Colchicine down-regulates lipopolysaccharide-induced granulocyte-macrophage colony-stimulating factor production in murine macrophages.

Activation of macrophages by LPS and taxol results in production of IL-1, IL-6, TNF-alpha, and granulocyte-macrophage CSF (GM-CSF), which are involved in regulating hemopoiesis, inflammation, and immune responses. Microtubules are proposed as a target site for LPS interaction(s), based on similarities between the effects of the tubulin-binding drug taxol and LPS. To clarify the role of microtubules in LPS-induced GM-CSF expression in macrophages, we examined whether microtubule depolymerizing agents affect GM-CSF production in macrophages. Pretreatment with colchicine impaired LPS induction of GM-CSF in RAW 264 cells, and studies using stable transfectants revealed that colchicine impaired the transcriptional responsiveness of a reporter gene driven by a GM-CSF promoter sequence. Colchicine inhibition of the GM-CSF response correlated with decreases in the mRNA levels of beta-tubulin; maximal inhibition of both events was observed 4 h after addition of colchicine. Microtubule agents inhibited LPS induction of IL-6 and TNF-alpha, while the induction of both IL-1beta and inducible nitric oxide synthase was unaltered, suggesting that LPS activates microtubule-dependent and -independent pathways. Interestingly, LPS stimulation of macrophages down-regulated levels of beta-tubulin transcripts, implying that LPS interacts with an element(s) of the microtubule network in vivo, activating pathways regulating transcription of beta-tubulin. The ability of both colchicine and LPS to modulate transcription of beta-tubulin suggests that this event does not per se underlie the inhibitory effect of colchicine on LPS-induced GM-CSF expression. These data led us to conclude that colchicine inhibits LPS induction of GM-CSF by affecting microtubule-dependent costimulatory signaling pathways that synergize with primary LPS-triggered responses.

IL-6 induction of protein-DNA complexes via a novel regulatory region of the inducible nitric oxide synthase gene promoter: role of octamer binding proteins.

Macrophage inducible nitric oxide synthase (iNOS) catalyzes the synthesis of NO. IL-6-stimulated macrophage differentiation of murine myeloid M1 cells is accompanied by iNOS gene induction and steady-state mRNA expression. Two regions within the iNOS promoter mediate transcriptional responsiveness to LPS and IFN-gamma. Region I contains several essential transcription factor binding motifs and promotes responsiveness to LPS, whereas region II potentiates the LPS response by IFN-gamma. Because region I possesses basal promoter activity and directly mediates iNOS gene activation, we attempted to identify the trans-acting factors involved in IL-6-stimulated induction of the murine iNOS gene through this region. Using an electrophoretic mobility shift assay and methylation interference, we show that IL-6 induced reciprocal changes in the binding activity of POU family members to the candidate nonconsensus octamer sequence of region I that correlated, temporally, with iNOS steady-state mRNA expression. Although DNA-protein binding activity of IL-6-stimulated whole-cell extracts also interacted with a radiolabeled canonical octamer motif, such DNA-protein complexes were not eliminated in competition assays using consensus nuclear factor kappaB or IL-6 oligonucleotides. Specifically, our studies show that octamer binding protein-1-related protein binding activity decreased, while binding of octamer binding protein-2-related proteins increased during differentiation. Mutation of the octamer motif disrupted both binding of the IL-6-induced protein-DNA interactions and transcriptional activation through region I, revealing that this motif is absolutely essential for IL-6 induction of iNOS. Thus, differential activation of octamer binding transcriptional modulators from the POU family may be a novel mechanism of IL-6-mediated iNOS gene regulation.

Expression of human glucocerebrosidase in murine macrophages: identification of efficient retroviral vectors.

