Differential expression of murine macrophage-mediated tumor cytotoxicity induced by interferons.

The requirements for interferon (IFN)-induced priming of murine peritoneal macrophages for cytolysis of tumor cell lines of distinct histological origin were investigated. Lysis of B16 melanoma targets required exposure of elicited macrophages to recombinant murine gamma interferon plus lipopolysaccharide (LPS) together, while sequential treatment of macrophages with IFN-gamma then LPS resulted in lysis of P815 mastocytoma targets. The kinetics of macrophage activation by IFN-gamma and LPS for lysis of P815 and B16 melanoma targets varied considerably, 8 h being sufficient for P815 targets but 24 h being required for B16 targets. Pretreatment of the macrophages with the antibiotic polymyxin B was able to inhibit completely the induction of tumor lysis of B16 targets but not of P815 targets. In addition, IFN-alpha/beta was able to prime macrophages for lysis of P815 targets but not of B16. Finally, the kinetics of priming macrophages with IFN-gamma for lysis of B16 targets had a profound effect on the subsequent exposure time requirement for LPS. The results indicate that the induction of murine macrophage-mediated tumor cytotoxicity can vary considerably depending on the amount and type of interferon used, the presence of a second signal, and the type of tumor target used.

Actin in B16 melanoma cells of differing metastatic potential. Effects of trypsin and serum.

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.

Induction of 32- and 34-kDa stress proteins by sodium arsenite, heavy metals, and thiol-reactive agents.

Challenge of human or murine melanoma cells with sodium arsenite, heavy metals (Zn2+, Cu2+ and Cd2+), or thiol-reactive agents (p-chloromercuribenzoate and iodoacetamide) induced the synthesis of four stress proteins with molecular masses of 100, 90, 72 (a doublet), and 32 (human) or 34 (murine) kDa. Enhanced expression of the 32- and 34-kDa polypeptides (p32 and p34) preceded or paralleled the synthesis of the other stress proteins. Hyperthermia, the calcium ionophore A23187, and amino acid analogs (L-azetidine-2-carboxylic acid and L-canavanine) induced the formation of the major stress proteins, but failed to increase synthesis of p32 and p34. Characterization of the dose and time dependence of p32 and p34 synthesis in human (A375) and murine (B16-F10) melanoma cells, respectively, indicated that these proteins were subject to similar regulatory mechanisms. Electrophoretic analysis of stressed cells pulsed with different metabolic precursors revealed that p32 and p34 were radiolabeled with [35S]methionine or 3H-amino acids but not by [3H]mannose or [35S]cysteine. Polyclonal antibodies raised against human p32 cross-reacted with murine p34. These data suggest that p32 and p34 are closely regulated human and murine gene products, respectively, whose synthesis can be modulated by thiol-reactive reagents. Induction of p32 and p34 by these agents, but not by heat shock, suggests that these proteins are a subset of stress-inducible gene products.

Induction of mammalian stress proteins by a triethylphosphine gold compound used in the therapy of rheumatoid arthritis.

In vitro exposure of cultured human, murine and rat cells to pharmacologic concentrations (10(-8) to 10(-6) M) of auranofin, 2,3,4,6,-tetra-O-acetyl-1-thio-beta-D-glucopyranosato-S- triethylphosphine gold(I) (Ridaura), a gold containing compound approved for the treatment of rheumatoid arthritis, results in the induction of several stress proteins. The enhanced synthesis of two polypeptides, p32 and p34, was particularly prominent. A similar response was observed in freshly collected human monocytes challenged with auranofin. In addition, oral administration of auranofin to rats induced enhanced synthesis of a 32-kDa protein in peritoneal exudate cells analyzed ex vivo at various times following drug treatment. These data suggest that increased synthesis of p32 and p34 might participate in mediating certain aspects of auranofin pharmacology.

Induction by immunomodulatory agents of a macrophage antigen recognized by monoclonal antibody 158.2 and correlation with macrophage function.

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.

Tumorigenic and metastatic properties of "normal" and ras-transfected NIH/3T3 cells.

To investigate the role of oncogene activation in the pathogenesis of malignant tumors, we have studied the tumorigenic and metastatic properties of NIH/3T3 secondary transfectants (designated A51) containing an activated c-Ha-ras-1 gene derived from the human T24 bladder carcinoma cell line and compared them with untransfected NIH/3T3 cells. Whereas subcutaneous implantation of NIH/3T3 cells in the supraclavicular region produced palpable tumors that failed to metastasize, NIH/3T3 cells inoculated in the footpad gave rise to malignant tumors that metastasized to the lung. Under identical conditions and irrespective of the site of implantation, A51 cells formed rapidly growing primary tumors that produced pulmonary metastases. In an assay for experimental metastasis, intravenously injected NIH/3T3 cells gave rise to pulmonary nodules only at high cell inocula and in long-term survivors (90 days after injection). In contrast, A51 cells formed multiple lung tumor colonies detectable 14 days after injection. These results indicate that "normal" untransfected NIH/3T3 cultures contain subpopulations of cells that express malignant properties and that transfection of NIH/3T3 cells with activated c-Ha-ras-1 accelerates formation of metastases.

