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Biomedical subjects

L Varesio

Publications and source records attributed to L Varesio.

At least 73 records · Page 4Linked to original sources
PubMed1990

Expression of human immunodeficiency virus long terminal repeat in the human promonocyte cell line U937: effect of endotoxin and cytokines.

Phagocytic macrophages are known to support noncytopathic, chronic infections of human immunodeficiency virus (HIV). Regulation of viral replication in such cells with either chronic low-grade or latent HIV infection is probably influenced by both viral and cellular factors acting on the viral long terminal repeat (LTR). This study identifies naturally occurring biological response modifiers which are able to affect the HIV-LTR linked to the chloramphenicol acetyl transferase (LTR-CAT) gene in a stable transfection of the human promonocyte cell line, U937, in the absence of other viral proteins. In this model system, endotoxin lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF-alpha) are able to independently stimulate expression of LTR-CAT. Granulocyte/macrophage-colony-stimulating factor can enhance the effect of TNF-alpha or LPS, but other cytokines tested had minimal or no effect on LTR-CAT. In addition to effects on cellular susceptibility and immune function, the ability of naturally occurring factors to affect HIV-LTR in its integrated state may have particular relevance to progression of active disease from latent infection.

Biological Factors↗
PubMed1990

Immortalization of macrophages from mouse bone marrow and fetal liver.

Fresh bone marrow (BM)-derived cells infected with the J2 recombinant retrovirus (carrying v-myc and v-raf/mil oncogenes) grow as immortal cell lines belonging to the monocytic lineage. BM cells cultured for 24 h in conventional medium are no longer able to grow following infection with the J2 virus. We investigated whether specific growth factors affected the proliferative response of BM cells to the J2 virus. If the BM cells were cultured for 24 h in the presence of concanavalin A or CSF-1 and then infected with the J2 virus, immortalization of BM cells was observed. Under these conditions, the cell lines that we obtained were shown to belong to the monocytic lineage. We investigated whether target cells for the J2 virus existed in other hematopoietic organs. We observed J2-induced proliferation in fetal liver (FL) but not in spleen or thymus. The cells proliferating in the FL had macrophage characteristics during the early passages. However, some macrophage markers were lost upon extensive in vitro culture. We conclude that we have identified conditions in which J2 virus consistently and selectively stimulates the growth of macrophages from murine bone marrow and a wider range of hematopoietic cells from fetal liver.

Animals↗
PubMed1990

Macrophage-colony-stimulating factor (CSF-1) induces proliferation, chemotaxis, and reversible monocytic differentiation in myeloid progenitor cells transfected with the human c-fms/CSF-1 receptor cDNA.

The c-fms protooncogene encodes the receptor for macrophage-colony-stimulating factor (CSF-1). Expression vectors containing either normal or oncogenic point-mutated human c-fms genes were transfected into interleukin 3 (IL-3)-dependent 32D cells in order to determine the effects of CSF-1 signaling in this murine clonal myeloid progenitor cell line. CSF-1 was shown to trigger proliferation in association with monocytic differentiation of the 32D-c-fms cells. Monocytic differentiation was reversible upon removal of CSF-1, implying that CSF-1 was required for maintenance of the monocyte phenotype but was not sufficient to induce an irrevocable commitment to differentiation. Human CSF-1 was also shown to be a potent chemoattractant for 32D-c-fms cells, suggesting that CSF-1 may serve to recruit monocytes from the circulation to tissue sites of inflammation or injury. Although c-fms did not release 32D cells from factor dependence, point-mutated c-fms[S301,F969] (Leu-301----Ser, Tyr-969----Phe) was able to abrogate their IL-3 requirement and induce tumorigenicity. IL-3-independent 32D-c-fms[S301,F969] cells also displayed a mature monocyte phenotype, implying that differentiation did not interfere with progression of these cells to the malignant state. All of these findings demonstrate that a single growth factor receptor can specifically couple with multiple intracellular signaling pathways and play a critical role in modulating cell proliferation, differentiation, and migration.

Animals↗
PubMed1990

Augmentation of GG2EE macrophage cell line-mediated anti-Candida activity by gamma interferon, tumor necrosis factor, and interleukin-1.

The 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.

Animals↗
PubMed1989

IL-2 induction of IL-1 beta mRNA expression in monocytes. Regulation by agents that block second messenger pathways.

