The role of tumor necrosis factor-alpha in liver toxicity, inflammation, and fibrosis induced by carbon tetrachloride.

Hepatic expression of the proinflammatory cytokine tumor necrosis factor-alpha (TNFalpha) occurs in many acute and chronic liver diseases, as well as following exposure to hepatotoxic chemicals, and is believed to help influence both the damage and repair processes that occur following these insults by regulating additional mediators. We examined the role of TNFalpha in transgenic mice deficient in TNF receptors (TNFR) utilizing carbon tetrachloride (CCl(4)) as a model hepatotoxic agent that allowed for the evaluation of necrosis, inflammation, and fibrosis. Hepatocyte damage, as evident by local areas of liver necrosis and elevated levels of serum transaminase, occurred to a similar degree in wild-type and TNFR-deficient knockout (KO) mice following acute exposure to CCl(4). In contrast, the inflammatory response, manifested as an inflammatory cell influx, as well as induction of chemokines and adhesion molecules that occurred in wild-type mice following treatment with CCl(4), was not as evident in TNFR-KO mice. This response was associated primarily with type-1 (TNFR1) rather than type-2 (TNFR2) receptor responses. Liver fibrosis resulting from chronic CCl(4) exposure was also markedly dependent upon TNFalpha as demonstrated by almost a complete histological absence of fibrosis in TNFR-deficient mice. This was further supported by marked reductions in procollagen and transforming growth factor beta synthesis in TNFR-deficient mice. Taken together, these results indicate that TNFalpha is responsible for regulating products that induce inflammation and fibrosis but not direct hepatocyte damage in CCl(4)-induced hepatotoxicity.

Regulation and role of interleukin 6 in wounded human epithelial keratinocytes.

Dermal wounding is accompanied by inflammation and the resulting proinflammatory cytokines, including interleukin (IL)-6, are thought to play an important role in the repair process. IL-6 is produced by normal human keratinocytes to various dermatological diseases and we have recently shown it is also required for normal wound repair. However, neither the events responsible for its induction nor its role in repair have been clearly identified. Using a recently developed in vitro wounding model, we demonstrate that IL-6 mRNA is expressed and immunoreactive IL-6 is released from cultures of human epidermal keratinocytes (NHEKs) following wounding. The transcription factors, NF kappa B and NF-IL-6 (C/EBP beta), which coordinately help regulate IL-6 expression, were activated following wounding and preceded the appearance of IL-6. Addition of IL-1 alpha to NHEK cultures increased IL-6 production and activated NF kappa B and C/EBP beta. Addition of the IL-1 alpha receptor antagonist inhibited both IL-6 mRNA expression and the transcription factors following wounding. Immunoreactive IL-1 alpha was detected in the medium following wounding in the absence of new message. Furthermore, addition of IL-6 to NHEK cultures decreased the expression of keratins 1 and 10, differentiation markers of keratinocytes, while proliferation was not affected. Taken together, these data indicate that constitutive keratinocyte-derived IL-1 alpha is a stimulus for IL-6 production in wounded epidermis, the response involves NF kappa B and C/EBP beta transcription factors, and IL-6 may be associated with modulation of keratinocyte differentiation rather than proliferation.

Epidemiologic associations between occupational and environmental exposures and autoimmune disease: report of a meeting to explore current evidence and identify research needs.

To advance understanding of autoimmunity associated with exposure to environmental factors, an "Exploratory Meeting Epidemiology on Occupational and Environmental Factors Associated with Autoimmunity" was organized in Bilthoven, the Netherlands, from May 10-12, 2000. Even if no firm conclusions can be drawn on a role of certain chemicals in the environment and in the work place in causing or exacerbating autoimmune responses and illnesses, many indications of this to occur exist. The aim of the meeting was to determine the optimal methodology for assessment of autoimmunity associated with occupational or environmental exposures in the human population, and to set up interdisciplinary and collaborative epidemiological studies to investigate the association of exposure to silica, hexachlorobenzene, ultraviolet radiation, and other agents with autoimmunity and autoimmune diseases in the human population. These agents were selected as carrying particular suspicion at present. It was concluded that there is a need for experimental studies in laboratory animals and for clinical investigations to improve scientific knowledge about the causes and mechanisms of environmentally-induced autoimmune disorders and their treatment; in addition there is a need for an interdisciplinary approach to epidemiological studies of the environmental and other causes of these disorders in human populations. Specific designs for epidemiological studies in this context, as well as laboratory assays for health outcomes, were reviewed. Several recommendations for the epidemiological approach to evaluating effects of environmental or occupational agents on autoimmunity were made. The prime recommendations are the following: 1) systematic descriptive epidemiological data on autoimmunity and autoimmune disorders are required; 2) the establishment of disease-reporting registries should be encouraged; 3) the development of internationally accepted standard diagnostic criteria for all autoimmune diseases should be encouraged; 4) the social impact of these disorders should be evaluated and estimations of direct and indirect economic costs should also be made; 5) the methods of exposure assessment used in epidemiological studies should be standardized; 6) laboratory methods for measurement of biological responses should be standardized; and 7) the inclusion of indicators of autoimmunity and autoimmune diseases and of relevant environmental exposures in ongoing epidemiological studies should be encouraged. The importance of studying environmental causes of autoimmune diseases and autoimmunity lies in the identification and prevention of risks to the public health, and in improving our knowledge of basic mechanisms of health and disease.

