Discriminatory effects of protein kinase inhibitors and calcium ionophore on endothelial ICAM-1 induction.

Discriminatory effects of protein kinase inhibitors and calcium ionophore on endothelial ICAM-I induction CHRISTINA L. MYERS, SUDHAI N. DESAI, JOSEPHINE SCHEMBRI-KING, GORDON L. LETTS, AND ROBERT W. WALLACE Boehringer Ingelheim Pharmaceuticals, Ridgefield, Connecticut 06877 Myers, Christina L., Sudhai N. Desai, Josephine Schembri-King, Gordon L. Letts, and Robert W. Wallace. Discriminatory effects of protein kinase inhibitors and calcium ionophore on endothelial ICAMinduction. Am. J. Physiol. 262 (Cell Physiol. 31): C365-C373,1992.-Intercellular adhesion molecule 1 (ICAM-1) is a proinflammatory adhesion glycoprotein induced by cytokines such as interleukin-lp (ILlp) and tumor necrosis factor-a (TNF-cu) as well as lipopolysaccharide (LPS). Little is known, however, concerning the intracellular regulatory mechanisms that modulate ICAMexpression in endothelial cells. We probed the involvement of protein kinase function and intracellular calcium ion upon ICAMexpression of human umbilical vein endothelial cells activated alternatively by TNF-cu, IL-l@, LPS, or phorbol 12myristate 13-acetate (PMA). Methodologies for the detection of ICAMincluded both enzyme-linked immunosorbent assay

as a surface receptor for the major group of rhinoviruses 6, 24). Endothelial ICAMhas been shown in vivo (2) and in vitro (19,ZO) to be markedly upregulated by proinflammatory cytokines such as interleukin-lp (IL-lp), tumor necrosis factor-a (TNF-cu), and interferon-y (IFN-T), as well as the tumor promotor and protein kinase C agonist phorbol 12-myristate 13-acetate (PMA) (12). ICAMexpression is dependent on protein and mRNA synthesis (2). While pharmacological modulation of the expression of the adhesion molecule during inflammation might be an attractive therapeutic objective, little is known regarding intracellular signals that are involved in triggering ICAMexpression. Therefore, we sought to investigate the role of intracellular calcium ion and protein and immunoprecipitation from biosynthetically labeled cells. kinase function in ICAMexpression on human umbilThe protein kinase inhibitor H-7 blocked induction of ICAMical vein endothelial cells (HUVEC) induced by the cy1 by all of the activators; nonlinear regression analysis revealed tokines IL-lp and TNF -cy and by lipopolysaccharide 50% inhibitory concentration (I&) values of 6-10 PM. Another (LPS). The involvement of these intracellular signaling kinase inhibitor, HA1004, did not block expression of the intermediates was probed with a divalent cation ionoadhesionmoleculeat concentrations up to 50 PM. In contrast, phore and multiple protein kinase inhibitors possessing the kinase inhibitor staurosporine dose dependently inhibited various selectivities for cyclic nucleotide-dependent proICAMexpression triggered by PMA (I& 67 t 4 nM) but, at tein kinases and calcium/phospholipid-dependent prosimilar concentrations, did not inhibit ICAMexpression induced by the other inflammatory stimuli. The divalent cation tein kinase C (PKC). We report that although protein kinase(s) plays a role in ICAMinduction by each of ionophore ionomycin (0.5 PM) interacted synergistically with these physiological activators, the mechanism(s) by PMA but not with cytokines or LPS in upregulating ICAM-1. which IL-l@, TNFcy, or LPS induces ICAMon HUWe conclude from these data that although PMA-induced ICAMexpression may be triggered through activation of VEC differs from that of PMA in that a PKC-mediated pathway is not the exclusive or predominant pathway protein kinase C, ICAMinduction by IL-l& TNF-(u, or LPS utilized by inflammatory stimuli. may involve distinct regulatory pathway(s). calcium/phospholipid-dependent protein kinase C; interleukinlb; lipopolysaccharide;phorbol ester; tumor necrosisfactor-a

MATERIALS

including leukocyte emigration out of the vasculature to sites of inflammation, are adhesion-dependent processes involving recognition between complementary cell surface adhesion proteins. One such protein is intercellular adhesion molecule 1 (ICAM-l), found on vascular endothelial cells and other nonhematopoietic cells such as fibroblasts and epithelial cells as well as some leukocytic cells such as macrophages and lymphocytes (2). ICAMis a ligand for lymphocyte function-associated antigen 1 (LFA-1) (15) and for Mac1 (l), glycoproteins expressed broadly among hematopoietic cell types. Interaction between ICAM- and LFAl/Mac-l is one of few documented examples of a heterophilic interaction between a member of the immunoglobulin family (ICAM-1) and members of the integrin family (LFA-1 and Mac-l) and is a critical step in the inflammatory process. ICAMhas also been identified

