors, Louis et al. [7] observed an association between this -308 polymorphism and the ex vivo TNF-alpha response to LPS. Subjects carrying an A allele had significantly higher TNF-alpha concentrations than G homozygotes. Mira and colleagues [8] reported a higher frequency of the A allele in patients with septic shock compared to a control group. Another group found a higher mortality rate from septic shock in carriers of an A allele [9]. These reports provided convincing evidence for a role of this polymorphism in the clinical course of septic shock.

Baldini et al. [2] discovered the CD14 genetic polymorphism at position -260. This polymorphism consists of a C/T single nucleotide exchange. In children, the plasma levels of soluble CD14 were significantly higher in T homozygotes compared with individuals homozygous for the C allele [2]. Two large clinical studies found this polymorphism associated with the risk for myocardial infarction [10, 11]. Individuals homozygous for the T nucleotide at position -260 had an increased risk for myocardial infarction, with an even higher association in patients without other risk factors such as hypertension or smoking [10]. In healthy blood donors, Hubacek et al. [10] found a higher monocyte CD14 density in TT carriers, a result that was challenged by our group [12]. We were unable to find an association between this polymorphism and the monocyte CD14 density, the plasma concentrations of soluble CD14 or the TNF-alpha concentrations after endotoxin stimulation of human whole blood.

In the present study, we found a linkage disequilibrium between the TNF-alpha -308 and the CD14 -260 promoter polymorphisms. Moreover, we studied the association between the genotypes and the TNF-alpha and IL-1beta response to stimulation of the LPS receptor CD14 in a whole blood assay.

METHODS

After approval by the local ethics committee and written informed consent, healthy Caucasian blood donors were studied. Venous blood samples were taken.

Genotyping for the CD14 -260 and the TNF-alpha -308 polymorphisms

Genomic DNA was isolated from the venous blood sample according to standard protocols. Twenty-80 ng genomic DNA (1 muL) were used. Real-time polymerase chain reaction (PCR) assays with specific fluorescence-labelled hybridisation probes were used for genotyping [13, 14]. The 10 muL PCR mixture contained 1 muL reaction buffer (LightCycler DNA master hybridization probes 10C buffer™, 1.75 mmol/l, Roche Diagnostics, Basel, Switzerland). For the CD14 -260 genotyping, 0.5 mumol/l of the primers (sense: 5'-GGTGCCAACAGATGAGGTTCAC, antisense: 5'-CTTCGGCT-GCCTCTGACAGTT) as well as 0.2 mumol/l of the detection probe specific for the T allele (5'-LC Red640-TTCCTGTTACGGCCCCCCT-p; the 3'-end was phosphorylated to block extension), and the anchor probe (5'-GGAGACACAGAACCCTAGATGCCCTGCA-fluoresceine) were used. The PCR conditions were: initial denaturation at 95^o C for 120 s, followed by 60 cycles of denaturation (95^o C for 0 s, 20^o C/s), annealing (55^o C for 10 s), and extension (72^o C for 10 s). The melting curve consisted of 1 cycle at 95^o C for 0 s, 45^o C for 10 s, and then increasing the temperature to 95^o C at a slope of 0.2^o C/s.

For the TNF -308 genotyping 0.5 mumol/l of the primers (sense: 5'-AAGGAAACAGACCACAGACCTG, antisense: 5'-GGTCTTCTGGGCCACTGAC) as well as 0.2 mumol/l of the detection probe specific for the G allele (5'-AACCCCGTCCCCATGCC) and the anchor probe (5'-CAAAACCTATTGCCTCCATTTCTTTTGGGGAC) were used. Sixty PCR cycles were run with one PCR cycle consisting of denaturation (95^o C for 0 s, 20^o C/s), annealing at 57^o C for 15 s, and extension (72^o C for 10 s).

The thermocycler was a LightCycler™ instrument (Roche Diagnostics).

Ex vivo lipopolysaccharide stimulation of whole blood TNF-alpha and IL-1beta release.

The TNF-alpha and IL-1beta responses of human whole blood to lipopolysaccharide (LPS)-stimulation were assessed as previously described [15]. Heparin-anticoagulated venous blood was diluted 1:1 (v/v) with RPMI 1640 (Gibco BRL, Karlsruhe, Germany). One hundred ng/ml endotoxin (E. coli O2:B22) were added. After 4 hours of incubation at 37^o C, the samples were centrifuged and the supernatants were stored at - 20^o C. The concentrations of TNF-alpha and IL-1beta were determined by measuring immunoreactivity using of chemiluminescence (Immulite™, DPC Biermann, Bad Nauheim, Germany).

