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The Journal of Immunology, 2005, 174: 1647-1656.
Copyright © 2005 by The American Association of Immunologists

Decoy Receptor 3 Increases Monocyte Adhesion to Endothelial Cells via NF-{kappa}B-Dependent Up-Regulation of Intercellular Adhesion Molecule-1, VCAM-1, and IL-8 Expression1

Chia-Ron Yang*, Shie-Liang Hsieh{dagger},{ddagger}, Feng-Ming Ho§ and Wan-Wan Lin2,*

* Department of Pharmacology, College of Medicine, National Taiwan University, {dagger} Department of Microbiology and Immunology, and Immunology Research Center, National Yang-Ming University, Taipei, Taiwan, and {ddagger} Genomics Research Center, Academia Sinica, Taipei, Taiwan; and § Department of Internal Medicine, Tao-Yuan General Hospital Department of Health, Taoyuan, Taiwan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Decoy receptor 3 (DcR3), a soluble receptor for FasL, LIGHT and TL1A, is highly expressed in cancer cells. We show that pretreatment of HUVECs with DcR3 enhances the adhesion of THP-1 and U937 cells and primary monocytes. A similar stimulatory effect of DcR3 on THP-1 adhesion was also observed in human microvascular endothelial cells (HMVECs). Flow cytometry and ELISA showed that DcR3-treated HUVECs exhibited significant increases in ICAM-1 and VCAM-1 expression. We also demonstrate the ability of DcR3 to stimulate the secretion of IL-8 by HUVECs. RT-PCR and reporter assays revealed that the expression of adhesion molecules and IL-8 are regulated at the level of gene transcription. Experiments with pyrrolidine dithiocarbamate indicated the involvement of an NF-{kappa}B signaling pathway. DcR3 was found to induce I{kappa}B kinase activation, I{kappa}B degradation, p65 nuclear translocation, and NF-{kappa}B DNA-binding activity. The enhancement by DcR3 of cell adhesion to HUVECs was not mimicked by the TL1A-Ab, which has been shown in our previous work to be a neutralizing Ab against TL1A, thereby inducing HUVECs angiogenesis. Moreover, DcR3-induced cell adhesion could be detected in human aortic endothelial cells (ECs) in which TL1A expression is lacking. Together, our data demonstrate that DcR3 increases monocyte adhesion to ECs via NF-{kappa}B activation, leading to the transcriptional up-regulation of adhesion molecules and IL-8 in ECs. This novel action appears not to be due to TL1A neutralization, but occurs through an as yet undefined target(s). This study implicates DcR3 in the relationship between inflammation and cancer development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The link between inflammation and the development of cancer has been recognized for some years (1) and the list of infectious agents associated with human cancers is continuing to grow. Examples include the association of Helicobacter pylori with gastric cancer (2), hepatitis C with hepatocellular carcinoma (3) and EBV-associated B cell lymphoma (4). It is known that the mechanisms of inflammation-associated tumor development by inflammatory cytokines and mediators include 1) stimulation of cellular proliferation, for example by matrix metalloproteinases (5); 2) inhibition of apoptosis (6); 3) up-regulation of cellular adhesion to aid in migration and homing during distant metastatic spread (7, 8); 4) stimulation of angiogenesis by COX-2-catalyzed PGE2 production (9) and chemokine IL-8 (10); and 5) cellular transformation, for example, by hepatitis B virus, EBV, and human papilloma virus (5). As a result, some inflammatory inhibitors have recently been applied in chemoprevention (11, 12).

Decoy receptor 3 (DcR3)3 is a lymphotoxin-like member of the TNFR superfamily. In addition to acting as the decoy receptor for Fas ligand (FasL) (13), DcR3 competes with HSV glycoprotein D for herpesvirus entry mediator, a receptor expressed by T lymphocytes (LIGHT) (14), and with TL1A (15). The binding of DcR3 to FasL, LIGHT, and TL1A neutralizes the proapoptotic actions induced by the Fas-FasL, LIGHT-lymphotoxin-{beta} receptor, and TL1A-DR3 interactions, respectively (14, 16, 17, 18). DcR3 is overexpressed in cells from malignant tumors, such as those of the esophagus, stomach, glioma, lung, colon, rectum, and pancreas (13, 16, 17, 19, 20, 21), and high serum levels of DcR3 have been detected in many cancer patients (22). It is, therefore, possible that overexpression of DcR3 may produce a relative advantage for tumor growth and survival. Supporting this notion, tumor cells engineered to release high amounts of DcR3 are protected from FasL-induced apoptotic cell death and chemotaxis, resulting in decreased immune cell infiltration in glioma xenografts (16). Moreover, high DcR3 expression levels are reported to be associated with poor prognoses in cancer patients (21).

