3 pre-B lymphocyte tumor cells

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Signal transduction triggered by lipid A-like molecules in 70Z/3 pre-B lymphocyte tumor cells Teresa A. Garrett, Meredith F.N. Rosser, Christian R.H. Raetz * Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA Received 23 September 1998; received in revised form 5 January 1999; accepted 5 January 1999

Abstract The lipid A (endotoxin) moiety of lipopolysaccharide (LPS) elicits rapid cellular responses from many cell types, including macrophages, lymphocytes, and monocytes. In CD14 transfected 70Z/3 pre-B lymphocyte tumor cells, these responses include activation of the MAP kinase homolog, p38, activation of NF-UB, and transcription of U light chains, leading to the assembly of surface IgM. In this work, we explored the specificity of the response with regard to lipid structure, and the requirement for p38 kinase activity prior to NF-UB activation in control and CD14 transfected 70Z/3 (CD14-70Z/3) cells. A p38-specific inhibitor, SB203580, was used to block p38 kinase activity in cells. CD14-70Z/3 cells were incubated with 1^50 WM SB203580, and then stimulated with LPS. Nuclear extracts were prepared, and NF-UB activation was measured using an electrophoretic mobility shift assay. SB203580 did not inhibit LPS induced NF-UB activation. In addition, LPS failed to activate p38 tyrosine phosphorylation in 70Z/3 cells lacking CD14, in spite of rapid NF-UB activation and robust surface IgM production with appropriate higher doses of LPS. LPS stimulation of p38 phosphorylation, NF-UB activation, and surface IgM expression were all blocked completely by lipid A-like endotoxin antagonists whether or not CD14 was present. Acidic glycerophospholipids and ceramides did not mimic lipid A-like molecules either as agonists or antagonists in this system. Our data support the hypothesis that lipid A-mediated activation of cells requires stimulation of a putative lipid A sensor that is downstream of CD14, but upstream of p38 and NF-UB. ß 1999 Published by Elsevier Science B.V. All rights reserved. Keywords: Endotoxin; Signal transduction; Lymphocyte tumor cell; Lipopolysaccharide; SB203580

1. Introduction Lipopolysaccharide (LPS) is the major glycolipid in the outer membrane of Gram-negative bacteria [1^ 5]. During severe Gram-negative infections, the lipid

* Corresponding author. Fax: +1-919-684-8885; E-mail: [email protected]

A moiety of LPS, also known as endotoxin, elicits rapid cellular responses from many cell types, including macrophages, lymphocytes, and monocytes. Cellular responses to lipid A include phagocytosis of bacteria, production of cytokines, such as tumor necrosis factor-K, interleukin-1 (IL-1) and IL-6, expression of surface IgM, and arachidonic acid release [6,7]. A full lipid A response leads to septic shock, an acute pathophysiological condition which can lead to organ failure, catastrophic drop in blood pressure, and death [8^10]. E¡ective treatments for lipid A-induced septic shock are not available, in

1388-1981 / 99 / $ ^ see front matter ß 1999 Published by Elsevier Science B.V. All rights reserved. PII: S 1 3 8 8 - 1 9 8 1 ( 9 9 ) 0 0 0 1 4 - 1

