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Biomedical subjects

L Varesio

Publications and source records attributed to L Varesio.

At least 91 records · Page 5Linked to original sources
PubMed1988

Differential in vitro modulation of suppressor and antitumor functions of mouse macrophages by lymphokines and/or endotoxin.

Peritoneal macrophages of normal mice exhibited natural suppressor activity, as indicated by their ability to inhibit the proliferation of spleen cells in response to stimulation with phytohemagglutinin (PHA) or concanavalin A (Con A). Their suppressor function could be modulated in vitro with a variety of treatment regimens. High-dose lipopolysaccharide (LPS) (LPSH; 10 micrograms/ml) or lymphokines (supernatant from Con A-stimulated spleen cells) plus low-dose LPS (LPSL; 10 ng/ml) caused a reduction in the suppressor activity of adherent peritoneal macrophages. In contrast, these same treatments induced the macrophages to become tumoricidal and cytostatic for tumor cells, indicating a major dissociation between the regulation of suppressor and cytotoxic activities of macrophages. The lack of correlation between these activities was further demonstrated by macrophages that had been activated in vitro by Corynebacterium parvum: these cells expressed high tumoricidal and cytostatic activities, and also strong suppressor activity. The suppressor function could be selectively downregulated by in vitro pretreatment with LPSH.

Adjuvants, Immunologic↗
PubMed1987

Erythroid differentiation and modulation of c-myc expression induced by antineoplastic drugs in the human leukemic cell line K562.

The human leukemia cell line K562 expresses constitutively high levels of c-myc mRNA and can be induced to differentiate along the erythroid lineage. Treatment of K562 cells with the antineoplastic drugs 1-beta-D-arabinofuranosylcytosine and daunomycin causes differentiation into hemoglobin-producing cells. The differentiation process is associated with an early block of cellular proliferation occurring during the first 24 h of treatment. RNA synthesis is progressively reduced to 20 to 30% of the control levels after 3 days of exposure to the drugs. Dot and Northern blot analyses were performed to evaluate the levels of c-myc or globin mRNA during the differentiation of K562. Daunomycin and 1-beta-D-arabinofuranosylcytosine, despite their distinct chemical nature, induced similar modulation of mRNA levels. Globin mRNA did not change during the first 24 h of culture and began to increase after 48 h of treatment with drugs, reflecting the kinetic of appearance of hemoglobin-producing cells. In contrast, a transient decrease of c-myc mRNA was observed after the first 24 h of drug treatment, followed by a return to normal levels of c-myc mRNA after 48 h of treatment. Thus, the expression of c-myc mRNA in K562 did not reflect their proliferative activity nor their stage of differentiation. We speculate that the transient down-regulation of c-myc mRNA may be an initial event in the erythroid differentiation of K562.

Cell Differentiation↗
PubMed1987

Interferon-alpha, -beta, and -gamma augment the levels of rRNA precursors in peritoneal macrophages but not in macrophage cell lines and fibroblasts.

We have studied the ribosomal RNA content in murine peritoneal macrophages activated to cytotoxicity with interferon-alpha (IFN-alpha), interferon-beta (IFN-beta), and interferon-gamma (IFN-gamma). Total RNA was purified from IFN-alpha, -beta, or -gamma activated macrophages, and Northern blot analysis was performed by using subcloned fragments specific for precursor rRNA (pre-rRNA) as probes. All types of interferon (alpha, beta, gamma) at the doses normalized for induction of cytotoxic activity caused accumulation of 45S, 41S, and 36S pre-rRNA. In contrast, the levels of 32S pre-RNA and 28S, 5.8S, and 18S mature rRNA were not affected by IFN treatment. Therefore, the accumulation of pre-rRNA in macrophages activated to cytotoxicity by IFN was not paralleled by changes in total content of mature rRNA. No accumulation of pre-rRNA upon IFN treatment was found in a murine fibroblast cell line (L929 cells) or in a macrophage cell line (GG2EE) immortalized from bone marrow of C3H/HeJ mice. Neither of those cell lines become cytotoxic in response to IFN. Overall, our data support the concept that IFN acts selectively on the mechanism controlling the levels of some pre-rRNA and that the mechanism of IFN action involves the post-transcriptional control of ribosomal gene expression.

Animals↗
PubMed1987

Selective inhibition of 28S ribosomal RNA in macrophages activated by interferon-gamma or -beta.