Growing evidence has demonstrated that members of the TNF superfamily are able to induce "reverse signals" after engagement with their receptors (23, 24, 25). Our recent studies suggested that DcR3, like other members of the TNFR superfamily, is capable of triggering "reverse signaling" to modulate monocyte differentiation and function (26, 27, 28, 29, 30). DcR3 was demonstrated to interfere with the differentiation and maturation of dendritic cells from monocytes, which leads to a skewing of the T cell response toward the Th2 phenotype (26, 29). This Th1-suppressing effect of DcR3 also supports its critical role in cancer development. In addition, we observed that DcR3 induces monocyte differentiation to osteoclasts rather than to macrophages (27, 28). Moreover, our recent work further demonstrates that DcR3 could stimulate monocyte adhesion to culture dishes through binding to an as yet unidentified molecule. This results in the induction of reverse signaling pathways involving PI3K, protein kinase C, Src tyrosine kinases, and focal adhesion kinase activation (30). All this evidence suggests that DcR3 is not only a decoy receptor for neutralizing cytokine ligands, but that it is also an effector molecule capable of triggering reverse signaling to modulate other physiological or pathological effects.

In view of the fact that DcR3 can trigger monocyte adhesion to culture dishes, we were interested in exploring whether DcR3 also plays a role in the initiation of an inflammatory response by tumor cells, in addition to its immune suppression. If so, it is conceivable to imply its involvement in the facilitation of tumor development as mentioned. For this purpose, we determined the effects of DcR3 on human endothelial cells in relation to monocyte adhesion, and elucidated the molecular mechanisms involved in this event. We demonstrate in our study that DcR3 exerts a direct effect on endothelial cells in activating the NF-{kappa}B signal pathway, which in turn increases ICAM-1 and VCAM-1 expression, IL-8 secretion, and monocyte adhesion. These observations suggest an important role for DcR3 in integration of the inflammatory process, immune suppression, and cancer progression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

Rabbit polyclonal Abs specific for ICAM-1 and VCAM-1, mouse mAbs specific for LFA-1 and Mac-1, and goat polyclonal Ab specific for VLA4 were purchased form Santa Cruz Biotechnology. Mouse monoclonal anti-human IL-8, recombinant human TNF-{alpha}, and ELISA kits for human TNF-{alpha} and IL-8 were purchased from R&D Systems. Human IgG1 was obtained from Calbiochem-Novabiochem. M199 medium, RPMI 1640 medium, and 0.25% trypsin/1 mM EDTA · 4Na were purchased from Invitrogen Life Technologies. The TL1A-Ab was prepared using female BALB/c mice immunized with a peptide sequence (TL1A aa 61–80, AQGEACVQFQALKGQEFAPS) predicted to be the extracellular domain of the TL1A protein. Ten boosts were administered at 2-wk intervals. After fusion to generate hybridomas, a mAb 6E6 (belonging to the subclass IgG1) was generated. Luciferase expression vectors containing the 5'-flanking region of the IL-8 gene (–133 to –50) and ICAM-1 (–1014) were kindly provided by Dr. N. Mukaida (Kanazawa University, Ishikawa, Japan) and Dr. T. Van Der Saag (Hubrecht Laboratorium, Utrecht, the Netherlands), respectively. TransAM NF-{kappa}B p65/NF-{kappa}B p50 transcription factor assay kits were purchased from Active Motive. 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein tetrakis(acetoxymethyl) ester (BCECF-AM), pyrrolidine dithiocarbamate (PDTC), and other chemicals were purchased from Sigma-Aldrich.

Cell culture

HUVECs were obtained by treating human umbilical cord veins with 0.05% collagenase for 8 min and then culturing in 75-cm2 flasks in M199 containing 20% FBS, 15 mg/ml endothelial cell (EC) growth supplement, 5 U/ml heparin, and 20 mM HEPES. Human microvascular ECs (HMVECs) were obtained from Cascade Biologics and cultured in medium 131 plus attachment factor. Human monocytic THP-1 and U937 cell lines were obtained from the American Type Culture Collection and cultured in RPMI 1640 medium containing 10% FBS. Human peripheral blood, obtained from healthy adult volunteers, was collected in syringes containing 1000 U/ml of preservative-free heparin. PBMC were isolated by density centrifugation using Ficoll-Hypaque and were resuspended in modified Eagle’s medium supplemented with 10% heat-inactivated FBS. Monocytes were subsequently purified using a Dynal monocyte negative isolation kit (Dynal Biotech) according to vendor’s instructions, and cultured in medium supplemented with 10% (v/v) heat-inactivated FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2 in air.

Generation of DcR3 protein

The recombinant DcR3.Fc fusion protein was produced as previously described (26). Recombinant DcR3 protein was cleaved from the recombinant DcR3.Fc fusion protein by papain as previously described (18). Briefly, 2.5 mg/ml DcR3.Fc was transferred into a Slide-A-Lyzer dialysis cassette (Pierce) and dialyzed against 100 mM sodium acetate (pH 5.5) at 25°C for 2 h followed by further dialysis at 4°C overnight. EDTA and cysteine were added to the reaction at final concentrations of 1 and 50 mM, respectively, followed by addition of papain-conjugated agarose; this was incubated at 37°C for 4 h. The papain-conjugated agarose was removed by centrifugation, and the cleaved Fc portion was removed by incubation of the Fc and DcR3 mixture with protein A-Sepharose 4 Fast Flow beads (Amersham Pharmacia Biotech).