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part because the cellular signaling events induced by LPS are not fully understood. The mode of lipid A recognition and initiation of intracellular signaling by cells is complex. The plasma LPS binding protein (LBP) and CD14, a glycosylphosphatidylinositol-anchored membrane protein, are utilized by certain responsive cells to initiate intracellular signaling [11^13]. LBP binds the lipid A moiety of LPS and transfers it to cells. LBP can either discharge the LPS to CD14, or with CD14 can insert the LPS into lipid bilayers [14^18]. Cells that express CD14 are especially sensitive to LPS, often responding to 0.1^1 ng/ml LPS. The mouse pre-B lymphocyte cell line, 70Z/3, does not normally express CD14, and yet responds maximally to 100^1000 ng/ml ReLPS [19]. LPS may bind directly to cells and initiate signaling in the absence of LBP and CD14. This is consistent with the view that LBP and CD14 function primarily to facilitate the delivery of lipid A to a plasma membrane receptor. In both the LBP-dependent and -independent signaling model, the putative lipid A sensing protein is unknown in 70Z/3 cells. A reasonable possibility is a transmembrane receptor with an extracellular lipid A binding site which, upon lipid A binding, initiates intracellular signaling. Very recent genetic studies with other systems strongly suggest that the tolllike receptor proteins TLR2 [59,60] and/or TLR4 [61] might function as such lipid A receptors. Alternatively, LPS that has inserted itself into the plasma membrane might initiate intracellular signaling by causing membrane bilayer distortion, perhaps detected by a signaling protein bound to the inner surface of the plasma membrane [18,20]. LPS can also be taken up by endocytosis or be transported across the plasma membrane, thereby gaining access to possible intracellular targets [21]. One of the earliest responses to LPS is activation of the MAP kinase homolog p38 [22^30]. Within 5 min of LPS stimulation, p38 is phosphorylated on tyrosine 182 and threonine 180 [22^25]. p38 is also phosphorylated in response to many other stimuli, including osmotic stress, phorbol myristate acetate, epidermal growth factor, IL-1, ultraviolet radiation, and tumor necrosis factor-K [25]. A slightly later cellular response to LPS is the activation of nuclear factors NF-UB [31^34] and Oct-2 [62^64]. NF-UB mediates expression of many genes involved in the

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LPS response [31,32]. In 70Z/3 cells, the major NFUB transcription product is the U light chain [35]. Expression of U light chains allows for the assembly of IgM molecules which are expressed on the surface of cells. Surface IgM can be detected on 70Z/3 cells after about 12 h of LPS exposure using a £uorescent labeled antibody. The signi¢cance of p38 activation in the response of 70Z/3 cells to LPS is unclear. In 70Z/3 cells lacking CD14, robust NF-UB activation and surface IgM expression are detected after stimulation with 100^ 1000 ng/ml ReLPS [35,36], but such cells do not appear to activate much p38, as judged by using relatively insensitive anti-phosphotyrosine antibodies [19,23,36]. When transfected with CD14, however, these cells respond to much less LPS, and in addition to NF-UB activation and IgM production, signi¢cant p38 activation is also observed [19,23,36]. These ¢ndings suggest that p38 activation is not needed to achieve NF-UB activation in the 70Z/3 system, consistent with the data of Beyaert et al. in TNF activated L929 cells [65]. In this work, we have characterized the role of p38 in the cellular responses of 70Z/3 and CD14-70Z/3 cells to LPS by using a more sensitive anti-phosphop38 antibody than was available in previous studies [19,23,36]. In addition, we utilized the speci¢c p38 inhibitor, SB203580 [37], to determine if p38 activity is required for activation of NF-UB in response to LPS. Our ¢ndings show that p38 is not activated in the absence of CD14, despite full activation of NFUB and IgM production at appropriate doses of LPS. The absence of p38 activation in 70Z/3 cells lacking CD14 is consistent with the inability of SB203580 to block NF-UB activation in CD14 transfected 70Z/3 cells. However, both p38 and NF-UB activation are blocked by the endotoxin antagonist B464-35-7, indicating that a common upstream lipid A `sensor' is required for both events. 2. Materials and methods 2.1. Materials SB203580 was provided by SmithKline Beecham [37]. The lipid A antagonist B464-35-7 was a gift of Dr. W. Christ, Eisai Research Institute, Andover,