Production of C3 as a marker of lymphokine-mediated macrophage activation.

C3 production was assayed using an enzyme-linked immunosorbent assay (ELISA) in cell-free supernatants harvested from thioglycollate-elicited macrophages exposed to a variety of macrophage stimulating and activating agents. Macrophage monolayers treated with the stimulating agents starch, glycogen, and zymosan secreted three- to four-fold less C3 (mean 12 ng/10(5) cells/12 hr) than macrophages exposed to lymphokines containing macrophage-activating factor (MAF) (mean C3 production 44 ng/10(5) cells/12 hr). The increased production of C3 in macrophages exposed to MAF parallels the ability of these macrophages to acquire tumoricidal capacity as monitored in an in vitro 72 hr tumor cell cytotoxicity assay using B16 melanoma cells. Macrophages previously rendered tumoricidal by exposure to MAF and which are refractory to further challenge by MAF following decay of their tumoricidal properties, do not produce C3 on rechallenge with MAF. Exposure of refractory macrophages to liposome-encapsulated MAF overcomes the refractory state and induces re-expression of the tumoricidal phenotype and C3 production. We conclude that quantitative detection of macrophage-generated C3 antigen provides a useful biochemical marker for monitoring the acquisition of tumoricidal properties in macrophages exposed to MAF and offers a sensitive assay for screening novel agents that activate macrophages via mechanisms similar to MAF.

Identification and characterization of a monoclonal antibody to an antigen expressed on activated macrophages.

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.

Experimental metastasis correlates with cyclic AMP accumulation in B16 melanoma clones.

Metastasis is a complex process whereby tumour cells from a primary neoplastic growth disseminate throughout the body and establish secondary tumour foci in distant organs. Biochemical traits associated with, or essential for, the expression of the metastatic phenotype have not yet been identified. In the course of examining stimulation of the B16 murine melanoma adenylate cyclase by melanocyte-stimulating hormone (MSH) and by the diterpene forskolin, we noted that tumour cell clones isolated from common parent cell populations differed widely in their responses to these agonists. We report here that the accumulation of cyclic AMP induced by MSH or forskolin shows a strong positive correlation with the ability of B16 melanoma clones to form pulmonary tumour colonies when injected intravenously (i.v.) into syngeneic mice ('experimental metastasis'). In parallel in vitro analyses of cyclic AMP metabolism and in vivo assays of experimental metastasis using replicate cell preparations, highly metastatic tumour cell clones consistently show greater than a 30-fold increase in cellular cyclic AMP when exposed to MSH or forskolin. By contrast, clones with limited metastatic abilities respond to the same agonists with only a two- to threefold increase in cellular cyclic AMP. These data suggest that cyclic AMP metabolism is linked with biochemical pathways that are responsible for the formation of experimental metastasis by the B16 melanoma.

Detection of a breast tissue-associated antigen by antiserum to Raji cell-bound circulating immune complexes of human breast cancer.

Rabbits tolerant to human immunoglobulin G were used to raise antisera against the Raji cell-bound circulating immune complexes from human breast cancer sera. After solid-phase adsorption treatment with glutaraldehyde-cross-linked normal human plasma, acetone-extracted normal liver tissue powder, and glutaraldehyde-fixed Raji cells, one antiserum reacted specifically with breast tissue extracts but not with extracts of other tissues, as examined by a counterimmunoelectrophoresis technique. Immunological reactivity of the treated antiserum was removed by incubation with normal, primary, or metastatic breast tumor tissue extracts. Incubation with normal human serum or extracts derived from tissues other than the breast showed no neutralizing effect on the antibodies. This specific antiserum reagent was used in a modification of the Raji cell radioimmunoassay. Raji cells were incubated with sera from cancer patients or normal controls and then reacted with 125I-labeled F(ab')2 fraction of the treated antiserum reagent. The amount of 125I-F(ab')2 bound was then determined. Although all sera exhibited elevated circulating immune complexes by the conventional Raji cell radioimmunoassay, 14 of 18 breast carcinoma sera demonstrated a significant uptake when compared with the normal population group as opposed to five (three lung and two colon) of 29 other cancer sera examined (p less than 0.001). An immunologically reactive breast tissue-associated antigen, purified from malignant breast tumor or normal breast tissue extracts with the use of antiserum reagent, exhibited an apparent molecular weight of 85,000 by sodium dodecyl sulfate; polyacrylamide gel electrophoresis and a pI value of 4.9 +/- 0.2. These results demonstrated that a breast tissue-associated antigen rather than a breast tumor-associated neoantigen, was involved in circulating immune complexes of breast cancer patients as detected by Raji cell immunoassay. It also implied the occurrence of disease-related autoimmunity in human breast cancer.