ARTICLE IN PRESS

Immunobiology 213 (2008) 621–627

0171-2985/$ - se

doi:10.1016/j.im

Abbreviation:

charide; PAMP

Peripheral bloo

length polymor�Correspond

fax: +497531 8

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www.elsevier.de/imbio

IL-10 release requires stronger toll-like receptor 4-triggering than TNF:

A possible explanation for the selective effects of heterozygous TLR4

polymorphism Asp(299)Gly on IL-10 release

Oliver Dehus, Sebastian Bunk, Sonja von Aulock, Corinna Hermann�

Biochemical Pharmacology, University of Konstanz, P.O.Box M668, 78457 Konstanz, Germany

Received 9 January 2008; received in revised form 5 March 2008; accepted 10 March 2008

Abstract

The toll-like receptor 4 Asp(299)Gly polymorphism results in an inactive receptor. Heterozygosis is associated withreduced LPS-inducible IL-10 protein and IL-10 mRNA from blood leukocytes and isolated monocytes, whilenumerous other mediators are not affected. We could exclude that this effect is due to the differences in the kinetics ofIL-10 release, in the expression of total surface TLR4 or in LPS-binding to monocytes between subjects heterozygousfor the Asp(299)Gly polymorphism or homozygous carriers of the wild-type allele. Furthermore, we could show thatIL-10 induction in general requires stronger LPS-triggering than TNF and is more sensitive to LPS inhibitors. Thelower number of responsive wild-type TLR4 receptors on monocytes of heterozygotes may explain why only IL-10release is affected.r 2008 Elsevier GmbH. All rights reserved.

Keywords: Cytokine induction; Human monocytes; IL-10 release; LPS; TLR4 Asp(299)Gly polymorphism

Introduction

The Asp(299)Gly polymorphism of toll-like receptor 4(TLR4), which mostly cosegregates with the Thr(399)Ilemutation in Europeans, was found to interrupt lipopo-lysaccharide (LPS)-induced TLR4 signaling in trans-fected THP-1 cells and to be associated with reducedresponsiveness to inhaled LPS in humans (Arbour et al.,2000). While two studies about the role of the TLR4

e front matter r 2008 Elsevier GmbH. All rights reserved.

bio.2008.03.001

LALF, Limulus anti-LPS factor; LPS, Lipopolysac-

S, Pathogen associated molecular patterns; PBMC,

d mononuclear cells; RFLP, Restriction fragment

phism.

ing author. Tel.: +497531 884524;

84117.

ess: Corinna.Hermann@uni-konstanz.de

Asp(299)Gly polymorphism in human systemic andperioperative endotoxemia demonstrated similar re-sponses of subjects with wild-type or heterozygouspolymorphic genotype (Calvano et al., 2006; Schipperset al., 2004), numerous associations with inflammatoryor infectious diseases, especially inflammatory boweldisease and Gram-negative infections have been re-ported (Hermann, 2007). However, in these studies, cell-based assays proving that the polymorphism carriers’ability to respond to immune stimuli is altered, aremostly lacking. Erridge et al. (2003) stimulated isolatedmonocytes with LPS from different Gram-negativebacteria and observed no deficits of the cells fromheterozygous TLR4 polymorphism carriers in releasingIL-1b . In a study reported by our group, analysis ofcytokine responses of blood leukocytes of 160 healthyvolunteers genotyped for the Asp(299)Gly polymorphism

ARTICLE IN PRESSO. Dehus et al. / Immunobiology 213 (2008) 621–627622

in an ex vivo whole blood test did not result indifferences in LPS-inducible release of inflammatorymediators like TNF, IL-6, IL-1b, IFNg, G-CSF,eicosanoids or serum cytokines, except for the releaseof the anti-inflammatory cytokine IL-10, which wassignificantly reduced in the group of subjects withheterozygous TLR4 alleles (von Aulock et al., 2003).

