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Am J Physiol Heart Circ Physiol 278: H853-H861, 2000;
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Vol. 278, Issue 3, H853-H861, March 2000

LPS tolerance in human endothelial cells: reduced PMN adhesion, E-selectin expression, and NF-kappa B mobilization

Cameron W. Lush, Gediminas Cepinskas, and Peter R. Kvietys

Department of Physiology, University of Western Ontario, London, Ontario N6A 5C1; and Vascular Biology Program, London Health Sciences Centre, London, Ontario, Canada N6A 4G5


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cytokine release from inflammatory (CD14+) cells is reduced after repeated stimulation with lipopolysaccharide (LPS; LPS tolerance). However, it is not known whether LPS tolerance can be induced in CD14- cells. The aim of the present study was to determine whether endothelial cells [human umbilical vein endothelial cells (HUVEC)] could be rendered tolerant to LPS with respect to LPS-induced polymorphonuclear neutrophil (PMN) adhesion. LPS stimulation (0.5 µg/ml; 4 h) of naive HUVEC increased PMN adhesion. Pretreatment of HUVEC with LPS (0.5 µg/ml) for 24 h resulted in a reduction in the proadhesive effects of a subsequent LPS challenge. The initial LPS stimulation increased 1) mobilization of the nuclear transcription factor NF-kappa B to the nucleus and 2) surface levels of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and E-selectin. In LPS-tolerant HUVEC, a second LPS challenge resulted in 1) less accumulation of NF-kappa B in the nucleus, 2) a reduction in E-selectin expression, and 3) unchanged ICAM-1 expression. LPS-tolerant cells were still capable of mobilizing NF-kappa B in response to stimulation with either interleukin-1beta or tumor necrosis factor-alpha , resulting in elevated E-selectin levels and increased PMN adhesion. These studies show for the first time that LPS tolerance can be induced in endothelial cells with respect to PMN adhesion. This tolerance is specific for LPS and is associated with an inability of LPS to mobilize NF-kappa B, resulting in less E-selectin expression.

interleukin-1beta ; tumor necrosis factor-alpha ; intercellular adhesion molecule-1; inflammation; cell culture; polymorphonuclear neutrophil


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

SEPSIS IS A generalized systemic inflammatory response that involves multiple organ systems. The release of the endotoxin, lipopolysaccharide (LPS), from the cell wall of gram-negative bacteria is generally regarded as the initiating event in the development of sepsis (26). LPS exerts its effects by stimulating myeloid cells to release endogenous mediators involved in inflammation [tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta ]. Furthermore, LPS activates circulating neutrophils and promotes neutrophil adhesion to nylon fibers and endothelial cells (2, 9). Finally, LPS can activate the endothelium, converting it to a proadhesive phenotype, i.e., increased surface levels of adhesion molecules capable of engaging circulating leukocytes. Taken together, these observations indicate that LPS can facilitate neutrophil adhesive interactions within the microcirculation of various organs. The resultant acute inflammation can lead to multiple organ failure and, ultimately, death.

The proinflammatory effects of LPS are believed to be mediated by activation of the nuclear transcription factor NF-kappa B in both myeloid cells and endothelial cells (1, 25). In quiescent cells, NF-kappa B (a heterodimer consisting of p50 and p65 subunits) is sequestered in the cytoplasm bound to its inhibitory protein Ikappa B. In LPS- or cytokine-activated cells, NF-kappa B is disassociated from Ikappa B by phosphorylation and degradation (via the ubiquitin-proteasome pathway) of Ikappa B. Subsequently, NF-kappa B is translocated to the nucleus where it transactivates genes encoding various inflammatory mediators (e.g., TNF-alpha and IL-1beta ) in myeloid cells and adhesion molecules [e.g., E-selectin, intercellular adhesion molecule (ICAM)-1, and vascular cell adhesion molecule-1] on endothelial cells (1). Thus activation of NF-kappa B potentially amplifies the inflammatory response.

Paradoxically, there is a growing body of evidence indicating that repeated treatment of animals with LPS renders them resistant to LPS (LPS tolerance; see Ref. 32). In vitro studies have shown diminished inflammatory responses (e.g., TNF-alpha mRNA expression and production) in LPS-tolerant myeloid cells like monocytes and macrophages (11, 12, 33, 36). This development of LPS tolerance is not due to a downregulation of the LPS receptor, CD14, on these cells. Rather, LPS tolerance in CD14+ cells is associated with an abnormal mobilization of NF-kappa B to the nucleus in response to LPS. The ability of myeloid cells to become resistant to LPS stimulation has led to the suggestion that LPS could be used for prophylaxis of severe sepsis in patients at risk.

