Marvin Fishman

Functional heterogeneity among peritoneal macrophages. II. Enzyme content of macrophage subpopulations.

Abstract Rabbit peritoneal exudate (PE) macrophages were separated into subpopulations on discontinuous density gradients of bovine serum albumin. Four such macrophage subpopulations, referred to as bands A, B, C, and D (from lightest to heaviest buoyant density), were examined for differences in enzyme content. With regard to three acid hydrolases—acid phosphatases, β-glucuronidase, and cathepsin D—cells in bands A and B had greater enzyme activity than cells in bands C and D. A similar distribution of activities was observed for acid p-nitrophenylphosphatase. Peroxidase activity was present only in band D. Lysozyme activity was greatest in band D cells and least in band A cells. Only small differences in cytochrome c oxidase activity were observed among the subpopulations. Arginase activity was found to be greater in cells from band A than cells in bands B, C, and D. Macrophage subpopulations derived from PE macrophages placed in tissue culture for 7 days and macrophage subpopulation cells cultured for 2 days showed differences in acid phosphatase content similar to those seen with freshly obtained subpopulations. These results extend previous work demonstrating heterogeneity among PE macrophages.

Functional heterogeneity among peritoneal macrophages: I. Effector cell activity of macrophages against syngeneic and xenogeneic tumor cells☆☆☆

Abstract Peritoneal exudate cells from immunized and nonimmunized animals were separated into subpopulations by centrifugation on discontinuous bovine serum albumin (BSA) density gradients. Cells in the several subpopulations were then tested for their cytostatic or cytotoxic activity against syngeneic and xenogeneic tumor cells. Nonimmune macrophages isolated at the 8 to 11% BSA interface were highly inhibitory to the growth of syngeneic and xenogeneic tumor cells during coculture for 24 to 48 hr. A second macrophage subpopulation of heavier density was not as effective in preventing tumor growth and frequently augmented it. Cytotoxic activity against (C58NT) D tumor cells could not be detected with macrophages or subpopulations of macrophages from immune as well as nonimmune animals, as determined by a 4-hr chromium release assay. The cytotoxic activity of the immune peritoneal exudate cells observed by this assay could be accounted for by the small percentage of lymphocytes present.

Functional heterogeneity among peritoneal macrophages: III. No evidence for the role of arginase in the inhibition of tumor cell growth by supernatants from macrophages or macrophage subpopulation cultures

Abstract The adherent population of peritoneal exudate cells (PE) obtained from rats and mice was analyzed for arginase activity in order to determine whether this enzyme has a role in tumor-growth-inhibitory activity. Freshly obtained tumor-growth-inhibitory rat PE cells had little or no arginase activity compared to the high levels of enzyme activity of mouse PE cells. Even after culturing, rat PE cells contained arginase activity 10 times less than that observed with comparable numbers of cultured or noncultured mouse cells. Subpopulations of mouse and rat PE macrophages, analyzed for arginase activity, showed that the light-density populations from cultured rat PE cells and noncultured mouse PE cells expressed arginase activities greater than that seen with heavy-density cells. However, the light-density rat PE cells expressed significantly less arginase activity than did the mouse cells. In attempts to test whether the inability of tumor cells to grow in supernatants or dialyzed supernatants from PE macrophage cultures is due to an arginine depletion, 200 μg/ml of the amino acid was added to the supernatants. The tumor-growth-inhibitory activities of such supernatants, as well as those from supernatants from highly active light-density rat PE macrophage cultures, were not abrogated by the addition of arginine. There was no correlation between the high levels of arginase activity of light-density PE macrophages and their antitumor activity and no evidence that the tumor-growth-inhibitory activity of rat or mouse PE macrophages in the macrophage-tumor models we studied was due to an arginine depletion.

Evidence that immune RNA is messenger RNA

Abstract Exposure of rabbit spleen cell cultures to i-RNA isolated from T2 phage-exposed rabbit peritoneal exudate cells induces the synthesis of antigen and allotype specific 19S proteins even in the presence of actinomycin D. The same i-RNA directs the synthesis of proteins with comparable properties in cell-free extracts prepared from mouse L cells, indicating that i-RNA functions as mRNA and contains the information required to code for the synthesis of IgM antibodies.

