Hematopoietic growth factors upregulate the p65 type II interleukin-1 receptor on bone marrow progenitor cells in vitro.

Having previously shown that interleukin-1 (IL-1) induces the expression of IL-1 receptors (IL-1Rs) on bone marrow (BM) cells in vivo through an indirect mechanism, we studied whether hematopoietic growth factors (HGFs) could induce the expression of IL-1R on BM cells in vitro. In vitro treatment of light-density murine BM (LDBM) cells with either IL-3, IL-6, granulocyte--colony-stimulating factor (CSF), or granulocyte-macrophage--CSF caused a 5- to 10-fold upregulation of IL-1R expression, whereas IL-1, IL-5, IL-7, and macrophage-CSF had no effect. Scatchard analysis showed one class of IL-1Rs on LDBM cells with an average of 66 +/- 20 sites per cells. After 24 hours of treatment with IL-3, the number of IL-1Rs increased to 413 +/- 125, without effecting the affinity. This effect required protein synthesis, but was independent of cell division. Purified lineage-negative progenitor cells (Lin-) did not express detectable levels of IL-1R, but 24 hours of treatment with IL-3, GM-CSF, and G-CSF stimulated IL-1--specific binding. Autoradiographic analysis of Lin- cells showed that IL-1R induction by IL-3 occurs on undifferentiated blast cells. Affinity labeling of Lin- cells treated with HGFs showed an increase in a 65-Kd IL-1 binding protein that did not bind or compete with an anti-type I IL-1R antibody, suggesting that these cells expressed type II IL-1R. These data suggest that IL-1 stimulation of myelopoiesis occurs by a mechanism involving IL-1R upregulation on hematopoietic progenitor cells by HGFs.     

Interleukin-6 and the soluble interleukin-6 receptor induce stem cell factor and Flt-3L expression in vivo and in vitro

OBJECTIVE: We recently established transgenic animals expressing either interleukin-6 (IL-6) or the soluble IL-6 receptor (sIL-6R) alone, or both components, IL-6 and the sIL-6R, in the liver. This animal model demonstrated that the expression of IL-6 in combination with its sIL-6R led to extramedullary expansion of hematopoietic progenitor cells in the spleen and liver. MATERIALS AND METHODS: We studied other relevant hematopoietic cytokines involved in the IL-6/sIL-6R-induced stimulation of hematopoiesis. RESULTS: Using immunohistochemistry, we showed that cell-associated stem cell factor (SCF) and Flt-3L expression were upregulated in liver and spleen only in double transgenic mice but not in IL-6 or sIL-6R single transgenic animals. Moreover, on murine NIH/3T3 fibroblasts and on human primary forskin fibroblasts, stimulation with the IL-6/sIL-6R complex, and to a lesser extent with IL-6 alone, led to induction of cellular SCF and Flt-3L expression. When human HTB-158 fibroblasts were stimulated with the IL-6/sIL-6R complex and subsequently cocultured with human umbilical cord CD34(+) cells, a significant upregulation in colony growth was found. CONCLUSIONS: We showed that IL-6 in combination with its soluble receptor stimulates cellular SCF and Flt-3L expression in vivo and in vitro. Cellular upregulation of SCF and Flt-3L by IL-6/sIL-6R might be used for the development of new stroma cell systems for ex vivo expansion of hematopoietic progenitor cells.     

Human cytomegalovirus alters interleukin-6 production by endothelial cells

In an effort to study whether human cytomegalovirus (HCMV) can disrupt the balanced cytokine network that controls human hematopoiesis, we investigated the ability of a laboratory strain HCMV (AD169) to alter the production of interleukin-6 (IL-6) by cultured endothelial cells (HUVECs). ECs are important components of human bone marrow stroma and produce factors that stimulate the proliferation and differentiation of human hematopoietic progenitors. HCMV was able to greatly increase production of both mRNA and protein for IL-6 in unprimed HUVECs. When we discriminated between viral pellet and cleared viral supernatants, the supernatants induced an increase in mRNA at 30 minutes and protein by 2 hours, whereas an increase in IL-6 caused by virus itself did not become evident until 12 hours. The possibility that IL-6 induction was simply caused by the presence in the viral stock of endotoxin, IL-1 alpha, IL-1 beta, tumor necrosis factor alpha, or IL-4, all known inducers of IL-6 in HUVECs, was ruled out by the addition of polymyxin B and appropriate neutralizing antibodies. These findings show that HCMV is capable of directly and indirectly modulating the production by HUVECs of IL-6, one of the cytokines involved in the process of hematopoiesis.     