It is unknown whether LPS tolerance (with respect to inflammatory responses) can be induced in endothelial cells (CD14- cells). This is an important question considering the fact that endothelial cells line the microvasculature of all organs and actively participate in the recruitment of leukocytes. In the present study, we show for the first time that endothelial cells can develop LPS tolerance with respect to neutrophil adhesion, a pivotal initial step in the inflammatory response. Furthermore, in LPS-tolerant cells, there is less nuclear accumulation of NF-kappa B during the second LPS challenge and, subsequently, less E-selectin surface level expression. Finally, to gain further insights into the mechanisms involved, we tested the responsiveness of LPS-tolerant endothelial cells to stimulation with other inflammatory mediators (IL-1beta and TNF-alpha ). IL-1beta and TNF-alpha were capable of 1) mobilizing NF-kappa B to the nucleus, 2) increasing E-selectin expression, and 3) increasing polymorphonuclear neutrophil (PMN) adhesion. These latter studies indicate that the development of LPS tolerance in endothelial cells involves mechanisms proximal to intracellular signaling processes for NF-kappa B activation.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Endothelial cells. Human umbilical vein endothelial cells (HUVEC) were harvested from umbilical cords by collagenase treatment (Worthington Biochem, Freehold, NJ) as previously described (30). The cells were grown in medium 199 (M199; GIBCO, Burlington, Canada) supplemented with 10% heat-inactivated FCS (Intergen, Purchase, NY), 2.4 mg/l thymidine (Sigma Chemical, Oakville, Canada), 10 IU/ml heparin sodium, antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin; GIBCO), 1.5 µg/ml fungizone (GIBCO), and 80 µg/ml endothelial mitogen (Biomedical Technologies, Stoughten, MA). The cell cultures were incubated in room air with 5% CO2 at 37°C and 95% humidity and were expanded by brief trypsinization with 0.25% trypsin in PBS containing 0.025% EDTA. First-passage cells were used for all experiments.

Neutrophils. Human neutrophilic PMN were isolated from the venous blood of healthy adults using standard dextran sedimentation and gradient separation on Histopaque-1077 (Sigma; see Ref. 30). This procedure yields a PMN population that is 95-98% viable (trypan blue exclusion) and 98% pure (acetic acid-crystal violet staining).

PMN adhesion assays. For the static adhesion assay, isolated neutrophils were suspended in PBS buffer and radiolabeled by incubating the cells at 5 × 107 cells/ml with 50 µCi Na51CrO4/ml PMN suspension at 37°C for 60 min. The cells were then washed with cold PBS to remove unincorporated radioactivity. Radiolabeled PMN (5 × 105/well) were added to HUVEC monolayers grown in 48-well plates (Costar), and 30 min later the percentage of added PMN that remained adherent after a wash procedure was quantitated as follows: %PMN adherence = lysate (cpm)/[supernatant (cpm) + wash (cpm) + lysate (cpm)], where cpm is counts per minute (30).

To determine PMN adhesion under flow conditions, HUVEC monolayers cultured on polystyrene slides were exposed to shear stress as previously described (31). Briefly, the flow chamber consisted of a slide with a confluent HUVEC monolayer that was attached to a polycarbonate base. These two flat surfaces were held ~250 µm apart by a Silastic rubber gasket (Dow Corning, Midland, MI). Flow across the monolayer was controlled with a syringe pump (Harvard Apparatus, South Natick, MA). The wall shear stress was calculated using the momentum balance for a Newtonian fluid. The viscosity of water at 37°C was used as an approximation of the viscosity of M199 (0.007 poise). The wall shear stress along the HUVEC monolayer is equal to tau  = 3 µQ/2ba2, where tau  is the wall shear stress, µ is the coefficient of viscosity, Q is the flow rate, a is the channel height, and b is the channel width. PMN (106 cells/ml) were perfused over the HUVEC monolayers for 10 min, and the number of adherent neutrophils were counted.

