International Immunology, Vol. 2, No 8
© 1990 Oxford University Press 0953-8178/90 $3.00
Activation of protein kinase C is crucial in the regulation of ICAM-1 expression on endothelial cells by interferon-7 Risto Renkonen, Arl Mennander, Jarkko Ustinov, and Pirkko Mattila Department of Bacteriology and Immunology and Transplantation Laboratory, University of Helsinki, Helsinki, Finland Key words: ICAM-1, IFN-y, intracellular messenger pathways, endothelial cells
ICAM-1 (CDS4) is expressed on endothelial cells and serves as an important llgand for the white cell adhesion molecule CD11a/CD18 (LFA-1). Many studies have demonstrated that Increased numbers of white cells binding to endothelial cells correlate with the level of ICAM-1 expression on endothelial cells. Several cytokines, Including IFN-7, Increase ICAM-1 expression in cultured human endothelial cells. We have analysed the second Intracellular messenger pathways involved In IFN-7-induced up-regulatlon of ICAM-1 expression In endothelial cells. IFN-7 induced a rapid activation of phosphollpase C, leading to a breakdown of phospholnosltoldiphosphate (PIPJ Into dlacyglycerol (DAG) and inosltoltrlphosphate (IP3). DAG Is a natural activator of the protein kinase C pathway. We were able to show that the effect induced by IFN-7 could be Inhibited by a protein kinase C inhibitor, H7, in a dose-dependent manner and mimicked by PMA, which stimulates protein kinase C. IFN-7 Induced a 5-fold translocatlon (activation) of protein kinase C from the cytosol into the endothelial cell membrane. Elevation of the IP3 levels led to activation of the calcium-dependent pathway. An inhibitor of calcium calmodulln, W7, decreased the IFN-7 Induced ICAM-1 expression, and addition of calcium lonophore to endothelial cells could replace IFN-7 in the up-regulation of ICAM-1. Finally, IFN-7 caused a significant Increase in the calcium flux of endothelial cells. cAMP and cGMP had no effect on the regulation of ICAM-1 expression on cultured human endothelial cells.
Introduction Leukocytes recirculating in the bloodstream can only exit the circulation and enter the sites of inflammation in tissues by first adhering to and then penetrating through the vascular endothehum (1 - 3). In lymph nodes the high endothelium lining the postcapillary venules is the site of exit (4). Recently similar high endothelium has been characterized at sites of chronic and acute inflammation in non-lymphoid organs, i.e. in synovias affected by rheumatoid arthritis (5), and during allograft rejection in the transplanted kidneys (6). Several receptors involved in this binding phenomenon have been characterized both on the white cell side and on the endothelial side (7). Intercellular adhesion molecule-1 (ICAM-1) is a single chain glycoprotein expressed by the endothelial cells (EC) (8,9). ICAM-1 is an EC surface ligand for the leukocyte LFA-1 (CD11a/CD18) adhesion molecule (10,11), but LFA-1 also has another ligand, ICAM-2 (12). Cultured endothelial cells express ICAM-1 in a natural state, but the expression can be enhanced by gamma-interferon (IFN-7), mterleukin 1 (IL-1) and tumor Correspondence to R Renkonen, as above Transmitting editor- S H E . K a u f m a n n
necrosis factor (TNF) with a concomitant increase in the lymphocyte binding (13,14) Recently also viruses have been demonstrated to use ICAM-1 when adhering to endothelium (15,16). The role of ICAM-1 is essential in the regulation of lymphocyte recirculation (17). Therefore we analysed the intracellular mechanisms involved in IFN->Hnduced up-regulation of ICAM-1 expression on endothelial cells. We show in this paper than IFN-7 splits the phosphomositoldiphosphate (PIP2) in the cell membrane to diacylglycerol (DAG) and inositoltriphosphate (IP3). DAG is a direct stimulator of the protein kinase C (PKC) pathway and IP3 liberates the intracellular calcium reservoires. PKC was found to be crucial in signal transduction in IFN-7 induced ICAM-1 expression in endothelial cells. PKC stimulators up-regulated the ICAM-1 expression on cultured endothelial cells, PKC inhibitors abolished the IFN-7 effect and, furthermore, IFN7 induced a significant translocation (i.e. activation) of PKC from cytosol the cell membranes. Also the calcium calmodulin pathway
Received 5 March 1990, accepted 18 May 1990
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Abstract
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Transduction of IFN-y signal in endotheiial cells
is involved, since calcium ionophore could increase the ICAM-1 expression, and a calcium calmodulin antagonist W7 inhibited the effect. IFN-y was also shown to increase the flux of Ca 2+ in endotheiial cells. Cyclic nucleotide protein kinases as well as their inhibitor HA 1004 had no effect on ICAM-1 expression. Taken together, these results indicate that both PKC and calcium calmodulin are important in the signal transduction after IFN-7 stimulation in cultured human umbilical cord vein endotheiial (HUVE) cells. Methods Endotheiial cell cultures
Antigen analysis The expression of surface antigens on endotheiial cells was analysed by flow cytometry. The cells were first exposed to an anti-ICAM-1 monoclonal antibody (LB2, 0.04 mg/ml) for 30 min, washed twice, exposed to a fluorescein isothiocyanate conjugated anti-mouse antibody (Coulter Immunology, Hialeah, FL), washed twice, resuspended in PBS, and analysed in the FACS.