1MateriaLs. RPM1 1640 medium (standard and methionine free), L-glutamine, 0.05% trypsin/0.53 mM EDTA, phosphatebuffered saline (PBS), N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) buffer solution, porcine heparin, and penicillin-streptomycin were purchased from GIBCO Laboratories (Grand Island, NY). Endothelial cell growth supplement was obtained from Bionetics Research (Rockville, MD). Bovine calf serum (iron supplemented) was bought from Hyclone Laboratories (Logan, UT). The following reagents were purchased from Sigma Chemical (St. Louis, MO): fatty acidfree bovine serum albumin (BSA), lipopolysaccharide from Escherichia cob 026:B6, paraformaldehyde, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), polymixin B sulfate, &mercaptoethanol, Sepharose 4B, tris(hydroxymethyl)aminomethane (Tris), bovine hemoglobin, sodium azide, and Triton X-100. PMA was purchased from Biomol (Plymouth Meeting, PA). Other supplies from Calbiochem (La Jolla, CA) were mouse monoclonal antibody to human coagulation factor VIII, 1-(5-isoquinolinesulfonyl)-2methylpiperazine dihydrochloride (H-7), N-@-guanidino-

VARIOUS

IMMUNE

RESPONSES,

0363-6143/92

$2.00

Copyright

0 1992

the American

AND METHODS

Physiological

Society

C365

Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (132.174.254.155) on November 30, 2018. Copyright © 1992 the American Physiological Society. All rights reserved.

C366

STIMULUS-SELECTIVE

MODULATION

ethyl)-5isoquinolinesulfonamide (HAlO04), staurosporine, and ionomycin. Glycerol, sodium fluoride, and sodium pyrophosphate were from Fisher Scientific (Fair Lawn, NJ). Dialyzed fetal bovine serum was obtained from Irvine Scientific (Santa Ana, CA). Sodium dodecyl sulfate (SDS), SDS-polyacrylamide gel electrophoresis (PAGE) molecular weight standards (40,000-200,000), and bromphenol blue were obtained from Bio-Rad Laboratories (Richmond, CA). Acetylated lowdensity lipoprotein labeled with l,l’-dioctadecyl-l-3,3,3’,3’tetramethylindocarbocyanine perchlorate was purchased from Biomedical Technologies (Stoughton, MA). ,&Galactosidaselabeled goat anti-mouse immunoglobulin G (IgG) was from Southern Biotechnology Associates; p-nitrophenyl-P-D-galactopyranoside was from Bethesda Research Laboratories (Gaithersburg, MD). Collagenase type II was supplied by Worthington Biochemical (Freehold, NJ). The following were dissolved in dimethyl sulfoxide (DMSO): PMA at 1 mg/ml, ionomycin at 1 mM, and staurosporine at 1 mM. The compounds were further diluted in RPM1 before use. All other compounds were diluted in water or RPMI. Human recombinant IFN-7 (2 x 10’ U/mg as measured by encephalomyocarditis virus infection in A549 cells) and human recombinant TNF-CY (6 x lo7 U/mg as measured by TNFinduced cytotoxicity on mouse L-929 cells) were provided by Dr. Gunther Adolf (Ernst-Boehringer Institiit fur ArzneimittelForschung, Vienna, Austria). Human recombinant IL-lp (gene engineered by British Biotechnology Limited, Oxford, UK) expressed in E. coLi (Ernst-Boehringer Institiit fur Arzneimittel-Forschung) was purified to homogeneity by Dr. Peter Grob (Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT). Affinity-purified polyclonal antisera to human TNF-cu and IL-lb were obtained from Endogen (Boston, MA). Mouse anti-human ICAM(R6.1 and R6.5) monoclonal antibodies (22, 23) were provided by Dr. Robert Rothlein (Boehringer Ingelheim Pharmaceuticals). The 14E5 hybridoma cell line which secretes a mouse anti-human endothelial cell monoclonal antibody was obtained from American Type Tissue Collection (Rockville, MD). Cell culture. HUVEC were isolated by the method of Jaffe et al. (10). Briefly, umbilical veins were cannulated, filled with 0.1% collagenase solution, and incubated for 20 minutes at 37°C. Contents were emptied into conical tubes containing media (see below) and centrifuged at 200 g for 5 min. The pelleted material was plated into Primaria Falcon flasks (Becton Dickinson, Oxnard, CA), and cells were allowed to attach for several hours, after which the medium was changed to remove blood components and nonadherent cells. Growth media consisted of RPM1 containing 20% heat-inactivated calf serum, 15 mM HEPES, 50 pg/ml endothelial cell growth supplement, 100 pg/ml heparin, 2 mM glutamine, and 100 units/ ml penicillin-streptomycin. At confluence, cells were passaged by brief trypsinization. Cells were characterized as endothelial by their cobblestone morphology, presence of factor VIII-associated antigen as determined by immunofluorescence (4), and uptake of acetylated low-density lipoprotein (27). Cells were used between passages 2 and 10. Cells retained all of the above criteria at all passages. Enzyme-linked immunosorbent assay (ELISA) for ICAMexpression. ICAMexpression was measured by an ELISA method described previously (22). Briefly, confluent HUVEC in Primaria brand 96-well microtiter plates were incubated overnight or for the indicated time in 10% serum/RPM1 with various concentrations of activators with or without other additions as noted in the text. After incubation, cells were washed with RPM1 and fixed with 1% paraformaldehyde for 15 min, then incubated with a “blocking” solution of 2% BSA in RPMI, followed by incubation with a 1:l mixture of R6.1 and R6.5 hybridoma-conditioned media. Next a 1:250 dilution

