In vitro assay of spontaneous cytotoxicity by human monocytes and macrophages against tumor cells

Abstract

The [ 3H] proline microcytotoxicity technique has been adopted to examine the spontaneous cytotoxic property of human circulating monocytes (M) derived from the peripheral blood and resident monocytes/macrophages (Mø) derived from effusion fluids. A leukocyte fraction is obtained by centrifugation in Ficoll-Hypaque (F-H) gradient. M are then isolated as adherent cells following incubation of the leukocyte fraction in plastic flasks containing 50% fetal calf serum (FCS) in culture medium. Mø are isolated from effusion fluids after first separating the Fc receptor bearing cells (as Fc receptor-7S EA resettes) in F-H gradient and then enriching them as adherent cells. The above techniques yield a M/Mø population of some 90–98% purity. The M/Mø non-selectively lyse tumor cells and allogeneic normal fibroblasts in the 48 h [ 3H] proline assay. The fibroblasts, however, exhibit considerable resistance to lysis, particularly at lower effector to target ratios. While appreciable levels of cytotoxicity are observed with resident Mø at 6 h, followed by a further increase in the level of cytotoxicity at 24–48 h, circulating M exhibit a consistent level of cytotoxicity only at 24–48 h. When circulating M and resident Mø from the same donor are examined concurrently for cytotoxicity, resident Mø are consistently found to show significantly higher levels of cytotoxicity when compared to circulating M. This may reflect a true functional heterogeneity within this lineage of effector cells or some degree of in vivo activation of resident Mø in malignant and non-malignant effusion fluids. Further studies of spontaneous cytotoxicity by M/Mø through CMC assay techniques, such as the [ 3H] proline microtoxicity technique, will be useful in the examination of the role of M/Mø in cell mediated immunity and immunosurveillance against cancer.