The Functional Interaction of the β2 Integrin Lymphocyte Function-Associated Antigen-1 with Junctional Adhesion Molecule-A Is Mediated by the I Domain

Line Fraemohs, Rory R. Koenen, Georg Ostermann, Bo Heinemann and Christian Weber
J Immunol November 15, 2004, 173 (10) 6259-6264; DOI: https://doi.org/10.4049/jimmunol.173.10.6259

Abstract

Binding of the β2 integrin LFA-1 (αLβ2) to junctional adhesion molecule-A (JAM-A) has been shown to enhance leukocyte adhesion and transendothelial migration. This is mediated by the membrane-proximal Ig-like domain 2 of JAM-A; however, the location of the JAM-A binding site in LFA-1 has not been identified. We have deleted the I domain in the αL subunit of LFA-1 and expressed this αL mutant in αl-deficient Jurkat J-β2.7 cells to demonstrate that the I domain of LFA-1 is crucial for their adhesion to immobilized JAM-A. This was substantiated by blocking the stimulated adhesion of wild-type Jurkat T cells or monocytic Mono Mac 6 cells to JAM-A using the I domain-directed mAb TS1/22 or the small molecule antagonist BIRT 377, which stabilizes the low-affinity conformation of the I domain. The immobilized LFA-1 I domain locked in the open high-affinity conformation was sufficient to support binding of transfected Chinese hamster ovary cells expressing JAM-A. Solid-phase binding assays confirmed a direct interaction of recombinant JAM-A with the immobilized locked-open I domain. These data provide the first evidence that the I domain of LFA-1 contains a functional binding site for JAM-A.

Leukocyte emigration from the bloodstream and into a site of tissue inflammation involves the sequential action of traffic signal and adhesion molecules mediating rolling, arrest, and diapedesis (1). The interaction between the β2 integrin LFA-1 (αLβ2, CD11a/CD18) and its ligands, the ICAMs 1–3, plays a critical role in leukocyte adhesion. It has been shown that junctional adhesion molecule-A (JAM-A, 5 formerly named JAM-1, (2)) serves as an additional ligand for LFA-1 (3). JAM-A, which is an Ig superfamily member that contains two Ig-like folds in the extracellular domain, is expressed on leukocytes (4) and at tight junctions of endothelial and epithelial cells, where homophilic JAM-A interactions promote cell-cell contact (5) and the organization of the junctional complex (6). Robust evidence has been provided for a key role of JAM-A in leukocyte transmigration (5) and inflammatory extravasation (7). Inhibition of transmigration with the mAb BV11 that specifically recognizes JAM-A homodimers implicated homophilic interactions involved in this process (8). In contrast, transmigration of human leukocytes has been shown to involve heterophilic interactions of JAM-A with its integrin receptor LFA-1 (3).

Like for other β2 integrins, the α subunit of LFA-1 contains an inserted domain (I domain), which has been implicated as the principal ligand-binding domain for interaction with the ICAMs (9, 10). The I domain consists of ∼200 aa residues and is inserted between β sheet 2 and 3 in the predicted β propeller of the α subunit (11). It possesses a dinucleotide-binding domain with a central open twisted β-sheet surrounded by α helices and a Mg2+/Mn2+ binding site termed the metal ion-dependent adhesion site on the ligand-binding face (12). Two different conformations of the αL I domain, open and closed, have been reported (12, 13). The open conformation represents the high-affinity state, whereas the closed conformation is the ligand-free, low-affinity state. Locking the I domain in the open conformation by the introduction of disulfide bonds resulted in 9000-fold increased affinity to ICAM-1, which could be reversed by disulfide reduction (13).

I domain-deleted LFA-1 is unable to bind its ligands ICAM-1 and ICAM-3 (10, 14). In contrast, I domain-deleted Mac-1 (αMβ2, CD11b/CD18) can bind at reduced levels to some Mac-1 ligands, including factor X and iC3b, but not fibrinogen. The β propeller domain of the αM subunit of Mac-1 seems to be important for these interactions, since binding could be inhibited with a mAb to the β propeller domain of αM (10).

