R-form LPS, the master key to the activation ofTLR4/MD-2-positive cells

R-form LPS, the master key to the activation ofTLR4/MD-2-positive cells

Michael Huber*1, Christoph Kalis*1, Simone Keck2, Zhengfan Jiang3,Philippe Georgel3, Xin Du3, Louis Shamel3, Sosathya Sovath3,Suzanne Mudd3, Bruce Beutler3, Chris Galanos2 andMarina A. Freudenberg2

1 Molecular Immunology, Institute for Biology III, Albert-Ludwigs University Freiburg,Freiburg, Germany

2 Max-Planck-Institute for Immunobiology, Freiburg, Germany3 Department of Immunology, The Scripps Research Institute, La Jolla, CA, USA

Lipopolysaccharide (endotoxin, LPS) is a major recognition marker for the detection ofgram-negative bacteria by the host and a powerful initiator of the inflammatoryresponse to infection. Using S- and R-form LPS from wild-type and R-mutants ofSalmonella and E. coli, we show that R-form LPS readily activates mouse cells expressingthe signaling receptor Toll-like receptor 4/myeloid differentiation protein 2 (TLR4/MD-2), while the S-form requires further the help of the LPS-binding proteins CD14 and LBP,which limits its activating capacity. Therefore, the R-form LPS under physiologicalconditions recruits a larger spectrum of cells in endotoxic reactions than S-form LPS.Wealso show that soluble CD14 at high concentrations enables CD14-negative cells torespond to S-form LPS. The presented in vitro data are corroborated by an in vivo studymeasuring TNF-a levels in response to injection of R- and S-form LPS in mice. Since theR-form LPS constitutes ubiquitously part of the total LPS present in all wild-typebacteria its contribution to the innate immune response and pathophysiology ofinfection is much higher than anticipated during the last half century.

Supporting information for this article is available athttp://www.wiley-vch.de/contents/jc_2040/2006/35593_pdf

Introduction

The interaction of highly conserved microbial constitu-ents with the innate immune system forms the basis ofrecognition of and reaction against intruding pathogens

in mammals. One such constituent is lipopolysaccharide(endotoxin, LPS), a major recognition marker commonto gram-negative bacteria [1–3], a large group compris-ing important human pathogens as well as commensals.Thus, the interaction of LPS with cells of the innateimmune system leads to the formation and release ofendogenous mediators initiating inflammatory andimmune responses essential for an antibacterial defense[3, 4]. This primarily protectivemechanismmay becomeovershadowed by an acute pathophysiological responsewith the typical clinical symptoms of septic shock thatfrequently follows the release of inflammatory media-tors, such as tumor necrosis factor (TNF)-a duringinfection [5–7].

Innate immunity

* Both authors contributed equally to this work.

Correspondence: Michael Huber, Molecular Immunology,Institute for Biology III, Albert-Ludwigs University Freiburg,79108 Freiburg, GermanyFax.: +49-761-5108-423e-mail: [email protected]

Received 12/10/05Revised 1/12/05

Accepted 9/1/06

[DOI 10.1002/eji.200535593]

Key words:CD14

� Lipopolysaccharide� Mast cells � TLR4

Abbreviations: LBP: LPS-binding protein � MC: mast cells �mCD14: membrane CD14 � MD-2: myeloid differentiationprotein 2, sCD14: soluble CD14

Eur. J. Immunol. 2006. 36: 701–711 Innate immunity 701

f 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji.de

Different cell types, such as macrophages, granulo-cytes and B cells, participate in the innate immuneresponse to LPS [8]. More recent data indicates thatmast cells (MC) that are strategically located at theinterface between host and environment also interactwith LPS [9], and thus take part in the innate immuneresponse. MC are primarily known for their deleteriouseffects in allergic reactions [10]. Binding of allergen toits specific IgE on the surface of MC results in animmediate release of preformed pro-inflammatorymediators (e.g., histamine, TNF-a and proteases) fromcytoplasmic granules (degranulation) [11] and a laterrelease of de novo synthesized arachidonic acid meta-bolites and various cytokines like interleukin 6 (IL-6),TNF-a, and chemokines [10, 11]. Unlike allergens, LPSinduces no degranulation of MC, but like allergens, itstimulates the de novo synthesis and release of cytokinesin these cells [9].