Stem cell factor, interleukin-3, and interleukin-6 promote retroviral-mediated gene transfer into murine hematopoietic stem cells.

The efficiency of retroviral-mediated gene transfer into hematopoietic stem cells (HSC) is dependent on the survival and self-renewal of HSC in vitro during retroviral infection. We have examined the effect of prestimulation of bone marrow with various cytokines, including the product of the Steel gene, Steel factor or stem cell factor (SCF) (the ligand for the c-kit receptor) on the efficiency of retroviral transduction of the human adenosine deaminase (hADA) cDNA into murine HSC. Bone marrow cells were prestimulated for 48 hours with hematopoietic growth factors, then cocultivated with the packaging cell line producing the ZipPGK-ADA simplified retrovirus for an additional 48 hours with continued growth factor exposure. Nonadherant cells from these cocultures were injected into lethally irradiated recipients. The content of day 12 colony-forming unit-spleen (CFU-S12) in SCF/interleukin 6 (IL-6)-prestimulated and cocultured bone marrow was more than threefold greater than that of IL-3/IL-6-prestimulated bone marrow cells. All mice receiving bone marrow cells infected with the PGK-ADA virus after prestimulation with IL-3/IL-6 or SCF/IL-6 demonstrated hADA expression in the peripheral blood after full hematopoietic reconstitution. While all recipients of IL-3/IL-6-prestimulated bone marrow expressed hADA at 4 months posttransplant, in three independent experiments examining a total of 33 mice, in most recipients of SCF/IL-6-prestimulated and infected bone marrow cells, the expression of human enzyme was higher than IL-3/IL-6 mice. Southern blot analysis of DNA from hematopoietic tissues from these same mice prepared at least 4 months posttransplantation also demonstrated a higher infection efficiency of HSC as measured by proviral integration patterns and genome copy number analysis. These results suggest that the higher level of hADA expression seen in mice receiving marrow prestimulated with SCF/IL-6 before retroviral infection is due to more efficient infection of reconstituting HSC. Other growth factor combinations were also studied; however, prestimulation with SCF/IL-6 or IL-3/IL-6 appeared optimal. Using retroviral-mediated gene transfer and viral integration patterns, Steel factor (SCF) in combination with IL-6 appears to increase the survival and self-renewal of reconstituting hematopoietic stem cells and proves useful in effecting expression of foreign genes in transplant recipients. Such pretreatment may also be useful in the application of retroviral transfer methods to human cells.     

Interleukin-18 stimulates hematopoietic cytokine and growth factor formation and augments circulating granulocytes in mice

Because interleukin-18 (IL-18) is similar to IL-1 and is known to be involved in the hematopoietic progenitor cell growth, the effect of IL-18 on circulating cell populations was examined. Repeated administration of IL-18 induced significant amounts of neutrophilia in mice. In parallel, high levels of interferon-gamma (IFN-gamma), IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were detected in the serum of these mice. Interestingly, the cytokine profiles as well as the cell populations in circulation altered around 2 weeks after the beginning of IL-18 administration. A weak but definite eosinophilia was observed concurrently with the appearance of serum IL-5. Consistent with these observations, IL-18 induced secretion of IFN-gamma, GM-CSF, and IL-6 from splenocytes in culture. IL-18 also induced low levels of IL-5 in the splenocyte culture, which was inhibited by IL-12. However, markedly high levels of IL-5 were secreted into the culture medium when splenocytes from IFN-gamma-deficient mice were stimulated by IL-18. CD4(+) T cells strongly responded to IL-18 to secrete IL-5 and GM-CSF. IL-18 stimulated secretion of IL-6 and expression of G-CSF mRNA in splenic adherent cells. Expression of IL-18 receptors was detected in CD4(+) T cells and splenic adherent cells (macrophages). These results show that IL-18 stimulates CD4(+) T cells and macrophages to secrete IL-5, GM-CSF, IL-6, and granulocyte-colony stimulating factor in the absence of IL-12, which in turn induces hematopoietic cell proliferation causing neutrophilia and eosinophilia in mice.     