Cell adhesion assay

HUVECs (5 x 104 cells in 100 µl of M199 medium) were grown in 96-well plates and treated for various time intervals at 37°C with DcR3 or TNF-{alpha}. THP-1 and U937 cells were labeled for 1 h at 37°C with 0.1 µg/ml BCECF-AM and washed twice with growth medium, and then 1 x 105 (100 µl) of the labeled cells were added to the EC monolayer, and cultures were incubated in a CO2 incubator for 1 h. Nonadherent cells were removed from the plate by gentle washing with PBS, and the number of adherent cells was determined by measuring the fluorescence intensity using a SpectraMax 340PC spectrophotometer (Molecular Devices) or by photography.

Flow cytometry

Cells were harvested and washed twice with FACS washing buffer (1% FCS and 0.1% NaN3 in PBS), followed by incubation with Abs at 4°C for 30 min. After washing with FACS washing buffer three times, the fluorescence of cells was analyzed using a FACScan flow cytometer (BD Biosciences).

RT-PCR analysis

Total RNA was isolated from HUVECs and human cell lines using RNAzol B Reagent (Tel-Test). Single-strand cDNA for use as a PCR template was synthesized from 10 µg of total RNA using random primers and M-MLV reverse transcriptase (Promega). The oligonucleotide primers used for the amplification are as follows: human ICAM-1 (GenBank accession no. BC015969) sense (864–882) 5'-TAT GGC AAC GAC TCC TTC T-3' and antisense (1084–1101) 5'-CCA AGG TGA CGC TGA ATG-3', which produced a product of 238 bp; and human VCAM-1 (GenBank accession no. M60335) sense (1693–1711) 5'-ATG ACA TGC TTG AGC CAG G-3' and antisense (1933–1952) 5'-AGT GTC AAA GAA GGA GAC AC-3', which produced a product of 260 bp. In all experiments, {beta}-actin was used as an internal control. The {beta}-actin primers used were sense (613–652) 5'-GAC TAC CTC ATG AAG ATC CT-3' and antisense (1103–1122) 5'-CCA CAT CTG CTG GAA GGT GG-3', which produced a product of 510 bp. Equal amounts of each RT product (1 µg) were PCR-amplified using Taq polymerase in 35 cycles consisting of 1 min at 95°C, 1 min at 55°C, and 1 min at 72°C. The amplified cDNA was run on 1% agarose gels and visualized under UV light following ethidium bromide staining.

Quantification of ICAM-1 and VCAM-1 expression

Levels of cell surface ICAM-1 and VCAM-1 expression were determined by an ELISA. After treatment with DcR3.Fc at 37°C for 12 h, cells were washed twice with PBS and fixed at room temperature with 1% (w/v) paraformaldehyde for 30 min. After washing with PBS, cells were then blocked with 1% BSA in TBS containing 0.05% (v/v) Tween 20 for 15 min before being incubated with anti-ICAM-1 or anti-VCAM-1 Ab (1:100) for 1 h and then with HRP-labeled anti-rabbit Ab (1:1000) for 30 min. After each incubation, cells were washed twice with PBS. Tetramethylbenzidine substrate (Science Products) was then applied to cells for 30 min, after which 2 M sulfuric acid was added to stop the reaction. The absorbance was measured at 450 nm.

Transfection and reporter gene assay

HUVECs seeded into 24-well plates were transfected with 1 µg of the ICAM-1 promoter plasmid (–1014) or the IL-8 promoter plasmid (–133) and 1 µg of the {beta}-galactosidase expression vector, using Lipofectin Reagent (Invitrogen Life Technologies) according to the manufacturer’s instructions. After 24 h, cells were treated with the indicated agents. Twenty-four hours later, the media were removed and cells were washed once with cold PBS. To prepare lysates, 50 µl of reporter lysis buffer (Promega) was added to each well and cells were scraped from the dishes. The supernatant was collected after centrifugation at 13,000 rpm for 30 s. Aliquots of cell lysates (5 µl) containing equal amounts of protein (10–20 µg) were placed into wells of an opaque black 96-well microtiter plate. An equal volume of luciferase substrate (Promega) was added to all samples, and the luminescence was measured in a microplate luminometer (Packard Instrument). The luciferase activity value was normalized to take account of the transfection efficiency as determined by the cotransfected {beta}-galactosidase expression vector (pCR3lacZ; Pharmacia Biotech). The level of induction of luciferase activity was determined as a ratio in comparison to cells with no stimulation.

Preparation of nuclear extracts

To prepare nuclear extracts, total cell lysates were first resuspended in buffer containing 10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, and 0.2 mM PMSF, followed by vigorous vortexing for 15 s before being allowed to stand at 4°C for 10 min; samples were then centrifuged at 2000 rpm for 2 min. The nuclear pellets were resuspended in 30 µM buffer containing 20 mM HEPES (pH 7.9), 25% (v/v) glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, and 0.2 mM PMSF for 20 min on ice, before centrifugation at 15,000 rpm for 2 min. Supernatants containing the solubilized nuclear proteins were stored at –70°C for subsequent immunoblot analysis.

Immunoblot analysis

Cells were lysed in lysis buffer, whole-cell extracts (120 µg) were electrophoresed on 10% SDS-PAGE, and proteins were transferred onto nitrocellulose membranes. Immunoblot detection was performed using the relevant rabbit antiserum or mouse mAb and the corresponding HRP-conjugated second Ab, following by detection using ECL reagents (Amersham Biosciences) and exposure to photographic film.