HUVEC nuclear protein extraction. Nuclear protein was extracted from HUVEC as previously described (7, 23). Cells were grown to confluence in 75-cm2 flasks, scraped, washed with cold PBS, and incubated in 100 µl of 0.3% Nonidet P-40, 10 mM Tris (pH 8.0), 60 mM NaCl, 1 mM EDTA, 0.5 mM dithiothreitol (DTT), 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride (PMSF; 4°C) for 5 min on ice. Samples were centrifuged at 4°C for 5 min at 500 g. The supernatant was then removed, and the pellets (nuclei) were suspended in 100 µl of 10 mM Tris (pH 8.0), 60 mM NaCl, 1 mM EDTA, and 0.5 mM DTT (4°C) and centrifuged at 500 g for 5 min at 4°C. The nuclei were then extracted in 30-50 µl of 20 mM HEPES, 0.75 mM spermidine, 0.15 mM spermine, 0.2 mM EDTA, 2 mM EGTA, 2 mM DTT, 20% glycerol, and 1 mM PMSF (4°C) in the presence of 0.4 M NaCl and were incubated on ice for 20 min. Finally, the samples were centrifuged for 10 min at 500 g (4°C), and the supernatants were collected and saved as the nuclear protein fraction. Samples were stored at -80°C.

Electrophoretic mobility shift assay . The double-stranded oligonucleotide containing consensus (5'-<UNL>AGGGACTTCC</UNL>GCT<UNL>GGGGACTTTCC</UNL>-3') binding sites for NF-kappa B (synthesized on site; Beckman-Oligo 1000M DNA synthesizer) were end-labeled with [gamma -32P]ATP (Amersham) by using T4-polynucleotide kinase (MBI Fermentas, Flamborough, ON), as described previously (7, 23) . One picomole of the labeled oligonucleotide was incubated with 5 µg of nuclear extract protein in the presence or absence of 50× excess of cold oligonucleotide. Samples were incubated for 30 min at room temperature and then run through a 4% nondenaturing polyacrylamide gel at 300 V for 1 h. The gel was dried and then exposed to X-ray film (Kodak) for 16 h in cassettes with intensifying screens.

ICAM-1/E-selectin ELISA. For assessment of ICAM-1 and E-selectin surface level expression, an ELISA was performed (20) on HUVEC grown in 96-well cell culture plates (Corning). HUVEC were fixed in 4% paraformaldehyde at 4°C for 30 min. The cells were then washed two times with cold PBS and were incubated with the mouse primary monoclonal antibody (MAb) against either human ICAM-1 (Dako) or human E-selectin (CL3) at a concentration of 10 µg/ml for 30 min at room temperature. After this treatment, immunocytochemical staining of HUVEC monolayers was performed using an avidin-biotin-conjugated peroxidase mouse IgG staining kit (Vectastain), and MAb binding was subsequently quantified with a microplate reader (model 3550-UV; Bio-Rad) at 450-nm wavelength.

Experimental protocols. To determine whether HUVEC could develop LPS tolerance with respect to acute inflammatory events, we used an experimental protocol that has previously been shown to be effective in inducing LPS tolerance with respect to tissue factor (TF) production (6). HUVEC were pretreated with either M199 or LPS (0.5 µg/ml, Escherichia coli serotype 055:B5, Sigma) for a period of 24 h. Subsequently, the cells were washed two times with M199 and stimulated again with LPS (0.5 µg/ml) for 4 h, and various end points relevant to inflammation were assessed. These included 1) PMN adhesion to HUVEC under static and flow conditions, 2) surface levels of the adhesion molecules ICAM-1 and E-selectin (ELISA), 3) functional role of ICAM-1 and E-selectin in PMN adhesion (immunoneutralization with monoclonal antibodies R6.5 and 7A9, respectively), and 4) translocation of the nuclear transcription factor NF-kappa B to the nucleus [electrophoretic mobility shift assay (EMSA)]. As a positive control, HUVEC were stimulated with IL-1beta (0.1 ng/ml) for all experiments.

To assess whether NF-kappa B was involved in LPS-induced adhesion, two different inhibitors were used: MG-132 (Calbiochem) and pyrrolidinedithiocarbamate (PDTC; Sigma). One hour before LPS stimulation (0.5 µg/ml), HUVEC were incubated with either 10 µM MG-132 or 100 µM PDTC. Subsequently, the cells were washed and exposed to LPS for 4 h, and PMN adhesion was assessed. To assess whether LPS-tolerant HUVEC were cross-tolerant to other inflammatory mediators (or if the phenomenon was specific to LPS), HUVEC were pretreated for 24 h with either LPS (0.5 µg/ml) or M199, washed two times with M199, and incubated with either IL-1beta (0.1 ng/ml) or TNF-alpha (10 ng/ml) for 4 h, and various indexes of acute inflammation were assessed. These included 1) PMN adhesion, 2) surface levels of ICAM-1 and E-selectin, and 3) NF-kappa B mobilization to the nucleus.

