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D H Pluznik

Publications and source records attributed to D H Pluznik.

At least 37 records · Page 2Linked to original sources
PubMed1991

IL-1 alpha induces granulocyte-macrophage colony-stimulating factor gene expression in murine B lymphocyte cell lines via mRNA stabilization.

IL-1 has been shown to induce granulocyte-macrophage (GM)-CSF production in several cell types including T lymphocytes, fibroblasts, and endothelial cells. We have previously demonstrated that bacterial LPS activates expression of the GM-CSF gene in murine B lymphocytes. Inasmuch as LPS is also an inducer of IL-1, we asked whether IL-1 could itself induce GM-CSF gene expression in B lymphocytes. The GM-CSF-dependent PT-18 cell line was used to detect GM-CSF biologic activity and a semi-quantitative polymerase chain reaction assay was developed to detect and measure GM-CSF mRNA. IL-1 alpha stimulated GM-CSF production by the murine B cell lines M12.4.1 and TH2.2 but not by the M12 line in a dose- and time-dependent fashion. GM-CSF mRNA expression in IL-1 alpha-treated M12.4.1 cells also increased in a dose- and time-dependent fashion. Nuclear run-on transcription assays revealed constitutive transcription of the GM-CSF gene in untreated M12.4.1 cells that did not change after IL-1 alpha treatment. Actinomycin D chase experiments indicated that IL-1 alpha increased GM-CSF mRNA stability in the M12.4.1 cells. These findings indicate that IL-1 alpha induces GM-CSF gene expression in murine B cell lines and that this event is posttranscriptionally controlled mainly at the level of mRNA stabilization.

Animals↗
PubMed1990

Post-transcriptional regulation of granulocyte-macrophage colony-stimulating factor synthesis in murine T cells.

The granulocyte-macrophage CSF (GM-CSF) gene is known to be controlled at a variety of levels in different cell types. We showed previously that GM-CSF production by lectin or phorbol ester (12-O-tetradecanoyl-phorbol-13-acetate (TPA]-treated T cells was unaffected by cyclosporin A whereas IL-2 and IL-3 expression were. Cyclosporin A is thought to inhibit transcription that suggests that IL-2 and IL-3 are regulated primarily at the transcriptional level while GM-CSF is not. The lack of coordinate gene expression is of particular interest because all three mRNA share the presence of adenosine uridine-rich sequences in the 3' untranslated region and these sequences are believed to act by modulating mRNA stability. We measured the level of GM-CSF mRNA in untreated cells and found it to be extremely low. GM-CSF mRNA levels increased approximately 60-fold within 6 h of TPA-treatment. Nuclear run-on transcription analysis of the same cells showed readily detectable GM-CSF transcription in unstimulated cells that increased less than twofold after TPA treatment. However, IL-2 transcription was insignificant before TPA addition. Actinomycin D chase experiments showed that GM-CSF transcripts in untreated cells have a very short half-life (approximately 45 min) although transcripts in TPA-treated cells have a half-life exceeding 3 h. These findings indicate that GM-CSF production in EL-4 cells treated with TPA is regulated predominantly by modulation of cytoplasmic mRNA half-life.

Animals↗
PubMed1990

Induction of interferon-beta and 2',5'-oligoadenylate synthetase mRNAs by interleukin 6 during differentiation of murine myeloid cells.