Immunoprecipitation and protein kinase assays

HUVECs grown in 60-mm dishes were washed twice with ice-cold PBS, lysed in 1 ml of lysis buffer containing 20 mM Tris-HCl (pH 7.5), 1 mM MgCl2, 125 mM NaCl, 1% (v/v) Triton X-100, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 25 mM {beta}-glycerophosphate, 50 mM NaF, and 100 µM sodium orthovanadate, and centrifuged at 14,000 rpm for 30 min. The supernatant was then immunoprecipitated with polyclonal Ab against I{kappa}B kinase (IKK){alpha}{beta} in the presence of A/G-agarose beads overnight. The beads were washed three times with lysis buffer and twice with kinase buffer (20 mM HEPES, pH 7.4, 20 mM MgCl2, 2 mM DTT). The kinase reactions were performed by incubating immunoprecipitated beads with 20 µl of kinase buffer supplemented with 20 µM ATP and 3 µCi of [{gamma}-32P]ATP at 30°C for 30 min. For the IKK{alpha}{beta} kinase assay, 2.5 µg of bacterially expressed GST-I{kappa}B{alpha} (aa 5–55) was added as a substrate. The reaction mixtures were analyzed by 12% SDS-PAGE followed by autoradiography.

ELISA

Cell culture supernatants were collected at various time points. Levels of TNF-{alpha} and IL-8 were quantified using commercial ELISA kits (R&D Systems), according to the vendor’s instructions.

Data analysis

Each experiment was performed in duplicate and its average was included for quantification. Data are expressed as the mean ± SEM of averages from at least three experiments. ANOVA was used to assess the statistical significance of the differences, and a value of p < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DcR3 increases monocyte adhesion to HUVECs

To determine the effect of DcR3 on monocyte adhesion to EC, HUVECs were treated for different time periods with DcR3.Fc, before the addition of BCECF-labeled THP-1 cells for 1 h. As shown in Fig. 1, DcR3.Fc (1–10 µg/ml) significantly increased the levels of THP-1 adhesion to HUVECs. This effect was detectable after 3 h of incubation and became more apparent as the incubation period was prolonged for up to 12 h (Fig. 1, A and B). To determine the efficacy of DcR3’s action, similar experiments were conducted with TNF-{alpha} and IL-1{beta}, which are known to be potent inflammatory cytokines (31, 32). We found that when HUVECs were incubated for 3 h, TNF-{alpha} and IL-1{beta} induced 2.6- and 2.8-fold increases in THP-1 adhesion, respectively, whereas DcR3 caused a 1.8-fold increase in adhesion compared with the IgG1-treated control group (Fig. 1C). We noted that the effect of DcR3 on THP-1 cell adhesion was additive with the effect of TNF-{alpha} and IL-1{beta}. DcR3-treated HUVECs also displayed increased adhesive ability to another monocyte cell line, U937 (Fig. 1C), and to primary monocytes (Fig. 1D). For U937 adhesion, the stimulating effects of DcR3, TNF-{alpha}, and IL-1{beta} on cell adhesion were 1.8-, 2.1-, and 1.8-fold compared with the control group, respectively (Fig. 1C). To understand whether the effect of DcR3 is EC specific or not, we also examined HMVEC. As shown in Fig. 1E, we found DcR3 treatment of HMVECs for 12 h resulted in a 1.7-fold increase in adhesion to THP-1 cells as compared with IgG-treated control group.



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  		FIGURE 1. Enhancement of THP-1 and U937 cell adhesion by DcR3.Fc. A, A total of 1 x 105 THP-1 cells, labeled with 0.1 µg/ml BCECF-AM, were added to HUVECs preincubated with DcR3.Fc or TL1A-Ab, each at 3 µg/ml, for 12 h, and the culture was continued at 37°C for 1 h. Following incubation, nonadherent cells were removed from the plate by gentle washing with PBS, and adherent cells were photographed. Images represent magnification at x40. B, BCECF-AM-labeled THP-1 cells were added to HUVECs preincubated with different concentrations of human IgG1 (hIgG1), DcR3.Fc, or TL1A-Ab for the time periods indicated, and the culture was continued at 37°C for 1 h. Following incubation, adherent cells were quantitated by measuring the absorbance at 538 nm. Data represent the mean ± SEM from a representative experiment. C, HUVECs were treated with human IgG1 (3 µg/ml), DcR3.Fc (3 µg/ml), TNF-{alpha} (20 ng/ml), IL-1{beta} (10 ng/ml), and/or TL1A-Ab (3 µg/ml) for 3 h. Then 1 x 105 BCECF-AM-labeled THP-1 or U937 cells were plated onto HUVECs and incubated for 1 h. D, A total of 1 x 105 primary human monocytes, labeled with 0.1 µg/ml BCECF-AM, were added to HUVECs preincubated with human IgG1 (3 µg/ml), DcR3.Fc (3 µg/ml), TNF-{alpha} (20 ng/ml), or TL1A-Ab (3 µg/ml) for 12 h and cultures were continued at 37°C for 1 h. E, Similar experiments to those shown in D were performed to look at the interaction between HMVECs and THP-1 cells. Data represent the mean ± SEM from five independent experiments. *, p < 0.05 as compared with the control group.