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MA. Fetal bovine serum was from Hyclone, and anti-p38 and anti-phospho-p38 antibodies were from New England Biolabs. 70Z/3 cells were provided by Carol Sibley, and CD14 transfected 70Z/3 cells (CD14-70Z/3) were provided by Theo N. Kirkland [35,38]. Salmonella minnesota R595 (ReLPS) was from List Biologicals. Acidic glycerophospholipids, C-16 ceramide, dihydro C-16 ceramide and C-6 ceramide were purchased from Sigma. RPMI 1640 medium with L-glutamine, penicillin, streptomycin, and Dulbecco's phosphate-bu¡ered saline (PBS) were from Gibco BRL. FITC-conjugated goat antimouse W chain antibody was purchased from Pierce. 2.2. Cell growth conditions 70Z/3 cells were grown in RPMI 1640 with L-glutamine supplemented with 10% Fetal bovine serum, 100 units/ml penicillin, 100 Wg/ml streptomycin, and 50 WM L-mercaptoethanol in a 5% CO2 atmosphere at 37³C. Cells were passaged by 10^20-fold dilution into fresh medium every 2^3 days. 2.3. Fluorescent activated cell sorting (FACS) analysis of surface IgM expression Cell activations by the indicated amounts of ReLPS, glycerophospholipids, and/or the antagonist B464-35-7 were carried out for 24 h at in 5% CO2 at 37³C with 6^15U105 cells at 2^5U105 cells per ml. Lipids were added to cells from a 1 mg/ml stock solution which was dispersed by sonic irradiation in sterile Dulbecco's PBS for 2 min just prior to use. Ceramide and ceramide analogs were dissolved in 95% ethanol prior to use, and diluted into growth medium so that the ¢nal ethanol concentration was less than 1%. Cells were harvested by centrifugation at 300Ug for 5 min, washed two times with complete growth medium, and incubated for 30 min at 4³C with a 1:50 dilution of FITC-conjugated goat antimouse W chain antibody, prepared according to the manufacturer's speci¢cations. Cells were washed two more times with complete medium and then ¢xed with 1% formaldehyde. Flow cytometry data were collected on a Becton Dickinson FACScan £uorescent activated cell sorter at the Duke Cell Sorter/ Flow Cytometry Laboratory. CellQuest software

was used to process the £ow cytometry data. Data is presented as the mean £uorescence of each sample divided by the mean £uorescence of the 10 Wg/ml control. 2.4. Analysis of NF-UB activation The indicated concentrations of ReLPS and/or B464-35-7 were added to a culture containing 1U107 cells at 4U105 cells/ml. After incubation at 37³C, in the presence of 5% CO2 for 30 min, cells were harvested, and nuclear extracts were prepared as described by Lawrence et al. [39]. Electrophoretic mobility shift assays (EMSAs) were performed according to Delude et al. [40]. Gels were dried, exposed to PhosphorImager screens, and quanti¢ed using Image Quant Software (Molecular Dynamics). 2.5. Detection of p38 by immunoblotting ReLPS and/or B464-35-7 was added to the cell suspension (3U106 cells/ml, 2 ml) in complete medium to the ¢nal concentrations, as indicated. The cells were incubated for 15 min at 37³C in the presence of 5% CO2 . Cells were harvested by centrifugation at 300Ug and resuspended in 135 Wl of ice-cold lysis bu¡er (1% Triton X-100, 20 mM Tris^HCl (pH 8.0), 137 mM NaCl, 50 mM NaF, 1 mM Na3 VO4 , 2 mM EDTA, 1 mM phenylmethanesulfonyl £uoride, 20 WM leupeptin, and 15 mU/ml aprotinin) [36]. Sample loading bu¡er was added (¢nal concentration of 50 mM Tris (pH 6.8), 2% SDS, 0.1 M DTT, 10% glycerol, 0.04% bromophenol blue), and each sample (180 Wl) was boiled for 5 min prior to loading 15 Wl onto a 12% SDS polyacrylamide gel. Mini-Protean gels (Bio-Rad) were electrophoresed in the Laemmli bu¡er system for 45 min at 200 V [41]. Proteins were transferred to Immobilon-P polyvinylidene £uoride (PVDF) membranes (Millipore) using a semi-dry transfer system (BioRad) for 40 min at 20 V with a Tris^glycine^methanol transfer bu¡er (25 mM Tris base, 192 mM glycine, 20% methanol). Membranes were probed with anti-p38 MAP kinase antibody or with anti-phospho-p38 MAP kinase antibody according to the manufacturer's recommendations (New England Biolabs).

