In vivo and in vitro effect of trehalose dimycolate on tumoricidal activity of peritoneal macrophages from rats

Trehalose dimycolate (TDM), a well-defined component of Mycobacterium tuberculosis cell wall has been tested in vivo and in vitro for its effect on the tumoricidal activity of Rat peritoneal macrophages. Macrophages could be rendered cytolytic against syngeneic tumor cells by an intraperitoneal injection of an aqueous suspension of TDM. However, as we failed to render them tumoricidal in vitro, we consider that the activation process is not due to a direct effect on macrophages.     

In vitro modulation of macrophage tumoricidal activity

Summary NaIO₄ treatment of mouse adherent peritoneal cells or lymphocyte-free cloned macrophages enhances their cytotoxic and tumoricidal activity. 5×10^–3 M NaIO₄ treatment of nontumoricidal BCG-activated macrophages renders them completely tumoricidal, whereas the same treatment of stimulated (peptone-normal) macrophages renders them weakly tumoricidal. Addition of LPS in nanogram quantities too low to enhance tumor cell killing by untreated peptone-normal macrophages causes NaIO₄-treated peptone-normal macrophages to be maximally tumoricidal. The activating action of NaIO₄, MAF, or LPS can be potently, but inconsistently, blocked or reversed by the reducing agent NaBH₄ or the aldehyde-reacting agent dimedone. NaIO₄ treatment of lymphocyte-free macrophage colonies does not make them cytotoxic, but NaIO₄-treated colony macrophages are cytotoxic for tumor cells when cultured in 10 ng/ml LPS (an amount of LPS inadequate to render untreated colony macrophages cytotoxic). Supernatants of NaIO₄-treated adherent peritoneal cells contain MAF activity. Thus, the NaIO₄-induced enhancement of peritoneal cell tumoricidal activity may result from both direct NaIO₄ activating effects on macrophages and indirect NaIO₄ effects through NaIO₄-induced MAF production.     

Induction of the tumoricidal activity of human and murine peritoneal macrophages under the action of antitumor chemical preparations

Platidiam, cyclophosphamide and adriamycin induced tumoricidal activity of peritoneal macrophages from patients with disseminated ovarian carcinoma when applied in the autologous tumor cells in vitro. This effect was not observed with 10 micrograms/ml concentration of 5-fluorouracil. The mice peritoneal macrophages after incubation in vitro with 0.01-1.0 micrograms/ml of aclarubicin showed cytostatic action on syngeneic and semisyngeneic P388 cells. The peritoneal macrophages from mice treated with 2.5 mu/kg of aclarubicin intraperitoneally 1-4 days before were cytotoxic for tumor cells too.     

In vitro tumor cell killing by peritoneal macrophages from mitomycin C-treated rats

Summary The local cellular response induced by intraperitoneal injection of mitomycin C was examined in terms of cell-mediated cytotoxicity for tumor cells. An in vitro cytolysis assay involving ¹²⁵I-iododeoxyuridine-labeled tumor target cells revealed that treatment of normal ACI/N rats (200 g) with a single intraperitoneal injection of mitomycin C (50, 100, or 200 g) induced tumoricidal macrophages in the peritoneal cavity. The tumoricidal activity was dependent on the dose of mitomycin C injected and it was detectable as early as 1 day after the intraperitoneal injection of mitomycin C. In addition to the increased tumoricidal activity, the functional activities of the peritoneal macrophages were found to be increased with respect both to uptake of 2-deoxy-d-glucose and to phagocytosis of latex beads. Additional experiments excluded the possibility that the tumor cell cytolysis was the result of direct cytotoxicity by mitomycin C that might have been incorporated in the peritoneal macrophages or of nutrient depletion in the medium during the cytolysis assay. Furthermore, endotoxin contamination of the mitomycin C, which might have produced the activated macrophages, was not detected. The mechanism by which mitomycin C injected intraperitoneally induced the tumoricidal macrophages locally remains uncertain; however, it is possible also in clinical situations.     

