Gene Expression in Human Endothelial Cells TRAIL Induces

The Journal of Immunology

TRAIL Induces Apoptosis and Inﬂammatory Gene Expression in Human Endothelial Cells1

Jie Hui Li,*† Nancy C. Kirkiles-Smith,*† Jennifer M. McNiff,†‡ and Jordan S. Pober2*†‡

Human TRAIL can efficiently kill tumor cells in vitro and kill human tumor xenografts in mice with little effect on normal mouse cells or tissues. The effects of TRAIL on normal human tissues have not been described. In this study, we report that endothelial cells (EC), isolated from human umbilical veins or human dermal microvessels, express death domain-containing TRAIL-R1 and -R2. Incubation with TRAIL for 15 h causes ϳ30% of cultured EC to die, as assessed by propidium iodide uptake. Death is apoptotic, as assessed by Annexin V staining, 4؅,6؅-diamidino-2-phenylindole staining, and DNA fragment ELISA. EC death is increased by cotreatment with cycloheximide but significantly reduced by caspase inhibitors or transduced dominant-negative Fas-associated death domain protein. In surviving cells, TRAIL activates NF-␬B, induces expression of E-selectin, ICAM-1, and IL-8, and promotes adhesion of leukocytes. Injection of TRAIL into human skin xenografts promotes focal EC injury accompanied by limited neutrophil infiltration. These data suggest that TRAIL is an inducer of tissue injury in humans, an outcome that may influence antitumor therapy with TRAIL. The Journal of Immunology, 2003, 171: 1526–1533.

RAIL (APO2 ligand) is a member of the TNF family (1, known as receptor-interacting protein (13). TRAIL ligation on hu- 2), and TRAIL-Rs belong to the TNFR family. The cy- man glioma tumor cells also leads to activation of transcription T toplasmic regions of TRAIL-R1 (DR4) (3) and factor AP-1 (14). Cumulatively, these observations suggest that TRAIL-R2 (DR5) (4, 5) contain a conserved death domain, a pro- TRAIL, like TNF, may in parallel activate transcription/translation- tein-protein interaction region, initially identified in CD95 (also independent pathways of apoptosis as well as antiapoptotic/proin- known as APO-1/Fas) and TNFR1 (CD120a), as well as in the flammatory pathways that depend upon NF-␬B- and/or AP-1-medi- cytoplasmic adapter molecules Fas-associated death domain ated new gene expression. The outcome of TRAIL binding to any (FADD)3 (also known as mediator of receptor-induced toxicity-1 given cell type may depend upon the relative strengths or kinetics of or MORT-1) and TNFR-associated death domain proteins, which activation of these competing responses (15, 16). interact with these receptors. Upon TRAIL binding, TRAIL-R1 or In a variety of rodent tumor models, including those involving -R2 recruit FADD, and FADD, in turn, recruits procaspase-8 (6, 7) human xenografted tumor cells, human TRAIL can selectively kill to form a death-inducing signaling complex. Within the death- the tumor cells without appreciable host toxicity (17–19). This inducing signaling complex, procaspase-8 undergoes autocatalytic wide therapeutic window has raised hopes that TRAIL can be use- activation, initiating a caspase cascade that leads to apoptotic cell ful in the treatment of human cancers. Human TNF initially death. Three other TRAIL-Rs, TRAIL-R3 (TRAIL receptor with- showed a favorable therapeutic window in mouse tumor models out an intracellular domain or TRID/DcR1/lymphocyte inhibitor of but has generally proven to be too toxic for human therapy (20). TRAIL or LIT) (8), TRAIL-R4 (7, 9), and osteoprotegerin (10), The increased toxicity of TNF observed in the clinic may be re- which do not have death domains, may function as decoy receptors lated to the fact that human TNF can act on human cells through to protect cells from TRAIL-induced apoptosis (5, 8). In some cell two receptors, TNFR1 (CD120a) and TNFR2 (CD120b) (21), but types, TRAIL binding to TRAIL-R1, -R2, and/or -R4 may also can act on mouse cells only through TNFR1 (22, 23). Mouse TNF, activate NF-␬B (11, 12), leading to transcription of genes known which can engage both TNFR types in rodents, is much more toxic to antagonize the FADD/caspase-8 death pathway and/or to pro- for mice than is human TNF (23). Mouse TRAIL-Rs are incom- mote inflammation. NF-␬B activation may be initiated by recruit- pletely characterized, and the capacity of human TRAIL to interact ment of an alternative death domain-containing adapter molecule, with different mouse receptors is unknown. Much of the antitumor efficacy of TNF is mediated through effects on vascular endothelial

