Protective effect of rhIL-11 in a murine model of acetaminophen-induced hepatotoxicity.

Acetaminophen intoxication results in hepatotoxicity associated with increased serum concentrations of hepatocellular leakage enzymes such as aspartate aminotransferase, lactate dehydrogenase, and alanine aminotransferase, centrilobular degeneration and necrosis, and activation of Kupffer cells. Recombinant human Interleukin-11 (rhIL-11) downregulates the production of proinflammatory mediators from activated macrophages and has direct effects on hepatocyte gene expression. Based on these biological activities of rhIL-11, the effect of pretreatment with rhIL-11 in a murine model of acetaminophen-induced hepatotoxicity was examined. Administration of 500 microg/kg acetaminophen to B6C3F1 mice resulted in progressive hepatotoxicity as demonstrated by elevated serum concentrations of hepatocellular leakage enzymes and TNFalpha and histopathology. Pretreatment with 250 or 500 microg/kg of subcutaneously administered rhIL-11 2 hours before acetaminophen administration reduced serum concentrations of hepatocellular leakage enzymes and TNFalpha by 40-50%. This was associated with a statistically significant decrease in mean severity score for centrilobular hemorrhage and necrosis from grade 3 to grade 2 for rhIL-11-treated animals compared to vehicle. These results indicate that treatment with rhIL-11 has a protective effect in a model of acetaminophen-induced liver damage.

Interleukin-11 modulates Th1/Th2 cytokine production from activated CD4+ T cells.

Recombinant human interleukin-11 (rHuIL-11) is a pleiotropic cytokine with effects on multiple cell types. rHuIL-11 reduces activated macrophage activity and downregulates production of proinflammatory mediators, such as tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO). In vitro and in vivo, rHuIL-11 inhibits production of key immunostimulatory cytokines, including IL-12 and interferon-gamma (IFN-gamma). rHuIL-11 has recently demonstrated immunomodulatory activity to downregulate IFN-gamma production, increase IL-4 production, and reduce inflammatory tissue injury in a human psoriasis clinical trial. The cellular mechanisms of these effects are not fully elucidated. We demonstrate here that expression of gp130 and IL-11 receptor (IL-11R) alpha mRNA, components of the IL-11R complex, are detected in human and murine CD4(+) and CD8(+) lymphocytes, suggesting that rHuIL-11 can directly interact with T cells. In a cell culture model of murine T cell differentiation, rHuIL-11 acts to inhibit IL-2 production as well as IL-12-induced IFN-gamma production and enhances IL-4 and IL-10 production. rHuIL-11 had no effect on T cell proliferation. The ability of rHuIL-11 to modulate cytokine production from activated CD4(+) T cells provides a mechanism through which rHuIL-11 may ameliorate such inflammatory diseases as psoriasis.

Molecular classification of psoriasis disease-associated genes through pharmacogenomic expression profiling.

Psoriasis is recognized as the most common T cell-mediated inflammatory disease in humans. Genetic linkage to as many as six distinct disease loci has been established but the molecular etiology and genetics remain unknown. To begin to identify psoriasis disease-related genes and construct in vivo pathways of the inflammatory process, a genome-wide expression screen of multiple psoriasis patients was undertaken. A comprehensive list of 159 genes that define psoriasis in molecular terms was generated; numerous genes in this set mapped to six different disease-associated loci. To further interpret the functional role of this gene set in the disease process, a longitudinal pharmacogenomic study was initiated to understand how expression levels of these transcripts are altered following patient treatment with therapeutic agents that antagonize calcineurin or NF-KB pathways. Transcript levels for a subset of these 159 genes changed significantly in those patients who responded to therapy and many of the changes preceded clinical improvement. The disease-related gene map provides new insights into the pathogenesis of psoriasis, wound healing and cellular-immune reactions occurring in human skin as well as other T cell-mediated autoimmune diseases. In addition, it provides a set of candidate genes that may serve as novel therapeutic intervention points as well as surrogate and predictive markers of treatment outcome.

Interleukin-11 therapy selectively downregulates type I cytokine proinflammatory pathways in psoriasis lesions.

