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The Toll-like receptor-4 (TLR4)4 (4) conveys LPS signals in monocytes (5, 6) .... New Haven, CT). LPS (E. coli 0111:B4), polymyxin B sulfate, N-acetyl- ..... receptor 1 (EDDR1, TrkE); JNK, c-Jun N-terminal kinase; LERK-3, ligand for EPH-.
Escherichia coli Braun Lipoprotein Induces a Lipopolysaccharide-Like Endotoxic Response from Primary Human Endothelial Cells1 Paul O. Neilsen,2* Guy A. Zimmerman,†‡ and Thomas M. McIntyre3*†‡ All bacteria contain proteins in which their amino-terminal cysteine residue is modified with N-acyl S-diacylglycerol functions, and peptides and proteins bearing this modification are immunomodulatory. The major outer membrane lipoprotein of Escherichia coli, the Braun lipoprotein (BLP), is the prototypical triacylated cysteinyl-modified protein. We find it is as active as LPS in stimulating human endothelial cells to an inflammatory phenotype, and a BLP-negative mutant of E. coli was less inflammatory than its parental strain. While the lipid modification was essential, the lipidated protein was more potent than a lipid-modified peptide. BLP associates with CD14, but this interaction, unlike that with LPS, was not required to elicit endothelial cell activation. BLP stimulated endothelial cell E-selectin surface expression, IL-6 secretion, and up-regulation of the same battery of cytokine mRNAs induced by LPS. Quantitative microarray analysis of 4400 genes showed the same 30 genes were induced by BLP and LPS, and that there was near complete concordance in the level of gene induction. We conclude that the lipid modification of at least one abundant Gram-negative protein is essential for endotoxic activity, but that the protein component also influences activity. The equivalent potency of BLP and LPS, and their complete concordance in the nature and extent of endothelial cell activation show that E. coli endotoxic activity is not due to just LPS. The major outer membrane protein of E. coli is a fully active endotoxic agonist for endothelial cells. The Journal of Immunology, 2001, 167: 5231–5239.

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he lipid A moiety of the LPS of Gram-negative bacteria is a powerful agonist for cells with appropriate receptors. One component of the LPS recognition system, CD14, may occur as a circulating, soluble component of the receptor signaling complex, or as a membrane-bound form that is localized to this environment by its phosphatidylinositol-linked tail. A circulating accessory protein, LPS-binding protein, enhances sensitivity of cells like endothelial cells to low concentrations of LPS (1–3). The Toll-like receptor-4 (TLR4)4 (4) conveys LPS signals in monocytes (5, 6), endothelial cells (7), or when transfected into epithelial cell lines (8, 9), and TLR4-deficient mice, in contrast to TLR2-deficient mice, are unable to mount a vigorous response to LPS (10). A second class of inflammatory bacterial endotoxins is the cell wall lipoproteins of Gram-positive bacteria, Gram-negative bacteria, and mollicutes (11), including mycoplasmas (12, 13). Pro-

Departments of *Pathology and †Medicine, and ‡Program in Human Molecular Biology and Genetics, University of Utah, Salt Lake City, UT 84112 Received for publication April 14, 2000. Accepted for publication August 27, 2001. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Institutes of Health Specialized Center of Research in Adult Respiratory Distress Syndrome (P50 HL50153), HL 44525, and the American Lung Association Asthma Research Center. The University of Utah Flow facility and the Microarray facility were supported by Cancer Center Support Grant P30 CA42014. 2 Current address: Echelon Research Laboratories, 420 Chipeta Way, Suite 180, Salt Lake City, UT 84108. 3 Address correspondence and reprint requests to Dr. Thomas M. McIntyre, 4130 Eccles Institute of Human Genetics, University of Utah, 15 North 2030 East, Salt Lake City, UT 84112-5330. E-mail address: [email protected] 4 Abbreviations used in this paper: TLR, Toll-like receptor; BLP, Braun lipoprotein; P3CSK4, S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-(S)Lys4; P3Cys, S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys; PMN, polymorphonuclear; sCD14, soluble CD14.

Copyright © 2001 by The American Association of Immunologists

cessed mature bacterial lipoproteins have no shared amino acid homology, but rather all share a lipid-modified N-terminal cysteine residue. This modified residue has a thioether-linked diglyceride, and Gram-negative and positive bacteria have an additional Nterminal amide-linked fatty acid that is missing in the lipoproteins isolated from mycoplasma (14). As with LPS, the lipid portion of bacterial lipoproteins is essential for their immune and inflammatory activities (12, 15–17). The prototypic Gram-negative bacterial lipoprotein (Braun lipoprotein (BLP)) was purified and characterized from Escherichia coli over 30 years ago (18). This major outer membrane protein stimulates B cell growth (15), and short synthetic lipid-modified peptides based on its sequence activate monocytes (19), neutrophils (20), and platelets (21). These lipopeptides are, however, less potent than their parent lipoprotein (22–24). Endothelial cells respond to live Gram-positive spirochetes or sonicated Borellia burgdorferi (25, 26), B. burgdorferi lipoproteins (27–29), and lipopeptides (23). TLR2 confers sensitivity to bacterial lipoproteins (30 –35), and TLR2-deficient cells are insensitive to Gram-positive bacterial products (10, 33). Recognition of endotoxic proteins and cell wall components by TLR2, like LPS recognition by TLR4, is enhanced by CD14 (30, 31, 36, 37), but this requirement is not absolute, as CD14-deficient mice have an unaltered response to Gram-positive organisms (38). Identification of TLR2 and TLR4, rather than CD14, as the specificity-conferring components of the signaling system (33) indicates that the response to endotoxic proteins may not be identical to the response to LPS. Moreover, it is as yet unknown whether all endotoxic proteins act in the same way, or through the same receptors. In this study, we report that primary cultures of human endothelial cells responded to exceedingly low concentrations of E. coli BLP, and that this protein contributes to their response to these bacteria. Endothelial cell activation by BLP and the synthetic lipopeptide, unlike LPS, need not depend on soluble CD14 (sCD14) presentation, but ultimately the response of the cells to BLP and LPS was 0022-1767/01/$02.00

