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Keywords:

  • CD14;
  • Lipopolysaccharide;
  • Mast cells;
  • TLR4

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

Lipopolysaccharide (endotoxin, LPS) is a major recognition marker for the detection of gram-negative bacteria by the host and a powerful initiator of the inflammatory response to infection. Using S- and R-form LPS from wild-type and R-mutants of Salmonella and E. coli, we show that R-form LPS readily activates mouse cells expressing the 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 physiological conditions recruits a larger spectrum of cells in endotoxic reactions than S-form LPS. We also show that soluble CD14 at high concentrations enables CD14-negative cells to respond to S-form LPS. The presented in vitro data are corroborated by an in vivo study measuring TNF-α levels in response to injection of R- and S-form LPS in mice. Since the R-form LPS constitutes ubiquitously part of the total LPS present in all wild-type bacteria its contribution to the innate immune response and pathophysiology of infection is much higher than anticipated during the last half century.

Abbreviations:
LBP:

LPS-binding protein

MC:

mast cells

mCD14:

membrane CD14

MD-2:

myeloid differentiation protein 2, sCD14: soluble CD14

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

The interaction of highly conserved microbial constituents with the innate immune system forms the basis of recognition of and reaction against intruding pathogens in mammals. One such constituent is lipopolysaccharide (endotoxin, LPS), a major recognition marker common to gram-negative bacteria 13, a large group comprising important human pathogens as well as commensals. Thus, the interaction of LPS with cells of the innate immune system leads to the formation and release of endogenous mediators initiating inflammatory and immune responses essential for an antibacterial defense 3, 4. This primarily protective mechanism may become overshadowed by an acute pathophysiological response with the typical clinical symptoms of septic shock that frequently follows the release of inflammatory mediators, such as tumor necrosis factor (TNF)-α during infection 57.

Different cell types, such as macrophages, granulocytes and B cells, participate in the innate immune response to LPS 8. More recent data indicates that mast cells (MC) that are strategically located at the interface between host and environment also interact with LPS 9, and thus take part in the innate immune response. MC are primarily known for their deleterious effects in allergic reactions 10. Binding of allergen to its specific IgE on the surface of MC results in an immediate release of preformed pro-inflammatory mediators (e.g., histamine, TNF-α and proteases) from cytoplasmic granules (degranulation) 11 and a later release of de novo synthesized arachidonic acid metabolites and various cytokines like interleukin 6 (IL-6), TNF-α, and chemokines 10, 11. Unlike allergens, LPS induces no degranulation of MC, but like allergens, it stimulates the de novo synthesis and release of cytokines in these cells 9.

Activation of cells by LPS is mediated by the Toll-like receptor 4 (TLR4), a member of the highly conserved protein family of TLR, which are specialized in the recognition of microbial components. In mice, defects in TLR4 result in LPS unresponsiveness 12. For functional interaction with LPS, TLR4 requires association with myeloid differentiation protein 2 (MD-2) 13, 14. According to current consensus activation of TLR4 is preceded by the transfer of LPS to membrane-bound (m) or soluble (s) CD14 by LPS-binding protein (LBP) 1519. This mechanism is believed to be true for LPS signaling generally. However, in a recent study we showed that Re-form LPS and lipid A, but not S-form LPS, are capable of inducing TNF-α responses also in the absence of CD14 20.

LPS, synthesized by most wild-type (WT) gram-negative bacteria (S-form LPS), consists of three regions, the O-polysaccharide chain, which is made up of repeating oligosaccharide units, the core oligosaccharide and the lipid A (Fig. 1A), which harbors the endotoxic activity of the entire molecule 1, 2, 21. R-form LPS synthesized by the so-called rough (R) mutants of gram-negative bacteria lacks the O-specific chain. Furthermore, the core-oligosaccharide may be present in different degrees of completion, depending on the class (Ra to Re) to which the mutant belongs 1, 2, 21, 22 (Fig. 1A). Notably, LPS from WT bacteria are always highly heterogeneous mixtures of S-form LPS molecules containing 1 to over 50 repeating oligosaccharide units and contain ubiquitously a varying proportion of R-form molecules lacking the O-specific chain (Fig. 1B). Many clinically relevant gram-negative bacteria synthesize this type of LPS. Further, gram-negative WT bacteria, such as Chlamydia and Neisseria synthesize LPS in which the number of sugar residues is highly reduced, and thus resemble R-form LPS, at least in their physicochemical properties. LPS are amphipathic molecules whose hydrophobicity decreases with increasing length of the sugar part 1, 2, 21, 22. Based upon these differences, S- and R-form LPS show marked differences in the kinetics of their blood clearance and cellular uptake as well as in the ability to induce oxidative burst in human granulocytes 23 and to activate the host complement system 24.

