Monocytes Upregulate Endothelial Cell Expression of Tissue Factor: A Role for Cell-Cell Contact and Cross-Talk

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1 Monocytes Upregulate Endothelial Cell Expression of Tissue Factor: A Role for Cell-Cell Contact and Cross-Talk By Emanuela Napoleone, Angelomaria Di Santo, and Roberto Lorenzet (TNF-a), well-known inducers of TF in HUVECs, were found in the supernatant from the coculture, and specific antibod- ies directed against either cytokine inhibited TF generation. The need of IL-1b and TNF-a synthesis in order to elicit TF expression was also suggested by the delay observed in TF mrna formation and TF activity generation when mono- cytes were incubated with HUVECs. IL-1b and TNF-a antigen levels in the coculture supernatant, and, consequently, HU- VEC TF expression, were inhibited in the presence of anti- CD18 monoclonal antibody. These findings emphasize the role of cell-cell contact and cross-talk in the procoagulant activity, which could be responsible for the thromboembolic complications observed in those vascular disorders in which monocyte infiltration is a common feature by The American Society of Hematology. Monocytes and endothelial cells interact at sites of vascular injury during inflammatory response, thrombosis, and development of atherosclerotic lesions. Such interactions result in modulation of several biological functions of the two cell types. Because both cells, on appropriate stimulation, synthesize tissue factor (TF), we examined the effect of human umbilical vein endothelial cell (HUVEC)/monocyte coculture on the expression of TF. We found that the coincubation resulted in TF generation, which was maximal at 4 hours, increased with increasing numbers of monocytes, and required mrna and protein synthesis. Supernatant from HU- VEC/monocyte coculture induced TF activity in HUVECs, but not in monocytes, indicating that HUVEC were the cells responsible for the activity, and that soluble mediators were involved. Interleukin-1b (IL-1b) and tumor necrosis factor-a T ISSUE FACTOR (TF) is an integral membrane glycoprotein tightly associated with phospholipids functioning as receptor and cofactor for coagulation factor VII and The effect of monocyte or its products influences endothelial cell migration and proliferation, and production of several endothelial molecules. Conversely, endothelial cells, its active form VIIa. On binding of factor VIIa to TF, the through generation of chemoattractants, adhesion molecules, complex acquires catalytic activity and converts factors IX and cytokines, deeply affect monocyte function. 23,25 Inand X to their active derivatives IXa and Xa, respectively, flammatory events, immune complex disorders, and rupture thus leading to thrombin generation and fibrin formation. 1,2 of atherosclerotic plaques lead to activation of coagulation TF, which under physiological conditions is not in contact followed by thrombus formation. In all instances, close assowith blood, can be found in the tissue adventitia surrounding ciation of monocytes to the endothelium, with consequent blood vessels, where, after vascular injury, it becomes avail- migration into the extracellular compartments, are comable for the interaction with factor VIIa, starts the coagula- monly observed. To better understand monocyte-endothelial tion process, and forms the fibrin clot that reduces or stops cell interactions, several groups have recently established the loss of blood. 3 and analyzed cocultures of the two cell types. Their results Among the cells in contact with the blood stream, quies- show that the close proximity of monocytes and endothelial cent monocytes and endothelial cells are not endowed with cells modulates the production of molecules such as prosta- TF activity, but synthesize and express TF on their mem- cyclin, plasminogen activator inhibitor, von Willebrand facbrane in response to a wide variety of stimulating agents. tor, procoagulants, mitogens, the macrophage-inflammatory Physiologically relevant agents that induce TF generation in protein-1a (MIP-1a), and monocyte chemoattractant promonocytes include: bacterial lipopolysaccharide (LPS), 4,5 the tein-1 (MCP-1) In the present study we investigated hydrolytic fragment of the fifth component of complement, 6 whether monocyte-endothelial cell coincubation can induce immune complexes, 7 P-selectin, 8 platelets, 9 and malignant the expression of TF. To test this hypothesis we used a cells. 10 Inducing agents for endothelial cell TF synthesis are: coculture system of monocytes and human umbilical vein LPS, 11,12 thrombin, 13 interleukin-1b (IL-1b) and tumor ne- endothelial cells (HUVECs). Our results indicate for the first crosis factor-a (TNF-a). 14 The expression of TF on mono- time that, in resting conditions, monocytes induce a procoagcyte and endothelial cell membrane, and the consequent acti- ulant activity of the TF type in endothelial cells, and that vation of coagulation, may be of clinical significance in a variety of pathologic conditions, such as septic shock, 15,16 immune inflammatory diseases, and cancer. 