Effector Mechanisms of Human Monocyte-mediated Tumor Cytotoxicity in Vitro: Biochemical, Functional, and Serological Characterization of Cytotoxins Produced by Peripheral Blood Monocytes Isolated by Counterflow Elutriation

Human peripheral blood monocytes, isolated in high purity by centrifugal counterflow elutriation from normal donors, release cell toxins, herein termed human monocyte toxins (HMTs) upon further stimulation in vitro. The principal form of HMTs produced by these human peripheral blood monocytes has been subjected to biochemical, functional, and serological characterization. By molecular sieving on Sephacryl S-200, HMTs can be resolved into two molecular weight classes. The larger, termed a, has a molecular weight of about 120, 000, and the smaller, termed 0, has a molecular weight of about 65, 000. The β class is by far the most predominant species and has been further characterized. Chro-matofocusing of β-HMT indicates a slightly acidic nature, since this species is eluted at pH 5.8. Functional characterization of β-HMT suggests that it is not a trypsin-like protease, since neither α, N-tosyl-L-lysylchloromethylketone nor α, N-tosyl-L-arginyl methyl ester are capable of causing significant inhibition of the cell-lytic activity of the molecule. Furthermore, cell lysis induced by β-HMT appears to be independent of oxygen-dependent mechanisms, since catalase is incapable of blocking lysis, and since hydrogen peroxide and superoxide anion are not produced in detectable amounts during lysis. Finally, β-HMT does not appear to be an arginase, since it is active in arginine-containing medium and further addition of arginine to the assay medium does not inhibit lysis significantly. β-HMT is serologically related to recombinant human tumor necrosis factor (rHuTNF), since its cell lytic activity can be blocked by a rabbit antiserum against rHuTNF. However, much higher levels of this antiserum are required to achieve neutralization than are required to neutralize a comparable number of cell lytic units of rHuTNF. Furthermore, the results of preliminary immunoprecipitation experiments using the rabbit anti-rHuTNF antiserum suggest that a peptide in the Mr 60, 000-70, 000 range is recognized by this serum, whereas no signal at Mr 17, 000 corresponding to rHuTNF is detectable. Thus, human peripheral blood monocytes can be triggered to release cell toxins, the principal form of which, β-HMT, appears to be functionally distinct from the cytotoxic proteases reported in the murine system and appears to be molecularly distinct from, but serologically related to rHuTNF.