Sensitivity to macrophage-mediated cytostasis is cell cycle dependent

Abstract We have examined the sensitivity of proliferating lymphoid cells in different phases of the cell cycle to macrophage-mediated cytostatic activity. These studies evaluated the ability of target cells enriched in individual cell cycle phases to pass into the next phase during brief (2–6 hr) periods of coculture with activated or nonactivated peritoneal macrophages. Both normal (concanavalin A-stimulated spleen cells) and neoplastic (Gross virus-induced thymic lymphoma) cells were analyzed. Spleen cells or lymphoma cells were first separated by centrifugal elutriation into populations highly enriched for G 1 , S, or G 2 /M phases of the cell cycle and cultured in the presence of nonactivated or activated macrophages for periods of 2, 4, or 6 hr. The cellular DNA content of recovered nonadherent target cells was then analyzed by flow cytometry after staining with propidium iodide. Macrophage contamination of target cell populations was insignificant under these conditions. Nonactivated macrophages did not affect target cell cycle traverse when compared with target cells cultured alone. Activated macrophage mediated cytostatic activity resulted in complete block of the transition of cells in G 1 phase into S phase and of the further accumulation of DNA by cells in early S phase. Cells already in mid to late S phase were able to continue DNA replication at rates nearly equivalent to control cells. There was no inhibition of the passage of cells through G 2 or mitosis. These effects were seen by as early as 2 hr of macrophage-target cell coculture and both normal and neoplastic cells exhibited identical patterns of cell cycle phase sensitivity.

Characterization of the recognition of target cells sensitive to or resistant to cytolysis by activated macrophages: II. Competitive inhibition of macrophage-dependent tumor cell killing by mitogen-induced, nonmalignant lymphoblasts

Abstract We have previously reported that tumoricidal rat macrophages can distinguish quiescent normal lymphocytes from concanavalin A (Con A)-stimulated lymphocytes and thymic lymphoma cells on the basis of their ability to compete for the macrophage-dependent cytolysis of a sensitive tumor cell line. The present study was undertaken to determine (a) whether recognition was related to the proliferative response induced by Con A stimulation and (b) whether the competition of cytolysis was dependent upon the binding of sensitive target cells to activated macrophages. These possibilities were tested by examining Con A-treated lymphocytes in different functional stages of the Con A response with respect to their ability to compete either for cytolysis or binding of a tumor cell line susceptible to both activities. The results show that the ability to compete for either function was acquired coincidentally with the Con A-induced proliferative response. This competitive activity was not due solely to the presence of Con A in the culture medium nor to culture of unstimulated lymphocytes but rather required a blastogenic response to the mitogen. Blast-transformed nonproliferative cells (96 hr post-Con A stimulation) were as competitive as cells which had been stimulated to reinitiate DNA replication by treatment with Interleukin 2. Thus, competition for cytolysis is a consequence of blastogenesis rather than proliferation per se and operates mechanistically by competing for the binding of target cells to activated macrophages, an event known to be a necessary prerequisite to cytolysis.

Tumor necrosis factor-α analyzed within individual macrophages by combined immunocytochemistry and computer-aided image analysis

Immunocytochemical staining procedures were combined with computer-aided image analysis to quantitate the relative intracellular production of tumor necrosis factor-alpha (TNF) within individual macrophages. Optimal conditions for time and methods for the activation of TNF production, fixation of cells for optimal immunocytochemical staining, and image analysis methods were determined. Thioglycolate elicited peritoneal macrophages were readily activated to significantly increased levels of intracellular TNF, as early as 1 hr after activation with lypopolysaccharide (LPS) + interferon-gamma: maximum intracellular TNF was evident after 2-3 hr. Both LPS and interferon-gamma was necessary to increase intracellular TNF. Normal alveolar macrophages also readily produced increased intracellular TNF, but normal peritoneal and splenic macrophages were poorly activated to TNF production. Acid stripping of receptor bound TNF allowed discrimination between intracellular TNF/integral membrane TNF, and TNF-receptor-bound TNF. Results stress the importance of studying these TNF forms early after activation. Applications for TNF quantitation by these means are discussed.