IL-10 is an important anti-inflammatory cytokinemainly produced by human monocytes, and IL-10dysfunction can result in excessive inflammation (Mooreet al., 2001). So far, there is no explanation how thisselective effect of the TLR4 Asp(299)Gly polymorphismon IL-10 release is mediated. Here we show that IL-10production is already reduced at the mRNA level, butthe reduced release of IL-10 protein is not due todelayed kinetics. Furthermore, we provide evidence thatIL-10 release requires stronger triggering of TLR4 thanTNF release, and therefore the lower number ofresponsive TLR4 receptors on monocytes of hetero-zygous carriers of the Asp(299)Gly polymorphism mayexplain why only IL-10 release is affected.

Material and methods

Volunteer population and TLR4 genotyping

The TLR4 Asp(299)Gly polymorphism was determinedin a population of 558 volunteers recruited at theUniversity of Konstanz, Germany, in the years 2000–04.DNA was prepared from EDTA anticoagulated blood(Sarstedt) by the QIAamp DNA Blood Mini Kit(Qiagen). Determination of the A(896)G TLR4 SNPwas performed by real-time PCR and melting pointanalysis according to Heesen et al. (2003). The hetero-zygous TLR4 polymorphism occurred with a frequency of7.2%. Subgroups of subjects with wild-type genotype andheterozygous TLR4 polymorphism were recruited fromthe 558 volunteers for the investigations described below.

Human whole blood and monocyte incubation

Differential blood cell counts were measured routi-nely with a Pentra60 to rule out acute infections (ABXTechnologies). Incubations of whole blood and ELISAmeasurements were carried out as described (vonAulock et al., 2003). Monocytes were isolated byMACS-negative selection (Miltenyi Biotec). Stimula-tions were performed with LPS from Salmonella abortus

equi (S.a.e.), or, where indicated, with LPS fromKlebsiella pneumoniae (Kl.pn.) (both from Sigma). Insome experiments Limulus anti-LPS factor (LALF, agenerous gift from F. Jordan, Charles River/Endosafe)was added. RNA from heparinized blood (Sarstedt) wasisolated with the QIAamp RNA Blood Mini Kit

(Qiagen) and used for reverse transcription. All experi-ments and measurements were carried out blindly withregard to the donors’ genotypes.

Quantitative real-time PCR

cDNA was quantified by quantitative real-time PCRon a LightCycler system (Roche) with LightCyclerFastStart DNA Master SYBR Green (Roche) usingspecific primers from Thermo Hybaid: TNF forward: 50-GAG TGA CAA GCC TGT AGC CCA TGT TGTAGC A-30, reverse: 50-GCA ATG ATC CCA AAGTAG ACC TGC CCA GAC T-30; GAPDH forward: 50-GAA GGT GAA GGT CGG AGT C-30, reverse: 50-GAA GAT GGT GAT GGG ATT TC-30; IL-10forward: 50-CAA GTT GTC CAG CTG ATC CTTCAT-30, reverse: 50-GGC AAC CTG CCT AAC ATG-30; Cyclophilin forward: 50-CTC CTT TGA GCT GTTTGC AG-30, reverse: 50-GAT GGC AAG CAT GTGGTG-30.

FACS analysis

For FACS analysis a FACS Calibur flow cytometer(Becton Dickinson) with Cell Quest software (BectonDickinson) was used. For assessment of the monocytes’LPS-binding capacity, EDTA blood was stained with in-house produced fluorescein-5 (6)-carboxamido caproicacid N-succinimidyl ester (FCHSE)-LPS and anti-CD14(BD Biosciences). FCHSE was used as backgroundcontrol. For investigation of TLR4 surface expression,5� 105 peripheral blood mononuclear cells (PBMC)were prepared with CPTTM Cell Preparation Tubes (BDBiosciences) and incubated with an anti-TLR4 antibody(a kind gift from Dr. Alexander Dalpke, University ofMarburg, Germany). An anti-mouse IgG-phycoerythrin(PE, DAKO) was used as label. Measurement of IgG-PE alone served as background control. Monocyteswere gated according to their forward and side scatter-ing properties.

Statistics

Statistical analysis was performed using the Graph-Pad Prism 4.0 program (GraphPad Software, SanDiego, USA). Data are given as mean7SEM. Signifi-cance of differences was assessed by t-test in case of twogroups only or by one-way ANOVA followed byBonferroni’s post-test. IC50 values were determinedaccording to a sigmoidal curve fit.

