A proteasome inhibitor reduces concurrent, sequential, and long-term IL-1{beta}- and TNF-{alpha}-induced ECAM expression and adhesion

Nilesh M. Dagia and Douglas J. Goetz

Department of Chemical Engineering, Ohio University, Athens, Ohio 45701

Submitted 17 March 2003 ; accepted in final form 2 June 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A promising approach for reducing aberrant leukocyte-endothelial adhesion during pathological inflammation is to inhibit endothelial cell adhesion molecule (ECAM) expression at the transcription level. Several compounds have been shown to decrease cytokine-induced upregulation of ECAMs primarily by modulating the activity of transcription factors [e.g., nuclear factor-{kappa}B (NF-{kappa}B)]. The majority of the in vitro studies have focused on the effect of transcription inhibitors on endothelial cells exposed to a single cytokine [primarily tumor necrosis factor-{alpha} (TNF-{alpha})] for a relatively short period of time (primarily 4-6 h). However, in the in vivo setting, multiple cytokines [e.g., interleukin-1{beta} (IL-1{beta}) and TNF-{alpha}] may be present for extended periods of time. Thus we studied the effects of a transcription inhibitor, the proteasome inhibitor lactacystin, on ECAM expression and myeloid (HL60) cell adhesion to human umbilical vein endothelial cells (HUVEC) activated by concurrent, sequential, and long-term (24 h) treatment with IL-1{beta} and TNF-{alpha}. We show, for the first time, that lactacystin inhibits 1) 4-h concurrent IL-1{beta}- and TNF-{alpha}-induced expression of E-selectin, VCAM-1, ICAM-1, and HL60 cell adhesion to HUVEC; 2) 4-h TNF-{alpha}-induced expression of E-selectin, VCAM-1, and HL60 cell adhesion to HUVEC that have become desensitized to IL-1{beta} activation; 3) 24-h TNF-{alpha}-induced expression of E-selectin and VCAM-1 but not ICAM-1; and 4) 24-h TNF-{alpha}-induced HL60 cell adhesion to HUVEC. Combined, our results demonstrate that a proteasome inhibitor can reduce concurrent, sequential, and long-term IL-1{beta}- and TNF-{alpha}-induced ECAM expression and myeloid cell adhesion.

endothelial cell adhesion molecules; inflammation; cytokines; proteasome inhibitor


OVER THE PAST 15 YEARS, it has become well established that the expression of surface molecules on the vascular endothelium is altered at sites of pathological inflammation [e.g., inflammatory bowel disease (18) and atherosclerosis (31)]. In particular, endothelial cell adhesion molecules (ECAMs) known to participate in leukocyte recruitment (e.g., E-selectin, VCAM-1, and ICAM-1) have been shown to be upregulated in such settings and to contribute, by virtue of their role in leukocyte adhesion, to disease progression and/or tissue damage (20). The expression of these ECAMs is influenced by the cytokine milieu in which the endothelial cells reside. For example, treating cultured human umbilical vein endothelial cells (HUVEC) with the proinflammatory cytokines interleukin-1{beta} (IL-1{beta}) or tumor necrosis factor-{alpha} (TNF-{alpha}) for 4 h elicits expression of E-selectin, VCAM-1, and ICAM-1 (5).

The cytokine-induced expression of these ECAMs is regulated at the gene level by the activity of transcription factors. For example, the promoter regions of genes encoding for E-selectin, VCAM-1, and ICAM-1 all have binding sites for the transcription factor nuclear factor-{kappa}B (NF-{kappa}B) (19, 24, 32, 39). NF-{kappa}B is present in the cytoplasm of unstimulated endothelial cells and is rendered inactive due to its association with the inhibitory protein I{kappa}B (21). IL-1{beta} or TNF-{alpha} stimulation of endothelial cells induces the phosphorylation of I{kappa}B (21). The phosphorylated form of I{kappa}B is then ubiquitinated and degraded via the proteasome-dependent pathway, eventually leading to its dissociation from NF-{kappa}B (21). The resulting nuclear translocation of the active NF-{kappa}B leads to transcription, translation, and expression of a large number of NF-{kappa}B-dependent genes, which include the genes encoding for E-selectin, VCAM-1, and ICAM-1 (21).

Several current or potential therapeutics for pathological inflammation work, at least in part, by modulating the activity of transcription factors (8, 26, 27, 29, 34, 38). For example, proteasome inhibitors can suppress the TNF-{alpha}-induced expression of ECAMs by inhibiting the degradation of phosphorylated I{kappa}B and thus blocking NF-{kappa}B activation (1, 17, 29). The inhibition of ECAM expression contributes to a reduction in leukocyte adhesion and transmigration across the endothelium (1, 17, 29). Although a variety of in vitro studies have used transcription inhibitors to elucidate the mechanism of ECAM gene regulation, the scope of these studies, in terms of the use of the inhibitors as anti-inflammatory agents, is limited. Specifically, the majority of the studies have focused on the action of an inhibitor given to the endothelial cells exposed to a single proinflammatory cytokine (in nearly all cases, TNF-{alpha}) for a relatively short period of time (i.e., <24 h with the primary focus on 4-6 h) (1, 17, 26, 29, 38). As therapeutic in the in vivo setting, the transcription inhibitors will need to act on endothelial cells in an environment with more than one cytokine and on endothelial cells that are exposed to cytokines for extended periods of time. Additionally, although certain cytokines (e.g., IL-1{beta} and TNF-{alpha}) have somewhat similar effects on the endothelium, their effects do not appear to be identical. In particular, it has been observed that HUVEC that have become refractory to IL-1-induced expression of E-selectin can be induced to express E-selectin by TNF-{alpha} (28).