10,20 More- From the Antonio Taticchi Unit for Atherosclerosis and over, expression of TF activity has been linked to the fibrin Thrombosis, Department of Vascular Medicine and Pharmacology, formation observed in unstable angina patients, 21 and TF- Istituto di Ricerche Farmacologiche Mario Negri Sud, Santa Maria producing cells have been identified in atherosclerotic Imbaro, Italy. plaques, thus suggesting a role for TF in the prothrombotic Submitted April 25, 1996; accepted August 29, Supported in part by the Italian National Research Council (CNR, state associated to atherosclerotic vessels. Monocyte-endothelial cell interactions play a key role in Rome, Italy), Progetto Finalizzato FATMA contract No. the inflammatory response, thrombosis, and development of , and Convenzione CNR-Consorzio Mario Negri Sud. Address reprint requests to Roberto Lorenzet, PhD, Consorzio atherosclerotic lesions. 23 Important initial steps for these in- Mario Negri Sud, Via Nazionale, S Maria Imbaro, Italy. teractions are recruitment of monocytes through soluble me- The publication costs of this article were defrayed in part by page diators and their consequent attachment to the vascular endo- charge payment. This article must therefore be hereby marked thelium, mediated by a complex array of adhesion advertisement in accordance with 18 U.S.C. section 1734 solely to molecules. 24 Such a close apposition might be predicted to indicate this fact. result in modulation of several biological functions by both 1997 by The American Society of Hematology. cell types /97/ $3.00/0 Blood, Vol 89, No 2 (January 15), 1997: pp AID Blood 0020 / 5h2b$$$ :30:31 blda WBS: Blood

2 542 NAPOLEONE, DI SANTO, AND LORENZET cytokines are the mediators responsible for the activity. A in serum-free RPMI 1640, and incubated in 0.5 ml of the same role for CD18 integrin adhesion molecules is suggested. medium with or without monocytes for different time intervals at 37 C in 7.5% CO 2. At the end of incubation, HUVECs were extensively washed, resuspended in 300 ml RPMI 1640, and disrupted MATERIALS AND METHODS by 3 freeze-thaw cycles. The disrupted cells were stored at 020 C Reagents and monoclonal antibodies (MoAbs). Medium 199 until procoagulant activity assay. (M199), newborn calf serum (NCS), penicilline, glutamine, 0.05% In selected experiments, HUVECs or monocytes were incubated trypsin/0.02% EDTA, were purchased from GIBCO-BRL (Grand for 2 hours with 10 mg/ml a-amanitin, a specific inhibitor of RNA Island, NY). Heparin, collagenase, polymixin B, and cycloheximide polymerase II. After washing, the a-amanitin treated HUVECs and were from Sigma Chemical Co (St Louis, MO). RPMI 1640 medium the a-amanitin treated monocytes were incubated for an additional and fetal calf serum (FCS) were from Biochrom (Berlin, Germany). period of 3.5 hours with monocytes and HUVECs, respectively. Endothelial cell growth factor (ECGF) was prepared according to The cells were then washed and treated as described below for Maciag et al. 32 Tissue-culture dishes were from Costar Data Packing procoagulant activity assay. Corp (Cambridge, MA). a-amanitin was from Calbiochem (San When cycloheximide (10 mg/ml) or polymixin B (1 ng/ml) were Diego, CA). Lymphoprep was from Nycomed (Oslo, Norway), and used, they were added at the beginning of incubation. In some experi- Percoll was from Pharmacia (Uppsala, Sweden). FVII deficient ments, HUVECs and monocytes, alone or together, were incubated plasma was obtained from Ortho Diagnostic System (Cologno for 4 hours. At the end of incubation, the supernatants were collected, Monzese, Italy), and the Limulus assay was from Whittaker Biopromonocytes sedimented by centrifugation (5 minutes at 16,000g) to eliminate ducts, Inc (Walkersville, MD). Recombinant human IL-1b and remonocytes and cellular debris, added to untreated HUVECs or combinant human TNF-a were from Peprotec (Rocky Hill, NJ). obtained from the same donors and cultured in parallel, The MoAbs against human TNF-a and human IL-1a were purwashed and incubated for 4 hours. At the end of incubation, the cells were chased from UBI (Lake Placid, NY); the MoAb against human IL- and the procoagulant activity was determined. 1b was from R&D System (Minneapolis, MN) and was used as In experiments in which MoAbs directed against IL-1a, IL-1b, purified IgG1 at 10 mg/ml. This concentration was found to be and TNF-a were used, they were incubated with HUVECs for 15 saturating. The ascite, specific for the b 2 subunit of the CD11/CD18 minutes at room temperature before addition of monocytes or super- integrin, was obtained from the hybridoma cells (clone TS1/ natant from HUVEC/monocyte coincubation. MoAbs to CD18 and ) purchased from American Type Culture Collection CD1 were added together with monocytes at different dilutions to (ATCC; Rockville, MD). Inhibition studies with this antibody were the endothelial monolayer. performed with 1/500 dilution (saturating concentration). The iso- Cell adherence. HUVECs and monocytes were cocultured for type matched control MoAb anti human CD1 was also from ATCC. 4 hours. At the end of incubation, the wells were rinsed 3 times to Cell culture. Endothelial cells were