PKC assay PKC activity was determined by a method developed by Iwasa era/. (19). The reaction mixture (150 ^m) contained 5 mM Mg acetate, 10 mM Tris-HCI, pH 7.5, 15 ^M FP]ATP, 200 ^g/ml histone H1 type Ill-S, 10 /tg phospholipid, 1 /ig diacylglycerol, and 70 or 700 pM CaCI2 for membrane or cytosolic fractions respectively. Phospholipid and diolein were first dissolved in a small volume of chloroform, then the chloroform was evaporated under a stream of nitrogen. The dry mixture was resuspended in 20 mM Tris-HCI, pH 7.5, by sonication for 5 min. Ten microlitres of the sample was added to the reaction mixture. After a 6 mm incubation at 30°C the reaction was stopped by precipitating aliquots in 25% trichloroacetic acid (TCA) and spotted down on a piece of Whatman ET31 paper. Whatman paper slips were washed three tmes with 10% TCA For negative control of each timepomt, 5 mM EDTA was added instead of CaCI2 and PS/DO. Reagents Stimulators of intracellular signal transduction N-6-2'-O-dibutyryladenosine 3':5'-cychc monophosphate (cAMP), N-2-2'-O-ti\butyrylguanosine 3':5' cyclic monophosphate (cGMP), calcium ionophore A23187, and phorbol 12-mynstate 13-acetate (PMA) were all purchased from Sigma. Recombinant IL-1a (1 3 x 107 U/mg) was purchased from Janssen Biochemica, Beerse, Belgium. Recombinant interferon-7 was received as a gift from Dr G. R. Arnold, Bender & Co., Vienna, Austria. In some experiments the endotheiial cells were treated with IFN-7 in combination with PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7), with a cyclic nucleotide protein kinase (A) inhibitor HA1004, or with a calcium calmodulin antagonist, /V-(6-ammohexyl)-5-chloro1-naphthalenesulphonamide hydrochloride (W7). All enzyme inhibitors were purchased from Seikagaku Kogyo, Tokyo, Japan.
Measurement of IP3 The endotheiial cells were treated with 6% percholonc acid, detached from the culture flasks mechanically, vortexed, and centrifuged at 2000 g for 15 mm. All procedures were done at + 4°C using siliconized glassware. The supernatants were diluted 1:20 in the assay buffer and the IP3 concentrations were determined with RIA kits (Amersham). Preparation of subcellular fractions Endotheiial cells growing in flasks were stimulated for 0, 15, 30, and 60 min with IFN-7(100U/ml). The cells were detached from the flasks with versene and immediately placed in a lysis buffer containing 10mM Tris-HCI, pH 7.5, 0.1% Nonidet-P40, 5mM Mg acetate, 1 ^M phenylmethylsulphonyl fluoride (PMSF), 250 mM sucrose, 125 ngln\\ aprotin The cells were frozen and melted slowly three times to disrupt the cell membranes. After the lysis EDTA (5 mM) and EGTA (2 mM) were added to prevent the degradation of PKC by calcium-dependent proteases. The
Results IFN-y up-regulates ICAM-1 expression In short-term culture 45 ± 10% cells express ICAM-1 on their cell surface measured with a monoclonal antibody (LB2) and fluorescence activated cell sorter. This expression can be enhanced by several cytokines, e.g. IFN-y, TNF, and IL-1. IFN7 (100 U/ml) increased significantly the ICAM-1 expression on endotheiial cells within 24 h (70 ± 9). Practically all endotheiial cells (96 ± 6%) became positive after 72 h of IFN-7 treatment (Fig. 1). Role of phospholipase C in the effect of IFN-y Phospholipase C (PLC) hydrolyses the PIP2, which is located in the cell membrane, into DAG and IP3. DAG is the natural stimulator of the PKC pathway, whereas IP3 increases cytosolic
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HUVE cells were isolated and cultured as described (18) In short, umbilical cord veins were cannulated, flushed with PBS, and incubated with 0 . 1 % collagenase (108 U/mg: Worthington Biochemical Corp, Freehold, NJ)in PBS at 37°C for 10 min. The veins were flushed with 50 ml of PBS, and the isolated cells were centrifuged (400 g, 7 mm) and resuspended in Medium 199 (Gibco, Grand Island, NY), supplemented with 20% human AB serum (Finnish Red Cross, Helsinki, Finland) and 50 /tl/ml gentamycin. The isolated endotheiial cells were plated in a 25 cm 2 culture flask (Nunc, Roskilde, Denmark). When the cells reached confluence at 4 - 6 days after isolation they were detached and divided 1:3 using 0.125% trypsin and 1:5000 versene (Gibco) for 5 min. Cell monolayers were confirmed to be endotheiial cells by immunofluorescent staining with anti-factor VIII antibody (Dakopatts, Glostrup, Denmark) and the purity of endotheiial cells used was always >95%. The cells were used at the second passage.
nuclei were removed by low speed centnfugation (1000 g) for 1 min. Supernatant containing cytosol and membrane fragments was ultracentnfuged at 150,000 g for 30 mm, after which the supernatant was aliquoted and kept at - 7 0 ° C . The pellet was resuspended in lysis buffer containing EDTA and EGTA at protein concentrations of 1 - 3 mg/ml, aliquoted, and kept at -70°C.
Transduction of IFN-y signal in endothehal cells
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Time (min) 1
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Fig. 1. Expression of ICAM-1 (analysed with a monoclonal antibody LB2) on cultured HUVE cells. The IFN-y stimulation was initiated at day 0 and the amount of ICAM-1 expression on the cell surface was quantitated by a fluorescence cell sorter. Mean ± SD of three experiments.
IFN-gamma PMA 0.1 uM
2+
Ca levels and leads to the stimulation of the calcium calmodulm pathway. To sort out the role of PLC activation in the signal transduction of IFN-7 in the endothelial cells we first analysed the production of IP3. IFN-7 (100 U/ml) caused a rapid and transient increase in the IP3 levels After 3 min incubation the IP3 level was 108 pM/106 cells, compared to 28 pM/106 cells in the natural state. The maximal response was seen after 10 min of incubation when the IP3 levels had risen to 440 pM/106 cells, corresponding to a 15.7-fold increase. Thereafter the IP3 levels began to decrease, even though the IFN-7 stimulation was continued and at 60 min the levels reached normal values (Fig. 2). After showing that the PLC was activated, we started to analyse the effects of the breakdown products, i.e. DAG and IP3, in the signal transduction during IFN-7 stimulation leading to increased expression of ICAM-1 on cultured endothelial cells. Second messengers mimicking IFN-y effect Since PLC activation was seen in the IFN-7 stimulation the next question was whether the PKC and/or calcium calmodulm pathways were involved in the signal transduction. In the first set of experiments a PKC stimulator, PMA, at concentrations of 1 0 and 0.1 jiM for 24 h, could totally replace IFN-7 in the induction of ICAM-1 expression on HUVE cells (Fig. 3). This effect was dose-dependent, since more dilute concentrations of PMA had no effect (data not shown). Since the elevation of cytosolic Ca 2+ levels is closely linked to the activation of PKC, we evaluated further the role of calcium in the ICAM-1 regulation. Calcium ionophore (A23187, 200 nM) leads to a prompt increase in the flux of Ca 2+ , and also increases the surface expression of ICAM-1 on HUVE cells within 24 h. The effect of ionophore alone was not as pronounced as the PMA or IFN-7 effect (Fig. 3). To evaluate the role of other intracellular second messengers, e.g. cAMP and cGMP, dibutyryl-derivates able to penetrate the cell membrane were added into the HUVE culture media for 24 h.
