Despite indirect evidence suggesting the existence of a extracellular heterophilic binding partner (Martìn-Padura et al., 1998), JAM-1 has only been described to interact in a homophilic manner both in cis (dimerization) and in trans. Such JAM-1/JAM-1 interactions appear to participate specifically in the formation and/or maintenance of intercellular junctions of endothelia and epithelia. The finding of JAM-1 expression in human hematopoietic cells, and in particular in Jurkat, J-β2.7 and CD4+CD45RO+ T cells used within this study, raised the important issue to
test whether homophilic binding between leukocytic JAM-1 and CHO cell-expressed JAM-1 was involved in the observed cell-cell adhesion phenomena.
Discussion 109
Several lines of evidence corroborated the conclusion that such homophilic interactions in trans
were not crucial for the firm arrest of leukocytes. i), the surface expression of JAM-1 in wild- type Jurkat cells and J-β2.7 cells did not correlate with the marked differences in their adhesion
behaviour to CHO-JAM transfectants on the one hand and CHO-vector transfectants or wild- type CHO cells on the other hand. The failure of αL-deficient but JAM-1 expressing J-β2.7 cells
to bind to JAM-1–transfected CHO cells revealed a crucial role for heterophilic but not for homophilic JAM-1 interactions in Jurkat/CHO-JAM cell binding. Moreover, reconstitution of JAM-1 binding-properties of J-β2.7 cells by their transfection with an αL-encoding cDNA
demonstrated the dependence on this integrin receptor. ii), leukocyte binding to CHO-JAM cells appeared to depend completely on stimulation by the phorbol ester PMA, suggesting that this cell adhesion required activated LFA-1 but not on the presence of leukocytic JAM-1 molecules. iii), preincubation of leukocytes with specific LFA-1 mAbs decreased their arrest on CHO-JAM cells to such an extent that a contribution of trans-homophilic JAM-1 binding to the observed cell adhesion could be convincingly excluded. iv), blocking leukocyte JAM-1 with anti–JAM-1 did not affect binding of Jurkat T cells to CHO-JAM cells nor binding of CD4+CD45RO+ memory T cells to unstimulated or cytokine-treated HUVECs, as described below. v), CHO cells with or without expression of JAM-1 showed equivalent binding to purified, immobilized LFA-1 receptor and to LFA-1–expressing Jurkat cells, respectively, and thus provided evidence against an involvement of homophilic JAM-1 interactions in the adhesive binding reported here. Notably, JAM-1 contamination of the LFA-1 preparation due to copurification was not detectable by protein analysis. vi), myeloid HL-60 cells, that did not show detectable JAM-1 surface expression in flow cytometry analysis in accordance with published data (Williams et al., 1999), showed a similar LFA-1–mediated binding pattern to CHO-vector or CHO-JAM cells than JAM-1–expressing Jurkat cells. This was in contrast to a report where HL-60 cells failed to bind recombinant human JAM-1 Fc chimera (Cunningham et al., 2000). However, this was most likely due to the fact that the monocytic cells were not stimulated and therefore did not express activated LFA-1. Finally, CHO-JAM transfectants in suspension failed to exert significant adhesive properties to confluent CHO-JAM cell monolayers under static conditions.
The latter finding was supported by a report demonstrating that CHO cells expressing murine JAM-1 do not aggregate in suspension (Martìn-Padura et al., 1998). Possible explanations for this observation could be changes in intracellular complexes involving JAM-1 and regulatory cytoplasmic proteins, and/or alteration in the extracellular JAM-1 conformation due to the cell detachment. A similar phenomenon has been demonstrated for the adherens junction (AJ) protein VE-cadherin, which is unable to promote aggregation of detached CHO cells (Breviario et al., 1995). However, this may not be relevant in this case since CHO cells in suspension expressing the homologous family member JAM-2 have been demonstrated to bind specifically to immobilized JAM-2–Fc chimera, thus identifying homophilic interactions in trans exerted by human JAM-2 (Cunningham et al., 2000). The results demonstrated here were also in
110 Discussion
accordance with findings that recombinant mouse as well as human JAM-1 Fc chimeras did not mediate homophilic adhesion to JAM-1 expressed by transfected COS or CHO cells in solid phase adhesion assays (Williams et al., 1999; Cunningham et al., 2000). Furthermore, they were consistent with the idea that a homophilic interaction of JAM-1 molecules may arise preferentially in regions of cell-cell contacts that exhibit a sufficiently high JAM-1 density, e.g. at intercellular junctions. However, the homophilic JAM-1 binding investigated under the experimental conditions of adhesion assays could be of relatively low affinity. Although there was no clear evidence for the involvement of such a type of molecular interaction in the cellular arrest of lymphoid Jurkat cells on CHO-JAM transfectants or of CD4+CD45RO+ T cells on HUVECs (as described below), it cannot be ruled out that during leukocyte recruitment JAM-1/JAM-1 interactions in trans may occur that are only of low stringency and therefore difficult to detect. Indeed, JAM-1–transfected CHO cell monolayers were shown to facilitate adhesion of freshly isolated human platelets (Naik et al., 2001), presumably via homophilic interactions, although other heterophilic receptors on the surface of platelets have not been excluded, e.g. by blocking antibodies. Alternatively, anti–JAM-1 may not have an inhibitory effect on homophilic interactions. With regards to the blocking properties of anti–JAM-1 in LFA-1–mediated leukocyte adhesion and transmigration, the epitope targeted by this antibody is likely to be located in the LFA-1–binding membrane-proximal Ig-like domain 2 rather than in the N-terminal region that is implicated in homophilic JAM-1 interactions (Bazzoni et al., 2000a; Kostrewa et al., 2001).