Critical Investigation of the CD14 Promoter Polymorphism : Lack of a Role for In Vitro Cytokine Response and Membrane CD14 Expression

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Critical Investigation of the CD14 Promoter Polymorphism: Lack of a Role for In Vitro Cytokine Response and Membrane

CD14 Expression

Sonja van Aulock,' Jan

RUpp,2

Katja Gueinzius,' Matthias Maass.' and Corinna Hermann"

Biochemical Pharmacology, University of Konstanz, Konstanz, Germany." and Institute of Medical Microbiology and Hygiene, University of Luebeck, Luebeck, Germany"

Bloodof volunteers, genotyped for the CD14 C(-159)---+T polymorphism, showed no difference in cytokine release when stimulated with nine CD14-dependent immune stimuli. Ananalysis of the published data on the proposed association of CD14 genotype with membrane CD14 density revealed no significant correlation, questioning a functional impact of the CD14 polymorphism.

Membrane-bound CD14 (mCDI4) recognizes bacterial compounds like lipopolysaccharide (LPS), Iipoteichoic acid (LTA), or peptidoglycan (PGN), while soluble CD14 (sCD14) mediates the response of endothelial and epithelial cells, which do not express CD14, to microbes. In 1999, a polymorphism of the cd14 gene within the promoter,

CC

-159)--+T [also referred to as

CC

-260)--+T], was described (2, 6, 14) and shown to be associated with a higher density of mCD14 (6) and higher sCD14 plasma concentrations (2). This was assumed to be the consequence of increased transcription of the CD14 gene in cases of the C---+T substitution, but this hypothesis was so far supported only by the findings of LeVan et al., who showed changes in the binding of the transcription factors Sp1, Sp2, and Sp3 in the CD14 promoter (11). Furthermore, the T allele variant of CD14 was identified as a risk factor for myocardial infarction (6, 14). Using a standardized whole-blood assay (4), we investigated whether blood leukocytes of volunteers with the TT variant show stronger inflammatory responses than leukocytes from carriers of the CC genotype when stimulated with CD14-dependent stimuli.

Heparinized blood from 160 healthy volunteers from the University of Konstanz, Germany (84 male, 76 female; mean age, 30; range, 20 to 70), was incubated with or without 1 ug/ml LPS from Salmonella enterica serovar Abortus-equi (Sigma). The release of interleukin-Lp (IL-l~), IL-6, IL-8, IL-lO, tumor necrosis factor alpha (TNF-ct), gamma inter- feron (IFN-')'), and granulocyte colony-stimulating factor (G-CSF) was measured by enzyme-linked immunosorbent assay (ELISA), and the serum levels of TNF-oc and of the proinflammatory marker "C-reactive protein" were determined by high-sensitivity ELISA (15). Genotyping of DNA (extracted from blood by using a QIAamp DNA blood mini- kit [QIAGEN]) for the CD14 polymorphism (14), revealed an incidence of a 24% CC, 51% CT, and 25% TT genotype.

Analysis of basal and LPS-induced cytokines dependent on genotype did not result in a significant difference between

*Corresponding author. Mailing address: Biochemical Pharmacol- ogy, University of Konstanz, P.Q. Box M655, 78457 Konstanz, Ger- many. Phone: 49 7531 884524. Fax: 49 7531 884156. E-mail: Corinna .hermannepuni-konstanz.de,

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the three groups for any parameter measured. Statistical analysis was done using GraphPad Prism 3.00 (GraphPad Software). Data showed Gaussian distribution and were an- alyzed by one-way analysis of variance, followed by Tukey's multiple-comparison test. Figure 1 shows the basal serum TNF-ct levels and LPS-inducible TNF-ct release in whole blood exemplarily.

Since mCD14 enhances the responsiveness of monocytes especially towards low concentrations of stimuli, 8 volunteers with CC, 12 with CT, and 9 with TT genotype were recruited again, and concentration-response curves of LPS, LTA (pre- pared in house, Bacillus subtilis DSM 1087), Staphylococcus aureus PGN (Sigma), Borrelia lysate (sonicated B. burgdoiferi sensu stricto N40; kindly provided byT. Kamradt, Berlin, Ger- many), Chlamydophila pneumoniae (TW183, purified elemen- tary bodies; a kind gift from Inge Muehldorfer, Altana Phanna, Konstanz, Gennany), UV-inactivated Escherichia coli (K-12, JM 109; kindly provided by Gerald Gruetz, University Clinic Charite, Berlin, Germany), Staphylococcus aureus (DSM 20233), mannan (prepared in house, Candida albicans DSM 1386), and the phorbol ester PMA (Sigma) were determined.

As indicated in Fig. 2, the LPS- and LTA-inducible TNF-oc releases were comparable between the CC, CT, and TT genotypes and showed no significant differences either at low or at high stimulus concentrations as determined by one-way analysis of variance followed byTukey's multiple-comparison test.