Gaucher's disease is an autosomal recessive disorder characterized by a functional deficiency in beta-glucocerebrosidase enzymatic activity and the resultant accumulation of the glycolipid glucocerebroside in macrophages. Due to the nature of the affected cells, Gaucher's disease is an excellent candidate for gene therapy of hematopoietic stem cells and autologous bone marrow transplantation of transduced cells using retroviral vectors containing the glucocerebrosidase (GC) gene. In order to identify a retroviral vector capable of high levels of expression of the GC gene in macrophages, we have used the murine myeloid leukemia cell line, M1, a cell line that can be differentiated with interleukin-6 (IL-6) from blasts to macrophages. Two vectors use the Moloney murine leukemia virus (MoMLV) enhancer/promoter (LG vector) or the myeloproliferative sarcoma virus (MPSV) enhancer/MoMLV promoter (MG vector), both located in the viral long-terminal repeat (LTR); the third vector uses the phosphoglycerate kinase (PGK) promoter located internally in the vector (PG vector). The amphotropic PA317 and GP+am12 packaging cell lines were used as virus producer cells, and the GP+am12 cell line demonstrated higher titers, higher levels of GC protein expression, and specific GC enzymatic activity as well as higher transduction efficiencies for all three vectors. The LG retroviral vector was the most efficient in transducing the M1 cells. On average, higher levels of RNA and protein expression were seen in the M1 clones transduced with the LG vector, and these levels increased after differentiation. Thus, the LG retroviral vector in which the expression of the GC gene is driven by the MoMLV LTR enhancer/promoter is the best vector of the three studied for future studies for gene therapy of Gaucher's disease and other hematopoietic disorders that involve macrophages.

Taxol induces the hematopoietic growth factor granulocyte-macrophage colony-stimulating factor in murine B-cells by stabilization of granulocyte-macrophage colony-stimulating factor nuclear RNA.

Taxol, a microtubule-stabilizing agent, has been shown to have antineoplastic activity against various tumors. In addition, it has been shown that taxol resembles bacterial lipopolysaccharide in its ability to activate macrophages. Recently we have shown that lipopolysaccharide induces the expression of the granulocyte-macrophage colony-stimulating factor (GM-CSF) in murine B-cell lines. In light of the similarity of taxol and lipopolysaccharide in their effects on macrophages, we tested whether taxol could also induce the expression of GM-CSF in B-cell lines. In the present study we used the murine B-lymphoma cell line M12.4.1. In unstimulated cells, no GM-CSF mRNA was detected, whereas in taxol-stimulated stimulated cells at a concentration of 30 microM, GM-CSF mRNA was induced 4-8 h after stimulation. This induction of GM-CSF mRNA was down-regulated by 10 ng/ml of interleukin 4. Actinomycin D chase experiments revealed that interleukin 4 did not affect the half-life of the taxol-induced GM-CSF cytoplasmic mRNA, nor did it alter GM-CSF gene transcription. Polymerase chain reaction analysis of nuclear RNA, utilizing probes specific for sequences in the first intron of GM-CSF, indicated that taxol enhances accumulation of nuclear precursor RNA and that interleukin 4 decreases this accumulation. The present study shows a novel activity of taxol in inducing the release of the hematopoietic growth factor GM-CSF from B-cells. Since GM-CSF is known to recruit macrophages and enhance their cytotoxicity against tumor cells, our observations suggest that part of the known antitumor activity of taxol may be due to synergistic effects of GM-CSF activity together with direct cytotoxic actions through microtubule stabilization.

Interferon-gamma antagonizes interleukin-6-induced expression of interleukin-4 receptors in murine myeloid cells by a transcriptional mechanism.

The murine myeloid leukemia cell line M1 induced by interleukin-6 (IL-6) is a model system to study the differentiation of blast cells to mature macrophages. We have recently shown that IL-6 induces the expression of the IL-4 receptor (IL-4R) in these cells. In the present study we investigate the mechanism of action of interferon-gamma (IFN-gamma), an antagonist of IL-4 in numerous cells and a cofactor in both induction and suppression of myelopoiesis, on the expression of IL-4R. Flow cytometry shows that IFN-gamma downregulates the IL-6-induced expression of IL-4R whereas it has no such effect on the high-affinity receptors for monomeric IgG2a (Fc gamma RI). As demonstrated by Scatchard analysis, the number of IL-4R decreases by more than 50% after IFN-gamma treatment whereas the receptor affinity remains unchanged. Northern analysis shows that this decrease is paralleled by a decrease in IL-4R mRNA but not Fc gamma RI or lysozyme mRNA. Nuclear run-on analysis shows that IFN-gamma suppresses the IL-6-induced transcription of the IL-4R gene, whereas actinomycin-D chase experiments showed no change of IL-4R mRNA stability. Furthermore, the production of soluble IL-4R protein is suppressed by IFN-gamma as well. These data explain how IL-4R can be modulated by IFN-gamma in myeloid cells and are consistent with the myelosuppressive capacity of IFN-gamma.