PMA 1.0 uM CoIoSOOnM cAMPO.lmM cAMPlAnM cGHPCUmM cGMPlAmM «
00
9)
TO
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ICAM-1 expressing cells (%) Fig. 3. Expression of ICAM-1 on endothelial cells incubated with various agents stimulating intracellular second messenger pathways After a 24 h incubation the expression of ICAM-1 was quantitated with LB2 monoclonal antibody in a fluorescence cell sorter. Both PMA and calcium ionophore are able to up-regulate ICAM-1 expression, whereas cAMP and cGMP are ineffective. Mean ± SD of three experiments
cAMP or cGMP had no effect on ICAM-1 expression on the endothelial cell surface at concentrations of 10- 3 —10- 9 M (Fig. 3). Inhibition of IFN-y signal transduction The above-presented results suggest that PKC and Ca 2 * would be essential in the signal transduction during regulation of ICAM-1 expression on HUVE cells during IFN-7 stimulation. In the next set of experiments we studied possible ways to down-regulate the IFN-7-induced ICAM-1 expression by interfering with different levels of protien kinase functions. The IFN-7 effect could be totally down-regulated with PKC inhibitor H7 at a concentration of 20 ftM. This effect was dose-dependent, i.e. 10 /iM of H7 still inhibited the IFN-7 effect but 2 /iM of H7 had no effect on IFN-7 induced up-regulation of ICAM-1 expression on HUVE cells (Table 1, Fig. 4). We also measured the mean fluorescence intensities (MFI) from the same preparations, which are indicated in Table 1. The calcium calmodulin antagonist W7 (20 ^M) also inhibited IFN-7-induced ICAM-1 expression, but not as effectively
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Time (days)
Fig. 2. Production of IP3 in the endothelial ceils. After IFN-7 (100 U/ml) stimulation the cells were lysed with a percholoric acid treatment and the supernatant was collected. IP3 levels were measured with an RIA kit and the results are expressed as pM/106 endothelial cells. One representative experiment is shown.
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Transduction of IFN-y signal in endothelial cells
Table 1 . Expression of ICAM-1 on HUVE cells incubated with IFN-7 (100 U/ml) in combination with different concentrations of H7, which is an enzyme inhibitor acting on PKC activation of positive cells Control IFN-7 IFN-7 + 2/iN A H7 IFN-7 + 10 ,i,M H7 IFN-r + 20 u,M H7
MFI
55 355 342 301 118
44.3 73.1 72.7 68.7 46.9
The amount of antigen on endothelial cells was measured 25 h after the initiation of the stimulus A dose-dependent effect of H7 is shown 20 iiM concentration blocks totally IFN-7-induced up-regulation of ICAM-1 expression One representative experiment out of two is presented.
30
ao
lime (min)
IFN-gamma (lOOU/ml) +H72 uM +H720 uM + W72 uM + W720 uM • HA 10042 uM + HA100420uM 80
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ICAM-1 expressing cella (%) Fig. 4. Effect of various enzyme inhibitors on the ICAM-1 expression on endothelial cells incubated in combination with IFN-7 (100 U/ml) for 24 h. H7 inhibits dose-dependently the IFN-7 effect. Also a calcium calmodulin antagonist, W7, decreases slightly the ICAM-1 expression, whereas HA 1004 (a protein kinase A inhibitor) has no effect. One representative experiment out of three is presented.