isolated from human umbilistained remove the nonadherent monocytes. The cells were then fixed and cal cord vein by digestion with 0.5% collagenase as previously described. by the Diff-Quik Staining Set from Baxter Dade (Dudingen, 33 For experiments, HUVECs were plated at a density of Switzerland). The number of monocytes adhering on HUVECs was cells/well in gelatin-coated 12-well plates, and grown to evaluated by counting in each of 20 microscopic fields per sample. confluency in 199 medium, supplemented with 20% NCS, 2 mmol/ Control of endotoxin contamination. Sterile pyrogen-free work- L L-glutamine, 100 mg/ml streptomycin, 100 U/mL penicillin, 5 ing conditions were observed to avoid any contamination by endomg/ml ECGF, and 10 mg/ml heparin, in humidified atmosphere of toxin. Solutions were prepared in glassware rendered pyrogen-free 92.5% air/7.5% CO 2 at 37 C. Cultured medium was refreshed every by heating at 180 C for 3 hours. Reagents were dissolved in sterile other day. The number of HUVECs at confluency was /well. pyrogen-free solvents, and, when routinely tested for endotoxin con- HUVECs were always used at the second passage, and cells were tamination by the Limulus assay, were found negative at the level shown to be von Willebrand factor-positive as judged by immuno- of 0.1 endotoxin U/mL, corresponding to 0.01 ng/ml. fluorescent staining. Procoagulant activity assay. Procoagulant activity was assessed Monocytes. Human monocytes were obtained from whole blood by a one-stage clotting assay. 8 Disrupted cells (100 ml) were mixed collected from healthy donors and anticoagulated with 0.1 vol of with 100 ml of normal human plasma at 37 C. After 30 seconds, 3.8% sodium citrate/0.15 mol/l NaCl. Leukocytes were sedimented 100 ml of 25 mmol/l CaCl 2 at 37 C was added to the mixture and at 180g for 15 minutes at 10 C. The sedimented cells were diluted the time to clot formation was recorded. Results were expressed in to the initial volume with citrate-saline (0.38% sodium citrate/0.15 arbitrary units (U) by comparison with a standard curve obtained mol/l NaCl), and sedimented again. This step was repeated to mini- using a human brain thromboplastin standard kindly donated by Dr mize platelet contamination. The cells were then layered onto L. Poller (Manchester, UK). This preparation was assigned a value Lymphoprep, and centrifuged at 700g for 20 minutes at 20 C. The of 1000 U for a clotting time of 20 seconds. The standard curve ring of mononuclear cells was collected and sedimented at 620g for was linear from to 0.01 U, corresponding to clotting times of 7 minutes at 8 C. The pellet was resuspended in citrate-saline and 20 and 511 seconds, respectively. In some instances, results were the cells were washed twice. Monocytes were purified using a discurve also expressed in nanograms of TF by comparison with a standard continuous Percoll density gradient; the mononuclear cell preparatein obtained by using a recombinant human TF, lipidated lipopro- tion was resuspended in RPMI 1640 medium supplemented with standard (American Diagnostica, through the courtesy of Dotion 10% heat-inactivated FCS, and was layered onto a 46% isosmotic menico Santo, Ortho Diagnostic). Percoll solution. After centrifugation at 20 C for 30 min at 550g, IL-1b and TNF-a determination. Conditioned medium from the cells at the RPMI 1640 medium/percoll interface were collected HUVECs and monocytes, alone or together, was sedimented by and sedimented at 620g. The pellet was resuspended in citrate-saline centrifugation to eliminate cellular debris, and the presence of IL-1b and the cells were washed two times. The final monocyte preparation and TNF-a was assessed by enzyme-linked immunosorbent assays was then resuspended in serum-free RPMI 1640 at the concentration (ELISA) as specified by the manufacturers (Amersham, Bucking- required. The Percoll isolated fraction contained approximately 85% hamshire, UK). The sensitivity of these ELISAs was 0.3 and 4.4 pg/ monocytes, as assessed by nonspecific esterase staining, of which ml for IL-1b and TNF-a, respectively. ú95% were viable as determined by trypan blue staining. Northern blot analysis. For northern blot analysis, HUVECs Coincubation studies. Confluent HUVECs were washed 3 times were cocultured with monocytes for different time intervals. At the AID Blood 0020 / 5h2b$$$ :30:31 blda WBS: Blood

3 MONOCYTES UPREGULATE ENDOTHELIAL TF 543 The procoagulant activity was characterized using congenital factor-deficient plasmas. The dependence on the presence of factor VII, factor X, and prothrombin indicates this activity to be attributable to TF (not shown). Virtually, no procoagulant activity was developed when HUVECs and monocytes were cultured alone, indicating that no endotoxin contamination was affecting our experimental conditions. Role of protein synthesis and DNA transcription. Incubation of HUVECs with monocytes in the presence of 10 mg/ ml cycloheximide virtually abolished TF activity generation, indicating the requirement for de novo protein synthesis. The role of DNA transcription for both cell types was investigated by using a-amanitin, a specific irreversible inhibitor of RNA polymerase II. In this set of experiments, HUVECs were incubated for 2 hours with 10 mg/ml a- amanitin. After washing, the a-amanitin treated HUVECs Fig 1. Effect of coincubation of monocytes (M) with HUVEC on were further incubated with monocytes that were not ex- TF activity. 