Psoriasis is a chronic inflammatory skin disease in which epidermal hyperplasia results from skin infiltration by type I T lymphocytes and release of associated cytokines. A multifunctional cytokine, rhIL-11, modulates macrophage and type I T-lymphocyte function in cell culture and shows anti-inflammatory activity in animal models. We are testing subcutaneous delivery of rhIL-11 to patients with psoriasis in a phase 1 open-label dose-escalation clinical trial. Tissue was obtained from lesional and uninvolved skin before and during treatment with rhIL-11 and was examined by histology/immunohistochemistry and quantitative RT-PCR. Expression of over 35 genes was examined in all patients, and multiple genetic markers of psoriasis were identified. Expression of numerous proinflammatory genes was elevated in psoriatic tissue compared with nonlesional skin. Seven of 12 patients responded well to rhIL-11 treatment. Amelioration of disease by rhIL-11, as shown by reduced keratinocyte proliferation and cutaneous inflammation, was associated with decreased expression of products of disease-related genes, including K16, iNOS, IFN-gamma, IL-8, IL-12, TNF-alpha, IL-1beta, and CD8, and with increased expression of endogenous IL-11. We believe that this is the first study in humans to indicate that type I cytokines can be selectively suppressed by an exogenous immune-modifying therapy. The study highlights the utility of pharmacogenomic monitoring to track patient responsiveness and to elucidate anti-inflammatory mechanisms.

Autocrine regulation of IL-12 receptor expression is independent of secondary IFN-gamma secretion and not restricted to T and NK cells.

The biological response to IL-12 is mediated through specific binding to a high affinity receptor complex composed of at least two subunits (designated IL-12Rbeta1 and IL-12Rbeta2) that are expressed on NK cells and activated T cells. The selective loss of IL-12Rbeta2 expression during Th2 T cell differentiation suggests that regulation of this receptor component may govern IL-12 responsiveness. In murine assays, down-regulation of IL-12Rbeta2 expression can be prevented by treatment with IFN-gamma, indicating that receptor expression and hence IL-12 responsiveness may be regulated, at least in part, by the local cytokine milieu. In this study, we report that cellular expression of both IL-12Rbeta1 and beta2 mRNA is increased in the lymph nodes of naive mice following systemic administration of murine rIL-12 (rmIL-12). Changes in IL-12R mRNA were associated with increased IFN-gamma secretion following ex vivo activation of lymph node cells with rmIL-12, indicating the presence of a functional receptor complex. Expression of IL-12R mRNA was not restricted to lymph node T cells, and its autocrine regulation was independent of secondary IFN-gamma secretion. Data from fractionated lymph node cells as well as rmIL-12-treated B cell-deficient mice suggest that IL-12-responsive B cells may represent an alternative cellular source for IFN-gamma production. However, the strength of the biological response to rmIL-12 is not governed solely by receptor expression, as rmIL-12-induced IFN-gamma secretion from cultured lymph node cells is accessory cell dependent and can be partially blocked by inhibition of B7 costimulation.

Interleukin-11 reduces T-cell-dependent experimental liver injury in mice.

Recombinant human interleukin-11 (rhIL-11) is a multifunctional cytokine that can reduce inflammation through the downregulation of multiple pro-inflammatory mediators from activated macrophages. rhIL-11 also inhibits production of several immunostimulatory cytokines such as IL-12 and interferon gamma (IFN-gamma) and has shown biological activity in multiple animal models of inflammatory disease consistent with immunomodulatory effects on macrophages and T cells. To further elucidate the anti-inflammatory activity of rhIL-11 in vivo, the effect of rhIL-11 in a model of Concanavalin A (Con-A)-induced T-cell-mediated hepatotoxicity was examined. Administration of a single dose of rhIL-11 before Con-A administration reduced centrilobular liver necrosis and enhanced survival. A dose-dependent reduction in serum levels of liver enzymes, tumor necrosis factor alpha (TNF-alpha), and IFN-gamma corresponded with this amelioration of liver damage. No significant change in infiltrating lymphocyte populations in the liver was observed following rhIL-11 treatment. Taken together, these results indicate that rhIL-11 ameliorates T-cell-mediated hepatic injury and suggests its therapeutic potential to treat inflammatory liver disease.

Hematopoietic, immunomodulatory and epithelial effects of interleukin-11.