The above considerations led us to further probe the effect of a transcription inhibitor, the proteasome inhibitor lactacystin (17), on IL-1{beta} and TNF-{alpha} induction of ECAMs and consequent myeloid-endothelial cell adhesion. Specifically, we focused on determining whether lactacystin can inhibit ECAM expression and myeloid cell adhesion to 1) endothelial cells activated by concurrent treatment with IL-1{beta} and TNF-{alpha}, 2) endothelial cells activated by sequential treatment with IL-1{beta} and TNF-{alpha} (i.e., heterocytokine stimulation of endothelial cells that have become refractory to homocytokine stimulation), and 3) endothelial cells exposed to TNF-{alpha} for a relatively long time period (i.e., 24 h).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials. Medium 199 (M199), RPMI 1640 (RPMI), Hanks' balanced salt solution (HBSS) with Ca2+ and Mg2+ (HBSS+) or without (HBSS-), heat-inactivated defined fetal bovine serum (FBS), L-glutamine, trypsin-versene, and penicillin/streptomycin all were obtained from Biowhittaker (Walkersville, MD). Endothelial mitogen was obtained from Biomedical Technologies (Stoughton, MA). Gelatin was purchased from Difco Labs (Detroit, MI). Heparin, dimethyl sulfoxide (DMSO) (endotoxin <50 pg/ml DMSO), bovine serum albumin (BSA), O-phenylenediamine dihydrochloride (OPD), and phosphate citrate buffer tablets with sodium perborate were obtained from Sigma Chemical (St. Louis, MO). Formaldehyde (37% wt/wt) was purchased from Fisher Scientific (Florence, KY). BSA was added to HBSS+ to generate a HBSS+, 0.5% BSA buffer. This is referred to as assay buffer. Recombinant human IL-1{beta} (endotoxin <100 pg/µg IL-1{beta}), recombinant human TNF-{alpha} (endotoxin <100 pg/µg TNF-{alpha}), and synthetic lactacystin were purchased from Cal-Biochem (San Diego, CA). IL-1{beta} was prepared as 25 ng/ml stock solution, and TNF-{alpha} was prepared as 5 µg/ml stock solution. Lactacystin was prepared as 40 mM stock solution in DMSO. Each was stored in small aliquots at -80°C.

Antibodies. Function-blocking murine MAb HEL3/2 (anti-human E-selectin; IgG1) was a generous gift from Dr. Raymond T. Camphausen (Wyeth Laboratories, Cambridge, MA). Function-blocking murine MAb 51-10C9 (anti-human VCAM-1; IgG1) and murine MAb G46-2.6 (anti-human MHC class-I antigens; IgG1) were obtained from BD Pharmingen (San Diego, CA). Murine MAb R6.5 (anti-human ICAM-1; IgG2a) was kindly provided by Dr. Robert Rothlein (Boehringer Ingelheim, Ridgefield, CT). Murine MAb 15.2 (anti-human ICAM-1; IgG1) was obtained from Ancell (Bayport, MN). Murine MAb TS1/22 (anti-human LFA-1; IgG1) was obtained from Endogen (Woburn, MA). Horseradish peroxidase-conjugated goat F(ab')2 anti-mouse IgG polyclonal secondary antibody, used to detect the primary MAbs in the enzyme-linked immunosorbent assay (ELISA), was purchased from CalBiochem.

Cell culture. HUVEC were purchased from Clonetics (San Diego, CA) and maintained in culture as described previously (14). In brief, the HUVEC were cultured in M199 supplemented with 8% FBS, 100 µg/ml heparin, 50 µg/ml endothelial mitogen, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. This is referred to as HUVEC medium. HUVEC were subcultured on gelatin precoated 96-well tissue culture plates (Corning, Corning, NY) for ELISA, viability, and apoptosis assays, and on gelatin precoated 35-mm tissue culture dishes (Corning) for adhesion assays. All assays were performed with confluent HUVEC monolayers. HL60 cells were cultured in RPMI supplemented with 8% FBS, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. For the adhesion assays, HL60 cells were washed, resuspended to 1 x 108 cells/ml in RPMI, and held on ice (<4 h) until the time they were used in the flow adhesion assay. Before perfusion through the parallel plate flow chamber, HL60 cells were diluted to 5 x 105 cells/ml in assay buffer.

Lactacystin followed by individual and concurrent IL-1{beta} and TNF-{alpha} treatment of HUVEC. HUVEC were pretreated with 20 µM lactacystin or 0.05% DMSO (carrier control for lactacystin). After a 1-h incubation, lactacystin and DMSO were removed and the HUVEC were treated individually or concurrently with IL-1{beta} and TNF-{alpha} for 4 h. Postcytokine treatment, the ELISA and the adhesion assays were performed. Note that imparting targeting (9, 36) to the delivery of transcription inhibitors will greatly enhance the therapeutic potential of these reagents (see latter part of DISCUSSION and Ref. 11). Thus, to simulate the targeted delivery, we chose to treat the HUVEC with lactacystin in a pulse fashion (1-h pretreatment followed by removal) rather than continuously. In preliminary experiments, we determined the concentration of IL-1{beta} or TNF-{alpha} needed to give the maximal level of HL60 cell adhesion to HUVEC at 4 h postcytokine activation. All subsequent experiments were carried out with IL-1{beta} and/or TNF-{alpha} concentrations at or above the concentration needed to achieve a maximal level of adhesion (0.25 ng/ml for IL-1{beta} and 25 ng/ml for TNF-{alpha}).

Lactacystin followed by sequential IL-1{beta} and TNF-{alpha} treatment of HUVEC. HUVEC were treated with one of the cytokines (either IL-1{beta} or TNF-{alpha}) for 24 h. After this incubation, the initial cytokine was removed and HUVEC were treated with a fresh dose of either the same cytokine (homocytokine activation) or the other cytokine (heterocytokine activation). After a 4-h incubation with the fresh (either homo- or hetero-) cytokine, the ELISA and the adhesion assays were performed. To test the effect of lactacystin on heterocytokine activation, IL-1{beta} that was used for the first activation was removed at 23 h and the HUVEC were treated for 1 h with 20 µM lactacystin or 0.05% DMSO. After the 1-h incubation, lactacystin and DMSO were removed and the HUVEC were treated with TNF-{alpha} for 4 h. After the 4-h TNF-{alpha} activation, the ELISA and the adhesion assays were performed.