5232 almost identical despite unrelated structures and, potentially, divergent receptors. The major outer membrane protein of E. coli is an effective endotoxic agonist for endothelial cells.

Materials and Methods Reagents and their sources were as follows: S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-Cys (P3Cys) (1) and S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys4 (P3CSK4), Boehringer Mannheim (Indianapolis, IN); HBSS, BioWhittaker (Walkersville, MD); human serum albumin, Baxter Healthcare (Glendale, CA); TNF-␣, IL-1␣, and anti-E-selectin Ab (BBIG-E4), R&D Systems (Minneapolis, MN); SB203580 and PD98,059, Alexis Biochemicals (San Diego, CA); genestein, Biomol (Plymouth Meeting, PA); secondary Abs, Biosource International (Camarillo, CA). E. coli strains K-12 and JE5505 were generously provided by the E. coli Genetic Stock Center (Yale University, New Haven, CT). LPS (E. coli 0111:B4), polymyxin B sulfate, N-acetylLeu-Leu-norleucinyl, and all other chemicals, unless otherwise noted, were from Sigma (St. Louis, MO). Recombinant human bacterial permeabilityincreasing factor was a generous gift of K. Huang (XOMA, Berkeley, CA). Human rsCD14 and anti-CD14 for Western blotting were obtained from Biometec (Greifswald, Germany; www.biometec.de).

Endothelial cell culture, neutrophil isolation, and adhesion assays Primary cultures of HUVECs and [111In]oxine-labeled neutrophils used in adhesion assays were as previously described (39). Briefly, 12- or 24-well plates (Costar Data Packaging, Cambridge, MA) of primary, 24-h postconfluent HUVEC monolayers were gently washed one to three times with warm HBSS containing 0.5% human albumin (HBSS/A). Agonists and their controls in HBSS/A were incubated with the cells for 4 h, the media removed (and stored at ⫺20°C for cytokine ELISA), and the cells washed once with HBSS/A. 111In-labeled neutrophils (0.25 ml 5.5 ⫻ 106/ml) were added to the monolayers for 5 min before unbound and loosely adherent neutrophils were collected in two washes and combined to calculate the percentage of tightly bound neutrophils by gamma counting (40). We tested the effects of signaling inhibitors by diluting them 1000-fold from DMSO stock solutions into warm M199 or HBSS/A. The monolayers were preincubated for 1 h at a final concentration of N-acetyl-Leu-Leu-norleucinyl (200 ␮M), genestein (1 ␮M), SB203580 (10 ␮M), and PD98,059 (10 ␮M) before the addition of LPS (100 ng/ml), BLP (100 ng/ml), or P3CSK4 (10 ␮g/ml) for 4 h.

BLP purification E. coli (DH5-␣) were grown in Luira Broth, pelleted, and stored frozen; alternatively, lyophilized cells (strain B) were purchased from Sigma. We used Inouye’s procedure for purification of the free form of murein lipoprotein from E. coli (41). Briefly, bacteria were resuspended in 1 ml S-buffer (10 mM sodium phosphate, pH 7.5, 5 mM EDTA) with 1 mM PMSF per gram wet bacteria and lysed on ice by sonication. Unbroken cells were removed (1000 ⫻ g, 15 min, 4°C); cell membranes were collected (40,000 ⫻ g, 40 min, 4°C) and resuspended in S-buffer; and then SDS was added to 4% and 2-ME to 0.5%. This was boiled for 30 min, stirred overnight at room temperature, and then centrifuged (50,000 ⫻ g, 30 min, 23°C) before collecting the supernatant. Contaminating proteins were precipitated at low pH and 5% acetone before BLP was collected as a precipitate from 30% acetone. It was renatured using 1% SDS. To remove LPS from the BLP, we extracted with phenol and the BLP was then recovered from the phenol phase by precipitation with 30% acetone. We repeated the acetone fractionation several times, and after the third or fourth acetone fractionation, one major band was observed on a silverstained polyacrylamide gel. The protein concentration was determined using the bicinchoninic acid protein assay kit (Pierce, Rockford, IL). The LPS content of our purified BLP at the dilutions we employed was less than 0.5 EU/␮g BLP, as determined by the Limulus Amebocyte Lysate assay (QCL1000; BioWhittaker). Each preparation of BLP was tested at submaximal levels of HUVEC stimulation with or without polymyxin B sulfate to bind and inactivate LPS (see Fig. 3). The effect of saponification on BLP or LPS was determined by adding 6 N KOH (50 ␮l) in methanol to 25 ␮g (in 50 ␮l) material. This material was stirred for 2 h at 50 – 60°C before neutralizing with HCl and adding to endothelial cells to quantitate polymorphonuclear (PMN) adhesion. Saponification in aqueous solution gave similar results.