Interleukin 11 (IL-11) is a pleiotropic cytokine with biological activities on many different cell types. Recombinant human IL-11 (rhIL-11) is produced by recombinant DNA technology in Escherichia coli. Both in vitro and in vivo, rhIL-11 has shown effects on multiple hematopoietic cell types. Its predominant in vivo hematopoietic activity is the stimulation of peripheral platelet counts in both normal and myelosuppressed animals. This activity is mediated through effects on both early and late progenitor cells to stimulate megakaryocyte differentiation and maturation. rhIL-11 has been approved for the treatment of chemotherapy-induced thrombocytopenia. The hematopoietic effects of rhIL-11 are most likely direct effects on progenitor cells and megakaryocytes in combination with other cytokines or growth factors. rhIL-11 also induces secretion of acute phase proteins (ferritin, haptoglobin, C-reactive protein, and fibrinogen) from the liver. The induction of heme oxidase and inhibition of several P450 oxidases have been reported from in vitro studies. In vivo, rhIL-11 treatment decreases sodium excretion by the kidney by an unknown mechanism and induces hemodilution. rhIL-11 also exhibits anti-inflammatory effects in a variety of animal models of acute and chronic inflammation, including inflammatory bowel disease, inflammatory skin disease, autoimmune joint disease, and various infection-endotoxemia syndromes. rhIL-11 has trophic effects on non-transformed intestinal epithelium under conditions of mucosal damage. The mechanism of the anti-inflammatory activity of rhIL-11 has been extensively studied. rhIL-11 directly affects macrophage and T cell effector function. rhIL-11 inhibits tumor necrosis factor-alpha (TNF alpha), interleukin 1beta (IL-1beta), interleukin 12 (IL-12), interleukin 6 (IL-6), and nitric oxide (NO) production from activated macrophages in vitro. The inhibition of cytokine production was associated with inhibition of nuclear translocation of the transcription factor, nuclear factor kappa B (NF-kappaB). The block to NF-kappaB nuclear translocation correlates with the ability of rhIL-11 to maintain or enhance production of the inhibitors of NF-kappaB, IkappaB-alpha and IkappaB-beta. In addition to effects on macrophages, rhIL-11 also reduces CD4+ T cell production of Th1 cytokines, such as IFN gamma induced by IL-12, while enhancing Th2 cytokine production. rhIL-11 also blocks IFN gamma production in vivo. The molecular effects of rhIL-11 have also been studied in a clinical trial. Molecular analysis of skin biopsies of patients with psoriasis before and during rhIL-11 treatment demonstrates a decrease in mRNA levels of TNF alpha, IFN gamma and iNOS. These activities suggest that in addition to its thrombopoietic clinical use, rhIL-11 may also be valuable in the treatment of inflammatory diseases. The clinical utility of the anti-inflammatory properties of rhIL-11 is being investigated in patients with Crohn's disease, psoriasis and rheumatoid arthritis. These diseases are believed to be initiated and maintained by activated CD4+ Th1 cells in conjunction with activated macrophages.

Recombinant human interleukin-11 does not affect functions of purified human neutrophils in vitro.

Recombinant human interleukin-11 (rHu-IL-11) is a multifunctional cytokine with thrombopoietic activity and demonstrated clinical efficacy in treating chemotherapy-induced thrombocytopenia. rHu-IL-11 also exhibits anti-inflammatory activity and is currently in clinical trials for the treatment of several inflammatory diseases. As neutrophils are involved in both innate immunity and an acute inflammatory response, the effect of rHU-IL-11 on the function of human peripheral blood neutrophils in vitro was examined. rHu-IL-11 was not cytotoxic and did not induce superoxide anion production or the release of granular enzymes from resting neutrophils. Phagocytosis and chemotaxis were unaffected. rHu-IL-11 treatment did not block the response of neutrophils to stimulation. Pretreatment with rHu-IL-11 did not reduce production of IL-8 following activation with lipopolysaccharide (LPS) or zymosan A particles. Pretreatment with rHu-IL-11 did not affect the release of lysozyme and beta-glucuronidase in response to A23187 or PMA-stimulated production of superoxide anion. These results indicate that rHu-IL-11 does not directly modulate key functions of neutrophils in vitro.

The therapeutic utility of Interleukin-11 in the treatment of inflammatory disease.