We have investigated the metabolism of RNA in mouse peritoneal exudate macrophages activated by interferon (IFN)-gamma or -beta. Both species of IFN induce cytotoxic activity in macrophages. We observed a decrease in the incorporation of [3H]-uridine into total RNA in macrophages treated with doses of IFN that induce cytotoxic activity. IFN-gamma was 100-fold to 1000-fold more potent that IFN-beta in inhibiting RNA synthesis. [3H]Uridine-labeled RNA was purified from IFN-activated and control macrophages and was size fractionated on agarose gels. Macrophages activated by either IFN-gamma or IFN-beta had an imbalanced accumulation of 28S ribosomal RNA compared with their accumulation of 18S ribosomal RNA. Pulse-chase experiments suggested that IFN induced a selective inhibition of the processing of 28S ribosomal RNA. These results provide the first evidence that IFN can modulate ribosomal gene expression at the post-transcriptional level. Moreover, they indicate that inhibition of 28S ribosomal RNA accumulation in macrophages is a molecular event triggered by IFN-gamma, as well as IFN-beta.

Animals↗
PubMed1987

Generation of a murine monoclonal antibody that detects the fos oncogene product.

A hybridoma producing a monoclonal antibody (MoAB) recognizing both the cellular and viral forms of fos has been generated by somatic cell hybridization techniques from spleen cells of mice immunized with a synthetic peptide corresponding to amino acids 128-152, a consensus region, of both the v-fos and c-fos oncogene products. Three proteins with molecular weights of 55,000, 44,000, and 42,000 were detected by immunoblotting. While MoAB 2G9C3 failed to immunoprecipitate fos from Finkel-Biskis-Jenkins murine osteosarcoma-virus-infected fibroblasts, both the 55,000 v-fos protein and the 39,000 cellular protein were coprecipitated using polyvalent rabbit antibodies to the same peptide. Whereas no cell surface membrane expression of fos was detected, after membrane permeabilization by a brief exposure to lysolecithin it was possible to specifically detect internal fos by immunofluorescence flow cytometry. Immunohistochemical staining of FBJ virus-infected cells revealed intense, nuclear staining.

Animals↗
PubMed1987

A murine macrophage cell line, immortalized by v-raf and v-myc oncogenes, exhibits normal macrophage functions.

In vitro immortalized cell lines with the morphology and phenotype of mature macrophages (M phi) have been generated by infecting freshly isolated bone marrow cells from C3H/HeJ mice with a recombinant retrovirus carrying v-raf and v-myc oncogenes. All of the clones obtained had M phi-like phenotypes, and one such clone, GG2EE, has been compared to normal M phi to ascertain the effects of immortalization on the expression of the biological functions of the lines. GG2EE cells expressed cytotoxic activity against L5178Y, P815 or RL male 1 target cells in response to stimulation with interferon-gamma (IFN-gamma) and heat-killed Listeria monocytogenes; in contrast, they failed to kill YAC-1 target cells. GG2EE cells did not constitutively express I-A or I-E antigens; nevertheless, I region-coded antigens could be induced by IFN-gamma treatment. GG2EE cells produced interleukin 1 upon stimulation with a T cell-derived lymphokine; they were weakly phagocytic, yet became highly phagocytic following IFN-gamma treatment. Since c-fos mRNA is augmented in peritoneal exudate M phi by protein kinase C activators but not by IFN-gamma, we evaluated the effects of calcium ionophore, phorbol myristate acetate, L-alpha-1-oleoyl-2-acetoyl-sn-3 glycerol (OAG) and IFN-gamma on the levels of c-fos mRNA in GG2EE cells. We found that calcium ionophore, PMA and OAG stimulation enhanced the expression of c-fos mRNA, but IFN-gamma treatment did not. The kinetics of c-fos induction in GG2EE cells were also comparable to those observed in peritoneal exudate M phi. Overall, the GG2EE cell line has the same biological properties as normal tissue M phi. Because it is capable of both constitutive and inducible M phi-like functions, this cell line provides a valuable tool for studying the molecular mechanisms controlling induction and/or expression of biological activities in M phi. It is striking that a cell line immortalized in vitro by two oncogenes, v-raf and v-myc, behaves, according to the criteria mentioned above, like a normal M phi population.

Animals↗
PubMed1987

Regulation of bone marrow cell survival in short-term cultures: a new macrophage function.