The interaction between LFA-1 and its ligand JAM-A has been found to depend on the membrane-proximal Ig-like domain 2 of JAM-A (3); however, the location of the JAM-A binding site in LFA-1 has not been defined. Using I domain deletion mutants of αL and I domain-specific inhibition, we demonstrate here that the I domain of LFA-1 is sufficient and required for functional interaction of LFA-1 with JAM-A.

Materials and Methods

Abs, reagents, and cell culture

mAb G25.2 (anti-αL, non-I domain specific, IgG2a) was obtained from BD Biosciences (San Jose, CA). mAb TS1/22 (anti-αL, I domain specific, IgG1) was purified from hybridoma supernatants by protein G affinity chromatography. Murine polyclonal Ab against JAM-A was as previously described (3), and a JAM-A mAb (clone M.Ab.F11) was purchased from Serotec (Oxford, U.K.). The LFA-1 antagonist BIRT 377 (15) was kindly provided by Dr. T. Kelly (Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT). PMA was obtained from Calbiochem (San Diego, CA). The fluorescent dye 2′,7′-bis-(2-carboxyethyl)-5-(or 6)-carboxyfluorescein acetoxymethyl ester (BCECF/AM) was purchased from Molecular Probes (Eugene, OR). Jurkat and J-β2.7 cells (16), Mono Mac 6 cells (17) and Chinese hamster ovary (CHO) cells (3) were maintained as described elsewhere.

I domain-deleted αL cDNA construct

An αL deletion mutant lacking the I domain (N129-K304) was produced by PCR using wild-type (wt) αL cDNA subcloned into the expression vector pAprM8 as template. Using the primers 5′-CAGTACATCAATGGGCGTGGATAG-3′ (pAprM8) and 5′-CGTTCGTAGATCTTGCCCTTGATACATTC-3′ (αL), a PCR fragment was amplified encoding an αL fragment upstream of the I domain (residues 1–128) including a 3′ BglII cloning site (underlined). The wt αL in pAprM8 was digested with HindIII and BglII to remove a fragment encoding for residues 1–304 of αL and, instead, the PCR fragment encoding for residues 1–128 was inserted as a HindIII/BglII fragment. Finally, the cDNAs encoding wt αL and the I domain-deficient mutant αLΔI were subcloned into the expression vector pcDNA3 (Invitrogen Life Technologies, Carlsbad, CA), which confers resistance to G418, and the constructs were sequenced to exclude mismatches.

J-β2.7 transfectants and flow cytometry

Stable transfection of αl-deficient Jurkat J-β2.7 cells (16) was generated as described previously (18). After electroporation of 107 cells with 10 μg of cDNA (αL, αLΔI, or vector), cells were cultured for 48 h under nonselective conditions. Transfected cells were selected with G418 sulfate (Calbiochem) at 0.8 mg/ml. G418-resistant cells were subcloned by limiting dilution in 96-well plates and analyzed by flow cytometry with mAb G25.2 to assess the level of LFA-1 expression. For FACS analysis, cells were incubated with specific Ab or mouse IgG isotype control for 30 min, washed, and stained with FITC-conjugated goat anti-mouse IgG mAb (Sigma-Aldrich, St. Louis, MO). Surface expression of proteins was analyzed immediately by flow cytometry in a FACSCalibur (BD Biosciences, San Jose, CA). Clones with comparable levels of LFA-1 expression were used in all studies.

Generation and purification of soluble proteins

Soluble JAM-A.Fc protein was stably expressed in CHO cells as a chimeric protein containing the extracellular part of human JAM-A fused to the Fc portion of human IgG1 as described elsewhere. 6 JAM-A.Fc protein was purified from the CHO cell supernatant by protein A affinity and anion exchange chromatography (HiTrap Protein A, MonoQ; Amersham Biosciences, Piscataway, NJ). Soluble ICAM-1.Fc was generated accordingly. Locked-open αL I domain (G128–Y307) in expression vector pET26b (Novagen, Madison, WI) was constructed by standard molecular biology techniques. In the locked-open αL I domain, the double mutation K287C K294C was introduced. I domain was expressed in Escherichia coli BL21(DE3), purified, and refolded as described previously (19). For refolding of the locked-open I domain, 0.1 mM CuSO4 and 0.1 mM o-phenanthroline were added to catalyze oxidative disulfide bond generation.