Lactacystin followed by long-term TNF-{alpha} treatment of HUVEC. HUVEC were pretreated with 20 µM lactacystin or 0.05% DMSO. After a 1-h incubation, lactacystin and DMSO were removed and the HUVEC were treated with either TNF-{alpha} or HUVEC medium alone for 24 h. After the 24-h incubation, the HUVEC incubated in medium alone were treated with TNF-{alpha} for 4 h. Post-TNF-{alpha} treatments, viability, apoptosis, ELISA, and/or adhesion assays were performed.

ELISA. ELISA was used to characterize the protein levels of adhesion molecules on HUVEC in a manner similar to that described previously (13). HUVEC were washed with cold HBSS+, fixed in 1% formaldehyde at 4°C for 20 min, washed with cold HBSS+, and incubated in cold M199 containing 8% FBS. Unless otherwise noted, from this point on all antibody dilutions and washes were carried out with M199 containing 8% FBS. Murine MAbs (primary MAbs) to ECAMs were added (10 µg/ml), and the HUVEC were incubated at 4°C for 20 min. After the incubation, the wells were washed and a peroxidase-conjugated polyclonal (secondary) antibody to mouse IgG was added (diluted 1:50). After a 20-min incubation at 4°C, the wells were washed and treated with OPD dissolved in phosphate citrate buffer containing sodium perborate. After a 10-min incubation at room temperature, the absorbance of the fluid in each well was determined at 450 nm using a microwell plate spectrophotometer (Molecular Devices, Sunnyvale, CA). In every experiment, each condition was run in triplicate wells.

Flow adhesion assays. A parallel plate flow chamber (Glycotech, Rockville, MD), similar to that described by McIntire, Smith, and colleagues (15), was used in this study. Our particular set up has been described previously (35). Temperature was maintained at 37°C with a heating plate. A 35-mm tissue culture dish containing a confluent HUVEC monolayer was loaded into the flow chamber. The flow chamber was mounted on an inverted microscope connected to a charge-coupled device videocamera, VCR, and monitor. After a brief rinse, HL60 cells (5 x 105 cells/ml in assay buffer) were drawn over the HUVEC monolayer at a shear stress of 1.8 dynes/cm2. To determine HL60 cell accumulation (HL60 cells/mm2 in figures), the number of HL60 cells adherent (either rolling or firmly adherent, i.e., not moving for 2 s) to the HUVEC monolayer in 8 different fields of view after 2.5 min of flow was determined, normalized to the area of the field of view, and averaged to give the result for that particular run. Such an assay was done n number of times (as indicated in the figure legends), and the values were averaged to give the results presented in the figures. In certain experiments, cytokine-activated HUVEC were pretreated with MAbs (10 µg/ml in HUVEC medium) 15 min before use in the adhesion assays.

Viability assay. A cell proliferation assay kit (Promega, Madison, WI), containing the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS tetrazolium salt), was used to assess the viability of the HUVEC. Viable cells reduced MTS to form a colored product. The protocol used was per the manufacturer's instructions. Briefly, a MTS/phenazine methosulfate (PMS) solution was added to the wells of a 96-well plate containing the HUVEC. After a 1-h incubation at 37°C, the absorbance of the fluid in each well was determined at 490 nm using the microwell plate spectrophotometer. In every experiment, each condition was run in at least six different wells.

Apoptosis assay. The Cell Death Detection ELISA Kit (Roche Applied Science, Indianapolis, IN) was used to assess apoptosis of HUVEC. The protocol followed was per the manufacturer's instructions. In brief, HUVEC monolayers were washed and treated with lysis buffer for 30 min at room temperature. The lysate was then centrifuged (200 g) for 10 min, and 20 µl of lysate supernatants were transferred to separate wells of a streptavidin-coated microplate. Subsequently, 80 µl of immunoreagent, containing a combination of anti-histone-biotin and anti-DNA-POD, was added to each well. After a 2-h incubation at room temperature, the wells were extensively washed and treated with 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) solution. After a 15-min incubation at room temperature, the absorbance of the fluid in each well was determined at 405 and 490 nm (reference reading) using the microwell plate spectrophotometer. In every experiment, each condition was run in triplicate wells.

Statistics. A single-factor analysis of variance (ANOVA) was used to assess the presence of statistical differences. If ANOVA indicated significant differences between conditions, a Bonferroni test was used for multiple pair-wise comparisons. P values <0.001 (for ELISA) and <0.05 (for adhesion, viability, and apoptosis assays) were considered statistically significant. Unless stated otherwise, all error bars represent SD.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lactacystin reduces 4-h concurrent IL-1{beta}- and TNF- {alpha}-induced expression of E-selectin, VCAM-1, ICAM-1, and HL60 cell adhesion. The proinflammatory cytokines IL-1{beta} and TNF-{alpha} may be present simultaneously at sites of inflammation. Thus we sought to determine the effect of lactacystin on 4-h concurrent IL-1{beta}- and TNF-{alpha}-induced expression of E-selectin, VCAM-1, and ICAM-1 on HUVEC. We chose to use HUVEC because these cells are widely used to investigate inflammation in vitro (e.g., citations of Ref. 12). As shown in Fig. 1, unactivated HUVEC did not appear to express E-selectin or VCAM-1 (Fig. 1, A and B) but did express ICAM-1 (Fig. 1C). Four-hour concurrent treatment of HUVEC with IL-1{beta} and TNF-{alpha} induced E-selectin and VCAM-1 expression and increased ICAM-1 expression (Fig. 1, A-C). Pretreatment of HUVEC with 20 µM lactacystin for 1 h before 4-h concurrent activation with IL-1{beta} and TNF-{alpha} significantly reduced the cytokine-induced expression of E-selectin, VCAM-1, and ICAM-1 (Fig. 1, A-C), relative to pretreatment with 0.05% DMSO (carrier control), which had no effect (Fig. 1, A-C).