ENDOTHELIAL CELL ACTIVATION BY E. coli LIPOPROTEIN ELISA, flow cytometry, CD14 mobility gel shift, and Western blotting IL-6 was quantified by sandwich ELISA using polyclonal capture, biotinylated detection mAb, and human rIL-6 as a standard (R&D Systems). Flow cytometry for E-selectin on endothelial cells was previously described (42). Analysis of CD14 interaction with BLP was examined through changes in CD14 mobility during nondenaturing PAGE. For this, 2 ␮g (20 ␮l 0.2 mg/ml stock in PBS) BLP or LPS (S. minnesota R595; List Biological Laboratories, Camerillo, CA) was incubated overnight at room temperature with 0.4 ␮g human rCD14 (Biometec, Greifswald, Germany). The complexes were resolved by electrophoresis on a native gradient 4 –20% mini-gel (Bio-Rad, Richmond, CA), and transferred to a polyvinylidene difluoride membrane. This was then probed with mouse antiCD14 mAb, (biG2; Biometec) and goat anti-mouse HRP before visualization with ECL (Amersham, Piscataway, NJ). For p38 activity, primary HUVEC in 12-well plates were washed twice with HBSS/A, and agonists in M199 were added for the stated times. The media were decanted, the cells washed once with cold media, and then 100 ␮l ice-cold lysis buffer (50 mM Tris, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM MgCl2, 20 mM NaF, 0.1% Triton X-100, 1 mM PMSF, 1 mM Na3VO4, 10 ␮g/ml leupeptin, and 10 ␮g/ml aprotinin) was added to the wells. Cells were scraped from the plate and then disrupted by passing 5–10 times through a 25gauge needle. We resolved the cellular proteins (50 ␮g protein per lane) by SDS-PAGE in a 9% gel and then transferred them to Immobilon (Millipore, Bedford, MA). p38 was detected with anti-phospho-p38 Ab (New England Biolabs, Beverly, MA) and visualized by ECL. Subsequently, the membrane was stripped (0.5% NaOH, 1% 2-ME, 50°C, 2 h), then reprobed with anti-p38 Ab (Santa Cruz Biotechnology, Santa Cruz, CA).

Array analysis of induced genes An Atlas human cDNA expression array (Clontech Laboratories, Palo Alto, CA) was used, according to the manufacturer’s instructions, to compare mRNA stimulated by endothelial treatment by BLP and LPS. For the quantitative microarray, two P150s of confluent HUVEC in serum-containing media were treated for 3 h with 100 ng/ml LPS (K-12-LCD25; List Biological Laboratories), PBS control, 200 ng/ml purified BLP, or SDS control. Plates without LPS contained 10 ␮g/ml polymyxin B. As a control, endothelial cells from the same culture were treated with agonists, and the bioactivity was assayed by PMN adhesion. Total RNA was isolated using TRIzol, according to the manufacturer’s instructions, and mRNA was purified with an Oligotex kit (Qiagen, Valencia, CA). mRNA was reverse transcribed and labeled with either Cy3 or Cy5 fluorescent probes before array and hybridization on a microchip, according to established protocols at the Microarray facility at the Huntsman Cancer Institute (University of Utah). The chip (chip A) employed was arrayed by the facility from 4610 supplied clones, human expression sequences tagged, and positive and negative controls, and so provides a cross section of expressed human genes.

Results E. coli bacterial lipoprotein activates endothelial-dependent leukocyte adhesion We treated primary cultures of human endothelial cells with various concentrations of purified E. coli BLP for 4 h and then determined whether these cells displayed an activated phenotype by quantifying leukocyte adhesion. We found (Fig. 1A) that bacterial lipoprotein stimulated endothelial cells to bind quiescent leukocytes in a concentration-dependent fashion. The half-maximal concentration for this was approximately 10 pM, and had achieved maximal endothelial cell activation by 1 nM, in which two-thirds of the added leukocytes were bound to the activated monolayer. We compared endothelial cell activation by BLP with a synthetic peptide bearing the tripalmitoyl-modified cysteinyl residue (P3CSK4), and found that the intact lipoprotein was approximately 100-fold more potent than the lipopeptide (Fig. 1A). Neither BLP nor P3CSK4 directly stimulated neutrophil adhesion over this short time in the absence of endothelial cells, so leukocyte adhesion reflects endothelial cell activation. The lipid-modified amino acid itself was inactive (Fig. 1A), but this lipid modification was necessary, as saponification to remove the esterified fatty acyl residues inactivated BLP, P3CSK4, and the positive control LPS (Fig. 1B).