*Interdepartmental Program in Vascular Biology and Transplantation, Boyer Center cells (EC) (24, 25). In cultured human EC, human TNF causes for Molecular Medicine, and Departments of †Pathology and ‡Dermotology, Yale apoptosis, especially in the presence of RNA or protein synthesis University School of Medicine, New Haven, CT 06510 inhibitors such as actinomycin D or cycloheximide (CHX), respec- Received for publication December 31, 2002. Accepted for publication May 22, 2003. tively. TNF also activates both NF-␬B and AP-1 in EC, leading to The costs of publication of this article were defrayed in part by the payment of page the expression of proinﬂammatory proteins, such as E-selectin charges. This article must therefore be hereby marked advertisement in accordance (CD62E), ICAM-1 (CD54), and IL-8. The balance of TNF re- with 18 U.S.C. Section 1734 solely to indicate this fact. sponses generally favors activation rather than apoptosis. We won- 1 This work was supported by National Institutes of Health Grant HL62188 (to J.S.P.). dered whether TRAIL would also act on human EC, and if so, 2 Address correspondence and reprint requests to Dr. Jordan S. Pober, Boyer Center for Molecular Medicine, Yale University School of Medicine, 295 Congress Avenue, what the responses to TRAIL signaling might be. New Haven, CT 06510. E-mail address: [email protected] In this study, we report the effects of recombinant human 3 Abbreviations used in this paper: FADD, Fas-associated death domain protein; TRAIL on cultured human EC, including both human umbilical FADD.DN, FADD dominant negative; EC, endothelial cell; HUVEC, human umbil- vein EC (HUVEC) and human dermal microvascular EC (HD- ical vein EC; HDVEC, human dermal microvascular EC; CHX, cycloheximide; DAPI, 4Ј,6Ј-diamidino-2-phenylindole; PDTC, pyrrolidine dithiolcarbamate; PI, pro- MEC), as well as upon resting human EC in human skin xenografts pidium iodide; Tc, threshold cycle. transplanted on SCID/beige mice. We ﬁnd that cultured human EC

Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 The Journal of Immunology 1527 express death domain-containing TRAIL-Rs, and that recombinant cytometry using a FACSort (BD Biosciences, Mountain View, CA). Ap- human TRAIL protein triggers FADD-caspase-8-dependent apo- optois of TRAIL-treated EC was assessed by an annexin V-FITC staining ptosis in EC. TRAIL also activates NF-␬B, resulting in E-selectin, kit (BD PharMingen) following the manufacturer’s protocol. Nuclear morphology during EC apoptosis was assessed by DAPI staining and fluores- ICAM-1, and IL-8 expression and increased leukocyte binding. cence microscopy, as described previously (28). DNA fragmentation was TRAIL is more potent than TNF as an inducer of apoptosis, but measured using a cell death detection ELISA kit (Roche) according to the less potent than TNF as an activator of EC inflammatory gene manufacturer’s instructions. expression. Importantly, TRAIL injected into human skin xenografts results in focal endothelial damage with little accompanying Quantitation of protein expression neutrophil recruitment. These data suggest that TRAIL may be an inducer of normal tissue injury in humans. We measured protein expression by immunoblotting or flow cytometric analysis as described previously (28). Immunoblots were analyzed by chemiluminescence and ␤-actin was used as protein loading control. For Materials and Methods flow cytometry, Ab-stained cells were analyzed using FACSort cytometer and CellQuest software. We calculated corrected mean fluorescence inten- Reagents and cells sities by subtracting the mean values of cells stained with the irrelevant Recombinant human TRAIL, TNF-␣, recombinant human Fas-Fc, recom- control mAb from that of cells stained with specific primary mAb. binant human TRAIL-R2-Fc, caspase-8 selective inhibitor Z-Ile-Glu-Thr- Asp-fluoromethyl ketone, and caspase-3 selective inhibitor Z-Asp-Glu- Val-Asp fluoromethyl ketone were from R&D Systems (Minneapolis, RT-PCR analysis Ј Ј MN). CHX, 4 ,6 -diamidino-2-phenylindole (DAPI), and pyrrolidine di- We performed total RNA extraction, reverse transcription, and PCR am- thiolcarbamate (PDTC) were from Sigma-Aldrich (St. Louis, MO). A cell plification as described previously (28, 29). The primers used for death detection DNA fragment ELISA kit and an annexin V-FITC staining TRAIL-Rs were as follows: TRAIL-R1, 5Ј-ATGGCGCCACCACCA kit were purchased from Roche (Indianapolis, IN) and BD PharMingen GCTAGA-3Ј and 5Ј-TGAGCAACGCAGACTCGCTGT-3Ј; TRAIL-R2, (San Diego, CA), respectively. 5Ј-CAGGTGTGATTCAGGTGAAGT-3Ј and 5Ј-GGACATGGCAGAGT ␬ ␣ The following Abs were used: rabbit anti-I B and anti-TRAIL-R1 CTGCAT-3Ј; TRAIL-R4 5Ј-CTCCCTTCTCATGGGACTTTGG-3Ј and (Santa Cruz Biotechnology, Santa Cruz, CA); mouse anti-TRAIL-R2 mAb 5Ј-CCACCAGTTGGTCCTGAATTG-3Ј. We separated and visualized the (eBioscience, San Diego, CA); rabbit anti-TRAIL-R4 and anti-FADD RT-PCR products by electrophoresis in 1.2% agarose gel using ethidium ␤ (Chemicon, Temecula, CA); mouse anti-human -actin (Novus Biologicals, bromide and UV illumination; identification of amplified bands was veri- Littleton, CO), mouse anti-human E-selectin (ascites preparations of H4/18) fied by DNA sequencing. (26); mouse anti-human ICAM-1 (Immunotech, Marseille, France); isotype We performed real-time RT-PCR using a Multicolor Real-Time PCR de- control Ab K16/16 (26); goat anti-rabbit IgG HRP-conjugated or anti-mouse tection system (Bio-Rad, Hercules, CA) using the SYBR Green PCR core IgG FITC-conjugated (Jackson ImmunoResearch, West Grove, PA). reagents (4304886), according to the recommended protocol, to determine the HUVEC and HDMEC were isolated and cultured as described (26) and threshold cycle (Tc). The primer sequences used were as follows: E-selectin, were used at passage levels 2 to 4. Such cultures are uniformly positive for 5Ј-GAATGTGTAGAGACCATCATAATAAT-3Ј and 5Ј-AGGAAGAATT von Willlebrand factor and CD31 and lack detectable contamination by GRAGCTGAAGTTT-3Ј; ICAM-1, 5Ј-CTGTTCCCAGGACCTGGCAAT-3Ј CD45-expressing leukocytes. HEK293, HeLa, and HL-60 cells were ob- and 5Ј-AGGCAGGAGCAACTCCTTTTTA-3Ј; IL-8, 5Ј-CTCTTGGCAGC tained from the American Type Culture Collection (Manassas, VA) and CTTCCTGATT-3Ј and 5Ј-ACTCTCAATCACTCTCAGTTCT-3; and ␤-ac cultured in DMEM medium containing 10% FBS (Life Technologies, tin, 5Ј-TGCACCACACCTTCTACAATGA-3Ј and 5Ј-CAGCCTGGATAG Grand Island, NY). CAACGTACAT-3Ј. In TRAIL-treated HUVEC or HDMEC, up-regulation of E-selectin, ICAM-1, and IL-8 were expressed as fold induction (FI), calculated as Ϫ Construction and transduction with recombinant retroviral vector follows: FI ϭ 2 7-[TRAIL (T target Ϫ T ␤-actin) Ϫ control (T target Ϫ ␤ c c c Tc -actin)]. Plasmid pcDNA3 AU1-FADDmt (human FADD dominant negative (FADD.DN) with AU1 epitope tag) (27), provided by Dr. V. Dixit (Ge- nentech, South San Francisco, CA), was subcloned into the pFB retroviral NF-␬B promoter reporter assay vector (Stratagene, La Jolla, CA) using PCR with primers 5Ј-CGACT- CACTATAGGGAGACCCA-3Ј and 5Ј-GCGGCCGCTCAGGACGCT- We transiently transfected HUVEC cells with NF-␬B promoter-firefly lu- TCGGAGGTA-3Ј, and topoisomerase cloning techniques (Invitrogen, ciferase and ␤-actin promoter-Renilla luciferase reporter plasmids using a Carlsbad, CA). The pFB vector was transfected into packaging cell line DEAE-dextran protocol (30). The transfected HUVEC were incubated with PA317 provided by Dr. G. Nolan (Stanford University, Palo Alto, CA) and 50 ng/ml TRAIL or TNF for 7 h, and their luciferase activity was deter- G418 (Life Technologies)-resistant cells were derived as a source of ret- mined using a Promega (Madison, WI) Dual-Luciferase reporter assay sys- rovirus stock. Transduction and G418 selection of HUVEC was accom- tem according to the manufacturer’s instruction, and the activity of the plished as described (28). In brief, this involved three serial transduction of NF-␬B promoter-reporter was normalized to that of the ␤-actin primary cultures with retrovirus-containing supernatants followed by se- promoter-reporter. lection with G418. Approximately 50% of transduced HUVEC survived selection with 0.5 mg/ml G418. Multiple FADD.DN-transduced HUVEC clones were pooled to avoid clonal variability. HL-60 cell adherence assay Similarly, human TRAIL cDNA was amplified using PCR with human We seeded HUVEC and HDMEC in 24-well plates, incubated cells with T blast cDNA and two primers, 5Ј-GGATCATGGCTATGATGGAG-3Ј Ј Ј TRAIL or TNF for 5 h, and then added HL-60 cells loaded with cal- and 5 -GCGGCCGCAGTTAGCCAACTAAAAAGGC-3 . The amplified cein-AM (Molecular Probes) at a ratio of 5:1 of leukocytes to EC. After 30 TRAIL cDNA fragment was confirmed by DNA sequencing and was sub- min, unbound HL-60 cells were removed by washing gently three times cloned into the pFB retroviral vector. The pFB vector was transfected into with 1 ml of PBS, and the retained calcein per well was determined by packaging cell PA317. G418 selection, retrovirus stock preparation, and fluorometry (Cytofluor2; PerSpective Biosystems, Framingham, MA). HEK293 transduction were performed as described as for HUVEC. TRAIL Binding was expressed by fluorescence and calculated as follows: (fluo- expression by the transduced HEK293 was determined by TRAIL-R2-Fc rescence associated with treated EC) Ϫ (fluorescence associated with staining and FACS analysis. untreated EC). Assessment of TRAIL-mediated apoptosis Human skin mouse model We treated 30,000 HUVEC or HDMEC plated in C6 plates with medium alone or with TRAIL for 3–15 h; CHX or caspase inhibitors were included We engrafted human skin on C.B-17 SCID/beige mice (Taconic Farms, during the entire treatment period. The doses of TRAIL were varied some- Germantown, NY) as described (31), and the grafts were healed in 4–6 wk what among experiments, because different HUVEC and HDMEC stocks before use. We then intradermally injected 20 ␮l of saline vehicle, TRAIL showed some variability in sensitivity. To assess cell viability, EC were (0.6 ␮g), or TNF (0.1 ␮g), and harvested the grafts 6 h later. The doses harvested, stained with 25 ␮g/ml propidium iodide (PI; Molecular Probes, used were determined as optimal from preliminary experiments. Grafts Eugene, OR) for 5 min at 37°C and then subjected to analytic flow were paraffin embedded, sectioned, and stained with H&E (31). The degree 1528 TRAIL-INDUCED EC INJURY AND ACTIVATION