Interferon-gamma destabilizes interleukin-1-induced granulocyte-macrophage colony-stimulating factor mRNA in murine vascular endothelial cells.

Vascular endothelial cells can affect hematopoiesis by releasing granulocyte-macrophage colony-stimulating factor (GM-CSF). In the present study, we show that GM-CSF production in murine aorta-derived vascular endothelial cells is regulated by two cytokines:interleukin-1 (IL-1), which induces the production of GM-CSF, and interferon gamma (IFN-gamma), which downregulates this production. The GM-CSF gene is constitutively transcribed in these cells and the transcription rate of the GM-CSF gene does not change with either IL-1 alone or IL-1 plus IFN-gamma. Whereas IL-1 treatment increases the GM-CSF mRNA half-life, simultaneous treatment with IL-1 plus IFN-gamma results in a decrease in the mRNA half-life. IL-1 also enhances IL-6 mRNA accumulation in these cells, although by increasing its transcription rate. In this case, IFN-gamma does not affect IL-6 mRNA expression. These data suggest that IL-1-induced GM-CSF expression in endothelial cells is regulated at the posttranscriptional level and that IFN-gamma specifically inhibits GM-CSF expression via destabilization of the mRNA. These suppressive effects of IFN-gamma provide evidence for an additional role of IFN-gamma in regulating GM-CSF production.

Evaluation of expression of transferred genes in differentiating myeloid cells: expression of human glucocerebrosidase in murine macrophages.

The retroviral vector LGSN, in which the human glucocerebrosidase (GC) cDNA is driven by the Moloney murine leukemia virus (MoMLV) long terminal repeat (LTR), was tested for expression in the murine myelomonocytic leukemia cell line M1 before and after induction of differentiation with interleukin-6 (IL-6). Southern analysis of the seven transduced clones selected for neomycin resistance in Geneticin (G-418 sulfate) demonstrated one to eight copies of intact provirus with rearrangements in only two clones. Absolute levels of human GC RNA and protein increased with increased copy numbers of provirus in the clones. Upon induction with IL-6 of the seven transduced clones to the macrophage phenotype, there was no significant change, overall, in RNA levels but some increase in human GC protein levels could be detected. Although this was the average trend, considerable clonal variation in RNA and protein levels was observed upon induction. Transduction of the M1 cells did not interfere with the ability of the cells to differentiate from blasts to macrophages as seen by the appearance of membrane receptors for the constant region of immunoglobulins (Fc gamma RI) and lysozyme production in the differentiated M1 cells. Thus, the M1 cell line can be used for testing retroviral vector expression in myeloid lineages at early and late stages of differentiation. This rapid in vitro testing of potential retroviral vectors will be beneficial for gene therapy of disorders that affect differentiated macrophages such as Gaucher's disease.

Ca2+ ionophore A23187-dependent stabilization of granulocyte-macrophage colony-stimulating factor messenger RNA in murine thymoma EL-4 cells is mediated through two distinct regions in the 3'-untranslated region.

We analyze the role of the Ca2+ ionophore A23187 in the induction of GM-CSF mRNA expression in EL-4 thymoma cells. Northern analysis shows that A23187 increases the half-life of GM-CSF mRNA. To identify potential Ca2+ response elements in the GM-CSF mRNA, we produced stable transfectants containing pRSV-CAT (EL-4cat) or hybrid constructs in which most of the GM-CSF 3'-untranslated region (EL-4gm) or the adenosine-uridine boxes alone (EL-4au) were placed in a downstream position from the CAT coding region. A23187 induces a 4.4-fold increase in CAT activity in EL-4cat cells and a 210-fold and 48-fold increase in CAT activity in EL-4gm and EL-4au cells, respectively. Actinomycin D chase experiments in transfected cells demonstrate that A23187 increases the half-life of CAT mRNA from 15 min to 3 h in EL-4au cells and more than 3 h in EL-4gm cells, suggesting that the effect of Ca2+ is mediated predominantly by the adenosine-uridine boxes with a smaller contribution from upstream regions. To map these upstream regions, we transfected cells with constructs containing mutations of the 3'-untranslated region. With two of these mutations, corresponding to a region located about 160 bases upstream of the adenosine-uridine boxes, CAT activity was induced only 50-fold compared to 200-fold in EL-4gm cells. These data indicate that two regions within the GM-CSF 3'-untranslated region interact to modulate Ca2+ effects on GM-CSF mRNA half-life.