Interleukin-6 treatment augments cutaneous wound healing in immunosuppressed mice.

It has been postulated that the inflammatory response that occurs after cutaneous wounding is a prerequisite for healing and that inflammatory cytokines, such as interleukin-6 (IL-6) are involved in this process. We showed previously that IL-6-deficient mice display delayed wound healing, which could be reversed by administration of a murine IL-6 expression plasmid or recombinant murine IL-6 (rMuIL-6). In the present study, we observed that delayed cutaneous wound healing, which occurs as a result of glucocorticoid-induced immunosuppression, can also be reversed by rMuIL-6, as evidenced by epithelialization, granulation tissue formation, and wound closure. In vehicle control mice, rMuIL-6 did not augment healing but rather delayed the process. Immunochemical studies indicated that the expression of matrix metalloproteinase-10 (MMP-10) was increased in dexamethasone-treated mice and that rMuIL-6 treatment reduced its expression, indicating that IL-6 may influence dermal matrix formation and, specifically, collagen synthesis. These results demonstrate that IL-6 can restore abnormal wound repair that occurs in immunodeficiency and suggest its use as a potential therapy.

Role of inflammation in chemical-induced hepatotoxicity.

The liver, which is the major organ responsible for the metabolism of drugs and toxic chemicals, is also the primary target organ for many toxic chemicals. Increasing evidence has indicated that inflammatory processes are intimately involved in chemical-induced hepatotoxic processes, and like other inflammatory diseases, such as autoimmunity, are responsible for producing mediators that can effect liver damage or repair. This review will summarize our current understanding of how inflammatory processes influence hepatic pathology and repair following exposure to established hepatotoxic chemicals including carbon tetrachloride, an industrial chemical, and acetaminophen, a widely used analgesic.

Quantitative relationship between arsenic exposure and AP-1 activity in mouse urinary bladder epithelium.

Because of the potential of arsenic for causing cancer in humans, and of the fact of widespread environmental and occupational exposure, deriving acceptable human-limit values has been of major concern to industry as well as to regulatory agencies. Based upon epidemiological evidence and mechanistic studies, it has been argued that a non-linear dose-response model at low-level exposures is more appropriate for calculating risk than the more commonly employed linear-response models. In the present studies, dose-response relationships and recovery studies employing a cancer precursor marker, i.e., activating protein (AP)-1 DNA-binding activity, were examined in bladders of mice exposed to arsenic in drinking water and compared to histopathological changes and arsenic tissue levels in the same tissue. While AP-1 is a functionally pleomorphic transcription factor regulating diverse gene activities, numerous studies have indicated that activation of the MAP kinase pathway and subsequently increased AP-1 binding activities, is a precursor for arsenic-induced cancers of internal organs as well as the skin. We observed previously that within 8 weeks of exposure AP-1 activation occurs in urinary bladder tissue of mice exposed to arsenic in the drinking water. In the present studies, C57BL/6 mice were exposed to sodium arsenite at various concentrations in the drinking water for 8 consecutive weeks. Minimal but observable AP-1 activity occurred in bladder tissue at exposure levels below which histopathological changes or arsenic tissue accumulation was detected. Marked AP-1 DNA-binding activity only occurred at exposure levels of sodium arsenite above 20 microg/ml, where histopathological changes and accumulation of arsenic in the urinary bladder epithelium occurred. Although the experimental design did not allow statistical modeling of the entire dose-response curve, the general shape of the dose-response curve is not inconsistent with the previously proposed hypothesis that arsenic-induced cancer follows a non-linear dose-response model.

Impaired cutaneous wound healing in interleukin-6-deficient and immunosuppressed mice.

It has been postulated that an inflammatory response after cutaneous wounding is a prerequisite for healing, and inflammatory cytokines, such as interleukin-6 (IL-6), might be intimately involved in this process. IL-6-deficient transgenic mice (IL-6 KO) displayed significantly delayed cutaneous wound healing compared with wild-type control animals, requiring up to threefold longer to heal. This was characterized by minimal epithelial bridge formation, decreased inflammation, and granulation tissue formation. Using electrophoretic mobility shift assays of wound tissue from IL-6 KO mice, decreased AP-1 transcription factor activation was shown compared with wild-type mice 16 h after wounding. In situ hybridization of wound tissue from wild-type mice revealed IL-6 mRNA expression primarily in the epidermis at the leading edge of the wound. Delayed wound healing in IL-6 KO mice was reversed with a single dose of recombinant murine IL-6 or intradermal injection of an expression plasmid containing the full-length murine IL-6 cDNA. Treatment with rmIL-6 also reconstituted wound healing in dexamethasone-treated immunosuppressed mice. The results of this study may indicate a potential use for IL-6 therapeutically where cutaneous wound healing is impaired.