FIGURE 1. TRAIL-R expression by human EC and control cells. a, RT-PCR assessment of TRAIL- R1, -R2, and -R4 mRNA expression by HeLa, HEK293, HUVEC, and HDMEC. b, FACS analysis of cell surface TRAIL R2 expression using anti- human TRAIL R2 immunostaining. The ﬁlled curve is human TRAIL-R2 stained; the open curve is the nonbinding isotype Ab control. c, Immunoblot analysis of TRAIL-R1 and -R4 using rabbit anti-human TRAIL-R1 Ab or rabbit anti-human TRAIL-R4 Ab; immunoblotting of ␤-actin was used as a loading control. All experiments were repeated at least three times with similar results.

of graft microvascular injury was evaluated from the H&E-stained section RT-PCR method, we could readily detect TRAIL-R1 and -R2 by a dermatopathologist (J. McNiff) blinded to the treatment protocols, mRNA in both cell types, as well as control cell lines HeLa or assessing EC morphology, thrombosis, and leukocyte infiltration. HEK 293 (Fig. 1a). TRAIL-R4 mRNA was detectable in HUVEC, Results HeLa, and HEK293, but not in HDMEC (Fig. 1a). We also mea- TRAIL-R expression by human EC and cell lines sured TRAIL-R2 cell surface protein expression by indirect im- The goal of this study was to determine the effects of TRAIL on munofluorescence and flow cytometry. As shown in Fig. 1b, HD- normal human EC in vitro and in vivo. We first examined whether MEC, HUVEC, HeLa, and HEK293 all express TRAIL-R2. cultured HUVEC and HDMEC express signaling TRAIL-Rs (i.e., Expression in HUVEC is somewhat greater than in HDMEC. TRAIL-R1, -R2, and -R4). Using a sensitive but nonquantitative Available Abs that react with TRAIL-R1 and -R4 do not work in

