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Cutting Edge: Combined Treatment of TNF- and IFN- Causes Redistribution of Junctional Adhesion Molecule in Human Endothelial Cells¹

Harunobu Ozaki^*, Kenji Ishii^2,*, Hisanori Horiuchi^*, Hidenori Arai^*, Takahiro Kawamoto^*, Katsuya Okawa, Akihiro Iwamatsu and Toru Kita^*

^* Department of Geriatric Medicine, Graduate School of Medicine, Faculty of Medicine, Kyoto University, Kyoto, Japan; and Central Laboratories for Key Technology, Kirin Brewery, Yokohama, Japan

	Abstract

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Proinflammatory cytokines such as TNF- and IFN- induce cell adhesion molecules in endothelial cells and promote transmigration of leukocytes across endothelial cells. However, when those two were administered together, leukocyte transmigration paradoxically decreased. We cloned a human and bovine homologue of the junctional adhesion molecule (JAM), a novel molecule at the tight junction, and examined the effects of proinflammatory cytokines on JAM in HUVECs. The combined treatment of TNF- plus IFN- caused a disappearance of JAM from intercellular junctions. However, flow cytometry, cell ELISA, and subcellular fractionation analysis demonstrated that the amount of JAM was not reduced. This suggested that JAM changed its distribution in response to proinflammatory cytokines. This redistribution of JAM might be involved in a decrease in transendothelial migration of leukocytes at inflammatory sites.

	Introduction

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Proinflammatory cytokines, such as TNF- and IFN- increase the expression of cell adhesion molecules, such as ICAM-1 and VCAM-1, in endothelial cells (ECs)³ and promote the transendothelial migration (TEM) of leukocytes (1, 2, 3). However, when those two cytokines were added in combination, TEM paradoxically decreased (4). This phenomenon was accompanied by the disappearance of platelet EC adhesion molecule-1 (PECAM-1) from intercellular junctions. PECAM-1 is a member of the Ig superfamily and is required for TEM (5, 6). Therefore, the reduction of adhesion molecules also might play important roles in TEM of leukocytes.

Recently, the junctional adhesion molecule (JAM), a novel member of the Ig gene superfamily, was identified and shown to be constitutively expressed at intercellular junctions of ECs (7). Padura et al. suggested that JAM might play a role in monocyte transmigration because anti-JAM mAb inhibited monocyte TEM in vitro and monocyte infiltration by chemokine in vivo. However, the effects of proinflammatory cytokines on JAM remain unknown.

In this study, we cloned human and bovine homologues of JAM independently from Padura et al. and investigated the effects of TNF- and IFN- on JAM in human ECs. We demonstrate that the treatment with TNF- plus IFN- induces redistribution of JAM on the cell surface. This redistribution might play an important role in regulating TEM of leukocytes in inflammation.

	Materials and Methods

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Cell culture and preparation

HUVECs were isolated, cultured, and used at passage 2 to eliminate detachment or retraction as described (4). Bovine arterial ECs were prepared as described previously (8).

Cloning of human, bovine, and murine JAM cDNAs

During purifying a novel membrane-associated protease in bovine aortic ECs, we purified a 40-kDa protein as a by-product, and the amino acid sequence was determined (9). One of the peptide sequences matched genes in expression sequence tag (EST) data banks (Fig. 1). An oligonucleotide primer (gtcccatacccattccgtgcctc) was designed based on the human EST clone AA152150, and RT-PCR was performed using the human placental cDNA library (Marathon-Ready cDNA; Clontech, Palo Alto, CA). A single clone was obtained and subjected to nucleotide sequence analysis. A human colon EC line cDNA library (Stratagene, La Jolla, CA) was screened with the human cDNA fragment, and the positive clones were sequenced. A mouse 7-day embryo cDNA library (Clontech) was screened with EST clone AA561790, and a bovine aortic EC cDNA library (Stratagene) was screened with murine cDNA. Nucleotide sequencing was performed by dideoxy method (Prism 310, Applied Biosystems, Foster City, CA).