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3. Results 3.1. 70Z/3 cell responses to ReLPS compared to other acidic glycerophospholipids and ceramides The 70Z/3 cell line is maximally responsive to signi¢cantly higher concentrations of ReLPS (100^1000 ng/ml) than are cells that express CD14 (0.1^1 ng/ ml). However, the maximal responses of both types of cells are the same. One possible explanation for this phenomenon is that 70Z/3 cells are not speci¢cally responding to ReLPS per se but rather to the presence of a high concentration of acidic phospholipids in the medium. To test this hypothesis, 70Z/3 cells were challenged with 10 Wg/ml of phosphatidylinositol (PI), phosphatidic acid (PA), lysophosphatidic acid (LPA), or cardiolipin (CL) for 24 h, and expression of IgM on the surface of the cells was analyzed by FACS analysis. As shown in Fig. 1A, only ReLPS was able to activate signi¢cant expression of surface IgM in 70Z/3 cells. Each lipid was also tested for its ability to antagonize ReLPS activation. None of the lipids were able to antagonize the surface IgM expression in response to ReLPS. It has been proposed that the lipid A moiety of LPS may stimulate cells by mimicking the proposed second messenger ceramide [42,43]. Numerous factors led to this conclusion including the fact that there is some similarity between ceramide and lipid

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Fig. 1. Acidic glycerophospholipids and ceramides neither stimulate IgM expression nor antagonize ReLPS activation of IgM expression in 70Z/3 cells. (A) Untransfected 70Z/3 cells were challenged with 10 Wg/ml of either ReLPS, phosphatidylinositol (PI), phosphatidic acid (PA), lysophosphatidic acid (LPA), or cardiolipin (CL) for 24 h, and then stained for surface IgM expression and analyzed as described in Section 2. Each acidic glycerophospholipid was also tested for its ability to antagonize ReLPS activation by challenging with 100 ng/ml ReLPS in the presence of 10 Wg/ml of each of the acidic glycerophospholipids. After 24 h, the cells were stained for surface IgM expression and analyzed as described above. (B) Untransfected 70Z/3 cells were challenged with 10 Wg/ml of either C-6 ceramide (C-6), C16 ceramide (C-16), or dihydro C-16 ceramide (dihydro C-16) as described for A. Because the ceramides were dissolved in 95% ethanol, ethanol (ETOH) at the same ¢nal concentration (1%) was also tested to be sure that it had no e¡ect on the cells. Each ceramide analog was further tested for its ability to antagonize ReLPS activation as in A.

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Fig. 2. Dose-dependent NF-UB activation and surface IgM production in 70Z/3 cells and CD14-70Z/3 cells. (A) 70Z/3 and CD14-70Z/ 3 were challenged with indicated amounts of ReLPS and analyzed for NF-UB activation as described in Section 2. (B) 70Z/3 and CD14-70Z/3 were challenged with indicated amounts of ReLPS and analyzed for surface IgM expression as in Fig. 1.

A at the molecular level [42,43]. However, the formal structural similarity is restricted to only one glucosamine unit of lipid A. Given that monosaccharides substructures of lipid A are poor activators of cells

[44], it may be more accurate to suggest that two molecules of ceramide mimic one molecule of lipid A. To test this, we asked whether ceramide and ceramide analogs are able to elicit cellular responses

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Fig. 3. ReLPS stimulation of tyrosine phosphorylation of p38 in CD14-70Z/3 cells but not in untransfected 70Z/3 cells. Each cell type was incubated either with or without 1 Wg/ml ReLPS for 15 min. Samples were then subjected to SDS^gel electrophoresis, and analyzed for phospho-p38 (upper blot) and total p38 (lower blot).

similar to ReLPS. 70Z/3 cells were treated with 10 Wg/ml C-16 ceramide, C-16 dihydroceramide, or the more cell permeable analog, C-6 ceramide. As is shown in Fig. 1B, none of these compounds were able to activate surface IgM production. Furthermore, the ceramide analogs were not able to potentiate or antagonize the ReLPS induced IgM response. These results suggest that the lipid A moiety of LPS induces a unique and speci¢c response, distinct from the proposed ceramide activated signaling pathway. We also quanti¢ed ReLPS activation of NF-UB and IgM expression in 70Z/3 and CD14-70Z/3 cells. Fig. 2 shows the dose-dependent activation of NFUB (panel A) after a 30 min ReLPS challenge, and IgM expression (panel B) after a 24 h ReLPS challenge. CD14-70Z/3 cells responded maximally to about 1^10 ng/ml ReLPS whereas 70Z/3 cells responded maximally to about 1000 ng/ml ReLPS. However, the maximum cellular response of each of these cell lines was very similar.