Interleukin 6 (IL 6) induces differentiation of murine myelomonocytic leukemia (M1) cells into mature macrophages. This process is monitored by the sequential appearance of surface markers, induction of intracellular enzymes, and changes in morphology as the cells progress from blast cells to mature macrophages. Differentiation is also associated with growth arrest and accumulation of the differentiating cells in the G0/G1 phase of the cell cycle. Interferon-beta (IFN-beta) is known to be involved in the growth arrest of M1 cells by inducing 2'5'-oligoadenylate synthetase (2',5'-AS). We therefore analyzed whether IL 6 has the potential to trigger the full differentiation program directly or whether its effect on M1 cells is mediated through IFN-beta or through the activation of genes that are typically induced by IFN-beta. We first tested whether IL 6 could induce IFN-beta mRNA. Using a reverse transcription/polymerase chain reaction procedure, we found that IFN-beta mRNA was induced by IL 6. By Northern analysis we determined that IL 6 also caused a significant increase in 2',5'-AS gene expression. IL 6, however, induced the expression of two mRNA species (1.7 and 2.4 kb), whereas IFN-beta mainly induced the expression of the 1.7-kb species. Enhancement of 2',5'-AS gene expression by IL 6 was observed even when protein synthesis was inhibited by cycloheximide. Furthermore, IL 6-induced growth arrest of M1 cells was not inhibited by anti-IFN-beta antibodies. Thus induction of 2',5'-AS gene expression is a primary response to IL-6 and not secondary to the induction of IFN-beta.

2',5'-Oligoadenylate Synthetase↗
PubMed1990

Alterations in T cell-derived colony-stimulating factors associated with GVH-induced immune deficiency.

Injection of parental C57BL/10 spleen cells into unirradiated immune-competent (B10 x B10.BR)F1 hosts has been demonstrated to produce a graft-vs.-host-induced immune deficiency in T cell-mediated functions, including mitogen or alloantigen stimulated proliferation or cytotoxic T cell generation. The production of T cell-derived lymphokines affecting hematopoiesis was also altered during GVH. During the first two weeks of GVH, IL-3 and particularly GM-CSF were produced spontaneously; in subsequent weeks, the spontaneous production dropped to normal or subnormal levels. CSF content in concanavalin A-stimulated splenic supernatants was reduced at weeks 1-2, and declined to less than 5% of normal levels by 3-4 weeks of GVH. This decline in CSF content was correlated with a decrease in immune function as assessed by concanavalin A-stimulated IL-2 production and by generation of cytotoxic T lymphocytes. Concurrent with the recovery of immune function during GVH weeks 8-15, mitogen-stimulated production of CSF returned to normal levels. In addition to the decrease in CSF production identified in acute suppressive GVH, CSF content in concanavalin A-stimulated splenic supernatants was also decreased in chronic stimulatory GVH, generated in the strain combination (B6 x B6bm1)F1----(B6bm1 x B6bm12)F1. This decrease in CSF production correlated with a decrease in self-restricted T helper cell function. Finally, a decrease in both immune function and CSF production capacity was observed in the acute GVH following allogeneic (minor histocompatibility loci) bone marrow transplantation into irradiated hosts.

Animals↗
PubMed1990

Factors contributing to the decrease in concanavalin A-induced colony-stimulating factors in acute suppressive graft-versus-host disorder.

We have recently demonstrated that IL-3 and GM-CSF content in concanavalin A--stimulated splenic cultures was severely depressed for several weeks in mice undergoing an acute graft-vs.-host reaction (GVHR). Several factors contributed to this decrease in CSF levels at different points in the first 10 weeks of GVH. Myeloid lineage cells, both GM-CFU and Mac-1+ cells, increased in the spleen during GVH weeks 3-6, and (125I)-GM-CSF binding by spleen cells increased 10 fold--hence CSF content in supernatants was probably reduced by local consumption during these weeks. During GVH weeks 3-12, levels of all T cells--and of L3T4+ T cells, in particular--were reduced, limiting production of T cell derived lymphokines. Finally, during the first six weeks of GVH, immune suppression was found in cocultures of normal F1 and GVH spleens. Furthermore, during weeks 3-5, nonspecific suppression was found, affecting CSF production not only by normal host spleens but also by parental donor or unrelated spleen cells as well.

Animals↗
PubMed1989

Effects of in vivo administration of anti-CD3 monoclonal antibody on T cell function in mice. II. In vivo activation of T cells.

