Prostaglandin E suppression of platelet-derived-growth-factor-induced Ito cell mitogenesis occurs independent of raf perinuclear translocation and nuclear proto-oncogene expression.

Ito cell mitogenesis occurs during liver injury and fibrogenesis in vivo coincident with the de novo expression of Ito cell PDGF beta receptor messenger RNA. PDGF-induced mitogenesis was studied in cultured rat hepatic Ito cells which resemble the myofibroblast associated with liver injury. Pretreatment with prostaglandin E markedly suppressed the PDGF response in a dose-dependent fashion. The PDGF-induced cascade was studied with or without PGE to determine the level of regulation which induced the observed suppression. PGE caused no apparent diminution in the abundance of the surface PDGF beta receptor nor its subsequent activation and tyrosine phosphorylation following PDGF stimulation. The cytoplasmic 'secondary messengers' mitogen-activated protein kinase pp42-44 and raf kinase, appeared to be comparably induced and therefore unaffected by PGE. Raf perinuclear translocation was also intact and comparable degrees of nuclear egr, fos, and jun expression occurred. Since other studies have suggested that many of these features of the PDGF cascade may be causally and sequentially linked, the data collectively suggests that the dominant PGE mitogenic suppressive effect resides at a raf-MAP parallel pathway or at a nuclear level distal to the induction of these early growth response genes.

Quantitative measurement of lymphocyte mediated growth inhibition of Candida albicans.

A rapid and reproducible assay has been developed to measure the capacity of lymphocytes to inhibit the growth of Candida albicans. Fungal growth inhibition was assessed optimally as the incorporation of [3H]uridine into preformed hyphae, following interaction of the hyphae with effector lymphocytes. The assay was sensitive to the detection of fungal growth inhibition by lymphocytes at low effector to target ratios and results correlated well with other methods for measurement of anti-C. albicans growth inhibition in vitro. Although the assay was developed for the measurement of lymphocyte mediated anti-fungal activity, other mammalian cell populations can be assayed for growth inhibition of C. albicans as well. The described assay utilizes the enzyme lyticase to reduce the surface binding of C. albicans. The use of this enzyme permits the efficient harvest of large numbers of experimental samples with a multiple automated sample harvester.

Growth inhibition of Candida albicans by interleukin-2-activated splenocytes.

Murine splenocytes, Percoll-enriched low-density lymphocytes, and interleukin-2 (IL-2)-activated lymphocytes were assessed for the capacity to limit the growth of the hyphal form of Candida albicans. No fungal-growth-inhibitory activity was exhibited for C. albicans by either splenocytes or Percoll-enriched lymphocytes. These cells were capable of cytotoxic activity for a natural killer cell-sensitive cell line. However, when cultured for several days with IL-2, splenocytes acquired the capacity to inhibit the growth of the fungus. The appearance of the antifungal activity coincided with the development of cytotoxic activity for the natural killer cell-insensitive cell line. Anti-C. albicans and antitumor activities of IL-2-activated lymphocytes were competitively and reciprocally inhibited by C. albicans and the natural killer cell-sensitive and -insensitive cell lines. The antifungal activity of the IL-2-activated lymphocytes was exhibited against a number of clinical isolates of C. albicans and related fungal species. IL-2-activated human peripheral blood lymphocytes also acquired the capacity to inhibit the growth of C. albicans. These data show that in vitro growth inhibition can be mediated by IL-2-stimulated lymphocytes which are neither fungal strain nor mammalian species restricted in their biological activity.

Nonstressed rat model of acute endotoxemia that unmasks the endotoxin-induced TNF-alpha response.