Activation of cells by LPS is mediated by the Toll-likereceptor 4 (TLR4), a member of the highly conservedprotein family of TLR, which are specialized in therecognition of microbial components. In mice, defects inTLR4 result in LPS unresponsiveness [12]. For func-tional interaction with LPS, TLR4 requires associationwith myeloid differentiation protein 2 (MD-2) [13, 14].According to current consensus activation of TLR4 ispreceded by the transfer of LPS tomembrane-bound (m)or soluble (s) CD14 by LPS-binding protein (LBP)[15–19]. This mechanism is believed to be true for LPSsignaling generally. However, in a recent study weshowed that Re-form LPS and lipid A, but not S-formLPS, are capable of inducing TNF-a responses also in theabsence of CD14 [20].

LPS, synthesized by most wild-type (WT) gram-negative bacteria (S-form LPS), consists of threeregions, the O-polysaccharide chain, which is madeup of repeating oligosaccharide units, the core oligo-saccharide and the lipid A (Fig. 1A), which harbors theendotoxic activity of the entire molecule [1, 2, 21]. R-form LPS synthesized by the so-called rough (R)mutantsof gram-negative bacteria lacks the O-specific chain.Furthermore, the core-oligosaccharide may be presentin different degrees of completion, depending on theclass (Ra to Re) to which the mutant belongs [1, 2, 21,22] (Fig. 1A). Notably, LPS fromWT bacteria are alwayshighly heterogeneous mixtures of S-form LPS moleculescontaining 1 to over 50 repeating oligosaccharide unitsand contain ubiquitously a varying proportion of R-formmolecules lacking the O-specific chain (Fig. 1B). Manyclinically relevant gram-negative bacteria synthesizethis type of LPS. Further, gram-negative WT bacteria,such as Chlamydia and Neisseria synthesize LPS in whichthe number of sugar residues is highly reduced, and thusresemble R-form LPS, at least in their physicochemicalproperties. LPS are amphipathic molecules whose

hydrophobicity decreases with increasing length ofthe sugar part [1, 2, 21, 22]. Based upon thesedifferences, S- and R-form LPS show marked differencesin the kinetics of their blood clearance and cellularuptake as well as in the ability to induce oxidative burstin human granulocytes [23] and to activate the hostcomplement system [24].

In the present study, using Salmonella and E. coli LPSpreparations, we show that R-form LPS and free lipid Ainduce strong TLR4-dependent IL-6 and TNF-a re-sponses in bone marrow-derived mouse MC, and that S-form LPS is in this respect virtually inactive. Acomparison of the activity of R- and S-form LPS forCD14 competent and CD14 lacking cells revealeddifferences in activity that were cell type specific andbased upon a differential requirement for CD14 and LBPby the two forms of LPS. The difference in stimulatorycapacity between R- and S-form LPSwas confirmed in anin vivo mouse model of TNF-a production.

Figure 1. Schematic representation of the different LPSchemotypes. (A) LPS comprises the lipid A moiety, the coreoligosaccharide, and the O-polysaccharide (O-antigen). De-pending on the completeness of the LPS molecule, different R-form LPS, SR-form LPS, and S-form LPS can be distinguished.KDO, 2-keto-3-desoxyoctonate; Hep, heptose; Glc, glucose; Gal,galactose; GlcNac, N-acetyl-glucosamine. (B) LPS from S.abortus equi and S. minnesota R595 was extracted, purifiedand size-separated by SDS-PAGE. The arrowhead depicts the R-form contained in the S-form preparation.

Michael Huber et al. Eur. J. Immunol. 2006. 36: 701–711702


Results

Activation of MC by LPS is primarily a propertyof R-form chemotypes

Highly purified S-form and different R-form (Re-Ra)LPS and free lipid A from Salmonella minnesota and E.coli were used for induction of IL-6 and TNF-a in in vitrodifferentiated bone marrow-derived MC. As shown inFig. 2A and D, R-form LPS (Re to Rc) and lipid A werepotent inducers of IL-6 in MC, while, S-form LPS was inthis respect practically inactive. R-form LPS with a morecomplete core (Rb and Ra) expressed intermediateactivity (Fig. 2A). Similar results were obtained whenthe TNF-a-inducing activity of the LPS and free lipid Apreparations was determined (Fig. 2B). MC thereforediscriminate between the different LPS forms withwhich they can react and become activated.