4 Ì 10 5 M were incubated with or without HUVEC (2 Ì posed to a-amanitin, for an additional period of 3.5 hours /well) for 4 hours at 37 C in 7.5% CO 2. At the end of incubation Similarly, monocytes treated with a-amanitin for 2 hours the cells were washed and resuspended in 300 ml RPMI TF were washed and further incubated for 3.5 hours with unactivity was measured in frozen and thawed samples, as described treated HUVECs. Blockade of DNA transcription in either end of incubation, HUVECs were extensively washed to remove monocytes. Total cellular RNA was isolated by the thiocyanate/ cesium chloride method. 34 Ten micrograms of each RNA sample was electrophoresed on a 1% agarose gel, and transferred to nitrocellulose (Zeta Probe Blotting Membranes, Bio-Rad, Hercules, CA) in 201 saline sodium citrate overnight, and backed at 80 C for 2 hours under vacuum. The cdna probe for TF (phtf8; ATCC) was labeled with 32 P by the random primer method and purified by gel filtration (Sephadex G-50). Filters were prehybridized in 7% sodium dodecyl sulfate (SDS), 0.5 mmol/l sodium phosphate (ph 7.2), 1 mmol/l EDTA, 1% bovine serum albumin (BSA). Hybridization was performed in the same buffer for 16 hours at 65 C. The filters were washed twice with.5% SDS, 40 mmol/l sodium phosphate (ph 7.2), 1 mmol/l EDTA, 0.5% BSA at 65 C for 30 minutes, and twice with 1% SDS, 40 mmol/l sodium phosphate (ph 7.2), 1 mmol/l EDTA at 65 C for 30 minutes. The filters were then exposed to Kodak X-OMAT AR x-ray film (Eastman-Kodak, Rochester, NY) by using an intensifying screen at 070 C for various times. As an internal control, filters were also hybridized and autoradiographed using a 32 P-labeled GAPDH. Statistical analysis. The results are given as mean values { SEM. Differences between groups were tested for significance using Student s t test for paired observations, unless otherwise indicated. ANOVA analysis followed by Dunnett s test was used for multiple comparisons. in Materials and Methods. Bars represent the mean of ten experiments Ô SEM. *P Ú.0001 versus both HUVEC and M incubated alone. cell type largely prevented generation of TF activity, indicat- ing that RNA synthesis by both HUVECs and monocytes is required for the activity (Fig 2). Neither treatment decreased cell viability, measured by trypan blue exclusion. RESULTS Effect of HUVEC/monocyte coincubation on TF expression. Coincubation of freshly isolated monocytes with HU- VECs at a ratio of 2:1 for 4 hours at 37 C resulted in expression of procoagulant activity (Fig 1). The amount of TF protein for HUVECs, monocytes, and HUVEC/monocytes, by extrapolation with a standard curve of serial dilutions of recombinant human TF, lipidated protein standard, corre- sponded to 0.48, 0.57, and ng/ cells, respec- tively. The activity increased steadily by increasing the number of monocytes incubated with HUVECs. Fig 2. Effect of a-amanitin on TF activity generation during HU- VEC/M coculture. a-amanitin (10 mg/ml) was incubated with HUVEC or M for 2 hours. At the end of incubation, HUVEC (2 Ì 10 5 /well) and M(4Ì10 5 /well) were washed and incubated for an additional period of 3.5 hours with untreated M and HUVEC, respectively. Cells were then processed for TF activity assay, as for Fig 1. Results are expressed in percent of TF activity. A 100% value was assigned to the activity expressed by the HUVEC/M coculture. Bars represent the mean of three experiments Ô SEM. **P Ú.0001 and *P Ú.01 when compared with HUVEC " M. AID Blood 0020 / 5h2b$$$ :30:31 blda WBS: Blood

4 544 NAPOLEONE, DI SANTO, AND LORENZET perimental conditions, HUVECs and monocytes were incubated alone or together for 4 hours. Conditioned medium was collected, sedimented by centrifugation, and the presence of IL-1b and TNF-a assessed by ELISA, while TF activity was tested on disrupted cells. Virtually, neither IL-1b nor TNFa could be detected in supernatants from HUVECs and monocytes cultured alone. By contrast, when HUVECs and monocytes were cultured together, antigens for IL-1b and TNF-a could be found in the conditioned medium, while HUVECs were expressing TF activity (Fig 4). To investigate whether these cytokines were responsible for HUVEC activation, specific neutralizing antibodies to IL-1b or TNF-a were incubated with HUVECs 15 minutes before incubation with monocytes. As shown in Fig 5A, both antibodies (at a concentration of 10 mg/ml) markedly reduced the generation of TF activity, suggesting that both cytokines were involved. Similarly, when MoAbs anti IL- 1b and TNF-a were incubated with HUVEC before addition of supernatant from HUVEC/monocyte coincubation, they could inhibit TF activity generation (Fig 5B). No further inhibition was obtainable when both MoAbs were present at the same time (not shown). In both instances, a MoAb against IL-1a did not affect TF activity. Fig 3. Effect of