Static cell adhesion assay

Adhesion assays under static conditions were performed as described previously (3), with the following modifications. Soluble JAM-A.Fc or ICAM-1.Fc at 15 μg/ml was immobilized overnight at 4°C in 96-well plates (Nunc, Roskilde, Denmark) coated with Fc-specific anti-human IgG (Sigma-Aldrich) at 20 μg/ml. Nonspecific sites were blocked with 0.5% BSA and BCECF/AM-labeled Jurkat cells or J-β2.7 transfectants in HHMC (HEPES-buffered HBSS, 1 mM Mg2+/1 mM Ca2+, 0.5% BSA) were allowed to adhere for 30 min at 37°C. Cells (1.5· 105 cells/well) were assayed for binding in the absence or presence of PMA (100 ng/ml) or 3 mM Mg2+/1 mM EGTA. Some cells were preincubated with TS1/22 (10 μg/ml) or BIRT 377 (10 μM) for 20 min at room temperature. Nonadherent cells were removed by a flick wash. Briefly, wells were repeatedly filled with HHMC and emptied by rigorously inverting the plate. The fluorescence of input and adherent cells was determined with a fluorescence plate reader (SpectraFluor Plus; Tecan, Crailsheim, Germany). Background binding to anti-human IgG was negligible and subtracted. Adhesion of monocytic Mono Mac 6 cells to immobilized JAM-A.Fc was performed accordingly with the following modifications: monocytes were preincubated for 20 min with 5% heat-inactivated human serum to block Fc receptors and 0.5 × 105 cells/well were used.

CHO transfectants and static adhesion to locked-open αL I domain

Adhesion assays under static condition were performed as described elsewhere (3) using CHO cells with stable expression of human JAM-A or transfected with vector control. Briefly, locked-open αL I domain or BSA as control was immobilized in 96-well Nunc Maxisorp plates at 15 μg/ml in 10 mM Tris (pH 9.0) overnight at 4°C. After blocking with 0.5% BSA, BCECF/AM-labeled CHO transfectants in HHMC were allowed to adhere for 30 min at 37°C. Some wells were preincubated with BIRT 377 (10 μM) or 10 mM DTT. Nonadherent cells were removed by a flick wash as described above. Background binding to BSA was negligible and subtracted.

Solid-phase binding assay

Ninety-six-well plates (Costar, Cambridge, MA) were coated with locked-open LFA-1 I domain (2 μg/ml) in 100 mM sodium carbonate buffer (pH 9.0)/10 mM Mg2+ and blocked with 3% BSA/10 mM Mg2+. Binding of JAM-A.Fc (0–2 μM) to immobilized I domain was performed in a total volume of 50 μl of TBS/0.5% BSA supplemented with either 2 mM Mg2+ or 2 mM EDTA. After washing, bound JAM-A.Fc was detected with peroxidase-conjugated anti-human Fc Ab (Sigma-Aldrich). Bound Ab was quantified using the tetramethylbenzidine substrate reagent kit (BD Pharmingen, San Diego, CA). Nonspecific binding to BSA-coated wells was subtracted to calculate specific binding. Normalized data were fitted to a simplified single site binding model using nonlinear regression as described previously (20).

Results

Expression of I domain-deleted LFA-1 in Jurkat J-β2.7 cells

The I domain of the αL subunit of LFA-1 is not essential for folding, heterodimerization with the β2 subunit, or surface expression (10). This allows investigation of its function by expression of an I domain-deleted mutant of the αL subunit of LFA-1. The I-less αL subunit, termed αLΔI, lacked the sequence N129-K304 (Fig. 1⇓A) with the length of the deletion based on x-ray crystal structures of αL (12). No linker sequence was added, since the N and C termini of the I domain are in close proximity (11). The cDNA constructs for αLΔI and wt αL were used for stable transfection of Jurkat J-β2.7 cells (16), which lack LFA-1 surface expression due to a deficiency in αL. Clones with comparable surface expression of αLΔI and wt αL were selected by flow cytometry (Fig. 1⇓B). The mAb G25.2, which recognizes an epitope outside the I domain, reacted equally well with the transfectants expressing αLΔI or wt αL. In contrast, the I domain-specific mAb TS1/22 bound to transfectants expressing wt αL but not αLΔI, confirming the deletion of the I domain in αLΔI. Furthermore, equivalent expression of JAM-A in transfectants was confirmed by flow cytometry (Fig. 1⇓B).