as H7. Concomitantly HA 1004, which is an inhibitor of protein kinase A, had no effect on IFN-7-induced ICAM-1 expression (Fig. 4). Activation of PKC by IFN-y To verify the direct role of PKC we wanted to evaluate whether IFN-7 actually is able to stimulate this enzyme in endothelial cells PKC is located in the cytosol of a resting cell, but upon activaton it is found associated to the cell membrane. This translocation of the enzyme from the cytosol to the membrane has been used as a marker of its activity. We separated the membranes of endothelial cells by ultracentrrfugation and measured the PKC activity. After a 30 min incubation with IFN-7 the membrane activity measured with histone phosphorylation had increased from 669 to 3528 c p.m., corresponding to a > 5-fold increase (Fig. 5). Finally we analysed the calcium flux from endothelial cells. We were able to show a significant 1.4-fold increase in the outflux of 45Ca2+ 5 min after the initiation of IFN-7 stimulation (data not shown). Discussion Lymphocyte homing is a crucial step in the initiation of local immunoresponses. The recirculating lymphocytes can exit blood circulation by first adhering to and then penetrating through the
microvascular endothelium (1 - 5 ) . Several homing receptors defined on the lymphocyte side as well as vascular adressins on the endothelial cell side involved in this homing have been defined so far. One of the first well characterized ligand pairs involved in the intercellular adhesion is the LFA-1 (CD11a/CD18) -ICAM-1 (CD54) pair (10,11). This receptor ligand pair does not express any organ-specificity, but has an important accessory role in the cell-cell adhesion. There are also other inducible endothelial molecules (vascular adressins) involved in homing, like ELAM-1, which has tissue specificity (2,13). ICAM-1 is a single chain 76 -114 kd glycoprotein (polypeptide backbone is 55 kd) on the endothelial cell surface, but it is also present on many white cells as well as fibroblasts and keratinocytes (11). ICAM-1 is involved in several cell-cell interactions, such as homoaggregation (20) and adhesion of white cells to epidermal keratinocytes (21) and fibroblasts (8). ICAM-1 mediates both lymphocyte and granulocyte adhesion to endothelial cells, but has a lesser role in monocyte adhesion (17). The expression of ICAM-1 can easily be up-regulated by several cytokines, such as IFN-7, IL-1 and TNF (8,13,14), within 4 - 2 4 h. Concomitantly with the increase of ICAM-1 on the endothelial cells, the lymphocyte binding increases. We have already initiated studies of the signal transduction in the lymphocyte binding to and penetrating through cultured endothelial monolayers (22). In this paper we have analysed the signal transduction mechanisms after IFN-7 stimulation of endothelial cells in correlation to regulation of ICAM-1 expression. The inositolphospholipid system is activated by a number of different cytokines, hormones, and growth factors (23). This is mediated through receptor-triggered hydrolysis of these inosrtolphospholipids by PLC. Several distinct isoenzymes of PLC have already been identified and some of them have been cloned completely (24). PLC hydrolyse PIP2 into IP3 and DAG. IP3 increases the cytosolic calcium levels and thereby activates the calcium calmodulin pathway. DAG is the natural stimulus for the PKC pathway. There is also evidence that certain lymphokines, e.g. IL-1 and IFN-7, can activate phosphatidyl choline, which
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Fig. 5. Direct activation of PKC measured by the membrane translocation assay. After various incubation periods with IFN-7 the cells were frozen and the membranes isolated. The PKC activity in the membranes was analysed with a histone phosphorylation technique After a 30 min incubation with IFN-7 the PKC activity was increased by > 5-fold in the membrane One representative experiment out of two is presented
LH
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Transduction of IFN-y signal in endothelial cells 723
At least three different subtypes of PKC have been identified. a, j3, and y. These subtypes have been sequenced and monoclonal antibodies against them are available (33). It has been suggested that there is a calcium binding site in the PKCa that has a high degree of structural homology to calcium calmodulin (34). IFN-7 has been shown to operate via the PKC-dependent but not the calcium calmodulm-dependent pathway in endothelial cells (35,36). On the other hand, the calcium calmodulin but not the PKC pathway was responsible for the signal transduction after IFN-7 stimulation in HL-60 cells (37). This discrepancy may relate to the transformation itself or to the different embryonal origins of these cells lines. Calcium ionophore A23187, which rapidly increases the level of intracellular Ca2+, was able to increase the expression of ICAM-1 molecules on HUVE cells. The addition of dibutyryl-cAMP and -cGMP, which are analogs of cAMP and cGMP able to penetrate in the cell, had no effect of ICAM-1. The role of PKC and cytosdic Ca2* was confirmed in enzyme inhibition studies. Selective inhibitors of PKC and a calcium calmodulin antagonist inhibited the IFN-7 effect of enhancing ICAM-1 expression on endothelial cells. Concomitantfy, the protein kinase A inhibitor had no effect. Calmodulin is a small intracellular protein involved among other things in Ca2 + transport. Recently several groups have demonstrated that W7 is a specific calcium calmodulin antagonist which is able to block calcium ionophore-induced IL-1 production (38,39). Different mediators of inflammation activate different routes of signal transduction or at least their phosphorylation substrates vary. We have shown earlier in the lymphocyte-endothelial binding assay that IL-1 operates via cAMP, whereas plateletactivating factor operates via the PKC pathway (40). IFN-7 increased lymphocyte penetration through intact cultured endothelial cell monolayers in vitro (41). Preliminary data indicate that this effect operates also via the PKC and not other pathways (J. P. Turunen and R. Renkonen, unpublished data). On the other hand, we have shown that while IL-1 also induced a 3- to 5-fold
penetration through the endothelial monolayers, this effect does not operate via PKC but through cAMP and protein kinase A. These observations show that different mediators of inflammation causing lymphocyte binding to and penetration through endothelial cell layers in vitro apply different intracellular signal transduction pathways. This once again demonstrates that there are clear differences in the intracellular signal transduction during lymphocyte binding to endothelial cells in vitro induced by different mediators of inflammation. Our results presented in this paper suggest that IFN-7enhanced ICAM-1 expression on HUVE cells is mediated via the PKC and calcium calmodulin pathways, but not by other second messenger pathways.
Acknowledgements This work was supported in part by grants from the Finnish Academy, Finnish Cancer Foundation, The Kidney Foundation, Helsinki, Research and Science Foundation of Farmos Ltd, and a grant from the University of Helsinki
Abbreviations DAG EC H7 HUVE ICAM IFN-7 IL-1 IP3 MFI PIP2 PKC PLC PMA PMSF TCA TNF W7
diacylglycerol endothelial cells 1 -{5-isoquinolinesulphonyf)-1 -methytp.perazine dihydrochlonde human umbilical cord vein endothelial intercellular adhesion molecule gamma-interferon interleukin 1 mosrtoltriphosphate mean fluorescence intensity phosphoinositoldiphosphate protein kinase C phospholipase C phorbol 12-myristate 13-acetate phenyimethyisulphonyl fluoride tnchloroacetic acid tumour necrosis factor /V-(6-aminohexyt)-5-chloro-1-naphthalenesulphon amide hydrochloride
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Transduction of IFN-y signal in endothelial cells
© 1990 Oxford University Press 0953-8178/90 $3.00
Activation of protein kinase C is crucial in the regulation of ICAM-1 expression on endothelial cells by interferon-7 Risto Renkonen, Arl Mennander, Jarkko Ustinov, and Pirkko Mattila Department of Bacteriology and Immunology and Transplantation Laboratory, University of Helsinki, Helsinki, Finland Key words: ICAM-1, IFN-y, intracellular messenger pathways, endothelial cells
ICAM-1 (CDS4) is expressed on endothelial cells and serves as an important llgand for the white cell adhesion molecule CD11a/CD18 (LFA-1). Many studies have demonstrated that Increased numbers of white cells binding to endothelial cells correlate with the level of ICAM-1 expression on endothelial cells. Several cytokines, Including IFN-7, Increase ICAM-1 expression in cultured human endothelial cells. We have analysed the second Intracellular messenger pathways involved In IFN-7-induced up-regulatlon of ICAM-1 expression In endothelial cells. IFN-7 induced a rapid activation of phosphollpase C, leading to a breakdown of phospholnosltoldiphosphate (PIPJ Into dlacyglycerol (DAG) and inosltoltrlphosphate (IP3). DAG Is a natural activator of the protein kinase C pathway. We were able to show that the effect induced by IFN-7 could be Inhibited by a protein kinase C inhibitor, H7, in a dose-dependent manner and mimicked by PMA, which stimulates protein kinase C. IFN-7 Induced a 5-fold translocatlon (activation) of protein kinase C from the cytosol into the endothelial cell membrane. Elevation of the IP3 levels led to activation of the calcium-dependent pathway. An inhibitor of calcium calmodulln, W7, decreased the IFN-7 Induced ICAM-1 expression, and addition of calcium lonophore to endothelial cells could replace IFN-7 in the up-regulation of ICAM-1. Finally, IFN-7 caused a significant Increase in the calcium flux of endothelial cells. cAMP and cGMP had no effect on the regulation of ICAM-1 expression on cultured human endothelial cells.