FIGURE 2. Recombinant TRAIL-triggered human EC death. TRAIL, CHX, or caspase inhibitors were included during the entire treatment period. a, Dose response of HUVEC susceptibility to TRAIL addition. HUVEC were treated with TRAIL at the concentrations indicated for 15 h, and the cell viability was determined by PI exclusion staining as described as in Materials and Methods. b, Effects of protein synthesis and caspase inhibitors on TRAIL-induced death. TRAIL-induced HUVEC death was determined by PI exclusion in the presence or absence of CHX (3 ␮g/ml) and caspase inhibitors Z-DEVD-FMK (40 ␮M) and Z-IETD-FMK (40 ␮M). c, Effects of TRAIL on HDMEC TRAIL-induced HDMEC death was assessed by PI exclusion in the presence or absence of CHX (3 ␮g/ml). d, FADD.DN expression by retroviral FADD.DN-transduced HUVEC (HUVEC.DN) or by HUVEC.mock was determined by immunoblotting using rabbit anti-human FADD Ab. The sensitivity of these two cell cultures to TRAIL-triggered death signal(s) was determined by PI exclusion staining after incubation with TRAIL for8hinthepresence of CHX (3 ␮g/ml). e, The sensitivity of HUVEC to membrane-bound TRAIL was determined by PI exclusion staining after coculture with HEK293.TRAIL effector, or control HEK293.pFB, at a ratio of 1:1 for 15 h. Killing speciﬁcity was determined in the presence or absence of TRAIL-R2-Fc or human Fas-Fc. Both TRAIL-R2-Fc and human Fas-Fc were added at a concentration of 1 ␮g/ml and were incubated with HUVEC from the beginning to the end of the assay. Because HUVEC were mixed with effectors at 1:1, the true number (percentage) of dead HUVEC may be twice that indicated in the Fig. 2e. All experiments were repeated at least three times with similar results. The Journal of Immunology 1529

flow cytometry, so we examined protein levels of these receptors a significant degree of apoptosis in either HUVEC or HDMEC by immunoblotting. TRAIL-R1 and -R4 proteins were detected in unless CHX is added (data not shown). HUVEC, HeLa, and HEK293 (Fig. 1c), but HDMEC expressed To determine whether FADD is the adapter protein in human only TRAIL-R1, consistent with the RT-PCR data (a). EC for the TRAIL-R-mediated death signaling, we prepared FAD- D.DN-transduced HUVEC (Fig. 2d). Most FADD.DN-expressing TRAIL-triggered apoptosis of human EC HUVEC survived treatment with 15 or 100 ng/ml TRAIL plus Next, we examined whether rTRAIL caused death of cultured CHX for 8 h, in contrast to mock-transduced HUVEC (Fig. 2d). HUVEC or HDMEC using PI exclusion and flow cytometry (28). Protection with FADD.DN is not complete, and the difference be- As shown in Fig. 2a, ϳ30% of HUVEC lose membrane integrity tween specific and mock-transduced cells somewhat narrowed at after treatment with 20 ng/ml TRAIL for 15 h, compared with later times (data not shown). Consistent with these data, HUVEC 7.8% of cells in replicate control cultures not treated with TRAIL. death was reduced by selective inhibitors of caspase-8 and -3 (Fig. Fifteen hours was determined in preliminary experiments to dis- 2, b and c). We concluded from these studies that TRAIL initiates play maximal cell death, and longer incubations lead to interfer- apoptosis of normal human EC via a FADD-caspase-8 pathway. ence with the assay by cell-derived debris. The dose-response TRAIL is normally a membrane-bound ligand expressed by curve varied somewhat in different HUVEC isolates, and cells monocytes, T lymphocytes, dendritic cells, and NK cells (15). To showed progressive loss of sensitivity to TRAIL with serial pas- address whether EC could be killed by membrane-bound TRAIL, sage (data not shown). HDMEC showed similar susceptibility to HUVEC were cocultured with HEK293-TRAIL, or with control TRAIL (Fig. 2c). Cell death of both cell types was increased in the HEK293-pFB at 1:1 ratios for 15 h. The viability of the mixed presence of the protein synthesis inhibitor CHX (Fig. 2, b and c). population was then determined by PI exclusion. Coculture of PI exclusion is highly quantitative but does not distinguish late HUVEC with HEK293-TRAIL resulted in a reproducible increase apoptotic from necrotic cell death. To determine whether TRAIL- of PI-stained cells (ϳ15%), compared with HUVEC culture alone triggered HUVEC death was apoptotic or necrotic, we incubated or with mock HEK293-pFB (ϳ5%) (Fig. 2e). Moreover, the HUVEC with 50 ng/ml TRAIL for 2.5, 5, or 10 h, and stained cells HEK293-TRAIL-mediated cytotoxicity was markedly inhibited by with Annexin V-FITC. At 5 or 10 h, ϳ15% of HUVEC showed 1 ␮g/ml TRAIL-R2-FC, but not by human Fas-Fc (Fig. 2e). Be- evidence of an early apoptotic phase which is Annexin V positive cause only 50% of the cultures are HUVEC, this increase in (Fig. 3a). In addition, TRAIL-induced HUVEC death is charac- HUVEC death is probably double that measured for the whole terized by nuclear condensation and fragmentation, a more specific populations, similar to the sensitivity of HUVEC to the soluble test of apoptosis (Fig. 3b), as shown by DAPI staining and fluo- rTRAIL used in our experiments. rescence microscopy. The results of a DNA fragment ELISA also supported the conclusion that death caused by TRAIL with or without CHX, is apoptosis (Fig. 3c). Although HDMEC do not TRAIL-triggered inflammatory gene expression in EC express TRAIL-R4, both HUVEC and HDMEC showed similar Because in the absence of CHX, 70% of EC are not killed by sensitivity to TRAIL (Fig. 2, b and c), which suggests that TRAIL, we also examined the activation response of these cells. TRAIL-R4 expressed on HUVEC, but not HDMEC, is not a major After treatment with TRAIL for 6–7 h, we observed an increase of inhibitor of TRAIL-mediated EC apoptosis. TNF does not induce E-selectin expression on HUVEC and HDMEC demonstrated by