Anti-CD3 mAb are known to be both immunosuppressive and mitogenic to T cells in vitro. However, only immunosuppression has been observed after in vivo administration of these mAb. The present study demonstrates that T cell activation does occur after in vivo administration of anti-CD3 mAb to mice, evidenced by increased IL-2R expression on T cells, CSF secretion, and extra-medullary hematopoiesis in the spleen. These effects required multivalent cross-linking of the mAb, since F(ab')2 fragments failed to induce them. However, the F(ab')2 fragments did induce modulation of CD3/TCR from the surface of T cells, demonstrating that TCR modulation is not sufficient to induce activation. In addition, interaction of the TCR with either intact or F(ab')2 fragments of the mAb led to increased expression of CD8 in vivo, suggesting that the F(ab')2 fragments of anti-CD3 mAb might be capable of inducing a T cell to undergo some, but not all, of the changes involved in reaching a fully activated state. Further study of the activating effects of anti-CD3 mAb might increase the understanding of the mechanisms of in vivo T cell activation and might also be exploited clinically to stimulate T cell function in immunocompromised states and to enhance hematopoiesis in myelodysplastic disorders.

Animals↗
PubMed1989

Interleukin 6 (interferon beta 2) and interferon alpha/beta present in postendotoxin serum induce differentiation of murine M1 myeloid leukemia cells.

Serum from lipopolysaccharide-treated mice (postendotoxin serum, PES) induces the differentiation of M1 myeloid leukemia cells into mature macrophages, as well as supporting the proliferation of the interleukin 6 (IL6)-dependent B9 hybridoma cells. The kinetics of appearance of these two activities in PES were identical. To determine whether these two activities are due to the presence of the same substance, we tested whether anti-IL6 antibodies could neutralize the differentiation-inducing activity of PES. We found that anti-IL6 antibodies completely neutralized the proliferation of B9 cells and resulted in a 60% neutralization of the differentiation-inducing activity of PES. Anti-interferon alpha/beta (INF alpha/beta) antibodies neutralized 70% of the differentiation-inducing activity of PES. These data suggest that the differentiation-inducing activity of PES is not limited to IL6, and that PES contains additional factors such as INF alpha/beta that are capable of inducing differentiation of M1 cells.

Animals↗
PubMed1989

Natural cytotoxic activity in a cloned natural killer cell line is mediated by tumor necrosis factor.

The interleukin-2-dependent mouse natural killer (NK) cell line NKB61A2 concomitantly exhibits NK and natural cytotoxic (NC) activities. This was determined by the cells' ability to lyse both the NK-sensitive YAC-1 lymphoma and the NC-sensitive WEHI-164 fibrosarcoma cell lines in a 4- and 18-hour 51Cr release assay, respectively. Cell-free supernatant from NKB61A2 cells grown in culture for 48 h had substantial lytic activity against WEHI-164. The mouse mast cell line PT18-A17 and the rat basophilic leukemia cell line RBL-2H3, which both express NC activity, also produced a soluble factor during culture which lysed WEHI-164 cells. This activity was increased in the basophilic/mast cells by crossbridging the surface IgE receptors. Similar results were obtained by triggering the basophilic NC cells with the calcium ionophore ionomycin and the tumor promoter phorbol-12-myristate-13-acetate (PMA). Such triggering of NKB61A2 cells, however, did not significantly increase their NC activity. Interestingly, both ionomycin and PMA had an inhibitory effect on the NK activity of NKB61A2. Recently it has been found that tumor necrosis factor (TNF) is a major mediator of NC activity. To determine if the soluble factor responsible for the NC activity of the NK clone was related to TNF, a rabbit polyclonal antiserum to mouse TNF was tested against the cell-free culture medium of NKB61A2, PT18-A17, RBL-2H3 and murine recombinant TNF (Mu-rTNF). The lytic activity of the culture medium from all these cells and the Mu-rTNF control was abrogated by this antibody. These data suggest that the murine cell line NKB61A2 has both NK and NC activities and that the NC activity is due to a factor immunologically similar to TNF. In addition, the enhancement of NC activity in the NK cell line is apparently under control by a separate pathway, different from that in the basophilic cells.

Antibodies, Monoclonal↗
PubMed1988

Differential expression of c-myc and the transferrin receptor in G1 synchronized M1 myeloid leukemia cells.