Stem cell factor enhances interleukin-3 dependent induction of 68-kD calmodulin-binding protein and thymidine kinase activity in NFS-60 cells

Stem cell factor (SCF) is known to act synergistically with other hematopoietic factors in increasing the colony formation of hematopoietic progenitor cells. We have shown that interleukin-3 (IL-3)- dependent proliferation of NFS-60 cells is associated with the induction of a specific calmodulin-binding protein of about 68 kD (CaM- BP68). To evaluate the relationship between proliferative stimulation and the induction of CaM-BP68 by cytokines, we examined whether the increased proliferative potential of NFS-60 cells in response to SCF is reflected in an increased induction of the CaM-BP68. We observed that SCF alone has a limited effect on proliferative stimulation and on the induction of CaM-BP68 in factor-deprived NFS-60 cells. However, when combined with IL-3, granulocyte colony-stimulating factor (G-CSF), or IL-6, it caused a significant increase in cytokine-dependent proliferative stimulation, as well as in the induction of CaM-BP68. Furthermore, an increase in IL-3-dependent induction of CaM-BP68 in the presence of SCF coincided with a corresponding increase in thymidine kinase activity, whose expression is linked to G1/S transition of the cells. At low concentrations SCF caused a synergistic increase in IL-3- dependent induction of both CaM-BP68 and thymidine kinase activity. In contrast to the changes in CaM-BP68 and thymidine kinase activity, no significant changes in DNA polymerase alpha were observed in factor- deprived NFS-60 cells in response to IL-3 and/or SCF. These observations suggest an increased expression of CaM-BP68 and thymidine kinase are associated with the synergistic effect of SCF on factor- dependent proliferation of hematopoietic progenitor cells.     

Production of interleukin-6 and interleukin-8 by nurse-like cells from rheumatoid arthritis patients after stimulation with monocytes

Abstract

It has been reported that nurse-like cells (NLCs) play a critical role in the pathogenesis of rheumatoid arthritis (RA). The interaction between NLCs established from RA patients (RA-NLCs), and freshly isolated blood monocytes was analyzed to further elucidate the pathogenesis of RA. RA-NLC lines were established from the synovium of RA patients. The RA-NLCs were cultured with monocytes freshly isolated from peripheral blood of healthy donors, and induction of interleukin (IL)-6 and IL-8 as well as the mRNA expression of these cytokines was examined. The levels of IL-6 were over 400 times higher in the supernatant from coculture of RA-NLCs and monocytes than in those from cultures of RA-NLCs alone. Anti-tumor necrosis factor (TNF)-α monoclonal antibody inhibited the induction of both cytokine in a dose-dependent fashion, although there was no detectable level of TNF-α in the supernatant from coculture. In addition, coculture of RA-NLCs and monocytes without direct cell contact did not induce cytokine production. To determine IL-6 producing cells, RA-NLCs and monocytes were separated into each fraction after coculture for 24?h. Cocultured RA-NLCs contained approximately 80 times higher IL-6 mRNA than the RA-NLCs cultured alone. The levels of IL-8 were also much higher (about 900 times) in the supernatant from coculture than in those from cultures of RA-NLCs alone. Cocultured RA-NLCs expressed IL-8 mRNA about 620 times higher than those cultured alone. These results indicate that NLCs produce high levels of IL-6 and IL-8 after cell–cell interaction with monocytes/macrophages via membrane-bound TNF-α, and that activation of NLCs by monocytes/macrophages may be involved in the pathogenesis of RA through maintenance of synovial inflammation.     

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.     