 
Next, to understand whether the stimulating effect of DcR3 is associated with its neutralization of TL1A, an endogenous angiostatic factor released from HUVECs (18), we tested the effect of TL1A-Ab. In a previous study, we identified TL1A-Ab as a neutralizing Ab against TL1A (18). In contrast to DcR3, we found that TL1A-Ab treatment of HUVECs (Fig. 1, B, C, and D) and HMVECs (Fig. 1E) had no effect on their adhesion to THP-1, U937, or primary monocytes.

DcR3 induction of ICAM-1 and VCAM-1 expressions in HUVECs

Because interactions between the monocyte integrins LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) and ICAM-1 (CD54), and between VLA-4 (CD49d/CD18) and VCAM-1 (CD106) are required for leukocyte-EC binding, we first investigated whether DcR3 can influence the expression of adhesion molecules on HUVECs and/or THP-1 cells. Flow cytometry with specific Abs revealed that HUVECs treated with DcR3 for 12 h expressed higher levels of ICAM-1 (1.6-fold) and VCAM-1 (2.8-fold) as compared with the human IgG-treated control group. Likewise, TNF-{alpha} increased ICAM-1 and VCAM-1 expression in HUVECs by 2.4- and 3.5-fold, respectively, as compared with the control group (Table I). These effects were confirmed using ELISA; in DcR3-treated cells, levels ICAM-1 and VCAM-1 were found to be increased by 2.1- and 2.9-fold, respectively, as compared with human IgG group, whereas in TNF-{alpha}-treated cells, they were elevated by 3.6- and 4.9-fold, respectively (Fig. 2B).


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  		Table I. DcR3.Fc increased ICAM-1 and VCAM-1 expression on HUVECa

 


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  		FIGURE 2. DcR3.Fc increases ICAM-1 and VCAM-1 expression in HUVECs. A, HUVECs were cultured in the presence of vehicle, DcR3.Fc (3 µg/ml), human IgG1 (hIgG1; 3 µg/ml), TL1A-Ab (3 µg/ml), or TNF-{alpha} (20 ng/ml) for 3 h. After incubation, total RNA was collected, and ICAM-1/VCAM-1 mRNA expression was detected by RT-PCR. B, HUVECs were treated with vehicle, DcR3.Fc (3 µg/ml), human IgG1 (3 µg/ml), or TNF-{alpha} (20 ng/ml) for 12 h. Surface expression of ICAM-1 and VCAM-1 were measured by ELISA using specific Abs, as described in Materials and Methods. C, HUVECs were cotransfected with 1 µg of reporter gene for the ICAM-1 promoter and 1 µg of {beta}-galactosidase ({beta}-gal)-lacZ for 24 h. Cells were then incubated with vehicle, human IgG1 (3 µg/ml), DcR3.Fc (3 µg/ml), TL1A-Ab (3 µg/ml), or TNF-{alpha} (20 ng/ml) for another 24 h. Luciferase activities were determined as described in Materials and Methods. Data represent the mean ± SEM from four independent experiments. *, p < 0.05 as compared with the control group.

 
Our previous work showed that DcR3 is able to influence actin reorganization in THP-1 cells, leading cell adhesion to culture dishes (30). In the present study we wished to determine whether increased THP-1 adhesion to DcR3-treated HUVECs, in a 1 h coculture system, is associated with this effect on THP-1 cells. To clarify this, we first examined the effects of DcR3 on adhesion molecule expression by THP-1 cells. We found that after a 12 h incubation DcR3 failed to alter the expression levels of ICAM-1, VCAM-1, and their corresponding integrins of LFA-1, Mac-1, and VLA-4 on THP-1 cells. In contrast, TNF-{alpha} increased the expression of these molecules to different extents in these cells (Table II). Furthermore, when THP-1 cells were preincubated with DcR3 for 1 h followed by coculture with HUVECs for another 1 h, we found that monocyte adhesion was only increased 1.1-fold compared with the human IgG-treated control group. This stimulating effect was less than the 1.8-fold increase seen in 3-h pretreated HUVECs (Fig. 1C), but equivalent to that observed when THP-1 and HUVECs were cocultured for 2 h (data not shown). These results suggest that short periods of treatment with DcR3 (1–2 h) is sufficient to slightly enhance monocyte adhesion to ECs, but this effect dose not result from the up-regulation of adhesion molecules on THP-1 cells. The influence of DcR3 on monocytes seems to make only a limited contribution in the experiments used in this study, i.e., 3–12 h preincubation of HUVECs with DcR3, to determine the stimulating effects of DcR3 on HUVECs. Thus, these results demonstrate that the DcR3-elicited monocyte-EC interaction in the current model primarily results from the up-regulation of ICAM-1 and VCAM-1 on HUVECs.


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  		Table II. The DcR3.Fc induced ICAM-1, VCAM-1, LFA-1, Mac-1, and VLA4 expression on THP-1a

 
In confirmation of the observed changes in protein expression, RT-PCR analysis indicated that DcR3 causes an increase in gene transcription for both ICAM-1 and VCAM-1 in HUVECs (Fig. 2A). The result of reporter assays to test for ICAM-1 promoter activity further support with this notion (Fig. 2C). In contrast, parallel experiments performed using TL1A-Ab revealed no changes in mRNA or protein levels for ICAM-1 or VCAM-1 (Fig. 2, A and C).