supernatant (sn) from HUVEC/M coincubation on Taken together, these results suggest that HUVEC/monocyte coincubation results in IL-1b and TNF-a synthesis, and that both cytokines, once released, stimulate TF expression by HUVECs. To rule out the possibility that subthreshold concentrations of LPS might prime cells to become respon- sive to other agonists, experiments aiming at excluding this possibility were carried out. In this set of experiments, polymixin B (100 ng/ml) was incubated with HUVECs and TF activity in HUVECs and M. HUVEC (2 Ì 10 5 /well) and M (4 Ì 10 5 / well) were incubated together for 4 hours at 37 C. At the end of incubation the supernatant was collected, sedimented by centrifuga- tion, and added to HUVEC or M obtained from the same donors and cultured in parallel. The cells were then washed and tested for TF activity, as described in Materials and Methods. Results are expressed in percent of TF activity. A 100% value was assigned to the activity expressed by the HUVEC/M coculture. Each column repre- sents the mean of seven experiments Ô SEM. *P Ú.01 when com- pared with HUVEC " M. HUVECs and monocytes were cocultured in the absence or in the presence of polymixin B (13.4 { 1.5 and 12.9 { 1.7, mean { SEM, n Å 4, respectively). Time course of TF induction. Time course experiments were performed in which the effect of monocytes on incuba- tion with HUVECs was compared with that of exogenously added recombinant IL-1b (ril-1b) or recombinant TNF-a (rtnf-a). HUVECs were incubated with monocytes, ril- 1b, or rtnf-a for different time intervals. At the end of incubation, the monolayer was thoroughly washed and the cells were tested for TF activity. As previously reported, 14 ril-1b and rtnf-a stimulate HUVEC TF activity (Fig 6B and C). The stimulation occurs in a dose-dependent fashion with both cytokines (not shown). The activity is already detectable within 2 hours, reaches a maximum at 4 hours, and rapidly declines to basal values within 16 hours. When monocytes are incubated with HUVECs, a shift in time is observed: no activity is detectable after 2 hours, a maximum is attained within 4 hours, sustained through 8 hours, and baseline is reached within 16 hours (Fig 6A). Northern blot analysis on TF mrna. A similar shift in the kinetics was also observed when TF mrna levels were studied. These experiments were conducted in parallel to those performed for determining the time course of TF activ- Effect of supernatant from HUVEC/monocyte coculture on TF expression. To ascertain whether direct cell-cell contact and/or secretory products were involved, HUVECs and monocytes were incubated separately or together for 4 hours. At the end of the incubation, the supernatants were collected, cleared of cells by centrifugation, and added to HUVECs or monocytes obtained from the same donors, and cultured in parallel. After 4 hours, the cells were washed and tested for TF activity. As can be seen in Fig 3, HUVECs incubated with the cell-free supernatant derived from the HUVEC/ monocyte coincubation expressed a TF activity that was sim- ilar to that generated by the intact cell system. Conversely, the same supernatant could not induce TF when incubated with monocytes. Incubations of monocytes with supernatant from HUVECs and of HUVECs with supernatant from monocytes were also ineffective (not shown). These results show that the TF activity that develops during HUVEC/ monocyte coincubation is solely attributable to HUVECs. Moreover, the activity is induced by soluble mediator(s) re- leased when the two cell types are incubated together. Involvement of IL-1b and TNF-a. Among the soluble mediators possibly involved, IL-1b and TNF-a, which are primarily produced by monocytes and well known inducers of endothelial cell TF, 14 were good candidates. In order to investigate whether these cytokines were present in our ex- monocytes for 4 hours at 37 C. In control experiments, this amount of polymixin B was sufficient to prevent the effect of 10 ng LPS/mL on TF expression by mononuclear cells. No differences in TF expression could be observed when AID Blood 0020 / 5h2b$$$ :30:31 blda WBS: Blood

5 MONOCYTES UPREGULATE ENDOTHELIAL TF 545 Fig 4. Presence of IL-1b and TNF-a during HU- VEC/M coculture. HUVEC (2 Ì 10 5 /well) and M (4 Ì 10 5 /well) were incubated together for 4 hours at 37 C. At the end of incubation the cells were washed, the supernatant was collected, and the presence of IL-1b and TNF-a was assessed by ELISA assay, as specified by the manufacturers (Amersham), while the cells were treated for TF activity assay as described in Materials and Methods. Bars represent the mean of five experiments Ô SEM. **P Ú.01, *P Ú.05, and P Ú.05 (by Wilcoxon rank-sum test) versus HUVEC and M cultured alone. ity, ie, HUVECs from the same umbilical cord and mono- creased to baseline value within 16 hours (Fig 6A, insert). cytes from the same donor were used on the same day. Rehybridization with a GAPDH as internal control showed Monocytes, ril-1b, or rtnf-a were incubated with HU- bands of similar intensity (not shown), confirming that equivalent VECs for different time intervals, after which the monolayer amounts of cellular RNA had been loaded onto each was extensively washed and the cells were treated in order lane, and that the increase in TF mrna was specific. to isolate mrna, as described in Materials and Methods. Role of adhesion molecules in TF generation. Monocyte Exposure of HUVECs to either ril-1b or rtnf-a resulted interactions with endothelium are mediated via adhesive in induction of a major 2.2-kb TF mrna, consistent with mechanisms. We explored the possibility that some of these the mature TF message (Fig 6, B and C, inserts). A minor mechanisms could be involved in TF expression. In our experimental TF transcript of 3.4-kb was also detected, in agreement with conditions, monocytes adhered to HUVECs after previous observations. 