Previous investigators have demonstrated that the tumor necrosis factor-alpha (TNF-alpha) response to endotoxin is inhibited by exogenous corticosterone or catecholamines both in vitro and in vivo, whereas others have reported that surgical and nonsurgical stress increase the endogenous concentrations of these stress-induced hormones. We hypothesized that elevated endogenous stress hormones resultant from experimental protocols attenuated the endotoxin-induced TNF-alpha response. We used a chronically catheterized rat model to demonstrate that the endotoxin-induced TNF-alpha response is 10- to 50-fold greater in nonstressed (NS) rats compared with either surgical-stressed (SS, laparotomy) or nonsurgical-stressed (NSS, tail vein injection) models. Compared with the NS group, the SS and NSS groups demonstrated significantly lower mean peak TNF-alpha responses at 2 mg/kg and 6 micrograms/kg endotoxin [NS 111.8 +/- 6.5 ng/ml and 64.3 +/- 5.9 ng/ml, respectively, vs. SS 3.9 +/- 1.1 ng/ml (P < 0.01) and 1.3 +/- 0.5 ng/ml (P < 0.01) or NSS 5.2 +/- 3.2 ng/ml (P < 0.01) at 6 micrograms/kg]. Similarly, baseline concentrations of corticosterone and catecholamines were significantly lower in the NSS group [84.5 +/- 16.5 ng/ml and 199.8 +/- 26.2 pg/ml, respectively, vs. SS group 257. 2 +/- 35.7 ng/ml (P < 0.01) and 467.5 +/- 52.2 pg/ml (P < 0.01) or NS group 168.6 +/- 14.4 ng/ml (P < 0.01) and 1,109.9 +/- 140.7 pg/ml (P < 0.01)]. These findings suggest that the surgical and nonsurgical stress inherent in experimental protocols increases baseline stress hormones, masking the endotoxin-induced TNF-alpha response. Subsequent studies of endotoxic shock should control for the effects of protocol-induced stress and should measure and report baseline concentrations of corticosterone and catecholamines.

Growth inhibition of Candida albicans by interleukin-2-induced lymph node cells.

Previous reports have demonstrated natural killer cells (NK) to exert growth inhibitory effects against certain fungi, but not against Candida albicans. In this investigation, interleukin-2 (IL-2)-induced lymph node cells with phenotypic and functional characteristics of NK were shown to inhibit the growth of C. albicans. Growth inhibition was evaluated by both the release of 51Cr by the fungus and the inhibition of microcolony growth of the fungus on Sabouraud's dextrose agar. Lymphoid cells derived from C57Bl/6 mice and immediately assessed for hyphal growth inhibition showed little or no activity. However, significant hyphal growth inhibition was produced by lymph node cells cultured with recombinant IL-2. Growth inhibitory activity was dependent upon the concentration of IL-2 and was mediated by nonadherent lymphocytes which lysed an NK-susceptible and to a lesser extent an NK-resistant cell line. Treatment of the IL-2-induced cells with anti-asialo GM1 but not anti-Thy-1 and complement abrogated growth inhibition of C. albicans. These results suggest that IL-2-induced lymph node cells with functional and phenotypic characteristics similar to those of activated NK, mediate in vitro growth inhibition of the hyphal form of C. albicans.

Lipoxygenase inhibitors block PDGF-induced mitogenesis: a MAPK-independent mechanism that blocks fos and egr.

Hepatic Ito cells proliferate during liver injury and fibrogenesis. Platelet-derived growth factor (PDGF)-induced [3H]thymidine incorporation was studied as Ito cells express the PDGF receptor after injury and activation. Pretreatment with either the nonspecific lipoxygenase inhibitor (nordihydroguaiaretic acid) or specific inhibitors of 5-lipoxygenase (SC-41661 and ICI-230487) inhibited PDGF-induced mitogenesis. Ito cells predominantly produce the leukotriene (LT) C4 >> LTB4. The PDGF-induced signal transduction cascade was studied to determine the potential mechanism of action of the lipoxygenase inhibitors. It was found that PDGF receptor abundance and receptor activation were not altered by lipoxygenase inhibition, suggesting that a postreceptor mechanism was involved. The two-key cytoplasmic serine-threonine kinases Raf and MAPK (mitogen-activated protein kinase), which are induced by PDGF and transmit the signal to the nucleus, were also not altered. Because Raf and MAPK can independently induce nuclear signaling, this suggests that the mechanism of action lies parallel or distal to these secondary messengers. Lipoxygenase inhibition did result in the suppression of PDGF-induced fos and egr expression. Collectively, this work suggests that lipoxygenase inhibition leads to the suppression of mitogenesis in part by disrupting the nuclear signaling that is required for protooncogene transcription at a step distal or parallel to MAPK activation.

Raf and mitogen-activated protein kinase regulate stellate cell collagen gene expression.

