Actin is present in cells in monomeric and polymeric (filamentous) forms. Filamentous actin is distributed in Triton-soluble (cytosolic) and Triton-insoluble (cytoskeletal core) fractions. We have used the DNase 1 inhibition assay and immunofluorescence to investigate the distribution of actin in monomeric and polymeric forms in cloned B16 murine melanoma cell lines of low and high metastatic capacity. The protease trypsin caused rounding up and detachment of both cell lines within 5 min. This was associated with almost complete depolymerization of cytosolic actin filaments but the Triton-insoluble cytoskeleton was not quantitatively affected by trypsin treatment. There were quantitative differences between the clones in their response to incubation in the presence or absence of 10% serum. The highly metastatic cell line contained 35% more actin when incubated in the presence of 10% serum, almost completely distributed to the Triton-insoluble cytoskeleton, an effect not seen in the low metastatic cells.

MA158.2, a rat monoclonal antibody with binding specificity for cells of the monocyte-macrophage lineage, reacts with an antigen (158.2) whose expression is enhanced on mononuclear cells activated to the tumoricidal phenotype by treatment with lymphokine supernatant containing macrophage activating factor (MAF). The functional relevance of enhanced expression of this antigen has been examined in mouse peritoneal macrophages treated with a variety of immunomodulatory agents and assayed for augmented macrophage-mediated defense reactions, including O-2 production, microbicidal, and tumoricidal activity. An interferon-gamma (IFN-gamma) preparation produced by recombinant DNA technology induced a dose-dependent increase in expression of the 158.2 antigen in inflammatory macrophages which was accompanied by acquisition of microbicidal activity against Listeria monocytogenes. However, these cells did not express tumoricidal activity and induction of this property required concomitant exposure to lipopolysaccharide (LPS). Similar results were obtained using macrophages elicited with pyran copolymer. Exposure to LPS alone induced enhanced expression of antigen 158.2 but did not elicit microbicidal activity. Macrophages challenged with IFN-alpha, IFN-beta, MDP, and bestatin did not exhibit increased 158.2 and also failed to acquire tumoricidal activity when treated concomitantly with LPS. Collectively, these data indicate that the MA 158.2 antibody recognizes an antigen expressed by macrophage populations displaying the so-called primed phenotype in which microbicidal activity is expressed but in which induction of tumoricidal activity requires the addition of a second signal such as LPS.

A hybridoma clone secreting a monoclonal antibody, designated MA158.2, that reacts with an antigen expressed on lymphokine-treated macrophages was produced by fusion of mouse myeloma cells with rat spleen cells immunized against C57BL/6 peritoneal macrophages rendered tumoricidal in vitro by incubation with the lymphokine macrophage-activating factor. The specificity of the antibody for activated macrophages and lack of reactivity with histologically diverse cell types was determined by radioimmune indirect binding and flow cytometry. MA158.2 antibody binds to mouse peritoneal macrophages elicited by nonspecific inflammatory agents and to tumoricidal macrophages elicited with Corynebacterium parvum. Resident peritoneal, splenic, and alveolar macrophages were only weakly positive. Several macrophage cell lines (P388D1, WEH1-231, J774, RAW 264.7), murine fibroblasts, and neutrophils did not bind detectable amounts of MA158.2. Radioimmune indirect binding analysis demonstrated that cell suspensions prepared from C57BL/6 mouse spleen, thymus, and lymph node as well as polymorphonuclear leukocytes, lymphocytes, and T- and B-cell murine lymphomas were MA158.2 negative. Expression of the reactive antigen on the macrophage cell surface was enhanced 3-fold following in vitro activation of elicited macrophages with macrophage-activating factor and the kinetics of activation to the tumoricidal state paralleled the increased expression of the antigen recognized by MA158.2. MA158.2 is a rat IgG2a antibody containing a single specific heavy and light chain that does not detect a polymorphic determinant. This monoclonal antibody will be a useful tool for monitoring the efficacy of agents in activating murine macrophages to the tumoricidal state and in analyzing the sequence of biochemical events that culminate in macrophage activation.