35 TF mrna, which was not detect- a 4 hour incubation (120 { 15 monocytes/mm 2, by Diff able in unstimulated HUVECs, accumulated to maximal levels Quick, as described in Materials and Methods). The number within 1 hour. A progressive decrease in TF mrna of adherent monocytes was greatly reduced in the presence levels followed, and by 8 hour postinduction, approximately of MoAb anti-cd18 (40 { 4 monocytes/mm 2 ), while MoAb 5% of maximal levels was detectable on the basis of band anti-cd1, used as control, had no effect (140 { 21 monocytes/mm analysis by Instant Imager (Packard, Downers Grove, IL). 2 ). Concomitantly, TF generation by HUVECs inanalysis Conversely, when monocytes were incubated with HUVECs cubated with monocytes for 4 hours was reduced by 60.8% the kinetic analysis showed that TF mrna attained a peak { 16.3% when the anti-cd18 was present during the cocul- at 4 hours, which was still consistent at 8 hours, and de- ture (Fig 7). No effect could be observed with anti-cd1. Since our results show that IL-1b and TNF-a are responsible for HUVEC stimulation, the reduced induction of TF expression should correlate with a diminished synthesis of the two cytokines. We explored this hypothesis by measuring IL-1b and TNF-a antigen levels in supernatants from the same wells in which TF activity was measured. As shown in Fig 7, the anti-cd18 MoAb reduced IL-1b and TNF-a levels by 54.6% { 6.5% and 71.3% { 5.8%, respectively. DISCUSSION Monocytes adhere to endothelial cells. Although maximal adhesion is achieved with cytokine-stimulated endothelial cells, 24,36 several reports have shown that monocytes spontaneously adhere to resting endothelial cells In the present report, we show that the coincubation of purified monocytes with confluent HUVECs results in TF activity generation in the absence of exogenously added stimuli. This activity Fig 5. Effect of anti IL-1a, anti IL-1b, and anti TNF-a MoAbs on correlates with the monocyte number and requires mrna TF activity by HUVEC cocultured with monocytes. The MoAbs, each at 10 mg/ml, were incubated with HUVEC 15 minutes before incubaand de novo protein synthesis. tion with monocytes or supernatant from HUVEC/M coculture. After When HUVECs or monocytes are exposed to supernatant 4 hours, the cells were washed and processed for TF activity assay, from HUVEC/monocyte cocultures, only HUVECs are able as described in Materials and Methods. Results are expressed in perto generate TF activity. This activity is in the same order of cent of TF activity. A 100% value was assigned to the activity exmagnitude as that expressed by HUVEC/monocyte coculpressed by HUVEC incubated with monocytes (A) or supernatant from HUVEC/M coculture (B). Bars represent the mean of four experiments Ô SEM. ***P Ú.001, **P Ú.005, and *P Ú.05 versus control. exclusively derived from HUVECs, and soluble ture. Thus the TF activity generated during the coculture is mediators AID Blood 0020 / 5h2b$$$ :30:31 blda WBS: Blood

6 546 NAPOLEONE, DI SANTO, AND LORENZET Fig 6. Time course in TF activity expression and TF mrna levels during incubation of HUVEC with monocytes, IL-1b, or TNF-a. HUVEC (2 Ì 10 5 /well) were incubated with monocytes (4 Ì 10 5 /well) (A), IL-1b (1 ng/ml) (B), or TNF-a (1 ng/ml) (C) for the indicated time intervals. At the end of incubation the cells were treated for TF activity assay as described in Materials and Methods. In a parallel set of samples total RNA was extracted from cells, and equal amounts of RNA were analyzed by Northern blot as described in Materials and Methods. Each point represent the mean of four experiments Ô SEM. **P Ú.01, and *P Ú.05 versus time zero. are involved, suggesting that exposure of monocytes to endo- which have the ability to shift the hemostatic balance of thelial cells may create a stimulatory environment for monocytes vascular endothelial cells toward a prothrombotic state, sufficient to induce synthesis and release of molecules could likely be the monocyte-derived products responsible by these cells. For example, it has been shown that monocytes for induction of TF activity in HUVECs. Indeed, our ELISA adherent to endothelial cells express monocyte-derived studies show that the antigens of IL-1b and TNF-a are pres- MIP-1a and MCP-1. 