OF

ICAM-

EXPRESSION

of the developing antibody (goat anti-mouse IgG P-galactosidase conjugate) was applied followed by incubation with the substrate (0.476 mg/mlp-nitrophenyl-P-D-galactopyranoside in 0.1 M Tris buffer, pH 8.5, containing 1 mM MgCl, and 0.0005% BSA). All incubations were at 37OC, and washing with RPM1 media was performed between each incubation except after the 2% BSA blocking solution. Chromophore development was determined by measuring optical density at 410 nm with a Dynatech Microtiter plate reader. Wells were read against blank controls containing cells incubated without primary (anti-ICAM1) and/or secondary (developing) antibodies. Reported data are derived from optical density readings which fall along the linear portion of the development curve. Viability assay. Quantitative calorimetric assays for mammalian cell survival and proliferation were performed as previously described (16, 25). This assay is based on the ability of mitochondrial succinate dehydrogenases to cleave MTT to the blue product formazan. Cells in 96-well microtiter plates were incubated overnight (-16 h) with various activators and/or other agents as noted in the text. Cells were washed and incubated with 1 mg/ml MTT in 10% serum/RPM1 for 4 h (100 pi/well). The product was solubilized with 60 ~1 of 10% Triton X-100 in 0.01 N HCl, aided by a period of incubation at 37°C and mixing by pipette. Bubbles were removed by adding 20 ~1 of 95% ethanol, and the resultant colored product was read on a Dynatech plate reader at 570 nm. Immunoprecipitation. HUVEC grown to confluency in 35mm-diameter Petri dishes were washed with PBS and incubated for 30 min in methionine-free RPM1 containing 10% dialyzed bovine serum and 20 mM HEPES. Various activators with or without H-7 or staurosporine were added as indicated in the text, followed by 10 ~1 of [‘“Slmethionine (-1,000 Ci/ mmol; Amersham, Arlington Heights, IL) to provide a final concentration of 100 &i/ml. Cells were incubated for 6 h at 37°C followed by washing with PBS and lysis at 4°C (10 mM Tris HCl, 0.02% NaN3, 1% Triton X-100, 10 mM sodium pyrophosphate, 140 mM NaF, 1% bovine hemoglobin). Insoluble material was sedimented by centrifugation. The lysate was incubated for 4 h with R6.5-coupled Sepharose 4B (23). The beads were washed five times with lysis buffer diluted 1:lO with 10 mM Tris saline, pH 8.0, five times with 10 mM Tris saline, and twice with 50 mM Tris HCl, pH 6.8. Protein was eluted from the beads by boiling in 0.5 M Tris. HCl, pH 6.8, containing 4% SDS, 5% ,&mercaptoethanol, 5% glycerol, and 0.04% bromphenol blue. SDS-PAGE was carried out according to the method of Laemmli (11) on 7.5% acrylamide gels. Fluorography was conducted with Enlightening autoradiography enhancer (New England Nuclear Research Products, Boston, MA) according to the manufacturer’s instructions. Immunoprecipitated ICAMwas identified based on its identical migration on SDS polyacrylamide gels with purified ICAMprovided by Dr. Steven Marlin, Boehringer Ingelheim Pharmaceuticals. Two-dimensional electrophoresis. HUVEC were labeled with [‘?]methionine and treated with various activators as described above. The cells were then solubilized in lysis buffer (10 mM Tris, pH 7.4, 0.3% SDS), and ribonuclease A and deoxyribonuclease I (Kendrick Labs, Madison, WI) were added at a final concentration of 50 and 100 pg/ml to degrade RNA and DNA, respectively. SDS boiling buffer was then added to give a final concentration of 5% SDS, 5% ,&mercaptoethanol, 10% glycerol, and 60 mM Tris, pH 6.8, and the sample was placed in a boiling water bath for 5 min. Equal aliquots of the sample were resolved by two-dimensional electrophoresis according to the method of O’Farrell(l8) by Kendrick Labs. First dimension isoelectric focusing was carried out in glass tubes (2 mm) using 2.0% Resolytes ampholines (pH 4-8, BDH from Hoeffer Scientific Instruments) for 9,600 volt-hours. The final pH gradient in the gel was measured by a surface electrode and


STIMULUS-SELECTIVE

MODULATION

ranged from pH 4.0 to 8.5. After equilibration for 10 min in SDS sample buffer (10% glycerol, 50 mM dithiothreitol, 2.3% SDS, and 0.0625 M Tris, pH 6.8), the tube gels were sealed to the top of 10% polyacrylamide gel slabs (0.75 mm) and electrophoresis was carried out for -4 h at 12.5 mA/gel. The following molecular mass markers were used as standards: myosin, 200 kDa; phosphorylase b, 97.4 kDa; BSA, 69 kDa; ovalbumin, 46 kDa; carbonic anhydrase, 30 kDa; and lysozyme, 14.3 kDa. After electrophoresis, the gels were fixed overnight in a solution of 10% acetic acid and 50% methanol, rehydrated for 1 h in 10% acetic acid, and dried onto filter paper. Autoradiography was carried out using Kodak X-OMAT AR film. RESULTS

