Suppression of fibroblast proliferation by activated macrophages: involvement of H2O2 and a non-prostaglandin E product of the cyclooxygenase pathway

Macrophages are considered promoters of fibroblast proliferation; however, suppression by activated macrophages may outweigh this effect. Activated murine peritoneal macrophages obtained by in vivo exposure to C. parvum or by in vitro LPS-activation of thioglycollate-induced macrophages, were tested for their effect on normal syngeneic dermal fibroblasts. C. parvum-activated macrophages, but not resident peritoneal macrophages suppressed fibroblast proliferation. Similarly, macrophages activated in vitro by LPS, but not those unexposed to LPS, suppressed fibroblast proliferation. Catalase partially protected fibroblasts from suppression by either activated macrophage population, suggesting involvement of H2O2 in the suppression. The effect of cyclooxygenase inhibitors on the suppression was also tested. Indomethacin, acetylsalicyclic acid, or eicosatetraynoic acid, all partially protected the fibroblasts from macrophage-mediated suppression. Prostaglandins E2, E1, and F2 alpha, added exogenously at concentrations as high as 10(-6) M, failed to suppress the proliferation of the fibroblasts. These findings suggest that a non-prostaglandin product of the cyclooxygenase pathway is involved in macrophage-mediated suppression of fibroblast proliferation.     

Microbiostatic effect of murine-activated macrophages for Toxoplasma gondii. Role for synthesis of inorganic nitrogen oxides from L-arginine

Recent studies show the importance of a single amino acid, L-arginine, as a necessary substrate for activated macrophage-mediated cytotoxic activity for tumor target cells and microbiostatic function for Cryptococcus neoformans. The present studies were carried out to determine the role of the L-arginine-dependent macrophage effector function on the microbiostatic effects of activated macrophages on the obligate intracellular protozoan, Toxoplasma gondii. A guanidino methylated derivative of L-arginine, NGmonomethyl-L-arginine (NGMMA), a competitive inhibitor of the L-arginine-dependent effector pathway, virtually abolished the normally potent microbiostatic effect of macrophages for Toxoplasma gondii after activation of the macrophages in vitro by IFN-gamma and LPS or in vivo by i.p. injection of killed Corynebacterium parvum. Addition of supplemental L-arginine to the culture medium overcame the capacity of NGMMA to block activated macrophage-mediated microbiostasis of Toxoplasma. The ability of NGMMA to inhibit the microbiostatic capacity of activated macrophages for Toxoplasma gondii correlated with almost total inhibition of synthesis of nitrite, nitrate, and L-citrulline from L-arginine. Therefore, as is the case for tumor target cells and C. neoformans, the synthesis of inorganic nitrogen oxides from a terminal guanidino nitrogen atom of L-arginine appears to be essential for murine cytotoxic activated macrophage mediated microbiostatic capacity for T. gondii.     

Generation of suppressor lymphocytes during sensitization in culture against a syngeneic tumor: affinity chromatography on insolubilized histamine

We investigated the effect of depletion of histamine-binding lymphoid cells on immunological properties of lymphocytes sensitized in culture against tumor cells. C57BL/6 spleen cells that were sensitized in vitro on monolayers of the syngeneic Lewis lung carcinoma (3LL) became cytotoxic to the tumor cells in vitro after 3 to 5 days of sensitization. Sensitized cells harvested after 4 days of sensitization occasionally enhanced tumor growth in vivo. Fractionation of the sensitized lymphocytes over insolubilized histamine-rabbit serum albumin-Sepharose (HRS) columns decreased or abolished the enhancing activity in vivo and specifically increased the in vitro cytotoxic activity of the depleted lymphocytes. A similar increase in the cytotoxic activity of HRS-fractionated cells was observed in an allogeneic combination of C57BL spleen cells sensitized against C3H fibroblasts. The effect of HRS chromatography on the in vitro cytotoxic activity increased with prolonged incubation of the depleted effector cells with the target cells.     

In vitro killing of schistosomula of Schistosoma mansoni by BCG and C. parvum-activated macrophages.

