Glucocorticoid inhibition of interleukin-1-induced interleukin-6 production by human lung fibroblasts: evidence for transcriptional and post-transcriptional regulatory mechanisms.

Interleukin (IL)-6 is a pleiotropic cytokine produced by a wide variety of cells including fibroblasts, macrophages, endothelial cells, and T and B lymphocytes. Regulated IL-6 production is an important part of normal biologic homeostasis, and abnormal IL-6 production has been associated with a large number of diseases including asthma and lung allograft rejection. Glucocorticoids are potent anti-inflammatory agents that are widely used to suppress pulmonary inflammation. To further understand the mechanisms underlying this inhibition, we determined whether glucocorticoid compounds regulate human lung fibroblast IL-6 production and characterized the mechanisms of the effects that were noted. These studies demonstrate that glucocorticoids inhibit IL-1-induced IL-6 production in a dose-dependent fashion. A greater than 95% decrease in IL-6 production was seen with 10(-6) and 10(-7) M dexamethasone, prednisolone, and hydrocortisone, and IC50 values for these agents were approximately 5 x 10(-10), 5 x 10(-9), and 10(-8) M, respectively. mRNA analysis demonstrated that these alterations in protein production were associated with proportionate decreases in IL-6 mRNA accumulation, and that this suppression of IL-6 mRNA could be reversed by the glucocorticoid receptor antagonist RU 486. Nuclear run-on studies demonstrated that glucocorticoids inhibit-IL-1-induced IL-6 gene transcription. However, the magnitude of this effect could not fully account for the potency of the glucocorticoid-induced alterations in IL-6 mRNA accumulation and protein production since 10(-6) M dexamethasone caused only a 50% decrease in IL-1-induced IL-6 gene transcription.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytokine-cytokine synergy and protein kinase C in the regulation of lung fibroblast leukemia inhibitory factor.

Studies were undertaken to characterize the cytokines and cytokine-cytokine interactions that stimulate human lung fibroblast leukemia inhibitory factor (LIF) production and the mechanisms of these regulatory effects were investigated. Unstimulated fibroblasts did not produce significant amounts of LIF, whereas recombinant interleukin-1 alpha (rIL-1 alpha), transforming growth factor-beta (TGF-beta), and recombinant tumor necrosis factor (rTNF) were dose-dependent stimulators of LIF production. TGF-beta and rIL-1 alpha also interacted in a synergistic fashion to further increase LIF elaboration. Under all conditions alterations in LIF production were associated with comparable alterations in LIF mRNA accumulation. The kinetics of mRNA induction, however, differed with rIL-1-induced LIF mRNA being readily detected after 2 h, TGF-beta 1 induction peaking after 16-24 h, and the induction caused by rIL-1 alpha plus TGF-beta 1 being most prominent after 2-4 h and decreasing with additional incubation. Protein synthesis was not required for LIF induction. In addition, even though A23187 was an effective stimulator of LIF production, the calmodulin antagonists W-7 and trifluoperazone dichoride (TFP) did not significantly alter the LIF-stimulatory effects of IL-1 and TGF-beta. PKC did appear to play an important role in this induction, however, since LIF was induced by PMA and cytokine induction of LIF production was markedly diminished by chronic phorbol ester preincubation, staurosporine, and H-7, but not by HA1004. These studies demonstrate that 1) rIL-1, TGF-beta, TNF, agents that increase intracellular calcium and agents that activate PKC, stimulate lung fibroblast LIF production; 2) rIL-1 and TGF-beta interact in a synergistic fashion to further increase fibroblast LIF production; and 3) rIL-1 and TGF-beta stimulate lung fibroblast LIF production via a pretranslational activation pathway that is largely PKC-dependent and protein synthesis-, cyclic nucleotide-, and calmodulin-independent. Cytokine-stimulated LIF production may play an important role in homeostasis and repair in the human lung.

IL-1 and transforming growth factor-beta regulation of fibroblast-derived IL-11.