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15-min LPS stimulation. In contrast, when CD1470Z/3 cells were analyzed, a robust increase in tyrosine phosphorylation of p38 is detected [19,36] (Fig. 3). p38 tyrosine phosphorylation was detected with a commercially available antibody speci¢c for phosphorylation of tyrosine 182. The lower panel of Fig. 3 shows that the total amount of p38 (as detected by a polyclonal antibody reactive with normal p38) is not appreciably di¡erent in any of the samples. The activation of p38 in wild-type 70Z/3 cells is not delayed, as p38 phosphorylation did not increase after a 1-h stimulation with 1000 ng/ml ReLPS (data not shown). To analyze this phenomenon further, a dose^response curve for ReLPS activation of p38 tyrosine phosphorylation was performed. As seen in Fig. 4, CD14-70Z/3 cells are able to phosphorylate p38 when challenged for 15 min with only 1 ng/ml ReLPS. However, 70Z/3 cells that express no CD14 display no induction of p38 phosphorylation even with a 10 Wg/ml ReLPS challenge. Thus, 70Z/3 cells lacking CD14 are able to respond fully to ReLPS with activation of NF-UB and production of surface IgM (Fig. 2) despite lack of detectable p38 activation (Figs. 3 and 4). 3.3. Antagonism of ReLPS induced responses by B464-35-7 It has been observed that certain lipid A analogs, such as the biosynthetic precursor lipid IVA and the lipid A from Rhodobacter sphaeroides, do not cause activation of human cells [45^50]. Furthermore, if these compounds are added to cells treated with an agonist, such as Salmonella ReLPS, the endotoxinmediated cell stimulation of the cells is diminished or abolished in a dose-dependent manner [45^50]. These

3.2. 70Z/3 cells do not activate p38 in response to ReLPS Activation of p38 in response to ReLPS was analyzed in 70Z/3 and CD14-70Z/3 cells. Fig. 3 shows that even with 1000 ng/ml of ReLPS, a dose that causes maximal activation of NF-UB and full IgM production in wild-type 70Z/3 cells, no increase in p38 tyrosine phosphorylation was detected after a

Fig. 4. Maximum p38 activation occurs in CD14-70Z/3 cells at 1 ng/ml ReLPS but not at all in untransfected 70Z/3 cells. Each cell type was incubated with the indicated concentrations of ReLPS for 15 min. Samples were then processed as in Fig. 3 using the anti-phospho-p38 antibody.

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T.A. Garrett et al. / Biochimica et Biophysica Acta 1437 (1999) 246^256 Fig. 5. Antagonism of ReLPS activation of NF-UB and surface IgM expression in untransfected 70Z/3 cells by B464-35-7. (A) Cells were incubated with no ReLPS (Uninduced), 100 ng/ml ReLPS (LPS), 10 Wg/ml B464-35-7 (B464-35-7), or 100 ng/ml ReLPS plus 10 Wg/ml B464-35-7 (B464-35-7+LPS). After a 30min incubation cells were processed for NF-UB analysis as described in Section 2. (B) Cells were activated as in A, except that activation was carried out for 24 h and the cells were analyzed for surface IgM production.

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observations have led to the development of E5531 [49], a synthetic lipid A-like molecule modeled on the nontoxic lipid A of Rhodobacter capsulatus. E5531 is a potent antagonist of lipid A-triggered signal transduction both in mouse and human models [49]. The analog, B464-35-7, is exactly the same as E5531 with the exception that the C3 and C3P alkyl chains are 11 carbons instead of 10 carbons long. We used B464-35-7 as a model antagonist and analyzed its ability to block ReLPS activation of 70Z/3 and CD14-70Z/3 cell responses. Antagonist assays of NF-UB activation and surface IgM expression in 70Z/3 cells are shown in Fig. 5. At 10 Wg/ml, B464-35-7 was able to antagonize fully the activation of cells by 100 ng/ml ReLPS (lanes marked `B464-35-7+ReLPS'), while not causing cellular activation by itself (lanes marked `B464-35-7'). We also analyzed the ability of B464-35-7 to antagonize the activation of p38 phosphorylation in CD14-70Z/3 cells (Fig. 6). B464-35-7 alone did not activate p38 phosphorylation. When added to the cells in the presence of ReLPS, B464-35-7 was able to antagonize completely the LPS-induced activation of p38 tyrosine phosphorylation.