Introduction Leukocytes recirculating in the bloodstream can only exit the circulation and enter the sites of inflammation in tissues by first adhering to and then penetrating through the vascular endothehum (1 - 3). In lymph nodes the high endothelium lining the postcapillary venules is the site of exit (4). Recently similar high endothelium has been characterized at sites of chronic and acute inflammation in non-lymphoid organs, i.e. in synovias affected by rheumatoid arthritis (5), and during allograft rejection in the transplanted kidneys (6). Several receptors involved in this binding phenomenon have been characterized both on the white cell side and on the endothelial side (7). Intercellular adhesion molecule-1 (ICAM-1) is a single chain glycoprotein expressed by the endothelial cells (EC) (8,9). ICAM-1 is an EC surface ligand for the leukocyte LFA-1 (CD11a/CD18) adhesion molecule (10,11), but LFA-1 also has another ligand, ICAM-2 (12). Cultured endothelial cells express ICAM-1 in a natural state, but the expression can be enhanced by gamma-interferon (IFN-7), mterleukin 1 (IL-1) and tumor Correspondence to R Renkonen, as above Transmitting editor- S H E . K a u f m a n n
necrosis factor (TNF) with a concomitant increase in the lymphocyte binding (13,14) Recently also viruses have been demonstrated to use ICAM-1 when adhering to endothelium (15,16). The role of ICAM-1 is essential in the regulation of lymphocyte recirculation (17). Therefore we analysed the intracellular mechanisms involved in IFN->Hnduced up-regulation of ICAM-1 expression on endothelial cells. We show in this paper than IFN-7 splits the phosphomositoldiphosphate (PIP2) in the cell membrane to diacylglycerol (DAG) and inositoltriphosphate (IP3). DAG is a direct stimulator of the protein kinase C (PKC) pathway and IP3 liberates the intracellular calcium reservoires. PKC was found to be crucial in signal transduction in IFN-7 induced ICAM-1 expression in endothelial cells. PKC stimulators up-regulated the ICAM-1 expression on cultured endothelial cells, PKC inhibitors abolished the IFN-7 effect and, furthermore, IFN7 induced a significant translocation (i.e. activation) of PKC from cytosol the cell membranes. Also the calcium calmodulin pathway
Received 5 March 1990, accepted 18 May 1990
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Abstract
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Transduction of IFN-y signal in endotheiial cells
is involved, since calcium ionophore could increase the ICAM-1 expression, and a calcium calmodulin antagonist W7 inhibited the effect. IFN-y was also shown to increase the flux of Ca 2+ in endotheiial cells. Cyclic nucleotide protein kinases as well as their inhibitor HA 1004 had no effect on ICAM-1 expression. Taken together, these results indicate that both PKC and calcium calmodulin are important in the signal transduction after IFN-7 stimulation in cultured human umbilical cord vein endotheiial (HUVE) cells. Methods Endotheiial cell cultures
Antigen analysis The expression of surface antigens on endotheiial cells was analysed by flow cytometry. The cells were first exposed to an anti-ICAM-1 monoclonal antibody (LB2, 0.04 mg/ml) for 30 min, washed twice, exposed to a fluorescein isothiocyanate conjugated anti-mouse antibody (Coulter Immunology, Hialeah, FL), washed twice, resuspended in PBS, and analysed in the FACS.
PKC assay PKC activity was determined by a method developed by Iwasa era/. (19). The reaction mixture (150 ^m) contained 5 mM Mg acetate, 10 mM Tris-HCI, pH 7.5, 15 ^M FP]ATP, 200 ^g/ml histone H1 type Ill-S, 10 /tg phospholipid, 1 /ig diacylglycerol, and 70 or 700 pM CaCI2 for membrane or cytosolic fractions respectively. Phospholipid and diolein were first dissolved in a small volume of chloroform, then the chloroform was evaporated under a stream of nitrogen. The dry mixture was resuspended in 20 mM Tris-HCI, pH 7.5, by sonication for 5 min. Ten microlitres of the sample was added to the reaction mixture. After a 6 mm incubation at 30°C the reaction was stopped by precipitating aliquots in 25% trichloroacetic acid (TCA) and spotted down on a piece of Whatman ET31 paper. Whatman paper slips were washed three tmes with 10% TCA For negative control of each timepomt, 5 mM EDTA was added instead of CaCI2 and PS/DO. Reagents Stimulators of intracellular signal transduction N-6-2'-O-dibutyryladenosine 3':5'-cychc monophosphate (cAMP), N-2-2'-O-ti\butyrylguanosine 3':5' cyclic monophosphate (cGMP), calcium ionophore A23187, and phorbol 12-mynstate 13-acetate (PMA) were all purchased from Sigma. Recombinant IL-1a (1 3 x 107 U/mg) was purchased from Janssen Biochemica, Beerse, Belgium. Recombinant interferon-7 was received as a gift from Dr G. R. Arnold, Bender & Co., Vienna, Austria. In some experiments the endotheiial cells were treated with IFN-7 in combination with PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7), with a cyclic nucleotide protein kinase (A) inhibitor HA1004, or with a calcium calmodulin antagonist, /V-(6-ammohexyl)-5-chloro1-naphthalenesulphonamide hydrochloride (W7). All enzyme inhibitors were purchased from Seikagaku Kogyo, Tokyo, Japan.