Adhesion receptor expression by hematopoietic cell lines and murine progenitors: modulation by cytokines and cell cycle status

Hematopoietic progenitor cells are incubated with cytokine combinations for in vitro expansion of stem cells and to enhance retrovirus-mediated gene transfer. Optimization of the engraftment of these treated cells would be critical to the success of stem cell transplantation or gene therapy. Previous studies demonstrated that a 48-hour incubation of donor BALB/c bone marrow with a mixture of four cytokines (IL-3, IL-6, IL-11, and SCF), resulted in expansion of primitive progenitor/stem cells but a loss of long-term engraftment in nonmyeloablated or myeloablated recipients. We have established the expression pattern for a number of adhesion receptors by normal hematopoietic progenitors and cell lines and the modulation in expression induced by cytokines or cell cycle progression to ascertain the molecular basis for such defective engraftment. Northern blot analysis demonstrated that the cytokine combination of IL-3, IL-6, IL-11, and SCF dramatically down-regulated alpha 4 integrin receptor expression in HL-60 cells. Synchronized FDC-P1 cells exhibited modulation of alpha 4 expression through cell cycle progression, both by quantitative RT-PCR and flow cytometry. Normal murine bone marrow lineage-depleted, Sca+ cells expressed a number of adhesion receptors, including alpha L, alpha 1, alpha 3, alpha 4, alpha 5, alpha 6, beta 1, L-selectin, CD44, and PECAM as assessed by flow cytometry, immunofluorescence, and RT-PCR. There was modulation of the expression of several of these receptors after incubation in the four cytokines for 24 and/or 48 hours: the proportion of cells expressing alpha L, alpha 5, alpha 6, and PECAM increased, whereas the proportion of cells expressing alpha 4 and beta 1 decreased, after cytokine incubation. There was a demonstrable concomitant decline in adhesion of these cells to fibronectin after the cytokine incubation, a finding that correlates with the decrease in expression of alpha 4. These changes in adhesion receptor expression and function with cytokines and during cell cycle transit may be critical to stem cell homing and engraftment after transplantation, as multiple receptors could be involved in the process of rolling, attachment to endothelium, endothelial transmigration, and migration within the marrow space.     

Production of interleukin-6 and interleukin-8 by nurse-like cells from rheumatoid arthritis patients after stimulation with monocytes

It has been reported that nurse-like cells (NLCs) play a critical role in the pathogenesis of rheumatoid arthritis (RA). The interaction between NLCs established from RA patients (RA-NLCs), and freshly isolated blood monocytes was analyzed to further elucidate the pathogenesis of RA. RA-NLC lines were established from the synovium of RA patients. The RA-NLCs were cultured with monocytes freshly isolated from peripheral blood of healthy donors, and induction of interleukin (IL)-6 and IL-8 as well as the mRNA expression of these cytokines was examined. The levels of IL-6 were over 400 times higher in the supernatant from coculture of RA-NLCs and monocytes than in those from cultures of RA-NLCs alone. Anti-tumor necrosis factor (TNF)-α monoclonal antibody inhibited the induction of both cytokine in a dose-dependent fashion, although there was no detectable level of TNF-α in the supernatant from coculture. In addition, coculture of RA-NLCs and monocytes without direct cell contact did not induce cytokine production. To determine IL-6 producing cells, RA-NLCs and monocytes were separated into each fraction after coculture for 24 h. Cocultured RA-NLCs contained approximately 80 times higher IL-6 mRNA than the RA-NLCs cultured alone. The levels of IL-8 were also much higher (about 900 times) in the supernatant from coculture than in those from cultures of RA-NLCs alone. Cocultured RA-NLCs expressed IL-8 mRNA about 620 times higher than those cultured alone. These results indicate that NLCs produce high levels of IL-6 and IL-8 after cell–cell interaction with monocytes/macrophages via membrane-bound TNF-α, and that activation of NLCs by monocytes/macrophages may be involved in the pathogenesis of RA through maintenance of synovial inflammation.     

Human interleukin-4 enhances stromal cell-dependent hematopoiesis: costimulation with stem cell factor