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5233 nM was a powerful agonist for the adhesive phenotype of endothelial cells, but a poor one for IL-6 expression. We next varied the time that the endothelial cells were exposed to BLP and P3CSK4 to find no immediate change after exposure to either agent. However, there was a subsequent rapid increase in adhesion starting by 1–2 h of exposure that reached a maximum by 150 –180 min of exposure (Fig. 2A). This time relationship is characteristic of E-selectin-mediated adhesion, which requires the de novo synthesis of both message and protein, and indeed we found that the P3CSK4 (Fig. 2B) and BLP (Fig. 2C) induced surface

FIGURE 1. BLP is a potent endothelial cell agonist. A, Endothelial celldependent PMN adhesion in relation to agonist concentration. HUVECs were isolated, grown to confluence, and 24 h later were treated for 4 h with the stated concentration of BLP, a pentapeptide bearing the lipid-modified cysteinyl residue (P3CSK4), or just lipid-modified cysteine. The calculated molecular mass of BLP, P3CSK4, or P3Cys is 6670, 1620, and 931 Da, so that 1 nM BLP, P3CSK4, or P3Cys is 6.7, 1.6, or 0.9 ng/ml. These agents were removed and the monolayers were washed before freshly isolated, unactivated human neutrophils (PMN) that had been labeled with 111In were introduced. After 5 min, loosely and unbound PMN were removed and the number of adherent cells were calculated by gamma counting. B, Effect of saponification on the bioactivity of BLP, the lipopeptide, and LPS. The lipid agonists were deacylated with base, as described in Materials and Methods, and reassayed for their ability to induce endothelial cell-dependent PMN adhesion as in A. C, IL-6 production by HUVEC in response to BLP or the lipopeptide. Supernatants were collected from HUVEC treated as above, and IL-6 content was determined by ELISA.

Thus, this posttranscriptional lipid modification of the major lipoprotein of E. coli is essential for its activity, but the protein component itself also contributes greatly to its activity. We examined a second inflammatory response of activated endothelial cells, IL-6 synthesis and secretion, to find that this response was also stimulated by BLP in a concentration-dependent fashion (Fig. 1C). Again, we found that the lipopeptide was less potent and less active than the intact lipoprotein, and that the modified cysteine alone was inactive (not shown). We observed that the concentration response profiles for leukocyte adhesion and IL-6 synthesis were identical when the lipopeptide was the agonist, but not when the endothelial cells were stimulated with BLP (Fig. 1, compare A with C). By example, BLP at concentrations below 1

FIGURE 2. BLP induces E-selectin expression on the surface of HUVEC. A, Time relationship of endothelial cell-dependent PMN adhesion. HUVEC were treated with BLP or P3CSK4 for the stated times before PMN adhesion was determined as in Fig. 1A. B and C, Endothelial cell surface E-selectin expression. HUVEC were treated with 10 ␮g/ml P3CSK4 or 100 ng/ml LPS (B) or 1 nM BLP (C) for 4 h before being harvested and stained with anti-E-selection for flow analysis.

5234 expression of E-selectin. In addition, both BLP and the lipopeptide caused surface expression of ICAM-1 and VCAM-1, and they stimulated the synthesis and secretion of IL-8 and monocyte chemoattractant protein-1 (not shown). BLP stimulation of endothelial cells is LPS independent We considered the possibility that some of the responses of endothelial cells to BLP, particularly those displayed at high concentrations, might be due to residual LPS in our preparations. To explore this, we added polymyxin B to bind and inactivate LPS. The negative control TNF (20 U/ml) was unaffected by this agent, while the response to LPS at 100 ng/ml was completely suppressed by polymyxin B (Fig. 3A). However, polymyxin B had no effect on leukocyte adhesion induced by BLP over a 10,000-fold concentration range. This is consistent with the complete suppression of surface E-selectin expression by polymyxin B when LPS was the agonist, while this LPS-binding agent had no effect on E-selectin expression when BLP was the agonist (not shown). Additionally,

FIGURE 3. Endothelial cell activation by BLP is not due to LPS contamination. A, Endothelial cell-dependent PMN adhesion. HUVEC were stimulated with 20 U/ml TNF-␣, 100 ng/ml LPS, or BLP (900, 9, or 0.09 nM) for 4 h before activation was assessed by PMN adhesion as in Fig. 1. Some endothelial cells were coincubated with 10 ␮g/ml polymyxin B to sequester LPS at a concentration able to block up to 100 ng LPS, as shown by the LPS-positive control in this experiment. B, IL-6 secretion. The supernatants from the cells used for the experiment presented in A were analyzed for IL-6 secretion by ELISA.

ENDOTHELIAL CELL ACTIVATION BY E. coli LIPOPROTEIN we found that recombinant bacterial permeability-increasing factor, which also binds LPS, blocked endothelial activation by LPS, but not by BLP (not shown). Finally, we found that IL-6 production in response to BLP was not inhibited by polymyxin B, although its production in response to the positive control LPS was abolished (Fig. 3B). There was, however, a small, but definite increase in IL-6 synthesis for unknown reasons when polymyxin B was included with BLP or TNF-␣, an increase that was not mirrored in the neutrophil adhesion assay.

FIGURE 4. Endothelial cell activation by BLP and P3 CSK4 is serum independent. HUVEC were treated for 4 h with the stated concentrations of LPS (A), BLP (B), or P3CSK4 (C) in HBSS in the presence or absence of 5% pooled human serum. PMN adhesion to endothelial cell monolayers was determined as in the preceding figures.

