TLR4 TLR4在人冠脉内皮细胞胞内发挥作用 LBP和sCD14在介导LPS-反应中的作用

The FASEB Journal express article 10.1096/fj.03-1263fje. Published online May 7, 2004.Toll-like receptor 4 functions intracellularly in human coronary artery endothelial cells: roles of LBP and sCD14 in mediating LPS-responsesStefan Dunzendorfer, Hyun-Ku Lee, Katrin Soldau, and Peter S. TobiasThe Scripps Research Institute, Department of Immunology, 10550 North Torrey Pines Road, La Jolla, CA 92037Corresponding author: Peter S. Tobias, The Scripps Research Institute, Department of Immunology IMM-12, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail:tobias@ABSTRACTEndothelial cells are activated by microbial agonists through Toll-like receptors (TLRs) to express inflammatory mediators; this is of significance in acute as well as chronic inflammatory states such as septic shock and atherosclerosis, respectively. We investigated mechanisms of lipopolysaccharide (LPS)-induced cell activation in human coronary artery endothelial cells (HCAEC) using a combination of FACS, confocal microscopy, RT-PCR, and functional assays. We found that TLR4, in contrast to TLR2, is not only located intracellularly but also functions intracellularly. That being the case, internalization of LPS is required for activation. We further characterized the HCAEC LPS uptake system and found that HCAEC exhibit an effective LPS uptake only in the presence of LPS binding protein (LBP). In addition to its function as a catalyst for LPS-CD14 complex formation, LBP enables HCAEC activation at low LPS concentrations by facilitating the uptake, and therefore delivery, of LPS-CD14 complexes to intracellular TLR4-MD-2. LBP-dependent uptake involves a scavenger receptor pathway. Our findings may be of pathophysiological relevance in the initial response of the organism to infection. Results further suggest that LBP levels, which vary as LBP is an acute phase reactant, could be relevant to initiating inflammatory responses in the vasculature in response to chronic or recurring low LPS. Key words: innate immunity • endothelium • endotoxinEndothelial cells (EC), which form the inner vascular lining, are important in the regulation of vascular tone, coagulation and fibrinolysis, cellular growth, differentiation, and last but not least, immune and inflammatory responses (1, 2). Moreover, these cells are targets for many endogenous and exogenous agents (e.g., lipopolysaccharide [LPS]), that activate the endothelium and result in production of proinflammatory mediators (3). These mechanisms play roles in Gram-negative sepsis, where the body may be overwhelmed with LPS and also in diseases that are linked to a low, but chronic, LPS burden. There is considerable evidence that endotoxin (LPS) contributes to the propagation of atherosclerosis (4, 5), which shares many features with inflammatory diseases (6).LPS is a major component of the outer surface of Gram-negative bacteria and a potent activator of cells of the immune and inflammatory system, including endothelial cells. In vivo, LPS-induced cell activation depends on the presence of at least four proteins: TLR4 (7–9), MD-2 (10), CD14 (11), and LPS binding protein (LBP) (12). The first three may be present as a complex on the surfaces of myeloid (and transfected) cells (13). Many cells, among them epithelial and endothelial cells, do not express the membrane bound form of CD14 (mCD14); their activation involves soluble CD14 (sCD14) (14). LPS·sCD14 complexes will form slowly in vitro (15), but, in vivo, plasma LBP (16–18) catalyzes LPS·CD14 complex formation (19). LPS·CD14 complexes, whether membrane bound or soluble, are necessary for LPS binding to TLR4-MD-2 (20). In mCD14 expressing cells, most LPS appears to be internalized quite independently of activation (21), and mCD14-negative cells also internalize LPS (22, 23). Although LPS internalization by phagocytes has generally been regarded as a disposal function (21), studies have also suggested that internalization might be required for LPS signaling in myeloid lineage cells (24, 25). However, a very recent publication clearly demonstrates that signaling starts on the