FIGURE 1.
FIGURE 1.

A, Schematic illustration of the wt αL and the I domain-deleted αL (αLΔI) subunits. Shown in gray are the β sheets W1-W7 in the predicted β propeller domain. The I domain of αL is inserted between β sheets W2 and W3. TM, Transmembrane region. In αLΔI the deletion includes residues N129–K304. B, Expression of JAM-A and αL epitopes on J-β2.7 clones stably transfected with wt αL, I domain-deleted αL (αLΔI), or vector alone. Cells were stained with mAbs G25.2 (anti-αL, non-I domain specific), TS1/22 (anti-αL, I domain specific), M.Ab.F11 (anti-human JAM-A), or isotype controls, followed by FITC-conjugated goat anti-mouse IgG and analysis by flow cytometry. Filled histograms, isotype control; open histograms, mAbs G25.2, TS1/22, or M.Ab.F11. Data shown are representative histograms.

The I domain of LFA-1 is necessary for adhesion to JAM-A

The binding of J-β2.7 transfectants was tested in adhesion assays on immobilized ICAM-1.Fc. After stimulation with PMA or Mg2+/EGTA, only the transfectants expressing wt LFA-1 but not those expressing I domain-deleted LFA-1 or vector controls showed substantial binding to ICAM-1.Fc (Fig. 2⇓A). These results are in accordance with findings that the I domain of LFA-1 is the principal ligand binding site for the interaction with ICAM-1 (9, 10). To determine whether the I domain of LFA-1 is required for binding to JAM-A, the adhesion of J-β2.7 transfectants to immobilized JAM-A.Fc was analyzed. As seen on ICAM-1.Fc, the wt LFA-1 transfectants showed firm adhesion to immobilized JAM-A.Fc both after stimulation of intracellular signal transduction pathways with PMA or extracellular activation with Mg2+, whereas transfectants expressing LFA-1 without the I domain or vector controls did not show adhesion to JAM-A.Fc (Fig. 2⇓B). These results imply that the I domain of LFA-1 is required for binding to JAM-A.

FIGURE 2.
FIGURE 2.

Effect of αL I domain deletion on adhesion of J-β2.7 transfectants to ICAM-1.Fc and JAM-A.Fc. J-β2.7 transfectants expressing wt αL or the I domain-deleted mutant αLΔI were allowed to adhere to immobilized ICAM-1.Fc (A) or JAM-A.Fc (B). Cells were stimulated with 3 mM Mg2+/1 mM EGTA or PMA (100 ng/ml) for 30 min. Nonadherent cells were removed by flick wash and adhesion was measured as percentage of input cells. Data represent mean ± SEM of at least three separate experiments done in triplicate.

LFA-1 binding to JAM-A is inhibited by an I domain-directed LFA-1 antagonist or blocking mAb

To further analyze the role of the LFA-1 I domain in the interaction with JAM-A, we used the LFA-1 I domain-specific mAb TS1/22 and the small molecule LFA-1 antagonist BIRT 377, both known to inhibit LFA-1 binding to ICAM-1 (15). BIRT 377 blocks interactions of LFA-1 and ICAM-1 by binding to an epitope in the I domain comprising P281 and stabilizing the I domain in a closed low-affinity conformation (21). Indeed, preincubation of Jurkat cells with TS1/22 or BIRT 377 almost completely inhibited their adhesion to immobilized ICAM-1.Fc after stimulation with PMA or Mg2+/EGTA (Fig. 3⇓A). These results are consistent with the notion that BIRT 377 and TS1/22 prevent the interaction of LFA-1 to ICAM-1 by interfering with the I domain. To determine whether the interaction of LFA-1 with JAM-A also involves the I domain, we studied the effect of mAb TS1/22 and BIRT 377 in adhesion assays on immobilized JAM-A.Fc. The preincubation of Jurkat cells with TS1/22 or BIRT 377 indeed reduced their adhesion to JAM-A.Fc following stimulation with PMA or Mg2+/EGTA to levels observed for unstimulated cells (Fig. 3⇓B). Isotype control or DMSO (solvent of BIRT 377) had no effect (data not shown). Thus, our results show that blocking I domain function with mAb TS1/22 or the small molecule LFA-1 antagonist BIRT 377 inhibits the interaction between LFA-1 and JAM-A, further substantiating that the I domain of LFA-1 is crucial for binding to JAM-A.

