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The role of junctional adhesion molecule-C (JAM-C) in oxidized LDL-mediated leukocyte recruitment

Tanja Keiper, Nadia Al-Fakhri, Emmanouil Chavakis, Athanasios N. Athanasopoulos, Berend Isermann, Stefanie Herzog, Rainer Saffrich^||, Karin Hersemeyer^**, Rainer M. Bohle, Judith Haendeler, Klaus T. Preissner^**, Sentot Santoso and Triantafyllos Chavakis^*,,1

^* Experimental Immunology Branch, NCI, NIH, Bethesda, Maryland, USA;
Department of Internal Medicine I, University Heidelberg, Heidelberg;
Institute for Clinical Chemistry and Pathobiochemistry, Justus-Liebig-University, Giessen,
Molecular Cardiology, Department of Internal Medicine III, J. W. Goethe University, Frankfurt,
^|| Department of Internal Medicine V, University Heidelberg, Heidelberg,
^** Institute for Biochemistry,
Institute for Pathology and
Institute for Clinical Immunology and Transfusion Medicine, Justus-Liebig-University, Giessen, Germany

¹Correspondence: Experimental Immunology Branch, NCI, NIH, 10 Center Dr., Rm. 4B17, Bethesda, MD 20892, USA. E-mail: chavakist{at}mail.nih.gov

SPECIFIC AIMS

The junctional adhesion molecule-C (JAM-C) was recently identified on platelets and endothelial cells as a counter-receptor for the leukocyte ß2-integrin Mac-1 (CD11b/CD18), thereby mediating interactions between vascular cells. As Mac-1 is important for the recruitment of inflammatory cells to the atherosclerotic vessel wall or in restenosis, the aim of the present work was to investigate the expression of its counter-receptor JAM-C in the atherosclerotic vessel wall and to study the effect of oxidized LDL (oxLDL) on the expression of JAM-C as well as the role of JAM-C in oxLDL-mediated inflammatory cell recruitment.

PRINCIPAL FINDINGS

1. JAM-C is up-regulated in the arteriosclerotic vessel wall
Immunostaining of normal human arteries revealed a weak expression of JAM-C in the endothelium and by individual smooth muscle cells in the media. In contrast, in arteriosclerotic vessels JAM-C was strongly expressed in endothelial cells and smooth muscle cells in the neointima and the media. To provide evidence for the role of JAM-C in the early atherogenic process, we have used ApoE–/– mice, which develop spontaneous early lesions. The spontaneous lipid rich lesions in 26-wk-old ApoE–/– mice displayed a strong staining for JAM-C compared with the faint staining of JAM-C in the artery wall of wild-type mice of the same age. These data strongly indicate that JAM-C is up-regulated during the arteriosclerotic process in both men and ApoE–/– mice.

2. JAM-C expression on endothelial and vascular smooth muscle cells is up-regulated by oxLDL
The observation that JAM-C in the atherosclerotic vessel wall was associated with smooth muscle cells in the neointima and in the media indicated that JAM-C may be expressed by these cells. Indeed, human arterial smooth muscle cells (HASMC) were found to contain JAM-C-protein and express JAM-C mRNA. The expression of JAM-C on HASMC and HUVEC was significantly up-regulated by oxLDL or enzymatically modified LDL (eLDL) in a time- and dose-dependent manner; JAM-C expression was not affected by TNF- or IL-1ß (Fig. 1 ).

View larger version (17K): [in this window] [in a new window]   Figure 1. oxLDL up-regulates JAM-C expression in HUVEC. A) Expression of JAM-C or ICAM-1 on HUVEC was quantified by cell ELISA. HUVEC were stimulated for 12 h without (open bars) or with oxLDL (10 µg/mL, filled bars), eLDL (20 µg/mL, dotted bars), IL-1ß (10 ng/mL, gray bars), or TNF- (10 ng/mL, hatched bars). JAM-C or ICAM-1 expression is shown as % of control (no stimulus) and represents mean ±SD (n=3) of a typical experiment. Similar results were obtained in 3 separate experiments. C, D) The expression of JAM-C on HUVEC was quantified by cell ELISA. B) HUVEC were stimulated for 12 h in the absence or presence of increasing concentrations of oxLDL. C) HUVEC were stimulated for 0–24 h in the absence (open circles) or presence of 10 µg/mL oxLDL (filled circles). JAM-C expression is shown as absorbance at 405 nm and represents mean ±SD (n=3) of a typical experiment. Similar results were obtained in 3 separate experiments.

3. JAM-C localization on endothelial cells is altered in the presence of oxLDL
JAM-C localization on endothelial cells has a polarized pattern. In quiescent endothelial cells JAM-C is found at cell-cell contacts colocalizing with the tight junction protein ZO-1. Upon 12 h stimulation of HUVEC with oxLDL, JAM-C localization on HUVEC was no longer restricted to interendothelial contacts. Although on oxLDL pretreated HUVEC the majority of JAM-C still localized to interendothelial contacts, a part of JAM-C distributed to sites of the endothelial cell surface distinct from interendothelial contacts.