surface of TLR4-transfected and TLR4 surface expressing cells when incubated with LPS-sCD14 complexes, even in the absence of LPS uptake (26). Other recent work suggests that internalization of LPS is required for activation of murine intestinal epithelial cells, where mCD14, but not TLR4, is found on the surface (27, 28).MATERIALS AND METHODSAntibodiesAnti-TLR4 polyclonal antibody (pAb) H80 and monoclonal antibody (mAb) HTA125 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-TLR2 2392 was a gift of Paul Godowski (Genentech), and anti-TLR4 HTA1216, HTA414, and HTA405 were gifts from Kensuke Miyake (Saga Medical School, Japan). Anti-LBP mAb 2B5 and anti-CD14 mAb 28C5 were produced in our laboratory (29). Polyclonal rabbit anti-human MyD88 was from Imgenex (San Diego, CA). The monoclonal mouse anti-human CD14 (clone M5E2), mouse anti-human CD144 (clone 55-7H1), mouse anti-human CD106 (VCAM-1; clone 51-10C9), rat anti-mouse CD31 (PECAM-1) (clone MEC13.3), rat anti-mouse CD144 (VE-cadherin; clone 11D4.1), and rat anti-mouse CD106 (VCAM-1; clone 429-MVCAM.A) used for immunofluorescence staining and PE-conjugated anti-human CD62E (E-selectin/ELAM; clone 68-5H11) were from PharMingen (San Diego, CA). All secondary antibodies used in fluorescence microscopy were Alexa Fluor conjugates (Molecular Probes, Eugene, OR). PE-conjugated secondary antibodies used in flow cytometry were purchased from PharMingen (San Diego, CA).Proteins, LPS, and other reagentsHuman recombinant sCD14 and LBP were expressed in and purified from BTI-TN-5B1-4 cells (Invitrogen, San Diego, CA) as recently described (23). Both proteins can be radiolabeled at an artificially introduced protein kinase A site. Macrophage-activating lipopeptide-2 (MALP-2) was from Alexis Biochemicals (San Diego, CA), recombinant mouse TNF-α was purchased from Serotec (Oxford, UK), and hIL-1β was from Sigma (St. Louis, MO). Compound 406 was generously provided by Professor S. Kusumoto (Osaka University, Japan). All LPS was purchased from List Biological Laboratories (Campbell, CA). Escherichia coli (O55:B5) LPSwas exclusively used for cell stimulation and tritium-labeled LPS (3H-LPS) from E. coli (K12 LCD25) was used in LPS uptake experiments. For use in fluorescence microscopy, LPS from Salmonella minnesota (Re595) was labeled with FITC (Molecular Probes) (19). Complexes of sCD14 with either LPS were preformed without LBP as described previously (30). Under these conditions, no LPS remains unbound. Complexed LPS (LPS·CD14, FITC-LPS·CD14, 3H-LPS·CD14) was prepared freshly each time and used right away.Cell cultureHCAEC and human umbilical vein endothelial cells (HUVEC) were purchased from Clonetics (San Diego, CA) and grown to confluence in full growth medium (EGM-2-MV) from the same company. HCAEC were from healthy young donors and were used for experiments from passages 4–8. There was no obvious difference in TLR2 surface expression or responsiveness to various stimuli between passage 4 and passage 8 cells. Medium was changed every day, and cells were passaged in a 1:3 ratio using trypsin/EDTA (0.025%/0.2 mM).Mouse endothelial cell cultureMouse microvascular and vascular EC were isolated from lung tissue and the aortas of C57/BL6 mice. Halothane-anesthetized animals were killed, and the lungs and the aorta were explanted under sterile conditions, minced, and digested for 1 h at 37°C in 1 mg/ml collagenase type II (Invitrogen). Cells were stained with rat anti-mouse CD31 (10 µg/ml), and goat anti-rat IgG conjugated to paramagnetic beads (Miltenyi Biotec, Auburn, CA) enabled further purification using MACS (magnetic cell sorting). Endothelial cells were then cultured and maintained on fibronectin-coated tissue culture plates in mouse brain microvascular growth medium (Cell Application Incorporation, San Diego, CA) supplemented with 15 ng/ml basic fibroblast growth factor (Sigma). Purity of the cells was >90% as determined by staining for mouse CD31, mouse CD106, and mouse CD144 and by uptake of Alexa Fluor 488-labeled AcLDL (Molecular Probes).Monocyte isolationHuman peripheral blood monocytes were purified by MACS (Miltenyi