Statistical analysis

Statistical analysis was performed with the SPSS for Windows Release 10.0.7 program. Values are given as median (minimum-maximum). Non-parametric tests (Wilcoxon Mann Whitney or Kruskal-Wallis test) were used to compare the cytokine concentrations between the genotypes. The association between the TNF-alpha -308 and the CD14 -260 promoter polymorphisms was assessed by Cramer's V test. P values < 0.05 were considered as statistically significant.

RESULTS

Complete TNF-alpha -308 and CD14 -260 genotyping, as well as TNF-alpha data were gathered for 132 individuals. Forty six subjects had the TNF-alpha -308 genotype AG, 86 were G homozygous. The CD14 -260 genotype frequencies were: 38 CC, 48 CT, and 46 T homozygous. There was a strong linkage disequilibrium between the two polymorphisms (p = 0.043), 42% of carriers of the TNF-alpha -308 genotype GG were homozygous for the CD14 T allele. Table 1 gives the genotype distribution.

Carriers of the TNF-alpha -308 genotype GA produced significantly higher TNF-alpha levels in response to endotoxin than G homozygotes (4,295 (175-7,610) versus 2,490 (401-6,990) pg/ml, p = 0.04). The CD14 -260 genotypes TT, CT, and CC did not differ in their ability to produce TNF-alpha (Table 2). However, there was a significant difference in the IL-1beta levels after endotoxin stimulation between the three CD14 -260 genotypes (p = 0.033) with significantly higher values in TT carriers compared to C homozygotes.

The TNF-alpha levels of the TNF-alpha -308 - CD14 -260 genotypes are given in Table 3. There was no difference in the TNF-alpha concentrations between all genotypes.

DISCUSSION

To our knowledge, this is the first report which shows a linkage disequilibrium between the TNF-alpha -308 G/A and the CD14 -260 C/T promoter polymorphisms. TNF-alpha -308 heterozygotes produced significantly higher TNF-alpha levels than G homozygotes in the human whole blood endotoxin stimulation assay. This result is in agreement with previous in vitro reporter gene assays [3, 4], as well as an ex vivo stimulation study [7]. Westendorpp et al. [1] found that 60% of the TNF-alpha response is determined genetically. However, so far there are conflicting results on the known TNF-alpha polymorphisms. In vitro and ex vivo studies demonstrated that the TNF-alpha -308 A allele is associated with a higher TNF production [3, 4, 7]. Warzocha et al. [16] were able to show an impact of this polymorphism on the outcome of non-Hodgkin's lymphoma. An association with the TNF-alpha levels in tear fluid of patients with scarring trachoma was also described [17]. These findings are in contrast with results obtained by others who did not find an association between the TNF-alpha -308 promoter polymorphism and TNF-alpha RNA in reporter gene assays [5, 6]. The results of the ex vivo whole blood stimulation assay used in our study also revealed a role of the promoter polymorphism for TNF-alpha synthesis. The TNF-alpha and the CD14 genes are located on human chromosome 6 [18] and 5 [19], respectively. We found a linkage disequilibrium between these two polymorphisms with 40% of the TNF-alpha -308 G homozygotes being CD14 -260 T homozygous. From our data, the functional implication of this linkage remains unclear. Moreover, the mechanism leading to a linkage between genes located on two different chromosomes remains to be elucidated.

We determined TNF-alpha and IL-1beta levels after endotoxin stimulation and analyzed the association with the TNF-alpha -308 and CD14 -260 genotypes. The lack of association between the CD14 -260 genotypes and the TNF-alpha response, as found in our study, is in line with a previous report [12]. Moreover, we could not show a significant difference in the TNF-alpha response between the TNF-alpha -308 -CD14 -260 genotypes. As a major result of our study, we found an association between the CD14 -260 polymorphism and the IL-1beta response to endotoxin. Carriers of the genotype CC had higher IL-1beta levels than T homozygotes. So far, two polymorphisms in the promoter region of the IL-11beta gene, at positions -31 and -511, have been described which are in near-complete linkage [20]. The IL-1beta -31 involving a TATA sequence affected DNA binding in electrophoretic mobility-shift assays. The IL-1beta -511 polymorphism was found to mediate its effect by linkage disequilibrium with the TATA box polymorphism. Our results suggesting an association between the CD14 -260 polymorphism and IL-1beta levels, could theoretically be related to differences in the CD14 receptor density in the various CD14 -260 genotypes. Hubacek and colleagues [10] reported a higher CD14 density on circulating monocytes of T homozygotes. In a previous study, we were unable to show any association between this polymorphism and the CD14 density on human monocytes or soluble CD14 levels [12]. It is therefore daring to speculate that the CD14 polymorphism is only a marker of another genetic factor which is the true mediator of IL-1beta concentrations.

CONCLUSION

REFERENCES

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