Results

The aim of this study was to investigate the selectivityof the effect of the TLR4 Asp(299)Gly polymorphism

ARTICLE IN PRESSO. Dehus et al. / Immunobiology 213 (2008) 621–627 623

on IL-10 release. Therefore, we had to compose a newlygenotyped study group (n ¼ 17 wild-type (+/+); n ¼ 10heterozygous polymorphics (7)) and to reconfirm thepreviously observed effect of the TLR4 Asp(299)Glypolymorphism on LPS-inducible IL-10 release. Again,like in the previous study (von Aulock et al., 2003),stimulation of the polymorphic heterozygotes’ wholeblood with LPS (S.a.e. 1 mg/ml) resulted in diminishedIL-10 release (+\+: 0.8170.08 ng/ml vs. 7: 0.5270.06 ng/ml, p ¼ 0.026), while the release of TNFwas not affected (+\+: 2.9670.38 ng/ml vs. 7: 3.267

control 1 10 100 10000

100

200

300TLR4 +\+

TLR4 +\-

LPS [ng/ml]

*

*

*IL-1

0 [p

g/5x

104 m

onoc

ytes

]

Fig. 1. Heterozygous Asp(299)Gly TLR4 polymorphism is

associated with reduced IL-10 but not TNF release from

isolated human monocytes. 5� 104monocytes/well were in-

cubated in the presence of LPS at the concentrations indicated

for 20 h. TNF and IL-10 were determined in the cell-free

supernatants by ELISA. TLR4 +\+ indicates the wild-type

(n ¼ 8) and TLR4 7 the heterozygous polymorphic genotype

(n ¼ 7). Data are means7SEM. *po0.05 indicates signifi-

cance versus the wild type.

TLR4 +\+0

400

800

1200

1600

TNF-

mR

NA

TLR4 +\-

Fig. 2. Heterozygous Asp(299)Gly TLR4 polymorphism is associate

20% human whole blood was incubated in the presence of 1 mg/ml L

and cDNA was analyzed by real-time PCR. TNF and IL-10 data wer

and whiskers blot as x-fold induction of mRNA. TLR4 +\+ ind

polymorphic genotype (n ¼ 8).

0.44 ng/ml, p40.05). The same effect was also observedusing purified monocytes stimulated with increasingconcentrations of LPS (Fig. 1). Analysis of mRNAexpression by real-time PCR confirmed that lower LPS-inducible IL-10 release occurs already at the IL-10mRNA level, while TNF mRNA levels were notinfluenced (Fig. 2). Since only the IL-10 release wasaffected by the TLR4 polymorphism, we investigatedwhether the reduced IL-10 levels were due to a shift inthe kinetics of IL-10 release of subjects with TLR4polymorphisms. For this purpose, we followed therelease of LPS-induced IL-10 in whole blood incuba-tions over a period of 28 h. We measured IL-10 byELISA after 5, 10, 15, 20, 25 and 30 h of stimulation, butno shift in the IL-10 release curve was apparent (Fig. 3).

Next, we assumed that the difference in the density ofthe total TLR4 surface expression might be responsiblefor the observed effects. When we compared the totalTLR4 surface expression of monocytes from six wild-type and nine heterozygous TLR4 polymorphic donorsby FACS analysis, we detected a higher density ofTLR4 on monocytes from heterozygous polymorphicdonors (median of relative fluorescence: (+\+):11.9271.00 vs. (7): 17.6072.00; p ¼ 0.042). To con-firm that the polymorphic TLR4 variant was tran-scribed, we investigated TLR4 mRNA by real-time PCRusing specific Hybprobes designed for genotyping,which were 100% specific for the wild-type gene, butpossessed one mismatch for the polymorphic TLR4variant. The LightCycler-performed melting point ana-lysis of the products revealed that indeed for carriers ofthe heterozygous polymorphisms both the wild-type(melting point 61 1C) and the polymorphic mRNAvariant (melting point 56 1C) are transcribed in equalshares (Fig. 4). To investigate whether the LPS-bindingcapacity of monocytes from homozygous wild-typesubjects is different from heterozygous subjects, we