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Figure 1. Schematic representation of the different LPS chemotypes. (A) LPS comprises the lipid A moiety, the core oligosaccharide, and the O-polysaccharide (O-antigen). Depending 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, purified and size-separated by SDS-PAGE. The arrowhead depicts the R-form contained in the S-form preparation.

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In the present study, using Salmonella and E. coli LPS preparations, we show that R-form LPS and free lipid A induce strong TLR4-dependent IL-6 and TNF-α responses in bone marrow-derived mouse MC, and that S-form LPS is in this respect virtually inactive. A comparison of the activity of R- and S-form LPS for CD14 competent and CD14 lacking cells revealed differences in activity that were cell type specific and based upon a differential requirement for CD14 and LBP by the two forms of LPS. The difference in stimulatory capacity between R- and S-form LPS was confirmed in an in vivo mouse model of TNF-α production.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

Activation of MC by LPS is primarily a property of 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-α in in vitro differentiated bone marrow-derived MC. As shown in Fig. 2A and D, R-form LPS (Re to Rc) and lipid A were potent inducers of IL-6 in MC, while, S-form LPS was in this respect practically inactive. R-form LPS with a more complete core (Rb and Ra) expressed intermediate activity (Fig. 2A). Similar results were obtained when the TNF-α-inducing activity of the LPS and free lipid A preparations was determined (Fig. 2B). MC therefore discriminate between the different LPS forms with which they can react and become activated.

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Figure 2. Re-form LPS is a potent MC activator. (A) MC (106/mL) were stimulated with 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 culture supernatants after 3 h of stimulation. (B) MC were stimulated with Re-LPS (1 μg/mL), S-LPS (10 μg/mL) or lipid A (1 μg/mL) from E. coli. TNF-α was determined in culture supernatants after 3 h of stimulation. (C) Expression of TLR4/MD-2 on MC from different mice strains was analyzed by FACS. Black-filled histograms show autofluorescence, gray histograms TLR4/MD-2-specific signal. (D) Anti-DNP IgE-loaded MC of Sn (1), ScN (2), Cr (3), and TCr-1 (4) mice were stimulated with Re- or S-form LPS of E. coli (1 μg/mL), or DNP-HSA (2 ng/mL), or a combination of DNP-HSA and LPS, or left unstimulated (-). IL-6 was determined in culture supernatants after 3 h of stimulation. In (A), (B) and (D) bars represent mean of duplicates ± SD. Comparable results were obtained in different experiments.

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

The IL-6 and TNF-α responses of MC to R-form LPS, like LPS responses generally, are mediated via TLR4/MD-2 signaling (Fig. 2C, D). Mouse MC, however, express relatively low levels of TLR4/MD-2 (Fig. 2C) compared to, for example, macrophages (see Supplementary Fig. 1 online). To investigate, whether an increase in TLR4/MD-2 expression on MC might improve the cytokine responses to S-form LPS, we used TLR4-rich MC derived from transgenic mice 25 (Fig. 2C, D). The higher TLR4/MD-2 expression correlated with a higher cytokine response of these cells to free lipid A (not shown) and Re-form LPS (Fig. 2D). However, it did not improve appreciably the poor response of the cells to S-form LPS (Fig. 2D).