We have studied the transcriptional activity, steady-state mRNA levels, and steady-state protein levels of the c-myc and transferrin receptor (TfR) genes in murine M1 myeloid leukemia cells arrested in G1 phase of the cell cycle by different methods. When cells are growth-arrested by density inhibition, a technique that places the majority of cells in early G1, c-myc protein, as detected by Western analysis, is expressed at 80% of the level seen in proliferating cells. Steady-state mRNA levels and, to a lesser extent, transcriptional activity of the c-myc gene, parallel the protein findings. Under these conditions, TfR gene expression is much lower than in normally cycling cells. We have previously demonstrated that density-inhibited M1 cells, released from density inhibition and treated with the DNA polymerase alpha inhibitor aphidicolin, remain in G1, but at a point temporally closer to S phase. Cells treated in this manner demonstrate reduced transcriptional activity and expression of the c-myc gene, but TfR gene expression approximates the level found in proliferating cells. These data suggest that neither c-myc nor TfR gene expression is constant throughout the G1 phase of the cell cycle in M1 cells. c-myc gene expression is highest in early G1 and falls to low levels by late G1, while the reverse is true for TfR gene expression.

Animals↗
PubMed1988

Granulocyte-macrophage colony-stimulating factor regulation in murine T cells and its relation to cyclosporin A.

The coordinated expression of the hematopoietic growth factors, interleukin 3 (IL3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in activated T cells suggests common synthetic pathways. However, cyclosporin A (CsA) appears to differentially effect the synthesis of these two lymphokines. When the supernatants from the concanavalin A-stimulated thymoma EL-4 or the T-cell hybrid 2B4 were assayed on the GM-CSF/IL3-dependent PT-18 and on the IL3-dependent DA-1 cell lines, IL3 activity could not be detected following CsA treatment, but substantial growth activity, which was identified as GM-CSF, was observed on the PT-18 cell line. CsA did not affect the kinetics of GM-CSF release, but inhibited the release of IL3 over a period of 40 h after the cells were stimulated and treated with CsA. In addition, CsA could be added up to 1 h after stimulation of the cells without affecting GM-CSF activity but inhibiting completely the IL3 activity. To substantiate the inability of CsA to inhibit GM-CSF activity, GM-CSF gene expression was evaluated. By Northern analysis GM-CSF mRNA was not inhibited by doses of CsA up to 1 microgram/ml. The data reveal that CsA can dissociate between the production of IL3 and GM-CSF, suggesting that these two CSFs are regulated by different mechanisms.

Animals↗
PubMed1987

Induction of granulocyte-macrophage colony-stimulating factor by lipopolysaccharide and anti-immunoglobulin M-stimulated murine B cell lines.

The present study was undertaken to elucidate whether B cell lymphoma and hybridoma cell lines can be stimulated by lipopolysaccharides (LPS) or by antibodies against immunoglobulin M (IgM) to produce granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF activity was assayed on the basophil/mast cell line PT-18 which is GM-CSF- and interleukin 3-dependent. Antibodies against murine recombinant GM-CSF were used to identify the colony-stimulating factor activity present in the supernatants of the stimulated B cell lines. When these cell lines were stimulated with LPS, two of five lymphoma and five of six hybridoma lines produced GM-CSF. Two cell lines, the B cell lymphoma M12.4.1 and the hybridoma TH2.2, were analyzed more extensively under serum-free conditions. In these two cell lines, the production of GM-CSF was dependent on the dose of LPS used and time of exposure. Antibodies against IgM stimulated the TH2.2 (IgM+) but not the M12.4.1 (IgM-) cells to produce GM-CSF. Northern blot analysis of the M12.4.1 and TH2.2 cells showed that mRNA of GM-CSF can be detected in LPS-stimulated but not in unstimulated cells. Our data show that transformed B cells can be stimulated to produce GM-CSF. The present data and previous studies on GM-CSF production by normal bone marrow-derived B cells suggest a possible participation of B cells in granulopoiesis.

Animals↗
PubMed1987

Differential regulation of colony-stimulating factors and interleukin 2 production by cyclosporin A.