Statistical analysis. All values are presented as means ± SE. For both adhesion and ELISA assays, treatments were performed in triplicate for each experiment. Statistical analysis (GraphPad Software) was performed using ANOVA and a paired two-sided Student's t-test (with Bonferroni corrections for multiple comparisons). P < 0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

LPS pretreatment reduces both PMN adhesion and E-selectin expression upon further stimulation . As shown in Fig. 1A, stimulation of endothelial cell monolayers with LPS (0.5 µg/ml) for 4 h resulted in a significant increase in PMN adhesion in a static adhesion system. HUVEC that were pretreated for 24 h with LPS (0.5 µg/ml) and then stimulated for 4 h with a second dose of LPS (0.5 µg/ml) exhibited a 47% reduction in PMN adherence compared with the LPS response in nonpretreated HUVEC. Similar results were obtained when PMN were interacted with HUVEC under shear stress (Fig. 1B). Taken together, these findings indicate that HUVEC can develop tolerance to LPS in terms of PMN adhesion under both static and flow conditions. With the knowledge of the number of PMN and endothelial cells that were interacted under static and flow conditions, a comparison of the LPS-induced PMN adhesion to HUVEC under these two conditions can be made (Table 1). In the static system, LPS-induced adhesion was equivalent to 5.4 ± 0.118 PMN/endothelial cell, whereas in the flow system LPS-induced adhesion was equivalent to 1.05 ± 0.082 PMN/endothelial cell at 0.6 dyn/cm2 and 0.31 ± 0.079 PMN/endothelial cell at 1.2 dyn/cm2. The static system, rather than the flow system, was employed to assess mechanisms involved in the development of LPS tolerance in HUVEC because 1) a greater number of PMN adhered to HUVEC in the static system than the flow system, and 2) the static system is more convenient to use than the flow system for the planned experiments.



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  		Fig. 1.   Effects of lipopolysaccharide (LPS) pretreatment of human umbilical vein endothelial cells (HUVEC) on the subsequent LPS-induced polymorphonuclear neutrophil (PMN) adherence to HUVEC under both static (A) and flow (B) conditions (0.6 and 1.2 dyn/cm2). Endothelial cells were preincubated with LPS (0.5 µg/ml) or medium 199 (M199; -) for a period of 24 h, washed, and then stimulated with LPS (0.5 µg/ml) for 4 h, and PMN adherence was determined (24 h LPS/4 h LPS). All values are expressed as means ± SE (n = 4). * Significant (P < 0.05) difference compared with the LPS response in nonpretreated cells (Student's paired t-test).


                              
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  		Table 1.   Comparison of the number of adherent PMN per endothelial cell under static and flow conditions

To determine whether this LPS tolerance was associated with changes in surface level expression of ICAM-1 and E-selectin on HUVEC, the surface levels of these adhesion molecules on tolerant and nontolerant HUVEC were assessed using an ELISA. As shown in Fig. 2A, HUVEC surface levels of ICAM-1 significantly increased after 4 h of LPS stimulation. After 24 h of LPS pretreatment, ICAM-1 levels remained significantly elevated and were not altered with further LPS stimulation for 4 h. E-selectin levels also significantly increased with 4 h of LPS stimulation (Fig. 2B). However, after the 24-h pretreatment, surface levels of E-selectin had returned to near control levels. Upon further stimulation with LPS, there was a blunted increase in E-selectin expression compared with those cells that received no pretreatment.



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  		Fig. 2.   Effects of LPS pretreatment of HUVEC on the subsequent surface level expression of intercellular adhesion molecule (ICAM)-1 (A) or E-selectin (B) in response to LPS stimulation. HUVEC were pretreated for 24 h with LPS (0.5 µg/ml), washed, and stimulated with LPS (0.5 µg/ml) for 4 h, and ICAM-1 or E-selectin surface expression was measured (ELISA). All absorbance values are expressed as means ± SE (n = 3). * Significant (P < 0.05) difference in the LPS response between LPS-pretreated and nonpretreated HUVEC (Student's t-test with Bonferonni corrections). OD, optical density.