Resistance to Schistosoma mansoni infection in the mouse has been induced either specifically by a primary infection with this parasite or nonspecifically by a variety of immunostimulants such as BCG. In the present study we developed an in vitro system to examine the effector mechanism of nonspecifically induced resistance. Activated macrophage monolayers obtained from BCG- or Corynebacterium parvum treated mice killed a respective mean 32 +/- 6% and 48 +/- 5% of schistosomula after 24 hr incubation. The killing of the parasites was verified by their inability to mature to adult worms upon injection into normal mice. The activated macrophage-mediated killing was related to cell:parasite ratio, and was partially lost if the macrophage monolayers were kept in cultures for 24 hr before incubation with the organism. Supernatants of macrophages cultured in the presence of schistosomula killed a mean of 51 +/- 3% of the organisms whereas those from cells cultured alone resulted in a mean killing of 25 +/- 3%. Furthermore, toxic supernatants could be generated equally well on incubation with S. mansoni schistosomula or Trichinella spiralis larvae. Our data show that activated macrophage monolayers through soluble mediators destroy a significant proportion of the multicellular parasite S. mansoni schistosomula in vitro.     

Immune response to a syngeneic mammary adenocarcinoma. II. In vitro generation of cytotoxic lymphocytes.

A method is described for the consistent in vitro generation cytotoxic cells by incubating Fischer 344 rat spleen cells on monolayers of a syngeneic mammary adenocarcinoma. Significant cytotoxicity by in vitro culture is generated as early as 3 days after initiation and effector cells are cytolytic only toward target cells of the sensitizing monolayer. Reciprocal sensitization with allogeneic fibroblasts as the immunizing monolayer yielded effector cells cytolytic for the fibroblasts but without effect on the mammary tumor. The consistency in the generation of cytotoxic cells by in vitro culture should permit its standardized use in following other related immune phenomena such as blocking by serologic factors and suppression, recritment of memory for cytotoxic function.     

Growth Kinetics of Yaba Tumor Poxvirus After In Vitro Adaptation to Cercopithecus Kidney Cells

Yaba tumor poxvirus has been adapted to continuous in vitro cultivation in monolayers of cercopithecus kidney cells. At 35 C, the minimum replicative cycle, after synchronous infection of CV-1 cells with multiplicity of infection of 135 focusforming units per cell, was 35 hr; however, maximum virus yields were not obtained until 75 hr postinfection (PI). Cytoplasmic incorporation of (3)H-thymidine [viral deoxyribonucleic acid (DNA) synthesis] was detected 3 hr PI and was preceded by synthesis of nonstructural associated antigens (YS). Synthesis of YS antigens was not inhibited by the DNA inhibitor, arabinofuranosyl cytosine (ARA-C). Synthesis of at least two virion structural antigens, although not detected by immunofluorescence until 2 hr after the onset of DNA synthesis, occurred in the presence of ARA-C, indicating potential translation of these structural antigens from parental DNA. The first progeny DNA was completed by 20 hr PI but was not detected in infectious form until 35 hr PI. The maximum rate of progeny DNA completion occurred between 20 and 30 hr PI. DNA synthesis continued 45 to 50 hr PI. The adapted virus retained its oncogenicity and, like the wild type, replicated better at 35 C than at 37 C. A synthetic step associated with viral DNA synthesis appears to be temperature-sensitive.     

Evidence for granulocyte-mediated macrophage activation after C. parvum immunization