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Fig. 1. Pretreatment of human umbilical vein endothelial cells (HUVEC) with 20 µM lactacystin for 1 h significantly reduces 4-h-concurrent interleukin-1{beta} (IL-1{beta})- and tumor necrosis factor-{alpha} (TNF-{alpha})-induced expression of E-selectin, VCAM-1, and ICAM-1 and HL60 cell adhesion. A-D: the protein levels of E-selectin (A), VCAM-1 (B), and ICAM-1 (C) on unactivated HUVEC and 4-h-concurrent IL-1{beta}- and TNF-{alpha}-activated HUVEC, with or without pretreatment with lactacystin, were determined by ELISA. A MAb to LFA-1 (TS1/22) served as a negative control (D). The level of absorbance, indicated by optical density (OD at 450 nm, y-axes), correlates with the level of endothelial cell adhesion molecule (ECAM) on the HUVEC. All values are means ± SD of triplicate wells. Results presented are representative of n = 4 separate experiments. E: lactacystin and DMSO-pretreated HUVEC were activated concurrently with IL-1{beta} and TNF-{alpha} for 4 h. Subsequently, HL60 cells were perfused over the HUVEC at 1.8 dynes/cm2 and the number of HL60 cells adherent to the HUVEC at the end of 2.5 min of flow was determined. All values are means ± SE of n>= 4 different runs. 1 h. Pre., pretreatment of HUVEC with 20 µM lactacystin (Lac), 0.05% DMSO (DMSO), or no pretreatment (-) for 1 h before concurrent activation with IL-1{beta} and TNF-{alpha}; 4 h. Act., treatment of HUVEC concurrently with IL-1{beta} and TNF-{alpha} (+) or no treatment (-) for 4 h before ELISA or adhesion assay. *P < 0.001; #P < 0.05.

 

To investigate the functional consequence of the above results, we studied the adhesion of HL60 cells to HUVEC under flow conditions. We chose to use HL60 cells, as opposed to freshly isolated leukocytes, because we were primarily interested in the effect of lactacystin on ECAM expression and adhesion as opposed to chemokine expression and transmigration. The HL60 cells we used exhibited little, if any, transmigration. As shown in Fig. 1E, a significant number of HL60 cells adhered to 4-h concurrent IL-1{beta}- and TNF-{alpha}-activated HUVEC, whereas very few, if any, HL60 cells adhered to unactivated HUVEC. Pretreatment of HUVEC with 20 µM lactacystin for 1 h before 4-h concurrent activation with IL-1{beta} and TNF-{alpha} significantly reduced HL60 cell adhesion (Fig. 1E), whereas pretreatment with 0.05% DMSO had little, if any, effect on HL60 cell adhesion (Fig. 1E). Combined, the results presented in this section clearly demonstrate, for the first time, that lactacystin can significantly reduce ECAM expression and HL60 cell adhesion to 4-h concurrent IL-1{beta}- and TNF-{alpha}-activated HUVEC.

Lactacystin inhibits 4-h TNF-{alpha}-induced expression of E-selectin and VCAM-1 and HL60 cell adhesion to HUVEC that have become refractory to IL-1{beta} activation. In addition to acting concurrently on the endothelium, IL-1{beta} and TNF-{alpha} may work together to sequentially activate the endothelium (28). Thus we next probed the effects of lactacystin on ECAM expression and HL60 cell adhesion to HUVEC activated by sequential treatment with these two cytokines. Previous studies have shown that HUVEC treated for 24 h with a cytokine (i.e., IL-1 or TNF-{alpha}) become desensitized, or refractory, to reinduction of E-selectin by a fresh dose of the same (homo) cytokine (28). The refractory HUVEC do, however, express E-selectin in response to reactivation by a heterocytokine (28). We sought to determine whether lactacystin could block the heterocytokine reactivation of HUVEC that have become refractory to homocytokine stimulation. Note that we focused on TNF-{alpha} activation of IL-1{beta}-refractory HUVEC because preliminary experiments indicated that IL-1{beta} treatment of TNF-{alpha}-refractory HUVEC had little effect on ECAM expression or myeloid cell adhesion (data not shown).

The level of E-selectin expression on HUVEC treated with IL-1{beta} for 24 h had returned (from the level at the 4-h IL-1{beta} timepoint) to near basal levels (Fig. 2A), whereas the levels of VCAM-1 and ICAM-1 (relative to unactivated HUVEC) (Fig. 2, B and C) remained elevated. Treatment of 24-h IL-1{beta}-activated HUVEC with a fresh dose of IL-1{beta} for 4 h caused a relatively small increase, compared with 4 h of IL-1{beta} activation alone, in E-selectin expression (Fig. 2A) and caused no increase in VCAM-1 or ICAM-1 expression (Fig. 2, B and C). The HUVEC had become desensitized, or refractory, to reactivation with IL-1{beta}. The IL-1{beta}-refractory HUVEC did, however, respond significantly to activation by TNF-{alpha}. Specifically, at 4 h post-TNF-{alpha} activation, the levels of E-selectin and VCAM-1 were both significantly increased (relative to the 24-h IL-1{beta} timepoint) (Fig. 2, A and B), whereas the level of ICAM-1 remained the same (Fig. 2C). Pretreatment of IL-1{beta}-refractory HUVEC with 20 µM lactacystin for 1 h before 4-h activation with TNF-{alpha} caused a significant reduction in TNF-{alpha}-induced expression of E-selectin and VCAM-1 (Fig. 2, A and B) compared with pretreatment with 0.05% DMSO, which had no effect (Fig. 2, A and B).