Stimulation of T lymphocytes with mitogens or antigens is followed by proliferation and lymphokine production. Although cyclosporin A (CsA), an immunosuppressive drug, has been shown to inhibit the production of certain lymphokines, including interleukin 2 (IL-2), interleukin 3 (IL-3), and gamma-interferon, its effect on the production of granulocyte/macrophage colony-stimulating factor (GM-CSF) has not been evaluated. In the current study, concanavalin A (Con A)-stimulated murine spleen cells secreted GM-CSF, IL-3, and IL-2, and in the presence of CsA (0.1-1.0 micrograms/ml), IL-2 and IL-3 activities were inhibited. In contrast, significant activity was detected when the CsA-treated culture supernatants were assayed on a cell line that is dependent on GM-CSF and/or IL-3. Similar CsA-resistant activity was observed when the EL-4 thymoma cells were stimulated with a phorbol ester [phorbol 12-myristate 13-acetate (PMA)] in the presence of CsA. The activity resistant to CsA was identified as GM-CSF by the ability of specific antibodies against murine recombinant GM-CSF to neutralize its activity. These findings indicate that GM-CSF, in contrast to IL-2 and IL-3, was not inhibited by CsA. In additional experiments, transfer blot of poly(A)+ RNA isolated from PMA-induced EL-4 cells in the presence or the absence of CsA was hybridized with GM-CSF and IL-2 cDNA probes. Expression of the GM-CSF gene in EL-4 cells was detected independent of CsA, whereas CsA inhibited the expression of the IL-2 gene. The present data show that production of IL-2 and IL-3, but not that of GM-CSF, is inhibited by CsA and suggest a differential control mechanism for lymphokine synthesis in T lymphocytes.

Animals↗
PubMed1987

Enhanced c-fos expression in differentiated monomyelocytic cells is associated with differentiation and not with the position of the differentiated cells in the cell cycle.

Enhanced expression of the proto-oncogene c-fos is reported to be involved in the differentiation of monomyelocytic cells into mature macrophages. Such differentiated cells usually accumulate in the G1 phase of the cell cycle. M1 murine myeloid leukemia cells, which can be synchronized in early and late G1 and can also differentiate into mature macrophages when stimulated with granulocyte colony-stimulating factor (G-CSF), were used to investigate whether the enhanced expression of c-fos in differentiated monomyelocytic cells is associated with the position of the cells in the cell cycle or with their state of differentiation. Our data show that the enhanced expression of the c-fos gene occurs only in fully differentiated cells and not in proliferating or G1-arrested cells. These data imply that the enhanced expression of c-fos in M1 cells is restricted to functionally differentiated cells and not related to the position of the differentiated cells in the cell cycle.

Animals↗
PubMed1986

T lymphocyte-dependent evolution of bacterial cell wall-induced hepatic granulomas.

Injection of streptococcal cell walls (SCW) i.p. into susceptible rats results in dissemination of SCW primarily to the liver, spleen, bone marrow, and peripheral joints. Within the liver, the SCW are phagocytized by the Kupffer cells, initiating a sequence of events leading to the formation of hepatic granulomas. The granulomas are characterized by large numbers of W3/13+, W3/25+ T lymphocytes and Ia+, esterase-positive macrophages. The generation of inflammatory mediators by these mononuclear cells appears to be central to the evolution of the granulomas and the subsequent fibrotic sequelae evoked by the SCW. In the absence of functional T lymphocytes (athymic rats), injection of SCW does not trigger lymphokine production, and organized granulomas do not develop in the livers. Furthermore, inhibition of T lymphocyte proliferation and lymphokine synthesis pharmacologically by cyclosporin A administration in euthymic animals inhibits SCW-induced hepatic granuloma development. Although macrophage function is apparently not impaired as evidenced by IL 1 and PGE2 production, a chronic inflammatory response to SCW cannot be sustained in the absence of T lymphocyte participation. These studies provide insight into the cellular and molecular mechanisms leading to formation and maintenance of chronic granulomatous lesions.

