1,721,069 research outputs found
Role of protein synthesis in the activation of cytotoxic mouse macrophages by lymphokines.
No full textThe role of protein synthesis during the activation of macrophages (Mφ) by lymphokines (LK) was studied. Peritoneal murine macrophages elicited by proteose-peptone (pMφ) were activated with LK (supernatants from normal mouse spleen cells pulsed with concanavalin A) and tested for cytotoxicity in an 18 hr assay against 111In-labeled L5178Y lymphoma target cells. Reversible (cycloheximide and puromycin) or poorly reversible (emetine and pactamycin) inhibitors of protein synthesis were added during activation, and their effects on pMφ-mediated cytotoxicity and pMφ protein synthesis were measured. Minimal concentrations of inhibitors, reducing the rate of protein synthesis by more than 90% without toxic effects on macrophages, were chosen. Exposure of pMφ to LK for 2 to 18 hr in the presence of reversible inhibitors of protein synthesis did not affect the induction of cytolytic activity, indicating that protein synthesis was not required during the activation period. In contrast, activation of macrophages for 2 hr in the presence of poorly reversible inhibitors of protein synthesis resulted in a considerable reduction of cytolytic activity. The impairment of cytotoxic activity was also evident when pMφ were treated with such drugs during the first 2 hr of an 18 hr exposure to LK or when LK-activated macrophages were treated for 2 hr with the drugs before the addition of the targets. These results demonstrate that active protein synthesis is not required during the exposure of pMφ to LK, but that new proteins have to be synthesized to allow the expression of the cytotoxic activity in LK-activated pMφ. © 1984
Inhibition of retroviral mRNA expression in the murine macrophage cell line GG2EE by biologic response modifiers
No full textWe immortalized the GG2EE macrophage (M phi) cell line by infection of freshly isolated bone marrow cells with the recombinant J2 retrovirus carrying v-raf and v-myc oncogenes. We investigated the expression of J2 virus mRNA in relationship with the proliferative ability and tumoricidal activity of GG2EE cells exposed to biologic response modifiers (BRM). Calcium ionophore (Ca2+I), picolinic acid (PA), or IFN-gamma were employed to activate GG2EE cells. Each BRM was due to inhibit the proliferation of GG2EE cells in a dose-dependent manner, whereas only Ca2+I or the combined treatment with PA plus IFN-gamma induced tumoricidal GG2EE cells. J2 virus mRNA expression was not affected by PA or IFN-gamma, but it was dramatically decreased by Ca2+I or PA plus IFN-gamma. These results indicated that the expression of J2 mRNA can be inhibited in GG2EE cells by appropriate BRM such as Ca2+I or IFN-gamma plus PA. In contrast, the expression of 2'5'-oligoadenylate synthetase mRNA was augmented to similar levels by treatment of the GG2EE cells with IFN-gamma alone or in combination with PA. The down-regulation of J2 mRNA expression was not associated with the antiproliferative activity of the BRM but rather with their ability to induce tumoricidal activity. These results suggest that the process of activation of tumoricidal macrophages also triggers a mechanism(s) of resistance to viral mRNA expression. Moreover, the finding that IFN-gamma or PA inhibit cell proliferation but not J2 mRNA expression indicates that the intracellular targets of these BRM are intact, independent from and unaffected by J2 virus expression
Tumor formation by a murine macrophage cell line immortalized in vitro by v-raf and v-myc oncogenes
No full textMurine bone marrow cells immortalized in vitro by the J2 recombinant retrovirus bearing the v-raf and v-myc oncogenes have the functional and phenotypic characteristics of macrophages. The present study was designed to determine whether these cells are tumorigenic in athymic or euthymic mice. One cloned cell line (GG2EE), that had been previously derived and characterized was used for this purpose. The results demonstrated that GG2EE cells were tumorigenic in allogeneic athymic BALB/c mice at doses of 1×104 to 1×107 cells per mouse regardless of the route administration. All mice utlimately died of progressive tumor growth. Conversely, the GG2EE cells were nontumorigenic or transiently tumorigenic in syngeneic euthymic C3H/HeJ mice. Further studies in BALB/c athymic mice demonstrated that the GG2EE cells were directly tumorigenic since ascites tumors (GG2EE-V) that developed expressed the H-2k surface phenotype of the injected GG2EE cells, excluding the possibility that the J2 virus constitutively produced by GG2EE cells caused in vivo transformation and therefore tumors of host cell origin. The in vivo passaged cells continued to express the M1/69, MAC-1, MAC-2, F4/80, Fc receptor and Ly5.1 antigens characterically expressed on the parental line. Biological properties including interferon-γ-induced Ia expression, phagocytosis, and activation for cytotoxicity were also retained following in vivo passage. These results demonstrated that J2 virus-immortalized GG2EE cells were directly tumorigenic in athymic mice in vivo and that the macrophage phenotype was maintained in these neoplastic cells. These observations suggest that this tumor model may be valuable for the study of macrophage function as well as therapeutic approaches to oncogen-expressing retrovirus-induced tumors. © 1988 Springer-Verlag
Requirement for protein synthesis for induction of macrophage tumoricidal activity by IFN-α and IFN-β but not by IFN-γ
No full textPeritoneal mouse macrophages elicited by proteose-peptone (pMΦ) were treated in vitro with IFN-α, IFN-β, or IFN-γ in the presence or absence of cycloheximide (Cy), a reversible inhibitor of protein synthesis, and were assayed for cytolytic activity against tumor cells. Inhibition of protein synthesis during treatment of pMΦ with IFN-α or IFN-β prevented the development of cytotoxic activity. In contrast, IFN-γ was fully capable of inducing cytotoxic pMΦ in the presence of Cy. Moreover, pMΦ treated with mixtures of IFN in the presence of Cy were activated for cytotoxicity only by IFN-γ together with IFN-α or IFN-β, but not by IFN-α plus IFN-β. These results indicate that the activation of pMΦ by IFN-γ is independent of new protein synthesis, whereas the activation of pMΦ by IFN-α and/or IFN-β requires active protein synthesis, suggesting that the mechanism of induction of cytotoxic pMΦ by IFN-γ differs from that by the other types of IFN
Augmentation of GG2EE macrophage cell line-mediated anti-Candida activity by gamma interferon, tumor necrosis factor, and interleukin-1.