TLR4/MD-2 expression levels determine MCsensitivity to R-form LPS

The IL-6 and TNF-a responses of MC to R-form LPS, likeLPS responses generally, are mediated via TLR4/MD-2signaling (Fig. 2C, D). Mouse MC, however, expressrelatively low levels of TLR4/MD-2 (Fig. 2C) compared

to, for example, macrophages (see Supplementary Fig. 1online). To investigate, whether an increase in TLR4/MD-2 expression on MC might improve the cytokineresponses to S-form LPS, we used TLR4-rich MC derivedfrom transgenic mice [25] (Fig. 2C, D). The higherTLR4/MD-2 expression correlated with a higher cyto-kine response of these cells to free lipid A (not shown)and Re-form LPS (Fig. 2D). However, it did not improveappreciably the poor response of the cells to S-form LPS(Fig. 2D).

Neither R- nor S-form LPS stimulated degranulationor augmented antigen-mediated degranulation of MC(see Supplementary Fig. 2 online). It has been shownthat co-stimulation of MC with antigen and LPS leads toa synergistic increase in cytokine release [26]. In ourstudy R-, but not S-form LPS induced a TLR4-dependentenhancement of IL-6 (Fig. 2D) and TNF-a (not shown)induction by suboptimal antigen concentrations.

Activation of MC by R-form LPS is independent ofLBP and CD14

Transfer of LPS to CD14 via LBP and a physicalassociation of CD14 with TLR4/MD-2, have beenidentified as necessary events in the activation of cellssuch as macrophages by LPS [15, 27, 28]. The expression

Figure 2. Re-form LPS is a potentMC activator. (A) MC (106/mL) were stimulatedwith Salmonella lipid A (1), LPS from S. minnesota R-form mutants: Re (2), Rd2 (3), Rd1 (4), Rc (5), Rb (6) and Ra (7), or S. minnesota S-form LPS (8). IL-6 was determined in culturesupernatants after 3 h of stimulation. (B) MC were stimulated with Re-LPS (1 lg/mL), S-LPS (10 lg/mL) or lipid A (1 lg/mL) from E.coli. TNF-a was determined in culture supernatants after 3 h of stimulation. (C) Expression of TLR4/MD-2 on MC from differentmice strains was analyzed by FACS. Black-filled histograms show autofluorescence, gray histograms TLR4/MD-2-specific signal.(D) Anti-DNP IgE-loadedMCof Sn (1), ScN (2), Cr (3), and TCr-1 (4)micewere stimulatedwith Re- or S-formLPS of E. coli (1 lg/mL), orDNP-HSA (2 ng/mL), or a combination of DNP-HSA and LPS, or left unstimulated (-). IL-6 was determined in culture supernatantsafter 3 h of stimulation. In (A), (B) and (D) bars represent mean of duplicates � SD. Comparable results were obtained in differentexperiments.



of CD14 onMC is still a matter of dispute [29–34]. To seeif the difference observed between the capacity of S- andR-form LPS to activate MC might be related to adifferential requirement for CD14 and LBP by the twoLPS, we investigated the expression of mCD14 on MC byFACS analysis using cells from CD14–/– mice as negativecontrol (Fig. 3A). Disruption of the CD14 gene impairedneither antigen-mediated degranulation nor IL-6 secre-tion in MC (Fig. 3B, C), showing that CD14 is notrequired for MC development in vitro. As shown inFig. 3A, mCD14 was not detectable on murine MC.Further, treatment with LPS, which was shown toenhance mCD14 expression in macrophages [35, 36],also failed to induce detectable mCD14 expression onMC (see Supplementary Fig. 3 online). We thencompared the IL-6 responses of MC from WT andCD14–/– mice to S- and R-form LPS and free lipid A. Theresults show that WT and CD14–/– cells exhibitedcomparable IL-6 responses to free lipid A and Re-LPSbut no response to S-form LPS (Fig. 3D), and thataddition of LBP to WT MC had practically no influenceon these responses (Fig. 3E). According to previousreports, mCD14-negative cells, which were non-respon-sive to LPS gained LPS sensitivity after supplementationwith soluble CD14 (sCD14) (reviewed in [17]). Surpris-ingly, addition of sCD14 in concentrations that wereshown to be physiological [37] alone or in combination

with LBP, neither enhanced the activity of R-form LPSnor endowed S-form LPS with stimulatory activity(Fig. 3E). These results are interpreted as strongevidence that, in MC, cytokine induction by R-formLPS and lipid A is independent of CD14 underphysiological conditions.