The involvement of macrophages (M phi) in the regulation of bone marrow (BM) cell survival in short-term cultures was studied. We developed a system to measure the survival of fresh BM cells in vitro, by evaluating 111indium (111In) release from prelabeled BM cells. 111In release was proportional to cell death and inversely related to the number of trypan blue excluding cells. Upon 24 hr of culture in conventional medium, more than 50% of BM cells died. In order to investigate whether BM cell death could be reduced by coculture with other cell types, 111In-labeled BM cells were incubated for 24 hr with peritoneal M phi, thymocytes (THY), or polymorphonuclear cells (PMN) and then assayed for their survival. We found that coculture of BM cells with M phi dramatically increased BM survival, whereas THY or PMN consistently failed to enhance BM survival. The ability to promote BM cell survival, here designated nurse activity, represented a novel function of M phi and was further characterized. The stage of activation of M phi did not influence their nurse activity, since M phi elicited in vivo by proteose-peptone, thioglycollate, or Bacillus Calmette-Guérin, as well as resident M phi unstimulated or activated in vitro with lipopolysaccharide, equally sustained survival of BM cells. BM-derived M phi (adherent cells from BM cultures maintained in 20% L-cell-conditioned medium for 14 days) were equally effective in exerting nurse activity. Moreover, nurse activity was also exerted across the histocompatibility barriers. Supernatants from M phi cultures or killed M phi were ineffective. We propose that the nurse effect of M phi on BM is a primitive function that may play an important role in the development of the hemopoietic system.

Animals↗
PubMed1987

Augmentation of c-fos mRNA expression by activators of protein kinase C in fresh, terminally differentiated resting macrophages.

Expression of c-fos mRNA was investigated in fresh, normal peritoneal macrophages (M phi), which are terminally differentiated, nonproliferating cells. The levels of c-fos mRNA were dramatically increased by stimulation with phorbol myristate acetate (PMA), calcium ionophore, or 1-oleoyl-2-acetoyl glycerol (OAG). Induction of c-fos mRNA by all the above agents followed similar kinetics, with a peak of mRNA 30 min after stimulation. These results demonstrate that c-fos mRNA can be augmented in fresh, terminally differentiated cells. Since the stimuli increasing c-fos mRNA are direct or indirect activators of protein kinase C, our data suggest that in M phi c-fos mRNA is controlled by protein kinase C activation. PMA, calcium ionophore, and OAG were biologically active in M phi. PMA and calcium ionophore induced respiratory burst and tumoricidal activity, respectively, whereas OAG and PMA were chemotactic for M phi. Interferons beta and gamma, potent M phi activators eliciting tumoricidal activity, did not alter the levels of c-fos mRNA. These results indicate that c-fos mRNA augmentation is a stimulus-specific rather than a function-specific response connected to activation of protein kinase C.

Calcimycin↗
PubMed1987

Antiproliferative activity of picolinic acid due to macrophage activation.

Activation and subsequent enhancement of cytotoxicity of mouse peritoneal macrophages (M phi) by picolinic acid (PLA) in vivo have been reported previously by the authors' group. The optimum dose was found to be 100 mg/kg. PLA-stimulated M phi lysed different tumor targets in vitro, MBL-2 lymphoma cells and Madison 109 lung carcinoma cells, with equal efficiency. Treatment with PLA was performed daily for 5 consecutive days with a dose of 100 mg/kg intraperitoneally in C57/BL mice, which previously had been inoculated with MBL-2 tumor cells. Treatment was initiated on the first day after tumor inoculation. Oral treatment with PLA (200 mg/kg) dissolved in the drinking water was also performed for 7 days. In addition, some groups received PLA treatment 1 or 2 days before tumor implantation but not afterwards, to elucidate the in vivo efficacy of M phi activation. Intraperitoneal therapy with PLA after tumor inoculation resulted in a highly significant increase in lifespan (46%); intraperitoneal pretreatment caused a significant increase (15%); orally administered PLA was without effect. Thus intraperitoneal treatment with PLA was found to have protective and therapeutic effects against the MBL-2 ascites tumor in vivo. These effects are most likely caused by macrophage activation.

Animals↗
PubMed1986

Selective augmentation by recombinant interferon-gamma of the intracellular content of S-adenosylmethionine in murine macrophages.

