Michael A. Chirigos

Functional and morphologic characteristics of interferon-treated macrophages

Abstract Resting macrophages were transformed as evidenced by functional criteria to activated cells by exposure to partially-purified or crude interferon (IF) preparations. Peritoneal macrophages were rendered cytotoxic for syngeneic lymphoblastic leukemia cells by either in vivo or in vitro IF treatment. Although IF was active at concentrations as low as 10 units/ml in the culture medium, mock IFs lacked activity at all concentrations tested. Phase contrast microscopy demonstrated that in vitro IF-treated macrophages exhibited increased vacuolization and prominent spreading on glass. In contrast, transmission and scanning electron microscopy failed to reveal major differences in the structure of macrophages exposed to IF in vivo. The data are discussed in terms of a novel form of tissue immunity endowed by IF-inducers.

Stimulation of Humoral and Cellular Antibody Formation in Mice by Poly Ir:Cr1

Summary Polyriboinosinic–polyribocytidilic acid stimulated formation of hemolytic antibodies in mice immunized with sheep erythrocytes. Similarly, poly Ir:Cr treatment of mice resulted in a marked reduction in survival of isografts. Temporarily, administration of poly Ir:Cr at the time of exposure of mice to antigen resulted in optimal enhancement of humoral and cellular antibody formation. Optimal enhancement of both humoral and cellular antibody formation was induced by 100-200 μg of poly Ir:Cr, while significant stimulation of hemolytic antibody formation was induced by a lower dose of 10 μg of poly Ir:Cr. We wish to thank Mr. Hugh Pettigrew of the Biometry Branch, National Cancer Institute, for the statistical evaluation of the experimental data.

A simple micro method for the direct determination of δ-amino[14Clevulinic acid production in murine spleen and liver homogenates

Abstract 1. 1. A simple and sensitive radiochemical procedure for the estimation of the activity of δ-aminolevulinate synthetase (succinyl-CoA: glycine succinyltransferase) in homogenates of normal murine spleen and liver is described. 2. 2. Homogenates of spleen or liver are incubated with α- keto [ 14 C] glutarate or [14C]succinate respectively, and the [14-amino14Clevulinic acid generated in the reaction is selectively adsorbed onto a Dowex 50 column run at pH 3.9. Since labeled α-amino acids and unreacted radioactive substrate are not reatained by the column, contamination of α-aminolevulinic acid by these substances does. δ- Amino- [ 14 C] levulinic can be recovered in yields of 97–100%. 3. 3. Maximal δ- amino[ 14 C]levulinic acid production was obtainedin spleen homogenates in the presence of α- keto[ 14 C]glutarate , glycine, EDTA, and pyridoxal phosphate. δ- Amino[ 14 C]leculinic acid utilization was minimal at homogenate concentrations of 2.5% or less. Varying degrees of inhibition of δ-amino14Clevulinic acid production were seen with malate, malonate, arsenite, ATP, CoA, and NAD+. 4. 4. [14]Succinate, glycine, EDTA, and pyridoxal phosphate produced the highest levels of δ- amino[ 14 C]levulinic acid in liver homogenates. δ- Amino[ 14 C]levulinic acid utilization was minimal at homogenate concentrations of 1% or less. Varying degrees of inhibition of δ- amino[ 14 ]levulinic acid production were seen with malate, malonate, arsenite, ATP, and CoA. 5. 5. The sensitivity of the procedure will allow the measurement of δ-amino-levulinate synthetase activity in normal spleen and liver and will permit more detailed investigation of mechanisms controlling heme synthesis. Smaller amounts of tissues can be used than have been previously employed by colorimetric methods. The method can be easily adapted to preparations of mitochondria and blood cells.

Stress-induced impairment of macrophage tumoricidal function.

&NA; Several studies have shown the effect of stress on immune cells and their functions. The purpose of the present work was to investigate the influence of acute immobilization stress on macrophage nonspecific tumoricidal activity. Peritoneal macrophages were activated by nonspecific immunopotentiators such as interferon or bacterial lipopolysaccharide (LPS) and the killing of MBL‐2 leukemic target cells was measured. Macrophages from mice submitted to stress showed decreased responsiveness to interferon or LPS. In addition, the role of corticosteroids as mediators in the phenomenon was also studied. Indeed, we observed that corticosteroids were able to inhibit macrophage cytotoxicity and could at least play some role. This data could contribute to a better understanding of the effect of stress on the host immunosurveillance against tumor development.