Biotec) according to the manufacturer’s protocol. Purity of the preparations and viability of the cells regularly exceeded 95% as determined by subsequent FACS analyses using FITC-anti-human CD14 and propidium iodide (2.5 µg/ml). The isolation procedure did not activate the cells and did not diminish their ability to be stimulated.Flow cytometryFor flow cytometry analysis of protein surface expression, HCAEC cultured in six-well plates were harvested with a cell scraper. Freshly prepared monocytes and HCAEC were washed twice and stained with various primary and isotype control antibodies (10 µg/ml) for 30 min at 4°C in PBS/5% FCS containing 0.1% sodium azide. After additional washing steps, cells were incubated with secondary PE-conjugated antibodies for a further 30 min at 4°C, washed and resuspended in PBS, and analyzed on a FACScan (BD Biosciences, San Diego, CA). HCAEC activation was determined by CD62E surface expression, which was quantified using PE-conjugated mouse anti-human CD62E or control PE-conjugated mouse IgG for 30 min at 4°C before FACS analyses.RT-PCRRNA was purified from LPS-stimulated or unstimulated HCAEC or monocytes using the Absolutely RNA RT-PCR Miniprep Kit from Stratagene (La Jolla, CA). mRNA levels of TLR2, TLR4, MD-2, CD14, LBP, and gp96 were determined by RT-PCR. Briefly, the first-strand cDNA was reverse-transcribed from 1 µg total RNA with random primers using the SuperScript first-strand Synthesis System from Invitrogen. The cDNA product was amplified by 30 cycles of 30 s at 94°C, 45 s at 55°C, and 2 min extension at 72°C using specific primer pairs. cDNA from monocytes or from stimulated HepG2 cells was used as control for CD14 or LBP, respectively. The RT-PCR products were separated in 1.2% (wt/vol) agarose gels and visualized with ethidium bromide.Immunofluorescence staining and fluorescence microscopyHCAEC were cultured in eight-well Nunc Lab-Tec II chambered coverglasses (Nalge Nunc, Naperville, IL) and after an incubation period of 4 h with either LPS (100 ng/ml), FITC-LPS (100 ng/ml), sCD14 (1.6 µg), LBP (2.5 µg), or preformed FITC-LPS·CD14 complexes (100 ng/ml), cells were fixed and permeabilized using the Cytofix/Cytoperm kit from PharMingen (San Diego, CA) according to the manufacturer’s protocol. Nonspecific binding sites were blocked with 0.5% BSA for 30 min at room temperature, and thereafter the following primary antibodies were added at the indicated concentration for 1 h at room temperature: H80 (20 µg/ml), HTA125 (10 µg/ml), 2B5 (10 µg/ml), anti-hCD14 (10 µg/ml), anti-hCD144 (10 µg/ml), anti-hVCAM-1 (10 µg/ml). Fluorescent wheat germ agglutinin (Oregon Green 488; Molecular Probes) was used for Golgi staining, and fluorescent phallotoxin was used for F-actin staining (both at 5 µg/ml). Cells were washed, and secondary fluorescence-labeled antibodies were added at 5 µg/ml for 1 h at room temperature. Nuclei were counterstained with DAPI (300 nM) for 3 min before cells were extensively washed and treated with SlowFade Light antifade reagent (Molecular Probes). Monocytes were stained in suspension. Thereafter, they were mounted to a cover glass with ProLong (Molecular Probes) mounting medium, thus maintaining their round shape. Images were acquired using the DeltaVision Optical Sectioning Microscope (Model 283). Following data acquisition using the Photometrics CH350L liquid-cooled CCD camera, which collects low-intensity signals with a linear response to light intensity, the data are then deconvoluted using DeltaVision software SoftWoRx 2.5 based on the Agard/Sadat inverse matrix algorithm. Following computational deconvolution, this system can provide high-resolution 3D images of cells, but each single optical section can still be viewed alone.Cell stimulation experimentsHCAEC and HUVEC were cultured in six-well plates, and after reaching confluence, EGM-2-MV was changed to DMEM/2% FCS or DMEM/2% depleted FCS at least 6 h before an experiment was started. Antibodies were added 30 min before cells were stimulated for 4 h at 37°C with LPS, LPS·CD14, or MALP-2 at various concentrations. Thereafter, the cells were harvested and stained for flow