DcR3 induction of IL-8 expressions in HUVECs

To investigate whether DcR3 can induce inflammatory cytokine production in HUVECs, which would contribute to cell adhesion via an autocrine feedback mechanism, we tested the effects of IL-8 and TNF-{alpha}. Our data showed that exogenous TNF-{alpha} (20 ng/ml), but not IL-8 (10 ng/ml), was able to enhance adhesion molecule expression in HUVECs (Fig. 3, A and B). In addition, ELISAs showed that DcR3 induces IL-8 release, whereas TNF-{alpha} production is induced by LPS, but not by DcR3 (Fig. 3, C and D). Increased IL-8 levels were further confirmed by activation of its promoter by DcR3 (Fig. 3E). This IL-8-inducing effect of DcR3 might be implicated in cell adhesion, because IL-8 can trigger a conformational change in LFA-1 and Mac-1 on rolling leukocytes, which greatly increases their adhesive properties (33).



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  		FIGURE 3. DcR3.Fc increases IL-8 expression in HUVECs. A, A total of 1 x 106 HUVECs were treated with IL-8 or TNF-{alpha} at the indicated concentrations for 12 h. After incubation, cells were treated with specific Abs for ICAM-1 or VCAM-1, and analyzed by flow cytometry. B, The fluorescence mean value of panel A was calculated. C and D, HUVECs were treated with vehicle, human IgG1 (hIgG1; 3 µg/ml), DcR3.Fc (3 µg/ml), or TL1A-Ab (3 µg/ml) for various periods as indicated. Cell culture medium was tested for TNF-{alpha} and IL-8 levels by ELISA. E, HUVECs were cotransfected with 1 µg of IL-8 promoter (–133) plasmid and 1 µg of {beta}-galactosidase ({beta}-gal)-lacZ for 24 h, and then cells were incubated with vehicle, human IgG1 (hIgG1; 3 µg/ml), DcR3.Fc (3 µg/ml), TL1A-Ab (3 µg/ml), or TNF-{alpha} (20 ng/ml) for another 24 h. Luciferase activities were determined as described in Materials and Methods. Data represent the mean ± SEM from four independent experiments. *, p < 0.05 as compared with the control group.

 
ICAM-1, VCAM-1, and IL-8 induction in HUVECs contributes to cell adhesion

Based on the results discussed, we suggest that the increased expression of ICAM-1, VCAM-1, and IL-8 induced, in HUVECs, by DcR3 are involved in promoting monocyte adhesion. To address this point, HUVECs were cultured in the presence of specific Abs for ICAM-1, VCAM-1, or IL-8 at the same time as DcR3 treatment. Fig. 4 demonstrates that DcR3’s ability to enhance THP-1 or U937 adhesion was attenuated by each Ab, and was abolished by their combination. These results indicate that DcR3 promotes monocyte adhesion to endothelial cells through costimulation of adhesion molecule (ICAM-1 and VCAM-1) expression and chemokine (IL-8) release in HUVECs.



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  		FIGURE 4. Effects of specific Abs for ICAM-1, VCAM-1, and IL-8 on DcR3.Fc-enhanced monocyte adhesion to HUVECs. HUVECs were preincubated with vehicle, DcR3.Fc (3 µg/ml), or TNF-{alpha} (20 ng/ml) for 3 h and then incubated with specific Abs for ICAM-1, VCAM-1, or IL-8 (10 µg/ml) for an additional 30 min. After incubation, BCECF-AM-labeled THP-1 or U937 cells were added to the HUVEC coculture for 1 h, and then adhesion was measured at an absorbance of 538 nm. Results represent the mean ± SEM from five independent experiments. *, p < 0.05 as compared with the corresponding control of DcR3 or cytokine alone.

 
The inability of DcR3 to increase monocyte adhesion to human aortic ECs

The results presented in Figs. 1, 2C, 3D, and 3E indicate that DcR3 has a TL1A-independent effect on HUVECs. To confirm this, we examined these events in another EC type, human aortic ECs, which we and others have reported previously do not express TL1A (15, 18). We found that DcR3 (3 µg/ml) caused an increase in THP-1 adhesion to human aortic ECs, with an efficacy of ~80% compared with TNF-{alpha} (20 ng/ml) (Fig. 5A). IL-8 production and ICAM-1 and VCAM-1 expression by human aortic ECs were also up-regulated by DcR3, with increases in protein levels being ~70% of those induced by TNF-{alpha} (Fig. 5, B and C). These data provide direct evidence that DcR3-induced THP-1 adhesion is not restricted to particular endothelial cell types and is independent of its decoy function as a neutralizer of TL1A.