Prevention of macrophage tumoricidal activity by agents known to increase cellular cyclic AMP

Abstract Agents known to increase intracellular levels of cyclic 3′5′-adenosine monophosphate (cyclic AMP) in a variety of tissues were examined for their affect on macrophage tumoricidal activity in vitro. Cholera toxin and prostaglandin E1 and E2 consistently inhibit the cytotoxicity of interferon-treated macrophages for MBL-2 lymphoblastic leukemia cells. These observations suggest a role for cyclic AMP in modulating macrophage functional activity. The finding that dibutyryl cyclic AMP inhibits tumor cell killing by interferon-activated macrophages further supports this view.

Direct activation in vitro of mouse peritoneal macrophages by pyran copolymer (NSC 46015).

Abstract Normal resting macrophages were transformed to cytostatic effector cells in the presence of pyran copolymer (NSC 46015) in the culture medium. Macrophage “activation” to inhibit MBL-2 leukemia cell growth was sharply dose-dependent and required >24 hr after exposure to pyran. The observed growth inhibition appeared to result from a modification of the macrophages themselves, since neither allogeneic macrophages nor drug alone interfered with MBL-2 cell growth. The primary mechanism of cytostasis did not involve phagocytosis or soluble macrophage product(s). Similar activation was observed for poly(I)·poly(C) and to a lesser extent for dextran sulfate. It is suggested that the antitumor activity of these polyanions is due to their function as direct macrophage stimulants.

Adjuvant treatment with levamisole in cancer a review of experimental and clinical data

Animal and human studies of adjuvant treatment with levamisole in cancer are reviewed and discussed. From the animal data it is concluded that the activity of levamisole is dose-dependent, more effective on slow-growing tumors, affects metastasis formation, preferentially is best when levamisole is used as an adjuvant to the usual cytoreductive treatments and that tumor enhancement is not expected. Clinical findings are put into perspective of the animal data and the most appropriate clinical situations are indicated.

Augmentation of specific macrophage-mediated cytotoxicity: Correlation with agents which enhance antitumor resistance

Abstract Purified peritoneal macrophages harvested from allograft-bearing mice were shown by criss-cross cytotoxicity assays to be specifically cytolytic for the appropriate target cells. This response peaked approximately 12 days after a subcutaneous inoculation of 1 × 10 7 allogeneic MBL-2 or L1210 leukemia cells. Intraperitoneal treatment of MBL-2 allograft-bearing mice with a number of adjuvants including living BCG, pyran copolymer, Corynebacterium parvum, Staphylococcus aureus membranes, and thymosin acted synergistically to potentiate specific macrophage reactivity. However, enhanced cytotoxicity was not observed with levamisole, tilorone, calf spleen extract, and bovine serum albumin. The significance of these adjuvant-activated macrophages as participants in host antitumor resistance is discussed.

Selective neutralization by antiinterferon globulin of macrophage activation by L-cell interferon, Brucella abortus ether extract, Salmonella typhimurium lipopolysaccharide, and polyanions

Abstract Addition of interferon (IF) inducers pyran copolymer, poly(I)-poly(C), an ether extract of Brucella abortus (Bru-Pel), or Salmonella typhimurium lipopolysaccharide (LPS) to cultures of peritoneal macrophages in vitro enhanced their cytotoxic activity for MBL-2 lymphoblastic leukemia cells. To evaluate the role of induced IF in the macrophage activation, highly specific rabbit anti-L-cell IF globulin was added to resting macrophage cultures at the same time as the macrophage-activating agents. Macrophage activation by these various biological and synthetic agents was totally neutralized by anti-IF globulin but not by normal rabbit globulin. Similarly, the anti-IF globulin inhibited the ability of chromatography-purified Newcastle disease virus-induced IF to render macrophages cytotoxic, and the degree of neutralization of IF titer corresponded with the inhibition of IF-induced macrophage-mediated cytotoxicity. In contrast, macrophage activation by concanavalin A-induced lymphokine, which contains an antigenically different IF, was not affected by high titers of the anti-L-cell IF antibodies. The results indicate that endogenously generated type I IF may play an important role in control of macrophage function.

In vitro stimulation of spleen cell cultures by poly I: poly C and levamisole.

Abstract Poly I: poly C and levamisole (LMS) were shown to stimulate DNA synthesis by spleen cell suspension cultures. Poly I: poly C was more effective than LMS at concentrations above 1 μg/ml; both agents were weakly stimulatory at concentrations 0.1–1.0 μg/ml. Levamisole augmented the DNA synthetic response to a supraoptimal concentration of phytohemagglutinin.