cytometry analyses. Human peripheral blood monocytes were resuspended in DMEM/2% FCS and seeded into 24-well tissue culture plates at 1 × 104cells/well. After addition of antibodies for 30 min, cells were stimulated with either LPS or MALP-2 (1 ng/ml) for 4 h at 37°C. Supernatants were harvested, and hIL-8 was measured with an ELISA (Biosource, Camarillo, CA). Mouse EC were stimulated with LPS, MALP-2, or cytokines for 12 h in culture medium containing 5% FCS. Thereafter, medium was measured for mouse macrophage inflammatory protein-2 (MIP-2), which is the murine homologue of human IL-8, by ELISA (R&D Systems, Minneapolis, MN). For stimulation of HCAEC with low LPS concentrations or with preformed LPS·CD14 complexes, it was necessary to deplete serum of sCD14 or LBP using anti-CD14 (mAb 28C5) and anti-LBP (mAb 2B5) as described previously (21, 23). Depleted FCS contained <1 ng/ml of remaining antibodies (measured by ELISA). Both CD14 and LBP were below the detection limit of Western blotting (1 ng of protein in our experiments).LPS uptake experimentsHCAEC, or mouse EC, were grown to confluence in six-well tissue culture plates, and medium was changed to DMEM/2% FCS before experiments. 3H-LPS (100 ng/ml) or 3H-LPS·CD14 (100 ng/ml) was incubated for 2 h with the cells of one well along with various antibodies, sCD14, and LBP. Thereafter, the adherent cells were washed twice with PBS and further treated with 250 µg/ml proteinase K (Sigma) in HBSS for 30 min at room temperature to remove cell surface proteins/receptors (22). The 3H counts remaining after proteinase K treatment were therefore considered to be intracellular. This treatment also detached the cells, which were harvested, washed twice in PBS, and lysed in 300 µl 2% SDS/50 mM EDTA. Liquid samples were transferred to scintillation vials and assayed for counts in Ecoscint scintillation fluid (National Diagnostics, Atlanta, GA). The same procedure was carried out in experiments where 32P-labeled LBP (250 ng/ml) uptake was investigated.Transient transfectionOne day after seeding into six-well plates, HCAEC (5×105 cells/well) were transiently transfected with cDNA for CD14 or TLR4 in pFLAG-CMV.1 (Sigma) using 6 µl/well Lipofectin Reagent (Invitrogen) and 1.6 µg/well DNA in 100µl Opti-MEM I (Gibco BRL, Gaithersburg, MD). After 3 h under serum-free conditions, EGM-2-MV was added and cells were analyzed the following day for surface expression of CD14 or TLR4. In several experiments, transfection efficiency was typically 5–10%.StatisticsData are expressed as mean and standard error of the mean (SE). Means were compared by Kruskal-Wallis ANOVA and by Mann Whitney U test. A difference with P < 0.05 was considered significant. Statistical analyses were calculated using the StatView software package (Abacus Concepts, Berkeley, CA).RESULTSFACS analyses of HCAEC do not detect surface TLR4To investigate cell surface expression of TLR2 and TLR4 protein, we performed several FACS analyses on monocytes and HCAEC and compared the results with the TLR2 and TLR4 mRNAexpression levels (Fig. 1). Nonpermeabilized HCAEC or monocytes were stained with 5 different anti-TLR4 (monoclonal HTA1216, HTA405, HTA414, HTA125, and polyclonal H80), anti-TLR2 (mAb 2392), or anti-CD14 (M5E2) primary antibodies. Dot blot assays with immobilized primary antibody confirmed that the secondary antibodies used were able to detect their primary partners (not shown). In contrast to human peripheral blood monocytes, which showed clear surface staining for TLR2, TLR4, and CD14, we could not detect TLR4 or CD14 on the surface of HCAEC, although the TLR4 mRNA expression was similar in monocytes and HCAEC. At an expression level of TLR2 mRNA (after induction with LPS-IFNγ or TNF-IFNγ) comparable to basal TLR4 mRNA levels, 92–98% of HCAEC stained positive for surface TLR2; even in unstimulated HCAEC, where almost no TLR2 mRNA was found, FACS still was able to detect surface TLR2. Although LPS-IFNγ enhanced TLR4 mRNA expression in HCAEC, TLR4 was still not detected on the surface. Figure 1 (HCAEC) shows a representative result of many experiments; in this experiment, HTA125 