Dissociation of early and late markers of murine myeloid differentiation by interferon-gamma and interleukin-6.

Murine myeloid leukemia M1 cells undergo terminal differentiation to mature macrophages after stimulation with interleukin-6 (IL-6). This process can be monitored by measuring the expression of early markers such as the high affinity receptor for monomeric IgG2a (Fc gamma RI) and Ia antigen followed by late markers such as lysozyme production and finally morphological changes from blast cells to mature macrophages. The same early markers that are expressed on M1 cells after induction with IL-6 are also expressed on monocytic cells after activation with interferon-gamma (IFN gamma). We used IL-6 and IFN gamma to investigate whether the early stages of M1 cell differentiation could be accomplished without commitment of the cells to terminal differentiation. Cytofluorometry shows that the expression of the same early differentiation markers (Fc gamma RI and Ia antigen) that are inducible by IL-6 on M1 cells can be induced by IFN gamma as well. However, stimulation with IFN gamma, in contrast to IL-6, does not induce the late differentiation markers such as lysozyme production, phagocytic activity, and morphological changes. Northern analysis supports these findings in that expression of Fc gamma RI mRNA is induced by either cytokine, whereas expression of mRNA for lysozyme is inducible by IL-6 only. Nuclear run-on analysis reveals that the changes in steady state mRNA levels of both Fc gamma RI and lysozyme are regulated by a transcriptional mechanism. These data suggest that early stages in the process of myeloid differentiation can be separately induced by IFN gamma and thus are independent from the later events induced by IL-6.

Soluble interleukin-4 receptor production by murine myeloid progenitor cells: induction by interleukin-6 and interferon-gamma.

Soluble cytokine receptors, molecules that can selectively modulate the effects of a single cytokine, have generated great interest both as indicators of disease and as targeted immunotherapeutic agents. However, cellular sources for soluble cytokine receptors are not well characterized and the regulation of soluble receptor production is not clear. We recently found that interleukin 6 (IL-6) induces the expression of membrane-bound interleukin 4 (IL-4) receptors (mIL-4R) in the murine myeloid leukemia M1 cell line. In the present study we show that IL-6 induces the expression and secretion of the soluble form of the IL-4 receptor (sIL-4R) in these cells as well, with the protein accumulating for up to 72 hours in the cell supernatants. This inducible production of sIL-4R protein is accompanied by the induction of mRNA specific for sIL-4R. In mature bone marrow (BM)-derived macrophages sIL-4R expression can be induced by exposure to either IL-6 or interferon-gamma (IFN gamma). The data suggest that myeloid cells exposed to inflammatory stimuli are one possible source for sIL-4R enabling them to modulate the effects of IL-4 on the immune response by the regulated production of the soluble receptor for this cytokine.

Interleukin 1 augments the expression of the interleukin 2 receptor alpha-chain in interleukin 6-stimulated myeloid cells by a transcriptional and posttranscriptional mechanism.