TLR4 +\+0

400

800

1200

1600

p = 0.012

IL-1

0-m

RN

A

TLR4 -\+

d with reduced IL-10 but not TNF mRNA. Five milliliters of

PS from S.a.e. for 6 h. RNA was prepared, reverse transcribed

e normalized to cyclophilin cDNA. Data are presented in a box

icates the wild-type (n ¼ 12) and TLR4 7 the heterozygous

ARTICLE IN PRESSO. Dehus et al. / Immunobiology 213 (2008) 621–627624

performed a FACS analysis. Monocytes from 37homozygous wild-type subjects and 18 heterozygoussubjects were incubated with 0.35 ng/ml FCHSE-labeledLPS. We observed similar LPS-binding to monocytes ofboth groups (median of relative fluorescence: (+\+):35.7071.74 vs. (+\): 30.9171.12).

0 5 10 15 20 25 300

1

2

3

4 TLR4 +\+TLR4 +\-

time [h]

IL-1

0 [n

g/m

l]

Fig. 3. The kinetics of IL-10 release is not affected by the

heterozygous Asp(299)Gly TLR4 polymorphism. One milli-

liter of 20% human whole blood was incubated in the presence

of 1 mg/ml LPS from S.a.e. for the time intervals indicated. IL-

10 was determined in the cell-free supernatants by ELISA.

Data are means 7 SEM. TLR4 +\+ indicates the wild type

(n ¼ 14) and TLR47 the heterozygous polymorphic genotype

(n ¼ 8).

Asp(299)Gly

0.0140.013

0.0120.0110.010

0.0090.008

0.0070.0060.005

0.0040.0030.002

0.0010.000

-0.001-0.002

50.0 51.0 52.0 53.0 54.0 55.0 56.0 57.0 58.0 59.0Tempe

Fluo

resc

ence

-d(F

2/F1

)/dT

Fig. 4. Carriers of the heterozygous Asp(299)Gly TLR4 polymorphis

RNA was prepared, reverse transcribed into cDNA and quantified b

point 61 1C) and/or TLR4 Asp(299)Gly polymorphic variant (melti

+\+ indicates the wild type and TLR4 7 the heterozygous polym

Since it must be assumed that the polymorphic TLR4variant, although not being defective in LPS binding, isdefective in LPS-responsiveness, we compared thesensitivity of TNF-and IL-10 release to the concentra-tion of LPS used for stimulation. When a concentrationresponse curve ranging from 10 pg/ml to 1 mg/ml LPS inwhole blood from healthy volunteers of only the wild-type genotype was performed, we found that IL-10release required significantly stronger LPS stimulationthan TNF release (Fig. 5). To avoid bacterial species-specific results, these experiments were performed withLPS from two different enterobacterial strains (S.a.e.

and Kl.pn.). The two LPS exhibited a similar potency tostimulate the release of TNF, IL-1b, IL-8, IL-10 andIFNg at the highest concentration employed (shown forTNF and IL-10 in Fig. 3), but while 30 pg/ml of LPSfrom both strains resulted in a significant release ofTNF, 100 pg/ml LPS (Kl.pn.) and 300 pg/ml LPS (S.a.e.)were necessary to induce significant IL-10 release. In linewith this observation, LPS (1 ng/ml)-inducible IL-10release was significantly more susceptible to inhibitionby the LPS-neutralizing factor LALF (Fig. 6).

Discussion

The only experimentally proven effect of the As-p(299)Gly polymorphism of TLR4 on inflammatoryresponses is a diminished release of the anti-inflamma-tory cytokine IL-10 upon in vitro stimulation of blood

wt

60.0 61.0 62.0 63.0 64.0 65.0 66.0 67.0 68.0 69.0 70.0rature (°C)

+\+

+\-

H2O

m transcribe the wild-type and the polymorphic TLR4 variant.

y real-time PCR. The presence of the TLR4 wild type (melting

ng point 56 1C) was analyzed by melting point analysis. TLR4

orphic genotype.