It has been previously demonstrated that at the peak of the peritoneal response to Corynebacterium parvum (Day 4), cytolytic macrophages can be characterized by the presence of intracellular bacteria. In the present study, the role of neutrophils in the activation of peritoneal macrophages by C. parvum was investigated. Inflammatory neutrophils isolated 5 hr after ip administration of C. parvum were transferred to normal, syngeneic mice and the peritoneal macrophages of recipients harvested 4 days later were tested for cytoxicity against HeLa cells. Neutrophils isolated from mice 5 hr after C. parvum immunization were effective in inducing cytolytic macrophages. Less than 100-fold as much bacteria was needed to induce comparable levels of cytotoxic activity when introduced inside granulocytes. Neutrophils obtained from mice 48 hr after C. parvum injection or mononuclear cells were not good macrophage activators. Viable neutrophils were not required as freeze-thawed cells were able to activate macrophages in recipient mice. The intracellular distribution of C. parvum changed dramatically with time. Initially almost all bacteria were found within neutrophils. By 24 hr, many macrophages contained either bacteria or granulocytes which had ingested C. parvum. Pyridine extracts of C. parvum, which do not activate peritoneal macrophages when injected directly into mice, did not induce neutrophils capable of activating macrophages. The residue of pyridine-extracted C. parvum did induce neutrophils that could activate macrophages when transferred. The results suggest that processing of the bacteria by inflammatory granulocytes may be an obligatory step in macrophage activation by this agent. The peak response occurred earlier than T-cell immunity is usually observed and it is suggested that direct activation of macrophages via ingestion of neutrophils may represent the earliest stage of macrophage activation by C. parvum.     

Induction of macrophage-mediated cytolysis of neoplastic cells by mycoplasmas

Unexpected cytolysis was encountered when nonactivated murine peritoneal macrophages were cultured with [³H]TdR-prelabeled syngeneic or allogeneic tumor cells at a 10:1 ratio. The level of specific cytolysis reached 70% within 48 hr of cocultivation. Similar killing was observed whether the macrophages were derived from untreated, thioglycollate-treated, or germ-free mice. Cytolytic activity was also demonstrated when bone marrow-derived or peritoneal macrophages from 9- and 5-day in vitro cultures, respectively, were employed rather than freshly harvested peritoneal macrophages. Thus, the macrophage-mediated killing was neither the result of in vivo preactivation nor a consequence of the presence of lymphocytes in the assay. Moreover, macrophages derived from different strains caused similar effects. Our study revealed that the neoplastic target cell cultures susceptible to cytolysis by nonactivated macrophages were contaminated with mycoplasma. A mycoplasma was isolated from the supernatant of a culture of the A9HT fibrosarcoma line, identified as Mycoplasma orale, and cultivated. Addition of viable mycoplasma from that isolate to mixed cultures of thioglycollate-elicited macrophages and [³H]TdR-prelabeled mycoplasma-free target cells resulted in specific cytolysis of transformed A9 cells, but not of normal mouse fibroblasts. The level of macrophage-dependent cytolysis correlated with the number of viable mycoplasma cells added and was higher than that attained by activation with LPS at optimal concentration. Similar specific cytolysis was observed with heat-killed mycoplasmas. Our results demonstrate that mycoplasmas may cause selective macrophage-mediated cytolysis of neoplastic but not of normal target cells, perhaps via activation of the macrophages. It is suggested that undetected infection of experimental systems by mycoplasmas may account for some reports on lysis of neoplastic cells by nonactivated macrophages.     

Macrophages activated as suspension cultures with lymphocyte mediators devoid of antigen become cytotoxic for tumor cells.

Normal peritoneal exudate cells (PEC) were activated as suspension cultures either in mediator-rich supernatants from o-chlorobenzoyl-bovine gamma-globulin (OCB-BGG) stimulated lymphocytes or in antigen-free Sephadex fractions from these supernants. After 24 hr incubation thration. The adherent cell fractions of PEC, recovered by trypsinization from monolayers and activated by this technique, were as cytotoxic as unfractionated PEC. Lymphocyte supernatants and antigen-free fractions of the supernatants induced comparable macrophage-mediated tumor cytotoxicity. Treatment of activated macrophages with trypsin did not alter their cytotoxic capacity.     