No full textThe expression of anti-Candida activity in the GG2EE macrophage cell line, generated by immortalization of fresh bone marrow with v-raf and v-myc oncogenes, was studied. GG2EE cells spontaneously inhibited the growth of an agerminative mutant of Candida albicans in vitro. The anti-Candida activity was maximal after 8 h of coculture and was proportional to the effector-to-target ratio. Gamma interferon (IFN-gamma), interleukin-1 (IL-1), and tumor necrosis factor (TNF) all significantly enhanced the anti-Candida activity of GG2EE cells. In contrast, IL-3, IL-4, and colony-stimulating factor 1 were ineffective. The augmentation of anti-Candida activity was not always concomitant with enhancement of phagocytosis, since IFN-gamma and colony-stimulating factor 1, but not IL-1 or TNF, augmented the phagocytic ability of GG2EE cells. Furthermore, the augmentation of anti-Candida activity in GG2EE cells did not correlate with the acquisition of antitumor activity. In fact, none of the cytokines alone were able to induce antitumor activity in GG2EE cells, which, however, could be activated to a tumoricidal stage by IFN-gamma plus heat-killed Listeria monocytogenes. These findings demonstrate that GG2EE cells exhibit spontaneous anti-Candida activity and that such activity is enhanced by TNF, IL-1, and IFN-gamma
Induction of cytotoxic macrophages by γ-interferon (γIFN) is associated with intracellular accumulation of the methyl donor S-adenosyl-methionine (SAM)
No full textAntimicrobial activity of a macrophage cell line generated in vitro with v-raf/v-myc oncogenes
No full textGeneration of macrophage cell line from fresh bone marrow cells with a myc/raf recombinant retrovirus
No full textWe have studied the effects of infection of fresh murine bone marrow (BM) cells by recombinant retroviruses carrying v-raf and v-myc oncogenes, either alone or in combination. Viruses containing v-raf or v-myc alone failed to induce BM proliferation in 24 out of 27 experiments performed so far, only the J2 virus containing both v-raf and v-myc oncogenes induced BM proliferation. Exogenous growth factors (GF) were not required to sustain the mitogenic effect of J2 virus. Infection with retroviruses carrying only v-raf or v-myc did not induce BM cell growth, indicating that co-expression of the two oncogenes was needed to provide the mitogenic signal(s) for BM proliferation. The kinetics of growth of the J2 virus-infected cells (J2 cells) were characteristically biphasic. The initial burst of proliferation was always followed by a quiescent phase culminating in cell death, which could not be reversed by addition of exogenous GF. In contrast, active proliferation of the quiescent monolayers could be restored by addition of dextran-based beads to the cultures, showing that the growth arrest of J2 cells was a reversible process. J2 cells actively growing in the presence of CT-beads could be expanded and cloned and subsequently grew continuously independent of the CT-beads. Eighteen clones obtained from different infections were all macrophages (M phi) by morphological criteria and all of them expressed the same membrane phenotype compatible with M phi, demonstrating that J2 virus infection leads to immortalization of the same BM-derived monocytic subpopulation. When injected in vivo, J2 cells produced histiocytic tumors in nude mice, but did not grow in immunocompetent syngeneic mice. The cells induced to proliferate in vitro in response to J2 virus infection appeared to be limited to the BM compartment, since spleen cells, thymocytes, peritoneal M phi and liver large granular lymphocytes did not grow in vitro in response to J2 virus. The immortalization of BM cells by J2 virus infection represents a novel reproducible experimental system to deliberately generate M phi lines, which proliferate in response to viral oncogenes and do not require exogenous GF to initiate or to sustain their continuous proliferation
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