ARTICLE IN PRESS

0.0

0.5

1.0

1.5

2.0

**

***

******

***

***

***

***

S.a.e.Kl.pn.

0

LPS [ng/ml]

TNF

[ng/

ml]

0.0

0.1

0.2

0.3

0.4

******

***

***

**

S.a.e.Kl.pn.

LPS [ng/ml]

IL-1

0 [n

g/m

l]

0.30.10.030.01 1 0 0.30.10.030.01 1 0 0.30.10.030.01 1 0 0.30.10.030.01 1

Fig. 5. Induction of significant amounts of IL-10 from whole blood requires a higher LPS concentration than TNF induction. One

milliliter of 20% human whole blood from 24 healthy volunteers of the wild type genotype was incubated in the presence of LPS at

the concentrations indicated for 20 h. TNF and IL-10 were determined in the cell-free supernatants by ELISA. Data are

means7SEM. *po0.05; **po0.01; ***po0.001 and indicate significance versus the control.

0

200

400

600

800

1000

1200

1400

IC50=62

0 104103102 10410310210log LALF [ng/ml]

***

TNF

[pg/

ml]

0

100

200

300

400

IC50=28

0 10log LALF [ng/ml]

**

IL-1

0 [p

g/m

l]

Fig. 6. IL-10 induction is more sensitive to LPS inhibition than TNF. One milliliter of 20% human whole blood from eight healthy

volunteers was incubated in the presence of 1 ng/ml LPS or 1 ng/ml LPS together with 10 ng/ml–10 mg/ml LALF for 20 h. TNF and

IL-10 were determined in the cell-free supernatants by ELISA. Data are means7SEM. **po0.01; ***po0.001.

O. Dehus et al. / Immunobiology 213 (2008) 621–627 625

leukocytes from subjects with heterozygous expressionof the polymorphic TLR4 variant with LPS (von Aulocket al., 2003). This previous observation was nowreconfirmed using a new study collective. It was shownthat it also translates to isolated monocytes and thata significant reduction of IL-10 formation in the caseof subjects with heterozygous TLR4 polymorphismalready occurs at IL-10 mRNA level, while the TNFmRNA levels remained unaffected. This, first of all,argues against an artifact of multiple testing in theprevious study (von Aulock et al., 2003), where IL-10was only one parameter measured among many.

The aim of this study was to investigate the under-lying mechanisms responsible for the selective effect of

the Asp(299)Gly polymorphism of TLR4 on cytokinerelease, which could not be explained by a delay in IL-10release. In vitro transfection experiments had proventhat the polymorphic variant of TLR4 is non-functionalfor LPS signaling (Arbour et al., 2000), and it is assumedthat the mutation at position 299, which is located in theextracellular LRR region of the TLR4 receptor, resultsin modified LPS binding. This is also supported by arecent study which provides evidence that the TLR4mutation affects interaction with receptor agonists orco-receptors rather than intracellular signaling (Rallab-handi et al., 2006). Therefore, it seemed likely thatpolymorphism carriers might suffer from impaired LPSresponsiveness. However, in our study, neither the total

ARTICLE IN PRESSO. Dehus et al. / Immunobiology 213 (2008) 621–627626

TLR4 surface expression nor the LPS-binding capacityof monocytes was reduced in heterozygous subjects,although it was confirmed that the polymorphicgene variant is transcribed. The latter finding suggeststhat some of the expressed TLR4 receptors wouldbe aberrant and not responsive. The unaltered LPS-binding capacity of monocytes could be explained bythe assumption that LPS initially binds to MD-2and is then presented to TLR4, which in the case ofthe Asp(299)Gly variant would not result in TLR4activation. The fact that subjects with a TLR4polymorphism showed an increased TLR4 surfaceexpression might even indicate that the expression ofthe polymorphic non-functional variant is partiallycompensated by a stronger expression of the wild-typeTLR4.