Measurement of IP3 The endotheiial cells were treated with 6% percholonc acid, detached from the culture flasks mechanically, vortexed, and centrifuged at 2000 g for 15 mm. All procedures were done at + 4°C using siliconized glassware. The supernatants were diluted 1:20 in the assay buffer and the IP3 concentrations were determined with RIA kits (Amersham). Preparation of subcellular fractions Endotheiial cells growing in flasks were stimulated for 0, 15, 30, and 60 min with IFN-7(100U/ml). The cells were detached from the flasks with versene and immediately placed in a lysis buffer containing 10mM Tris-HCI, pH 7.5, 0.1% Nonidet-P40, 5mM Mg acetate, 1 ^M phenylmethylsulphonyl fluoride (PMSF), 250 mM sucrose, 125 ngln\\ aprotin The cells were frozen and melted slowly three times to disrupt the cell membranes. After the lysis EDTA (5 mM) and EGTA (2 mM) were added to prevent the degradation of PKC by calcium-dependent proteases. The
Results IFN-y up-regulates ICAM-1 expression In short-term culture 45 ± 10% cells express ICAM-1 on their cell surface measured with a monoclonal antibody (LB2) and fluorescence activated cell sorter. This expression can be enhanced by several cytokines, e.g. IFN-y, TNF, and IL-1. IFN7 (100 U/ml) increased significantly the ICAM-1 expression on endotheiial cells within 24 h (70 ± 9). Practically all endotheiial cells (96 ± 6%) became positive after 72 h of IFN-7 treatment (Fig. 1). Role of phospholipase C in the effect of IFN-y Phospholipase C (PLC) hydrolyses the PIP2, which is located in the cell membrane, into DAG and IP3. DAG is the natural stimulator of the PKC pathway, whereas IP3 increases cytosolic
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HUVE cells were isolated and cultured as described (18) In short, umbilical cord veins were cannulated, flushed with PBS, and incubated with 0 . 1 % collagenase (108 U/mg: Worthington Biochemical Corp, Freehold, NJ)in PBS at 37°C for 10 min. The veins were flushed with 50 ml of PBS, and the isolated cells were centrifuged (400 g, 7 mm) and resuspended in Medium 199 (Gibco, Grand Island, NY), supplemented with 20% human AB serum (Finnish Red Cross, Helsinki, Finland) and 50 /tl/ml gentamycin. The isolated endotheiial cells were plated in a 25 cm 2 culture flask (Nunc, Roskilde, Denmark). When the cells reached confluence at 4 - 6 days after isolation they were detached and divided 1:3 using 0.125% trypsin and 1:5000 versene (Gibco) for 5 min. Cell monolayers were confirmed to be endotheiial cells by immunofluorescent staining with anti-factor VIII antibody (Dakopatts, Glostrup, Denmark) and the purity of endotheiial cells used was always >95%. The cells were used at the second passage.
nuclei were removed by low speed centnfugation (1000 g) for 1 min. Supernatant containing cytosol and membrane fragments was ultracentnfuged at 150,000 g for 30 mm, after which the supernatant was aliquoted and kept at - 7 0 ° C . The pellet was resuspended in lysis buffer containing EDTA and EGTA at protein concentrations of 1 - 3 mg/ml, aliquoted, and kept at -70°C.
Transduction of IFN-y signal in endothehal cells
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Time (min) 1
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Fig. 1. Expression of ICAM-1 (analysed with a monoclonal antibody LB2) on cultured HUVE cells. The IFN-y stimulation was initiated at day 0 and the amount of ICAM-1 expression on the cell surface was quantitated by a fluorescence cell sorter. Mean ± SD of three experiments.
IFN-gamma PMA 0.1 uM
2+
Ca levels and leads to the stimulation of the calcium calmodulm pathway. To sort out the role of PLC activation in the signal transduction of IFN-7 in the endothelial cells we first analysed the production of IP3. IFN-7 (100 U/ml) caused a rapid and transient increase in the IP3 levels After 3 min incubation the IP3 level was 108 pM/106 cells, compared to 28 pM/106 cells in the natural state. The maximal response was seen after 10 min of incubation when the IP3 levels had risen to 440 pM/106 cells, corresponding to a 15.7-fold increase. Thereafter the IP3 levels began to decrease, even though the IFN-7 stimulation was continued and at 60 min the levels reached normal values (Fig. 2). After showing that the PLC was activated, we started to analyse the effects of the breakdown products, i.e. DAG and IP3, in the signal transduction during IFN-7 stimulation leading to increased expression of ICAM-1 on cultured endothelial cells. Second messengers mimicking IFN-y effect Since PLC activation was seen in the IFN-7 stimulation the next question was whether the PKC and/or calcium calmodulm pathways were involved in the signal transduction. In the first set of experiments a PKC stimulator, PMA, at concentrations of 1 0 and 0.1 jiM for 24 h, could totally replace IFN-7 in the induction of ICAM-1 expression on HUVE cells (Fig. 3). This effect was dose-dependent, since more dilute concentrations of PMA had no effect (data not shown). Since the elevation of cytosolic Ca 2+ levels is closely linked to the activation of PKC, we evaluated further the role of calcium in the ICAM-1 regulation. Calcium ionophore (A23187, 200 nM) leads to a prompt increase in the flux of Ca 2+ , and also increases the surface expression of ICAM-1 on HUVE cells within 24 h. The effect of ionophore alone was not as pronounced as the PMA or IFN-7 effect (Fig. 3). To evaluate the role of other intracellular second messengers, e.g. cAMP and cGMP, dibutyryl-derivates able to penetrate the cell membrane were added into the HUVE culture media for 24 h.