Enhanced levels of sCD14 render MC responsiveto S-form LPS

sCD14 levels are strongly up-regulated under inflam-matory conditions in both humans and mice [37–44].Therefore, we investigated also the effects of highconcentrations of sCD14 (4 lg/mL recombinant sCD14)on the IL-6 production of MC in response to differentamounts of Re-form and S-form LPS (Fig. 4). These highconcentrations of sCD14 enhanced the IL-6 response ofMC to R-form and enabled activation by S-form LPS.

Requirement for LBP in the CD14 dependentactivation ofmacrophages by LPS is dependent onthe LPS chemotype

In contrast to MC, mouse bone marrow-derivedmacrophages express mCD14 (see SupplementaryFig. 4 online). Therefore, here we investigated theLPS-induced activation of macrophages focusing on

Figure 3.Differential requirements for LBP and CD14 among different immune cells. (A) Expression ofmCD14 onMC fromWTandCD14–/–micewas analyzed by FACS. Black-filled histograms showautofluorescence, gray histogramsCD14-specific signal.WTandCD14–/– MC expressed comparable amounts of FceR1 and c-kit on their surfaces (not shown). (B) Anti-DNP IgE-loaded WT (blackbars) and CD14–/– MC (white bars) were stimulated with indicated amounts of DNP-HSA. After 30 min of stimulation b-hexosaminidase release was determined. Similar results were obtained with independently generated MC. (C) Anti-DNP IgE-loaded WT (black bars) and CD14–/– MC (white bars) were stimulated with DNP-HSA or left unstimulated (control). IL-6 wasdetermined in culture supernatants after 3 h of stimulation. Similar results were obtainedwith independently generated MC. (D)WTandCD14–/–MCwere stimulatedwith lipidA (2 lg/mL), Re-formor S-formLPS from S.minnesota. IL-6was determined in culturesupernatants after 3 h of stimulation. (E) MCwere stimulatedwith Re-formor S-form LPS from S. minnesota in presence or absenceof LBP (0.5 lg/mL), CD14 (0.5 lg/mL), or a combination of both. IL-6 was determined in culture supernatants after 3 h ofstimulation. The used concentrations of CD14 and LBP represent physiological plasma levels in healthy mice [37]. Bars in (B–E)represent mean of triplicates � SD.



possible differences in the requirement for LBP andCD14 by S-form and R-form LPS. Re-form LPS was anexcellent stimulus for WT and CD14–/– cells. Supple-mentation with additional exogenous LBP had amoderate enhancing effect on the IL-6 response;however, only in cells expressing CD14 (Fig. 5A, B).Under LBP-free conditions, S-form LPS was clearly lessactive than Re-form LPS in both cell types (Fig. 5A, B).Supplementation with additional LBP strongly en-hanced the response of WT macrophages to S-formLPS (Fig. 5A), but had no effect on CD14–/– cells

(Fig. 5B). Thus, in vitro, S-form LPS is highly dependenton the assistance of LBP and CD14 for optimal inductionof IL-6 (Fig. 5A, B) and TNF-a (not shown) inmacrophages, while Re-form LPS in this respect isclearly less dependent on the two LPS-binding proteins.

Role of LBP and CD14 in the activation ofsplenocytes by LPS

We compared the mitogenic responses of murine Blymphocytes to S- and Re-form LPS and the role of LBPand CD14 in these responses. Spleen cells from WT andCD14–/– mice were stimulated with each of the twoforms of LPS in the presence or absence of murinerecombinant LBP. Re-form LPS induced a high, LBP-independent mitogenic response in both WT andCD14–/– splenocytes (Fig. 5C). S-form LPS was clearlyless active, but its mitogenic activity for both cell typeswas comparable and not enhanced by LBP (Fig. 5C),suggesting that B cells do not express mCD14. For thisreason we compared mCD14 expression on B cells of WTand CD14–/– mice by FACS analysis using two differentCD14-specific antibodies. CD14 was, as expected, absentfrom CD14–/– cells, but also absent from WT cells,showing that splenic B cells do not express this receptor(see Supplementary Fig. 5 online). Addition of solubleCD14 up to 0.5 lg/mL alone or in combination with LBP(up to 1 lg/mL) had no influence on the activity ofeither form of LPS (data not shown). However, additionof soluble CD14 in pathophysiological concentrations