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  		FIGURE 5. DcR3.Fc increases THP-1 adhesion, IL-8 secretion, and ICAM-1 and VCAM-1 expression in human aortic ECs (HAECs). A, A total of 1 x 106 human aortic ECs were treated with vehicle, human IgG1 (hIgG1; 3 µg/ml), DcR3.Fc (3 µg/ml), TL1A-Ab (3 µg/ml), or TNF-{alpha} (20 ng/ml) for 12 h, then BCECF-AM-labeled THP-1 cells were added for 1 h, after which adhesion was measured at an absorbance of 538 nm. B, Cell culture medium was tested for IL-8 levels by ELISA. C, Cells were treated with specific Abs for ICAM-1 and VCAM-1, and analyzed by flow cytometry. Mean values of the fluorescence intensity are indicated. Data represent the mean ± SEM from four independent experiments. *, p < 0.05 as compared with the control group.

 
NF-{kappa}B plays a role in DcR3-induced cell adhesion

Because the promoter regions of the ICAM-1 and VCAM-1 genes contain NF-{kappa}B binding sites (34), we next asked whether NF-{kappa}B plays a role in DcR3-induced monocyte adhesion to HUVECs. To answer this question, several approaches were used. Firstly, we used an NF-{kappa}B inhibitor, PDTC and found that this effectively abolished the adhesive responses caused by DcR3 and TNF-{alpha} (Fig. 6A). DcR3- and TNF-{alpha}-induced IL-8 secretion (Fig. 6B) as well as up-regulation of ICAM-1 (Fig. 6C) and VCAM-1 (Fig. 6D) expression were also abrogated by PDTC. Furthermore, DcR3 treatment of HUVECs markedly increased nuclear translocation of the NF-{kappa}B subunit, p65 (Fig. 7A), and the DNA-binding activity of NF-{kappa}B (Fig. 7B). We also demonstrated that I{kappa}B{alpha} degradation (Fig. 7C) and IKK complex activation (Fig. 7D), which are upstream events following NF-{kappa}B activation, were elicited by DcR3. All these data suggest that the NF-{kappa}B signaling cascade is an essential component in the involvement of adhesion molecule expression and IL-8 gene transcription in the DcR3-mediated adhesion response.



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  		FIGURE 6. PDTC abolishes DcR3.Fc-induced THP-1 adhesion, IL-8 secretion, and ICAM-1 and VCAM-1 expression in HUVECs. HUVECs were pretreated with PDTC (100 µM) for 30 min, and then incubated with DcR3.Fc (3 µg/ml), or TNF-{alpha} (20 ng/ml) for 12 h. A, Following incubation, BCECF-AM-labeled THP-1 cells were added to HUVECs and incubated for 1 h, and then adhesion was measured at an absorbance of 538 nm. B, Cell culture medium was then tested for IL-8 levels by ELISA. C and D, Cells were treated with specific Abs for ICAM-1 (C) or VCAM-1 (D), and analyzed by flow cytometry. Mean values of fluorescence intensity are indicated. Data represent the mean ± SEM from four independent experiments. *, p < 0.05 as compared with the control group.

 


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  		FIGURE 7. DcR3.Fc induces NF-{kappa}B activation in HUVECs. A, HUVECs were treated with vehicle, DcR3.Fc (3 µg/ml), human IgG1 (hIgG1; 3 µg/ml), TL1A-Ab (3 µg/ml), or TNF-{alpha} (20 ng/ml) for 1 h. Nuclear extracts were prepared and Western blot analysis of p65 was conducted. B, HUVECs were incubated with vehicle, DcR3.Fc (3 µg/ml), human IgG1 (3 µg/ml), TL1A-Ab (3 µg/ml), or TNF-{alpha} (20 ng/ml) for 3 h. Then nuclear extracts were prepared and analyzed for NF-{kappa}B activity using TransAM transcription factor assay kits. C, HUVECs were treated with DcR3.Fc (3 µg/ml) or LPS (1 µg/ml) for different time periods. I{kappa}B{alpha} degradation was determined by immunoblotting with an I{kappa}B{alpha}-specific Ab. D, Cells were incubated with DcR3.Fc (3 µg/ml) or LPS (1 µg/ml) for 0–60 min, and cell lysates were then immunoprecipitated with Abs specific for IKK{alpha} and IKK{beta}. One set of immunoprecipitates was subjected to kinase assays (KA) as described in Materials and Methods using GST-I{kappa}B{alpha} (5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 ) as a substrate (top). The other set of immunoprecipitates was subjected to SDS-PAGE and analyzed by Western blotting (WB) with an anti-IKK{alpha} Ab (bottom). The presence of equal amounts of the immunoprecipitated kinase complex in each assay was confirmed by immunoblotting for IKK{alpha}. The results shown are representative of three experiments with similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There is accumulating evidence to support a close link between inflammation and cancer progression. Infections may be responsible for the development over 15% of all malignancies worldwide (35). In addition, infectious agents may induce immunosuppression or directly transform cells by inserting active oncogenes into the host genome, inhibiting tumor suppressors or stimulating mitosis (35, 36), whereas inflammatory mediators may also play a pathological role, perhaps contributing to the initiation as well as the promotion of carcinogenesis.

The inflammatory component of a developing neoplasm may include a diverse leukocyte population, such as monocytes, macrophages, and dendritic cells. However, these leukocytes seem to have a dual role in neoplasm development. For example, tumor-associated macrophages are a significant component of inflammatory infiltrates in neoplastic tissues and are derived from monocytes. They may kill neoplastic cells following activation by IL-2, IFN, and IL-12 (37, 38), but they also produce a number of potent angiogenic and lymphangiogenic growth factors and cytokines, all of which are mediators that can potentiate neoplastic progression (39). Tumor-associated macrophages also produce increased amounts of the immunosuppressive cytokine, IL-10, serving to blunt the antitumor response by CTLs (40).