was used for TLR4 and anti-TLR2 2392 for TLR2 detection. Both antibodies worked very well in monocytes (see Fig. 1, Monocytes). However, upon transient transfection and overexpression of TLR4 or CD14 in HCAEC, some surface expression (~1% of cells) was seen in FACS analyses (see Fig. 1, HCAEC transfected), establishing that the detection system works in HCAEC.HCAEC express RNA for TLR2, TLR4, MD-2, and gp96, but not CD14 or LBPTo assess mRNA expression in HCAEC, RT-PCR analyses was performed. Agarose gel electrophoresis revealed clear bands at the expected fragment sizes for TLR2, TLR4, and MD-2. Stimulation of the HCAEC for 12 h with 10 ng/ml of E. coli LPS (O55:B5) did not significantly change TLR4 or MD-2 mRNA expression, but enhanced TLR2 mRNA. More important, neither stimulated nor unstimulated HCAEC were positive for LBP or CD14. As expected, mRNA for the chaperon protein gp96 was found in three different HCAEC preparations and in monocytes (Fig. 2).TLR4 is intracellular in HCAEC and on the surface of monocytesExpression of TLR4 mRNA in the absence of cell surface TLR4 led us to search for intracellular TLR4 in HCAEC by 3D-deconvolution confocal microscopy. In HCAEC, which are nontransfected cells, TLR4 was found exclusively around the nucleus (Fig. 3A–D). After stimulation of the cells, VCAM-1 was found intracellularly and surface expressed (Fig. 3C). Nonimmune rabbit IgG was used as control and revealed uniform background staining (not shown). In monocytes, use of the same anti-TLR4 antibody (H80), resulted in bright surface staining and showed colocalization of TLR4 to membrane CD14 (Fig. 3E).Anti-TLR4 antibodies do not block HCAEC response to LPSHCAEC activation was determined by measuring CD62E surface expression (Fig. 4). Preliminary experiments showed that CD62E is minimally expressed on resting cells, but is rapidly induced and highly expressed upon stimulation. HCAEC were incubated with various antibodies 30 min before stimulation with LPS (10 ng/ml) for 4 h. All anti-TLR4 antibodies used failed to inhibit LPS-induced activation of HCAEC. Even long-term preincubation (up to 12 h) of HCAEC with anti-TLR4 was not able to block LPS activation (not shown). This is in contrast to monocytes, where HTA405 or HTA125 significantly, but incompletely, inhibited LPS-induced IL-8 secretion. The effects of HTA405 on HCAEC or monocyte activation by LPS werefurther explored at a wide range of LPS and mAb concentrations (see Table 1). In no case werethe results qualitatively different from Figure 4. As anticipated from published work, the abilityof LPS to stimulate cells was abolished by anti-LBP (mAb 2B5) or anti-CD14 (mAb 28C5) inendothelial cells as well as in monocytes. Anti-TLR2 (mAb 2392) completely blocked MALP-2-induced cell activation in both cell types (Fig. 4). The fact that anti-TLR2 did not inhibit LPS-induced cell activation excludes the possibility that TLR2 agonist impurities in the LPSpreparation were responsible for activation of the cells. Similarly, lack of an effect of 2B5 onMALP-2-initiated activation suggests that there is not an LPS contaminant in MALP-2. In contrast to anti-TLR4, Compound 406 (lipid 4A) dose-dependently (0.1–100 µg/ml) inhibitedLPS-induced HCAEC activation (not shown).TLR4 is required for LPS signaling but not for LPS uptake in ECWe cultured pulmonary microvascular and aortic EC derived from TLR4 –/– C57/BL6 mice andcompared their response to LPS and other proinflammatory stimuli with cells from wild-typeanimals (Fig. 5). Compared with cultured HCAEC, the mouse cells did not respond well withCD62E expression to any stimulus, and therefore we measured MIP-2 secreted into the medium.Even after 12 h incubation with 10 ng/ml of E. c oli LPS (O55:B5), the TLR4 –/– cells were stillcompletely unresponsive to this stimulus, whereas the response to murine TNF (10 ng/ml),MALP-2 (100 ng/ml), or human IL-1 (10 ng/ml) was independent of TLR4 genotype. The wild-type cells were activated by all stimuli. Results shown (Fig. 5A) are from experiments withmurine aortic EC; lung microvascular EC responded similarly (data not shown). When TLR4 –/–cells were