Induction of tumor cell resistance to macrophage-mediated lysis by preexposure to non-activated macrophages☆

Thioglycollate-elicited peritoneal exudate (non-activated) macrophages do not lyse tumor cells and in contrast to activated macrophages bind less target cells. However, a non-lethal encounter of tumor cells with non-activated macrophages resulted in a pronounced effect on the subsequent tumor cell binding to and lysis by activated macrophages. Our results have shown that binding of tumor cells by non-activated macrophages was Ca2+ and temperature dependent; had a requirement for a Pronase-sensitive structure on macrophage surface membranes; was saturable; and was 2-3X less than that observed for activated macrophages. Experiments were conducted in which syngeneic tumor cells were incubated with a monolayer of non-activated macrophages and then assayed for selective binding and sensitivity to lysis. The important observations were that as a result of a 3-hr incubation with non-activated macrophages at an EC: TC ratio of 5:1 there was an increase in the number of tumor cells that bound to both activated and non-activated macrophages; a loss of selective binding in which the ratio of tumor cells bound to activated/non-activated macrophages (normally greater than 2) was lowered to 1.0; and a concomitant decrease in the susceptibility of tumor cells to macrophage-mediated cytolysis. The induction of tumor cell resistance to macrophage kill required an exposure to an excess number of non-activated macrophages, was reversible upon culturing with or without macrophages for 24 hr and required cell-cell contact. Our results reinforce the importance of selective binding between tumor cells and activated macrophages as an initial phase in tumor cell killing and also illustrates an active role for non-activated macrophages in vivo in allowing tumor cells to escape the immune surveillance by activated macrophages.

Macrophage-mediated cytostatic activity blocks lymphoblast cell cycle progression independently in both G1 phase and S phase☆

Recent work has shown that macrophage-mediated cytostatic activity inhibits cell cycle traverse in G1 and/or S phase of the cell cycle without affecting late S, G2, or M phases. The present report is directed at distinguishing between such cytostatic effects on G1 phase or S phase using the accumulation of DNA polymerase alpha as a marker of G1 to S phase transition. Quiescent lymphocytes stimulated with concanavalin A undergo a semisynchronous progression from G0 to G1 to S phase with a dramatic increase in DNA polymerase alpha activity between 20 and 30 hr after stimulation. This increase in enzyme activity was inhibited, as was the accumulation of DNA, when such cells were cocultured with activated murine peritoneal macrophages during this time interval. However, if mitogen-stimulated lymphocytes were enriched for S-phase cells by centrifugal elutriation and cocultured with activated macrophages for 4-6 hr, DNA synthesis was inhibited but the already elevated DNA-polymerase activity was unaffected. Similar results were obtained when a virally transformed lymphoma cell line was substituted as the target cell in this assay. These results show that both G1 and S phase of the cycle are inhibited and suggest that inhibition of progression through the different phases may be accomplished by at least two distinct mechanisms.

Hydrogen peroxide release by rat peritoneal macrophages in the presence and absence of tumor cells

Abstract The ability of mineral oil-elicited rat peritoneal macrophages to release hydrogen peroxide (H 2 O 2 ) to the extracellular medium was measured in the presence and absence of rat lymphoma cells grown in tissue culture, and in the presence of phorbol myristate acetate (PMA). Horseradish peroxidase (HRP)-catalyzed oxidation of scopoletin or phenol red was used to measure H 2 O 2 release during incubation of cells in monolayer culture for periods up to 24 h. Macrophages appeared to release H 2 O 2 with or without PMA, although PMA greatly increased the amount of H 2 O 2 released in short (1 to 4 h) incubations. Tumor cells did not replace PMA as a triggering agent for H 2 O 2 release. Instead, tumor cells inhibited H 2 O 2 release. The probable basis for inhibition was competition between macrophages and tumor cells for the supply of oxygen (O 2 ). Tumor cells did not inhibit H 2 O 2 release when the O 2 concentration was held constant. The rates at which macrophages took up O 2 and released H 2 O 2 were proportional to the O 2 concentration, as measured with the O 2 electrode. Rates of H 2 O 2 release could be calculated from the difference in the rate constants for O 2 uptake measured in the presence of two different extracellular H 2 O 2 -consuming systems (HRP-scopoletin vs catalase). PMA-stimulated uptake of O 2 and release of H 2 O 2 were highest in a small subpopulation of macrophages, obtained at the lowest-density position on gradients of bovine serum albumin. These cells also released H 2 O 2 in the absence of PMA. Tumor cells had no effect on the rate constants for O 2 uptake and H 2 O 2 release by the unfractionated macrophages or the macrophage subpopulations.