PMA 1.0 uM CoIoSOOnM cAMPO.lmM cAMPlAnM cGHPCUmM cGMPlAmM «
00
9)
TO
80
ICAM-1 expressing cells (%) Fig. 3. Expression of ICAM-1 on endothelial cells incubated with various agents stimulating intracellular second messenger pathways After a 24 h incubation the expression of ICAM-1 was quantitated with LB2 monoclonal antibody in a fluorescence cell sorter. Both PMA and calcium ionophore are able to up-regulate ICAM-1 expression, whereas cAMP and cGMP are ineffective. Mean ± SD of three experiments
cAMP or cGMP had no effect on ICAM-1 expression on the endothelial cell surface at concentrations of 10- 3 —10- 9 M (Fig. 3). Inhibition of IFN-y signal transduction The above-presented results suggest that PKC and Ca 2 * would be essential in the signal transduction during regulation of ICAM-1 expression on HUVE cells during IFN-7 stimulation. In the next set of experiments we studied possible ways to down-regulate the IFN-7-induced ICAM-1 expression by interfering with different levels of protien kinase functions. The IFN-7 effect could be totally down-regulated with PKC inhibitor H7 at a concentration of 20 ftM. This effect was dose-dependent, i.e. 10 /iM of H7 still inhibited the IFN-7 effect but 2 /iM of H7 had no effect on IFN-7 induced up-regulation of ICAM-1 expression on HUVE cells (Table 1, Fig. 4). We also measured the mean fluorescence intensities (MFI) from the same preparations, which are indicated in Table 1. The calcium calmodulin antagonist W7 (20 ^M) also inhibited IFN-7-induced ICAM-1 expression, but not as effectively
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Time (days)
Fig. 2. Production of IP3 in the endothelial ceils. After IFN-7 (100 U/ml) stimulation the cells were lysed with a percholoric acid treatment and the supernatant was collected. IP3 levels were measured with an RIA kit and the results are expressed as pM/106 endothelial cells. One representative experiment is shown.
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Transduction of IFN-y signal in endothelial cells
Table 1 . Expression of ICAM-1 on HUVE cells incubated with IFN-7 (100 U/ml) in combination with different concentrations of H7, which is an enzyme inhibitor acting on PKC activation of positive cells Control IFN-7 IFN-7 + 2/iN A H7 IFN-7 + 10 ,i,M H7 IFN-r + 20 u,M H7
MFI
55 355 342 301 118
44.3 73.1 72.7 68.7 46.9
The amount of antigen on endothelial cells was measured 25 h after the initiation of the stimulus A dose-dependent effect of H7 is shown 20 iiM concentration blocks totally IFN-7-induced up-regulation of ICAM-1 expression One representative experiment out of two is presented.
30
ao
lime (min)
IFN-gamma (lOOU/ml) +H72 uM +H720 uM + W72 uM + W720 uM • HA 10042 uM + HA100420uM 80
©
X
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ICAM-1 expressing cella (%) Fig. 4. Effect of various enzyme inhibitors on the ICAM-1 expression on endothelial cells incubated in combination with IFN-7 (100 U/ml) for 24 h. H7 inhibits dose-dependently the IFN-7 effect. Also a calcium calmodulin antagonist, W7, decreases slightly the ICAM-1 expression, whereas HA 1004 (a protein kinase A inhibitor) has no effect. One representative experiment out of three is presented.
as H7. Concomitantly HA 1004, which is an inhibitor of protein kinase A, had no effect on IFN-7-induced ICAM-1 expression (Fig. 4). Activation of PKC by IFN-y To verify the direct role of PKC we wanted to evaluate whether IFN-7 actually is able to stimulate this enzyme in endothelial cells PKC is located in the cytosol of a resting cell, but upon activaton it is found associated to the cell membrane. This translocation of the enzyme from the cytosol to the membrane has been used as a marker of its activity. We separated the membranes of endothelial cells by ultracentrrfugation and measured the PKC activity. After a 30 min incubation with IFN-7 the membrane activity measured with histone phosphorylation had increased from 669 to 3528 c p.m., corresponding to a > 5-fold increase (Fig. 5). Finally we analysed the calcium flux from endothelial cells. We were able to show a significant 1.4-fold increase in the outflux of 45Ca2+ 5 min after the initiation of IFN-7 stimulation (data not shown). Discussion Lymphocyte homing is a crucial step in the initiation of local immunoresponses. The recirculating lymphocytes can exit blood circulation by first adhering to and then penetrating through the
microvascular endothelium (1 - 5 ) . Several homing receptors defined on the lymphocyte side as well as vascular adressins on the endothelial cell side involved in this homing have been defined so far. One of the first well characterized ligand pairs involved in the intercellular adhesion is the LFA-1 (CD11a/CD18) -ICAM-1 (CD54) pair (10,11). This receptor ligand pair does not express any organ-specificity, but has an important accessory role in the cell-cell adhesion. There are also other inducible endothelial molecules (vascular adressins) involved in homing, like ELAM-1, which has tissue specificity (2,13). ICAM-1 is a single chain 76 -114 kd glycoprotein (polypeptide backbone is 55 kd) on the endothelial cell surface, but it is also present on many white cells as well as fibroblasts and keratinocytes (11). ICAM-1 is involved in several cell-cell interactions, such as homoaggregation (20) and adhesion of white cells to epidermal keratinocytes (21) and fibroblasts (8). ICAM-1 mediates both lymphocyte and granulocyte adhesion to endothelial cells, but has a lesser role in monocyte adhesion (17). The expression of ICAM-1 can easily be up-regulated by several cytokines, such as IFN-7, IL-1 and TNF (8,13,14), within 4 - 2 4 h. Concomitantly with the increase of ICAM-1 on the endothelial cells, the lymphocyte binding increases. We have already initiated studies of the signal transduction in the lymphocyte binding to and penetrating through cultured endothelial monolayers (22). In this paper we have analysed the signal transduction mechanisms after IFN-7 stimulation of endothelial cells in correlation to regulation of ICAM-1 expression. The inositolphospholipid system is activated by a number of different cytokines, hormones, and growth factors (23). This is mediated through receptor-triggered hydrolysis of these inosrtolphospholipids by PLC. Several distinct isoenzymes of PLC have already been identified and some of them have been cloned completely (24). PLC hydrolyse PIP2 into IP3 and DAG. IP3 increases the cytosolic calcium levels and thereby activates the calcium calmodulin pathway. DAG is the natural stimulus for the PKC pathway. There is also evidence that certain lymphokines, e.g. IL-1 and IFN-7, can activate phosphatidyl choline, which