FIGURE 3.
FIGURE 3.

Interaction between LFA-1 and JAM-A is attenuated by LFA-1 antagonist and mAb TS1/22. Adhesion of Jurkat cells to immobilized ICAM-1.Fc (A) or JAM-A.Fc (B) and adhesion of monocytic Mono Mac 6 cells to JAM-A.Fc (C) after stimulation with 3 mM Mg2+/1 mM EGTA or PMA (100 ng/ml). Some cells were preincubated with BIRT 377 (10 μM) or TS1/22 (10 μg/ml) for 30 min before adding to the plate. Nonadherent cells were removed by flick wash and adhesion was measured as percentage of input cells. Preincubation with DMSO or isotype control had no significant effect on binding (data not shown). Data represent mean ± SEM of at least three separate experiments done in triplicate.

To analyze whether interactions of JAM-A and LFA-1 also occur in leukocytes other than lymphocytes, e.g., monocytes, we used monocytic Mono Mac 6 cells in static adhesion assays on immobilized JAM-A.Fc. As seen with Jurkat cells, Mono Mac 6 cells showed adhesion to JAM-A.Fc after stimulation with Mg2+ or PMA (Fig. 3⇑C). As revealed by an almost complete inhibition with a blocking LFA-1 mAb or with the I domain antagonist BIRT 377 (Fig. 3⇑C), this binding was dependent on LFA-1 and mediated by the I domain rather than homophilic interactions of JAM-A.

Recombinant isolated locked-open LFA-1 I domain binds JAM-A

The recombinant LFA-1 I domain has previously been shown to support binding to its ligand ICAM-1 (13). We therefore hypothesized that the isolated LFA-1 I domain is sufficient for binding to JAM-A. LFA-1 on resting cells does not bind spontaneously to its ligands, but needs to assume an open conformation as a result of stimulation. To study the interaction between isolated locked-open I domain and JAM-A, we purified and refolded a recombinant form of the LFA-1 I domain, which was locked in the open high-affinity conformation by the introduction of a disulfide bond to achieve maximum affinity (13). CHO cells were stably transfected with JAM-A (CHO/JAM-A) or vector control (CHO/vector). Using a polyclonal JAM-A Ab, surface expression of JAM-A was detected on CHO/JAM-A but not CHO/vector transfectants by flow cytometry (data not shown). In adhesion assays on immobilized substrate, CHO transfectants expressing JAM-A but not vector controls showed substantial binding to the locked-open I domain (Fig. 4⇓A). Preincubation of the locked-open I domain with LFA-1 antagonist BIRT 377 did not inhibit binding (Fig. 4⇓B). This reflects the fact that BIRT 377 binds to the closed (but not to the open) conformation of the LFA-1 I domain and prevents conversion to the high-affinity conformation (21). To confirm that JAM-A only binds to the activated I domain, the disulfide bond in the locked-open I domain was reduced with DTT. This indeed diminished the adhesion of CHO/JAM-A transfectants to background levels (Fig. 4⇓B). Our data reveal that the isolated LFA-1 I domain is sufficient to support binding to JAM-A and that this binding was specifically mediated by the open conformation of LFA-1.

FIGURE 4.
FIGURE 4.

CHO transfectants expressing JAM-A binds to locked-open αL I-domain. A, Adhesion of CHO/vector and CHO/JAM-A transfectants to immobilized locked-open αL I domain. B, Adhesion of CHO/JAM-A transfectants to immobilized locked-open αL I domain. Some wells were preincubated with BIRT 377 (10 μM) or 10 mM DTT. Data represent mean ± SEM of at least three separate experiments done in triplicate.

Biochemical evidence for a direct interaction of JAM-A with the LFA-1 I domain

Since the functional results from cell-based adhesion studies suggested a direct molecular interaction between JAM-A and the LFA-1 I domain, we performed solid-phase binding assays with recombinant and purified proteins to demonstrate the binding of soluble JAM-A.Fc to the immobilized locked-open LFA-1 I domain. Soluble JAM-A.Fc bound to the high-affinity form of the I domain was detected by HRP-conjugated anti-Fc Ab. The experiments indeed confirmed that soluble JAM-A directly bound to locked-open I domain and further revealed that the binding was dose dependent and saturable with a dissociation constant (Kd) of ∼0.5 μM, as indicated by fitting of the binding curve (Fig. 5⇓). The interaction was cation dependent, since binding could be almost completely inhibited by addition of EDTA (Fig. 5⇓).