Cell adhesion, mediated by specific cell surface molecules, plays a central role in inflammation. The recruitment of monocytes from the circulation into the extravascular space involves several steps (41, 42, 43). Among them, interactions of the monocyte integrins LFA-1 and Mac-1 with ICAM-1, and of VLA-4 with VCAM-1 are crucial steps in forming stable attachments. In addition, the chemokine IL-8 promotes both leukocyte chemotaxis and angiogenesis (44, 45) and has also been proven to greatly increase the adhesive capacities of monocytes, by triggering conformational changes in LFA-1 and Mac-1 on rolling monocytes (33). As a result, monocytes attach firmly to the endothelium, and rolling is arrested (41). The expression of ICAM-1 and VCAM-1 by ECs are strongly up-regulated by inflammatory cytokines, such as IL-1 and TNF-{alpha} (41). Tumor cells not only take advantage of the trophic factors produced by inflammatory cells, but may also use the same adhesion molecules and chemokines to aid in migration and homing during distant metastatic spread (1). Indeed, previous studies have revealed significantly higher serum concentrations of ICAM-1 and VCAM-1 in patients with gastric cancer (46), prostate cancer (47), breast cancer (48), and lung cancer (49). These studies demonstrated that cell adhesion molecules play important roles in the growth and migration of cancer.

To establish inflammatory environments that will accelerate cancer development, tumor cells may produce various cytokines and chemokines to attract leukocytes. In this study, we demonstrate that the tumor-associated molecule, DcR3, contributes to this process. Recent studies, including our own, have demonstrated that DcR3, regardless of whether or not it is fused with an Fc, has the same binding affinity to FasL, LIGHT, and TL1A (18, 26, 50). In this study, using a DcR3.Fc fusion protein, we found that DcR3 increases the ability of human primary monocytes and the monocyte cell lines THP-1 and U937 to adhere to HUVECs, human aortic ECs and/or HMVECs. We also found that it can up-regulate ICAM-1, VCAM-1, and IL-8 expression in both HUVECs and human aortic ECs, and that all these factors contribute to enhanced monocyte adhesion. All these results suggest that DcR3 action in EC appears to be a general phenomenon. In addition, our previous study showed that DcR3-treated THP-1 cells undergo reorganization of actin resulting in enhanced adherence to culture plates (30). Our current study, however, showed that DcR3 treatment of HUVECs induced a greater extent of monocyte adhesion than DcR3 treatment of the THP-1 cells themselves (data not shown). Thus, although the stimulating effect of DcR3 on monocytes may help the adhesion response initially (in the first 1–2 h), over longer time periods (3 h or more), we believe that interactions between ICAM-1-LFA-1, Mac-1 and VCAM-1-VLA-4 make a more important contribution to the adherence of monocytes to endothelial cells. This is in view of our observation that induction of ICAM-1, VCAM-1 and IL-8 by DcR3 greatly increases EC adhesive capacity.

This study also suggests that DcR3.Fc can modulate monocyte adherence to EC via a ligand distinct from FasL, LIGHT, and TL1A. Although TL1A, but not FasL or LIGHT, is expressed by HUVECs (18), a TL1A-specific Ab was not found to mimic the effects of DcR3. Nevertheless, like other members of the TNFR family (23, 24, 25, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61), DcR3 may act as an effector molecule in EC and transduce reverse signaling, giving rise to NF-{kappa}B activation. To date, the identity of the novel ligand(s) for DcR3 remains unclear and needs further investigation. However, reverse signaling has been shown to mediate several biological effects of DcR3 in monocytes, macrophages, and dendritic cells (26, 28, 30, 60, 62).

In conclusion, our results suggest that DcR3 increases monocyte adhesion to endothelial cells by up-regulating the expressions of adhesion molecules and chemokines. This also raises the possibility that DcR3 not only functions as a decoy receptor for neutralizing immune system attack of tumor cells, but also plays a role in the causal relationships linking inflammation, innate immunity, and tumorigenesis.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the National Science Council of Taiwan (NSC93-2752-B002-006-PAE and NSC93-2320-B002-044). Back

2 Address correspondence and reprint requests to Dr. Wan-Wan Lin, Department of Pharmacology, College of Medicine, National Taiwan University, Number 1, Jen-Ai Road, Section 1, Taipei 100, Taiwan. E-mail address: wwl{at}ha.mc.ntu.edu.tw Back

3 Abbreviations used in this paper: DcR3, decoy receptor 3; BCECF-AM, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein tetrakis(acetoxymethyl) ester; FasL, Fas ligand; EC, endothelial cell; HMVEC, human microvascular endothelial cell; PDTC, pyrrolidine dithiocarbamate, LIGHT, lymphotoxin-like, exhibits inducible expression, and competes with HSV glycoprotein D for herpesvirus entry mediator expressed by T lymphocytes; IKK, I{kappa}B kinase. Back

Received for publication May 24, 2004. Accepted for publication November 15, 2004.


   References
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 Introduction
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