Neither R- nor S-form LPS stimulated degranulation or augmented antigen-mediated degranulation of MC (see Supplementary Fig. 2 online). It has been shown that co-stimulation of MC with antigen and LPS leads to a synergistic increase in cytokine release 26. In our study R-, but not S-form LPS induced a TLR4-dependent enhancement of IL-6 (Fig. 2D) and TNF-α (not shown) induction by suboptimal antigen concentrations.

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

Transfer of LPS to CD14 via LBP and a physical association of CD14 with TLR4/MD-2, have been identified as necessary events in the activation of cells such as macrophages by LPS 15, 27, 28. The expression of CD14 on MC is still a matter of dispute 2934. To see if the difference observed between the capacity of S- and R-form LPS to activate MC might be related to a differential requirement for CD14 and LBP by the two LPS, we investigated the expression of mCD14 on MC by FACS analysis using cells from CD14–/– mice as negative control (Fig. 3A). Disruption of the CD14 gene impaired neither antigen-mediated degranulation nor IL-6 secretion in MC (Fig. 3B, C), showing that CD14 is not required for MC development in vitro. As shown in Fig. 3A, mCD14 was not detectable on murine MC. Further, treatment with LPS, which was shown to enhance mCD14 expression in macrophages 35, 36, also failed to induce detectable mCD14 expression on MC (see Supplementary Fig. 3 online). We then compared the IL-6 responses of MC from WT and CD14–/– mice to S- and R-form LPS and free lipid A. The results show that WT and CD14–/– cells exhibited comparable IL-6 responses to free lipid A and Re-LPS but no response to S-form LPS (Fig. 3D), and that addition of LBP to WT MC had practically no influence on these responses (Fig. 3E). According to previous reports, mCD14-negative cells, which were non-responsive to LPS gained LPS sensitivity after supplementation with soluble CD14 (sCD14) (reviewed in 17). Surprisingly, addition of sCD14 in concentrations that were shown to be physiological 37 alone or in combination with LBP, neither enhanced the activity of R-form LPS nor endowed S-form LPS with stimulatory activity (Fig. 3E). These results are interpreted as strong evidence that, in MC, cytokine induction by R-form LPS and lipid A is independent of CD14 under physiological conditions.

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Figure 3. Differential requirements for LBP and CD14 among different immune cells. (A) Expression of mCD14 on MC from WT and CD14–/– mice was analyzed by FACS. Black-filled histograms show autofluorescence, gray histograms CD14-specific signal. WT and CD14–/– MC expressed comparable amounts of FcϵR1 and c-kit on their surfaces (not shown). (B) Anti-DNP IgE-loaded WT (black bars) and CD14–/– MC (white bars) were stimulated with indicated amounts of DNP-HSA. After 30 min of stimulation β-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 was determined in culture supernatants after 3 h of stimulation. Similar results were obtained with independently generated MC. (D) WT and CD14–/– MC were stimulated with lipid A (2 μg/mL), Re-form or S-form LPS from S. minnesota. IL-6 was determined in culture supernatants after 3 h of stimulation. (E) MC were stimulated with Re-form or S-form LPS from S. minnesota in presence or absence of LBP (0.5 μg/mL), CD14 (0.5 μg/mL), or a combination of both. IL-6 was determined in culture supernatants after 3 h of stimulation. The used concentrations of CD14 and LBP represent physiological plasma levels in healthy mice 37. Bars in (B–E) represent mean of triplicates ± SD.

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Enhanced levels of sCD14 render MC responsive to S-form LPS

sCD14 levels are strongly up-regulated under inflammatory conditions in both humans and mice 3744. Therefore, we investigated also the effects of high concentrations of sCD14 (4 μg/mL recombinant sCD14) on the IL-6 production of MC in response to different amounts of Re-form and S-form LPS (Fig. 4). These high concentrations of sCD14 enhanced the IL-6 response of MC to R-form and enabled activation by S-form LPS.