As previously reported using static adhesion assays (5, 13), both MAb against either ICAM-1 or E-selectin were effective in partially inhibiting the PMN adhesion induced by stimulation of untreated HUVEC with LPS for 4 h (Fig. 3A). However, in LPS-tolerant HUVEC (HUVEC exposed to LPS for 24 h) only the MAb directed to ICAM-1 was effective in inhibiting the PMN adhesion to HUVEC induced by the secondary LPS stimulation (Fig. 3B). The E-selectin MAb had no effect on the LPS-induced adhesion in tolerant HUVEC. These findings are consistent with the observation that there is reduced E-selectin expression in LPS-tolerant HUVEC, although ICAM-1 levels remain elevated (see Fig. 2).



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  		Fig. 3.   Effects of blocking monoclonal antibodies (MAb) on PMN adhesion to naive (A) and tolerant (B) HUVEC. A: naive HUVEC monolayers were challenged with LPS for 4 h, washed, and incubated with either ICAM-1 MAb (35 µg/ml) or E-selectin (E-sel) MAb (10 µg/ml) for 20 min. Subsequently, PMN adherence was determined. All values are expressed as means ± SE (n = 3-5). * Significant (P < 0.05) difference compared with the LPS response (Student's t-test with Bonferonni corrections). B: tolerant HUVEC monolayers (24 h pretreatment with LPS) were challenged with LPS for 4 h, washed, and incubated with either ICAM-1 MAb (35 µg/ml) or E-selectin MAb (10 µg/ml) for 20 min. Subsequently, PMN adherence was determined. All values are expressed as means ± SE (n = 3-5). * Significant (P < 0.05) difference compared with the LPS response in tolerant HUVEC (LPS/LPS; Student's t-test with Bonferonni corrections).

LPS tolerance in HUVEC is associated with a reduced ability to mobilize NF-kappa B. Because cytokine-induced increases in adhesion molecule expression on HUVEC have been shown to be dependent on NF-kappa B activation (8, 21-23), we assessed whether activation of NF-kappa B was involved in LPS-induced PMN adhesion to HUVEC in our system. Naive HUVEC were preincubated with two different inhibitors of NF-kappa B activation (MG-132 or PDTC) for 1 h before LPS stimulation for 4 h. As shown in Fig. 4, HUVEC that were pretreated with MG-132 or PDTC exhibited significantly less PMN adherence upon LPS stimulation compared with nonpretreated cells. As a control, cells were treated with the vehicles for both inhibitors (PBS for PDTC and DMSO for MG-132), a procedure that had no effect on PMN adhesion to HUVEC (data not shown).



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  		Fig. 4.   Effect of inhibition of nuclear factor (NF)-kappa B activation on LPS-induced PMN adherence to naive HUVEC. Endothelial cells were pretreated with the NF-kappa B inhibitor MG-132 (10 µM; A) or pyrrolidinedithiocarbamate (PDTC, 100 µM; B) for 1 h, washed, and then stimulated for 4 h with LPS (0.5 µg/ml), and PMN adhesion was determined. All values are expressed as means ± SE (n = 4). * Significant (P < 0.05) difference compared with the LPS response in nonpretreated cells (Student's paired t-test).

On the basis of the fact that NF-kappa B appears to play an important role in the LPS-induced PMN adhesion to HUVEC, in the next series of experiments we assessed LPS-induced NF-kappa B mobilization to the nucleus in tolerant and nontolerant HUVEC. Figure 5 shows that, after 4 h of LPS stimulation, NF-kappa B was mobilized to the nucleus of HUVEC (lane 2). In cells that received the LPS pretreatment (tolerant HUVEC), the level of NF-kappa B in the nucleus had returned to near control levels 24 h later (lane 1 vs. lane 4). Further stimulation of tolerant HUVEC with LPS (lane 3) resulted in minimal mobilization of NF-kappa B. Competition studies were also performed (data not shown) whereby nuclear extracts were preincubated with unlabeled consensus sequence oligonucleotides (50× concentrated), resulting in the absence of bands upon incubation with labeled probe.


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  		Fig. 5.   Mobilization of NF-kappa B to the nucleus of tolerant and nontolerant HUVEC. Cells were rendered tolerant by pretreatment with LPS (0.5 µg/ml) for 24 h. After stimulation for 4 h with LPS (0.5 µg/ml), nuclear extracts were obtained and incubated with 32P-labeled human NF-kappa B consensus oligonucleotides, electrophoresed, and exposed to X-ray film. Densitometric analysis [OD/mm2, corrected for control background-(-/-)] revealed the following results: -/LPS = 215; LPS/LPS = 56; LPS/- = 17. Qualitatively similar experiments were obtained in 3 additional experiments.