Our results clearly indicate that IL-10 induction ingeneral requires stronger LPS stimulation of monocytesthan TNF induction and that IL-10 release is moresusceptible to inhibition of LPS by a neutralizing agent.Divergent sensitivities of the TNF and IL-10 ELISA,which would influence these results, were excluded.Taken together, this means that significant IL-10expression requires a higher density of activatedreceptor complexes than TNF and thus is moresusceptible to a lack of functional receptors, like in thecase of the TLR4 polymorphism. Although both TNFand IL-10 are released in response to TLR4 stimulation,the signal transduction pathways, which initiate genetranscription, differ. While pro-inflammatory cytokineslike TNF are induced via a synergistic interplay of theNF-kB pathway and activation of the MAPK-kinasesERK1/2, JNK and p38, IL-10 induction is dependent onp38 and the transcription factor Sp1, but does notinvolve ERK1/2 and NF-kB (Adib-Conquy et al., 2003;Hoebe and Beutler, 2004; Ma et al., 2001), which mayalready explain why TNF induction is more sensitive toLPS stimulation than IL-10.

However, we investigated several key parametersknown to be relevant for IL-10 induction includingp38 on the basis of phosphorylated p38 by Western blotanalysis, as well as the role of the MyD88 independentTRIF/IRF pathway (Hacker et al., 2006), and theinduction of cyclo-oxygenase-2 and PGE2 (Williams etal., 2000). We observed no difference between homo-zygous wild-type and heterozygous polymorphic sub-jects in any of these experiments (unpublished data). ForTNF induction it is believed that LPS binding to theTLR4 receptor complex alone is sufficient to induceTNF (Latz et al., 2002), though this has not beeninvestigated for IL-10 so far. One might speculate thatIL-10 induction requires further processes like inter-nalization of the LPS/receptor complex and intracellularprocessing. Preliminary results obtained with LPScoated to surfaces support this hypothesis but werenot finally conclusive.

Given the pivotal role of LPS and its receptor TLR4in bacterial immune recognition, this study gives a firstexplanation of specific inflammatory alterations inheterozygous Asp(299)Gly polymorphic subjects. Theresulting pro-inflammatory phenotype could hence be arisk factor for excessive inflammation. Consistent withthis, the TLR4 Asp(299)Gly polymorphism has beenconvincingly linked with inflammatory bowel diseaseand ulcerative colitis (Franchimont et al., 2004), inwhich IL-10 reduction is known to play a decisive role(Kuhn et al., 1993). Therefore, it would be of majorinterest to investigate LPS-inducible IL-10 levels inpatients with inflammatory bowel disease carrying theAsp(299)Gly polymorphism.

References

Adib-Conquy, M., Moine, P., Asehnoune, K., Edouard, A.,

Espevik, T., Miyake, K., Werts, C., Cavaillon, J.M., 2003.

Toll-like receptor-mediated tumor necrosis factor and

interleukin-10 production differ during systemic inflamma-

tion. Am. J. Respir. Crit. Care Med. 168, 158–164.

Arbour, N.C., Lorenz, E., Schutte, B.C., Zabner, J., Kline,

J.N., Jones, M., Frees, K., Watt, J.L., Schwartz, D.A.,

2000. TLR4 mutations are associated with endotoxin

hyporesponsiveness in humans. Nat. Genet. 25, 187–191.

Calvano, J.E., Bowers, D.J., Coyle, S.M., Macor, M., Reddell,

M.T., Kumar, A., Calvano, S.E., Lowry, S.F., 2006.

Response to systemic endotoxemia among humans bearing

polymorphisms of the Toll-like receptor 4 (hTLR4). Clin.

Immunol. 121, 186–190.

Erridge, C., Stewart, J., Poxton, I.R., 2003. Monocytes

heterozygous for the Asp299Gly and Thr399Ile muta-

tions in the Toll-like receptor 4 gene show no deficit

in lipopolysaccharide signalling. J. Exp. Med. 197,

1787–1791.

Franchimont, D., Vermeire, S., El Housni, H., Pierik, M., Van

Steen, K., Gustot, T., Quertinmont, E., Abramowicz, M.,

Van Gossum, A., Deviere, J., Rutgeerts, P., 2004. Deficient

host-bacteria interactions in inflammatory bowel disease?

