Cytochalasin B III. Inhibition of Human Polymorphonuclear Leukocyte Phagocytosis 1

Abstract

Cytochalasin B (CB), a metabolic product of Helminthosporium dematioideum has been shown to inhibit cytoplasmic division of mammalian (1-4) and Xenopus laevis eggs (5). Nuclear division proceeds normally in cells in the presence of CB. This results in disassociation of cytoplasmic and nuclear division and multinucleated cells are produced (1). Cytoplasmic division (cytokinesis) can be thought of as occurring in three phases, i.e., division of the nucleus, furrowing of the membranes, and fusion of membranes. It has been demonstrated that nuclear division proceeds normally (1-4) and furrowing is not affected (5) in the presence of CB. Therefore, fusion of membranes seems to be the target of drug action. Since membrane fusion also occurs in the process of phagocytosis we were interested in testing the effect of CB on this function of polymorphonuclear leukocytes. We report here the inhibition of bacterial phagocytosis by human polymorphonuclear leukocytes in the presence of Cytochalasin B.

Materials and Methods. Cytochalasin B. The CB was stored in a stock solution of 1 mg/ml in dimethylsulfoxide (DMSO). Dilutions were made in Hank's balanced salt solution (BSS). Phagocytosis assays utilized a modified Maal⊘e method previously described (6, 7). This consisted, essentially, of mixing 5 × 10⁶ human leukocytes, separated from venous blood by dextran sedimentation and differential centrifugation with bacteria at a 1:1 bacteria:leukocyte ratio. Leukocytes and bacteria were suspended in the BSS containing 0.1% gelatin. Opsonin was provided by adding pooled adult human serum at a final concentration of 10%. A total volume of 1 ml was incubated in 12-75-mm plastic tubes (Falcon Plastics) and rotated end over end at 10 rpm on a Roto rack (Fisher Scientific Co.). The phagocytic mixtures were sampled immediately after mixing and after 30-, 60-, and 120-min incubation by placing 0.001 ml in 1 ml of distilled water and adding 0.1 ml to Penassay agar (Difco Labs) for pour plates and colony counts.