FIGURE 3. TRAIL-mediated killing of HUVEC is apoptotic. a, Annexin V-FITC staining. At the indicated time points, HUVEC cells treated by 50 ng/ml TRAIL were stained with annexin V-FITC, as analyzed by FACS. The data show a time-dependent increase in annexin V-positive cells. b, Examination of nuclear changes. HUVEC were incubated with or without 50 ng/ml TRAIL for 10 h, and nuclear morphology was examined by DAPI staining as described in Materials and Methods. Note the nuclear condensation and fragmentation in the TRAIL-treated group, which is indicative of apoptosis. c, Assessment of apoptosis by DNA fragment ELISA. HUVEC cells were incubated with 50 ng/ml TRAIL for 15 h in the presence or absence of CHX (3 ␮g/ml). HUVEC were harvested and fragmented DNA was quantiﬁed with a DNA fragment ELISA kit. Absorbance at 450 nm indicates the amounts of fragmented DNA released from apoptotic HUVEC. All experiments were repeated at least three times with similar results. 1530 TRAIL-INDUCED EC INJURY AND ACTIVATION

FIGURE 4. Inflammatory gene expression and function by TRAIL-stimulated EC. a and b, Human EC were stimulated with TRAIL, heated TRAIL, and TNF for 6 h, and their E-selectin expression levels were evaluated by flow cytometry. The filled curve is stained with anti-human ␤-selectin Ab; the open curve is the nonbinding isotype Ab control in HUVEC (a), a HDMEC (b). ICAM-1 (c) expression on HUVEC after stimulation for 6 h with 50 ng/ml TRAIL. d, HUVEC and HDMEC were incubated with or without 50 ng/ml TRAIL for 3 h. mRNA for E- selectin, ICAM-1, and IL-8 were assessed using an iCycler iQTM multicolor real-time PCR detection system, as described in Materials and Methods. Fold induction of E-selectin, ICAM-1, and IL-8, normalized relative to expression in the absence of TRAIL. e, HL-60 cell adherence of TRAIL-activated EC. TRAIL- or TNF-activated HUVEC and HDMEC were incubated with calcein-AM-labeled HL-60 cells for 30 min, and after washing, retained calcein was measured using the fluorescence multiwell plate reader Cyt- ofluor2, and the induced specific HL-60 binding was calculated. All experiments were repeated at least three times with similar results.

FACS (the time point was selected because of preliminary exper- pression in HDMEC (Fig. 4b). Although our preparations of TRAIL iments for the optimal level of E-selectin protein expression) (Fig. contained only very low levels of endotoxin contaminant (tested by 4a). This TRAIL response is consistently smaller than that induced by the manufacturer), we excluded the possibility that LPS contami- TNF (Fig. 4a). TRAIL was also effective at inducing E-selectin ex- nation in the rTRAIL reagent was responsible for the E-selectin