We have previously shown that in mixed cultures of PBL incubation with human rIL-2 induces the rapid expression of IL-1 alpha and IL-1 beta mRNA. Because studies have demonstrated that IL-2R can be expressed on the surface of human peripheral blood monocytes, we chose to investigate whether IL-1 beta mRNA could be directly induced in purified human monocytes by treatment with Il-2 and, if so, to analyze the second messenger pathways by which it may be controlled. Human monocytes do not spontaneously express IL-1 beta mRNA, but can express the gene as soon as 1 h after treatment with IL-2. The level of IL-1 beta mRNA induced by IL-2 at 5 h in human monocytes was about one-fourth that induced by LPS. LPS induction of IL-1 beta mRNA in human monocytes can be blocked by either an inhibitor of protein kinase C (PKc) 1-(5-isoquinolinesulfonyl)-2-methylpiperazine or an inhibitor of calcium/calmodulin (CaM) kinase N-(6-aminohexyl) 5-chloro-1-naphthalenesulfonamide, suggesting that both PKc and CaM kinase are involved in transducing signals initiated by LPS. In contrast, IL-2 induction of IL-1 beta mRNA expression is blocked only by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, suggesting that PKc, and not CaM kinase, is activated by IL-2. These data suggest that overlapping but distinct second messenger pathways are involved in the transduction of signals initiated by IL-2 and LPS.

Animals↗
PubMed1989

Heterogeneity of hematopoietic cells immortalized by v-myc/v-raf recombinant retrovirus infection of bone marrow or fetal liver.

The J2 recombinant retrovirus expressing v-myc/v-raf (also known as MYC/RAF1) immortalized macrophages from the bone marrow of lipopolysaccharide-responsive mouse strains, producing the ANA-1 cell line from C57BL/6 mice and the INF-3A cell line from C3H/HeN mice. In contrast, J2 recombinant retrovirus infection of the fetal liver from C57BL/6-Ly-5a mice immortalized a cell line (GGD) that did not exhibit the characteristics of mature macrophages. The GGD cell line was classified as leukocytic on the basis of its expression of the Ly-6B.2, Fc gamma R, and Ly-5.2 antigens. Our results indicate that the J2 recombinant retrovirus selectively immortalizes macrophages from the bone marrow of C57BL/6 and C3H/HeN mice but immortalizes cells without definitive macrophage characteristics from murine fetal liver under the same culture conditions.

Animals↗
PubMed1989

A functional protein kinase C is required for induction of 2-5A synthetase by recombinant interferon-alpha A in Daudi cells.

Treatment of Daudi B lymphoblastoid cells with interferon (IFN)-alpha or -beta has been reported (Yap, W. H., Teo, T. S., and Tan, Y. H. (1986) Science 234, 355-358) to cause a transient increase in the level of diacylglycerol, which is the endogenous activator of protein kinase C (PK-C). To assess the role for PK-C in the induction of 2-5A synthetase mRNA and activity by IFN-alpha in Daudi cells, we have examined the effects of PK-C inhibitors and activators. We have found that the PK-C inhibitor, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), strongly inhibits the induction of 2-5A synthetase mRNA and activity by recombinant IFN-alpha A (rIFN-alpha A) and that this inhibition appears to be at a post-transcriptional level. The inhibition by H7 could not be attributed to a generalized decrease in macromolecular synthesis or to inhibition of the binding or internalization of rIFN-alpha A. Moreover, pretreatment of Daudi cells for 24 h with phorbol esters to down-regulate or desensitize PK-C substantially inhibited the subsequent induction of 2-5A synthetase mRNA and activity by rIFN-alpha A. These data suggest that PK-C activity is required for the induction of 2-5A synthetase by rIFN-alpha A. However, phorbol esters, which are potent PK-C activators, did not induce 2-5A synthetase. Taken together, our data indicate that a functional PK-C is required for 2-5A synthetase induction by rIFN-alpha A at a post-transcriptional level in Daudi cells but that activation of PK-C is not sufficient for induction of this enzyme.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Modulation of CD3- large granular lymphocyte functions by agonist and antagonists of protein kinase C: effects on NK and lymphokine-activated killer activity and production of IFN-gamma.