Hepatic stellate cells become activated into myofibroblast-like cells during the early stages of hepatic injury associated with fibrogenesis. The subsequent dysregulation of hepatic stellate cell collagen gene expression is a central pathogenetic step during the development of cirrhosis. The cytoplasmic Raf and mitogen-activated protein (MAPK) kinases were found to differentially regulate alpha I(I) collagen gene expression in activated stellate cells. This suggests an unappreciated branch point exists between Raf and MAPK. A MAPK-stimulatory signal was mapped to the most proximal NF-1 and Sp-1 binding domains of the 5'-untranslated region of the collagen gene. A Raf-inhibitory signal was mapped to a further upstream binding domain involving a novel 60-kDa DNA-binding protein (p60). The cell-specific expression and induction of p60 in stellate cells during the early stages of hepatic fibrogenesis in vivo suggest a central role for this pathway during liver injury and stellate cell activation.

Bile acid stimulation of early growth response gene and mitogen-activated protein kinase is protein kinase C-dependent.

Hepatic stellate cells are exposed to elevated bile acid levels during hepatic injury and fibrogenesis. Upon activation, the stellate cell becomes a major effector cell during the development of hepatic fibrosis and cirrhosis. Bile acids may function as costimulatory signalling molecules. This hypothesis was tested in vitro using rat-derived hepatic stellate cells. Bile acids were studied at concentrations that occur during cirrhosis in vivo. Conjugated and unconjugated bile acids rapidly induced egr and fos gene expression as well as cytoplasmic mitogen-activated protein kinase (MAPK) activation. Protein kinase C was required for both egr induction and MAPK activation. These studies imply that bile acids could contribute to the perpetuation of hepatic fibrosis by helping to keep the stellate cell in an activated state.

1,25-Dihydroxyvitamin D3 activates Raf kinase and Raf perinuclear translocation via a protein kinase C-dependent pathway.

1,25-Dihydroxyvitamin D3's (D3) potential mitogenic mechanism of action was pursued in cultured rat hepatic Ito cells, a fibrogenic effector cell which proliferates in vivo during liver injury and fibrogenesis. D3 stimulated Ito cell DNA synthesis and potentiated platelet-derived growth factor-induced mitogenesis. D3's enhancement of [3H]thymidine incorporation was associated with nuclear Egr expression. Recent studies have causally linked the activated proto-oncogene c-Raf with downstream Egr induction. The serine-threonine kinase Raf protein is phosphorylation-activated by a large array of agonists including plasma membrane and cytoplasmic tyrosine kinases but has not previously been associated with the steroid superfamily of mediators. To consider potential prenuclear acute pathways of D3-induced stimulation, the activation of Raf was examined following D3 exposure. D3 induced Raf activation as assessed via (a) enhanced Raf phosphorylation following in vivo 32P labeling, (b) enhanced kinase function utilizing exogenous histone 1 protein as substrate, and (c) the shift in Raf physical localization changing from a diffuse cytoplasmic distribution to a perinuclear domain. A similar activation of Raf kinase was found in 3T3 cells exposed to D3 with enhanced histone phosphorylation detectable within 1 min following stimulation. The proximal cascade leading to Raf kinase activation may involve a protein kinase activity was severely attenuated by stimulated kinase activity was severely attenuated by previous phorbol ester treatment for 20 h or staurosporine pretreatment.

Posttranslational inhibition of Ito cell type I collagen production by triiodothyronine.

Increased Ito cell collagen production occurs during in vivo liver fibrogenesis. Regulation of the overproduction of collagen was studied in cultured rat hepatic Ito cells, which resemble the myofibroblast associated with liver fibrosis. Previous studies suggest that the steroid hormones, retinoic acid, and glucocorticoids may have antifibrogenic properties in vitro and in vivo when used at pharmacological doses. Their potential roles at physiological levels are not well understood. The current study examined the potential regulation of the overproduction of type I collagen in cultured rat hepatic Ito cells by another steroid hormone, 3,5,3'-triiodo-L-thyronine (T3). T3 induced a 3.4-fold reduction in type I collagen production. The effect was dose dependent and was maximal with physiological levels of T3 (10(-9) M). The effect of T3 was independent of any suppression in total protein synthesis. The mechanism of the suppressive effect of T3 on collagen production was explored and was found to be at a posttranslational level. This study suggests that the inhibitory effects of T3 on type I collagen production are likely caused by enhanced intracellular turnover of type I collagen.