incubated with 3H-LPS (100 ng/ml) for 2 h with various concentrations of LBP and 3H-LPS uptake was measured, no difference was found compared with wild-type cells, suggesting that uptake occurs independently of TLR4 phenotype (Fig. 5B). Moreover, mouse ECLPS uptake mimicked the LPS uptake observed in human cells (HCAEC).LBP facilitates the uptake of LPS and LPS·CD14 complexes into HCAECBecause previous data show that sCD14 and LBP are required for LPS-induced HCAECactivation, we further investigated the role of these proteins in LPS uptake. HCAEC wereincubated for 2 h with 100 ng/ml of 3H-LPS along with an increasing amount of human LBP(Fig. 6A). LBP dose-dependently stimulated 3H-LPS uptake into cells. The maximum of theeffect was seen close to equimolarity of LPS with LBP and resulted in a ~10-fold increase in theamount of LPS transported into the cells. Because LPS·CD14 complexes are necessary for cellactivation, we were curious whether LBP would enhance uptake of complexes preformedwithout LBP, and found that LBP did enhance 3H-LPS·CD14 complex uptake ~5-fold. LBP-enhanced LPS and LPS·CD14 complex uptake was sensitive to an anti-LBP antibody (mAb2B5), but was never blocked completely and a low basal uptake remained. This suggests thatLBP is not absolutely necessary for the uptake of LPS and LPS·CD14 complexes. This basaluptake was insensitive to either anti-TLR4 (mAb HTA405), anti-LBP (mAb 2B5), or anti-CD14(mAb 28C5) (data not shown).Similar patterns of uptake are seen when LPS, sCD14, and LBP were added simultaneously andtherefore CD14 complexation of LPS was catalyzed by LBP (Fig. 6B). Thus LPS in the absenceof LBP, alone or in the presence of sCD14, is only minimally internalized as compared with LPSin the presence of LBP. When LBP catalyzed complexation of LPS to sCD14 is inhibited by anti-CD14 (mAb 28C5), uptake follows the LPS-LBP pattern (10-fold increase). When complexation of LPS to either sCD14 or LBP is inhibited by anti-LBP (mAb 2B5), the effects of both LBP and CD14 are abolished. Finally, data obtained with 32P-LBP (Fig. 6C) show that LBP is internalized with LPS and that the uptake follows the same pattern as observed when LPS uptake was measured.LPS, LBP, and CD14 all colocalize with intracellular TLR4In fact, LPS complexes are found intracellularly. HCAEC were incubated for 4 h with FITC-LPS (100 ng/ml) alone or with sCD14 (1.6 µg/ml), LBP (2.5 µg/ml), or both. Thereafter, cells were stained for TLR4, LBP, or CD14 (Fig. 7). Confocal microscopy showed clear colocalization of FITC-LPS and LBP (Fig. 7A), FITC-LPS and intracellular TLR4 (Fig. 7B), LBP and TLR4 in the presence of sCD14 (Fig. 7C), and finally sCD14 and TLR4 in the presence of LBP (Fig. 7D). Under serum-free conditions (without sCD14), where no cell activation occurs, no colocalization of either FITC-LPS (Fig. 7E) or LBP (Fig. 7F) with intracellular TLR4 was observed. Colocalization of LBP and CD14 could not be shown because both are detected by mouse antibodies and these could not be distinguished by labeled second antibodies. LBP or CD14 were never detected intracellularly when added without LPS; instead, large aggregates were found in the microscopic field outside the cells. When HCAEC were stimulated for 2 h with LPS (10 ng/ml), the intracellular adaptor molecule MyD88, which is involved in TLR4 signaling, was recruited to a perinuclear region (Fig. 7G). This was not the case in the absence of sCD14 (Fig. 7H).LBP enhances HCAEC activation at low LPS concentrations via facilitated LPS·CD14 uptakeBecause the preceding experiments revealed a role for LBP in LPS uptake, we were curious whether this would have an impact on cell activation. To exclude additional LPS complex formation during the experiments, CD14-depleted FCS was used for experiments with preformed LPS·CD14 complexes and, because we wanted to see effects of added recombinant LBP, LBP-depleted FCS was used for experiments with uncomplexed LPS. Neither CD14 nor LBP at the concentrations used in our experiments activated cells without LPS.Figure 8A shows that 10 ng/ml of LPS with 250 ng/ml LBP enabled a full response of the cells and