Enhancement of cAMP levels and of protein kinase activity by tumor necrosis factor and interleukin 1 in human fibroblasts: role in the induction of interleukin 6.

Although tumor necrosis factor (TNF) and interleukin 1 (IL-1) affect many cell functions, the molecular mechanisms of TNF and IL-1 action are not understood. Our present study shows that exposure of human FS-4 fibroblasts to TNF or IL-1 caused a rapid accumulation of intracellular cAMP and an increase in protein kinase activity. Intracellular cAMP levels peaked 3-5 min after the addition of TNF or IL-1 and returned to basal level by 15 min. Increased phosphorylation of histone HII-B protein was demonstrated with extracts prepared from TNF- or IL-1-treated cells, suggesting an increase in cAMP-dependent protein kinase activity. No evidence was obtained for protein kinase C activation in TNF-treated FS-4 cells. TNF, IL-1, and forskolin all stimulated interleukin 6 (IL-6) mRNA levels in FS-4 cells. The protein kinase inhibitor H-8, inhibiting preferentially cAMP-dependent kinase activity, reduced forskolin-stimulated IL-6 mRNA induction more strongly than TNF- or IL-1-driven IL-6 mRNA induction. These results suggest that activation of cAMP-dependent protein kinase by TNF and IL-1 is important in some actions of these cytokines. In addition, our data on IL-6 induction by TNF and IL-1 suggest that other, yet unidentified, signal transduction mechanisms contribute to TNF and IL-1 actions on gene expression in human fibroblasts.     

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.     

Human granulocyte-monocyte colony-stimulating factor and interleukin 3 stimulate monocyte cytotoxicity through a tumor necrosis factor- dependent mechanism

Human colony-stimulating factors (CSF) exert multiple effects on the proliferation, differentiation, and function of myeloid lineage cells. In this study, the effects of three recombinant human CSFs (granulocyte- monocyte CSF [GM-CSF], interleukin 3 [IL-3], and granulocyte CSF [G- CSF]) on antibody-independent monocyte tumoricidal activity were investigated by using WEHI 164 fibrosarcoma cells as monocyte-sensitive targets. None of the CSFs directly induced monocyte cytotoxicity, although both GM-CSF and IL-3 were found to significantly enhance monocyte killing in response to a second stimulatory event (endotoxin). No effect was seen with G-CSF. Antitumor necrosis factor antibody completely abolished CSF-enhanced monocyte cytotoxicity, which suggests that this effect was mediated through increased release of tumor necrosis factor (TNF). As previously shown for GM-CSF, IL-3 was found to induce cytoplasmic accumulation of TNF messenger RNA (mRNA) after 18 hours of exposure. These results suggest that GM-CSF and IL-3 may stimulate monocyte killing indirectly by enhancing expression of TNF mRNA, thereby leading to augmented TNF protein secretion in response to a second activation signal.     

Induction of the transcription factor IRF-1 and interferon-beta mRNAs by cytokines and activators of second-messenger pathways

Nuclear protein IRF-1 (interferon regulatory factor 1) was earlier shown to bind to cis-acting regulatory elements present on interferon (IFN)-alpha/beta genes and some IFN-inducible genes. Here we show that in both human FS-4 and murine L929 cells, steady-state levels of IRF-1 mRNA were increased by treatment with tumor necrosis factor (TNF), interleukin 1 (IL-1), poly(I).poly(C), or IFN-beta. IRF-1 mRNA induction was also demonstrated in cells treated with calcium ionophore A23187 or with phorbol 12-myristate 13-acetate, but not with epidermal growth factor, dibutyryl-cAMP, or the adenylate cyclase activator forskolin. To determine whether stimulation of IRF-1 mRNA levels correlates with IFN-beta induction, we compared IRF-1 and IFN-beta mRNA levels in cells exposed to various stimuli. In L929 cells, treatment with poly(I).poly(C) under conditions that failed to induce significant levels of IFN-beta mRNA led to a very low induction of IRF-1 mRNA, but "priming" cells with IFN prior to the addition of poly(I).poly(C) greatly increased both IRF-1 and IFN-beta mRNAs. In FS-4 cells an increase in IFN-beta mRNA (examined by the polymerase chain reaction) was seen after treatment with TNF, IL-1, A23187, or poly(I).poly(C), but not with IFN-beta, epidermal growth factor, dibutyryl-cAMP, or forskolin. Thus, all treatments that increased steady-state levels of IFN-beta mRNA also enhanced IRF-1 mRNA levels. However, treatment with IFN-beta, which caused a marked stimulation in IRF-1 mRNA, failed to produce a detectable increase in IFN-beta mRNA. It appears that IRF-1 may be necessary but not sufficient for IFN-beta induction. The ability of TNF and IL-1 to increase both IRF-1 and IFN-beta mRNAs may be responsible for some similarities in the actions of TNF, IL-1, and the IFNs.     