Interleukin-11 (IL-11) is a pleiotropic cytokine that exhibits anti-inflammatory and mucosal protective effects in a variety of animal models of acute and chronic inflammation, such as mucositis, inflammatory bowel disease and autoimmune joint disease. This reduction in inflammation and epithelial damage is mediated in part through effects of recombinant human (rh) IL-11 on macrophage effector function and epithelial cell growth. In vitro studies indicate that rhIL-11 inhibits tumour necrosis factor (TNF)-alpha, IL-1beta, IL-12, IL-6, and nitric oxide production from activated macrophages. Analysis of the effects of rhIL-11 on transcription factors that activate pro-inflammatory cytokines demonstrate that the level of induced nuclear factor kappa B (NF-kappaB) binding activity in the nucleus of rhIL-11-treated peritoneal macrophages is significantly reduced. Studies of normal intestinal epithelial cells indicate that rhIL-11 reduces the rate of cellular proliferation. Analysis of cell-cycle progression demonstrates that growth inhibition of epithelial cells by rhIL-11 correlates with delayed entry into S phase and suppression of pRB phosphorylation. IL-11 also protects intestinal crypt stem cells from radiation- or chemotherapy-induced insults. Such immunomodulatory and epithelial activities may contribute to the protective effects of this cytokine and support the clinical utility of rhIL-11 in the treatment of mucositis, as well as a variety of chronic inflammatory diseases, such as Crohn's disease and rheumatoid arthritis.

IL-11 regulates macrophage effector function through the inhibition of nuclear factor-kappaB.

Recombinant human IL-11 (rhIL-11) is an anti-inflammatory cytokine that can reduce the production of inflammatory mediators such as TNF-alpha, IL-1beta, IL-12, IL-6, and nitric oxide. Inhibition of proinflammatory cytokine production from activated macrophages was associated with a reduction in the levels of LPS-induced TNF-alpha, IL-1beta, IL-6, and IL-12 p40 mRNA. Analysis of rhIL-11 effects on transcription factors that activate proinflammatory cytokines demonstrated that the level of LPS-induced NF-kappaB binding activity in the nucleus of rhIL-11-treated peritoneal macrophages was significantly reduced. The block to NF-kappaB nuclear translocation correlated with the ability of rhIL-11 to maintain or increase protein levels of the inhibitors of NF-kappaB, IkappaB-alpha, and IkappaB-beta following LPS treatment. Furthermore, rhIL-11-treatment of LPS macrophages resulted in significant elevation of IkappaB-alpha and IkappaB-beta mRNA levels. These results suggest that the anti-inflammatory activity of rhIL-11 is mediated in part by inhibition of NF-kappaB-dependent transcriptional activation. Furthermore, these studies demonstrate for the first time the regulation of IkappaB-beta by an anti-inflammatory cytokine. Given the finding that inappropriate activation of NF-kappaB contributes to multiple inflammatory conditions, the ability of rhIL-11 to inhibit the binding activity of this pleiotropic transcription factor indicates that rhIL-11 has therapeutic potential in a wide range of diseases.

Recombinant human IL-11 attenuates the inflammatory response through down-regulation of proinflammatory cytokine release and nitric oxide production.

To elucidate the molecular mechanisms regulating the anti-inflammatory activities of recombinant human (rh)IL-11, the ability of rhIL-11 to reduce serum levels of inflammatory mediators such as TNF-alpha, IL-1beta, IL-12, and IFN-gamma in LPS-treated mice and to down-regulate macrophage function in culture was investigated. In a mouse model of endotoxemia, pretreatment with rhIL-11 blocked LPS-induced elevation of TNF-alpha, IL-1beta, and IFN-gamma serum levels, but had no effect on IL-12 p40, IL-6, or IL-10 serum levels. The effects of rhIL-11 on the production of inflammatory mediators in vivo may occur in part through direct interactions with macrophages. rhIL-11 pretreatment of thioglycollate-elicited peritoneal macrophages resulted in greater than 60% inhibition of LPS-induced production of TNF-alpha, IL-1beta, IL-12 p40, and nitric oxide. The activity of rhIL-11 was not mediated through induction of IL-10, IL-6, or TGF-beta1. These results indicate that the ability of rhIL-11 to modulate the inflammatory response is not dependent on known anti-inflammatory cytokines and substantiate a role for this cytokine in the attenuation of inflammatory conditions.

A novel developmental regulatory motif required for stage-specific activation of the epsilon-globin gene and nuclear factor binding in embryonic erythroid cells.