FIGURE 5. TRAIL-induced NF-␬B activation. a, HUVEC, HDMEC, and HEK293 cells were treated with TRAIL, as indicated, and cell lysates were size fractionated by SDS-PAGE and analyzed for I␬B␣ by immunoblotting. ␤-actin was used as a loading control. Upper bands,I␬B␣; lower bands, ␤-actin. b, TRAIL-induced NF-␬B reporter activity was measured in HUVEC transiently transfected with pBIIXLuc and p␤-actin-Rluc. The firefly and Renilla luciferase activities were measured, and relative luciferase activity was calculated as described. c, HUVEC were treated with 50 ng/ml TRAIL for 6 h, in the presence or absence of PDTC, and the cell surface E-selectin expression levels were evaluated by flow cytometry. The filled curve is stained with E-selectin Ab, and the open curve is the nonbinding isotype Ab control. All experiments were repeated three times with similar results. The Journal of Immunology 1531 induction in EC by heating TRAIL at 80°C for 10 min before addition ng/ml TNF (Fig. 5b). We also find that the NF-␬B inhibitor PDTC to EC. This treatment, which does not inactivate LPS actions on EC- prevented TRAIL-induced E-selectin expression in HUVEC (Fig. (data not shown), abolished induction of E-selectin expression in EC 5c), confirming a link between NF-␬B activation and the proin- by TRAIL (Fig. 4a). The response to TRAIL was not confined to flammatory response of TRAIL in these cells. induction of E-selectin. ICAM-1 was also up-regulated in HUVEC surviving TRAIL treatment (Fig. 4c). We also examined the mRNA TRAIL-induced EC injury and inflammation levels of E-selectin, ICAM-1, and IL-8 in TRAIL-stimulated human To examine the effects of TRAIL on human EC in vivo, we in- EC using real-time quantitative RT-PCR. Incubation with 50 ng/ml tradermally injected TRAIL into stable human skin xenografts on TRAIL for 3 h increased expression of E-selectin, ICAM-1, and IL-8 SCID/beige mice. Histological analysis confirmed that both TNF mRNAs in both HUVEC and HDMEC (Fig. 4d). and TRAIL resulted in epidermal and vessel injury (Fig. 6, b–d). The functional consequence of adhesion molecule and chemo- Vascular damage was seen as either loss of EC or formation of kine expression by EC is the binding and activation of leukocytes. microthrombi (Fig. 6, b–d). This was accompanied by local infiltra- To determine whether the degree of activation observed in TRAIL- tion of neutrophils and mononuclear cells (Fig. 6, b–d). The extent of treated EC could be biologically significant, we tested the adhesion leukocyte recruitment was much greater in TNF-injected skin than in of HL-60 leukocytes to HUVEC, and to HDMEC, pretreated with TRAIL-injected skin. Heat-treated TRAIL did not induce inflamma- vehicle only, with TRAIL, or with TNF. As shown in Fig. 4e,50 tion or endothelial injury, excluding the possibility of an LPS con- ng/ml TRAIL pretreatment significantly increased the binding of leu- taminant producing these responses (data not shown). kocytes to EC over control-treated cells, but once again, the effect of TRAIL was less than that produced by 50 ng/ml TNF (Fig. 4e). Discussion TNF-mediated induction of proinflammatory genes in EC is de- TRAIL-Rs are widely expressed on many cell types, but the effects pendent upon the activation of NF-␬B via degradation of I␬B␣.As of TRAIL on normal human tissues have not been described pre- demonstrated by immunoblot, TRAIL causes I␬B␣ degradation in viously, and the physiological function(s) of this novel TNF family HUVEC, HDMEC, and HEK 293 cells (Fig. 5a). TRAIL-induced member remains unknown (15, 32). We studied the expression of NF-␬B activation in HUVEC was more directly examined using an TRAIL-Rs and responses to TRAIL in normal human EC in vivo NF-␬B promoter reporter gene assay. Stimulation with 50 ng/ml and in vitro. Following incubation with TRAIL for 15 h, ϳ30% of TRAIL for 7 h caused an increase of luciferase activity driven by cultured EC lose membrane integrity (Fig. 2, a–c). EC death is NF-␬B, although this response was smaller than that caused by 50 increased by cotreatment with CHX but significantly reduced by