The biochemical mechanisms involved in the activation and killing of tumor targets by large granular lymphocytes (LGL) have not yet been clearly defined. This laboratory has investigated these processes by analyzing the effects of protein kinase C (PKC) inhibitors (1-(5-isoquinolinesulfonyl)2-methyl-piperazine-dihydrochloride and retinol) on LGL cytotoxicity and IFN-gamma production. We now report that PKC inhibitors block the LGL functions of 1) NK activity, 2) IFN-gamma production, and 3) LAK activity induced by IL-2. Complete inhibition of cytotoxic activity occurs rapidly because only 2.5 h treatment of the LGL with the inhibitors was required. However, the inhibition of NK activity by the PKC inhibitors could be reversed by IL-2 or the synthetic diacylglycerol, L-gamma-1-oleyl-2-acetol-sn-3-glycerol (OAG), but not by IFN-alpha. The reversal of inhibition observed with OAG indicates that, in these studies, (1-(5-isoquinolinesulfonyl)2-methyl-piperazine-dihydrochloride is inhibiting PKC activity and not the activity of other cellular kinases. Furthermore, inhibition of LGL functional activity with PGE2 could not be reversed with OAG, supporting the contention that PG inhibition of NK activity is mediated by a pathway that does not directly involve PKC. These results indicate, in addition to IL-2-mediated events, that basal NK activity is under PKC regulatory control.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Cytokine gene expression during the generation of human lymphokine-activated killer cells: early induction of interleukin 1 beta by interleukin 2.

Culture of human peripheral blood leukocytes with interleukin 2 (IL-2) stimulates their differentiation into lymphokine-activated killer (LAK) cells, with a broad range of cytotoxicity against fresh tumor cells and tumor cell lines (Grimm et al., J. Exp. Med., 155: 1823-1841, 1982). We chose to utilize a molecular approach to determine whether IL-2 stimulates the expression of cytokine genes by the mixed cell population which may be involved in the generation or regulation of lytic activity. Northern blot analysis performed with total cellular RNA from LAK cells cultured for varying periods of time with IL-2 revealed that the genes which code for cytokines [interleukin 1 (IL-1)alpha and beta, gamma-interferon, tumor necrosis factor alpha, and lymphotoxin] were not spontaneously expressed. As soon as 2 h after IL-2 treatment, IL-1 alpha and IL-1 beta mRNAs were expressed. Both nonadherent and adherent populations of LAK cells express IL-1 beta mRNA; however, the adherent population produced more IL-1 beta mRNA and maintained its expression for a prolonged period of time. Other cytokine mRNAs (gamma-interferon, tumor necrosis factor alpha, and lymphotoxin) were expressed later than the IL-1 mRNAs with maximal levels between Days 2 through 7. Our results indicate that LAK cell populations can generate a variety of cytokines which may be involved in the generation of lytic activity.

Cell Separation↗
PubMed1989

Morphological change and cellular differentiation induced by cisplatin in human neuroblastoma cell lines.

In 1986 we reported on the capacity of cis-diaminedichloroplatinum(II) (cisplatin, CDDP) to induce erythroid cellular differentiation in the K562 cell. To continue our study of the differentiating activity of cisplatin, we treated two human neuroblastoma cell lines with different doses of the drug in vitro. Both cell lines showed changes in morphology; however, only one achieved a fully differentiated neuronal phenotype (cisplatin concentration 1 micrograms/ml). The differentiated neuroblastoma cells exhibited extensive neurite outgrowth that reached maximal elongation after 5 days of culture, forming several interconnections. Cisplatin could induce neuronal differentiation, as did retinoic acid, a neuroblastoma-differentiating agent. The results show that cisplatin should be a candidate for further in vitro and in vivo studies of induced differentiation.

Antigens, Neoplasm↗
PubMed1989

Generation of macrophage cell line from fresh bone marrow cells with a myc/raf recombinant retrovirus.

We 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.

Animals↗
PubMed1988

In vitro proliferation of human large granular lymphocytes with v-raf/v-myc recombinant retrovirus.

The effect of infection with a retrovirus carrying v-raf/v-myc oncogenes (J2 virus) on the in vitro proliferation of human large granular lymphocytes (LGL) was investigated. LGL infected with J2 virus (J2LGL), unlike uninfected cells, grew with a proliferation peak eight days after infection. Such cells retained the morphology and functional properties typical of LGL. Furthermore, 5% of J2LGL produced virus the day after infection, whereas non-virus production was detectable five days later. These data indicate that J2 virus provides a transient mitogenic signal for LGL.