LPS-tolerant HUVEC are not cross-tolerant to TNF-alpha or IL-1beta . To further probe for possible mechanisms involved in the development of LPS tolerance in HUVEC, PMN adhesion to LPS-tolerant and nontolerant HUVEC induced by cytokines was examined. It was found that HUVEC pretreated for 24 h with LPS (0.5 µg/ml) and then stimulated with either IL-1beta (0.1 ng/ml) or TNF-alpha (10 ng/ml) demonstrated no significant differences compared with nonpretreated cells in terms of PMN adherence (Fig. 6). Furthermore, the IL-1beta - and TNF-alpha -induced increases in the surface level expression of both ICAM-1 and E-selectin on HUVEC were similar in LPS-tolerant and nontolerant HUVEC (Fig. 7). Finally, as shown in Fig. 8, an EMSA of the nuclear extracts from IL-1beta (0.1 ng/ml)- or TNF-alpha (10 ng/ml)-stimulated HUVEC indicated that there was little difference in the level of mobilization of NF-kappa B in LPS-pretreated and nonpretreated cells (lane 2 vs. lane 3).


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  		Fig. 6.   Effects of cytokines on PMN adhesion to LPS-tolerant and nontolerant HUVEC. HUVEC were pretreated for 24 h with LPS (0.5 µg/ml), washed, and stimulated with either interleukin (IL)-1beta (0.1 ng/ml) or tumor necrosis factor (TNF)-alpha (10 ng/ml) for 4 h, and PMN adherence was determined. All values are expressed as means ± SE (n = 3).




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  		Fig. 7.   Effects of LPS pretreatment of HUVEC on the subsequent surface level expression of ICAM-1 (A) or E-selectin (B) in response to cytokine stimulation. HUVEC were pretreated for 24 h with LPS (0.5 µg/ml), washed, and stimulated with either IL-1beta (0.1 ng/ml) or TNF-alpha (10 ng/ml) for 4 h, and ICAM-1 or E-selectin surface expression was measured (ELISA). All absorbance values are expressed as means ± SE (n = 4-5).



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  		Fig. 8.   Nuclear mobilization of NF-kappa B in LPS-tolerant HUVEC in response to cytokine treatment. Cells were pretreated with LPS (0.5 µg/ml) for 24 h followed by stimulation for 4 h with IL-1beta (0.1 ng/ml; A) or TNF-alpha (10 ng/ml; B). Nuclear extracts were obtained and incubated with 32P-labeled human NF-kappa B consensus oligonucleotides, electrophoresed, and exposed to X-ray film. Densitometric analysis [OD/mm2, corrected for control background (-/-)] revealed the following results: -/IL-1beta  = 158 and LPS/IL-1beta  = 126 (A); -/TNF-alpha  = 151 and LPS/TNF-alpha  = 170 (B). Qualitatively similar experiments were obtained in 2 additional experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

During gram-negative bacterial infections, the release of large amounts of endotoxin (LPS) can result in a systemic inflammatory response (fever, tachycardia, hypotension, leukopenia) that can lead to eventual multiple organ failure and death. It is generally believed that LPS induces a systemic inflammatory response by stimulating the secretion of various proinflammatory cytokines (TNF-alpha and IL-1beta ) from inflammatory cells of the monocytic lineage and by activating the endothelium (inducing a proadhesive phenotype). These events facilitate neutrophil sequestration and infiltration in various organ systems. If unchecked, this inflammatory reaction leads to organ dysfunction and ultimate failure. One therapeutic approach that has received widespread attention is to exploit the fact that various inflammatory cells can be rendered resistant to LPS stimulation after repeated challenges (LPS tolerance). Tolerance to LPS in vivo is a well-established phenomenon (2, 3, 10, 24, 35) characterized by reductions in 1) cytokine secretion by inflammatory cells and 2) neutrophil adhesion to endothelial cells of various organs. In vitro approaches have been applied to dissect out the mechanisms involved in the development of LPS tolerance in monocytes and macrophages. However, to date, there is little known regarding the role of the endothelium in the development of LPS tolerance despite its important role in the inflammatory process. Thus, in the present study, we developed an in vitro model of LPS tolerance in endothelial cells (HUVEC) whereby pretreatment with LPS resulted in a reduced PMN adhesion to HUVEC upon further stimulation with LPS.

