Soluble MD-2 enhances the phagocytosis of Gram-negative bacteria by polymorphonuclear neutrophils. Fluorescent E coli were incubated with freshly isolated human circulating leukocytes for 5, 10 and 20 minutes in 2% plasma or in the presence of absence of 10 or 20 μg/mL of recombinant human soluble MD-2 in albumin 0.1%. Polymorphonuclear neutrophils were gated by FACS according to their forward and side-scatter characteristics and CD14 expression. At the end of incubation times, extracellular fluorescence was quenched using Trypan blue. The percentage of cells becoming fluorescent (E coli internalization) was determined using another gate (M1). The geometric mean of cells becoming fluorescent was also measured. Cytochalasin D was used as an inhibitor of phagocytosis. Representative experiments of 4 similar experiments performed with 3 different blood donors. (A) Flow cytometric analysis of fluorescent E coli phagocytosis by human polymorphonuclear neutrophils. (B) Time course of phagocytosis of fluorescent E coli by polymorphonuclear neutrophils expressed as a phagocytic index (PI). The PI was defined as follows: (geometric mean of fluorescence of m1-gated cells x percent positive cells)/100. 0.1% human serum albumin (HSA) only (no sMD-2), ■; 10 μg/mL sMD-2 in 0.1% HSA, ○; 20 μg/mL in 0.1% HSA, ▴; 2% human plasma, ◇; 10 μg/mL sMD-2 in 0.1% HSA and cytochalasin D, □.