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Figure 4. Pathophysiological levels of sCD14 support MC stimulation by S-form LPS. Wild-type MC (106 cells/mL) were left unstimulated or stimulated with the indicated amounts of Re-form (S. minnesota R595) LPS, or S-form LPS from S. abortus equi for 20 h in the presence or absence of recombinant CD14 (4 μg/mL). Subsequently, IL-6 protein levels in the supernatant were assessed by ELISA. Each bar is the mean of triplicates ± SD. Note that the used concentrations of CD14 are pathophysiological concentrations as present in mouse plasma 37.

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Requirement for LBP in the CD14 dependent activation of macrophages by LPS is dependent on the LPS chemotype

In contrast to MC, mouse bone marrow-derived macrophages express mCD14 (see Supplementary Fig. 4 online). Therefore, here we investigated the LPS-induced activation of macrophages focusing on possible differences in the requirement for LBP and CD14 by S-form and R-form LPS. Re-form LPS was an excellent stimulus for WT and CD14–/– cells. Supplementation with additional exogenous LBP had a moderate 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 less active than Re-form LPS in both cell types (Fig. 5A, B). Supplementation with additional LBP strongly enhanced the response of WT macrophages to S-form LPS (Fig. 5A), but had no effect on CD14–/– cells (Fig. 5B). Thus, in vitro, S-form LPS is highly dependent on the assistance of LBP and CD14 for optimal induction of IL-6 (Fig. 5A, B) and TNF-α (not shown) in macrophages, while Re-form LPS in this respect is clearly less dependent on the two LPS-binding proteins.

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Figure 5. S-form LPS requires CD14 and LBP for activation of macrophages. Wild-type (A) and CD14–/– (B) bone marrow-derived macrophages (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) of LBP (0.3 μg/mL). IL-6 protein levels in the supernatant were assessed by ELISA. Each point is the mean 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 (μg/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) of LBP (0.3 μg/mL). Mitogenic activity was measured by incorporation of [3H]thymidine and scintillation counting. Each point is the mean of triplicates ± SD.

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Role of LBP and CD14 in the activation of splenocytes by LPS

We compared the mitogenic responses of murine B lymphocytes to S- and Re-form LPS and the role of LBP and CD14 in these responses. Spleen cells from WT and CD14–/– mice were stimulated with each of the two forms of LPS in the presence or absence of murine recombinant LBP. Re-form LPS induced a high, LBP-independent mitogenic response in both WT and CD14–/– splenocytes (Fig. 5C). S-form LPS was clearly less active, but its mitogenic activity for both cell types was comparable and not enhanced by LBP (Fig. 5C), suggesting that B cells do not express mCD14. For this reason we compared mCD14 expression on B cells of WT and CD14–/– mice by FACS analysis using two different CD14-specific antibodies. CD14 was, as expected, absent from 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 soluble CD14 up to 0.5 μg/mL alone or in combination with LBP (up to 1 μg/mL) had no influence on the activity of either form of LPS (data not shown). However, addition of soluble CD14 in pathophysiological concentrations (4 μg/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 and splenocytes

As shown in Fig. 1B, S. abortus equi S-form LPS (like all S-form LPS) is a heterogenous mixture of true S- and R-form molecules. Here, we isolated R-form fraction from S. abortus equi LPS and compared its activity to that of parental LPS in MC, macrophages and splenocytes. In MC, the parental S-form LPS induced no detectable IL-6, while the R-form fraction isolated from the same LPS preparation induced a strong IL-6 secretion (Fig. 6A). This suggests strongly that the rudimentary activity of S-form LPS preparations observed occasionally in MC experiments is due to the portion of R-form LPS they contain. The R-form fraction was also considerably more active than the parental LPS in inducing IL-6 production in macrophages (Fig. 6B) and mitogenic responses in splenocytes (Fig. 6C).

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Figure 6. (A–C) R-form LPS constitutes the active fraction of WT S-form LPS. (A) MC (106/mL) or (B) macrophages (5 × 105/mL) were stimulated with the original S-form LPS of S. abortus equi or the R-form fraction isolated thereof. IL-6 was determined in culture supernatants after 20 h of stimulation. Each point is the mean 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 incorporation. Each point is the mean of triplicates ± SD. (D) R-form LPS is 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 μg/mL). IL-6 was determined in supernatants after 4.5 h of culture. Each bar is the mean of triplicates ± SD.