Cell Division↗
PubMed1988

Differential inhibition of IL-1 and TNF-alpha mRNA expression by agents which block second messenger pathways in murine macrophages.

IL-1 and TNF-alpha are induced in macrophages by LPS; however, it is unclear whether similar mechanisms control the expression of both genes. Here, we report on the detection of differential regulation of LPS induced IL-1 and TNF-alpha mRNA expression and protein production in murine macrophages based on the use of inhibitors of second messenger pathways. Northern blot analysis was performed with total RNA obtained from murine (C57Bl/6) peritoneal macrophages stimulated in vitro with LPS with or without an inhibitor of protein kinase C (PKc)(1-(5-isoquinolinesulfonyl)-2-methylpiperazine hydrochloride; H7) or an inhibitor of calmodulin (CaM)-dependent kinase (N-(6-amino-hexyl)-5-chloro-1-naphthalene-sulfonamide hydrochloride; W7). Northerns were analyzed with probes for IL-1 alpha and IL-1 beta and TNF-alpha. The expression of the three cytokine mRNA by LPS was inhibited in a dose response manner by H7. In contrast, the expression of IL-1 mRNA, but not TNF-alpha mRNA, was blocked by treatment with W7. Parallel studies monitoring biologic activities of these two cytokines confirm the mRNA data. PKc inhibitors, H7 and retinal, block both IL-1 and TNF-alpha protein production and inhibitors of CaM kinase, W7, N-(6-aminobutyl)-5-chloro-2-naphthalenesulfonamide, calmidazolum, and trifluoperazine dichloride inhibit only IL-1 production. These data suggest that both PKc and CaM kinase dependent pathways are involved in the induction of IL-1 mRNA by LPS. In contrast, TNF-alpha expression appears to be PKc dependent but not CaM kinase dependent.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1988

Inhibition of retroviral mRNA expression in the murine macrophage cell line GG2EE by biologic response modifiers.

We 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.

Adjuvants, Immunologic↗
PubMed1988

Characterization of a murine monoclonal antibody that detects a C-terminal fragment of the raf oncogene product.

A murine mAb, STEGI 1, was generated against a 30-kDa raf protein purified from an Escherichia coli expression vector. Immunoblot analysis confirmed that this antibody recognized the original immunizing protein as well as a 44- to 48-kDa protein from several raf-transformed cell lines. Immunoprecipitation experiments isolated a 48-kDa protein from a cell line transfected with a c-raf construct as well as from normal NIH 3T3 fibroblasts. Parallel experiments with polyvalent antiserum prepared against E. coli-derived v-raf (C terminus)-precipitated proteins with apparent Mr of 48 and 74 kDa, as had been described previously. Immunofluorescence flow cytometry of raf-transformed cell lines revealed intense intracytoplasmic staining. This staining was specifically inhibited by preincubation of STEGI 1 with purified raf 30-kDa protein. It should now be possible to more easily assess the role of the raf oncogene product in malignant transformation.

Amino Acid Sequence↗
PubMed1988

Protein kinase C inhibitors block the activation of macrophages by IFN-beta but not by IFN-gamma.

We investigated the role of protein kinase C (PK-C) in the activation of cytotoxic peritoneal murine macrophages (M phi) by IFN-gamma or by IFN-beta. Two potent inhibitors of PK-C, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) and retinal, were used. We found that both drugs inhibited in a dose-dependent manner the activation of cytotoxicity induced by IFN-beta, suggesting the requirement for intact PK-C activity in this process. In contrast, neither H-7 nor retinal inhibited the activation of cytotoxic M phi by IFN-gamma, indicating that IFN-gamma acts through a PK-C-independent pathway. The effectiveness of both drugs in inhibiting PK-C in intact M phi was evaluated by measuring the inhibition of induction of c-fos mRNA by L-alpha-1-oleoyl-2-acetoyl-sn-3-glycerol, a process that has been shown to be dependent on PK-C activation. We have found a strict correlation in the dose-dependent inhibition by both drugs of c-fos mRNA induction and activation of M phi by IFN-beta. These results indicate that different pathways of activation are triggered by IFN-gamma and IFN-beta, the former being independent from and the latter dependent on intact PK-C activity.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1988

Tumor formation by a murine macrophage cell line immortalized in vitro by v-raf and v-myc oncogenes.

Murine 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 x 10(4) to 1 x 10(7) 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-gamma-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.

Animals↗