Members of the human beta-globin gene family are expressed at discrete stages of development and therefore provide an important model system for examining mechanisms of temporal gene regulation. We have previously shown that expression of the embryonic beta-like globin gene (epsilon) is mediated by a complex array of positive and negative upstream control elements. Correct developmental stage- and tissue-specific gene expression is conferred by synergistic interactions between a positive regulatory element (termed epsilon-PRE II) which is active only in embryonic erythroid cells and at least two other regulatory domains upstream of the epsilon-globin gene promoter. A nuclear factor highly enriched in cultured embryonic erythroid cells and in mouse embryonic yolk sac binds to a novel, evolutionarily conserved sequence within epsilon-PRE II. We show here that binding of this factor to the conserved element within epsilon-PRE II is critical for transcriptional activity. Point mutations that interfere with protein binding to epsilon-PRE II abolish transcriptional activation of the constitutive epsilon-globin promoter. Adult erythroid nuclei (from cultured cells or adult mouse liver) also contain a factor that binds to this region, but the complex formed migrates more rapidly during nondenaturing electrophoresis, suggesting either that distinct proteins bind to epsilon-PRE II or that a single protein is differentially modified in these cells in a way that modulates its activity. Several lines of evidence suggest that the binding factors in embryonic and adult erythroid cells are distinguished by posttranscriptional differences.

Upstream regulatory region of the human embryonic beta-like globin gene, epsilon.

The sequence of the human embryonic beta-like globin gene (epsilon) upstream regulatory region has been reported previously. In the course of our own work, we found a significant number of discrepancies between our sequence and the data base sequence, which we show here to contain large clusters of errors within functional epsilon-globin regulatory domains.

Developmental regulation of the human embryonic beta-like globin gene is mediated by synergistic interactions among multiple tissue- and stage-specific elements.

The stage-specific regulation of mammalian embryonic globin genes has been an experimentally elusive problem, in part because of the developmentally early timing of their expression. We have carried out a systematic analysis of truncation and internal deletion mutations within the 5'-flanking region of the human embryonic beta-like globin gene (epsilon) in erythroid and nonerythroid cell lines. Within a 670-bp region upstream from the constitutive promoter are multiple positive and negative control elements. Of these, a positive regulatory element (epsilon-PRE II) which is active only in embryonic erythroid cells is of particular interest. Remarkably, although it is inactive on its own, in the presence of other sequences located further upstream, it confers tissue- and developmental stage-specific expression on a constitutive epsilon-globin or heterologous promoter. The activity of epsilon-PRE II is also modulated by another positive regulatory domain located further downstream to direct erythroid cell-specific, but little or no embryonic stage-specific, transcription. A nuclear factor highly enriched in embryonic erythroid cells binds specifically within a 19-bp region of epsilon-PRE II. Nuclei from adult erythroid cells also contain a factor that binds to this region but forms a complex of faster electrophoretic mobility. We speculate that interactions between epsilon-PRE II and other upstream control elements play an important role in the developmental regulation of the human embryonic beta-like globin gene.

IGH minisatellite suppression of USF-binding-site- and E mu-mediated transcriptional activation of the adenovirus major late promoter.

The 50bp repeat unit of the minisatellite within the DH-JH interval of the human immunoglobulin heavy chain locus binds a nuclear factor present in a wide variety of cell types. The binding site contains the myc/HLH motif, CACGTG, and represents a 15 of 17 base match for the USF/MLTF binding site adjacent to the adenovirus major late promoter (MLP). Unlike the USF/MLTF site, the IGH minisatellite possesses no enhancer activity. However, it can significantly suppress, in cis and in trans, USF-site-mediated transcriptional activation of the MLP. In murine myeloma cells, the IGH minisatellite can suppress, in trans, MLP activation by the murine heavy chain gene enhancer, E mu. These activities potentially represent a DNA-based form of squelching.

Members of the rel/NF-kappa B family of transcriptional regulatory proteins bind the HRAS1 minisatellite DNA sequence.

The 28 base pair repeat unit of a minisatellite 1000 bp downstream from the human HRAS1 gene (VTRHRAS1) bound four proteins (p45, p50, p72 and p85) in nuclear extracts from a variety of human cell lines which were indistinguishable from several members of the rel/NF-kappa B family of transcriptional regulatory factors. VTRHRAS1 bound the constitutively expressed, but not the inducible, forms of these proteins. Analysis of partially purified binding factors from different cell lines demonstrated qualitative differences in the p50 subunit; phosphocellulose fractionation also revealed considerable heterogeneity in the p72 and p85 subunits. These results suggest the possibility that the HRAS1 minisatellite, in serving as a tandem array of rel/NF-kappa B binding sites, may function in the transcriptional regulation of HRAS1 and nearby genes.