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Fig. 2. Pretreatment of IL-1{beta}-refractory HUVEC with 20 µM lactacystin for 1 h significantly reduces 4-h TNF-{alpha}-induced expression of E-selectin and VCAM-1 and HL60 cell adhesion. A-C: OD (450 nm). HUVEC were treated with various sequences of IL-1{beta}, lactacystin, and TNF-{alpha} before ELISA (A-C) or adhesion assays (D). Reading down the column underneath a given set of bars gives the sequence of treatments for that particular set of bars. A-C: the protein levels of E-selectin (A), VCAM-1 (B), and ICAM-1 (C) were determined by ELISA. A MAb to LFA-1 (TS1/22) served as a negative control (data not shown) and gave results similar to those shown in Fig. 1D. All values are means ± SD of triplicate wells. Results presented are representative of n>= 2 separate experiments. D: HL60 cell adhesion to the HUVEC was determined at 1.8 dynes/cm2. All values are means ± SE of n >= 3 different runs. 23 or 24 h. Act at t = 0, treatment of HUVEC with IL-1{beta} (IL-1) for 23 or 24 h or no treatment (-); 1 h. Treat. at t = 23 h, treatment of HUVEC with 20 µM lactacystin (Lac), 0.05% DMSO (DMSO), or no treatment (-) for 1 h before activation with fresh IL-1{beta} or TNF-{alpha}; 4 h. Act. at t = 24 h, treatment of HUVEC with IL-1{beta} (IL-1), TNF-{alpha} (TNF), or no treatment (-) for 4 h before ELISA or adhesion assay. *P < 0.001; #P < 0.05.

 

To investigate the functional consequence of the above results, we studied the adhesion of HL60 cells to the HUVEC. As shown in Fig. 2D, HL60 cell adhesion to 24-h IL-1{beta}-activated HUVEC was significantly less than the level of HL60 cell adhesion to 4-h IL-1{beta}-activated HUVEC and only slightly above the level of HL60 cell adhesion to unactivated HUVEC. Treatment of 24-h IL-1{beta}-activated HUVEC with a fresh dose of IL-1{beta} for 4 h had little effect on HL60 cell adhesion relative to the 24-h IL-1{beta} timepoint (Fig. 2D). The HUVEC had become refractory to IL-1{beta}-induced HL60 cell adhesion. Treatment of IL-1{beta}-refractory HUVEC with TNF-{alpha} for 4 h did, however, significantly increase the level of HL60 cell adhesion relative to the 24-h IL-1{beta} timepoint (Fig. 2D). Pretreatment of IL-1{beta}-refractory HUVEC with 20 µM lactacystin for 1 h before 4-h activation with TNF-{alpha} significantly reduced TNF-{alpha}-induced increased HL60 cell adhesion (Fig. 2D) relative to treatment with 0.05% DMSO, which had no effect (Fig. 2D).

Combined, the results presented in this section clearly demonstrate, for the first time, that lactacystin can inhibit ECAM expression and HL60 cell adhesion to HUVEC activated by sequential treatment with IL-1{beta} and TNF-{alpha}. Specifically, lactacystin can inhibit 4-h TNF-{alpha}-induced expression of E-selectin, VCAM-1, and myeloid cell adhesion to HUVEC that have become refractory to IL-1{beta} activation.

Lactacystin inhibits 24-h TNF-{alpha}-induced expression of E-selectin and VCAM-1 but not ICAM-1 and inhibits 24-h TNF-{alpha}-induced HL60 cell adhesion to HUVEC. Because there have been few, if any, investigations into the effect of transcription inhibitors on HUVEC exposed to cytokines for long periods of time (i.e., 24 h), we probed the effect of lactacystin on 24-h TNF-{alpha}-induced expression of ECAMs and myeloid cell adhesion to HUVEC. [Note that because HL60 cells exhibit very little adhesion to 24-h IL-1{beta}-activated HUVEC (Fig. 2D), we chose to focus on 24-h TNF-{alpha}-activated HUVEC.] In preliminary experiments, we determined the effect of 24-h TNF-{alpha} treatment, by itself or subsequent to a 1-h pretreatment with lactacystin, on HUVEC viability and apoptosis. Note that in these experiments and throughout this study, the lactacystin was removed from the HUVEC before the addition of TNF-{alpha}. As shown in Fig. 3, treatment of HUVEC for 24 h with TNF-{alpha} or pretreatment of HUVEC with lactacystin for 1 h, followed by treatment with TNF-{alpha} for 24 h, had no significant effect on viability (Fig. 3A) or apoptosis (Fig. 3B) relative to untreated HUVEC. These results are consistent with a previous report demonstrating that pretreatment of HUVEC with a combination of lactacystin and its aqueous derivative, {beta}-lactone, followed by 12-h treatment with TNF-{alpha}, has no effect on metabolic activity (2). Thus we proceeded to determine the effect of lactacystin on 24-h TNF-{alpha}-activated HUVEC.