Various cell activators induce ICAMon HUVEC. ICAMexpression on the surface of endothelial cell monolayers, as measured by ELISA, was detectable but modest on unactivated cells (Table 1). Upon stimulation with either TNF-CY, IL-l& IFN-7, or LPS, the level of ICAMexpression increased significantly. Of all the activators that induce ICAMexpression, IFN-7 was the least efficacious; the largest induction was in response to treatment with TNF-a. In contrast to ICAM1 expression, the various activators induced only small changes in the surface expression of another antigen defined by l4E5 monoclonal antibody (6), suggesting that the activating agents increase ICAMexpression specifically rather than by generally increasing overall protein biosynthesis. Maximal stimulation of ICAM- 1 expression as observed upon TNF-cu stimulation could not be augmented by costimulation with any one or more of the other activators. Figure 1 shows that induction of ICAMby TNF-a, IL-lp, or LPS was concentration dependent, with 50% induction occurring at lo-30 units/ml TNF-cw, 0.03-0.1 rig/ml IL-l& and 30-100 rig/ml LPS. Treatment of the endothelial cells with PMA also induced the expression of ICAM-1, with half-maximal induction occurring between 1 and 3 rig/ml. Induction was also time dependent over a period of several hours; ICAM- expression peaked at 8 h and remained elevated for up to 72 h in the continued presence of cytokine or LPS (data not shown). Table 1. Induction of ICAMby various cell activators

on HUVEC ODm

Treatment ICAM-

Control TNF-cu IL-l@ IFN-7 LPS TNF-cu TNF-cu TNF-cu

+ IL-l@ + IL-l@ + IL-lp

+ LPS + LPS

+ IFN-7

0.178kO.028 1.23OkO.078 0.876t0.063 0.414t0.030 0.729&O. 109 1.163t0.127 1.202t0.272 1.231kO.206

14E5

0.647 0.604 0.581 0.568 0.568 0.640 0.577 0.536

Values are means k SD of 4 [intracellular adhesion molecule 1 (ICAM-I)] and 2 replicate wells (14E5). OD410, optical density at 410 nm. Confluent human umbilical vein endothelial cells (HUVEC) were treated for 16 h with the indicated activators [tumor necrosis factor-a (TNF-a), 100 units/ml; interleukin-lj3 (IL-lb), 1 rig/ml; interferon-y (INF-r), 250 units/ml; lipopolysaccharide (LPS), 1 pg/ml]. Enzymelinked immunosorbent assay (ELISA) was carried out as described in MATERIALS AND METHODS. Data are representative of 2 experiments.

OF

ICAM-

EXPRESSION

C367

As shown in Table 2, ICAMexpression in response to TNF-cu or IL-lp could be antagonized only by the cytokine-neutralizing antibodies specific for the cytokine that was used as a stimulus. Neutralizing antibodies to IL-l@, TNF-cr, or the combination were ineffective in blocking PMA-induced ICAMexpression. This suggests that the cytokines and PMA are not stimulating the production and secretion of a common stimulus, such as TNF-cu, which could subsequently act through an autocrine mechanism. Prior heat inactivation of the cytokines (99OC, 1 h) abolished their ability to induce ICAMexpression. In addition, expression of ICAMin response to LPS, but not in response to PMA or various cytokines, could be selectively abolished by coincubation with 100 pg/ml polymixin B sulfate (data not shown). Effect of stimulation by various activators on the total composition of cellular proteins. To determine the effect of TNF-a, IL-l& PMA, and LPS on the composition of proteins in HUVEC, we labeled the cells with [3”S]methionine and subjected the cellular proteins to twodimensional electrophoresis. Figure 2 shows that there are few changes in the overall protein composition in cells treated with either TNF-cu, IL-lo, or LPS. In fact, we observed increased amounts of only two proteins which had molecular weights between 14,000 and 30,000 and p1 values between 6.5 and 7.5. PMA treatment had a more significant effect on the overall protein pattern; the same two proteins increased in intensity along with an additional five proteins whose intensity did not increase when the cells were stimulated with the cytokines or LPS. The identities of these proteins are not known. Figure 2F shows the migration of immunoprecipitated ICAMon the two-dimensional electrophoretic system. As expected for a glycoprotein, ICAMexhibits charge heterogeneity during isofocusing. This characteristic, along with its relative low abundance compared with the other cellular proteins, makes it impossible to identify ICAMwith certainty in the gels containing total cellular proteins. However, it is clear from this experiment that the increase in cell surface ICAM-1, as measured by ELISA, is not due to a generalized increase in overall protein biosynthesis. ICAMinduction by TNF-cu, IL-l& LPS, or PMA is inhibited by protein kinase inhibitors. To assessthe importance of protein kinase function on ICAMexpression, cells were incubated with various protein kinase inhibitors (H-7, HA1004, or staurosporine) immediately before and during activation with cytokine, LPS, or PMA. Treatment of HUVEC with 50 PM H-7, a PKC antagonist, resulted in virtually complete inhibition of ICAMinduction by all four activators (Fig. 3). The effect of H-7 was dose dependent; nonlinear regression analysis of dose-response curves (data not shown) (9) provided half-maximal inhibitory concentrations (I&,) relative to ICAM-I upregulation as follows: 10 t 1 PM (TNF-a), 6 t 1 PM (IL-lo), 8 t 1 PM (LPS), and 7 t 1 PM (PMA) (n = 3 for all stimuli). Another kinase inhibitor that is more specific for cyclic nucleotide-dependent kinases, HA1004, exhibited minimal effects on ICAMexpression induced by any of the activators, including PMA (Fig. 3). This is consistent with the inability of


C368

STIMULUS-SELECTIVE

MODULATION

OF

TNF-a

ICAM-

EXPRESSION

IL-l@

1.8,

1.8,

I

I

0.6

3.0 Concentration

Concentration

(units/ml)