Figure 5. S-form LPS requires CD14 and LBP for activation of macrophages. Wild-type (A) and CD14–/– (B) bone marrow-derivedmacrophages (106 cells/mL) were stimulated for 20 h with the indicated concentrations (ng/well 0.2 mL) of S. minnesota R-form(595) LPS (upper panels) or S. abortus equi S-form LPS (lower panels) in the absence (closed symbols) or presence (open symbols) ofLBP (0.3 lg/mL). IL-6 protein levels in the supernatant were assessed by ELISA. Each point is themean of triplicates� SD. (C)Wild-type (left panels) and CD14–/– (right panels) splenocytes (106 cells/mL) were stimulated for 20 h with the indicated concentrations(lg/well 0.2 mL) of S. minnesota R-form (595) LPS (upper panels) or S. abortus equi S-form LPS (lower panels) in the absence (closedsymbols) or presence (open symbols) of LBP (0.3 lg/mL). Mitogenic activity was measured by incorporation of [3H]thymidine andscintillation counting. Each point is the mean of triplicates � SD.

Figure 4. Pathophysiological levels of sCD14 support MCstimulation by S-form LPS. Wild-type MC (106 cells/mL) wereleft unstimulated or stimulated with the indicated amounts ofRe-form (S. minnesota R595) LPS, or S-form LPS from S. abortusequi for 20 h in the presence or absence of recombinant CD14(4 lg/mL). Subsequently, IL-6 protein levels in the supernatantwere assessed by ELISA. Each bar is the mean of triplicates �SD. Note that the used concentrations of CD14 are pathophy-siological concentrations as present in mouse plasma [37].



(4 lg/mL, [37]) enhanced the activity of both LPS-chemotypes (data not shown).

R-form fraction isolated from S-form LPSis a potent activator of MC, macrophages andsplenocytes

As shown in Fig. 1B, S. abortus equi S-form LPS (like allS-form LPS) is a heterogenous mixture of true S- and R-form molecules. Here, we isolated R-form fraction fromS. abortus equi LPS and compared its activity to that ofparental LPS in MC, macrophages and splenocytes. InMC, the parental S-form LPS induced no detectable IL-6,while the R-form fraction isolated from the same LPSpreparation induced a strong IL-6 secretion (Fig. 6A).This suggests strongly that the rudimentary activity of S-form LPS preparations observed occasionally in MCexperiments is due to the portion of R-form LPS theycontain. The R-form fraction was also considerably moreactive than the parental LPS in inducing IL-6 productionin macrophages (Fig. 6B) and mitogenic responses insplenocytes (Fig. 6C).

R-form LPS is the only appreciable MC stimulusin gram-negative bacteria

We investigated if the differences in stimulatory activityfor MC observed between S- and R-form LPS were alsotrue for the respective S- and R-form parent bacteria.Using killed S. minnesota S- and Re-form bacteria forstimulation of WT MC, we show that the two types ofbacteria exhibited differences in the induction of IL-6response that were very comparable to those observedbetween the respective isolated S- and R-form LPS(Fig. 6D). Gram-negative bacteria contain, in addition toLPS, a number of highly conserved constituents, such aslipopeptides, peptidoglycan or unmethylated CpG DNA,which, after their isolation, also activate innate immunecells, although with a weaker potency. To evaluate therelative contribution of such components to theactivation of MC with bacteria, we investigated theactivity of Re- and S-form bacteria for MC generatedfrom TLR4-deficient mice. As shown in Fig. 6D, TLR4-deficient MC, in contrast to WT cells, exhibited nodetectable IL-6 secretion when stimulated with eitherform (Re or S) of bacteria. Thus, in the activation of MCby whole gram-negative bacteria, R-form LPS is thedominant stimulus, while S-form LPS and otherconstituents like lipopeptides, peptidoglycan or DNAmake no appreciable contribution.

R-form LPS induces higher levels of TNF-a thanS-form LPS in vivo

Here we analyzed the in vivoTNF-a responses of WTandCD14-deficient mice to Re- and S-form LPS after i.v.injection. R-form LPS induced a strong dose-dependentTNF-a response in WT and a lower one in CD14–/– mice(Fig. 7). In contrast, S-form LPS induced no detectable

Figure 6. (A–C) R-form LPS constitutes the active fraction ofWTS-form LPS. (A) MC (106/mL) or (B) macrophages (5 � 105/mL)were stimulated with the original S-form LPS of S. abortus equior the R-form fraction isolated thereof. IL-6 was determined inculture supernatants after 20 h of stimulation. Eachpoint is themean of triplicates � SD. n.d, not detectable. (C) Splenocytes(2 � 106/mL) were stimulated with original S-form LPS of S.abortus equi or the R-form fraction isolated thereof for 72 h.Mitogenic activity was measured by [3H]thymidine incorpora-tion. Each point is the mean of triplicates � SD. (D) R-form LPSis the dominant MC stimulus in gram-negative bacteria. MC(106/mL) from Sn and ScN mice were stimulated with killed S-and Re-form of S. minnesota bacteria (25 lg/mL). IL-6 wasdetermined in supernatants after 4.5 h of culture. Each bar isthe mean of triplicates � SD.

