The Toll-Like Receptor 4 Asp299Gly and Thr399Ile Polymorphisms Influence the Late Inflammatory Re- sponse in Human Endotoxemia, Claudia Marsik, 1,2 Bernd Jilma, 1* Christian Joukhadar, 1 Christine Mannhalter, 2 Oswald Wagner, 2 and Georg Endler 2 ( 1 Department of Clinical Pharmacology and 2 Institute of Medical and Chemical Laboratory Diagnostic, Medical University of Vienna, Vienna, Austria; * address correspondence to this author at: Department of Clinical Pharmacology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Wien, Austria; fax 43-1-40400, ext. 2998, e-mail Bernd.Jilma@meduniwien.ac.at) Toll-like receptor 4 (TLR4) is the principal receptor for bacterial endotoxin recognition, and functional variants in the gene confer endotoxin hyporesponsiveness in humans (1). Recognition of endotoxin leads to the activation of intracellular signaling pathways, up-regulating a wide array of inflammatory modulators, which contribute to early host-cell response (2, 3). Recently, a common polymorphism in TLR4 (4 ) that is associated with hyporesponsiveness to inhaled endotoxin in humans was identified (5–7 ). This missense mutation (Asp299Gly) in the fourth exon of the TLR4 gene alters the extracellular domain of this receptor. An additional mis- sense polymorphism (Thr399Ile) in the extracellular domain of the TLR4 receptor co-segregates with the Asp299Gly substitution in more than 95% of the Cauca- sian population (8). Recent studies showed that genetic variants of TLR4 may contribute to differences in risk in vivo (9) and may modulate in vitro response to bacterial lipopolysaccharide (LPS) (8) related to inflammatory markers. On the basis of these studies, we hypothesized that genetic variants of TLR4 might also contribute to the large interindividual differences in inflammatory marker concentrations after LPS challenge. The aim of our study was to investigate the associa- tion between TLR4 polymorphisms (Asp299Gly and Thr399Ile) and values for inflammatory markers in hu- man experimental endotoxemia. The study protocols were approved by the Ethics Com- mittee of the Medical University Vienna, and all participants gave written informed consent before entering the study. Complete data and DNA samples were available for 74 healthy male volunteers. These volunteers participated in several clinical trials receiving placebo in addition to endotoxin, including recently published studies (10, 11) and several unpublished trials. All volunteers were 19 –35 years of age with a body mass index between the 15th and 85th percentiles. The LPS model has been described in detail previously (11 ). All volunteers received 2 ng of LPS (National Refer- ence Endotoxin, Escherichia coli; USP) per kilogram of body weight as a bolus infusion over 2 min. Blood samples were collected by venipuncture into Vacutainer Tubes containing EDTA as anticoagulant (Bec- ton Dickinson) before LPS infusion and thereafter at the times indicated in Fig. 1. Plasma samples were processed immediately by centrifugation at 2000g at 4 °C for 15 min and stored at -80 °C before analysis. Plasma concentra- tions of interleukin-6 (IL-6) (11 ), IL-1, and tumor necro- sis factor (TNF) were measured by a high-sensitivity enzyme immunoassay (R&D-Systems), and all samples from individual participants were run in the same assay. Plasma concentrations of prothrombin fragment (F 1 + 2 ) and D-dimer were measured by ELISAs from Behring and Roche Diagnostics, respectively (12 ). Supersensitive C-reac- tive protein (CRP) values were determined by nephelometry (Tina-quant ® CRP; Roche Diagnostics) (11 ). Blood counts were performed with an XE 2001 cell counter (Sysmex). Total RNA was prepared with the QIAamp RNA Blood Mini Kit (Qiagen) according to the manufacturer’s in- structions. mRNA was directly transcribed into cDNA by use of the RT-Reagent Kit (Applied Biosystems) and stored at -80 °C until analysis. Tissue factor (TF) mRNA was quantified with an ABI Prism 7700 (Applied Biosystems) using primers designed by Primer Express Software (Applied Biosystems) as described previously (10, 13, 14). Genomic DNA was prepared from frozen whole blood by use of a blood DNA isolation reagent set according to standard procedures (11 ). Subsequent allele-specific PCR amplification for the TLR4 Asp299Gly and Thr399Ile alleles was performed according to a previously described protocol (15 ). Genotypes were assigned by independent investigators who were unaware of the participants’ iden- tities. For statistical analysis, we used the SPSS 10.0 software package (SPSS). The means (SE) are given. The nonpara- metric Mann–Whitney U-test was used. Because only one individual was homozygous for Asp299Gly and Thr399Ile, he was included in the heterozygous group. A 2-tailed P value 0.05 was considered statistically signif- icant. Correlation of continuous variables was determined by the Spearman correlation coefficient. Among the 74 persons who received LPS, 63 (85%) were wild type for TLR4 299Asp and 399Thr, 10 (14%) were heterozygous for the less frequent 299Gly allele, and 1 (1%) was homozygous for the TLR4 299Gly allele. In our study population, the Asp299Gly and Thr399Ile polymor- phisms were in complete linkage [consistent with the 95% linkage dysequilibrium in Caucasians (15 )]. Genotype frequencies were in Hardy–Weinberg equilibrium. Basal and peak values of selected inflammatory mark- ers were not different between wild-type TLR4 individu- als and carriers of the 299Gly mutant allele. However, volunteers with the Asp299Gly allele had lower concen- trations of some of the inflammatory cytokines, acute- phase reactants, and other mediators of inflammation relatively late after the onset of endotoxemia (Fig. 1 and Table 1). IL-6 concentrations were lower at 6 h, and CRP and IL-1a concentrations were lower at 24 h after LPS infu- sion in participants heterozygous or homozygous for Asp299Gly (Fig. 1). Additionally, carriers of the Asp299Gly polymorphism had significantly higher mono- cyte counts 24 h after LPS infusion (Fig. 1). 2178 Technical Briefs