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Fig. 3. Lactacystin pretreatment has no significant effect on the viability or apoptosis of 24-h-TNF-{alpha}-activated HUVEC. A: the viability of unactivated and 24-hTNF-{alpha}-activated HUVEC, with or without pretreatment with lactacystin, was determined. Wells containing only HUVEC medium (without HUVEC) served as a negative control. The level of absorbance correlates with the number of viable HUVEC on the monolayer. The level of absorbance for unactivated HUVEC was set at 100% in every experiment and was used to normalize the other data. Results presented are an average of 2 different experiments. No significant differences were detected between the left 3 bars. B: the level of apoptosis (as measured by histone release) of unactivated and 24-h-TNF-{alpha}-activated HUVEC, with or without pretreatment with lactacystin, was determined. Wells containing DNA-histone complex solution served as a positive control (right bar). The level of absorbance correlates with apoptosis. The level of absorbance for unactivated HUVEC was set at 100% in every experiment and was used to normalize the other data. Results presented are an average of 3 different experiments. No significant differences were detected between the left 4 bars. HUVEC, the wells contained HUVEC (+) or did not contain HUVEC (-). 1 h. Pre., pretreatment of HUVEC with 20 µM lactacystin (Lac), 0.05% DMSO (DMSO), or no pretreatment (-) for 1 h before activation with TNF-{alpha}. 24 h. Act., treatment of HUVEC with TNF-{alpha} (TNF) or no treatment (-) for 24 h before the viability or apoptosis assay. Error bars represent ± SE.

 

HUVEC treated with TNF-{alpha} for 24 h expressed a level of E-selectin that was slightly higher than the basal level (Fig. 4A) and expressed elevated levels of VCAM-1 and ICAM-1, relative to unactivated HUVEC (Fig. 4, B and C). Note that E-selectin expression at 24 h post-TNF-{alpha} activation was distinctly less than the level seen at 4 h post-TNF-{alpha} treatment (Fig. 4A vs. Fig. 2A, right-most bar). Pretreatment of HUVEC with 20 µM lactacystin for 1 h before 24-h activation with TNF-{alpha} significantly reduced the induced expression of E-selectin and VCAM-1 relative to pretreatment with 0.05% DMSO alone (Fig. 4, A and B) but had no effect on ICAM-1 expression (Fig. 4C). To investigate the functional consequence of the above results, we studied the adhesion of HL60 cells to the HUVEC. The level of HL60 cell adhesion to 24-h TNF-{alpha}-activated HUVEC was significantly higher than the level of adhesion to unactivated HUVEC (Fig. 4D). Pretreatment of HUVEC with 20 µM lactacystin for 1 h before 24-h activation with TNF-{alpha} significantly reduced HL60 cell adhesion relative to pretreatment with 0.05% DMSO alone, which had no effect (Fig. 4D).



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Fig. 4. Pretreatment of HUVEC with 20 µM lactacystin for 1 h significantly inhibits 24-h-TNF-{alpha}-induced expression of E-selectin and VCAM-1 but not ICAM-1 and significantly inhibits 24-h-TNF-{alpha}-induced HL60 cell adhesion. A-C: the protein levels of E-selectin (A), VCAM-1 (B), and ICAM-1 (C) on unactivated HUVEC and 24-h-TNF-{alpha}-activated HUVEC, with or without pretreatment with lactacystin, were determined by ELISA (OD 450 nm). A MAb to LFA-1 (TS1/22) served as a negative control (data not shown) and gave results similar to those shown in Fig. 1D. All values are means ± SD of triplicate wells. Results presented are representative of n = 5 separate experiments. D: lactacystin- and DMSO-pretreated HUVEC were activated with TNF-{alpha} for 24 h. Subsequently, HL60 cells were perfused over the HUVEC at 1.8 dynes/cm2, and the number of HL60 cells adherent to the HUVEC at the end of 2.5 min of flow was determined. All values are means ± SE of n >= 2 different runs. 1 h. Pre., pretreatment of HUVEC with 20 µM lactacystin (Lac), 0.05% DMSO (DMSO), or no pretreatment (-) for 1 h before activation with TNF-{alpha}. 24 h. Act., treatment of HUVEC with TNF-{alpha} (TNF) or no treatment (-) for 24 h before ELISA or adhesion assay. *P < 0.001; #P < 0.05.

 

To further probe and substantiate the above results (i.e., Fig. 4), we sought to determine whether lactacystin applied transiently for 1 h could reduce ECAM expression in response to TNF-{alpha} treatment applied 24 h later. Thus we pretreated HUVEC for 1 h with lactacystin and subsequently incubated the HUVEC in HUVEC medium for 24 h (as opposed to HUVEC medium with TNF-{alpha}, as was done in Fig. 4). After the 24-h rest period, we treated the HUVEC with TNF-{alpha} for 4 h and subsequently conducted an ELISA assay. As shown in Fig. 5 (left panels), lactacystin reduced 4-h TNF-{alpha} (applied 24 h postlactacystin)-induced E-selectin and VCAM-1 expression but had little effect on ICAM-1 expression. In line with other reports (1, 17), we confirmed (Fig. 5, right panels) that 1-h pretreatment with lactacystin immediately before 4-h TNF-{alpha} activation (as opposed to having a 24-h rest period before application of TNF-{alpha}, as was done in Fig. 5, left panels) inhibited E-selectin, VCAM-1, and ICAM-1 expression.



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Fig. 5. Pretreatment of HUVEC with lactacystin for 1 h followed by 24-h-incubation in HUVEC medium before 4-h-TNF-{alpha} activation reduces the induced expression of E-selectin and VCAM-1 but not ICAM-1 (left). In contrast, 1-h pretreatment of HUVEC with lactacystin immediately before 4-h-TNF-{alpha} activation reduces the induced expression of E-selectin and VCAM-1, as well as ICAM-1 (right). Expression was measured as OD (450 nm). HUVEC were treated with various sequences of lactacystin, HUVEC medium, and TNF-{alpha}. Reading down the column underneath a given set of bars gives the sequence of treatments for that particular set of bars. Subsequent to the treatments, the protein levels of E-selectin, VCAM-1, and ICAM-1 were determined by ELISA. A MAb to LFA-1 (TS1/22) served as a negative control (data not shown) and gave results similar to those shown in Fig. 1D. All values are means ± SD of triplicate wells. Results presented are representative of n >= 4 separate experiments. 1 h. Pre., pretreatment of HUVEC with 20 µM lactacystin (Lac), 0.05% DMSO (DMSO), or no pretreatment (-) for 1 h. 24 h. Rest, incubation (+) of HUVEC with HUVEC medium or no incubation (-) for 24 h before activation with TNF-{alpha}. 4 h. Act., treatment of HUVEC with TNF-{alpha} (TNF) or no treatment (-) for 4 h before the ELISA. *P < 0.001.