30,31 Molecules synthesized and released ent in conditioned medium from HUVEC/monocyte coculby monocytes could then be responsible for TF synthesis ture, and that their amount is sufficient to provoke TF synthesis and expression by HUVECs. This hypothesis is corroborated by HUVECs, as assessed in dose-response experiments by experiments in which a-amanitin, a specific inhibitor of in which recombinant IL-1b and TNF-a were incubated with RNA polymerase II, was used to block RNA synthesis by HUVECs. These findings are in agreement with previous HUVECs and monocytes, alternatively. The largely reduced studies, which suggested the presence of IL-1b 26,28,41 and TF activity observed under this condition clearly shows that TNF-a 28 during cocultures of HUVECs with monocytes or both cell types are contributing with RNA and protein syn- mixed leukocyte suspensions. Moreover, TF activity, both in thesis for HUVECs to express TF on their membrane. the intact cell system and in HUVECs exposed to coculture The monocyte secretory cytokines IL-1b and TNF-a, supernatant, could be diminished by inhibitory antibodies AID Blood 0020 / 5h2b$$$ :30:31 blda WBS: Blood

7 MONOCYTES UPREGULATE ENDOTHELIAL TF 547 Fig 7. Effect of anti-cd18 on TF activity, IL-1b, and TNF-a levels during HUVEC/M cocultures. HUVEC (2 Ì 10 5 /well) were incubated with monocytes (4 Ì 10 5 /well) in the presence or in the absence of anti-cd18 or anti-cd1 for 4 hours. At the end of incubation, superna- tants were collected and IL-1b and TNF-a antigen levels were mea- sured by ELISA assay. After washing, HUVEC monolayers were tested for TF activity as described. Results are expressed in percent of TF activity, and percent of IL-1b and TNF-a antigen levels. A 100% value was assigned to the activity and antigen levels expressed by HUVEC/M coculture in the absence of anti-cd18. Bars represent the mean of three experiments Ô SEM. **P Ú.005 and *P Ú.05 versus control (100%). directed against IL-1b and TNF-a, establishing a direct involvement of IL-1b and TNF-a in TF synthesis by HU- VECs. Activation of HUVECs with monocytes resulted in TF activity and mrna levels which were delayed in their ex- pression when compared with activation with ril-1b and rtnf-a. This set of experiments, together with the results observed with a-amanitin, are consistent with the hypothesis that, to elicit HUVEC procoagulant response, synthesis of soluble mediators has to take place. Monocyte interactions with endothelium are dependent on the recognition by sur- face adhesion molecules, expressed on both monocyte and endothelial cell membrane. The leukocyte b 2 integrin family (CD11/CD18) is important for the binding of monocytes to resting endothelium; antibodies directed against CD18 inhibit monocyte adherence to endothelial cells as was also found in our experimental conditions. In the presence of anti- CD18, adherence of monocytes to HUVECs was reduced by Ç60%. This inhibition in binding was accompanied by a marked reduction in TF activity generation. Since in our experiments HUVEC TF synthesis was caused by monocyte secretory cytokines IL-1b and TNF-a, and inhibition of monocytes binding to HUVECs by anti- CD18 resulted in diminished TF activity, CD18-mediated adhesion could represent a regulatory pathway for monocyte activation and cytokine expression. Indeed, the finding that, in the presence of anti-cd18, IL-1b and TNF-a antigen levels are reduced during the cocultures, seems to sustain this hypothesis. This finding is consistent with the observation that engagement of members of the adhesion molecule family initiates signal transduction leading to expression of a number of immediate-early genes. Release of cytokines from monocytes can be triggered following engagement of surface receptors with MoAbs immobilized on plastic. 42,43 However, the engagement of b2 integrins with specific antibodies as surrogate ligands seems not to provide the necessary signals for mrna expression or protein secretion. 44,45 Although receptor engagement might mimic the receptorligand interaction that mediates cell-cell adhesion, it is conceivable that during cell-cell interaction, more than a single adhesion molecule or signal could take place. Indeed, the anti-cd18 antibody only partially blocks monocyte adhesion and cytokine production, suggesting the involvement of other adhesion mechanisms or soluble signals as costimulatory agents in the response. A previous report showed that binding of monocytes to HUVEC could not elicit TNF-a secretion in the absence of LPS. 45 However, in this case, gluteraldehyde-fixed HUVEC monolayers were used, a condition which could, at least in part, affect HUVEC/monocyte interaction. Moreover, a different sensitivity of the assays used for TNF-a detection (ELISA v L929 cytotoxicity assay) should be taken into account. TF is widely considered to play a leading role in fibrin deposition during inflammatory and thrombotic disorders. Endothelial cells and monocytes are the only cells in the blood stream capable of expressing TF. In order to prevent disseminated intravascular coagulation, TF expression must be localized and limited at sites of endothelial cell injury or perturbation, where cell activation takes place. In this respect, a recent report has shown that adhesion of monocytes on cytokine-treated endothelial cells results in monocyte TF generation. 