Interleukin-4 (IL-4) has distinct hematopoietic activities, primarily as a costimulant with other cytokines to enhance colony formation of hematopoietic progenitors. We investigated the influence of IL-4 on stromal cell-supported long-term cultures (LTCs) of normal human bone marrow. Addition of IL-4 to LTCs of unseparated bone marrow or highly enriched CD34+ cells resulted in a significant increase of myeloid progenitors in the nonadherent, as well as in the stromal cell-adherent cell populations. In contrast, the total cell number was not influenced by IL-4, suggesting a selective effect on primitive progenitor cells. Cord blood cells or CD34+ bone marrow cells were incubated with stem cell factor (SCF) and/or IL-4 in stromal cell-free cultures. In these experiments, a twofold to fivefold increase of myeloid progenitor cells was observed in the presence of SCF and IL-4 as compared with SCF alone. Preincubation of the stromal cell cultures with IL-4 resulted in an enhanced adherence of CD34+ cells to the stromal layer. Secretion of hematopoietic growth factors produced by the stromal cells, such as granulocyte-macrophage colony-stimulating factor (G-CSF), and IL-1, was inhibited by IL-4. Thus, the increase of hematopoietic progenitors in LTCs, as observed in the presence of IL-4, can be at least partially explained by a costimulation of SCF and IL-4 on primitive progenitor cells and by an enhancement of hematopoietic cells to stroma. The downregulation of CSFs by IL-4 might prevent the expansion of the mature hematopoietic cell compartment.     

Functional expression of Fas antigen (CD95) on hematopoietic progenitor cells

We investigated the expression of an apoptosis-associated antigen (Fas) (CD95) on hematopoietic progenitor cells in the presence or absence of interferon-gamma (IFN-gamma) and/or tumor necrosis factor-alpha (TNF- alpha). CD34+ cells freshly isolated from bone marrow did not express Fas. However, IFN-gamma and/or TNF-alpha induced the expression of both the mRNA of Fas and Fas itself in a dose-dependent fashion on the surface of CD34+ cells after 48 hours of serum-free culture. IFN-gamma and TNF-alpha had a synergistic effect on the induction of Fas, when both cytokines were added to the culture. The TNF-alpha-induced Fas expression is mediated by p55 TNF-alpha receptor. CD34+ cells cultured in medium alone or with stem cell factor (SCF) showed some slight expression of Fas. When anti-Fas antibody (IgM) was added to CD34+ cells after the induction of Fas expression, CD34+ cells underwent apoptosis, as shown by a decrease in the number of viable cells, morphologic changes, the induction of DNA fragmentation, and a decrease in the number of colony-forming cells (CFC) including colony-forming unit granulocytes/macrophages (CFU-GM) and burst-forming unit erythroids (BFU-E). These observations indicate that IFN-gamma and/or TNF-alpha, well known as negative hematopoietic regulators, induce functional Fas on hematopoietic progenitor cells. The suppression of hematopoiesis by negative hematopoietic regulators may be mediated in part by Fas induction.     

Expression and secretion of vascular endothelial growth factor-A by cytokine-stimulated hematopoietic progenitor cells. Possible role in the hematopoietic microenvironment

In the hematopoietic microenvironment, bone marrow endothelial cells may play an important role in trafficking and maintenance of progenitor and stem cells due to adhesive interactions and paracrine secretion of hematopoietic growth factors. However, it is unknown whether progenitors in turn modulate endothelial proliferation and function.We analyzed mRNA expression (Northern blot) and release of vascular endothelial growth factor-A (VEGF-A), which specifically acts on endothelial cells, by cytokine-stimulated peripheral blood-derived CD34+ hematopoietic progenitor cells.While unstimulated CD34+ cells expressed VEGF-A mRNA weakly without cytokine release in vitro, incubation for 24 hours with a single cytokine (e.g., kit ligand [KL]) resulted in increased VEGF-A mRNA expression and significant secretion of VEGF-A into the supernatant. The amount of VEGF released was substantially augmented by incubation with a combination of cytokines (e.g., KL, IL-3, GM-CSF, G-CSF), or by exposure to hematopoietic cytokines for a longer time period. In addition, we show that VEGF induced the release of hematopoietic growth factors (GM-CSF) by bone marrow endothelial cells and that in vitro stromal cell-derived factor-1 (SDF-1) driven transendothelial progenitor cell migration was increased by the presence of VEGF, which might be due to pore formation (increased endothelial fenestration).In vivo, release of VEGF by progenitor cells may result in a paracrine loop supporting proliferation of both endothelium and progenitors and may facilitate transendothelial migration during cytokine-induced progenitor cell mobilization.     