Increased susceptibility to Escherichia coli infection in mice pretreated with Corynebacterium parvum

The contribution of activated macrophages to protection against Escherichia coli was studied in mice treated intravenously with Corynebacterium parvum 7 days before infection. C. parvum-treated mice showed increased phagocytic activity and enhanced resistance to Listeria infection. In contrast, these mice showed increased susceptibility to a subsequent challenge with E. coli that correlated closely with a reduction in the LD50 of lipopolysaccharide (LPS) in these mice. The peritoneal macrophages obtained from C. parvum-treated mice had a strong ability to phagocytize and kill E. coli in in vitro experiments. A rapid decline in the number of bacteria in the liver of C. parvum-treated mice was observed in the early period of infection. However, the number of bacteria in liver and spleen increased progressively to a lethal dose from 6 hr after infection. At this time, a significant increase in beta-glucuronidase, a lysosomal acid hydrolase, was found in the serum of these mice. In vitro experiments revealed that the peritoneal macrophages from C. parvum-treated mice were highly susceptible to the cytotoxic effect of LPS after 6 hr of incubation with LPS. It is suggested that the hypersensitivity of activated macrophages to the cytotoxic effect of endotoxin derived from E. coli may be partly responsible for the increased susceptibility of C. parvum-treated mice to E. coli infection.     

Evidence for the involvement of cytolytic macrophages in rejection of SV40-induced tumors

The potential role of cytolytic macrophages in in vivo resistance to tumors induced by simian virus 40 (SV40) was evaluated in two experimental systems. First, a cell line produced by sequential in vivo passage of SV40-transformed fibroblasts through syngeneic C3H/HeJ mice was found to develop both increased neoplastic character and resistance to macrophage-mediated lysis, suggesting in vivo selection pressure against the macrophage-sensitive phenotype. In the second approach, SV40-transformed cells from C3H.OL mice, a strain that fails to produce SV40-specific cytolytic T lymphocytes (CTL), were cloned, and the cloned cells were tested for susceptibility to macrophage cytolysis in vitro. Two clones SV-COL-E8 and SV-COL-F5, which represent the extremes of macrophage susceptibility and resistance, respectively, were tested for progressive growth in syngeneic C3H.OL recipients. Progression in vivo was found to correlate with resistance to macrophage cytolysis in vitro. Other in vitro measures of the neoplastic phenotype, cell division rate and anchorage-independent growth, did not predict the relative abilities of clones E8 and F5 to form tumors. Likewise, the cells were indistinguishable in their sensitivity to cytolysis by allogeneic CTL and by natural killer cells. Finally, the presence of activated macrophages in the peritoneum of mice rejecting a challenge of syngeneic SV40-transformed cells was confirmed in both CTL responder and nonresponder strains. These studies suggest that cytolytic macrophages are indeed generated during rejection of SV40-induced mouse tumors and that, in the absence of an effective anti-SV40 CTL response, resistance of the transformed cell to macrophage-mediated cytolysis can be a determining factor in in vivo tumor growth.     

Defective tumoricidal capacity of macrophages from A/J mice. I. Characterization of the macrophage cytotoxic defect after in vivo and in vitro activation stimuli.

Macrophages from A/J mice fail to develop tumoricidal activity after any of several in vivo or in vitro treatments that activate cells from C3H/HeN mice. Peritoneal macrophages from A/J mice treated i.p. with viable Mycobacterium bovis, strain BCG, killed Corynebacterium parvum, or pyran copolymer fail to develop in vitro tumoricidal activity; varying the numbers of macrophages from treated mice added to target cells, or the dose and time of treatment, or the treatment schedule of these in vivo activation stimuli did not evoke cytotoxic activity. Moreover, cytotoxic activity by macrophages from A/J mice was not observed with any of four target cell lines derived from three different mouse strains. In vitro treatment of peritoneal exudate macrophages from A/J mice with lymphokine-rich supernatants, bacterial endotoxins, or T cell mitogens was also ineffective; varying the numbers of treated macrophages added to target cells, the dose of in vitro activation stimuli, or the time of treatment did not evoke cytotoxic activity. Thus, A/J mice exhibit a profound defect in macrophage tumoricidal capacity to both in vivo and in vitro activation stimuli over a wide range of experimental conditions.     