Retinoic acid suppresses the response to platelet-derived growth factor in human hepatic Ito-cell-like myofibroblasts: a post-receptor mechanism independent of raf/fos/jun/egr activation.

Activated Ito-cell-like myofibroblasts proliferate in vivo during human liver injury and subsequent fibrogenesis. To examine the associated regulatory mechanisms, human liver myofibroblasts were characterized after culture purification from mixed liver-cell isolates obtained from perfused normal human livers. The cells resembled rat Ito-cell-derived myofibroblasts expressing desmin and alpha-smooth-muscle actin filaments as well as the interstitial collagens type I and III. [3H]Thymidine incorporation was inducible with platelet-derived growth factor (PDGF) and was suppressible with retinoic acid (RAc) in a concentration-dependent fashion. RAc suppression did not alter PDGF alpha- or beta-receptor abundance or activation. In addition, RAc functioned via a pathway distal or independent of cytoplasmic raf activation (i.e. phosphorylation, kinase function and perinuclear translocation) and nuclear fos, jun and egr expression, as these steps were similarly unaffected by RAc treatment. Since normal Ito cells contain abundant amounts of vitamin A which is lost during activation, these data suggest that retinoids could contribute to the maintenance of the quiescent non-proliferative state by suppressing mitogenesis at a post-cytokine receptor step distal from or independent of fos/jun/egr [e.g. via changes in activator protein-1 (AP-1) binding].

Growth inhibition of Candida albicans hyphae by CD8+ lymphocytes.

We have shown previously that IL-2-activated splenocytes can inhibit the growth of Candida albicans hyphae in vitro. Herein we demonstrate that plastic nonadherent lymphocytes that are CD8+ mediate the antifungal activity. Enrichment for CD8+ cells markedly enhanced the antifungal activity of the IL-2-activated lymphocyte population for C. albicans and the cytotoxic activity of the lymphocytes for an NK-resistant cell line. Depletion of CD8+ cells reduced the lymphocyte population's antifungal activity and cytotoxic activity for the NK-resistant cell line. Enrichment for NK1.1+ cells markedly reduced the antifungal activity of the lymphocyte population for C. albicans and increased the cytotoxic activity of the lymphocytes for an NK-sensitive cell line. Depletion of NK1.1+ cells increased the lymphocyte population's antifungal activity and cytotoxic activity for the NK-resistant cell line. Generation of the antifungal lymphocytes in culture required IL-2 and was not replaced with IFN-gamma. These data show that IL-2-activated CD8+ T lymphocytes exert the greatest amount of antifungal effect against the hyphal form of C. albicans, whereas IL-2- or IFN-gamma-activated NK cells have little or no effect against the hyphae.

Gamma-linolenic acid suppression of hepatic Ito cell mitogenesis: post-PDGF receptor prostaglandin-independent mechanism.

Ito cell mitogenesis occurs during liver injury and fibrogenesis in vivo. Platelet-derived-growth factor (PDGF)-induced mitogenesis was studied in cultured rat hepatic Ito cells, which resemble the myofibroblast associated with liver injury. Pretreatment with gamma-linolenic acid (GLA), an essential fatty acid prostanoid precursor, markedly suppressed the PDGF response in a dose-dependent reversible fashion. Prostaglandins E1 and E2 were found to be the predominant prostanoids formed by cultured Ito cells. GLA depressed endogenous PG production, suggesting that the antimitogenic effect was independent of GLA conversion to a prostanoid metabolite. The PDGF-induced cascade was studied with and without GLA to determine the level of regulation that induced the observed suppression. GLA caused no apparent diminution in the abundance of the surface PDGF-beta receptor nor its subsequent activation and tyrosine phosphorylation after PDGF stimulation. Raf kinase activation and Raf perinuclear translocation were also intact despite the presence of GLA. PDGF induction of nuclear Egr and Fos also occurred with or without GLA. Activation of the serine threonine kinase c-Raf has previously been found to be sufficient to activate egr and fos and to induce mitogenesis. Therefore, the GLA suppressive effect is likely to be operative at a parallel non-Raf pathway or distal to Raf-induced early gene expression.