View larger version (66K): [in this window] [in a new window]   FIGURE 1. Deduced amino acid sequences of cDNA clones encoding the human, bovine, and murine JAM. Conserved cysteine residues are boxed. Putative N-glycosylation sites are printed by boldface type and are indicated by a triangle. The amino acid sequence that was detected in the EST clone was underlined. Putative phosphorylation sites are printed by boldface type and are indicated by an asterisk. The transmembrane domain is indicated by vertical blankets. Dashes indicate insertions to maximize homology. (GenBank accession number of human JAM, AF111713; bovine JAM, AF111714)

Rabbit Ab against the human JAM extracellular domain (ECD) (JAM (ECD)) was generated by immunizing rabbits with bacterial fusion protein GST-JAM (ECD). Rabbit Ab against the human JAM cytoplasmic domain was generated by immunizing rabbits with a synthetic peptide, CSQPSARSEGEFKQ. These Abs recognized JAM transiently expressed in Chinese hamster ovary (CHO) cells by Western blot and immunofluorescence microscopy. Mouse mAb against the human JAM (ECD), designated 3D8, was raised against JAM (ECD):IgG1 hinge fusion protein (10) and screened by cell ELISA against the stable CHO cell line that expresses human JAM.

Other Abs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) if not described.

Immunofluorescence microscopy

Cultured cells were fixed with 4% paraformaldehyde in PBS and blocked with 2% goat serum and 0.1% BSA in PBS. For staining in a nonpermeabilized condition, fixed cells were incubated with anti-JAM (ECD), anti-PECAM-1 mAb (158-2B3; Neo Markers, Union City, CA), anti-VCAM-1 mAb (10C9; PharMingen, San Diego, CA) followed by Alexa 488-conjugated secondary Abs (Molecular Probes, Eugene, OR). For staining in permeabilized conditions, paraformaldehyde-fixed cells were permeabilized with 0.1% Triton X-100 in PBS and then incubated with anti-JAM (3D8) followed by Alexa 488-conjugated secondary Ab and phalloidin-rhodamine (Molecular Probes).

Flow cytometry

HUVECs were dispersed by PBS with 1 mM EDTA, washed with PBS containing 1% BSA, and stained with rabbit anti-JAM (ECD), anti-PECAM-1 mAb 158-2B3, anti-VCAM-1 mAb 10C9, or anti-JAM (3D8) for 1 h at 4°C followed by incubating with Alexa 488-labeled secondary Abs. Flow cytometry was performed by using FACS (FACS Vantage; Becton Dickinson, Mountain View, CA).

Cell ELISA

HUVECs fixed with 4% paraformaldehyde were incubated with anti-VCAM-1 mAb (10C9), anti-JAM (3D8), or each isotype-matched control IgG followed by HRP-conjugated anti mouse Ab (Amersham-Pharmacia, Piscataway, NJ). The HRP activity was detected by development with tetramethylbenzidine (Dako, Carpinteria, CA). OD was measured at A_{450 nm}.

Fractionation of cell-surface proteins

Cell-surface proteins were biotin labeled with 1 mg/ml NHS-sulfo-biotin (Pierce, Rockford, IL) as described by Yip et al. (11). For fractionation of biotinylated cell-surface proteins from intracellular proteins, postnuclear supernatants were bound to streptavidin-agarose (Sigma, St. Louis, MO). The bound proteins were washed with Triton lysis buffer (20 mM Tris, pH 7.4, 10 mM EDTA, 1% Triton X-100, and protease inhibitors), and eluted into SDS sample buffer. The same amount of proteins (v/v) from the initial total-cell lysate, the eluate from streptavidin-beads (biotinylated surface proteins), and the flow through (unbiotinylated proteins) were then subjected to Western blot analysis. Protein concentration was determined by the Bradford method.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cloning of human, bovine, and murine JAM

Microsequensing analysis of a 40-kDa protein from bovine aortic ECs revealed an amino acid sequence of five peptides. We searched EST banks for cDNA homology deduced from the peptides and found three sequences (AA564790, AA152150, and AA304161) that were homologous to one of the sequences. Based on these sequences, we cloned human, bovine, and murine full-length cDNAs as described in Materials and Methods.

