Although ICAM-3 is implicated in both adhesion and signal transduction events of leukocytes, its low affinity for LFA-1 compared to other ligands of LFA-1 has puzzled many investigators. Here we investigated the role of ICAM-3 in supporting LFA-1-mediated ICAM-1 binding and subsequently cell signaling. We observed that although ICAM-3 binds poorly to LFA-1 expressed on resting T cells, it specifically facilitates and increases LFA-1-mediated adhesion to the high affinity ligand of LFA-1, ICAM-1. We demonstrate that low-affinity binding of LFA-1 to ICAM-3 together with ICAM-1 alters the cell surface distribution of LFA-1 dramatically, inducing large clusters of LFA-1 that facilitate ICAM-1 binding after LFA-1 activation. We found that LFA-1-mediated ICAM-1 cell-cell interactions such as T cell proliferation greatly depend on low affinity LFA-1/ICAM-3 interactions that enhance stable LFA-1/ICAM-1 cell-cell contact. Taken together, these data demonstrate that low affinity LFA-1 binding to ICAM-3 regulates strong LFA-1/ICAM-1-mediated adhesion by driving LFA-1 into clusters to facilitate cell-cell interactions that take place in the immune system.

The integrin leukocyte function-associated antigen 1 (LFA-1) is pivotal for cell adhesion and signalling within the immune system. Here, Minke Binnerts and Yvette van Kooyk discuss evidence from mutational and crystallographic studies showing that binding of LFA-1 to its ligands intercellular adhesion molecule 1 (ICAM-1), -2 and -3 might be structurally distinct.

We have identified amino acid residues within the evolutionarily conserved I domain of the alpha-chain (CD11a) of the leukocyte integrin leukocyte function-associated antigen (LFA) 1 that are critical for intercellular adhesion molecule (ICAM) 3 (CD50) binding. ICAM-3, a ligand of LFA-1, is thought to mediate intercellular adhesion essential for the initiation of immune responses. Using a panel of human/murine I domain chimeras and point mutants, we observed that the Ile-Lys-Gly-Asn motif, located in the NH2-terminal part of the CD11a I domain, is required for ICAM-3 but not ICAM-1 binding. These findings demonstrate that the I domain of CD11a contains distinct functional subdomains for ligand specific binding. An aspartic acid located at position 137, which is essential to ICAM-1/LFA-1 interactions (Edwards, C.P., M. Champe, T. Gonzalez, M.E. Wessinger, S.A. Spencer, L.G. Presta, P.W. Berman, and S.C. Bodary. 1995. J. Biol. Chem. 270:12635-12640), was also critical for ICAM-3 binding, whereas Ser at position 139 did not effect ICAM-1 or ICAM-3 binding. A synthetic peptide containing the Ile-Lys-Gly-Asn motif inhibited ICAM-3-dependent adhesion and proliferation of T cells at micromolar concentrations, suggesting that this peptide interferes with immune recognition. These observations underscore the importance of ICAM-3 in leukocyte function, and may lead to development of a new category of immunosuppressive agents.

Several studies indicate that the I domain located in the alpha chain (CD11a) of leukocyte function-associated antigen-1 (LFA-1; CD11a/CD18) plays an essential role in ligand recognition. me recently identified three distinct epitopes (IdeA, IdeB, and IdeC) within the CD11a I domain, recognized by antibodies that block binding of LFA-1 to intercellular adhesion molecules (ICAM) 1, 2, and 3. In the present study, we used a series of human/ murine CD11a I domain chimeras, to localize a fourth I domain epitope (IdeD), recognized by three independently derived anti-CD11a antibodies that selectively block the binding of LFA-1 to ICAM-3, but not to ICAM-1. The IdeD epitope depended on human CD11a residues Asp(182) and Ser(184) and was not present in CD11b or CD11c. Although mutation of Asp(182) and Ser(184) failed to abolish ICAM-3 adhesion of LFA-1 transfectants, alignment of these residues with the crystal structure of the CD11a I domain suggested that the IdeD epitope is located in close proximity to residues (Ile(126) and Asn(129)) recently implicated in the ICAM-3 binding site (1). Interestingly, the IdeB and IdeC epitopes appeared to be in close proximity of a divalent cation binding pocket within the CD11a I domain that regulates both ICAM-1 and ICAM-3 adhesion. Taken together, these data indicate that distinct regions of the CD11a I domain contain epitopes for antibodies that either selectively inhibit binding of LFA-1 to ICAM-3, or interfere with both ICAM-1 and ICAM-3 binding of LFA-1.

LFA‐1 (CD11a/CD18) mediates leukocyte adhesion by binding to one of its ligands: ICAM‐1, ICAM‐2 or ICAM‐3. Here, we investigated whether stimuli known to induce adhesion to ICAM‐1 were also capable of inducing LFA‐1‐mediated adhesion of T lymphocytes to ICAM‐2 and ‐3 transfectants. We observed that phorbol 12‐myristate 13‐acetate, Mn 2+ , cross‐linking of CD3 or activating antibodies against LFA‐1 enhanced LFA‐1‐mediated T cell adhesion to ICAM‐2 and ‐3, although to a lesser extent than to ICAM‐1. These results indicate that, similar to what has been reported for adhesion to ICAM‐1, activation of LFA‐1 is also required for adhesion to ICAM‐2 and ‐3. Furthermore, the results suggest that ICAM‐1 is the major ligand for LFA‐1 on activated T lymphocytes. Interestingly, we observed that in contrast to activating antibodies against CD18, activating antibodies against CD11a were incapable of inducing adhesion of LFA‐1 to all three ligands. The antibody MEM‐83 stimulated binding to ICAM‐1, while at the same time inhibiting the interaction of LFA‐1 with ICAM‐2 and ‐3. The antibody NKI‐L16 selectively induced adhesion to ICAM‐1 and ‐2, but not to ICAM‐3. Our results suggest that different conformations of LFA‐1 are required to support adhesion to ICAM‐1, ‐2 or ‐3, and that ligands may bind on different sites of the LFA‐1 molecule.