Allogeneic recognition and killing capacity of immune macrophages in mixed macrophage cultures (MMC).

Stimulation of DNA and RNA synthesis did not occur in mixed macrophage cultures (MMC) consisting of macrophages growing in different allogeneic combinations, compared with syngeneic cultures. Incubation of immune macrophages with either macrophages bearing those alloantigens used for immunization or unrelated alloantigens led to suppression of ³HTdR incorporation. Specific killing, studied by ⁸⁶Rb uptake, was effected by immune macrophages growing in contact with target macrophages bearing the sensitizing alloantigens. Repeated immunization was found to be important for optimal macrophage cytotoxic capacity. Cell crowding was important for maximum killing effect, and no killing occurred when immune macrophages were separated from the specific allogeneic target cells. Immune spleen cells were capable of arming nonimmune macrophages and rendering them cytotoxic. This suggests that macrophage cytotoxicity may be due to a product(s) derived from lymphocytes and attached to the macrophage surface.     

Rejection of murine ovarian cancer following treatment with regional immunotherapy: correlations with a neutrophil-mediated activation of cytostatic macrophages

Rejection of the murine ovarian teratocarcinoma (MOT) in C3HeB/FeJ mice, following intraperitoneal (ip) treatment with Corynebacterium parvum (C. parvum), is abrogated by injections of silica. We, therefore, investigated whether C. parvum-elicited macrophages affect MOT targets in vitro. Tumor-cytostatic, but not cytolytic, macrophages were detected in normal and tumor-challenged mice treated with C. parvum. The dose responsiveness and kinetics of macrophage activation strongly correlated with tumor rejection. A pyridine extract of C. parvum, possessing greatly diminished tumor rejection properties, was significantly less effective in activating macrophages. Cytostatic macrophage activation and prevention of tumor outgrowth also followed treatment in C3H/HEJ mice, a strain with a known deficiency in cytolytic macrophage function. Peritoneal neutrophils, obtained 6 hr after treatment with C. parvum, were capable of activating cytostatic macrophages when reinjected ip into normal mice. These results indicate a critical role for tumor cytostatic macrophages in this immunotherapy model and suggest their activation is mediated by inflammatory neutrophils.     

Effect of calcitonin on DNA synthesis in experimental wounds.

3H-thymidine incorporation and intracellular distribution in epidermal cells and fibroblasts of scar tissue from wounded rabbits were studied following synthetic salmon calcitonin administration. Both scintillation counting and electron microscopic autoradiography revealed a marked increase in 3H-thymidine incorporation within nuclear dense chromatin of epidermal and fibroblasts cells, mainly in 2-day wounded rabbits and at 2.5 hr. Then, 3H-thymidine uptake decreased moderately. These findings demonstrate an important role of calcitonin on DNA synthesis.     

In vitro sensitization of human lymphoid cells to antigens on cultured melanoma cells. I. Culture conditions resulting in augmented cell-mediated cytotoxicity

Culture conditions have been established that result in the sensitization of normal human peripheral blood lymphoid cells on allogeneic melanoma monolayers. Optimal culture conditions require 2 to 8 × 10⁶ mitomycin C treated stimulator melanoma cells to sensitize 5 to 10 × 10⁶ responder lymphoid cells. Neither rocking nor refeeding of the culture is necessary for the sensitization procedure. Stimulator cells grown in either fetal calf serum or human serum will serve as effective stimulator or target cells. Peak cytotoxic activity was detected at 44 hr in a microcytotoxicity assay, although some cytotoxic activity was detectable at 24 hr.     

