Adhesion to solid-phase purified ICAM-1 or rosetting with iC3b-opsonized sheep RBCs (SRBCs) by cotransfected COS-7 cells. COS-7 cells transfected with the wt, mut-1, or mut-1 β₂ subunit plus the wt LFA-1 α subunit (A) were activated with the KIM185 mAb and added (10⁵ cells) to triplicate ICAM-1-coated microtiter wells for 15 minutes at 37°C. After removing nonadherent cells by 3 gentle PBS washes, the number of cells retained in each well was directly counted by microscopy (see Figure 2). The mean value for LFA-1/wtβ₂ cells was set at 100% for each experiment, and all other values for that experiment were expressed as a percentage of that standard. As shown, binding to ICAM-1 by cells transfected with mut-1 β₂ was no different than the background level observed for untransfected cells, consistent with nearly absent LFA-1 expression (see Figure 2). Binding by cells expressing LFA-1 with mut-2 β₂ was diminished by about 50% compared with wt β₂ (P < .05; n = 4), indicating diminished functional activity of LFA-1 containing mut-2 β₂. Treatment with mAb R3.1 after activation with KIM185 completely abrogated binding to ICAM-1, confirming the LFA-1 dependence of binding. (B) SRBCs opsonized with iC3b as described in “Patients, materials, and methods” (5 × 10⁷ cells) were mixed with COS-7 cells that had been transfected with the wt Mac-1 α subunit plus the wt, mut-1, or mut-2 β₂ subunit (10⁵ COS-7 cells). After 30-minute incubation at 37°C, as described, the cell mixtures were examined by light microscopy for rosette formation, defined as at least 3 SRBCs bound to one COS-7 cell. As shown, COS-7 cells transfected with mut-1 β₂ exhibited very low levels of rosetting compared with cells transfected with wt β₂. In contrast, COS-7 cells transfected with mut-2 β₂ formed rosettes to the same extent as cells transfected with wt β₂, indicating that the mut-2 β₂ subunit had no effect on Mac-1-mediated binding. Treatment of COS-7 cells with mAb 60.1 after activation with KIM185 virtually abrogated rosetting, confirming the Mac-1 dependence of this function. Error bars indicate SEM.