The degrees of identity at the amino acid level among human and the corresponding bovine or murine clones were 75% and 68%, respectively (Fig. 1). The amino acid sequence of the murine clone was identical with that of a recently identified murine adhesion molecule, JAM, except one amino acid (268 ThrArg) (7). Therefore, we concluded that the cDNAs were human and bovine homologues of murine JAM.

JAM disappears from intercellular junctions of HUVECs treated with TNF- plus IFN- in combination

We examined the localization of JAM and the effects of inflammatory cytokines on the distribution of JAM in HUVECs. HUVECs were treated with 100 U/ml TNF- and 200 U/ml IFN-, separately or in combination (4, 12, 13), and the localization of JAM was examined by immunofluorescence microscopy with anti-JAM (ECD). In untreated HUVECs, most of the JAM was expressed at intercellular junctions (Fig. 2Aa). In contrast, in TNF--treated HUVECs, JAM was stained less intensively at intercellular junctions (Fig. 2Ab). IFN- also slightly reduced the JAM expression (Fig. 2Ac). In a sharp contrast, after treatment with TNF- plus IFN- in combination, JAM was almost completely disappeared from intercellular junctions, which was accompanied with elongated morphology of ECs (12) (Fig. 2Ad). The disappearance of JAM occurred as early as 8 h after cytokine treatment (data not shown). We also found that this phenomenon is reversible 48 h after replacement of the medium (data not shown). The same combination of cytokine treatment also markedly reduced PECAM-1 expression from intercellular junctions (Fig. 2A, e and f) (4, 14). In contrast, VCAM-1 expression was markedly induced by the same treatment of HUVECs (Fig. 2A, g and h) (3). These results indicated that JAM disappeared from intercellular junctions by the treatment with the combination of TNF- plus IFN-. Double staining with anti-JAM mAb (3D8) and rhodamine-conjugated phalloidin demonstrated that a rearrangement of actin fibers was also induced by TNF- plus IFN- (Fig. 2B) (12). This result also indicated that a change in fluorescence staining is not because of any loss or change in the endothelial monolayer.

View larger version (77K): [in this window] [in a new window]   FIGURE 2. Localization of JAM in HUVEC treated with inflammatory cytokines. A, HUVECs were either untreated (a, e, g) or treated with 100 U/ml TNF- for 24 h (b), 200 U/ml IFN- for 24 h (c), or 100 U/ml TNF- plus 200 U/ml IFN- for 24 h (d, f, h). HUVECs were fixed and stained in a nonpermeabilized condition for JAM (a, b, c, d) with anti-JAM (ECD) and PECAM-1 (e, f) with mAb 158-2B3 and VCAM-1 (g, h) with 51-10C9 and followed by Alexa 488-cojugated secondary Ab against rabbit or mouse IgG. B, HUVECs were either untreated (a, b) or treated with 100 U/ml TNF- plus 200 U/ml IFN- for 24 h (c, d). Fixed HUVECs were permeabilized and stained for JAM with anti-JAM mAb 3D8 followed by Alexa 488-cojugated secondary Ab and for F-actin with rhodamine-conjugated phalloidin. Fluorescence for JAM (a, c) and F-actin (b, d) was detected. Note that the intensity of the immunofluorescence signal of JAM and PECAM-1 always seemed more intensive in overlapped areas of elongated HUVECs than in nonoverlapped areas (original magnification, x400).