Suppression of stellate cell type I collagen gene expression involves AP-2 transmodulation of nuclear factor-1-dependent gene transcription.

The regulation of collagen gene expression was studied in culture-activated rat hepatic stellate cells, the fibrogenic effector cell involved in hepatic fibrogenesis. Treatment of cells with a 5-lipoxygenase-specific inhibitor caused a reduction in alphaI(I) collagen mRNA transcript abundance, which suggested that leukotriene production was involved in maintaining the activated cell's high level of collagen mRNA production. The underlying mechanism involved a decrease in collagen gene transcription. Suppression of gene transcription was localized to an nuclear factor-1 (NF-1) binding domain in the proximal promoter and an AP-2 binding domain adjacent to it. Gel retardation assays demonstrated that an increase in AP-2 binding adjacent to the NF-1 site was likely to be the transmodulator responsible for the suppression of the NF-1-dependent gene expression. The data suggest that post-translational alterations in AP-2 activity are responsible for this unappreciated mechanism of regulating the collagen gene.

Protein kinase C and mitogen-activated protein kinase are required for 1,25-dihydroxyvitamin D3-stimulated Egr induction.

Recent studies have demonstrated that 1,25-dihydroxyvitamin D3 (D3) can activate Raf kinase and induce Egr expression in cultured rat hepatic Ito cells (Lissoos, T. W., Beno, D. W. A., and Davis, B. H. (1993) J. Biol. Chem. 268, 25132-25138). Since Raf is an upstream activator of mitogen-activated protein kinase (MAPK), the current study evaluated the ability of D3 to activate MAPK. D3-activated MAPK and induced its cytoplasmic to perinuclear translocation in Ito cells. MAPK activation was found to be protein kinase C-dependent, which was analogous to previous studies of D3 and Raf activation. To further explore the D3 cascade, a series of transient transfections were performed using dominant negative raf and MAPK mutant plasmids which effectively block Ras-induced Raf and MAPK activity, respectively. D3 induced a marked increase in the expression of a chloramphenicol acetyltransferase reporter gene linked to the Egr promoter (egr-CAT). When the dominant negative Raf plasmid was co-transfected, there was no significant reduction in egr-CAT. In contrast, when the dominant negative MAPK plasmid was co-transfected, egr-CAT induction was completely abolished. These results suggest that 1) D3 stimulates MAPK via a protein kinase C-dependent pathway, 2) D3-induced Egr expression can occur via a pathway independent of Ras-induced Raf, and 3) D3 absolutely requires MAPK activity for Egr expression.

Administration of prostaglandin E1 analog reduces rat hepatic and Ito cell collagen gene expression and collagen accumulation after bile duct ligation injury.

Recent studies suggest that prostaglandin E may have the ability to suppress cytokine responsiveness. We examined the effects of prostaglandin E administration on several parameters of acute and chronic liver injury induced by bile duct ligation. Enisoprost, a prostaglandin E1 analog, was found to suppress early hepatic and Ito cell type I collagen gene expression without diminishing the induction of the fibrogenic cytokine transforming growth factor-beta. Overall liver inflammation and cell proliferation were not altered, suggesting that prostaglandin E acts distal to the initial injurious event(s). During later phases, drug administration reduced total collagen accumulation and type I collagen periductular infiltration associated with early nodule formation.

Differential regulation of interleukin-8 and intercellular adhesion molecule-1 by H2O2 and tumor necrosis factor-alpha in endothelial and epithelial cells.