As previously reported (9), treatment of HUVEC for 4 h with LPS induced an increase in PMN adhesion (Fig. 1). However, if the HUVEC were pretreated with LPS for 24 h and rechallenged with LPS, there was a reduced adhesive response. The results of these experiments indicate that endothelial cells can become tolerant to the proinflammatory effects of LPS in terms of PMN adhesion. The only other study to indicate that HUVEC can become tolerant to LPS is that of Busso et al. (6). They showed that pretreatment of HUVEC with LPS renders HUVEC hyporesponsive in terms of TF expression, a coagulation factor. Thus the significance of our study is that we have developed a model to study the development of tolerance to LPS in HUVEC that is more directly relevant to the recruitment of neutrophils to inflamed tissues.

The decreased adhesiveness of LPS-tolerant HUVEC is most likely due to an impairment of the typical increase in surface levels of E-selectin in response to LPS stimulation (Fig. 2). After an LPS challenge, surface levels of ICAM-1 increase and remain elevated for 24 h, and a subsequent LPS challenge has no effect. By contrast, the LPS-induced increase in E-selectin returns to near control levels by 24 h, and further stimulation with LPS results in a blunted increase in surface levels of E-selectin. Furthermore, our results using functional blocking MAb directed to E-selectin and ICAM-1 indicate that, although both adhesion molecules contribute to the LPS-induced increase in PMN adhesion to naive HUVEC (Fig. 3A), only ICAM-1 contributes to the PMN adhesion in LPS-tolerant HUVEC (Fig. 3B). To our knowledge, this is the first demonstration that the development of LPS tolerance in HUVEC involves an impairment of E-selectin expression and function.

Activation of the nuclear transcription factor NF-kappa B is believed to play an important role in the LPS- or cytokine-induced increases in adhesion molecule expression on endothelial cells and leukocyte adherence (21, 22). Our observations that 1) LPS stimulated NF-kappa B mobilization to the nucleus of naive HUVEC and 2) two different proteasome inhibitors (PDTC and MG-132) blunted the LPS-induced increase in PMN adhesion to naive HUVEC (Fig. 4) provide additional support for this contention. More importantly, our observations indicate that the development of LPS tolerance in HUVEC involves an impairment in the ability of HUVEC to mobilize NF-kappa B in response to LPS stimulation. We show that the secondary LPS stimulation of LPS-tolerant HUVEC resulted in a severely blunted mobilization of NF-kappa B to the nucleus (Fig. 5). Our findings are consistent with previous studies on tolerance development in monocytes and macrophages that indicate that LPS-tolerant cells are unable to mobilize NF- kappa B to the nucleus in response to further LPS stimulation (14, 16, 27, 35). Conversely, Ziegler-Heitbrock et al. (34) found that tolerant monocytes were still capable of mobilizing NF-kappa B to the nucleus but that it was inactive since it consisted primarily of p50 homodimers. Taken together, the majority of previous studies on tolerance development in inflammatory cells and the present study using endothelial cells support the contention that LPS-tolerant cells have an impaired ability to mobilize NF-kappa B to the nucleus in response to a subsequent LPS challenge.

Previous studies on myeloid cells indicate that LPS-tolerant cells, although unresponsive to LPS, do respond to other cytokines with respect to NF-kappa B mobilization to the nucleus (14, 16). By contrast, one recent study indicated that LPS-tolerant macrophages are totally depleted of their pool of NF-kappa B and thus would be unresponsive to further stimulation by any relevant agent (4). The findings of the present study using endothelial cells are more consistent with the former observations. We show that LPS-tolerant HUVEC are capable of mobilizing NF-kappa B to the nucleus in response to IL-1beta or TNF-alpha stimulation (Fig. 8). Furthermore, we show that the mobilized NF-kappa B is functionally active in inducing a proadhesive phenotype (i.e., p50/p65 heterodimer). The increase in E-selectin expression and PMN adhesion in response to IL-1beta or TNF-alpha challenge was similar in naive and tolerant HUVEC (Figs. 6 and 7). These data suggest that LPS tolerance in our model appears to be LPS specific, i.e., TNF-alpha and IL-1beta are still capable of stimulating LPS-tolerant endothelial cells. These observations also indicate that the development of LPS tolerance in HUVEC with respect to PMN adhesion occurs at some point upstream to NF-kappa B activation. It is possible that LPS and cytokines such as TNF-alpha and IL-1beta use different intracellular signaling pathways to mobilize NF-kappa B. Kohler and Joly (14) found that, although LPS failed to induce Ikappa B kinase activity in LPS-tolerant cells, TNF-alpha was able to rapidly induce its activity, indicating the use of distinct pathways. Although the exact signal transduction pathways in HUVEC for each mediator (LPS, TNF-alpha , or IL-1beta ) remain to be clarified, our data suggest that at least some components of these pathways are distinct from one another.