TNF- and/or IFN- treatment did not change the amount of JAM on the cell surface of HUVECs

Next, to examine whether the amount of JAM on the cell surface is reduced in response to the cytokine treatment, we performed FACS analysis (Fig. 3). Unexpectedly, the expression of JAM and PECAM-1 was not decreased after the same cytokine treatment. In contrast, VCAM-1 expression was clearly increased by TNF- and/or IFN-. Furthermore, prolonged treatment with TNF- plus IFN- for up to 48 h did not change the cell-surface JAM expression (data not shown). FACS analysis using anti-JAM mAb (3D8) showed a comparable result with that using anti-JAM (ECD) (data not shown). The results of Figs. 2 and 3 suggested that the cytokine treatment might have changed the structure of intercellular junctions so that Abs could not reach. Therefore, to determine whether JAM is still present at intercellular junctions or changed its distribution to cell surface, JAM on the cell surface was determined by cell ELISA (Fig. 4). The expression of JAM in HUVECs after the treatment was almost equal to that in untreated cells (Fig. 4A). In contrast, VCAM-1 expression was clearly increased by the same treatment (Fig. 4B).

View larger version (45K): [in this window] [in a new window]   FIGURE 3. FACS of cytokines-treated HUVECs. Cell-surface expression of JAM, PECAM-1, and VECAM-1 on HUVECs under a baseline condition or treated with 100 U/ml TNF-, 200 U/ml IFN-, and 100 U/ml TNF-a plus 200 U/ml IFN- were assessed by FACS.

View larger version (19K): [in this window] [in a new window]   FIGURE 4. Cell ELISA of cytokines-treated HUVECs. Cell-surface expression of JAM (A) and VCAM-1 (B) on HUVECs under a baseline condition or after the treatment with 100 U/ml TNF- and 200 U/ml IFN- for 24 h were assessed by cell ELISA. Each experiment was performed in triplicate.

Neither subcellular localization nor total amount of JAM was affected by TNF- and/or IFN- treatment

The results of immunofluorescence microscopy, FACS, and cell ELISA indicated that JAM loses localization at intercellular junctions and redistributes on the cell surface by proinflammatory cytokines similarly to PECAM-1 (14). Therefore, to confirm this, we examined the change in the amount of JAM in the cell surface and intracellular fractions by cell-surface biotinylation and separating them from intracellular proteins with avidin-agarose (Fig. 5A). Western blotting revealed that JAM was predominantly distributed in the cell-surface fraction of unstimulated ECs. In contrast, extracellular signal-regulated kinase was exclusively localized in the intracellular fraction (15) and PECAM-1 was detected in both fractions (16).

View larger version (30K): [in this window] [in a new window]   FIGURE 5. A, Separation of cell-surface and intracellular proteins. HUVECs were biotinylated, and the postnuclear extracts were adsorbed with streptavidin-agarose. The same amount of proteins (v/v) from the initial total cell lysate (T), the eluate from streptavidin-beads (cell-surface proteins; CS), and the flow through (intracellular proteins; IC) were then subjected to Western blot with anti-JAM (ECD) (lanes 1–3), anti-PECAM-1 (lanes 4 and 5), and anti-ERK Abs (lanes 6 and 7). As rabbit anti-ERK1 Ab cross-reacts with ERK2, two bands were recognized (arrowheads: upper, ERK1; lower, ERK2). B, TNF- plus IFN- did not change the subcellular localization of JAM. HUVECs with or without treatment of TNF- plus IFN- were subjected to cell-surface biotinylation followed by fractionation with avidin-agarose as described in A. The equal amount of protein (v/v) was analyzed by Western blot with a rabbit Ab against human JAM cytoplasmic domain (lanes 1–4), anti-PECAM-1 Ab (lanes 5–8), and anti-VCAM-1 Ab (lanes 9–12).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, we cloned human and bovine JAM and demonstrated that the combined treatment of TNF- plus IFN- caused redistribution of JAM as well as PECAM-1. JAM disappeared from intercellular junctions of HUVECs by immunofluorescence microscopy (Fig. 2). However, flow cytometry, cell ELISA, and subcellular fractionation analysis showed that the amount of JAM on the cell surface was not changed by the treatment ( Figs. 3–5). Therefore, we concluded that JAM changed its distribution on the cell surface by the treatment, and we suggest that the redistribution of JAM might play a role in decreasing TEM of leukocytes.

