Junctional adhesion molecule C mediates leukocyte adhesion to rheumatoid arthritis synovium

Abstract

Objective: Leukocyte infiltration into the rheumatoid arthritis (RA) synovium is a multistep process in which leukocytes leave the bloodstream and invade the synovial tissue (ST). Leukocyte transendothelial migration and adhesion to RA ST requires adhesion molecules on the surface of endothelial cells and RA ST fibroblasts. This study was undertaken to investigate the role of junctional adhesion molecule C (JAM-C) in mediating leukocyte recruitment and retention in the RA joint.

Methods: Immunohistologic analysis was performed on RA, osteoarthritis (OA), and normal ST samples to quantify JAM-C expression. Fibroblast JAM-C expression was also analyzed using Western blotting, cell surface enzyme-linked immunosorbent assay, and immunofluorescence. To determine the role of JAM-C in leukocyte retention in the RA synovium, in vitro and in situ adhesion assays and RA ST fibroblast transmigration assays were performed.

Results: JAM-C was highly expressed by RA ST lining cells, and its expression was increased in OA ST and RA ST endothelial cells compared with normal ST endothelial cells. JAM-C was also expressed on the surface of OA ST and RA ST fibroblasts. Furthermore, we demonstrated that myeloid U937 cell adhesion to both OA ST and RA ST fibroblasts and to RA ST was dependent on JAM-C. U937 cell migration through an RA ST fibroblast monolayer was enhanced in the presence of neutralizing antibodies against JAM-C.

Conclusion: Our results highlight the novel role of JAM-C in recruiting and retaining leukocytes in the RA synovium and suggest that targeting JAM-C may be important in combating inflammatory diseases such as RA.

Figure 1

Immunohistologic analysis of junctional adhesion molecule C (JAM-C) in synovial tissue (ST) samples from normal (NL) subjects, patients with osteoarthritis (OA), and patients with rheumatoid arthritis (RA). Frozen sections of normal, OA, and RA ST were stained for JAM-C expression. A and B, Representative photomicrographs of JAM-C (A) and IgG control (B) expression in RA ST (original magnification × 400). C, Mean and SEM JAM-C expression in normal ST, OA ST, and RA ST endothelial cells (ECs). JAM-C was more highly expressed in OA ST (98%) and RA ST (94%) ECs than in normal ST (83%) ECs. D, Mean and SEM JAM-C expression in normal ST, OA ST, and RA ST lining cells. JAM-C was more highly expressed in normal ST (99%) and OA ST (95%) lining cells than in RA ST (85%) lining cells.

Figure 2

JAM-C expression in RA ST fibroblasts. A, Mean and SEM total number of pixels showing JAM-C expression in RA ST fibroblasts left untreated or cultured with tumor necrosis factor α (TNFα) for 24 hours. Western blots showed a 35-kd protein band consistent with the molecular weight of JAM-C; however, no differences were observed after stimulation with TNFα. B, Mean and SEM fold change in expression of JAM-C on the cell surface of RA ST fibroblasts left untreated or cultured with TNFα, determined by cell surface enzyme-linked immunosorbent assay. Surface expression of JAM-C on RA ST fibroblasts did not change after stimulation with TNFα. C, Surface expression and localization of IgG control and JAM-C on RA ST fibroblasts, examined using immunofluorescence and confocal microscopy. JAM-C was expressed throughout the surface, and no difference in expression was noted between adjacent fibroblasts in the presence or absence of TNFα. NS = nonstimulated (see Figure 1 for other definitions).

Figure 3

JAM-C mediates adhesion of untreated or phorbol myristate acetate (PMA)–stimulated U937 cells to RA and OA ST fibroblasts. The percentage of maximal adhesion was defined as the number of adherent cells on the ST sections divided by the number of adherent cells on the control sections. A, Inhibition of U937 cell adhesion to RA ST fibroblasts by both of the neutralizing antibodies against JAM-C, F26 (79% of maximal adhesion) and H33 (86% of maximal adhesion), and by an anti–Mac-1 antibody (79% of maximal adhesion). B, Additive inhibitory effect on U937 cell adhesion to RA ST fibroblasts of F26 combined with anti–Mac-1 (66% of maximal adhesion). C, Inhibition of PMA-stimulated U937 cell adhesion to RA ST fibroblasts at 37°C by F26 (71% of maximal adhesion). D, Inhibition of PMA-stimulated U937 cell adhesion to OA ST fibroblasts at 37°C by F26 (77% of maximal adhesion). Bars show the mean and SEM. See Figure 1 for other definitions.

Figure 4

JAM-C mediates adhesion of U937 cells to RA synovium. Stamper-Woodruff in situ assays were performed using frozen RA ST sections and fluorescence-labeled U937 cells. Left, Mean and SEM percentage of maximal binding, defined as the number of adherent cells on the test sections divided by the number of adherent cells on the control sections, of U937 cells by IgG, F26, H33, and anti–P-selectin. U937 cell adhesion was inhibited to a similar degree by both of the anti–JAM-C antibodies, F26 (49% of maximal binding) and H33 (60% of maximal binding), and by anti–P-selectin (41% of maximal binding). Right, Most U937 cells adhered to the blood vessels and synovial lining (arrows) (original magnification × 400). See Figure 1 for definitions.

Figure 5

JAM-C mediates U937 cell adhesion in a model of myeloid RA ST fibroblast transmigration. U937 cell migration through RA ST fibroblast monolayers grown on Transwell inserts, in response to incubation with stromal cell–derived factor 1α for 2 or 4 hours, was studied. Both neutralizing anti–JAM-C antibodies, F26 and H33, enhanced migration of U937 cells through the fibroblast monolayer. Bars show the mean and SEM fold change in migrated cells in test wells compared with migration in IgG control–treated wells. See Figure 1 for definitions.

Figure 6

Mean percentage of U937 cells expressing junctional adhesion molecule A (JAM-A), JAM-B, and JAM-C. Flow cytometric analysis was performed on U937 cells immunostained with antibodies against JAM-A, JAM-B, or JAM-C, or IgG control. U937 cells expressed JAM-A, but not JAM-B or JAM-C.