LPS

1 .o

0.6-

PMA

1

0 Concentration

(rig/ml)

0.1

(rig/ml)

0.3 Concentration

Table 2. Effect of neutralizing antibodies to IL-lp and TNF-cu upon endothelial ICAA4-1 induction by PMA Treatment

OLo

Control Anti-TNF-cu Anti-IL-l@ IL-l@ (1 rig/ml) +Anti-IL-lp +Anti-TNF-cu TNF-cu (100 units/ml) +Anti-IL-16 +Anti-TNF-tu PMA (10 rig/ml) +Anti-IL-18 +Anti-TNF-cu +Anti-IL-lp and anti-TNF-cu

0.057&0.012 0.059t0.013 0.082kO.035

0.755Iko.090 0.121t0.011 1.022kO.072 1.182&0.123 1.102t0.107 0.233t0.033

1.106t0.077 1.084kO.125 1.134kO.077 1.316t0.074

Values are means _+ SD of 4 replicate wells. Similar results were obtained in another experiment. PMA, phorbol 12-myristate 13-acetate. Other definitions are as in Table 1. Neutralizing antibodies were used at a final dilution of l:20.

cyclic nucleotide analogues (dibutyryl-CAMP, dibutyrylcGMP, %bromo-CAMP, and &bromo-cGMP, 1 mM each), a phosphodiesterase inhibitor (3-isobutyl-l-methylxanthine, 1 mM), and an adenylate cyclase activator (forskolin, 100 PM) to stimulate endothelial ICAMexpression (data not shown). In contrast, treatment of HUVEC with staurosporine inhibited PMA-induced ICAMexpression (I&, 67 t 4 nM, n = 3) but failed to diminish TNF-W-, IL-& or LPS-stimulated ICAM1 expression even at 100 nM (Fig. 3). The effect of varying the time of administration of H7 with respect to cell activation,by IL-lp is shown in Fig. 4. Maximal inhibition by H-7 was associated with administration during the first hour of an 8-h induction period; inhibition was dramatically diminished when administered later (~2 h) in the induction period. Similar

1

3

10

Fig. 1. Dose-dependent induction of intercellular adhesion molecule 1 (ICAM1) on human umbilical vein endothelial cells (HUVEC) by various activators. HUVEC were stimulated with the indicated concentrations of tumor necrosis factor-a (TNF-c~), interleukin-lb (ILlfi), lipopolysaccharide (LPS), or phorbol 12-myristate 13-acetate (PMA) for 16 h. Cell surface ICAMwas detected by enzyme-linked immunosorbent assay (ELISA) as described in MATERIALS AND METHODS. Data are reported in units of optical density (OD 410; means & SD) derived from 4-8 replicate wells. Similar results were obtained in 2 other experiments.

30

(rig/ml)

results were obtained when TNF-cw or PMA was utilized as activator (data not shown). Effect of H-7 and staurosporine on the biosynthesis of ICAM-I. To ascertain whether the protein kinase inhibitors were exerting their effects proximal or distal to de novo synthesis of ICAM-1, cells were labeled with [“5S]methionine as described in MATERIALS AND METHODS and ICAMwas immunoprecipitated using a specific antibody. Figure 5 shows autoradiograms of the total cellular proteins (Fig. 5A ) and immunoprecipitated ICAM(Fig. 5B) from labeled cells stimulated with TNF-cw in the absence or presence of H-7 or staurosporine. Treatment with TNF-(U induced the biosynthesis of ICAM-1, and the induction could be blocked by treating the cells with cycloheximide. As expected, treatment with cycloheximide completely inhibited new protein biosynthesis as indicated by a complete loss of :“S-labeled cellular proteins (Fig. 5A, lane 3) and immunoprecipitated ICAM(Fig. 5B, lane 3). H-7 inhibited the biosynthesis of ICAM- as effectively as cycloheximide (Fig. 5B, lanes 4 and 5) but had no discernible effect on overall protein biosynthesis (Fig. 5A, lanes 4 and 5). ICAMbiosynthesis was not blocked when the TNF-cu-stimulated cells were treated with staurosporine, although the amount of ICAM- was slightly decreased (Fig. 5B, lanes 6 and 7). This may be due to the slight inhibitory effect of staurosporine on overall protein biosynthesis (Fig. 5A, lanes 6 and 7). A number of protein bands consistently coprecipitated with ICAM-1; their identity is unknown. The effect of H-7 and staurosporine on ICAMbiosynthesis in cells stimulated with cytokines as well as LPS and PMA was also determined. Table 3 shows that H-7 was very effective in inhibiting the biosynthesis of ICAMirregardless of the cell stimulus. Staurosporine, on the other hand, was also very effective in inhibiting ICAMbiosynthesis in response to PMA stimulation,


STIMULUS-SELECTIVE

MODULATION

OF ICAM-

EXPRESSION

C369

6946-

c

20097-

D

6946-

L

976946-

F

:*

Fig. 2. Effect of various activators on total protein composition of HUVEC. Cells were labeled with [“S]methionine m the presence of various activators for 6 h, and the cellular proteins were resolved by two-dimensional electrophoresis. A: no stimulus; B: TNF-ol (100 units/ml); C: IL-l@ (1 rig/ml); D: PMA (10 rig/ml); E: LPS (1 pg/ml); F: immunoprecipitated ICAM-1. Mobility of the molecular weight standards (X10-“) is indicated along left. Approximate pH gradient generated during isofocusing is indicated along bottom. Autoradiographs in A-E received equal exposure times; autoradiograph in F received twice the exposure time of those in A-E. Arrows indicate protein spots that are more intense than observed in the unstimulated sample (A).