The Journal of Immunology Endothelial cell recognition of BLP is serum independent Serum improves cell activation by LPS by supplying at least two, depending on the target cell, components, LPS-binding protein and sCD14. Endothelial cell activation by the lipoprotein isolated from B. burgdorferi has a similar requirement for serum components (43). As shown in Fig. 4A, the addition of small amounts of serum dramatically altered the sensitivity of endothelial cells to LPS. This

5235 was not true for BLP, in which the addition of human serum had little effect on the sensitivity of endothelial cells to this already potent agonist (Fig. 4B). PMN adhesion in response to the less effective P3CSK4 lipopeptide also was unaffected by the presence of serum (Fig. 4C). BLP activates endothelial cells by CD14-dependent and independent mechanisms The preceding serum-free experiments show that endothelial cells need not interact with BLP through sCD14, but were not informative about BLP/sCD14 complex recognition as an additional mechanism of ligand presentation. Complex formation with sCD14 is an essential component of LPS endothelial cell activation because the anti-CD14 Ab MY4 abolished the effect of LPS on endothelial cells (Fig. 5A). When serum was present, we found (Fig. 5B) that stimulation of endothelial cells by low concentrations of BLP was strongly suppressed by MY4. This dependence on sCD14 decreased as the concentration of BLP increased, until it was completely independent of sCD14. In contrast, endothelial cell activation by P3CSK4 was largely unaffected by MY4 at any concentration (Fig. 5C). It appears that sCD14 will interact with the protein component of BLP when it is present, and this complex, like free BLP, is a potent endothelial cell agonist. We determined whether BLP could form a complex with sCD14 using a gel mobility shift assay. The data show (Fig. 6) that increasing the concentration of LPS, the positive control, increased the migration of human rsCD14. The addition of purified BLP to sCD14 also altered its mobility, although in the opposite direction, as the formation of a larger complex with this 6670-Da protein decreased the mobility of sCD14 compared with its free form. We conclude from the complete shift of sCD14 to this slower migrating complex in the presence of BLP that there is a stable and avid interaction of BLP with CD14. Endothelial cell responses to BLP mimic LPS signaling We determined whether BLP stimulated endothelial cells like LPS, and examined p38 activation because it is partially responsible for LPS signaling through TLR4 in these cells (44). BLP, like LPS, induced a time-dependent increase in phospho-p38 (Fig. 7), although BLP did not require the addition of serum for this event. We found the p38 inhibitor SB203580, at a concentration that blocked this increase in phospho-p38 immunostaining (not shown), reduced PMN adhesion to LPS-activated endothelial cell (Fig. 7). This inhibitor caused an equivalent reduction in the number of PMN adhering to endothelial cells when the endothelial monolayer was treated with BLP, so both endotoxins partially relied on p38 signaling. We explored events further downstream by determining whether there were significant differences in the types of genes induced by

FIGURE 5. CD14 increases sensitivity to low concentrations of BLP. Endothelial cells were incubated with the stated concentration of serum (A) or 1% pooled human serum (B and C) with or without 1 ␮g blocking anti-CD14 mAb MY4. After a 30-min preincubation, LPS (A) at 100 ng or the stated concentration of BLP (B) or P3CSK4 (C) was added for a 4-h incubation. After agonist removal and washing of the monolayer, PMN adhesion was determined as before. Incubation of MY4 with endothelial cells alone had no effect on subsequent PMN adhesion.

FIGURE 6. BLP binds sCD14 and alters its mobility during electrophoresis. Human rsCD14 was incubated with E. coli BLP or various concentrations of LPS for 18 h at room temperature. This mixture was resolved by electrophoresis in a nondenaturing gradient gel before the proteins were transferred to polyvinylidene difluoride membrane for detection by Western blotting with an anti-CD14 mAb, as described in Materials and Methods.

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ENDOTHELIAL CELL ACTIVATION BY E. coli LIPOPROTEIN Table I. Atlas array of similar and differentially induced mRNA from stimulated HUVECa Induction of mRNA

FIGURE 7. p38 is partly responsible for IL-1, LPS, and BLP stimulation of endothelial cell-dependent PMN adhesion. A, Composite p38 and phospho-p38 Western blot. HUVEC were treated for 10 or 30 min with: 5% human serum; 50 U/ml TNF-␣; 10 ng/ml LPS with or without 5% human serum albumin; 650 ng/ml BLP. Cell lysates were separated by SDS-PAGE, blotted onto Immobilon, and probed with anti-phospho-p38 Ab for visualization using ECL. The blot was stripped and reprobed for p38 with an anti-peptide polyclonal Ab. B, Effect of SB203580 on endothelial cell-dependent PMN adhesion. HUVEC were treated, or not, for 30 min with 10 ␮M SB203580 to inhibit p38 activity, and then stimulated with 10 ng/ml IL-1, 100 ng/ml LPS, or 100 ng/ml BLP for 4 h before PMN adhesion was determined.