One additional point to be made is that the development of LPS tolerance involves different mechanisms from the development of tolerance to other stressors, i.e., ischemia-reperfusion (15, 29). In a recent study, we showed that HUVEC could develop tolerance to anoxia-reoxygenation (A/R; an in vitro counterpart to ischemia-reperfusion) with respect to PMN adhesion to HUVEC (7). Two major differences are worth addressing. First, the A/R-induced adhesion to HUVEC occurs within 30 min and does not require activation of NF-kappa B (7). This is in contrast to the LPS-induced adhesion to HUVEC, which occurs hours after challenge and requires NF-kappa B activation (Fig. 4). Second, the development of A/R tolerance requires NF-kappa B activation, i.e., if NF-kappa B activation or translocation is prevented, A/R tolerance with respect to PMN adhesion does not develop (7). This is in contrast to the development of LPS tolerance, which involves an inability of the second LPS challenge to activate/translocate NF-kappa B (Fig. 5). Thus the development of A/R tolerance involves intracellular events downstream of NF-kappa B activation (transcription of relevant genes), whereas the development of LPS tolerance involves as yet uncharacterized intracellular events upstream of NF-kappa B activation.

From a clinical/therapeutic perspective, three aspects of the present study may appear, at first glance, to be relatively disappointing. First, the PMN adhesion to LPS-tolerant HUVEC is not completely prevented in response to the second stimulation with LPS (Fig. 1). Second, the development of LPS tolerance involves modulation of E-selectin, rather than ICAM-1 (Fig. 2). Third, the LPS-induced tolerance appears to be specific for LPS, i.e., IL-beta and TNF-alpha can still induce a proadhesive phenotype in LPS-tolerant HUVEC (Figs. 6-8). However, there are several reasons for why our findings may be of significance to the development of LPS tolerance in vivo. First, in terms of PMN adhesion, we would argue that any degree of prevention of PMN adhesion to endothelium of organs during sepsis would be of benefit to the host. Second, although ICAM-1/CD18 adhesion interactions are important for eventual emigration of PMN into the interstitium, a prerequisite for ICAM-1/CD18 adhesive interactions is the tethering of PMN to endothelial cells via the selectins. The importance of selectin-mediated tethering of PMN to the endothelium is exemplified by the protection afforded by MAb to the selectins against tissue injury induced during inflammation (18, 19, 28). Finally, with respect to LPS tolerance being ineffective in preventing IL-1beta and TNF-alpha induction of a proadhesive phenotype in endothelial cells, it must be pointed out that LPS is generally considered to be the initiating mediator in sepsis, and interference with LPS-mediated events could provide the host with protection against the sequence of events induced by LPS. Finally, our recent preliminary studies indicate that pretreatment of animals with LPS only partially prevents polymicrobial sepsis (peritonitis)-induced PMN accumulation in the heart, but completely reverses sepsis-induced myocardial dysfunction (17).

In conclusion, our findings indicate that endothelial cells can develop tolerance to the proinflammatory effects of LPS in terms of PMN adhesion. This reduced proadhesive phenotype in tolerant cells can be attributed to reduced surface level expression of the neutrophil adhesion ligand E-selectin. Our data also indicate that this reduction in E-selectin expression is due to a lack of mobilization of NF-kappa B in response to LPS stimulation in tolerant HUVEC. These events may play a significant role in the reduced neutrophil sequestration and infiltration in various organ systems noted in LPS tolerance models in vivo. Thus future attempts at the development of therapeutic modalities for the amelioration of sepsis-induced multiple organ failure should also take into consideration the contribution of the endothelium to the development of LPS tolerance.


    ACKNOWLEDGEMENTS

We thank Dr. Trevor Archer, London Regional Cancer Centre for the synthesis of nuclear factor-kappa B oligonucleotides and Ronald Noseworthy for technical assistance. Dr. Robert Rothlein generously provided monoclonal antibody (MAb) R6.5, Dr. Paul Kubes supplied us with MAb 7A9, and Dr. Donald Anderson provided us with MAb CL3.


    FOOTNOTES

This work was supported by Grants MT-13940 and MT-13668 from the Medical Research Council of Canada.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: P. R. Kvietys, London Health Sciences Centre, Vascular Biology Program, 375 South St., Rm. C210, London, Ontario, Canada N6A 4G5 (E-mail: pkvietys{at}julian.uwo.ca).

Received 12 May 1999; accepted in final form 13 October 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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