In vitro cytotoxicity of peritoneal macrophages activated with Mycobacterium smegmatis

Incorporation of 3H-TdR into EL4 leukemic cells in vitro was inhibited by peritoneal exudate cells (PEC) harvested from syngeneic C57BL/6J mice given an intraperitoneal (i.p.) injection of 1x10(7) viable Mycobacterium smegmatis ATCC 607 (Smeg) 4 days before. This phenomenon was also observed in the following five systems of PEC from animals and syngeneic tumor cells: C57BL/6J mice and B16 melanoma; DBA/2 mice and P815 mastocytoma; SWM/Ms mice and K5 fibrosarcoma; BALB/c, nu/nu mice and KKN-1 fibrosarcoma; and strain 2 guinea pigs and line-10 hepatoma. The in vitro cytotoxicity of the PEC activated by viable Smeg was much higher than those activated by dead-Smeg, viable BCG or proteose peptone. The activity of the adherent fraction of the PEC was stronger than that of the nonadherent one, and not influenced by either anti-theta or anti-mouse lymphocyte rabbit sera. The PEC induced with Smeg 4 days before contained a large population of mononuclear cells (88.9%) and a significant level of polymorphonuclear cells (PMN) (3.2%), and showed a much higher cytotoxicity than the PEC induced with Smeg 3 hr before, which contained a much larger population of PMN (71.9%), suggesting that PMN were not the effector cells in this system. In vitro and in vivo treatment with macrophage-inhibitors such as carrageenan, trypan blue and cytochalacin B, reduced the activity of the PEC. All of these facts suggested macrophages as the effector. Viable macrophages were required for the growth inhibition of EL4 in vitro: gamma-ray irradiated or freeze-thawed macrophages were ineffective. Kinetic studies revealed that inhibition of 3H-TdR incorporation into EL4 cells started within 3 hr of incubation together with the activated macrophages at an effector to target (E/T) ratio of 5, and the incorporation decreased gradually with the lapse of incubation time. On the other hand, 51Cr release from labelled EL4 was undetected when the E/T ratio was 5 but detected at on E/T of 10 or more. Even at the higher E/T ratio, at least 10 hr were needed until the release of 51Cr, suggesting that the activated macrophages produced growth inhibition of tumor cells followed by cell destruction.     

Interaction of rat peritoneal macrophages with homologous, sialidase-treated lymphocytes in vitro

The interaction in vitro between rat peritoneal macrophages and homologous, sialidase-treated lymphocytes was investigated. Lymphocytes were isolated from blood, thymus, and spleen on a density gradient. Total sialic acids obtained by acid hydrolysis were 10 nmol/10(8) lymphocytes, composed of 29% N-acetyl-neuraminic acid and 71% N-glycoloylneuraminic acid. Sialidase treatment released maximally 33% of membrane sialic acids. Lymphocytes were bound to peritoneal macrophages to an extent which increased in parallel with the amount of sialic acids released, whereas binding of untreated lymphocytes was not significant. This interaction was inhibited by free galactose and substances containing terminal galactose residues. Asialoorosomucoid with its oligoantennary sugar chains proved to be a 10(5) times more potent inhibitor of the interaction than lactose. The addition of homologous serum had no influence on binding. Electron microscopy revealed that vital lymphocytes were tightly bound to macrophages and only damaged lymphocytes appeared to be phagocytozed. The experiments demonstrate that the interaction between rat peritoneal macrophages and sialidase-treated lymphocytes is mediated by a macrophage receptor specific for galactose. This sugar is demasked on the surface of lymphocytes after the removal of terminal sialic acids. The role of this mechanism in cell recognition, elimination and homing of lymphocytes is discussed.     

In vitro effect of diacetoxyscirpenol and deoxynivalenol on microbicidal activity of murine peritoneal macrophages

Diacetoxyscirpenol and deoxynivalenol, two trichothecene mycotoxins shown previously to exert immunosuppressive effects on the immune system were examined for their in vitro effects on some functions of murine peritoneal macrophages. The cells were pre-incubated for 4 hr with the mycotoxin concentrations of 0.1 ng/ml–1 g/ml. At concentrations that did not affect the cell viability (Specific Lactate Dehydrogenase test), diacetoxyscirpenol and deoxynivalenol suppress microbicidal activity of phagocytic cells. The diacetoxyscirpenol concentrations, which reduce phagocytosis (2ng/ml), microbicidal activity (1 ng/ml), superoxide anion production (1 ng/ml) and phagosome-lysosome fusion (0.1 ng/ml), indicate that the inhibition of killing mechanism arise from both oxidative and non-oxidative pathways. Phagocytosis, microbicidal activity and superoxide anion production are inhibited by deoxynivalenol at 1 ng/ml whereas phagosome-lysosome fusion is reduce above 100 ng/ml. These results suggest that microbicidal activity inhibition by deoxynivalenol did not depend on non-oxidative pathway (phagosome-lysosome fusion) impairment.     