BLP and LPS. An array of 588 cell cycle and activation-dependent genes revealed that mRNA for a number of adhesion proteins, signaling molecules, and cytokines was increased in response to both of these bacterial products (Table I). There were a few of this sample of regulatory genes that were stimulated to a greater extent by LPS than by the lipoprotein, and there was one example in which BLP was a better agonist than LPS. In general, however, endothelial cell activation by bacterial lipoprotein resembled activation by LPS. Comparison of individual blots in the above experiment makes precise quantitation difficult, so we reexamined this issue with a competitive mRNA microarray hybridization that can be precisely quantified. Table II shows that mRNA for 30 known genes and expression sequences tagged of the 4400 arrayed cDNAs were induced at least 2-fold compared with vehicle-stimulated control endothelial cells. We sequenced the first 10 entries in Table II and verified their identity. This quantitative approach using representative human genes, rather than the targeted ones represented by the blot employed above, shows that the complement of genes and the extent to which they are induced by BLP are nearly identical to induction by LPS. Bacterial lipoprotein contributes to endothelial cell activation by E. coli The BLP is the single most abundant lipoprotein in E. coli, although many other less abundant proteins are so modified (11). We

BLP ⫽ LPS

LPS ⬎ BLP

BLP ⬎ LPS

CRABP II DDR1 E-selectin Ezrin GCSF receptor ICAM-1 IL-8 IL-6 Inhibin ␤ JNK1 JNK2 LERK-3 MKK6 MCP-1 MIP2-␣ NKEFB RACH1 TAFII31 VCAM-1 Protein C inhibitor

HSP27 KIP2 TRK-B Y-box protein

Caspase 10

a In separate experiments, HUVEC were stimulated with either 1 ␮g/ml LPS for 4 h, 100 ng/ml BLP for 3 h, or vehicle control for the same time. mRNA was harvested and hybridized to an Atlas Human cDNA Expression Array I blot. This table represents mRNAs that were strongly induced; many more mRNAs were moderately induced. CRAB II, cellular retinoic acid-binding protein II; DDR1, discoidin domain receptor 1 (EDDR1, TrkE); JNK, c-Jun N-terminal kinase; LERK-3, ligand for EPHrelated kinases; MKK6, MAP kinase kinase 6; MIP2-␣, macrophage inflammatory protein 2-␣; MCP-1, monocyte chemotactic protein 1; NKEFB, natural killer-enhancing factor B; TAFII31, transcription initiation factor TFIID subunit; KIP2, cyclindependent kinase inhibitor 1B; HSP27, heat shock 27-kDa protein I.

determined whether this single protein might contribute to endothelial cell stimulation by comparing a BLP-negative strain with its parental strain. We found that the whole cell lysate of the JE5505 K12 mutant lacking BLP was less active as an endothelial cell agonist than the strain containing this outer membrane and periplasmic lipoprotein (Fig. 8). Thus, a single lipoprotein can contribute significantly to the recognition of, and endotoxic response to, E. coli.

Discussion The BLP of the outer membrane of E. coli, the first lipoprotein with the N-terminal lipid-modified cysteinyl residue to be identified (18), contributes to cytokine synthesis and the endotoxic shock induced by E. coli (45). How this bacterial lipoprotein accomplishes this is unknown, but we do know that the lipoprotein isolated from B. burgdorferi, the causative agent of Lyme disease, also activates endothelial cells (23, 26, 29). This lipoprotein interacts with sCD14, enhancing its activation of endothelial cells, an effect that is mimicked by a lipopeptide derived by proteolysis from the parent lipoprotein (43). In this study, we show that BLP isolated from E. coli activates endothelial cells, and is far more potent in doing so than the outer surface proteins of B. burgdorferi, as picomolar rather than nanomolar concentrations elicit activity. While the triacylcysteinyl modification is essential for the activity of both lipoproteins, this disparity in potencies, as well as the lower potency of the lipopeptides that bear this lipid modification, point to a role for the apolipoprotein in receptor recognition. We find that BLP is a true endotoxic protein, as it precisely reproduces the response of endothelial cells to LPS. We purified BLP and then insured that the resulting material was free of biologically relevant amounts of LPS by assaying LPS levels by a Limulus assay, and by showing that polymyxin B at

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Table II. Quantitative microarray analysis of endothelial cell genes induced by LPS or BLP Fold Inductiona Gene Name

ESTs N-methylpurine-DNA glycosylase GRO2 oncogene VCAM-1 ESTs IL-6 Superoxide dismutase 2, mitochondrial ESTs Colony-stimulating factor 1 (M-CSF) IFN-␥-induced cell line; protein 10 (IP-10) IL-8 Parathyroid hormone NFKB 1 (p105) ESTs Human mRNA for metallothionein from cadmium-treated cells Monocyte chemotactic protein-1 ESTs Myosin regulatory L chain 2, smooth muscle isoform ESTs Human IAP homolog C (MIHC) mRNA, complete cds Monocyte chemotactic protein 3 precursor Human osteoprotegerin (OPG) mRNA, complete cds Homo sapiens CEV14 mRNA, partial cds ESTs Human IAP homolog C (MIHC) mRNA, complete cds ESTs, Highly similar to DNA-directed RNA polymerase I 190 kD polypept Human orphan G protein-coupled receptor (RDC1) IFN-inducible 56-KDa protein GRO1 oncogene (melanoma growth stimulating activity, ␣) ESTs Hexabrachion (tenascin C, cytotactin)