I.,--’

;

t

but considerably less effective in cells stimulated by the cytokines and LPS. The inhibition by staurosporine observed in response to the latter group of stimuli may be due to the general protein synthesis-inhibiting effects of staurosporine as observed in Fig. 5, while the inhibition observed in PMA-stimulated cells may be due to antagonism of PKC. Ionomycin acts synergistically with PMA. The role of calcium ion in the induction of ICAMwas investigated with the use of the ionophore ionomycin. Ionomycin (0.5 PM) by itself produced little or no effect on basal (unstimulated) ICAMexpression (Table 4). However, the ionophore synergistically augmented expression of the adhesion molecule induced by a suboptimal concentration (1 rig/ml) of PMA. Enhanced expression in the presence of ionophore approached or slightly exceeded the expression obtained with an optimal concentration (10 rig/ml) of the phorbol stimulus (Table 4). In contrast, ionomycin exerted negligible effects in conjunction with suboptimal concentrations of TNF-(u, IL-l& or LPS. Similar results were obtained with the calcium ionophore A23187 (1 PM; data not shown). Viability assays. At the highest concentrations used, treatment with some of the protein kinase inhibitors and

;

b

ionophore was associated with slight impairment of cellular viability as determined by mitochondrial reductase activity (16). In the absence of activation, 18-27% losses in reductase activity were associated with staurosporine (100 nM) treatment. None of the other compounds exhibited significant effects. Although none of the activators alone resulted in viability changes, compromised viability was more evident with selected treatment in the presence of cell activation. Staurosporine treatment in combination with cytokine, PMA, or LPS resulted in up to 50% reduction of mitochondrial dehydrogenase activity. HA1004 treatment resulted in decrements as great as 40%, and ionophore treatment resulted in decrements up to 32%. DISCUSSION

The induction of ICAMin HUVEC observed in this study exhibited similar kinetics and sensitivities to proinflammatory cytokines, LPS, and PMA as previously described (12,19,20). Specificity was demonstrated by selective inhibition of the cytokine-, LPS-, or PMAinduced response by, respectively, the corresponding neutralizing antibody to cytokine, by polymixin B which renders LPS biologically inert through binding, and by


c370

STIMULUS-SELECTIVE

MODULATION

OF

TNFa

ICAM-

EXPRESSION

IL-18 1401

Fig. 3. Effect of various kinase inhibitors on endothelial ICAMexpression induced by TNF-cu, IL-l& LPS, or PMA. HUVEC were stimulated (16 h) with TNF-cu (100 units/ml), IL-lfl (1 rig/ml), LPS (1 ,ug/ml), or PMA (10 rig/ml) in the absence or presence of l-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7; 50 PM), N-(2guanidinoethyl)-5-isoquinolinesulfonamide (HA1004; 50 PM), or staurosporine (stsp; 100 nM). ICAMexpression was determined by ELISA. Results are reported relative to expression induced by each activator in the absence of inhibitor and represent the means t SE of 3 experiments.

"s 80 4 C60

No

No Addition

LPS

PMA 125 1

t

No

H7

No

Addition

o0.8-

+

g 0.6+

/

A---

/---- _+-------+

/ 0.4;

1'

0.2 +

0.0blv t-

H7

HA1004

8t8p

Addition

/

0'

n’

____ - --+

0 Elapsed

Fig. 4. Inhibition of administration. times after addition mined by ELISA addition; l , IL-lp

---_~---.

2 time

;

-

4 (hours)

before

-

--i 6

H7 addition

of ICAMexpression by H-7 varies with the time H-7 (50 PM) was administered to HUVEC at various of IL-lp (1 rig/ml); ICAMexpression was deterafter 8 h of activation with the cytokine. l , No only; 0, IL-l@ plus H-7.

staurosporine and H-7, inhibitors of PKC. Our data suggest that ICAM- induction by PMA is not secondary to potential phorbol-induced endothelial production of extracellular TNF-cw or IL-lfl activity. However, we cannot rule out the possible existence of a TNF-a or IL-lfi autocrine pathway that acts intracellularly, or the induction of some other unknown common stimulus which acts through an autocrine mechanism. Combinations of activators at optimal concentrations did not significantly augment ICAMexpression over that elicited by the more efficacious agent alone (Table l), suggesting that the intracellular signaling and biosynthetic pathways triggered by the various activators are not entirely separate but converge at some common point. Is PKC activation necessary for inflammatory stimuli to elicit ICAMexpression ? H-7 blocked expression of ICAMin response to the cytokines, LPS, and PMA; however. staurosporine inhibited onlv the ICAM-