 

Combined, the results presented in this section clearly demonstrate, for the first time, that lactacystin inhibits E-selectin, VCAM-1 expression, and HL60 cell adhesion to HUVEC exposed to TNF-{alpha} for a relatively long period (i.e., 24 h) but does not have an effect on ICAM-1. These results are in contrast to lactacystin's effect on 4-h TNF-{alpha} activation of HUVEC immediately after treatment with lactacystin, where it is observed that lactacystin reduces expression of E-selectin and VCAM-1, as well as ICAM-1 (Refs. 1 and 17, and Fig. 5, right panels).


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The central role and increased expression of ECAMs have important implications for reducing aberrant leukocyte adhesion to the endothelium during pathological inflammation. Indeed, there has been an intense focus on the development of anti-adhesion-based therapeutics (16, 25). One class of compounds that has received considerable attention is those that diminish leukocyte adhesion by inhibiting ECAM expression at the transcription level. Over the past decade, several transcription inhibitors have been identified as potential anti-inflammatory agents (8, 26, 29, 34, 38). The majority of the in vitro studies have investigated the effects of transcription inhibitors under a narrow window of conditions (1, 17, 26, 29, 38). In this study, we extensively probed the effects of a transcription inhibitor, the proteasome inhibitor lactacystin, on IL-1{beta} and TNF-{alpha} activation of HUVEC. Specifically, we looked at the effects of lactacystin on ECAM expression and myeloid cell adhesion to endothelium exposed to concurrent, sequential, and long-term treatment with IL-1{beta} and TNF-{alpha}.

To our knowledge, there have been few, if any, investigations into the effect of lactacystin on concurrent or heterocytokine stimulation of endothelial cells. Our work clearly demonstrates that lactacystin can significantly reduce 4-h concurrent IL-1{beta}- and TNF-{alpha}-induced E-selectin, VCAM-1, and ICAM-1 expression (Fig. 1, A-C) and myeloid cell adhesion to HUVEC under physiological levels of fluid shear (Fig. 1E). These findings complement previous studies that have revealed that lactacystin can inhibit 4-h TNF-{alpha}-induced ECAM expression and leukocyte adhesion (1, 17). The data shown in Fig. 2 demonstrate that although 24-h IL-1{beta}-activated HUVEC are desensitized to reactivation by IL-1{beta} (i.e., IL-1{beta}-refractory HUVEC), they do respond to reactivation by TNF-{alpha} (heterocytokine activation), a result consistent with other reports (28). Clearly, lactacystin can inhibit 4-h TNF-{alpha}-induced expression of E-selectin and VCAM-1 (Fig. 2, A and B) and myeloid cell adhesion to IL-1{beta}-refractory HUVEC (Fig. 2D). These results reveal a role for the proteasome and suggest involvement of NF-{kappa}B in TNF-{alpha} stimulation of IL-1{beta}-refractory HUVEC. Interestingly, we found that treatment of 24-h TNF-{alpha}-refractory HUVEC with IL-1{beta} for 4 h elicited very little, if any, increase in E-selectin, VCAM-1, and ICAM-1 expression and myeloid cell adhesion (data not shown). The differences between this result and that reported previously (28) may be explained, in part, by the differences in concentrations of IL-1{beta} and TNF-{alpha} used in the assays.

Before the present study, there has been little, if any, investigation into the effects of transcription inhibitors on HUVEC exposed to cytokines for long periods of time (i.e., 24 h). We found that lactacystin can significantly reduce 24-h TNF-{alpha}-induced expression of E-selectin and VCAM-1 (Fig. 4, A and B) and 24-h TNF-{alpha}-induced myeloid cell adhesion to HUVEC (Fig. 4D). Lactacystin did not, however, suppress 24-h TNF-{alpha}-induced ICAM-1 expression (Fig. 4C). It appears that lactacystin applied transiently for 1 h continued to exert an effect at the 24-h time point because E-selectin and VCAM-1 expression was suppressed in the experiments described in Figs. 4 and 5, left panels. Thus the lack of ICAM-1 suppression at the 24-h time point suggests that TNF-{alpha} induction of ICAM-1 can occur independently of the proteasome and possibly independently of NF-{kappa}B activation. In support of this, we note that induction pathways parallel to NF-{kappa}B activation [i.e., c-Jun (22, 30)] have been identified, inhibition of the proteasome pathway has been shown to induce c-Jun-dependent transcription on fibroblast cells (23), and ICAM-1 expression can be induced through an NF-{kappa}B-independent mechanism by overexpression of c-Jun and c-Fos (37). Thus although our study clearly demonstrates that a proteasome inhibitor can reduce long-term TNF-{alpha} induction of E-selectin and VCAM-1 and myeloid cell adhesion, it also suggests a proteasome-independent mechanism for TNF-{alpha} induction of ICAM-1 that is detectable at 24 h postactivation but not at 4 h postactivation. This novel and rather unexpected finding highlights the fact that the effects of transcription inhibitors on short-term exposure of endothelial cells to cytokines such as IL-1{beta} and TNF-{alpha} cannot necessarily be extrapolated to what will occur for long-term exposure.