Synergism between erythropoietin and interleukin-3 in the induction of hematopoietic stem cell proliferation and erythroid burst colony formation

The influence of recombinant erythropoietin (Ep) and interleukin-3 (IL- 3) on the proliferation and differentiation of murine hematopoietic stem and progenitor cells was investigated in serum-deprived cultures. The differentiation of progenitor cells, purified by collecting blast cell colonies from spleen cell cultures of 5-fluorouracil-treated mice, was evaluated by scoring the number and type of colonies appearing after eight days in semisolid culture. IL-3 induced the formation of both erythroid and granulocyte-macrophage colonies in a concentration- dependent fashion, the plateau being reached at 300 U/mL. However, concentrations of IL-3 alone that had little or no effect (less than or equal to 10 U/mL) induced maximal numbers of erythroid bursts in the presence of Ep (1.5 IU/mL). In the presence of Ep alone, no colonies were seen. Proliferation of quiescent hematopoietic stem cells, purified by cell sorting and evaluated by spleen colony assay (CFU-S), was investigated by measuring the total cell number and CFU-S content and the DNA histogram at 20 and 48 hours of liquid culture. Almost no cells or CFU-S survived 20 hours of incubation without the addition of IL-3. The presence of either IL-3 (400 U/mL) or the combination of EP and IL-3 (10 U/mL), supported the maintenance of nearly 40% of sorted CFU-S for 48 hours. Approximately 10% of these cells were in the S phase of the cell cycle at 20 hours and an increase in the total cell number per culture, but not in the CFU-S content, was detected at 48 hours. These data indicate that IL-3 exerts a differentiative and proliferative effect on early stem and progenitor cells, which is concentration dependent. At IL-3 concentrations, which had little or no activity alone, Ep acted synergistically to induce both proliferation of stem cells and differentiation of erythroid progenitors.     

Effect of hyperoxia on human macrophage cytokine response

In the development of lung damage induced by oxidative stress, it has been proposed that changes in alveolar macrophages (AM) function with modifications in cytokine production may contribute to altered repair processes. To characterize the changes in profiles of cytokine production by macrophages exposed to oxidants, the effects of hyperoxia (95% O2) on interleukin (IL)-1 beta, IL-6, IL-8, and tumour necrosis factor-alpha (TNF-alpha) expression were studied. Experiments were first performed using AM obtained from control subjects and children with interstitial lung disease. Results showed that a 48 h O2 exposure was associated with two distinct patterns of response: a decrease in TNF-alpha, IL-1 beta and IL-6 expression, and an increase in IL-8. To complete these observations we used U937 cells that were exposed for various durations to hyperoxia. We confirmed that a 48 h O2 exposure led to similar changes with a decrease in TNF-alpha, IL-1 beta and IL-6 production and an increase in IL-8. Interestingly, this cytokine response was preceded during the first hours of O2 treatment by induction of TNF-alpha, IL-1 beta and IL-6. These data indicate that hyperoxia induces changes in the expression of macrophages inflammatory cytokines, and that these modifications appear to be influenced by the duration of O2 exposure.     

Retinoic acid inhibits interleukin-6-induced macrophage differentiation and apoptosis in a murine hematopoietic cell line, Y6.

We established a radiation-induced murine hematopoietic cell line, Y6, that could be induced to differentiate into macrophages by interleukin-6 (IL-6). IL-6 also induced growth inhibition and apoptosis in Y6 cells. Retinoic acid (RA) inhibited such effects of IL-6 on Y6 cells. The inhibitory effect of RA on the effects of IL-6 was not caused by the downregulation of the IL-6 receptor, because RA neither affected the expression of IL-6 receptor mRNA nor the expression of IL-6 receptor molecule on the cell surface. Furthermore, RA did not inhibit the IL-6-induced expression of junB mRNA, indicating that the expression of functionally active IL-6 receptor and the signal transduction pathway activating the junB gene are not inhibited by RA. IL-6-induced macrophage differentiation of Y6 cells was preceded by the downregulation of the c-myc gene, which was also prevented by RA. Because the inhibitory effect of RA on Y6 cells was reversible and seemed not to require de novo protein synthesis, the RA receptor by itself might be directly involved in the inhibition of the IL-6 signal transduction pathway. The results indicated that the IL-6 signal transduction pathways leading to the induction of macrophage differentiation and junB gene expression can be dissected by RA.