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

We investigated if the differences in stimulatory activity for MC observed between S- and R-form LPS were also true for the respective S- and R-form parent bacteria. Using killed S. minnesota S- and Re-form bacteria for stimulation of WT MC, we show that the two types of bacteria exhibited differences in the induction of IL-6 response that were very comparable to those observed between the respective isolated S- and R-form LPS (Fig. 6D). Gram-negative bacteria contain, in addition to LPS, a number of highly conserved constituents, such as lipopeptides, peptidoglycan or unmethylated CpG DNA, which, after their isolation, also activate innate immune cells, although with a weaker potency. To evaluate the relative contribution of such components to the activation of MC with bacteria, we investigated the activity of Re- and S-form bacteria for MC generated from TLR4-deficient mice. As shown in Fig. 6D, TLR4-deficient MC, in contrast to WT cells, exhibited no detectable IL-6 secretion when stimulated with either form (Re or S) of bacteria. Thus, in the activation of MC by whole gram-negative bacteria, R-form LPS is the dominant stimulus, while S-form LPS and other constituents like lipopeptides, peptidoglycan or DNA make no appreciable contribution.

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

Here we analyzed the in vivo TNF-α responses of WT and CD14-deficient mice to Re- and S-form LPS after i.v. injection. R-form LPS induced a strong dose-dependent TNF-α response in WT and a lower one in CD14–/– mice (Fig. 7). In contrast, S-form LPS induced no detectable TNF-α response in CD14–/– mice and its TNF-α-inducing activity in WT mice was lower compared to R-form LPS. This demonstrates that also in vivo the activation capacity of R-form is superior to that of S-form LPS and supports the results obtained in our in vitro studies.

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Figure 7. S-form in comparison to R-form LPS is highly CD14 dependent and a less potent inducer of TNF-α secretion in vivo. Wild-type (left panel) and CD14–/– (right panel) mice were injected with the indicated amounts of S. minnesota Re-form (R595) LPS or S. abortus equi S-form LPS i.v. in 0.15 M glucose solution (0.2 mL/20 g body weight) and TNF-α levels measured. The graph shows the cumulative data from seven experiments. Each point represents the mean of two to eight mice ± SD. In CD14–/– mice TNF-α levels in response to S-form LPS were not detectable.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

The message of this study is that the S-form LPS, which has been considered for half a century now as the classical form of endotoxically active LPS, activates a narrower spectrum of TLR4/MD-2-expressing cells than R-form LPS and with a lower potency, both in vitro and in vivo. We show here that S-form LPS is practically devoid of stimulatory activity for MC, while R-form LPS is a potent activator. This difference is based on a differential requirement for CD14 in the activation of cells by the two types of LPS. While the S-form requires CD14, R-form LPS can activate cells, regardless of the presence or absence of this LPS-binding protein. This explains why R-form LPS in contrast to S-form strongly stimulates MC, which we show here to lack CD14. Moreover, R-form LPS induces higher TNF-α responses than S-form in vivo, demonstrating that the different activation capacities of the two LPS chemotypes are also present in vivo.

sCD14 provides help during activation of CD14-negative cells by LPS 17, 27, 4549. Interestingly, as shown here, in the case of murine cells high concentrations of sCD14 are required that are above the normal plasma levels 37. Such concentrations were found in P. acnes-treated 37 or S. typhimurium-infected (unpublished results) mice. This suggests that the up-regulation of sCD14 in the course of an inflammatory response 3741, 43, 44 ensures the contribution of MC and other CD14-negative LPS target cells to the antibacterial defense. This mechanism, however, might provoke also an enhanced risk of endotoxin shock 50, or potentiate acute allergic reactions.

The present results also provide retrospectively an explanation for why R-form LPS and free lipid A induce strongly oxidative burst in human granulocytes, while S-form LPS is totally inactive in this respect 23. In human granulocytes, CD14 occurs intracellularly and is only sporadically expressed on the cell surface 51. Thus, in normal granulocytes, CD14, which is essential for the activity of S-form LPS, is not readily available on the cell surface.