Fig. 6. Antagonism of ReLPS stimulated p38 tyrosine phosphorylation in CD14-70Z/3 cells by B464-35-7. CD14-70Z/3 cells were treated with the indicated concentrations of ReLPS and/or B464-35-7. After a 15-min incubation cells were analyzed for phospho-p38 by immunoblotting as described in Section 2.

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[29,51,52]. Uptake was also indicated by the cytotoxicity of SB203580 in cells exposed for prolonged periods of time (12^24 h) [29,51,52] (our results, data not shown). The results of Fig. 7 further support the view that NF-UB activation occurs independently of p38 activation. While p38 is most certainly activated in response to LPS in CD14 transfected 70Z/3 cells (Figs. 3 and 4), NF-UB activation is not dependent upon prior activation of p38. 4. Discussion

Fig. 7. No inhibition of NF-UB activation in ReLPS stimulated CD14-70Z/3 cells treated with SB203580. The structure of the p38 kinase inhibitor, SB203580, used in this study is shown in panel A. It is a member of a class of pyridinyl imidazoles which inhibit cytokine production in response to a number of cell stimuli [37,51,52]. In panel B, cells were incubated with the indicated concentrations of SB203580 (10 mM stock dissolved in ethanol) for 1 h at 37³C in 5% CO2 . The 0 WM sample has the maximum amount of ethanol added as a control (0.5%), which has no e¡ect. After this preincubation ReLPS was added (1 ng/ml). The cells were incubated for an additional 30 min at 37³C in 5% CO2 . Cells were then processed for analysis of NFUB activation as described in Section 2.

3.4. E¡ect of the p38 inhibitor, SB203580, on activation of NF-UB in CD-14 70Z/3 cells A p38 inhibitor, designated SB203580, has been reported [37]. This compound (Fig. 7) is a member of a class of pyridinyl imidazoles that inhibit ReLPS stimulated synthesis of cytokines in human monocytes by blocking p38 kinase activity [29,51,52]. We utilized this compound to probe the role of p38 kinase activity in the activation of NF-UB by ReLPS in CD14-70Z/3 cells. CD14-70Z/3 cells were pretreated for 1 h with the indicated amounts of inhibitor and then challenged with 1 ng/ml ReLPS for 30 min. As is shown in Fig. 7, the addition of SB203580 had no e¡ect on the activation of NF-UB by ReLPS. According to previous studies, the conditions of the experiment should lead to rapid uptake of the compound

In this work, we have characterized the responses of the mouse pre-B lymphocyte cell line, 70Z/3, to ReLPS and the lipid A antagonist B464-35-7. We have also investigated the responses of CD14 transfected 70Z/3 cells. Wild-type 70Z/3 cells robustly activate NF-UB and surface IgM expression in response to a 100^1000 ng/ml ReLPS challenge (Fig. 2). When transfected with CD14, cells are about 1000-fold more responsive (sensitive to 0.1^1 ng/ml), and in addition they activate the MAP kinase homolog, p38, by phosphorylation of tyrosine and threonine [22^25]. (Figs. 2^4). However, untransfected 70Z/3 cells do not activate p38 in response to ReLPS (Figs. 3 and 4). Using the SmithKline Beecham p38 inhibitor, SB203580, we have demonstrated that NF-UB activation, though temporally downstream of p38 activation, does not require p38 kinase activity (Fig. 7). This result suggests that LPS recognition initiates a complex set of parallel signaling events. The few signaling molecules that have been directly implicated in LPS-dependent signaling (p38, NF-UB) in this system do not appear to function in sequence, and do not all depend upon the presence of CD14. We have also shown that the cellular activation of both cell lines can be fully antagonized by the lipid A antagonist B464-35-7 (Figs. 5 and 6). This antagonist and its parent compound, E5531, hold great promise as new drugs for the treatment of the complications of Gram-negative sepsis. The observation that the lipid A antagonists block all cellular responses to LPS, whereas the p38 inhibitors do not, suggests that the former could be more e¡ective in the treatment of endotoxemia than the latter. In addition, the antagonism of lipid A cellular activation by this class