Treatment of mouse peritoneal macrophages with IFN-gamma augmented the intracellular content of S-adenosylmethionine, as measured by quantitative high-performance liquid chromatography. Accumulation of S-adenosylhomocysteine, a competitive product of S-adenosylmethionine, was not detectable, either by direct measurement of absorbance or by radioisotopic techniques in IFN-gamma-treated macrophages. However, accumulation of S-adenosylhomocysteine was observed after treatment of macrophages with known inhibitors of S-adenosylhomocysteine catabolism. No inhibition of phospholipid methylation was observed upon IFN-gamma treatment, indicating that no reduction of the S-adenosylmethionine to S-adenosylhomocysteine ratio is induced by IFN-gamma in murine macrophages. The increased content of S-adenosylmethionine was associated with the acquisition of tumoricidal activity by macrophages upon IFN-gamma treatment. LPS also augmented the cellular content of S-adenosylmethionine and activated macrophages to become cytotoxic, suggesting a common mechanism of action for IFN-gamma and LPS in macrophage activation. Treatment of macrophages with cycloleucine, an agent that induces depletion of cellular S-adenosylmethionine, made the macrophages refractory to induction of cytolytic activity by IFN-gamma, suggesting a critical role for S-adenosylmethionine in macrophage activation.

Adjuvants, Immunologic↗
PubMed1986

Comparison of five short-term assays that measure nonspecific cytotoxicity mediated to tumor cells by activated macrophages.

Five different short term assays (less than 48 h) used to measure macrophage-mediated, nonspecific cytotoxicity were compared under similar conditions in the same laboratory using the same reagents. The purpose was to determine the extent to which results were comparable. Three of the assays were dependent on the release of a radioisotope to measure cytotoxicity, one was dependent on cell counting, and the last was dependent on flow cytometric quantification of remaining viable tumor target cells after they had been exposed to macrophages. The variables examined were the following: three different populations of macrophages; four different kinds of target cells; two types of radioisotopes; and two different agents that trigger the expression of cytolytic activity by primed macrophages. Recombinant gamma interferon was used as the priming agent in all the experiments. There was unexpectedly good agreement between the results of the various assays. No differences were found among the different macrophage populations, the isotopes or the triggering agents. Perhaps the most important finding was that differences in target cell susceptibility to killing by activated macrophages, which were apparent in assays of less than 24 h duration, disappeared when the same kinds of targets were compared in assays of greater than 40 h duration. The results of this study are an important first step toward standardizing the way in which macrophage-mediated, nonspecific cytotoxicity is measured in short-term assays, laboratory to laboratory.

Cell Line↗
PubMed1986

Posttranscriptional control of human gamma interferon gene expression in transfected mouse fibroblasts.

Human gamma interferon genomic DNA was introduced into NIH 3T3 fibroblasts by calcium phosphate precipitation and was not expressed in these cells at the cytoplasmic mRNA or protein level. Treatment of the transfected cells with cycloheximide (1 microgram/ml) induced the accumulation of cytoplasmic gamma interferon mRNA and biologically active human gamma interferon. Analysis of the nuclear enriched RNA from untreated cells indicated that human gamma interferon mRNA was present, suggesting that cycloheximide may act by inhibiting a specific nuclease or may enhance the processing or transport of the RNA from the nucleus to the cytoplasm.

Animals↗
PubMed1985

The strain of mouse and assay conditions influence whether MuIFN-gamma primes or activates macrophages for tumor cell killing.

Investigators from two different laboratories have compared several variables in the short-term macrophage-mediated cytotoxicity assays used by each group to study the role of MuIFN-gamma in macrophage activation. The findings suggest that the capacity of MuIFN-gamma to activate macrophages without the need for a second triggering stimulus is related to assay conditions and, most especially, the strain of mouse used to provide the macrophages.

Animals↗
PubMed1985

Hyporesponsiveness to augmentation of murine natural killer cell activity in different anatomical compartments by multiple injections of various immunomodulators including recombinant interferons and interleukin 2.

Augmentation of natural killer (NK) cell activity has been observed after the single administration of a wide variety of biological response modifiers (BRM); however, multiple injections of BRM have resulted in hyporesponsiveness to NK augmentation in both preclinical and clinical studies. In these studies, hyporesponsiveness to augmentation of NK cell activity occurred after multiple injections of interferon (IFN recombinant human IFN-alpha A/D and recombinant IFN-gamma) and interleukin 2 and was found to be systemic (lungs, liver, blood, and spleen). In contrast, hyporesponsiveness to augmentation by multiple injections of maleicanhydride divinyl ether (MVE-2) or Propionibacterium acnes was limited to the spleen and peripheral blood lymphocytes, with continued augmentation of NK cell activity in the peritoneum, lungs, and liver. Despite the hyporesponsiveness to augmentation of NK activity by multiple IFN injections, NK activity could still be augmented by a single injection of another BRM. The NK cell hyporesponsiveness induced in the spleen by MVE-2 was also reversed by a single administration of IFN or polyinosinic-polycytidylic and poly-L-lysine solubilized by carboxymethyl cellulose but not by OK-432 or P. acnes. These results demonstrate that the nature of the hyporesponsiveness to NK augmentation, which is induced by multiple treatments with BRM, varies with the type of agent. The noncytokine BRM that were studied induced hyporesponsiveness only in specific lymphoid compartments but not in major nonlymphoid organs, whereas cytokine BRM induced a systemic hyporesponsiveness. The hyporesponsive state induced by the different types of BRM, also varied in regard to the pattern of susceptibility to augmentation of NK activity by unrelated BRM.