In vitro cytochemical and cytophysiological study of in vivo activated mouse peritoneal macrophages

The morphological, cytochemical (acid phosphatase activity) and cytophysiological (phagocytosis) features of mouse peritoneal macrophages activated in vivo by two bacterial agents. Corynebacterium parvum parvum and Polidin, were investigated in vitro. Both immunostimulants induced an increase in cytochemical and phagocytic activities of the activated peritoneal macrophages but in different degrees, the changes being more extensive in the case of Corynebacterium parvum-activated macrophages.     

In vitro multiplication and apparently indefinite subcultures of normal mouse resident peritoneal macrophages

Multiplication of Balb/C mouse resident peritoneal macrophages was observed, when all the cells collected in the washing fluid of the peritoneal cavity were seeded in appropriate culture medium. This multiplication began only after the appearance of a confluent monolayer of flat cells, referred to as "mesothelial" cells. The macrophages produced passed from the "mesothelial" cell layer to the suspension and could be passaged indefinitely. Each culture underwent an identical cycle of events which always included the appearance of a confluent monolayer of "mesothelial" cells. The cells transferred were characterized by their ability to phagocytose yeasts, and by the presence of Fc receptors and cytoplasmic non-specific esterases.     

In vitro cytochemical and cytophysiological study of in vivo BCG-activated mouse peritoneal macrophages

The morphological, cytochemical (acid phosphatase activity) and cytophysiological (phagocytosis) features of mouse peritoneal macrophages activated in vivo by BCG, were investigated in vitro. BCG induced an increase in cytochemical and phagocytic activities of the activated peritoneal macrophages.     

Separation of soluble amoebicidal and tumoricidal activity of activated macrophages.

Macrophage-conditioned medium (M phi CM) prepared from mouse peritoneal macrophages activated in vivo with bacillus Calmette-Guérin (BCG) or Propionibacterium acnes and triggered with lipopolysaccharide in vitro contained tumoricidal and amoebicidal activity. The murine fibroblast cell line L929 was used as the indicator of tumoricidal activity and Naegleria fowleri amoeba was used to detect amoebicidal activity in M phi CM. The protease inhibitor, soybean trypsin inhibitor, decreased tumoricidal activity but had little effect on amoebicidal activity in M phi CM. Anti-TNF alpha antiserum inhibited tumoricidal activity in M phi CM. The antiserum reduced amoebicidal activity in BCG-activated M phi CM but had no effect on amoebicidal activity in P. acnes-activated M phi CM. Recombinant TNF alpha, rIL-1 alpha, or rIL-1 beta independently did not affect cytolysis of amoebae. Also, rTNF alpha had no effect on the growth of amoebae. Preparative flat-bed electrofocusing of BCG-activated M phi CM yielded fractions that exhibited different amoebicidal and tumoricidal activity profiles. Three domains of activity were analyzed (acidic, neutral, and basic). Anti-TNF alpha antiserum eliminated tumoricidal activity, but not amoebicidal activity, in fractions from the acidic domain. A combination of anti-TNF alpha and anti-IL-1 alpha antisera failed to eliminate amoebicidal activity in fractions from the basic domain. These results indicate that different factors are responsible for macrophage amoebicidal and tumoricidal activity. The amoebicidal factors in M phi CM affected cytolysis of several species of amoebae.     