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Fig. 5. Direct activation of PKC measured by the membrane translocation assay. After various incubation periods with IFN-7 the cells were frozen and the membranes isolated. The PKC activity in the membranes was analysed with a histone phosphorylation technique After a 30 min incubation with IFN-7 the PKC activity was increased by > 5-fold in the membrane One representative experiment out of two is presented
LH
40
4000-1
Transduction of IFN-y signal in endothelial cells 723
At least three different subtypes of PKC have been identified. a, j3, and y. These subtypes have been sequenced and monoclonal antibodies against them are available (33). It has been suggested that there is a calcium binding site in the PKCa that has a high degree of structural homology to calcium calmodulin (34). IFN-7 has been shown to operate via the PKC-dependent but not the calcium calmodulm-dependent pathway in endothelial cells (35,36). On the other hand, the calcium calmodulin but not the PKC pathway was responsible for the signal transduction after IFN-7 stimulation in HL-60 cells (37). This discrepancy may relate to the transformation itself or to the different embryonal origins of these cells lines. Calcium ionophore A23187, which rapidly increases the level of intracellular Ca2+, was able to increase the expression of ICAM-1 molecules on HUVE cells. The addition of dibutyryl-cAMP and -cGMP, which are analogs of cAMP and cGMP able to penetrate in the cell, had no effect of ICAM-1. The role of PKC and cytosdic Ca2* was confirmed in enzyme inhibition studies. Selective inhibitors of PKC and a calcium calmodulin antagonist inhibited the IFN-7 effect of enhancing ICAM-1 expression on endothelial cells. Concomitantfy, the protein kinase A inhibitor had no effect. Calmodulin is a small intracellular protein involved among other things in Ca2 + transport. Recently several groups have demonstrated that W7 is a specific calcium calmodulin antagonist which is able to block calcium ionophore-induced IL-1 production (38,39). Different mediators of inflammation activate different routes of signal transduction or at least their phosphorylation substrates vary. We have shown earlier in the lymphocyte-endothelial binding assay that IL-1 operates via cAMP, whereas plateletactivating factor operates via the PKC pathway (40). IFN-7 increased lymphocyte penetration through intact cultured endothelial cell monolayers in vitro (41). Preliminary data indicate that this effect operates also via the PKC and not other pathways (J. P. Turunen and R. Renkonen, unpublished data). On the other hand, we have shown that while IL-1 also induced a 3- to 5-fold
penetration through the endothelial monolayers, this effect does not operate via PKC but through cAMP and protein kinase A. These observations show that different mediators of inflammation causing lymphocyte binding to and penetration through endothelial cell layers in vitro apply different intracellular signal transduction pathways. This once again demonstrates that there are clear differences in the intracellular signal transduction during lymphocyte binding to endothelial cells in vitro induced by different mediators of inflammation. Our results presented in this paper suggest that IFN-7enhanced ICAM-1 expression on HUVE cells is mediated via the PKC and calcium calmodulin pathways, but not by other second messenger pathways.
Acknowledgements This work was supported in part by grants from the Finnish Academy, Finnish Cancer Foundation, The Kidney Foundation, Helsinki, Research and Science Foundation of Farmos Ltd, and a grant from the University of Helsinki
Abbreviations DAG EC H7 HUVE ICAM IFN-7 IL-1 IP3 MFI PIP2 PKC PLC PMA PMSF TCA TNF W7
diacylglycerol endothelial cells 1 -{5-isoquinolinesulphonyf)-1 -methytp.perazine dihydrochlonde human umbilical cord vein endothelial intercellular adhesion molecule gamma-interferon interleukin 1 mosrtoltriphosphate mean fluorescence intensity phosphoinositoldiphosphate protein kinase C phospholipase C phorbol 12-myristate 13-acetate phenyimethyisulphonyl fluoride tnchloroacetic acid tumour necrosis factor /V-(6-aminohexyt)-5-chloro-1-naphthalenesulphon amide hydrochloride
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leads to generation of DAG without formation of IP3 (25). This was not the case in IFN-7-stimulated endothelial cells since IP3 increased within minutes in our assays. We demonstrate in this paper that IFN-7 activates PLC rapidly and transiently, leading to activation of both the calcium calmodulin and the PKC pathways. Previously ICAM-1 has been reported to be up-regulated by PMA, which is a PKC activator. We wanted to analyse in detail the IFN-7 effect at the level of intracellular second messengers. The PKC stimulator PMA was able to independently up-regulate ICAM-1 expression on endothelial cells. PKC has a key role in signal transduction (26,27). It is stimulated by several phorbolesters, e.g. PMA. H7 is an enzyme inhibitor that inhibits PKC-mediated phosphorylation (28,29). H7 also inhibits several PMA-induced effects in a dose-dependent manner (30) These enzyme inhibitors are not as specific as previously had been thought (31,32) Therefore we confirmed our results demonstrating that H7 could inhibit the IFN-7 effect by another PKC inhibitor, sphingosine (data not shown). This inhibitor could also decrease the IFN-y induced ICAM-1 expression on endothelial cells. The role of PKC was also verified by PMA, which could mimic the IFN-7 effect on ICAM-1 expression, and IFN-7 led directly to translocation of PKC to cell membranes.
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Transduction of IFN-y signal in endothelial cells