FIGURE 5.
FIGURE 5.

Dose-dependent binding of recombinant JAM-A.Fc to immobilized locked-open LFA-1 I domain. Specific binding of JAM-A.Fc at indicated concentrations was analyzed in the presence of either 2 mM Mg2+ or 2 mM EDTA. Bound JAM-A.Fc was detected with HRP-conjugated anti-human Fc Ab. The fraction of bound ligand is given as mean ± SEM of three to five separate experiments. Solid lines represent a nonlinear fit of the data as described in Materials and Methods.

Discussion

In this study, we have examined the role of the αL I domain in the functional interaction of LFA-1 with JAM-A. By expression of I domain-deleted αL in αL-deficient Jurkat J-β2.7 cells, we found that the I domain is required for interaction with JAM-A. Furthermore, binding of wt Jurkat cells to immobilized JAM-A could be inhibited with a blocking mAb specific for the I domain and the LFA-1 antagonist BIRT 377, which stabilizes the closed low-affinity conformation of the I domain. These data provide the first evidence that the I domain of LFA-1 contains the functional binding site for the interaction with JAM-A.

By introduction of a disulfide bond in the I domain, we have locked the αL I domain in the open conformation, shown to have high affinity for ICAM-1 (13, 22). Locked-open I domain was sufficient to mediate adhesion of CHO transfectants expressing JAM-A. Binding was specifically mediated by the open conformation, since reduction of the disulfide bond with DTT abolished adhesion. Conformational change in the αL I domain is therefore of importance not only for the interaction with ICAM-1, but also for the interaction with JAM-A. In the interaction with ICAM-1, a change in the conformation of the LFA-1 I domain has been shown to control the transition from rolling adhesion to firm adhesion, since only the low-affinity closed conformation mediates rolling, whereas the high-affinity open conformation supports firm arrest (23). This extends the importance of conformational change in the αL I domain for adhesive function in flow to proinflammatory conditions where JAM-A is expressed as a LFA-1 ligand on the apical surface of endothelial cells (24) or surface-adherent platelets.

Further evidence for an I domain-mediated interaction between LFA-1 and JAM-A was provided by solid-phase binding experiments demonstrating specific binding of soluble JAM-A.Fc to the immobilized locked-open I domain. The equilibrium dissociation binding constant for this interaction is estimated to be 0.5 μM, which is within the range of dissociation constants (0.13–0.55 μM) published for the LFA-1/ICAM-1 interaction (13, 25). The finding that the interaction between JAM-A.Fc and the immobilized locked-open I domain is dependent on divalent cation demonstrates that considerable similarity exists in the interactions of LFA-1 with JAM-A and ICAM-1, -2, and -3. Thus, a coordination of binding involving divalent metal ions appears to be the principal mechanism mediating the interaction between LFA-1 and its ligands. By analogy to the interaction between LFA-1 and ICAM-1 which requires the acidic amino acid residue E34 on ICAM-1 (26), a yet to be identified acidic residue present on JAM-A, e.g., E163 or D169, may be involved in the interaction with LFA-1.

Being strategically located at the intercellular junctions, the JAM proteins play an important role in inflammation and during diapedesis (5, 7). Homophilic JAM-A interactions between leukocytes and the endothelium may contribute to transmigration in addition to heterophilic interaction of JAM-A with its integrin receptor LFA-1 (3), possibly by a molecular zipper-like mechanism (27). To address the issue of whether JAM-A-LFA-1 interactions also occur in leukocytes other than lymphocytes, we have now performed adhesion assays using monocytic cells on purified JAM-A.Fc. The results clearly indicate that monocytes can also be stimulated to bind to JAM-A. This adhesion is comparable to that of lymphocytes, dependent on LFA-1 and mediated via the I domain, as shown by inhibition experiments. Thus, an unspecific contribution of other monocytic adhesion receptors can be excluded and the interaction of JAM-A and the LFA-1 I domain is not restricted to lymphocytes. It has been shown that the transmigration of murine neutrophils and monocytes involves JAM-A, as shown by using an inhibitory mAb (5, 7). In accordance with these data, a blocking Ab to JAM-A also inhibits transendothelial migration of neutrophils transmigration in a human system (3). Moreover, JAM-A redistribution away from the junctions does not negatively regulate transmigration of human neutrophils or monocytes (28).