46 The different experimental conditions could help understanding the apparent discrepancy with our results; indeed, these other investigators were using cytokine-activated endothelial cells, while, in our study, no exogenously added stimulus was used to activate HUVECs. Wharram et al 28 observed TF expression by HUVECs dur- ing HUVEC/M coculture, although LPS or/and aggregated IgG had to be present to produce a significant amount of TF. We have used an extreme precaution in our experiments to minimize LPS contamination, and no detectable amounts of this trigger could be found in our system. Moreover, polymixin B could not prevent TF activity generated during the cocultures, supporting the hypothesis that the observed TF activity was not attributable to LPS contamination. Al- though the different results could be explained, at least in part, by differences in calibration systems, such as the different thromboplastin standard used for calculating TF activity arbitrary units, it should be noted that these investigators were using a ratio of 1 M:8-10 HUVECs, at an M concentration of approximately 75,000/mL, approximately one tenth of that found in blood. In our experimental conditions, the ratio of 2 M:1 HUVEC used could account for the results obtained. Also pertinent to our results, lately, it has been reported that TF antigen could be detected during coculture of HU- VECs with monocytes, although most of it was claimed to be expressed on monocytes. 47 The use of subconfluent HUVECs, which are still undergoing mitosis, and the detec- AID Blood 0020 / 5h2b$$$ :30:31 blda WBS: Blood

8 548 NAPOLEONE, DI SANTO, AND LORENZET tion of TF antigen instead of activity are some of the different 14. Bevilacqua MP, Pober JS, Majeau GR, Fiers W, Cotran RS, experimental conditions that may render it difficult to comagulant Gimbrone MA Jr: Recombinant tumor necrosis factor induces procopare activity in cultured human vascular endothelium: Character- their results with ours. We have thus reported here the original observation that, ization and comparison with the actions of interleukin 1. Proc Natl Acad Sci USA 83:4533, 1986 in the absence of exogenously induced activation, HUVEC/ 15. Østerud B, Flægstad T: Increased tissue thromboplastin activmonocyte interaction results in upregulation of TF expresity in monocytes of patients with meningococcal infection: Related sion in HUVECs. On the basis of these results, it is conceiv- to an unfavourable prognosis. Thromb Haemost 49:5, 1983 able to speculate that, in pathological conditions, such as 16. Semeraro N, Triggiani R, Montemurro P, Cavallo LG, Copostoperative venous thrombosis, in which blood flow is lucci M: Enhanced endothelial tissue factor but normal thrombomodulin reduced and circulating cells more easily interact with vascular in endotoxin-treated rabbits. Thromb Res 71:479, 1993 cells, adherence of monocytes to an unperturbed endothecreased 17. de Prost D, Ollivier V, Ternisien C, Chollet-Martin S: In- lial layer would create a cell-cell functional cross-talk reanticoagulant monocyte procoagulant activity independent of the lupus in patients with systemic lupus erythematosus. Thromb sulting in endothelial cell TF expression; this trigger would eventually lead to fibrin deposition and thrombus formation. Haemost 64:216, Idell S, James KK, Levin EG, Schwartz BS, Manchanda N, ACKNOWLEDGMENT Maunder RJ, Martin TR, McLarty J, Fair DS: Local abnormalities in coagulation and fibrinolytic pathways predispose to alveolar fibrin The authors thank their colleagues at the Department of Vascular deposition in the adult respiratory distress syndrome. J Clin Invest Medicine and Pharmacology for critical reading of this manuscript 84:695, 1989 and fruitful discussions, A. Di Castelnuovo for statistical analysis, 19. Nawroth PP, Handley DA, Esmon CT, Stern DM: Interleukin R. Bertazzi for assistance, and the G.A. Pfeiffer Memorial Library 1 induces endothelial cell procoagulant while suppressing cell-surstaff for their help in the preparation of the manuscript. face anticoagulant activity. Proc Natl Acad Sci USA 83:3460, Semeraro N, Montemurro P, Conese M, Giordano D, Stella REFERENCES M, Restaino A, Cagnazzo G, Colucci M: Procoagulant activity of 1. Nemerson Y: Tissue factor and hemostasis. 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10 : Monocytes Upregulate Endothelial Cell Expression of Tissue Factor: A Role for Cell-Cell Contact and Cross-Talk Emanuela Napoleone, Angelomaria Di Santo and Roberto Lorenzet Updated information and services can be found at: Articles on similar topics can be found in the following Blood collections Hemostasis, Thrombosis, and Vascular Biology (2485 articles) Information about reproducing this article in parts or in its entirety may be found online at: Information about ordering reprints may be found online at: Information about subscriptions and ASH membership may be found online at: Blood (print ISSN , online ISSN ), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC Copyright 2011 by The American Society of Hematology; all rights reserved.