Figure 7. S-form in comparison to R-form LPS is highly CD14dependent and a less potent inducer of TNF-a secretion in vivo.Wild-type (left panel) and CD14–/– (right panel) mice wereinjected with the indicated amounts of S. minnesota Re-form(R595) LPS or S. abortus equi S-form LPS i.v. in 0.15 M glucosesolution (0.2 mL/20 g body weight) and TNF-a levels measured.The graph shows the cumulative data fromseven experiments.Each point represents the mean of two to eight mice � SD. InCD14–/– mice TNF-a levels in response to S-form LPS were notdetectable.



TNF-a response in CD14–/– mice and its TNF-a-inducingactivity in WT mice was lower compared to R-form LPS.This demonstrates that also in vivo the activationcapacity of R-form is superior to that of S-form LPSand supports the results obtained in our in vitro studies.

Discussion

The message of this study is that the S-form LPS, whichhas been considered for half a century now as theclassical form of endotoxically active LPS, activates anarrower spectrum of TLR4/MD-2-expressing cells thanR-form LPS and with a lower potency, both in vitro andin vivo. We show here that S-form LPS is practicallydevoid of stimulatory activity for MC, while R-form LPSis a potent activator. This difference is based on adifferential requirement for CD14 in the activation ofcells by the two types of LPS. While the S-form requiresCD14, R-form LPS can activate cells, regardless of thepresence or absence of this LPS-binding protein. Thisexplains why R-form LPS in contrast to S-form stronglystimulates MC, which we show here to lack CD14.Moreover, R-form LPS induces higher TNF-a responsesthan S-form in vivo, demonstrating that the differentactivation capacities of the two LPS chemotypes are alsopresent in vivo.

sCD14 provides help during activation of CD14-negative cells by LPS [17, 27, 45–49]. Interestingly, asshown here, in the case of murine cells high concentra-tions of sCD14 are required that are above the normalplasma levels [37]. Such concentrations were found in P.acnes-treated [37] or S. typhimurium-infected (unpub-lished results) mice. This suggests that the up-regulationof sCD14 in the course of an inflammatory response[37–41, 43, 44] ensures the contribution of MC andother CD14-negative LPS target cells to the antibacterialdefense. This mechanism, however, might provoke alsoan enhanced risk of endotoxin shock [50], or potentiateacute allergic reactions.

The present results also provide retrospectively anexplanation for why R-form LPS and free lipid A inducestrongly oxidative burst in human granulocytes, while S-form LPS is totally inactive in this respect [23]. In humangranulocytes, CD14 occurs intracellularly and is onlysporadically expressed on the cell surface [51]. Thus, innormal granulocytes, CD14, which is essential for theactivity of S-form LPS, is not readily available on the cellsurface.

A varying part of the LPS isolated from all S-formWTbacteria is of the R-form type. As shown in the presentstudy the R-form fraction isolated from S-form LPS is astrong, CD14-independent activator of MC and other celltypes. We propose, therefore, that the low activity of S-form LPS preparations for MC observed in this study,

and for MC and other cell types devoid of CD14elsewhere [26, 30, 52, 53], is due to the portion of R-form LPS they contain.

The presence of CD14 enhances the response of cellsto both LPS chemotypes. A recent study by Kim et al.[54] proposed that a large hydrophobic pocket found onthe N-terminal side of the CD14 structure is responsiblefor the binding of the lipid portion of LPS. The CD14-independent activation by R-form LPS suggests that it iseither capable of binding directly to the extracellularportion of TLR4/MD-2 or that it integrates into the cellmembrane and subsequently binds to the receptorcomplex. The difference in physicochemical propertiesbetween R- and S-form LPS may form the basis for thedifferences in LBP and CD14 requirement. It isconceivable that the lack of the long polysaccharidechains, which increases the hydrophobicity of R-formLPS, allows a better incorporation and mobility of theLPS in the mammalian cell membrane, thus providing abetter access to the signaling receptor. This would alsoexplain why the highly hydrophobic lipid A, which iscompletely devoid of core sugar constituents, is at leastas powerful an activator as R-form LPS. Alternatively,the observed difference between R- and S-form LPS inthe requirement for LPS-binding proteins might berelated to possible differences in the structure of theirlipid A moiety [55]. The information on the structureand biological activity of lipid A so far has been obtainedfrom studies on the lipid A of R-form LPS. Lipid A frompure genuine S-form LPS is not available yet.