LPS

BLP

15 ⫾ 3.1 15 ⫾ 0.28 13 ⫾ 0.45 12 ⫾ 2.7 11 ⫾ 1.5 7.1 ⫾ 1.2 5.1 ⫾ 0.77 5.0 ⫾ 0.41 4.0 ⫾ 0.56 3.9 ⫾ 0.45 3.7 ⫾ 2.9 3.3 ⫾ 0.81 3.0 ⫾ 0.33 3.0 ⫾ 0.24 2.9 ⫾ 0.30 2.8 ⫾ 0.04 2.7 ⫾ 0.64 2.7 ⫾ 0.25 2.6 ⫾ 0.82 2.5 ⫾ 0.12 2.5 ⫾ 0.10 2.4 ⫾ 0.34 2.3 ⫾ 0.72 2.3 ⫾ 0.22 2.3 ⫾ 0.16 2.3 ⫾ 0.17

24 ⫾ 0.77 15 ⫾ 0.45 14 ⫾ 2.7 16 ⫾ 2.5 19 ⫾ 0.26 5.8 ⫾ 0.10 5.3 ⫾ 1.7 7.1 ⫾ 0.03 3.7 ⫾ 0.85 1.4 ⫾ 0.63 14 ⫾ 5.7 4.8 ⫾ 1.1 2.9 ⫾ 0.07 4.5 ⫾ 0.84 1.8 ⫾ 0.16 4.8 ⫾ 0.67 2.5 ⫾ 0.23 3.0 ⫾ 0.57 2.2 ⫾ 1.1 2.1 ⫾ 0.08 3.1 ⫾ 0.58 2.3 ⫾ 0.67 4.2 ⫾ 1.1 2.6 ⫾ 0.03 2.3 ⫾ 0.09 2.73 ⫾ 0.19

2.3 ⫾ 0.22 2.2 ⫾ 0.23 2.0 ⫾ 0.03 2.0 ⫾ 0.11 2.0 ⫾ 0.01

2.9 ⫾ 0.18 1.2 ⫾ 0.04 2.6 ⫾ 0.52 1.4 ⫾ 0.01 1.6 ⫾ 0.01

UniGeneID

Imageid

Hs.91288 Hs.70390 Hs.81791 Hs.85092 Hs.48284 Hs.91288 Hs.59475

326660 824246 841361 1009036 1015315 310406 505249 320727 141500 491243 326092 321068 418291 321780 309388 1072789 308478 1042286 301462 201890 322873 325973 470234 345850 685141 357255

Hs.23016 Hs.85121 Hs.789 Hs.47353 Hs.84261

43181 325364 323238 377185 484707

Hs.79396 Hs.75765 Hs.109225 Hs.93913 Hs.73830 Hs.118258 Hs.82813 Hs.2248 Hs.624 Hs.37045 Hs.83428 Hs.16940 Hs.118786 Hs.340 Hs.5855 Hs.9615

a Two-P150s of confluent HUVEC in serum-containing media were treated for 3 h with 100 ng/ml LPS (K-12-LCD25, List Biological Laboratories), PBS control, 200 ng/ml purified BLP, or SDS control. Plates without LPS contained 10 ␮g/ml polymyxin B. As a control, two 12-well plates from the same culture were treated with agonists for 3.5 h and the bioactivity was assayed by 5-min PMN adhesion assay.

concentrations that abolish endothelial cell activation by even high levels of LPS had no effect on the events induced by BLP. We can be sure, then, that the BLP itself is biologically active and that it independently displays inflammatory properties.

FIGURE 8. E. coli lacking BLP are less inflammatory than the parental strain. E. coli K12 or a BLP-negative mutant strain JE5505 were heat killed, sonicated, and adjusted to equivalent protein concentrations (0.01, 0.1, and 1 ␮g/ml). These were added in the presence of 10 ␮g/ml polymyxin B to endothelial cells for 4 h before PMN adhesion was quantitated as before. The positive control was 10 ng/ml LPS, while the negative control was the HBSS/A buffer.

Endothelial cells express the LPS receptor TLR4 (34), whose defect is responsible for the insensitivity of the C3H/HeJ mouse strain to LPS (46 – 48). A separate receptor confers sensitivity to endotoxic proteins in this LPS-insensitive mouse strain because they have unimpaired responses to E. coli lipoproteins (16, 49, 50). TLR2 functions mainly, although not exclusively (5), as a receptor for other bacterial wall components (30 –33), and this can occur in a serum-independent way (36, 51). The response of TLR2-deficient mice to LPS is unimpaired, while responses to various bacterial wall components are lost (10). Thus, bacterial products can signal independently of LPS, and this proceeds through a different receptor. We found that endothelial cells are activated by picomolar levels of BLP, nanomolar amounts of a synthetic lipopeptide, and not at all by free lipid-modified cysteine. Activation by BLP was completely dependent on the N-terminal lipid modification, but the significant gain in potency over the lipopeptide or B. burgdorferi outer surface protein (29) suggests that either the protein is preferentially recognized by the endothelial cell receptor, that it does a better job of presenting the required N-terminal lipid modification, or that these similarly modified proteins act via distinct receptors. The spirochete lipoprotein exists as N- and C-terminal globular domains with a central ␤-sheet that lacks a hydrophobic core (52). In contrast, the 58-aa BLP folds into two amphipathic helices (53) that may aid in its recognition. Previous work (54) has shown that synthetic peptides based on BLP sequences are B cell mitogens, and that their activity is augmented by conjugation to the N-terminal triacylcysteine residue.