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of compounds is suggestive of a key lipid A recognition event in cells that initiates intracellular signal transduction. The structural speci¢city of the LPS response was further demonstrated in the experiments of Fig. 1. No naturally occurring acidic glycerophospholipid or ceramide analog was able to activate or antagonize surface IgM expression in 70Z/3 cells. The discovery of Rhodobacter lipid A as an antagonist of LPS activation suggests that other Gramnegative microorganisms or mutants may have lipid As that, like Rhodobacter, are nontoxic and antagonistic. Recently, lipid A from an Escherichia coli msbB mutant, which lacks a myristoyl moiety, was found to antagonize LPS-induced E-selectin expression in human endothelial cells [53]. Further understanding the biosynthesis of lipid A may enable the engineering of novel lipid A analogs in vivo. For instance, like Rhodobacter, Pseudomonas incorporates hydroxydecanoate at the 3 and 3P positions of its lipid A. The gene encoding LpxA, the acyltransferase responsible for incorporating those acyl chains, has recently been cloned from Pseudomonas aeruginosa [54]. Through heterologous expression of Pseudomonas lpxA in an E. coli mutant with a temperature sensitive lesion in its own chromosomal lpxA, Dotson et al. showed that a hybrid lipid A with hydroxydecanoate at the 3 and 3P positions was formed [54]. This lipid A has an acylation pattern similar (but not identical) to Rhodobacter capsulatus lipid A [49]. Although the lipid A from this strain has not yet been tested for its ability to agonize or antagonize animal cells, this clearly demonstrates the possibility of re-engineering E. coli lipid A in vivo. This approach might lead to the discovery of novel lipid A analogs that are even more potent antagonists than the Rhodobacter-based lipid As. Our ¢ndings further accentuate the need to identify the point at which the complex LPS triggered signaling cascade is initiated, i.e., the putative LPS signaling receptor. The presence of such a protein is supported by the picomolar potency of lipid A, the structural speci¢city of the response and the existence of lipid A nonresponsive mutants [55,61]. Unfortunately, this receptor has yet to be identi¢ed by direct binding assays, in part, because lipid A is too hydrophobic to be used in receptor binding assays. Recent

genetic studies have implicated TLR2 [59,60] and/or TLR4 [61] as lipid A signaling receptors. However, lipid A binding to TLR2 or TLR4 and their speci¢city for agonists versus antagonists [45^50, 66] remain to be demonstrated. Nevertheless, a model that invokes an extracellular lipid A receptor is appealing. Platelet activating factor, a di¡erent lipid signaling molecule, functions via an extracellular, G-protein linked receptor that has been expression cloned [56]. Likewise, lysophosphatidic acid and sphingosine 1-phosphate receptors have been cloned [57,58]. They are all members of the seven transmembrane receptor family with extracellular lipid binding domains [56^58]. However, the signals initiated by LPS are not occurring via any of these receptors. Lysophosphatidic acid (Fig. 1) and sphingosine 1-phosphate (data not shown) do not activate surface IgM expression in 70Z/3 cells, and conversely, LPS is not a potent agonist for the platelet activating factor receptor [67]. Alternative modes of LPS signaling are possible. It has been suggested that lipid A is inserted into the plasma membrane in a LBP catalyzed process [16^ 18]. This raises the possibility that recognition and signaling may initiate inside the bilayer, perhaps by a physical disruption of a lipid domain, or by a lateral interaction with the membrane spanning domain of a protein like TLR2 or TLR4. It has also recently been observed that FITC-LPS is transported to the inside of certain cells [21]. Once the ¢rst step of lipid A recognition and signal initiation is better de¢ned, it should be easier to elucidate the multiple pathways of lipid A-triggered signaling, which appear to be branched in the 70Z/3 system. Acknowledgements This research was supported by NIH Grant GM51796 to C.R.H.R. T.A.G. was supported by predoctoral fellowship DGE092-53851 from the National Science Foundation. M.F.N.R. was supported by a Cell and Molecular Biology Program NIH/NIGMS training grant 5 T32 GM07184-23. We would also like to thank Dr. W. Christ of Eisai and Dr. J.C. Lee of SmithKline Beecham for providing B464-357 and SB203580, respectively.

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