expression elicited by PMA. If PKC is a necessary step in the signaling pathway for the cytokines and LPS, one would expect staurosporine to also inhibit ICAMexpression in response to these stimuli at a concentration similar to that needed to inhibit PMA-mediated expression. One explanation for our results is that activation of PKC results in ICAMexpression through a mechanism that is at least partially distinct from that through which the inflammatory stimuli induce ICAM-1. This interpretation is consistent with the results obtained with calcium ionophore. Synergistic activation by calcium ion and phorbol esters is a characteristic of PKCmediated physiological reactions (17). We observed such synergy when calcium ionophores were combined with phorbol esters, but no synergistic effect was observed when the ionophore was applied in combination with TNF-a, IL-l& or LPS. Inhibition by H-7 of ICAMexpression elicited by inflammatory stimuli suggests that protein phosphorylation is involved in these signaling pathways. Although H-7 is generally considered a PKC antagonist, it also inhibits a variety of other protein kinases by competing at the ATP binding site (8), one of the most highly conserved regions between different kinases (7). Thus it is possible that H-7 may block ICAMexpression induced by inflammatory stimuli by inhibiting a kinase distinct from PKC. This is consistent with recent experiments in our laboratory in which H-7 blocked ICAMexpression in response to inflammatory stimuli after downregulation of PKC (unpublished observations). Ritchie et al. (21) recently reported that stimulation of endothelial cells with TNF-Q did not trigger the translocation of PKC to the membrane fraction or the phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) protein, a major substrate for PKC. However. both PKC translocation and phosphoryl-


STIMULUS-SELECTIVE

MODULATION

OF

ICAM-

c371

EXPRESSION

Table

3. Effect of H-7 or staurosporine biosynthesis in HUVEC

A

Experiment

I

on ICAMExperiment

Treatment

CPm

Control TNF-cu (100 units/ml) +H-7 IL-16 (1 rig/ml) +H-7 PMA (10 rig/ml) +H-7 LPS (1 dml) +H-7

768 2,495 435 3,661 226 3,488 399 3,071 501

2

Treatment

CPm

Control TNF-cu (100 units/ml) +Staurosporine IL-10 (1 rig/ml) +Staurosporine PMA (10 rig/ml) +Staurosporine LPS (1 rg/ml) +Staurosporine

757 6,410 3,527 5,292 3,180 4,748 1,475 2,391 1,978

Biosynthesis was quantitated as radioactivity associated with munoprecipitated ICAMfrom biosynthetically labeled HUVEC described in MATERIALS AND METHODS. l-(5-Isoquinolinesulfonyl)-2methylpiperazine dihydrochloride (H-7), 50 PM; staurosporine, nM. cpm, counts per minute.

Table

4. Effect of ionophore ionomycin

of ICAM-

Treatment

on induction

Control Ionomycin PMA (1 rig/ml) +Ionomycin PMA (10 rig/ml) IL-10 (0.1 rig/ml) +Ionomycin IL-10 (1.0 rig/ml) TNF-(Y (10 units/ml) +Ionomycin TNF-(U (100 units/ml)

1

Experiment OD,,,

0.105kO.021 0.159rtO.017 0.332k0.057 0.943kO.043 0.760+0.044 1.164+0.065 1.170+-0.128 1.516kO.128 0.877kO.088 1.105+0.070 1.511f0.136

Treatment

ation of the MARCKS protein occurred in response to PMA stimulation. Moreover, they also found that staurosporine could block PMA-induced ICAMexpression but was ineffective in inhibiting TNF-a-induced ICAM1 expression. They concluded that the pathways for TNF-a- and PMA-stimulated ICAMexpression in endothelial cells are distinct. Our results are consistent with their findings and extend their results by including data which indicate that the signaling pathways triggered by LPS and IL-l/? are also distinct from the PMAmediated pathways. The results of this study are relevant to the study of Magnuson et al. (14) describing endothelial activation by LPS, TNF-a, and IL-lb in terms of cellular adhesiveness toward neutrophils. The authors’ observation that H-7 but not HA1004 inhibited inflammatory stimulusprovoked endothelial adhesiveness for neutrophils cor-

2 OD,,,

Control Ionomycin PMA (1 rig/ml) +Ionomycin PMA (10 rig/ml) LPS (100 rig/ml) +Ionomycin

Values are means f SD of 4-8 replicate wells representative of 3 sets of experiments repeating mycin used at 0.5 PM. Fig. 5. Effect of H-7 and staurosporine on total protein and ICAMbiosynthesis in TNF-cu-stimulated HUVEC. Endothelial cells were labeled for 6 h with [%]methionine in the presence of TNF-or and various compounds. Total cellular proteins (A) and immunoprecipitated ICAM(B) were then resolved by SDS polyacrylamide gel electrophoresis and visualized by autoradiography. Lane 1, no addition; lane 2, TNF-a (100 units/ml); lane 3, TNF-cu plus cycloheximide (1 pg/ ml); lane 4, TNF-(U plus H-7 (25 PM); lane 5, TNF-(U plus H-7 (50 PM); lane 6, TNF-(U plus staurosporine (50 nM); lane 7, TNF-(U plus staurosporine (100 nM). Arrowhead points to the ICAMprotein band.