IL-11 and IL-6 are fibroblast-derived cytokines with overlapping biologic properties. To determine whether IL-11 and IL-6 are similarly regulated, we characterized the effects of rIL-1 and TGF-beta (beta 1 and beta 2) on human lung fibroblast IL-11 production and compared this regulation with that of IL-6. Unstimulated fibroblasts did not produce significant amounts of IL-11, whereas rIL-1 alpha and TGF-beta were dose-dependent stimulators of IL-11 protein production, mRNA accumulation, and gene transcription. rIL-1 alpha and TGF-beta also interacted in a synergistic fashion to further increase IL-11 protein production and mRNA accumulation. The effects of rIL-1 and TGF-beta individually were not altered by the cyclic nucleotide-dependent protein kinase inhibitor HA1004, protein kinase C (PKC) inhibition with staurosporine, or chronic phorbol ester preincubation, or the calmodulin antagonists W7 and TFP. The effects of rIL-1 alpha and TGF-beta in combination were also unaltered by HA1004, staurosporine, and chronic phorbol ester exposure. A23187, however, did induce IL-11 mRNA accumulation and W7 and TFP did reverse the synergistic stimulation caused by rIL-1 and TGF-beta in combination. In contrast with the regulation of IL-11, TGF-beta did not effectively stimulate IL-6 mRNA accumulation, rIL-1 alpha was a more potent stimulator of IL-6 than IL-11 production, and rIL-1-induced IL-6 mRNA accumulation was augmented by W7 and TFP. These studies demonstrate that: 1) rIL-1, TGF-beta, and agents that increase intracellular calcium stimulate lung fibroblast IL-11; 2) the IL-11 stimulatory effects of rIL-1 and TGF-beta are, at least partially, transcriptionally mediated and are the result of signal transduction pathways that are largely PKC, cyclic nucleotide, and calmodulin independent; and 3) rIL-1 and TGF-beta interact in a synergistic fashion to further increase fibroblast IL-11 production and that this synergy is mediated by a largely PKC- and cyclic nucleotide-independent and calmodulin-dependent activation pathway. Importantly, they also demonstrate that rIL-1 and TGF-beta stimulate lung fibroblast IL-6 and IL-11 production via distinct and differentially regulatable activation pathways.

Retinoic acid inhibition of IL-1-induced IL-6 production by human lung fibroblasts.

IL-6 is a multi-functional cytokine that plays an important role in normal biologic homeostasis and disease pathogenesis. Retinoids are vitamin A analogs that regulate the function of a wide variety of inflammatory and structural cells. To further understand the biology of retinoids and IL-6 we determined whether all-trans-retinoic acid (RA) and other retinoids regulate lung fibroblast IL-6 production. RA did not stimulate fibroblast IL-6 production. Instead, it inhibited the production of IL-6 by IL-1-stimulated cells. This effect was dose-dependent with an IC50 of 10(-7) M RA and significant inhibition being noted with doses of RA as low as 10(-8) M. These inhibitory effects could not be explained by cytotoxicity or a shift in the kinetics of IL-6 production. They also did not appear to involve alterations in the early events in IL-1-induced IL-6 production, because RA inhibited IL-6 production even when added 6 h after IL-1 and RA did not inhibit IL-1 binding to cell surface IL-1 receptors. RA inhibition of IL-6 protein production was associated with a comparable decrease in IL-6 mRNA accumulation and gene transcription. 13-cis-retinoic acid, retinol, retinaldehyde, all-trans etretin, Ro 13-6298, and 9-cis retinoic acid also inhibited IL-1-induced IL-6 production. However, 4-hydroxyphenyl retinamide and etretinate did not share this property. The inhibitory effects of these analogues may be mediated by nuclear retinoic acid receptors as mRNA encoding RAR-alpha, RAR-gamma, and RXR-alpha were present, and RAR-beta was induced by RA in human lung fibroblasts. These studies demonstrate that RA and other retinoid analogs inhibit IL-1-induced IL-6 production and that this effect is analog-specific and, at least partially, transcriptionally mediated.

Correlation between the induction of heat shock protein 70 and enhanced viral reactivation in mammalian cells treated with ultraviolet light and heat shock.

Enhanced viral reactivation (EVR) is considered to be one manifestation of an inducible response to DNA damage in mammalian cells analogous to the SOS response in Escherichia coli. EVR is characterized by the increased survival of ultraviolet (UV)-irradiated virus in cells which have been pretreated with DNA-damaging agents or by another type of cellular stress, heat shock (HS). In this study, we have analyzed the induction of nuclear proteins from Vero cells treated with either UV or HS, with the goal of identifying the protein(s) which mediate the EVR response. Results of 2-dimensional protein gel electrophoresis and fluorographic analysis of [35S]methionine-labeled nuclear proteins showed that UV-irradiation caused the increased synthesis of five proteins at 4-9 h after treatment. At 19-24 h, one of these proteins was still being synthesized at a higher level in UV-irradiated cells, and there were nine additional proteins whose syntheses were enhanced over control levels. In contrast, HS induced only one Mr 72,000 nuclear protein whose synthesis was maximal during the 4-9-h labeling period and corresponded to one of the proteins induced by UV at 19-24 h. Subsequent Western and Northern blot analyses have confirmed that this protein is a member of the heat shock protein (hsp) 70 family. Elevated nuclear levels of this protein correlated temporally with the maximum EVR response induced by each treatment (4 h after HS and 24 h after UV). Since the kinetics of EVR is different following UV and HS and parallels the difference in the induction of nuclear levels of hsp70 following each treatment, the results suggest that hsp70 may be involved in mediating the EVR response. In addition, this protein may also play a role in the recovery of DNA synthesis in UV-irradiated cells.