We have recently shown that interleukin 6 (IL-6) induces transient expression of the alpha-chain of the interleukin 2 receptor (IL-2R alpha) in the murine leukemia myeloid M1 cell line. Others have reported that IL-6 and interleukin 1 (IL-1) synergistically enhance the expression of IL-2R alpha in T cells. Thus, in the present study, we investigated whether IL-1 affects the kinetics of IL-6-induced IL-2R alpha expression in M1 cells. By cytofluorometry, we find that surface expression of IL-2R alpha at 24 h after induction by IL-6 is strongly enhanced by IL-1. However, IL-1 does not change the transient kinetics of expression of IL-2R alpha. Binding data and Scatchard analysis support these results and show an increase from 3100 to 17,620 low-affinity IL-2 binding sites per cell without any change in affinity after induction of M1 cells by the combination of IL-6 and IL-1. By Northern analysis, we find that the increase in IL-2R alpha surface expression after treatment with IL-6 and IL-1 occurs in parallel with an increase in IL-2R alpha but not IL-2R beta mRNA expression. By nuclear run-on analysis and actinomycin-D chase experiments, we find that the increase in IL-2R alpha mRNA expression is due to both an increase in IL-2R alpha gene transcription and to an increase in IL-2R alpha mRNA stability. These data suggest that the IL-6-induced expression of IL-2R alpha can be specifically up-regulated by IL-1, however, without affecting the transient nature in expression of IL-2R alpha.

Binding of sequence-specific proteins to the adenosine- plus uridine-rich sequences of the murine granulocyte/macrophage colony-stimulating factor mRNA.

Adenosine+uridine (AU)-rich sequences in the 3' untranslated region (3'UTR) of the mRNA of many cytokines and oncogenes play an important role in mediating RNA degradation. Among the cytokines containing such AU-rich sequences in their 3'UTR is the hematopoietic growth factor granulocyte/macrophage colony-stimulating factor (GM-CSF). GM-CSF gene expression in T cells is regulated by modulation of mRNA half-life. Transfection studies using murine EL-4 thymoma cells have demonstrated that degradation depends on the presence of specific elements in the 3'UTR, including the AU-rich sequences. A number of AU-binding factors have recently been discovered, suggesting that specific regulation may occur through specific protein-mRNA interaction(s). We present evidence from gel-shift analyses and label-transfer experiments that murine cells contain proteins that bind specifically to AU-rich sequences. Three major proteins of 33, 39.5, and 42 kDa are detected. Phorbol ester treatment of cells does not alter the abundance or apparent binding affinity of the proteins. The 33-kDa protein is present in the cytoplasm of murine and human cells, whereas the 39.5- and 42-kDa proteins are present in murine extracts only. Constitutively expressed AU-binding proteins of the type that we describe may function by directing mRNA degradation in the absence of a stimulus to the contrary.

Transient expression of the IL-2 receptor alpha-chain in IL-6-induced myeloid cells is regulated by autocrine production of prostaglandin E2.

The alpha-chain of the interleukin 2 receptor (IL-2R alpha) is expressed on monocytes and macrophages after activation by bacterial lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). In the present study, we investigated whether the expression of IL-2R alpha is associated with the process of differentiation of myeloid cells to mature macrophages and how this expression is regulated. The murine myeloid M1 cell line, which can be induced by leukemia inhibitory factor (LIF) or interleukin 6 (IL-6) to differentiate from blast cells to mature macrophages, was used as a model system for myeloid differentiation. Bone marrow (BM)-derived macrophages were used as mature myeloid cells. Cytofluorometry revealed that IL-2R alpha is transiently expressed during M1 cell differentiation, with peak levels 24 h after induction by LIF or IL-6, whereas the high affinity receptor for monomeric IgG2a (FcR), a surface marker typical for macrophage differentiation, continues to rise up to 72 h. BM-derived macrophages already express FcR but not IL-2R alpha. IL-2R alpha expression is induced on these cells after treatment by IL-6 for up to 48 h. Treatment of IL-6-induced M1 cells with indomethacin permitted a sustained expression of IL-2R alpha beyond 24 h, and this effect was reversed by the addition of prostaglandin E2 (PGE2). Northern analysis showed that in M1 cells the expression of mRNA for IL-2R alpha, but not for IL-2R beta, is also transient, indicating that cell surface expression of IL-2R alpha is regulated at the mRNA level. These data show that inducers of macrophage differentiation such as LIF and IL-6 can induce a transient expression of the IL-2R alpha-chain in differentiating murine myeloid M1 cells and that autocrine production of PGE2 is involved in the control of the transient expression of this receptor. However, induction of expression of IL-2R alpha by IL-6 appears to be independent of differentiation because it can be induced on fully differentiated BM-derived macrophages as well.