Inflammatory cytokines are known to play pivotal roles in TEM of leukocytes by inducing several cell adhesion molecules in ECs. For example, Bradley et al. reported that prolonged treatment of HUVECs with TNF- causes induction of VCAM-1, ICAM-1 and -2, ß₁ and ß₃ integrins, and redistribution of them from the apical surface to intercellular junctions (17). The authors speculated that the expression of cell adhesion molecules on the apical surface might first facilitate attachment to ECs and subsequently redistribution to cell junctions might enhance transmigration. Interestingly, however, when ECs were treated with TNF- plus IFN- in combination, this increase in TEM of leukocytes was almost nullified (Ref. 4 and our unpublished observation). The authors have shown that this phenomenon correlates with a decrease of PECAM-1 at intercellular junctions. The mechanisms of a decrease of PECAM-1 at intercellular junctions by TNF- plus IFN- have been controversial. Rival et al. has reported that this decrease is due to the inhibition of synthesis (4), while Romer et al. has shown that PECAM-1 redistributed from the junction to the cell surface (14). Our results of flow cytometry, cell ELISA, and cell-surface fractionation gave evidence to support the latter report.

Here, we demonstrated that a new member of the Ig gene superfamily, JAM, is also regulated by inflammatory cytokines. TNF- or IFN- alone caused slight redistribution of JAM in HUVECs (Figs. 2 and 3), and combination of those two markedly induced the redistribution of JAM as well as PECAM-1. Given that JAM is localized at intercellular junctions and involved in TEM, it is conceivable that the redistribution of JAM also contributes to negative regulation of transmigration of leukocytes in addition to PECAM-1. Based on our study, we propose a novel family of cell adhesion molecule consisting of JAM and PECAM-1 that are involved in negative regulation of leukocyte TEM. Both molecules are predominantly expressed at intercellular junctions of ECs (6, 7) and redistribute in response to proinflamatory cytokines. However, different sublocalization in intercellular junctions, JAM at an apical side (7) and PECAM-1 at a basolateral side (18), suggests their different roles in TEM. Further study will help to reveal the physiological significance of JAM in vivo.

	Acknowledgments

 
We thank Dr. Brian Seed for the plasmid pCd5lneg1 that was used to generate JAM (ECD):IgG1 hinge fusion protein, Dr. Noriaki Kume for helpful discussion, and Xiaoming Hu and Yukio Ohshima for excellent technical assistance.

	Footnotes

 
¹ This study was supported by the Ministry of Education, Science, Sports, and Culture, Research Grants 09281104 and 09044293, the Takeda Medical Research Foundation (1998, 1999), and the HMG-CoA Reductase Research Foundation.

² Address correspondence and reprint requests to Dr. Kenji Ishii, Department of Geriatric Medicine, Graduate School of Medicine, Faculty of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. E-mail address:

³ Abbreviations used in this paper: ECs, endothelial cells; ECD, extracellular domain; EST, expression sequence tag; JAM, junctional adhesion molecule; PECAM-1, platelet endothelial cell adhesion molecule-1; TEM, transendothelial migration; CHO, Chinese hamster ovary.

Received for publication March 11, 1999. Accepted for publication April 26, 1999.

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				M. Aurrand-Lions, C. Lamagna, J. P. Dangerfield, S. Wang, P. Herrera, S. Nourshargh, and B. A. Imhof Junctional Adhesion Molecule-C Regulates the Early Influx of Leukocytes into Tissues during Inflammation J. Immunol., May 15, 2005; 174(10): 6406 - 6415. [Abstract] [Full Text] [PDF]

				G. Ostermann, L. Fraemohs, T. Baltus, A. Schober, M. Lietz, A. Zernecke, E. A. Liehn, and C. Weber Involvement of JAM-A in Mononuclear Cell Recruitment on Inflamed or Atherosclerotic Endothelium: Inhibition by Soluble JAM-A Arterioscler. Thromb. Vasc. Biol., April 1, 2005; 25(4): 729 - 735. [Abstract] [Full Text] [PDF]

				L. Fraemohs, R. R. Koenen, G. Ostermann, B. Heinemann, and C. Weber The Functional Interaction of the {beta}2 Integrin Lymphocyte Function-Associated Antigen-1 with Junctional Adhesion Molecule-A Is Mediated by the I Domain J. Immunol., November 15, 2004; 173(10): 6259 - 6264. [Abstract] [Full Text] [PDF]