Furthermore, no significant differences were observed for IL-

1~, IL-8, IL-lO, and IFN-~. For PGN and Borrelia lysate and for mannan, 10 and 100 ng/rnl, respectively, were necessary to induce significant cytokine release. However, the responses were comparable between the three groups (e.g.,for TNF-ct [in ng/ml], PGN, 1.7± 004for CC versus 1.3± 0.3 for TT; Borrelia lysate, 0.9± 0.2 for CC versus 1.2± 0.5 for TT; mannan, 0.38

± 0.09 for CC versus 0.56± 0.13 for TT).For stimulation with C. pneumoniae, E. coli, and S. aureus, the minimal cytokine- inducing concentrations were 5 X 10⁶, 5 X 10³, and 5 X 10⁵ bacteria per rnl, respectively. Again, no significant differences in the release of IL-113, IL-8, IL-lO, TNF-ct, and IFN-')' were observed (for TNF-oc [in ng/ml],C.pneumoniae, 0.35et0.07 for CC versus 0.37± 0.08 for TT;E. coli, 4 ± 0049for CC versus 2.6 ± 0.53 for TT; S. aureus, 1.5± 0.37 for CC versus 1.2± First publ. in: Clinical and Diagnostic Laboratory Immunology 12 (2005), 10, pp. 1254-1256

Konstanzer Online-Publikations-System (KOPS) URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-79362

URL: http://kops.ub.uni-konstanz.de/volltexte/2009/7936

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FIG. 2. Effect of CD14C(-159)~Tpolymorphism on TNF-O' release induced by concentrations of LPS and LTA. Twenty percent human whole blood was incubated overnightwith (a) LPS and (b) LTA in the concentrations indicated and TNF-O' release was determined in the cell-free supernatants by ELISA. Data are given as means± SD.

For CC,n = 8; CT,n = 12;TT, n = 9.

1255

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FIG. 1. Effect of CD14 C(-159)~T polymorphism on basal and LPS-inducible cytokine release. (a) Basal TNF-O' was determined from serum of 160 genotyped volunteers by ELISA. (b) Twenty percent human whole blood was incubated overnightwith 1 fLg LPS/ml for 24 h and TNF-O' release was measured in the cell-free supernatants by ELISA. The median is indicated by a horizontal line. For CC,n = 39;

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0.20 for IT). This also held true for stimulation with the CD14-independent stimulus phorbol myristate acetate.

So far, only a few studies have investigated LPS-inducible TNF-oc release of the different CD14 genotypes using monocytes (5), peripheral blood mononuclear cells (10) or blood (3).

Kondo et al. reported increased LPS-induced TNF-oc release for the IT genotype (10), and Eng et al. claimed a tendency toward higher TNF-oc production in the TT group when stimulated with LPS and a significant difference in response to C.

pneumoniae (3). In contrast, Heesen et al. observed no differ- ence between the three genotypes (5), which is in line with our findings.Itis important to note that in the studies by Kondo et al.

(10) and Eng et al. (3), the standard deviations are large, and in the former report, the IT group already had sevenfold-higher basal TNF-oc levels than did the CC or CT group (10).

Our finding that monocytes from volunteers with the CC genotype were as sensitive as from donors with the IT genotype, with presumably higher mCD14 expression, was surpris- ing. We therefore readdressed the current knowledge on the phenotype of this polymorphism. The initial report on the CD14 polymorphism found a higher density of mCD14 among volunteers with the TT genotype (6). This was confirmed by only one group (3), while two other groups found no association between genotype and mCD14 expression (5, 8). As a

simple evaluation, we normalized the data from each of the four studies to the mean mCD14 density of the CC group in the respective study: compared to the 70 CC subjects, the 67 TT subjects showed on average 10% higher mCD14 (109.9%).

Statistical significance could not be tested since all studies used different or no ways of expressing variance. A power analysis assuming homogeneity of variances indicates that with the given coefficient of variation (CV) of 25%, a significance level of 5% and a power of 80%, this difference would become significant only at group sizes larger than 100 (S.-Plus 6.2; MathSoft). The association of the IT genotype with myocardial infarction sug- gested a stronger inflammatory response in subjects with the IT genotype due to increased CD14 levels. However, our analysis does not support an association of the homozygous IT genotype with a relevant increase in mCD14 expression or consequently increased sensitivity to inflammatory stimuli. Itshould also be noted that the number of studies which do not find a correlation of the CD14 IT genotype with the progression of coronary artery disease (1, 7-9, 12, 13, 16) is increasing.

This work was supported by a grant from the Deutsche Forschungs- gemeinschaft, FOR 434/2.

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Critical Investigation of the CD14 Promoter Polymorphism : Lack of a Role for In Vitro Cytokine Response and Membrane CD14 Expression