sCD14 (160 ng/ml) showed no additional effect. When LPS and sCD14 were added without LBP under these conditions, a minimal stimulation was observed, probably due to incomplete LBP depletion of the serum. Stimulation under each condition was blocked by an anti-LBP antibody, thus confirming the necessity of LBP for LPS·CD14 complex formation during the 4 h course of these experiments.In contrast, 10 ng/ml of preformed LPS·CD14 complexes stimulated the cells without any effect of anti-LBP or additional LBP (Fig. 8C), suggesting that LBP is not absolutely required (Fig. 6A) for sufficient uptake of preformed LPS·CD14 complexes at 10 ng/ml. However, uptake did occur, because a blocking anti-TLR4 (mAb HTA405), which would block surface TLR4, failed to inhibit activation even when added together with anti-LBP (mAb 2B5).At a low LPS concentration (0.1 ng/ml) (Fig. 8B), addition of sCD14 (100 ng/ml) did not stimulate cells. Providing LBP (200 ng/ml) enabled a response of the cells, which was further enhanced by additional sCD14. Activation was again blocked by anti-LBP (mAb 2B5), which would also inhibit rapid LBP catalyzed formation of stimulatory LPS·CD14 complexes. Preformed LPS·CD14 complexes at 0.1 ng/ml (Fig. 8D) did not stimulate cells in CD14-depleted FCS. When additional LBP was provided, cell stimulation was observed, which cannot be explained by LBP catalyzed LPS·sCD14 complex formation, because preformed complexes were added in CD14 depleted medium. Again, stimulation was insensitive to anti-TLR4 (mAb HTA405) and therefore mediated by intracellular TLR4.Thus, at 0.1 ng/ml of LPS, LBP enhances the uptake of LPS·CD14 complexes, preformed in vitro (Fig. 8D) or formed by LBP-mediated catalysis (Fig. 8B), and this LBP-enhanced uptake results in HCAEC activation.LBP-stimulated LPS uptake into HCAEC involves a scavenger receptor pathwaySome scavenger receptors have been shown to be lipid A or LPS uptake receptors (31–33). Because HCAEC were found to be CD14 negative, we were curious whether scavenger receptor blocking agents would affect the LPS uptake in these cells. HCAEC were pretreated with dextran sulfate (MW 8000), polyinosinic acid (PIA), polyadenylic acid (PAA), or polycytidylic acid (PCA) for 30 min before measurement of 3H-LPS uptake with or without additional LBP. Both dextran and PIA reduced the LBP-enhanced 3H-LPS uptake to the level of basal 3H-LPS uptake, whereas the polycationic substances PAA and PCA lacked this effect (Fig. 9). Unfortunately, both dextran sulfate and PIA activated HCAEC to express surface CD62E even in the absence of added LPS (data not shown).DISCUSSIONTo begin a study of regulation and modification of TLR2 and TLR4 expression in HCAEC under various conditions, we performed FACS analyses on those cells, but despite the use of several different anti-TLR4 antibodies, we never observed positive surface staining for TLR4. Nevertheless, the cells responded well to both TLR2 and TLR4 agonists, and mRNA for both TLR2 and TLR4 was expressed. TLR2 mRNA was only weakly detectable in resting cells, but the TLR2 protein was still found on the HCAEC surface by FACS, confirming the high sensitivity of this method. After stimulation of the cells, TLR2 mRNA levels corresponded to TLR2 surface expression. However, this was not observed for TLR4. Despite high expression of TLR4 mRNA in both monocytes and HCAEC, TLR4 protein was never seen on the surface of HCAEC. The reliability of the detection system was confirmed in monocytes, which stained well for TLR2 and TLR4 on the surface, and in transfected HCAEC, where some TLR4 surface protein could be detected by FACS after transfection and overexpression of human TLR4.With the knowledge that TLR4 has been shown to reside in the Golgi apparatus in murine intestinal epithelial cells (28) and that LPS is transported to the Golgi in human epithelial HeLa cells (34), we further investigated HCAEC using confocal microscopy and found TLR4 located in a perinuclear region but not on the surface, where VCAM-1 was observed after cell stimulation. This is in contrast to monocytes, where TLR4 was clearly seen to colocalize with。