CD11c/CD18 binds selectively to peptides derived from the ICAM-4 sequence. (A) A total of 56 overlapping synthetic peptides 15 amino acid-long that corresponded to the protein sequence of the extracellular part of ICAM-4 were synthesized as immobilized spots on a cellulose membrane. The reactivities of the peptides with purified CD11c/CD18 integrin were tested. The CD11c/CD18 mAb CBRp150/4G and peroxidase-conjugated rabbit antimouse antibody were used to detect bound CD11c/CD18 integrin. The figure shows the reactivity of the overlapping peptides selected with the soluble CD11c/CD18 integrin. Negative control was carried out in the absence of the integrin. According to these results 2 peptides were chosen for solid-phase synthesis: P-D1 derived from the ICAM-4 Ig-like domain 1 and P-D2 derived from the domain 2. Both of the peptides included an amino acid shown to be involved in adhesion to CD11c/CD18 according to our mutational studies (see Figure 4). (B) The adhesion of the CD11c/CD18 transfectants to biotinylated versions of the selected ICAM-4 peptides and a control peptide (Pcontr2) captured by streptavidin microplates. Indicated amounts of the peptides were coated per well, and the cell adhesion was performed as described in “Materials and methods.” Sequences of the synthesized ICAM-4–derived peptides and the peptides used as control are listed in the figure. (C) The effects of the defined ICAM-4 peptides and the control peptide on adhesion of the CD11c/CD18 transfectants to coated ICAM-4Fc are shown. The cells were pretreated or not with the control peptide (Pcontr1), P-D1, P-D2 or the P-D1 and P-D2 peptides together at different final concentrations. A representative binding experiment with results expressed as the percentage of input cells bound are shown. Control peptides (▪), P-D2 (▴), P-D1 (○), P-D1 + P-D2 (◊).