				T. Vincent, R. F. Pettersson, R. G. Crystal, and P. L. Leopold Cytokine-Mediated Downregulation of Coxsackievirus-Adenovirus Receptor in Endothelial Cells J. Virol., August 1, 2004; 78(15): 8047 - 8058. [Abstract] [Full Text] [PDF]

				K. Zen, B. A. Babbin, Y. Liu, J. B. Whelan, A. Nusrat, and C. A. Parkos JAM-C Is a Component of Desmosomes and a Ligand for CD11b/CD18-mediated Neutrophil Transepithelial Migration Mol. Biol. Cell, August 1, 2004; 15(8): 3926 - 3937. [Abstract] [Full Text] [PDF]

				G. Bazzoni and E. Dejana Endothelial Cell-to-Cell Junctions: Molecular Organization and Role in Vascular Homeostasis Physiol Rev, July 1, 2004; 84(3): 869 - 901. [Abstract] [Full Text] [PDF]

				N. Reymond, A.-M. Imbert, E. Devilard, S. Fabre, C. Chabannon, L. Xerri, C. Farnarier, C. Cantoni, C. Bottino, A. Moretta, et al. DNAM-1 and PVR Regulate Monocyte Migration through Endothelial Junctions J. Exp. Med., May 17, 2004; 199(10): 1331 - 1341. [Abstract] [Full Text] [PDF]

				G. T. Mercier, J. A. Campbell, J. D. Chappell, T. Stehle, T. S. Dermody, and M. A. Barry A chimeric adenovirus vector encoding reovirus attachment protein {sigma}1 targets cells expressing junctional adhesion molecule 1 PNAS, April 20, 2004; 101(16): 6188 - 6193. [Abstract] [Full Text] [PDF]

				K. Ebnet, A. Suzuki, S. Ohno, and D. Vestweber Junctional adhesion molecules (JAMs): more molecules with dual functions? J. Cell Sci., January 1, 2004; 117(1): 19 - 29. [Abstract] [Full Text] [PDF]

				M. Bruewer, A. Luegering, T. Kucharzik, C. A. Parkos, J. L. Madara, A. M. Hopkins, and A. Nusrat Proinflammatory Cytokines Disrupt Epithelial Barrier Function by Apoptosis-Independent Mechanisms J. Immunol., December 1, 2003; 171(11): 6164 - 6172. [Abstract] [Full Text] [PDF]

				M. U. Naik, D. Vuppalanchi, and U. P. Naik Essential Role of Junctional Adhesion Molecule-1 in Basic Fibroblast Growth Factor-Induced Endothelial Cell Migration Arterioscler. Thromb. Vasc. Biol., December 1, 2003; 23(12): 2165 - 2171. [Abstract] [Full Text] [PDF]

				J. C. Forrest, J. A. Campbell, P. Schelling, T. Stehle, and T. S. Dermody Structure-Function Analysis of Reovirus Binding to Junctional Adhesion Molecule 1: IMPLICATIONS FOR THE MECHANISM OF REOVIRUS ATTACHMENT J. Biol. Chem., November 28, 2003; 278(48): 48434 - 48444. [Abstract] [Full Text] [PDF]

				K. Ebnet, M. Aurrand-Lions, A. Kuhn, F. Kiefer, S. Butz, K. Zander, M.-K. M. z. Brickwedde, A. Suzuki, B. A. Imhof, and D. Vestweber The junctional adhesion molecule (JAM) family members JAM-2 and JAM-3 associate with the cell polarity protein PAR-3: a possible role for JAMs in endothelial cell polarity J. Cell Sci., October 1, 2003; 116(19): 3879 - 3891. [Abstract] [Full Text] [PDF]

M. U. Naik, S. A. Mousa, C. A. Parkos, and U. P. Naik Signaling through JAM-1 and {alpha}v{beta}3 is required for the angiogenic action of bFGF: dissociation of the JAM-1 and {al