5238 We show that BLP and sCD14 physically interact to form a complex that was stable to electrophoresis, but that endothelial cell activation by BLP need not occur through this sCD14/BLP complex. Endothelial cells themselves do not express the phosphatidylinositol-modified form of CD14 (55) that is expressed on plasma membranes of cells such as monocytes, and therefore are insensitive to low concentrations of LPS. Endothelial cells can, however, use the soluble form of CD14 found in blood to raise their sensitivity to LPS. sCD14 is secreted by LPS-activated monocytes, particularly from monocytes isolated from septic patients (56). Changes in circulating sCD14 levels are therefore positioned to affect LPS signaling to endothelial cells. Accordingly, we found that endothelial cell activation by LPS was increased 1000-fold by the presence of serum, and that the blocking Ab MY4 prevented endothelial cells’ response to all but the highest concentrations of LPS. In contrast, BLP was fully active as an endothelial cell agonist in the absence of added serum. In fact, there was a slight diminution in the potency of BLP by the addition of human serum. The less active lipopeptide was similarly unaffected by the addition of serum. These observations differ from those obtained in monocytes with a different lipopeptide, P3Cys-Ala-Gly, in which cytokine production was dependent on serum, although it was independent of CD14 (57). This again suggests that the peptide or protein moiety affects recognition of this class of lipoproteins. One of the effects of serum, which was not apparent when simply comparing the level of activation in its presence or absence, was that the nature of the interaction of BLP with endothelial cells was changed. When serum was present, all of the ability of BLP to activate endothelial cells was inhibited by blocking sCD14 with the mAb MY4. Only when the amount of BLP was vastly increased, in the presence of a fixed amount of serum, did sCD14independent recognition system recover. This property was not displayed by the lipopeptide, in which the presence of anti-CD14 Ab had no effect on endothelial cell activation. We conclude from this that sCD14 avidly interacts with BLP, a conclusion supported by the physical evidence in Fig. 6. Thus, even though endothelial cells recognize free BLP, in the presence of serum this becomes unavailable until supersaturating concentrations are attained. The complete loss of sCD14-independent recognition suggests that either sCD14 displays a higher affinity for BLP than endothelial cell receptors for BLP or that this complex is quite stable. It is likely that the lipid portion of BLP is involved in the lipoprotein high affinity interaction with sCD14 (43). However, we suggest the protein component also is involved in complex formation, as the lipopeptide bearing the triacylcysteinyl modification did not form a stable complex with sCD14 that was inhibitable with the CD14 Ab. We found only a few discernible differences in the genes induced in response to BLP or LPS. Of the 30 genes induced at least 2-fold by LPS as measured by microarray, 29 were induced by BLP. This competition microarray protocol is a quantitative technique, and we found that the level of gene induction of this cohort of genes was similar between the two agonists. There was a single exception to this generalization, in which induction by BLP was greater than in response to LPS, and one exception when the converse was true. IFN-␥-inducible protein-10, a CC chemokine (58), was statistically significantly stimulated by LPS, but not by BLP. The 1.4-fold change found in BLP-treated cells compared with control cells is not significantly different from unresponsive genes, and so IFN-␥-inducible protein-10 is not downstream of the BLP signaling cascade. Additionally, there was a significant quantitative difference in the level of IL-8 induction between cells activated with BLP and LPS. BLP induced a 14-fold increase in IL-8 mRNA compared with the still significant 4.8-fold induction by

ENDOTHELIAL CELL ACTIVATION BY E. coli LIPOPROTEIN LPS. We supported the results of the above microarray approach with an Atlas blot. Different genes are arrayed in the two approaches, with the Atlas blot focusing on inflammatory and cell regulatory genes. Again, BLP and LPS tended to induce the same genes, and to a similar level. The minor differences found by both approaches suggest that BLP and LPS are not completely equivalent, even though the majority of downstream events and outcomes are shared. BLP is the most abundant outer membrane and periplasmic protein of E. coli, and therefore is the most abundant lipoprotein of E. coli. C3H/HeJ mice that are completely unresponsive to lipoprotein-free LPS (59), due to an inactivating point mutation in TLR4, still undergo lethal shock when exposed to heat-killed E. coli (45). However, shock in this model is dampened when E. coli devoid of BLP are substituted for wild-type bacteria. We find that this BLP null strain of E. coli is also less active as an endothelial cell agonist (45), showing that the BLP is an endotoxic protein whose actions on endothelial cells and in a septic shock model resemble those of LPS.

Acknowledgments We thank Dee Biddle and Craig Osborne for their expert technical assistance, and Gopal Marathe for advice. We are indebted to Donelle Benson Jessica Phibbs, Jennifer Eyre, and Margaret Vogel for supplying all of the primary human cells used in these experiments.

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