Macrophage heterogeneity in tumor resistance: Cytostatic and cytotoxic activity of corynebacterium parvum-activated and proteose peptone-elicited rat macrophages against Moloney sarcoma tumor cells

Peritoneal macrophages from proteose peptone and Corynebacterium parvum (CP)-treated Lewis and Brown Norway rats were separated into subpopulations by centrifugation on discontinuous gradients of Ficoll. Four macrophage subpopulations were prepared and tested for cytostatic and cytotoxic activity against syngeneic and allogeneic Moloney sarcoma tumor cells. Macrophages were cocultured with tumor cells for 48 hr, whereupon either the inhibition of [¹²⁵I]iododeoxyuridine uptake was measured (cytostasis) or the tumor monolayers were observed for cytotoxic effects. CP-Activated macrophages from heavy-density portions of the gradient (8–10% and 10%-pellet) were highly cytostatic and cytotoxic to both the syngeneic and allogeneic tumor cells while macrophages from the light-density portions (4–6 and 6–8% Ficoll bands) were not. Proteose peptone-stimulated macrophages from the heavy-density portions of the gradient were cytostatic but not cytotoxic to the tumor cells. The effector macrophages from the CP-activated pool were large, well-differentiated cells as determined by electron microscopic examinations and had enhanced phagocytic activity when contrasted with the noncytotoxic, less dense macrophages.     

Macrophage stimulation by bacterial lipopolysaccharides. III. Selective unresponsiveness of C3H/HeJ macrophages to the lipid A differentiation signal.

Peritoneal macrophages from the endotoxin-unresponsive C3H/HeJ substrain of mice were entirely refractory to activation in vitro by protein-free LPS, a defect that was not overcome by co-culture of spleen cells from the responder C3H/St substrain with LPS resistant C3H/HeJ macrophages. The defect in responsiveness appears confined to the lipid A activation signal since C3H/HeJ macrophages were fully activated after in vitro treatment by lipid A protein (LAP)--LPS complex, isolated LAP, and BCG. Moreover, after exposure to allogeneic tumor cells in vivo, C3H/HeJ macrophages were cytotoxic for tumor target cells in vitro. By contrast, macrophages from the responder C3H/St strain were fully activated by protein-free LPS to become cytolytic for tumor cells in vitro. C3H/HeJ macrophages, therefore, exhibit a highly selective defect characterized by unresponsiveness to the lipid A activation signal of protein-free LPS and resistance to the toxic effects of high concentrations of LPS that were lethal to the responder C3H/St strain.     

Requirement for macrophages in lipopolysaccharide-stimulated mixed thymocyte cultures.

Cortisone-resistant thymocytes are stimulated by allogeneic thymocytes in the presence of lipopolysaccharide or small numbers of syngeneic, allogeneic or third party macrophages, as measured by ³H-thymidine uptake and by the generation of specifically cytotoxic effector cells. Lipopolysaccharide (LPS) has no stimulating effects on macrophage-depleted mixed thymocyte cultures. It also has no stimulating effect on syngeneic thymocytes in the absence of allogeneic thymocytes.Macrophages and lipopolysaccharide are only required in the induction phase of mixed thymocyte reactions. The possible role of both lipopolysaccharide and macrophages in lymphoproliferative reactions involving pure thymocyte populations is discussed.     

Cell-mediated cytotoxicity against virus-infected target cells in humans. II. Interferon induction and activation of natural killer cells.

The mechanisms by which human lymphocytes lyse virus-infected allogeneic fibroblast cultures were analyzed with particular consideration of the role of anti-viral antibodies and interferon. Human cells infected with viruses were able to induce high levels of interferon upon contact with human lymphocytes. Interferon, whether produced by lymphocytes after direct infection with virus or induced upon exposure of lymphocytes to virus-infected fibroblasts, appeared to be responsible for enhancing the cytotoxic efficiency of the natural killer cell against the infected target. Activation of cytotoxic lymphocytes occurred as early as 6 hr after addition of interferon and increased up to 24 hr. Antibody-dependent cell-mediated cytotoxicity (Ab-CMC) could be easily induced by sensitization of infected target cells with antiviral antibodies and could be detected at 4 hr from the beginning of the cytotoxic test, before the effect of interferon on the natural killer cell was evident. However, the antibody-dependent effector cell was inactive after 4 hr of incubation. F(ab')2 fragments of rabbit anti-human IgG completely inhibited Ab-CMC but did not at all affect the spontaneous cytotoxic activity of the effector cells against virus-infected target.