MC are found in almost all connective tissues andthus frequently encounter gram-negative bacteria dur-ing infection. Since there is ample evidence that MC[56], TLR [57] and infectious agents [58, 59] play acrucial role in allergic, inflammatory and chronicdisorders, we expect that MC-derived cytokines suchas TNF-a and IL-6 induced by R-form LPS contribute tothe development of these pathologies. In contrast, thecytokine responses of MC to R-form LPS underphysiological conditions are very likely beneficial. Suchresponses are expected to occur at the interface of thehost with the environment, such as the mucosal surfacesof the respiratory, urogenital and gastrointestinal tract,where MC encounter commensal bacteria. Recently,evidence was presented for the requirement of com-mensals and corresponding TLR in the induction ofcytoprotective cytokines in the colon ofmice [60]. In thislocation, cytokines, including TNF-a and IL-6, play animportant role in the maintenance of epithelial home-ostasis and protection from injury. In the face of thepresent results, it is conceivable that MC and also othermCD14-negative cells contribute to this function byresponding to the R-form LPS of gram-negative bacteria.

In summary, this study demonstrates that R and SLPS chemotypes differ in their capacity to activate



immune cells. Under physiological conditions, the S-form is limited in its activating capacity to cells thatexpress mCD14, while the R-form has the potential tostimulate all cells that express TLR4/MD-2. Thesefindings have important implications for our under-standing of how cells are activated by LPS and open newperspectives for a more discriminative analysis of thesecomplex phenomena. Moreover, the amount and varietyof cells participating in the innate immune response togram-negative bacteria has likely been underestimatedso far.

Materials and methods

Reagents

S-form LPS from S. minnesota, S. abortus equi, and E. coli O8were extracted from parent bacteria and further purified asdescribed [61–63]. R-form LPS from S. minnesota mutants: R595 (Re), R3 (Rd), R7 (Rd), R5 (Rc), R345 (Rb) and R60 (Ra)were extracted and purified as described [62, 64]. Lipid A fromS. minnesota R595 was prepared as described [65]. Re-formLPS of E. coli, serotype 515 (liquid) and lipid A from E. coli,serotype 515 (liquid) were from Alexis Deutschland GmbH(Gr�nberg, Germany). S. minnesota (S-form) and S. minnesotamutant R 595 (Re-form) bacteria for stimulation of MC wereprepared as described [66]. The following mAb were used:commercial IgE with specificity for DNP (SPE-7; Sigma,Deisenhofen, Germany), anti-TLR4/MD-2 (clone MTS510;Alexis Deutschland GmbH), anti-RP105 (clone RP/14;eBioscience, San Diego, CA) and PE-conjugated anti-CD14antibody (clone rmC5–3; Pharmingen, San Diego, CA). Anti-CD14 (clone G5A10 [67]) was a gift from R.Landmann(Kantonspital, Department Forschung, Basel, Switzerland). Toanalyze the maturation of bone marrow-derived MC, PE-conjugated anti-c-kit (clone 2B8, Pharmingen) and FITC-conjugated anti-IgE antibody (clone 23G3, Southern Biotech-nology Associates Inc., Birmingham, AL)were used. Antibodiesto CD14, TLR4/MD-2 and RP105 were labeled using the Alexaflour 647 mAb labeling kit (Molecular Probes, Leiden, TheNetherlands) according to the manufacturer's instructions.Recombinant murine CD14 and LBP were purchased fromBiometec (Greifswald, Germany). Endotoxin content in theserecombinant protein fractions was determined to be less than10 pg LPS/1 lg protein using the Limulus test [68]. DNP-HSAcontaining 30–40 mol DNP/mol albumin was purchased fromSigma.

Mice

129/Sv, C57BL/6, CD14-deficient C57BL/6 [37, 69], C57BL/10ScSn (Sn), TLR4-deficient C57BL/10SnCr (Cr), TLR4-deficient C57BL/10ScN (ScN) and TLR4-transgenic C57BL/10SnCr (TCr-1) [25] mice were bred under specific pathogen-free conditions in the animal facilities of the Max-Planck-Institute for Immunobiology. Animals of both sexes, 6–8 weeksold, were used. The use of all experimental animals wasapproved by the Regierungspr�sidium-Freiburg (G-03/50 andT-04/24).