Although the expression of ECAMs clearly correlates with myeloid cell adhesion (Figs. 1, 2, and 4), a closer inspection of the data suggests that the relationship between lactacystin treatment and myeloid cell adhesion might be rather complex. For example, at the 4-h timepoint, lactacystin appears to reduce the IL-1{beta}- and TNF-{alpha}-induced E-selectin expression by ~32% (Fig. 1A) while inhibiting ~82% of the HL60 cell adhesion (Fig. 1E). In this context, we have made several other observations. First, function-blocking MAb experiments suggest that E-selectin and VCAM-1 (ICAM-1 was not tested) are involved in HL60 cell adhesion to 4-h concurrent IL-1{beta}- and TNF-{alpha}-activated HUVEC (data not shown). Second, HL60 cells that were adherent to 4-h concurrent IL-1{beta}- and TNF-{alpha}-activated HUVEC were predominantly (93 ± 1.74%; average ± SD; n = 4) firmly adherent. In contrast, less than half (48 ± 6.22%; average ± SD; n = 4) of the HL60 cells that were adherent to lactacystin-pretreated 4-h concurrent IL-1{beta}- and TNF-{alpha}-activated HUVEC were firmly adherent (i.e., the majority were rolling). Third, a significant number of HL60 cells exhibited a transient interaction (an interaction that lasted for a fraction of a second) with lactacystin-pretreated 4-h concurrent IL-1{beta}- and TNF-{alpha}-activated HUVEC (data not shown). Such interactions were not observed when HL60 cells were perfused over unactivated HUVEC (data not shown). Due to the transient nature of these interactions, the majority of the transiently adhesive HL60 cells were no longer adherent to the lactacystin-pretreated 4-h-concurrent IL-1{beta}- and TNF-{alpha}-activated HUVEC when the accumulation (i.e., the data in Fig. 1E) was measured.

Combined, the above observations suggest that the net accumulation of myeloid cells on the HUVEC involves a multistep cascade that relies on the interaction of the myeloid cells with E-selectin as well as another ECAM(s). Thus a plausible explanation of the observation in Fig. 1 is that although lactacystin does not completely eliminate 4-h-concurrent IL-1{beta}- and TNF-{alpha}-induced E-selectin expression (Fig. 1A), it does reduce induced expression of E-selectin, VCAM-1, and ICAM-1 (Fig. 1, A-C). These reductions, in aggregate, might be sufficient to cause a significant inhibition in the overall accumulation of HL60 cells on 4-h-concurrent IL-1{beta}- and TNF-{alpha}-activated HUVEC (Fig. 1E). Although this explanation is consistent with the data, it is quite conceivable that lactacystin has other effects on HUVEC (e.g., effects on receptor presentation) that may also contribute to the reduction in myeloid cell adhesion. Further studies are needed to completely understand the relationship between lactacystin treatment and the consequent effects on myeloid adhesion.

Overall, the results presented in this study bode well for using transcription inhibitors as anti-inflammatory agents to reduce aberrant leukocyte adhesion. However, it is important to note that these inhibitors can have other effects on endothelial cells in addition to suppression of ECAM expression (16, 25). The nonspecific effect of transcription inhibitors becomes even more problematic if they are administered systemically and could thus potentially affect all cells, in addition to the target endothelial cells. For example, NF-{kappa}B is a ubiquitous transcription factor, and genetic deletions of NF-{kappa}B subunits have been shown to cause lethality (3) and suppress immune response (33). Detrimental side effects caused by the nonspecific action of transcription inhibitors administered systemically lead to the idea of adopting targeting approaches to selectively deliver the transcription inhibitors to sites of inflammation. For example, a targeted drug delivery scheme could be employed wherein transcription inhibitors are incorporated into drug carriers that bear a ligand for a selectively expressed ECAM (4, 6, 7, 9, 11, 36). Ideally, once administered, the carriers would selectively bind to endothelium within inflamed tissue via the ligand-ECAM chemistry and not bind to other segments of the endothelium or other tissue. In this manner, the transcription inhibitors could be targeted to the site of pathological inflammation, thereby reducing the unwanted side effects due to the nonspecific action of the inhibitors administered systemically.

E-selectin is an attractive target for selective drug delivery (4, 6, 9, 10, 36) because it appears to be expressed at high levels on endothelium at sites of inflammation but minimally expressed on endothelium within normal tissue (10, 36). The present work provides an in vitro model to test the biological effect of E-selectin-targeted transcription inhibitors. Specifically, the results of our study clearly indicate that treating IL-1{beta}-refractory HUVEC with TNF-{alpha} elicits a second wave of E-selectin expression that can support myeloid cell adhesion (Fig. 2). Thus one can test the feasibility of the targeting approach by attempting to inhibit the TNF-{alpha}-elicited second wave of E-selectin expression and myeloid cell adhesion by using a transcription inhibitor-loaded drug carrier targeted to the first wave of E-selectin elicited by the initial cytokine activation with IL-1{beta}.

In summary, we have shown, for the first time, that the proteasome inhibitor lactacystin can 1) reduce 4-h-concurrent IL-1{beta}- and TNF-{alpha}-induced expression of E-selectin, ICAM-1, VCAM-1, and HL60 cell adhesion to HUVEC; 2) inhibit 4-h TNF-{alpha}-induced expression of E-selectin, VCAM-1, and HL60 cell adhesion to HUVEC that have become desensitized to IL-1{beta} activation; 3) inhibit 24-h TNF-{alpha}-induced expression of E-selectin and VCAM-1 but not ICAM-1; and 4) inhibit 24-h TNF-{alpha}-induced HL60 cell adhesion to HUVEC. Combined, our results demonstrate that a proteasome inhibitor can reduce concurrent, sequential, and long-term IL-1{beta}- and TNF-{alpha}-induced ECAM expression and myeloid cell adhesion.


    DISCLOSURES
 
This work was supported by an individual research grant from the Whitaker Foundation and the National Science Foundation [BES 9733542 (0096303)].


    FOOTNOTES
 

Address for reprint requests and other correspondence: D. J. Goetz, Dept. of Chemical Engineering, Ohio Univ., 172 Stocker Center, Athens, OH 45701 (E-mail: goetzd{at}ohio.edu).

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. Section 1734 solely to indicate this fact.


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