Changes in hematopoiesis-supporting ability of C3H10T1/2 mouse embryo fibroblasts during differentiation

To investigate the functional change of stromal cells along with differentiation, we used a differentiation-inducible mouse embryo fibroblast cell line, C3H10T1/2 (10T1/2). Stably determined preadipocyte and myoblast cell lines were established after a brief exposure of 10T1/2 cells to 5-azacytidine. These cell lines terminally differentiated into adipocytes and myotubes, respectively, under appropriate conditions. The hematopoiesis-supporting ability of each 10T1/2-derived cell line was examined by coculture with FACS-sorted murine hematopoietic stem cells (Thy-1lo c-kit+ Lin-). The number of granulocyte-macrophage progenitors (CFU-GM) was slightly reduced after 7 days of culture with parent 10T1/2 fibroblasts, whereas a marked increase in CFU-GM number was observed when the cells were cultured on preadipocytes. Mature adipocytes and myogenically determined cell lines, on the other hand, did not support CFU-GM growth. Further, Northern analysis showed that the preadipocyte cell line acquired the ability to produce a significant amount of stem cell factor (SCF), interleukin-6 (IL-6), leukemia inhibitory factor, and macrophage colony- stimulating factor mRNAs in response to IL-1 or lipopolysaccharide stimulation. Terminal adipocytic differentiation resulted in reduced ability to express these cytokine mRNAs. Similarly, highest IL-6 activity was detected in the supernatant of preadipocyte culture, whereas adipocytes did not secrete IL-6 even after IL-1 stimulation. Interestingly, hematopoiesis-nonsupporting myoblasts and myotubes also expressed abundant SCF mRNA, suggesting that SCF, per se, may not be sufficient for stem cell growth and survival. The 10T1/2-derived cell lines could provide a valuable tool to aid in the analysis of stromal cell development and the search for novel stromal factors.     

Effects of tumour necrosis factor-alpha on growth hormone and interleukin 6 mRNA in ovine pituitary cells.

The inflammatory cytokines tumour necrosis factor-alpha (TNF alpha), interleukin 1 (IL-1) and interleukin 6 (IL-6) have been demonstrated to influence pituitary hormone synthesis directly and via the hypothalamus. Furthermore, IL-6 is produced by some anterior pituitary cells suggesting a paracrine/autocrine role for this cytokine. We show that TNF alpha induces dispersed ovine pituitary cells to produce increased levels of growth hormone (GH) and IL-6 mRNA and secreted IL-6 in a dose and time dependent manner. TNF alpha at concentrations between 1-1000 U/ml increased GH and IL-6 mRNA, relative to control levels, by 5 h post-stimulation. For IL-6, TNF alpha increased specific mRNA at 5 h and 12 h but not 24 h post-stimulation. TNF alpha also induced secreted IL-6 to levels above that spontaneously secreted at all time points from 5 h to 48 h. Levels of common glycoprotein alpha-subunit and follicle stimulating hormone-beta (FSH beta) subunit mRNA were unaffected by TNF alpha. We conclude that TNF alpha can regulate both GH and IL-6 synthesis in dispersed ovine pituitary cells. The implications for paracrine/autocrine control of pituitary hormone synthesis in acute and chronic disease are discussed.     

Interleukin-1 alpha upregulates tumor necrosis factor receptors expressed by a human bone marrow stromal cell strain: implications for cytokine redundancy and synergy

To explore the biochemical and physiologic basis of the overlapping effects of interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor alpha (TNF-alpha) on myeloid cytokine production, we have studied the dynamics of granulocyte colony-stimulating factor (G-CSF) and granulocyte-monocyte colony-stimulating factor (GM-CSF) production as well as IL-1 receptor and TNF receptor expression in a clonally derived bone marrow stromal cell strain (CDCL). IL-1 alpha and TNF alpha act in a synergistic manner to stimulate G-CSF and GM-CSF production by CDCL, resulting in an increase in CSF secretion that is 250-fold greater than that observed with either cytokine alone. This synergism in protein secretion is paralleled by synergistic increases the steady-state level of GM- and G-CSF mRNA, with supra-additive levels achieved by 24 hours. Coincident with this synergistic induction of myeloid CSFs, treatment of CDCL cells with IL-1 alpha induces a 300% increase in the expression of TNF receptors. IL-1 alpha induction of TNF receptors reaches a peak after 6 hours and gradually returns to baseline level by 24 hours. IL-1 alpha does not affect TNF receptor ligand binding affinity. A kinetic study comparing IL-1/TNF synergistic induction of growth factor secretion with IL-1 alpha induction of TNF receptors shows that these events occur in parallel. In contrast with the induction of TNF receptors by IL-1 alpha, treatment with TNF alpha has no effect on either the number of IL-1 receptors expressed by CDCL cells or IL-1 receptor ligand binding affinity. Brief treatment of IL-1 alpha/TNF alpha-stimulated CDCL cells with cycloheximide before receptor induction reduces the synergistic increase in growth factor mRNA by 40% to 60% compared with cells not treated with CHX. Taken together, these results raise the possibility that IL-1 alpha cross-induction of TNF receptors may contribute to the biochemical mechanisms underlying the synergistic stimulation of G-CSF and GM-CSF production by IL-1 alpha and TNF alpha.