Interleukin-4 inhibits interleukin-1 alpha-induced granulocyte-macrophage colony-stimulating factor gene expression in a murine B-lymphocyte cell line via downregulation of RNA precursor.

Interleukin-4 (IL-4) regulates the growth of B cells. When combined with colony-stimulating factors (CSFs) and selected cytokines, IL-4 has a synergistic effect on the clonal growth of bone marrow cells. Recently, we have shown that IL-1 alpha and lipopolysaccharide induce expression of the granulocyte-macrophage CSF (GM-CSF) gene in murine B-cell lines. In the present study, we show that IL-4 inhibits the production of GM-CSF in the IL-1 alpha-stimulated murine B-cell line M12.4.1. IL-4 did not change the transcription rate of the GM-CSF gene, and caused only a slight decrease in cytoplasmic GM-CSF messenger RNA (mRNA) half-life in cells treated with IL-1 alpha. PCR analysis of nuclear RNA with probes specific for GM-CSF intron sequences suggests that IL-1 alpha enhances accumulation of nuclear precursor RNA and that decreased GM-CSF expression after IL-4 treatment is mainly due to intranuclear destabilization of the primary transcript. Under the same experimental conditions, IL-4 did not affect expression of the IL-4 receptor mRNA and did increase the mRNA concentration of the low-affinity receptor for IgE (Fc epsilon RII). These data suggest that the suppressive effect of IL-4 is specific for GM-CSF mRNA expression, and thus provide evidence for an additional role of IL-4 in the regulation of GM-CSF expression in B cells.

Concanavalin A-induced granulocyte-macrophage colony-stimulating factor production in a murine T-cell line is posttranscriptionally controlled.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor (HGF) that regulates the proliferation and differentiation of cells of the myeloid lineage. It can be produced by a variety of cells. One of the major sources of GM-CSF is activated T cells, which transiently produce this HGF. We used the EL-4 thymoma cell line as a model system to address the molecular basis for GM-CSF regulation in T cells. Both concanavalin A (ConA) and the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) induce GM-CSF expression in EL-4 cells. However, the biological activity of GM-CSF in the supernatants of the TPA-stimulated cells was higher than that of ConA-stimulated cells. To elucidate this difference in biological activity levels, we examined how ConA regulates GM-CSF gene expression in EL-4 cells and compared it to the better-characterized regulation by TPA. Peak mRNA levels of GM-CSF occur 6 h after stimulation with either of these two agents. GM-CSF mRNA levels after ConA treatment are lower and decrease significantly after 10 h compared to TPA treatment, which causes much higher levels that persist for at least 24 h. Neither agent alters GM-CSF gene transcription. Actinomycin D chase experiments show that ConA increases the GM-CSF mRNA half-life from less than 30 to 90 min, whereas TPA prolongs it to greater than 3 h. These results indicate that GM-CSF mRNA induction by ConA (in common with TPA) is regulated predominantly via RNA stabilization and that the difference in prolongation of the mRNA half-life provides the primary explanation for the lower levels of GM-CSF mRNA induced by ConA compared to TPA.

Correlation in the expression of type IV collagenase and the invasive and chemotactic abilities of myelomonocytic cells during differentiation into macrophages.

Monomyelocytic phagocytes originate in the bone marrow and while differentiating into macrophages migrate to inflammatory foci and target tissues by egress from the capillary blood vessels. During such diapedesis, the cells must traverse tissue barriers such as basement membrane, which has type IV collagen as its principal structural element. We studied whether the expression of type IV collagenase activity, invasion through basement membrane, and the response to inflammatory chemoattractants are related to each other and to the process of differentiation of murine M1 myeloid leukemia cells into macrophages. M1 cells stimulated with mouse lung-conditioned medium (MLCM) or interleukin 6 (IL6) differentiate into macrophages by 72 h, as determined by expression of Fc receptors, induction of lysozyme, and morphological changes from blast cells to mature macrophages. During this process of differentiation the invasive ability of the cells and the amount of type IV collagenase in the supernatants from the invading cells continuously increased up to 72 h. Zymographic analysis of supernatants of the invading cells revealed a single 100-kd metalloproteinase with gelatinolytic activity. Chemotaxis towards arachidonic acid metabolites, which are present in inflamed tissues, was detected only in differentiated cells. Studies with thioglycolate (TG)-elicited peritoneal macrophages gave results similar to those obtained with differentiated M1 cells, showing that the ability to invade basement membrane, the expression of type IV collagenase, and the chemotactic response to inflammatory chemoattractants all increased with the differentiation of myeloid cells and reached their highest expression in fully differentiated cells.