Adjuvants, Immunologic↗
PubMed1985

Imbalanced accumulation of ribosomal RNA in macrophages activated in vivo or in vitro to a cytolytic stage.

Previous studies have shown that peritoneal murine macrophages activated in vivo and in vitro to a tumoricidal stage have a depressed rate of RNA synthesis. In attempting to clarify the differences in RNA metabolism between noncytotoxic and tumoricidal macrophages, we have studied the relative accumulation of various species of RNA in macrophages activated in vivo and in vitro with the use of agarose gel electrophoresis. Macrophages activated in vitro to a cytotoxic stage with supernatants containing lymphokines (LK) and traces of lipopolysaccharide (LPS) have an imbalanced accumulation of mature ribosomal RNA (rRNA), with a decreased accumulation of 28S rRNA compared to 18S rRNA. In contrast, macrophages primed in vitro with LK free of detectable endotoxins that exhibit suppressive rather than tumoricidal activity do not manifest a decreased 28S:18S rRNA ratio. The conclusion that the decreased 28S:18S rRNA ratio was associated with the activation of macrophages to a cytolytic stage was supported by the finding that cytotoxic macrophages activated in vivo by i.p. injection of Propionibacterium acnes (formerly designated C. parvum) also demonstrated a decreased accumulation of 28S comparable with that observed in in vitro-activated macrophages. Moreover, activated macrophages that lost their cytolytic activity upon prolonged in vitro culture had an augmented accumulation of 28S rRNA. These results provide the first direct evidence that the expression of cytolytic activity is associated with modulation of a specific class of RNA. The unbalanced accumulation of rRNA appears to be a late molecular event in the activation process occurring during the transition from primed to cytotoxic macrophages, because inflammatory and primed macrophages had normal rRNA accumulation. A model of macrophage activation accounting for these results is proposed.

Animals↗
PubMed1984

Role of protein synthesis in the activation of cytotoxic mouse macrophages by lymphokines.

The role of protein synthesis during the activation of macrophages (M phi) by lymphokines (LK) was studied. Peritoneal murine macrophages elicited by proteose-peptone (pM phi) 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 pM phi-mediated cytotoxicity and pM phi 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 pM phi 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 pM phi 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 pM phi to LK, but that new proteins have to be synthesized to allow the expression of the cytotoxic activity in LK-activated pM phi.

Animals↗
PubMed1984

Lymphokines inhibit macrophage RNA synthesis.

The effects of lymphokine (LK) preparations on the incorporation of [3H]uridine into macrophage RNA were investigated. Supernatants from murine spleen cells activated in vitro by alloantigens or Con A, and shown to contain macrophage-activating factor (MAF), were used as the source of LK. It was observed that such LK preparations contain factor(s) causing a profound inhibition of [3H]uridine incorporation into the RNA of proteose-peptone-elicited peritoneal macrophages. Such RNA-labeling inhibitory factor (RIF) was absent in control supernatants from nonstimulated cultures, and showed activation curves similar to that of MAF. RIF activity was not due to altered permeability of macrophages to [3H]uridine nor to the changes in the specific activity of the pool of RNA precursors, but rather reflected an altered metabolism of RNA. The inhibition of RNA synthesis was dependent upon the presence of nanogram amounts of LPS as a costimulator. Moreover, the response to RIF appeared to be genetically controlled since macrophages from C3H/HeJ mice were not affected by RIF, while C3H/HeN mice were fully responsive. In parallel cultures of macrophages, LK were also tested for their MAF activity, and a strong similarity between the biological conditions in which MAF and RIF activities were expressed could be demonstrated. The assay for RIF provides a new and convenient parameter for measuring macrophage-sensitive LK activity that might be very useful for monitoring purification or for screening of T-cell hybridoma supernatants.

Animals↗