A varying part of the LPS isolated from all S-form WT bacteria is of the R-form type. As shown in the present study the R-form fraction isolated from S-form LPS is a strong, CD14-independent activator of MC and other cell types. 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 CD14 elsewhere 26, 30, 52, 53, is due to the portion of R-form LPS they contain.

The presence of CD14 enhances the response of cells to both LPS chemotypes. A recent study by Kim et al. 54 proposed that a large hydrophobic pocket found on the N-terminal side of the CD14 structure is responsible for the binding of the lipid portion of LPS. The CD14-independent activation by R-form LPS suggests that it is either capable of binding directly to the extracellular portion of TLR4/MD-2 or that it integrates into the cell membrane and subsequently binds to the receptor complex. The difference in physicochemical properties between R- and S-form LPS may form the basis for the differences in LBP and CD14 requirement. It is conceivable that the lack of the long polysaccharide chains, which increases the hydrophobicity of R-form LPS, allows a better incorporation and mobility of the LPS in the mammalian cell membrane, thus providing a better access to the signaling receptor. This would also explain why the highly hydrophobic lipid A, which is completely devoid of core sugar constituents, is at least as powerful an activator as R-form LPS. Alternatively, the observed difference between R- and S-form LPS in the requirement for LPS-binding proteins might be related to possible differences in the structure of their lipid A moiety 55. The information on the structure and biological activity of lipid A so far has been obtained from studies on the lipid A of R-form LPS. Lipid A from pure genuine S-form LPS is not available yet.

MC are found in almost all connective tissues and thus frequently encounter gram-negative bacteria during infection. Since there is ample evidence that MC 56, TLR 57 and infectious agents 58, 59 play a crucial role in allergic, inflammatory and chronic disorders, we expect that MC-derived cytokines such as TNF-α and IL-6 induced by R-form LPS contribute to the development of these pathologies. In contrast, the cytokine responses of MC to R-form LPS under physiological conditions are very likely beneficial. Such responses are expected to occur at the interface of the host with the environment, such as the mucosal surfaces of the respiratory, urogenital and gastrointestinal tract, where MC encounter commensal bacteria. Recently, evidence was presented for the requirement of commensals and corresponding TLR in the induction of cytoprotective cytokines in the colon of mice 60. In this location, cytokines, including TNF-α and IL-6, play an important role in the maintenance of epithelial homeostasis and protection from injury. In the face of the present results, it is conceivable that MC and also other mCD14-negative cells contribute to this function by responding to the R-form LPS of gram-negative bacteria.

In summary, this study demonstrates that R and S LPS chemotypes differ in their capacity to activate immune cells. Under physiological conditions, the S-form is limited in its activating capacity to cells that express mCD14, while the R-form has the potential to stimulate all cells that express TLR4/MD-2. These findings have important implications for our understanding of how cells are activated by LPS and open new perspectives for a more discriminative analysis of these complex phenomena. Moreover, the amount and variety of cells participating in the innate immune response to gram-negative bacteria has likely been underestimated so far.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

Reagents

S-form LPS from S. minnesota, S. abortus equi, and E. coli O8 were extracted from parent bacteria and further purified as described 6163. R-form LPS from S. minnesota mutants: R 595 (Re), R3 (Rd), R7 (Rd), R5 (Rc), R345 (Rb) and R60 (Ra) were extracted and purified as described 62, 64. Lipid A from S. minnesota R595 was prepared as described 65. Re-form LPS of E. coli, serotype 515 (liquid) and lipid A from E. coli, serotype 515 (liquid) were from Alexis Deutschland GmbH (Grünberg, Germany). S. minnesota (S-form) and S. minnesota mutant R 595 (Re-form) bacteria for stimulation of MC were prepared 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-CD14 antibody (clone rmC5–3; Pharmingen, San Diego, CA). Anti-CD14 (clone G5A10 67) was a gift from R.Landmann (Kantonspital, Department Forschung, Basel, Switzerland). To analyze the maturation of bone marrow-derived MC, PE-conjugated anti-c-kit (clone 2B8, Pharmingen) and FITC-conjugated anti-IgE antibody (clone 23G3, Southern Biotechnology Associates Inc., Birmingham, AL) were used. Antibodies to CD14, TLR4/MD-2 and RP105 were labeled using the Alexa flour 647 mAb labeling kit (Molecular Probes, Leiden, The Netherlands) according to the manufacturer's instructions. Recombinant murine CD14 and LBP were purchased from Biometec (Greifswald, Germany). Endotoxin content in these recombinant protein fractions was determined to be less than 10 pg LPS/1 μg protein using the Limulus test 68. DNP-HSA containing 30–40 mol DNP/mol albumin was purchased from Sigma.