FIGURE 6. TRAIL-induced tissue injury and inflammation in human skin. H&E-stained sections of human skin xenografts from C.B-17 SCID/beige mice 6 h after intradermal injection with vehicle saline alone (a), TNF (0.1 ␮g) (b), or TRAIL (0.6 ␮g) (c and d). a, After saline treatment, normal tissue shows healthy vessels, intact EC, and no fibrin deposition. b, After 0.1 ␮g TNF treatment, the epidermis shows striking keratinocyte cell necrosis, and damaged EC; c and d, after 0.6 ␮g TRAIL treatment; arrows show damaged vessels. Only minimal inflammation is seen, judged by scant neutrophil infiltration, loss of some EC, and partial thrombosis (c). Necrotic keratinocytes are observed in epidermis, and superficial dermal vessels show patchy loss of EC with fibrin deposition. All experiments were repeated three times with similar results. 1532 TRAIL-INDUCED EC INJURY AND ACTIVATION selective caspase inhibitors or transduced FADD.DN (Fig. 2, b–d). Such information could shed light on the species difference of Moreover, experiments with Annexin V staining, DAPI nuclear tissue injury produced by human TRAIL. staining, and a DNA fragment ELISA demonstrate that TRAIL- The TRAIL pathway represents a potentially promising target triggered HUVEC death is predominantly apoptotic (Fig. 3). Most for anticancer therapy (17–19, 39), and the finding reported in this EC do not die in response to TRAIL in the absence of CHX, study that TRAIL induces apoptosis and inflammatory gene ex- prompting us to examine other related responses. We found that pression in EC does not exclude this possibility. TNF exerts its TRAIL-treated EC also show NF-␬B-dependent activation re- primary antitumor action in vivo via damage of tumor vessels. Our sponses and inflammatory gene expression that results in adhesion finding suggests TRAIL may also have indirect antitumor effects in of leukocytes (Figs. 4 and 5). Most importantly, TRAIL induces addition to direct cytotoxicity toward tumor cells. TNF appears EC damage and tissue injury in normal human skin in vivo. These more active on tumor vessels than normal vessels, and it will be responses are qualitatively similar to those induced by TNF (Fig. interesting to test whether TRAIL also shows such different sen- 6). However, compared with TNF, TRAIL is a stronger inducer of sitivity. A proinflammatory effect of a cytokine is also not neces- apoptosis in vitro (Fig. 2, a–c, e) but a weaker activator of NF-␬B sarily a problem for tumor therapy. The inflammatory response (b). In vivo, the TRAIL response also caused less EC activation provides signals that stimulate APCs, such as dendritic cells to and inflammation than TNF but resulted in comparable levels of express molecules that promote binding, stimulation, and activa- EC injury (Fig. 6). The dose responses of both HUVEC and HD- tion of lymphocytes (40). In other words, the EC injury response MEC to TRAIL varied somewhat among isolates, similar to the we have observed in human skin after TRAIL treatment (Fig. 6c) previously noted variability of HUVEC cultures to TNF or IL-1 could facilitate host immune responses by acting as an adjuvant. (33). In addition, apoptotic responses diminished somewhat with In summary, we have found that recombinant human TRAIL passage level. This is also true for the apoptosis response to TNF can both injure and activate human EC in vitro and in vivo. Com- (J. Li and J. Pober, unpublished observation). The explanation for pared with TNF, TRAIL is potent at causing injury but less effec- these changes is unknown. tive at stimulating inflammation. These findings need to be con- Previous investigations have reported the toxic effects of TRAIL sidered in planning and interpreting human therapeutic trials with on normal human cells, including hepatocytes (34), brain cells rTRAIL as a treatment for cancer and for evaluating the physio- (35), and erythropoietic cells (36). In this regard, the effects on EC logical role of endogenous TRAIL in immune and inflammatory are not unique. However, the effects of injury to EC may be more reactions. biologically significant, because this can be amplified in vivo through secondary thrombosis and consequent tissue ischemia Acknowledgments (Fig. 6, c and d). Our observations that HUVEC and HDMEC are We thank our colleagues Jae Choi, David Enis, Martin S. Kluger, and susceptible to TRAIL death signals differ from those reported pre- Keyvan Mahboubi for their critical suggestions for this manuscript. We are viously by others (17, 18). The basis of this difference is unclear also grateful to Louise Benson, Gwendolyn Davis, and Lisa Grass for ex- but could result from the methods used to assess cell death, from cellent technical assistance with cell culture. the state of the EC cultures or from the potency of the TRAIL preparations used. We detected death by loss of membrane integ- References rity, by Annexin V binding, by nuclear condensation, and by DNA 1. Pitti, R. M., S. A. Marsters, S. Ruppert, C. J. Donahue, A. Moore, and A. Ashkenazi. 1996. Induction of apoptosis by Apo-2 ligand, a new member of fragmentation, which may be more sensitive than crystal violet the tumor necrosis factor cytokine family. J. Biol. Chem. 271:12687. staining used by other investigators (17, 18). We also do not know 2. Wiley, S. R., K. Schooley, P. J. Smolak, W. S. Din, C. P. Huang, J. K. Nicholl, the characterization of EC cultures used in the previous published G. R. Sutherland, T. D. Smith, C. Rauch, C. A. Smith, et al. 1995. 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