In this study we have identified and cloned a new member of the IgSF that localizes at intercellular junctions in the epithelium and endothelium of different origins. For its type of cellular distribution and its adhesive properties (see below), we propose for this newly described protein the name JAM, junctional adhesion molecule.
At a molecular level, JAM is a type I integral membrane protein and presents, at the extracellular region, two domains with intrachain disulfide bonds typical of Ig loops of the V-type. Searches of available DNA and protein data bases revealed no direct sequence similarities with any known protein with the exception of A33 antigen, which is a recently cloned intestine-specific Ig protein (30
). The V-V is a novel arrangement of Ig domains, and JAM is not a member of any subfamily of the IgSF (8
The distribution of two anti JAM mAbs (BV11 and BV12) in cultured endothelial and epithelial cells shows strict localization at cell–cell contacts. At confocal and immunoelectron microscopy in cell culture and in vivo sections, JAM is found to be concentrated at the apical region of intercellular junctions in correspondence to TJ. In addition, codistribution with TJ markers such as cingulin or occludin can be observed.
JAM cDNA transfection into CHO cells leads to localization of the protein at cell–cell contacts. This localization is apparent only in confluent monolayers and when neighboring cells express JAM. In mixed cultures when JAM transfectants are in contact with control transfectants, the protein remains diffuse, indicating that JAM clustering is due to homotypic interaction.
JAM expression reduces the paracellular permeability of cell monolayers, suggesting that this molecule promotes cell–cell adhesion. In the same experimental system, other molecules known to mediate homophilic adhesion such as VE- or N-cadherin induce a similar reduction in paracellular permeability when transfected in CHO cells. This effect seems specific since transfection of a truncated mutant of VE-cadherin that was expressed on the cell membrane with an efficiency comparable to that of the wild-type form (39
) was ineffective.
In contrast to these findings, in preliminary experiments JAM-CHO transfectants did not aggregate in suspension (not shown). A possible explanation for this discrepancy is that detachment of the cells could alter JAM conformation and/or cause a slight digestion of biologically active epitopes so that the molecule loses its adhesive properties. Interestingly, VE-cadherin presents a similar behavior since it is poorly active in promoting homotypic aggregation of cells in suspension, but significantly reduces paracellular permeability (10
). For VE-cadherin, the effect on permeability requires its anchorage to catenins and the actin cytoskeleton since a truncated mutant of the molecule is ineffective (39 and the present paper). We still do not know whether JAM is linked to the cell cytoskeleton or whether this association is required for its adhesive activity. If this requirement for JAM association to the cytoskeleton is the case, it is possible that when cells are in suspension and the organization of the cytoskeleton is modified, JAM activity is inhibited.
The distribution of JAM at TJ tempts us to speculate that this protein participates in the assembly and organization of these structures, and directly interacts with TJ components. However, further studies are required to prove this possibility directly.
When compared with occludin, JAM presents a different biological behavior. It has been reported that transfected occludin concentrates at intercellular junctions and confers adhesion mostly in cells that already express organized AJ or TJ (6
). JAM seems to act independently from preorganized junctional structures. The CHO cells used in this work do not express cadherins (10
), and do not present organized TJ at electron microscopy (I. Martìn-Padura, unpublished results). However, when transfected with JAM, the cells are able to concentrate this protein at intercellular contacts. This behavior is similar to that of other junctional proteins such as PECAM or VE-cadherin. It is possible that JAM participates in the first steps of intercellular contact formation through homophilic clustering, and that only a second time gets incorporated in TJ.
A major finding of the present study is that a JAM mAb, BV11, was able to inhibit monocyte transmigration through endothelial monolayers in vitro and to block monocyte infiltration in a skin inflammatory model in vivo.
This effect was not mediated by unspecific Fc receptor binding since F(ab′) fragments of the mAb retain the activity, and since another mAb of the same isotype able to bind EC was inactive. In addition, the effect was specific for BV11 since another available mAb directed to JAM (BV12), but recognizing a different epitope (see Materials and Methods), was ineffective.
BV11 inhibited not only spontaneous monocyte transmigration but also transmigration induced by adding monocyte-specific chemokines (MCP-1 or MCP-3) or by activating the endothelial cell monolayers with LPS. This result suggests that BV11 exerts a specific and general effect on transmigration. Indeed, BV11 does not influence monocyte chemotaxis through empty filters, and does not change monocyte adhesion to endothelial cells, indicating that its activity is not due to inhibition of monocyte reactivity to chemokines or to adhesion molecules.
The data obtained in vitro using chemotaxis assays were confirmed in an in vivo model of skin inflammation. In this model the mAb BV11 was strongly effective, and essentially blocked monocyte transmigration to MCP-3. The effect of the mAb seems specific since the same dose of an irrelevant mAb of the same isotype was ineffective.
In addition, mice deficient of the complement component C1q (58
) were equally responsive to mAb BV11 (M. Romano and P. Fruscella, unpublished data), excluding in this way a role of complement activation in the observed inhibition.
The mechanism of action of mAb BV11 and of JAM in general is still unknown. A possibility is that JAM, for its localization at intercellular contacts, binds monocytes and directs their migration through the intercellular cleft. Other proteins such as the matrix protein hevin have been suggested to modulate transendothelial migration of lymphocytes by facilitating their motility (27
). A similar mechanism has also been hypothesized for another junctional immunoglobin such as PECAM-1, which is located in a less apical domain of the interendothelial cleft (5
). PECAM is unable to induce leukocyte adhesion, but appears to be required for their extravasation (38
). It is possible that these two proteins collaborate in promoting cell movement through the interendothelial junctions by acting at different regions of the intercellular cleft.
An important question is related to JAM counterreceptor on monocytes. mAb BV11 does not recognize monocytes, and does not change endothelial cell or JAM-CHO transfectant paracellular permeability, strongly suggesting that this mAb does not exert its activity by inhibiting homotypic binding. It is conceivable that monocytes recognize this protein through a heterotypic type of interaction inhibited by mAb BV11. Other immunoglobulins, including PECAM, have been found to express both homophilic and heterophilic types of interactions (32
In conclusion, in this paper we report the identification and cloning of a new member of the immunoglobulin family with a so far undescribed V-V structure that we called JAM. This protein concentrates at endothelial and epithelial junctions, and codistributes with TJ components. The mAb BV11 directed to JAM inhibits monocyte transmigration in vitro and in vivo, strongly suggesting that this molecule is relevant in the control of monocyte infiltration in inflammatory conditions.
The mechanism of action of JAM requires further investigation, but the data reported strongly support the concept that manipulation of the molecular organization of endothelial junctions could be an effective approach in controlling endothelial permeability and monocyte traffic.