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 weeks old, were used. The use of all experimental animals was approved by the Regierungspräsidium-Freiburg (G-03/50 and T-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 various mouse 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 FcϵR1-positive MC were preloaded overnight with IgE (0.2 μg/mL) and subsequently stimulated in RPMI 1640 (106 cells/mL) in duplicates or triplicates with the agents under test. Culture supernatants for IL-6 (and TNF-α) measurements were collected 3–20 h later as indicated.

IL-6 induction in macrophages

Cultured macrophages derived from bone marrow precursor cells were grown in the presence of L-cell-conditioned medium in teflon bags as described previously 74. After 10 days of culture 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 resuspended in serum-free DMEM (105 cells/0.2 mL/well), placed in 96-well plates (Nunc, Roskilde, Denmark) and cultured for 24 h at 37°C in a humidified atmosphere containing 8% CO2. Thereafter macrophage supernatants were removed and fresh DMEM (0.2 mL) added. The macrophages were then stimulated in triplicates with different amounts of LPS and culture supernatants 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-α

IL-6 levels in culture supernatants were estimated by ELISA using the MP5–20F3 rat anti-mouse IL-6 antibody (PharMingen, San Diego, USA) as capturing antibody and the MP5–32C11 biotinylated rat anti-mouse IL-6 antibody (PharMingen) as detection reagent for IL-6, according to the instructions of the supplier. TNF-α levels in culture supernatants was measured in a cytotoxicity test using a TNF-sensitive L929 cell line of fibroblasts in the presence of actinomycin D as described previously 75. The detection limit of the assay was 3.2 pg TNF-α/mL supernatant and 32 pg/mL plasma. Rabbit anti-mouse TNF-α (Genzyme, Boston, MA) was used as an inhibitor to test the specificity of the assay.

Degranulation assay

For degranulation studies, MC were preloaded with 0.2 μg/mL IgE anti-DNP overnight. The cells were then washed and resuspended in Tyrode's buffer, adapted to 37°C for 30 min, and stimulated for 30 min at 37°C with the indicated concentrations of antigen (DNP-HSA). The degree of degranulation was determined by measuring the release of β-hexosaminidase 76.

Mitogenic response of splenocytes

Pooled single cell suspensions from three mice/strain were prepared as described 25, in FCS-free DMEM. Triplicates of cells (4 × 105 cells/0.2 mL serum-free DMEM per well) were placed in 96-well round-bottom plates, stimulated with LPS and [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. A minimum of ten thousand cells was acquired for each sample. Nonspecific binding was blocked by incubation with 10% normal mouse serum. Cells were analyzed on a FACSCalibur machine (Becton Dickinson, San Jose, CA).

Induction of TNF-α 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 M glucose solution (0.2 mL/20 g body weight). Plasma was collected 1 h later and the TNF-α levels detected using a cytotoxicity test.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

The authors thank Kerstin Gimborn, Hella Stübig, Jasmin Ippisch and Nadja Goos for their excellent technical assistance. This study was partly supported by the Landesstiftung Baden-Württemberg P-LS-AL/3 (MH and MAF) and the DFG, SP "Angeborene Immunität" (FR 448/4–3) and by grants from the US National Institutes of Health (AI050241).

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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

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