Animals↗
PubMed1986

Natural cytotoxic (NC) cell activity in basophilic cells: release of NC-specific cytotoxic factor by IgE receptor triggering.

A murine interleukin 3 (IL 3)-dependent basophilic mast cell line, PT-18 (A17), and a rat basophilic leukemic cell line, RBL-2H3, were shown to be capable of selective natural cytotoxic (NC) but not natural killer (NK) cell activity. The basophilic cell types could also be augmented in their NC activity by bridging of their surface IgE receptors. IgE-mediated triggering of the basophilic cells was accomplished by coating the cells with IgE and exposing the IgE-bound cells to specific antigen or to anti-IgE monoclonal antibody. Another method of triggering was by direct binding of basophilic cells to anti-IgE receptor monoclonal antibody. Basophilic cells, triggered by these methods, not only displayed increased NC activity but also released a soluble factor capable of selectively lysing NC tumor targets, WEHI-164, but not three of the NK-sensitive targets, YAC-1, RLM1, and RBL-5. Normal C3H/HeJ mouse embryonic fibroblasts were also not lysed. Dose response and time course of the cytotoxic factor release from triggered RBL-2H3 cells were similar to those of tritiated serotonin release. As with serotonin or histamine release, the NC-specific cytotoxic factor (NCCF) was not released in the absence of extracellular calcium. Therefore, NCCF appears to be released along with other mediators during the triggering of basophilic cells by bridging of IgE receptors. The m.w. of the native form of this factor, determined by a gel filtration method, was about 43,000.

Animals↗
PubMed1986

Colony stimulating factor-induced differentiation of murine M1 myeloid leukemia cells is permissive in early G1 phase.

Granulocyte-colony stimulating factor (G-CSF) induces differentiation of M1 murine myeloid leukemia cells into mature granulocytes/macrophages and also causes accumulation of the cells in the G1 phase of the cell cycle. We examined, therefore, whether synchronization of M1 cells in the G1 phase could affect G-CSF-induced differentiation as quantitated by expression of Fc fragment receptors (FcR) and lysozyme activity. Cells were arrested in early G1 by density inhibition in the absence of serum and in late G1 by addition of aphidicolin. Cells synchronized in early G1, when stimulated with G-CSF, showed enhanced expression of FcR and lysozyme activity. Eighty percent of the cells expressed FcR 18 hr after addition of G-CSF while, in exponentially growing cells, this percentage was reached 72 hr after addition of G-CSF. Cells synchronized in late G1 did not show enhanced expression of differentiation markers. These results imply that with respect to G-CSF-induced differentiation, the G1 phase can be separated into an early permissive and a later nonpermissive stage.

Animals↗
PubMed1986

Synergistic action of lipopolysaccharide and tumor-promoting phorbol esters: two-signal requirement for colony-stimulating factor production by murine bone marrow cells.

In contrast to mature spleen cells and macrophages, which produce colony-stimulating factor (CSF) in response to lipopolysaccharide (LPS), murine bone marrow (BM) cells do not respond to LPS alone. However, when BM cells are treated with LPS and the tumor-promoting phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) simultaneously, they generate significant levels of CSF. The non-tumor-promoter 4-O-methyl-TPA will not replace TPA, and if BM cells from C3H/HeJ mice are employed, no CSF is produced after stimulation with LPS and TPA. Lipid A is as effective as LPS in stimulating BM cells in the presence of TPA, but other mitogens, such as phytohemagglutinin (PHA), concanavalin A (Con A), or pokeweed mitogen (PWM), are ineffective. B-lymphocytes may be the main source of the CSF from BM cells, since BM cells adherent to surfaces coated with goat antimouse immunoglobulin produced CSF in amounts similar to those produced by unseparated BM cells after stimulation with LPS and TPA. Finally, the CSFs produced by BM cells under these experimental conditions were identified as belonging to the GM-CSF and G-CSF subclasses. We interpret these results as suggesting that B cells present in the BM, as opposed to mature spleen cells and macrophages, require at least two signals for CSF production.

Animals↗