SDS-PAGE and silver staining of LPS

Analysis of LPS preparations was carried out by SDS-PAGE(12.5%) [70] followed by a silver staining technique [71].

Cytokine induction in MC

MC derived from bone marrow precursor cells of the variousmouse strains, were grown in the presence of 1% X63Ag8–653-conditioned medium as a source of IL-3 [72] as described [73].Differentiated, c-kit and FceR1-positive MC were preloadedovernight with IgE (0.2 lg/mL) and subsequently stimulatedin RPMI 1640 (106 cells/mL) in duplicates or triplicates withthe agents under test. Culture supernatants for IL-6 (and TNF-a) measurements were collected 3–20 h later as indicated.

IL-6 induction in macrophages

Cultured macrophages derived from bone marrow precursorcells were grown in the presence of L-cell-conditioned mediumin teflon bags as described previously [74]. After 10 days ofculture the cells were washed twice with a serum-free, high-glucose formulation of Dulbecco's modified Eagle medium(DMEM). For induction of IL-6, macrophages were resus-pended in serum-free DMEM (105 cells/0.2 mL/well), placedin 96-well plates (Nunc, Roskilde, Denmark) and cultured for24 h at 37�C in a humidified atmosphere containing 8% CO2.Thereafter macrophage supernatants were removed and freshDMEM (0.2 mL) added. The macrophages were then stimu-lated in triplicates with different amounts of LPS and culturesupernatants for IL-6 measurements were collected 24 h later.They were stored in aliquots at –80�C until use.

Determination of IL-6 and TNF-a

IL-6 levels in culture supernatants were estimated by ELISAusing the MP5–20F3 rat anti-mouse IL-6 antibody (PharMin-gen, San Diego, USA) as capturing antibody and theMP5–32C11 biotinylated rat anti-mouse IL-6 antibody (Phar-Mingen) as detection reagent for IL-6, according to theinstructions of the supplier. TNF-a levels in culture super-natants was measured in a cytotoxicity test using a TNF-sensitive L929 cell line of fibroblasts in the presence ofactinomycin D as described previously [75]. The detectionlimit of the assay was 3.2 pg TNF-a/mL supernatant and32 pg/mL plasma. Rabbit anti-mouse TNF-a (Genzyme,Boston, MA) was used as an inhibitor to test the specificityof the assay.

Degranulation assay

For degranulation studies, MCwere preloaded with 0.2 lg/mLIgE anti-DNP overnight. The cells were then washed andresuspended in Tyrode's buffer, adapted to 37�C for 30 min,and stimulated for 30 min at 37�C with the indicatedconcentrations of antigen (DNP-HSA). The degree of degra-nulation was determined by measuring the release of b-hexosaminidase [76].



Mitogenic response of splenocytes

Pooled single cell suspensions from three mice/strain wereprepared as described [25], in FCS-free DMEM. Triplicates ofcells (4 � 105 cells/0.2 mL serum-free DMEM per well) wereplaced in 96-well round-bottom plates, stimulated with LPSand [3H]thymidine incorporation measured as described [25].

FACS analysis

TLR4/MD-2 complex on cells was detected with Alexa 647-conjugated anti-TLR4/MD-2, CD14 with Alexa 647- or PE-conjugated anti-CD14, RP105 with Alexa 647-conjugated anti-RP105. Aminimum of ten thousand cells was acquired for eachsample. Nonspecific binding was blocked by incubation with10% normal mouse serum. Cells were analyzed on aFACSCalibur machine (Becton Dickinson, San Jose, CA).

Induction of TNF-a production in vivo

Mice were injected with different amounts of S. minnesota Re-form (R595) LPS or S. abortus equi S-form LPS i.v. in 0.15 Mglucose solution (0.2 mL/20 g body weight). Plasma wascollected 1 h later and the TNF-a levels detected using acytotoxicity test.

Acknowledgements: The authors thank Kerstin Gim-born, Hella St�big, Jasmin Ippisch and Nadja Goos fortheir excellent technical assistance. This study waspartly supported by the Landesstiftung Baden-W�rt-temberg P-LS-AL/3 (MH and MAF) and the DFG, SP"Angeborene Immunit�t" (FR 448/4–3) and by grantsfrom the US National Institutes of Health (AI050241).

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R-form LPS, the master key to the activation ofTLR4/MD-2-positive cells

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