The LFA-1-dependent adhesion is regulated not only by ligand affinity through conformational changes, but also by avidity as a result of cell surface clustering. Specific interactions of the αL and β2 cytoplasmic domains with components of the cytoskeleton (29), such as talin (30), or interactions with specific regulators such as cytohesin-1 (31) appear to be involved in integrin regulation. Similarly, JAM-A expressed at tight junctions of endothelial and epithelial cells interacts via the cytoplasmic PDZ-binding domain with ZO-1 and AF-6 (32), which are both directly linked to the actin cytoskeleton (33, 34). This allows JAM-A to be anchored to the actin filament system and to be involved in the reorganization of the actin cytoskeleton during leukocyte migration. Thus, the interactions of both binding partners with cytoskeletal regulators appear to be essential for a transmembrane signal transduction after cellular stimulation, e.g., with chemokines, as well as in postligand binding events.

Several reports have confirmed the importance of heterophilic interactions between members of the JAM family and integrins. Endothelial JAM-B has been shown to interact with leukocyte JAM-C but also with the integrin VLA-4 (α4β1, CD49d/CD29) (35). Interactions between JAM-C on platelets and leukocyte Mac-1 (αMβ2) may favor the formation of platelet-leukocyte aggregates (36), which may promote the development of atherothrombotic processes (37). In addition, homotypic interactions of JAM-A in trans have been implicated in the adhesion of platelets to inflamed endothelium in flow (38). Thus, JAM-A expressed on circulating platelets may play a role in inflammatory thrombosis or may contribute to the development and progression of atherosclerotic lesions. In contrast, such interactions may be involved in physiological hemostasis and the clearance of thrombotic material.

By orchestrating and stabilizing the assembly of junctional complexes, cytoskeletal and homophilic interactions of JAM-A are pivotal in maintaining vascular integrity and controlling paracellular permeability (5, 6). Besides affecting these physiological functions of JAM-A, inhibitory Abs directed against JAM-A may trigger platelet secretion and aggregation via engagement of FcRs (39). Thus, inhibitors specifically interfering with the interactions between the LFA-1 I domain and JAM-A may provide sufficient selectivity to block inflammatory recruitment and diapedesis of leukocytes without unwanted and deleterious side effects. Our findings that the functional interaction with JAM-A is mediated by the open conformation of the LFA-1 I domain may give rise to a novel target site for the development of anti-inflammatory therapies, e.g., by small molecule antagonists.

Acknowledgments

We thank M. Roller and G. Dudda for technical assistance.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This study was supported by Deutsche Forschungsgemeinschaft (Grant WE1913/2-3), State of Brandenburg, and the European Union.

  • ↵2 This work in part fulfills requirements for the doctoral thesis of L.F.

  • ↵3 R.R.K. and G.O. contributed equally to this paper.

  • ↵4 Address correspondence and reprint requests to Dr. Christian Weber, Kardiovaskuläre Molekularbiologie, Universitätsklinikum Aachen, Pauwelsstrasse 30, 52074 Aachen, Germany. E-mail address: cweber{at}ukaachen.de

  • ↵5 Abbreviations used in this paper: JAM-A, junctional adhesion molecule-A; CHO, Chinese hamster ovary; wt, wild type; BCECF/AM, 2′,7′-bis-(2-carboxyethyl)-5-(or 6)-carboxyfluorescein-acetoxymethyl ester.

  • ↵6 G. Ostermann, L. Fraemohs, T. Baltus, A. Schober, M. Lietz, A. Zernecke, and C. Weber. Involvement of junctional adhesion molecule-A (JAM-A) in mononuclear cell recruitment on inflamed or artherosclerotic endothelium: inhibition by soluble JAM-A. Submitted for publication.

  • Received May 4, 2004.
  • Accepted September 14, 2004.

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