The reactive oxygen intermediate H2O2 can function as a signaling molecule to activate gene expression. In this study, we demonstrate that oxidant stress induced by tumor necrosis factor alpha (TNFalpha) or H2O2 differentially regulates intercellular adhesion molecule-1 (ICAM-1) and interleukin-8 (IL-8) gene expression in endothelial and epithelial cells. Northern blot analysis revealed that TNFalpha induced both ICAM-1 and IL-8 expression in either the A549 lung epithelial cell line or the human microvessel endothelial cell line (HMEC-1). In contrast, H2O2 selectively induced only ICAM-1 in HMEC-1 and only IL-8 in A549. This cell type-specific pattern of IL-8 expression was also observed in several other endothelial and epithelial cells. TNFalpha induced greater IL-8 gene expression as compared with H2O2, but the kinetics of induction were similar. The induction of epithelial IL-8 message was accompanied by a corresponding increase in functional IL-8 protein secretion as determined by a neutrophil motility assay. The increased neutrophil motility stimulated by conditioned media from H2O2- or TNFalpha-exposed A549 cells was completely inhibited by an anti-IL-8 antibody. TNFalpha and H2O2 also induced a differential pattern of CC chemokine expression in A549. While TNFalpha induced both RANTES and MCP-1, H2O2 induced only MCP-1. These data suggest that epithelial cells under oxidant stress contribute to the inflammatory cytokine network by selective production of IL-8, MCP-1, and RANTES, which may critically influence the site-specific recruitment of leukocyte subsets.

Cystic fibrosis colonopathy.

An epidemic of fibrosing colonopathy, a new disease caused by the prolonged administration of excessive doses of pancreatic enzymes, was first reported in 1994. More than 60 cases were known to occur worldwide before dosage guidelines were enforced. Predisposing factors were young age, previous intestinal surgery, meconium ileus equivalent, and use of H2 blockers, corticosteroids, and DNase. Abnormal features included foreshortened colon, strictures, marked submucosal fibrosis, ascites, and nodular hyperplasia of the liver. Histologic examination showed eosinophilia, mild cryptitis, epithelial regeneration, and widespread interruption of the muscularis mucosa. These findings are distinct from, but share many of the features of, those of Crohn's's disease and ischemic bowel disease. The pathogenic mechanisms remain unknown.

Endotoxin-induced reduction in biliary indocyanine green excretion rate in a chronically catheterized rat model.

Using a nonstressed chronically catheterized rat model in which the common bile duct was cannulated, we studied endotoxin-induced alterations in hepatic function by measuring changes in the maximal steady-state biliary excretion rate of the anionic dye indocyanine green (ICG). Biliary excretion of ICG was calculated from direct measurements of biliary ICG concentrations and the bile flow rate during a continuous vascular infusion of ICG. Despite significant elevations in mean peak serum tumor necrosis factor-alpha (TNF-alpha) concentrations (90.9 +/- 16.2 ng/ml), there was no effect on mean rates of bile flow or biliary ICG clearance after administration of 100 microg/kg endotoxin at 6 or 24 h. Significant differences from mean baseline rates of bile flow and biliary ICG excretion did occur after administration of 1,000 microg/kg endotoxin (mean peak TNF-alpha 129.6 +/- 24.4 ng/ml). Furthermore, when rats were treated with up to 16 microg/kg of recombinant TNF-alpha, there was no change in mean rates of bile flow or ICG biliary clearance compared with baseline values. These data suggest that the complex regulation of biliary excretion is not mediated solely by TNF-alpha.

Staphylococcal enterotoxin B potentiates LPS-induced hepatic dysfunction in chronically catheterized rats.

Most models of liver dysfunction in sepsis use endotoxin (lipopolysaccharide; LPS) to induce a pathophysiological response. In our study published in this issue (Beno DWA, Uhing MR, Goto M, Chen Y, Jiyamapa-Serna VA, and Kimura RE. Am J Physiol Gastrointest Liver Physiol 280: G858-G865, 2001), the adverse effect of LPS on hepatic function in vivo was only significant at relatively high LPS doses despite high tumor necrosis factor-alpha concentrations. However, many patients with sepsis are exposed to multiple bacterial toxins that may augment the immune response, resulting in increased hepatic dysfunction. We have developed a model of polymicrobial sepsis by parentally administering a combination of staphylococcal enterotoxin B (SEB) and LPS. Using this model, we demonstrate that SEB (50 microg/kg) potentiates the effect of LPS-induced hepatic dysfunction as measured by decreased rates of biliary indocyanine green clearance and bile flow. These increases were most pronounced with doses of 10 and 100 microg/kg LPS, doses that by themselves do not induce hepatic dysfunction. This may explain the seemingly increased incidence and severity of liver dysfunction in sepsis, and it suggests that the exclusive use of LPS for replicating septic shock may not be relevant for studies of hepatic dysfunction.