Figure 5
KIM-1 mediates phagocytosis of apoptotic necrotic cells but not other phagocytotic targets, zymosan or latex beads.
(A) Graph showing percentage of KIM1-PK1 or pcDNA-PK1 cells that have internalized fluorescent apoptotic LLC-PK1 cells after 1 hour incubation with apoptotic fluorescent green labeled cells at 37°C or 4°C (on ice). At 4°C, binding but not internalization occurs. KIM1-PK1 cells showed much less fluorescence at 4°C than at 37°C (**P = 0.006; n = 3 per condition; error bars indicate SD). (B) Graph showing phagocytosis (black bars) by KIM1-PK1 cells as assessed by flow cytometry (left axis; percentage fluorescent cells) or binding plus phagocytosis (white bars) as assessed by spectrophotometry (right axis; relative fluorescence intensity). Labeled apoptotic thymocytes were incubated with KIM1-PK1 cells that had been pretreated with cytochalasin D (30 μM), nocodazole (30 μM), or vehicle. Total (bound plus phagocytosed) thymocytes were equivalent in each group; however, phagocytosis was inhibited by cytochalasin D and nocodazole. (C) Fluorescence images of KIM1-PK1 cells following coincubation with fluorescence-labeled zymosan particles (left, 0.5 mg/ml), latex beads (center, Fluorosphere; 1:800 dilution), or heparin (right, 25 μg/ml) for 1 hour at 37°C. Note no uptake of any of these particles. (D) Preincubation of KIM1-PK1 cells with anti–Kim-1 antibodies (AWE2), but not IgG, reduced phagocytic index (number of apoptotic LLC-PK1 cells/KIM1-PK1 phagocyte) as assessed by flow cytometry (50 μg/ml, left panel). Right panels show photomicrographs of KIM1-PK1 cells after internalization and binding of CMFDA-labeled apoptotic LLC-PK1 cells that had been pretreated (1 hour) with soluble mKIM1-Fc (bottom right panel, 0.8 μg/ml) or IgG-treated (top right panel). Pretreatment with mKIM1-Fc inhibited phagocytosis. Original magnification, ×40 (C); ×10 (D).