Regulation of interleukin-4 receptors on murine myeloid progenitor cells by interleukin-6.

Interleukin-4 (IL-4) is a T-cell-derived cytokine that regulates induction of proliferation of resting B cells and acts on various other immunocompetent cells, such as monocytes/macrophages and mast cells, as well as hematopoietic progenitor cells. On hematopoietic progenitor cells, cooperation with another cytokine (such as granulocyte-macrophage colony-stimulating factor [GM-CSF], G-CSF, IL-3, or IL-6) is required to render the cells responsive to IL-4. The present study was undertaken to determine if such an interaction entails induction of IL-4 receptor (IL-4R) expression. Using the murine myeloid leukemia M1 cell line and mature, bone marrow (BM)-derived macrophages, we investigated whether IL-4R expression can be induced during differentiation. We detected no high-affinity IL-4R on the surface of either cell, but with exposure to IL-6 a significant induction of IL-4R was measured on both cell types by fluorescence-activated cell sorter analysis. This increase in IL-4R was first noted 6 hours after exposure of the cells to IL-6 and continued to increase up to 48 hours. By RNase protection analysis we found that the expression of IL-4R mRNA also appeared within 6 hours, continuing to increase up to 48 hours. Nuclear run-on assays showed that this increase in steady-state level of IL-4R mRNA results from a transcriptional activation of the IL-4R gene. These data suggest that regulation of IL-4R expression by IL-6 is under transcriptional control.

Identification of sequences within the murine granulocyte-macrophage colony-stimulating factor mRNA 3'-untranslated region that mediate mRNA stabilization induced by mitogen treatment of EL-4 thymoma cells.

Phorbol esters (TPA) and concanavalin A (ConA) are known to induce granulocyte-macrophage colony-stimulating factor (GM-CSF) production in murine thymoma EL-4 cells by mRNA stabilization. The role of the 3'-untranslated region (3'-UTR) in GM-CSF mRNA stabilization induced by TPA and ConA in EL-4 cells was examined by transfection studies using chloramphenicol acetyltransferase (CAT) constructions. The GM-CSF 3'-UTR contains a 63-nucleotide region at its 3' end with repeating ATTTA motifs which is responsible for mRNA degradation in a variety of cell types (Shaw, G., and Kamen, R. (1986) Cell 46, 659-666). We produced constructs containing most of the GM-CSF 3'-UTR (303 nucleotides, pRSV-CATgm) or the 3'-terminal AT-rich region (116 nucleotides, pRSV-CATau) and measured CAT enzyme activity and CAT mRNA after transient transfection into EL-4 and NIH 3T3 cells. Low levels of CAT activity were seen in both cells with either plasmid compared with levels of CAT activity obtained with pRSV-CAT. TPA treatment caused an approximately 10-fold increase in CAT activity and mRNA in EL-4 cells transfected with pRSV-CATgm. No increases were seen in EL-4 cells transfected with pRSV-CATau or pRSV-CAT. No response to TPA was detected in transfected NIH 3T3 cells, indicating that the response to TPA is relatively cell-specific. There was no increase in CAT activity after ConA treatment in EL-4 or NIH 3T3 cells transfected with any of the constructs suggesting that the GM-CSF 3'-UTR lacks elements that can respond alone to ConA. Nuclear run-on and actinomycin D chase experiments in EL-4 cells showed that TPA induces CAT activity via mRNA stabilization. By linker-substitution mutagenesis we show that TPA inducibility depends on a 60-nucleotide region of the 3'-UTR whose 5' end is located 160 nucleotides upstream of the 5' end of the AU-rich region.

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.

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.