In vitro suppression of the phagocytic response of fish macrophages by tetracyclines

Tetracycline (TC) and oxytetracycline (OTC) caused a dose-dependent suppression of the chemiluminescence (CL) emitted by phagocytes from the kidney of rainbow trout, Salmo gairdneri , using zymosan or latex beads as the stimulus. Compared to the control response without antibiotics, partial but significant suppression was found after exposing the cells to OTC concentrations of 0.1–50.0 μg ml⁻¹. Cells exposed to 100 or 500 μg ml⁻¹ OTC showed CL responses below the base levels elicited by control cells. Comparable results were obtained with cells exposed to TC and stimulated by the same stimuli. The kinetics of the CL response and the suppressive effects of the antibiotics were similar in cells from individual fish but the magnitude of responses varied. No acclimation occurred following extended exposure to the drugs.     

In vitro interactions of murine peritoneal macrophages and sarcoma cells. II. Induction of DNA synthesis in macrophages

Unstimulated peritoneal macrophages grown in vitro for 5 days do not incorporate thymidine. Sarcoma (AA) cells introduced to the culture on the fifth day die and disintegrate whereas the macrophages become markedly stimulated and autoradiography shows that they are triggered to synthesize DNA. In syngeneic cocultures up to 28% of the macrophages incorporate thymidine after 5 days of coculture.     

Ultrastructure and phagocytic activity of rat peritoneal macrophages exposed to low temperatures in vitro

Hypothermia affects various components of the immune system, leading to impaired immune resistance. To examine the in vitro effect of low temperature on the ultrastructure and phagocytic function of rat peritoneal macrophages, cells were incubated at 4, 10, 24, and 37 degrees C for 60 min. Subsequently, their ultrastructure and capacity to engulf latex particles and generate superoxide anions were evaluated. The results showed a close inverse relationship between incubation temperature and ultrastructural changes, i.e., the lower the temperature, the higher the number of altered cells. In addition, at lower temperatures the number of cells capable of phagocytosis was reduced; the cells engulfed fewer particles per cell and generated less superoxide anions. These findings may be relevant for explaining the increased susceptibility to bacterial infections under hypothermic conditions.     

In vitro determination of phagocytic indices of Candida berkhout species by rat peritoneal macrophages

The aims of this investigation were to study and describe the behaviour of 13 different species of Candida, as compared with C. albicans, by means of phagocytosis assays in vitro.Tests were carried out with rat peritoneal macrophages in contact with quantified suspensions of live yeasts. Phagocytic indices, candidacidal activity and filamentation rat were tested microscopically after 3 h incubation at 37 ° C.The phagocytic indices obtained allowed us to separate the fungi into four groups. Candida albicans and tropicalis belong to Group I; diddensii and shehatae, among others, belong to Group II; sake, krusei, viswanathii, etc., Group III; and C. glaebosa and haploid strains of Pichia ohmeri (C. guilliermondii var. membranaefaciens), Group IV. These data would suggest a possible correlation between pathogenesis and phagocytic indices.There were no evidences of any phagocytes ability to kill yeasts. Candidacidal activity was absent in the species assayed. Yeast lysis may have been observed if our assays would have taken longer than 3 h.     

In vitro reactivity of splenic lymphocytes from normal and UV-irradiated mice against syngeneic UV-induced tumors.

Skin tumors induced in mice by chronic ultraviolet (UV) irradiation are highly antigenic and are frequently immunologically rejected upon transplantation to normal syngeneic recipients. In this study we characterized this immune response with an in vitro microcytotoxicity test. Cytotoxic activity was present in the spleen cells of mice given a single injection of syngeneic UV-induced fibrosarcoma cells. After removal of adherent spleen cells, the remaining splenic lymphocytes were specifically cytotoxic for the immunizing tumor and showed no cross-reactivity with other syngeneic UV-induced or methylcholanthrene-induced tumors of similar histologic type. The level of cell-mediated reactivity against UV-induced tumors was quite high compared to that obtained with syngeneic tumors induced by methylcholanthrene, and the cytotoxicity was attributable to a population of theta antigen-bearing lymphocytes. With this in vitro test, we compared the response of normal mice, which reject a syngeneic tumor challenge, with that of UV-irradiated mice, in which the syngeneic UV-induced tumors grow progressively. After tumor cell inoculation, lymphocytes form the unirradiated (regressor) mice showed a high degree of cytotoxicity that reached a maximum level 8 days after injection. In contrast, no reactivity could be detected in the spleens of tumor-challenged UV-irradiated (progressor) mice.