4. JAM-C mediates leukocyte adhesion to oxLDL-stimulated endothelial cells
Upon oxLDL stimulation of HUVEC, both adhesion to HUVEC and transmigration of THP-1 cells and monocytes isolated from peripheral blood increased 2-fold. JAM-C did not participate in the Mac-1-dependent adhesion of THP-1 monocytes to quiescent HUVEC, while Fc-JAM-C blocked adhesion of THP-1 cells and monocytes to oxLDL prestimulated endothelial cells under both static and physiologic flow conditions. In contrast, anti-Mac-1 and anti-ICAM-1 inhibited adhesion of THP-1 cells and monocytes both to unstimulated and oxLDL-stimulated endothelial cells under static and flow conditions (Fig. 2 ). Whereas adhesion of monocytes to quiescent HUVEC was blocked almost completely by mAb to ICAM-1, this mAb inhibited monocyte adhesion to oxLDL-stimulated HUVEC partially, and only the combination of ICAM-1 and JAM-C blockade nearly abolished monocyte adhesion to oxLDL pretreated HUVEC (Fig. 2) . Thus, JAM-C mediates adhesion of THP-1 monocytic cells and monocytes solely to oxLDL-stimulated endothelial cells, whereas ICAM-1 is involved in monocyte adhesion to both quiescent and oxLDL-stimulated HUVEC. In contrast, JAM-C blockade with Fc-JAM-C resulted in inhibition of the Mac-1-dependent THP-1 transmigration through quiescent and oxLDL-stimulated HUV] EC and its effect were comparable to the inhibition of THP-1 transmigration provided by ICAM-1 blockade. Together, JAM-C serves as both an adhesion and transmigration receptor on oxLDL-stimulated HUVEC.

View larger version (20K): [in this window] [in a new window]   Figure 2. JAM-C mediates THP-1 monocytic cell adhesion to oxLDL-stimulated endothelial cells. A) The adhesion of MCP-1-stimulated THP-1 cells to quiescent HUVEC (open bars) or oxLDL prestimulated (12 h, 10 µg/mL) HUVEC (filled bars) was studied under static conditions and is shown in the absence (–) or presence of blocking mAb against Mac-1, blocking mAb against ICAM-1, Fc control, Fc-JAM-C, or blocking mAb against PECAM-1 (CD31) (each at 20 µg/mL) or combinations thereof, as indicated. Cell adhesion is represented as % of control. Data are mean ±SD (n=3) of a typical experiment and similar results were observed in 3 separate experiments. B) The transmigration of THP-1 cells toward 50 ng/mL MCP-1 across quiescent HUVEC (open bars) or oxLDL prestimulated (12 h, 10 µg/mL) HUVEC (filled bars) is shown in the absence (–) or presence of blocking mAb against Mac-1, blocking mAb against ICAM-1, Fc control, Fc-JAM-C, or blocking mAb against PECAM-1 (CD31) (each at 20 µg/mL) or combinations thereof, as indicated. Transmigration is represented as % of control. Data are mean ±SD (n=3) of a typical experiment and similar results were observed in 3 separate experiments.

CONCLUSIONS AND SIGNIFICANCE

The present work provides novel information on oxLDL-mediated up-regulation of JAM-C in vascular cells and in particular, JAM-C prominently contributes to leukocyte attachment and transmigration in oxLDL-stimulated endothelial cells.

At first JAM-C was found to be up-regulated in atherosclerotic vessels in men and in the spontaneous early lesions of ApoE–/– mice. A strong expression of JAM-C was found in endothelial cells as well as in smooth muscle cells in the neointima and in the media vs. only a weak expression of JAM-C in normal arteries. In vitro, JAM-C expression on vascular smooth muscle cells was described here for the first time.

Several proinflammatory cytokines as well as oxLDL are involved in the up-regulation of adhesion molecules on endothelial and smooth muscle cells during atherogenesis. JAM-C on endothelial and smooth muscle cells was up-regulated only by oxLDL or eLDL but not by TNF- or IL-1ß as opposed to ICAM-1, which was up-regulated by all stimuli. Together, we provide here the first information regarding the regulation of the expression of JAM-C on vascular cells.

In contrast to quiescent endothelial cells, where JAM-C localized strictly to cell-cell contacts, ZO-1, JAM-C localization on oxLDL-stimulated endothelial cells was no more restricted to intercellular contacts. In accordance with these findings, JAM-C mediated both Mac1-dependent adhesion of monocytes to, and their transmigration through, oxLDL-stimulated endothelial cells whereas JAM-C on quiescent endothelial cells only participated in Mac-1-dependent transmigration. Thus, upon oxLDL stimulation endothelial JAM-C functions as both an adhesion and a transmigration receptor for leukocytes.

The present work underscores our earlier findings that JAM-C on platelets interacts with leukocyte Mac-1 to mediate a firm leukocyte-platelet interaction. Such leukocyte-platelet interactions are important at sites of vascular injury, where the endothelial cell lining has been denuded, such as in atherosclerotic or postangioplasty restenotic lesions; at these sites leukocyte infiltration can occur through interactions with deposited platelets. Together with our previous data, the present findings point to a potential participation of endothelial JAM-C in the enhanced inflammatory cell recruitment during initiation of atherosclerosis. Thus, JAM-C may provide a novel target in such inflammatory vascular pathologies in order to device therapeutic interventions in atherothrombosis and post-angioplasty restenosis.

View larger version (41K): [in this window] [in a new window]   Figure 3. A putative role for JAM-C in atherosclerosis? A) JAM-C is up-regulated in the atherosclerotic vessel. B) JAM-C on endothelial and vascular smooth muscle cells is up-regulated by oxLDL. C) JAM-C participates in the oxLDL-mediated leukocyte recruitment. Left panel: under quiescent conditions JAM-C is mainly localized in the interendothelial contacts and does not mediate leukocyte adhesion to endothelial cells. Right panel: Upon stimulation with oxLDL JAM-C expression is enhanced and JAM-C localizes both in the interendothelial contacts as well as at sites of the endothelial surface distinct from interendothelial contacts. JAM-C can mediate leukocyte adhesion to oxLDL-stimulated endothelial cells. This mechanism together with the up-regulation of ICAM-1 (as well as other receptors, such as VCAM-1 or selectins; not depicted here) results in increased recruitment of inflammatory cells. L: leukocyte; EC: endothelial cell.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4196fje;

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