100

by PMA, LPS, and cytokines Experiment

4

imas

0.271kO.054 0.226kO.015 0.475zko.057 1.559kO.089 1.796kO.057 1.042+0.045 1.084+0.082

of ELISA. Data are all conditions. Iono-

relates with our related findings regarding inhibition of ICAMexpression under similar conditions. Our data are also similar to those of Lane et al. (12) who reported that both H-7 and staurosporine inhibit PMA-stimulated ICAMexpression on endothelial cells. However, it is in conflict with more recent data from the same group (13) which indicate that H-7 and staurosporine also inhibit ICAMexpression elicited by IL-l, LPS, and TNF. The reason for these different results is not known; however, it should be noted that Lane et al. (13) reported that micromolar concentrations of staurosporine were required to inhibit ICAMexpression in response to inflammatory mediators. These levels are much higher than what is usually required to inhibit PKC-mediated cellular reactions. At micromolar concentrations staurosporine may have numerous effects on the cells; our data indicate that at 100 nm, staurosporine is beginning to inhibit overall cellular protein biosynthesis as determined by the incorporation of [35S]methionine into newly synthesized protein (Fig. 5A). Although many of the probes used in this study were associated with modest changes in cell viability in the presence of stimulus, such changes were not likely responsible for the observed effects on ICAMexpression. Changes in cell viability as measured by mitochondrial


C372

STIMULUS-SELECTIVE

MODULATION

deh .ydrogenase acti .vity did not correlate with changes in exP ression of the adhesion molecule, either quantitatively or qualitatively. The latter is evidenced by the ability of ionophore in combination with PMA to augment ICAMexpression despite a slight decrease in cell viability. In addition, other investigators have reported that there is no significant impairment of protein synthesis by H-7 in LPS-treated HUVEC (14); moreover, the I& values determined for the kinase inhibitors H-7 and staurosporine in th is report are in the range of concentrations rep lorted to exert biological effects in other in vitro systems that are not due to altered cell viability (3, 26). In some cases we found that there was a more pronounced effect of staurosporine on ICAMbiosynthesis as measured by immunoprecipitation of [ ‘%I methioninelabeled ICAMthan as determined by ELISA. The immunoprecipitation technique provides an indication of the total level of newly synthesized ICAMin the cell while the ELISA reports only the ICAMon the cell surface. One possible explanation is that a rather high level of inhibition of total ICAMbiosynthesis is required before a significant reduction of cell surface ICAM-I is observed. Alternatively, the results may be due to a somewhat variable effect of staurosporine on total cellular protein biosynthesis which would also reduce nonspecifically the level of ICAMobtained in the immunoprecipitates. What is the signaling mechanism by which inflammatory mediators trigger ICAMexpression on endothelial cells? Our results suggest that PKC is not a necessary intermediate in the pathway, nor does it appear that cyclic nucleotide-dependent phosphorylation or elevated intracellular calcium is by itself sufficient to trigger ICAMexpression. However, our results with H-7 suggest that one or more cellular protein kinases may play a key role. Identification of these critical elements of the signal transduction pathways for ICAMexpression in response to inflammatory stimuli will be a challenging and important focus for future research.

OF

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18. 19.

We gratefully acknowledge the generous provision of RRl/land R6.5coupled Sepharose by Dr. Steven Marlin, Dept. of Immunology, Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT. We are also indebted to John Sabo for the nonlinear regression analysis of H-7 dose-response data for the determination of IC& values. Address for reprint requests: C. L. Myers, Boehringer Ingelheim Pharmaceuticals, PO Box 368, 90 East Ridge, Ridgefield, CT 06877. Received

26 December

1990; accepted

in final

form

4 September

20.

1991. 21.

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Synergistic effects of a calcium ionophore and activators of protein kinase C on epithelial paracellular permeability.

Synergistic action of calcium ionophore A23187 and protein kinase C activator bryostatin 1 on human B cell activation and proliferation.

GRP78 and GAPDH.

Influence of calcium and ionophore 23187 on tubular phosphate reabsorption.

Effects of calcium ionophore (A-23187) on glucose oxidation and iodide transport in dog thyroid slices.

Effects of the calcium ionophore A23187 on pancreatic acinar cell membrane potentials and amylase release.

Effects of tamoxifen and protein kinase C inhibitors on hyperthermic cell killing.

Effects of protein kinase C inhibitors on viral entry and infectivity.

Studies on the mechanism of renin release from isolated superfused rat glomeruli: effects of calcium, calcium ionophore and lanthanum.

Effects of ionophore A23187 and external calcium concentrations on renal gluconeogenesis.

Pharmacology of protein kinase inhibitors.

Induction of zinc metallothionein by calcium ionophore in vivo and in vitro.

Effect of calcium ionophore on unfertilized oocytes after ICSI cycles.

Effects of selenium, calcium and calcium ionophore on human oocytes in vitro maturation in a chemically defined medium.

Physiological responses of sea urchin eggs to stimulation by calcium ionophore A23187 analysed with protease inhibitors.

Effects of protein kinase inhibitors on regeneration in vitro of adult frog sciatic sensory axons.

Effects of protein kinase inhibitors on pig oocyte maturation in vitro.

Optimization of an imidazopyridazine series of inhibitors of Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1).

Neurotrophic effects of epidermal growth factor on cultured brain neurons are blocked by protein kinase inhibitors.

Differential effects of isoquinolinesulfonamide protein kinase inhibitors on CA1 responses in hippocampal slices.

Effects of hemodialysis on protein-bound calcium.

Protein kinase C, calcium and phospholipid degradation.

The influence of calcium ionophore and activators of protein kinase C on steroid production by preovulatory ovarian follicles of the goldfish.

Effects of Ca2+ and ionophore A23187 on protein synthesis in intact rabbit reticulocytes.