The toll-like receptor (TLR)-4 Asp299gly polymorphism is

associated with Crohn’s disease and ulcerative colitis. Gut

53, 987–992.

Hacker, H., Redecke, V., Blagoev, B., Kratchmarova, I., Hsu,

L.C., Wang, G.G., Kamps, M.P., Raz, E., Wagner, H.,

Hacker, G., Mann, M., Karin, M., 2006. Specificity in Toll-

like receptor signalling through distinct effector functions

of TRAF3 and TRAF6. Nature 439, 204–207.

Heesen, M., Wessiepe, M., Kunz, D., Vasickova, K., Blomeke,

B., 2003. Rapid and reliable genotyping for the Toll-like

receptor 4 A896G polymorphism using fluorescence-labeled

hybridization probes in a real-time polymerase chain

reaction assay. Clin. Chim. Acta 333, 47–49.

Hermann, C., 2007. Variability of host-pathogen interaction.

J. Endotoxin Res. 13, 199–218.

Hoebe, K., Beutler, B., 2004. LPS, dsRNA and the interferon

bridge to adaptive immune responses: Trif, Tram, and other

TIR adaptor proteins. J. Endotoxin Res. 10, 130–136.

ARTICLE IN PRESSO. Dehus et al. / Immunobiology 213 (2008) 621–627 627

Kuhn, R., Lohler, J., Rennick, D., Rajewsky, K., Muller, W.,

1993. Interleukin-10-deficient mice develop chronic enter-

ocolitis. Cell 75, 263–274.

Latz, E., Visintin, A., Lien, E., Fitzgerald, K.A., Monks, B.G.,

Kurt-Jones, E.A., Golenbock, D.T., Espevik, T., 2002.

Lipopolysaccharide rapidly traffics to and from the Golgi

apparatus with the toll-like receptor 4-MD-2-CD14 com-

plex in a process that is distinct from the initiation of signal

transduction. J. Biol. Chem. 277, 47834–47843.

Ma, W., Lim, W., Gee, K., Aucoin, S., Nandan, D.,

Kozlowski, M., Diaz-Mitoma, F., Kumar, A., 2001. The

p38 mitogen-activated kinase pathway regulates the human

interleukin-10 promoter via the activation of Sp1 transcrip-

tion factor in lipopolysaccharide-stimulated human macro-

phages. J. Biol. Chem. 276, 13664–13674.

Moore, K.W., de Waal Malefyt, R., Coffman, R.L., O’Garra,

A., 2001. Interleukin-10 and the interleukin-10 receptor.

Annu. Rev. Immunol. 19, 683–765.

Rallabhandi, P., Bell, J., Boukhvalova, M.S., Medvedev, A.,

Lorenz, E., Arditi, M., Hemming, V.G., Blanco, J.C.,

Segal, D.M., Vogel, S.N., 2006. Analysis of TLR4

polymorphic variants: new insights into TLR4/MD-2/

CD14 stoichiometry, structure, and signaling. J. Immunol.

177, 322–332.

Schippers, E.F., van ‘t Veer, C., van Voorden, S., Martina,

C.A., le Cessie, S., van Dissel, J.T., 2004. TNF-alpha

promoter, Nod2 and toll-like receptor-4 polymorphisms

and the in vivo and ex vivo response to endotoxin. Cytokine

26, 16–24.

von Aulock, S., Schroder, N.W., Gueinzius, K., Traub, S.,

Hoffmann, S., Graf, K., Dimmeler, S., Hartung, T.,

Schumann, R.R., Hermann, C., 2003. Heterozygous toll-

like receptor 4 polymorphism does not influence lipopoly-

saccharide-induced cytokine release in human whole blood.

J. Infect. Dis. 188, 938–943.

Williams, J.A., Pontzer, C.H., Shacter, E., 2000. Regulation

of macrophage interleukin-6 (IL-6) and IL-10 expre-

ssion by prostaglandin E2: the role of p38 mitogen-

activated protein kinase. J. Interferon Cytokine Res. 20,

291–298.