The role of tumor necrosis factor alpha in chemical-induced hepatotoxicity.

Only recently have toxicologists come to understand the role of inflammation, and TNFalpha specifically, in classical toxicological processes. This relationship appears fairly complex, as inflammation and proliferation may well be only one facet of a time- and dose-dependent continuum of toxicological and repair processes. Not surprisingly, considerable efforts are being undertaken using our newly found understanding of molecular control to develop specific and safe chemical, biological, and molecular regulators of TNFalpha for potential therapeutic use. Their effectiveness in controlling environmental or occupational diseases has yet to be established.

Mechanisms of arsenic carcinogenicity: genetic or epigenetic mechanisms?

Environmental and occupational exposure to arsenic is associated with increased risk of skin, urinary bladder, and respiratory tract cancers. The mechanisms responsible for arsenic carcinogenesis have not been established. Arsenic does not act through classic genotoxic and mutagenic mechanisms, as do other metals such as cadmium or chromium. Increasing evidence indicates that arsenic acts at the level of tumor promotion by modulating the signaling pathways responsible for cell growth.

Arsenic mediates cell proliferation and gene expression in the bladder epithelium: association with activating protein-1 transactivation.

Although the mechanism of action has not yet been defined, epidemiological studies have demonstrated an association between elevated arsenic levels in drinking water and the incidence of urinary bladder transitional cell carcinomas. In the current studies, we demonstrate that mice exposed to 0.01% sodium arsenite in drinking water develop hyperplasia of the bladder urothelium within 4 weeks of exposure. This was accompanied by the accumulation of inorganic trivalent arsenic, and to a lesser extent dimethylarsinic acid, in bladder tissue, as well as a persistent increase in DNA binding of the activating protein (AP)-1 transcription factor. AP-1 transactivation by arsenic also occurred in bladders of transgenic mice containing an AP-1 luciferase reporter. Consistent with these in vivo observations, arsenite increased cell proliferation and AP-1 DNA binding in a human bladder epithelial cell line. Gene expression studies using RNase protection assays, reverse transcription-PCR, and cDNA microarrays indicated that arsenite alters the expression of a number of genes associated with cell growth, such as c-fos, c-jun, and EGR-1, as well as cell arrest, such as GADD153 and GADD45. The proliferation-enhancing effect of arsenic on uroepithelial cells likely contributes to its ability to cause cancer.

TNF-alpha regulates transforming growth factor-alpha expression in regenerating murine liver and isolated hepatocytes.

TNF-alpha is a pleotropic proinflammatory cytokine that has been implicated as a contributing factor in a number of disease processes, primarily through its ability to induce the expression of inflammatory and cytotoxic mediators. TNF-alpha is also involved in cell growth accompanying the healing process in multiple organ systems and influences liver repair following hepatotoxic damage or regeneration following partial hepatectomy. In this respect, TNF-alpha is a known mitogen for hepatocytes. In this paper we describe a novel role for TNF-alpha in the modulation of expression of TGF-alpha, the latter being a complete hepatocyte mitogen. TNF-alpha directly up-regulates TGF-alpha mRNA by up to 7-fold in isolated mouse hepatocytes, whereas neutralization of TNF-alpha significantly decreased liver mRNA and protein expression of TGF-alpha following chemical-induced hepatotoxicity. That TNF-alpha directly stimulated TGF-alpha was suggested by the inability of either anti-IL-6 Abs or cycloheximide to inhibit TNF-alpha-induced TGF-alpha expression in hepatocytes. However, in the presence of anti-TGF-alpha neutralizing Abs, the mitogenic activity of TNF-alpha is abrogated. Using cells transfected with the TGF-alpha promoter, and an RNA polymerase inhibitor, it was shown that TNF-alpha modulates TGF-alpha expression through both pre- and posttranscriptional events. Taken together, these data suggest that TNF-alpha participates in liver repair and regeneration, in part, by directly inducing the expression of TGF-alpha.

Immunotoxicology: role of inflammation in chemical-induced hepatotoxicity.

The liver, which is the major organ responsible for the metabolism of drugs and chemicals, is also the primary target organ for many toxic chemicals. Increasing evidence has indicated that inflammatory processes are intimately involved in chemical-induced hepatotoxic processes, and like other inflammatory diseases, such as autoimmunity, are responsible for producing mediators which can effect liver damage or repair. This review will summarize the authors' current understanding of how inflammatory processes influence hepatic pathology and repair following exposure to established hepatotoxic chemicals including carbon tetrachloride (CCl4), an industrial chemical, and acetaminophen (APAP), a widely used analgesic.

Tumor necrosis factor alpha and toxicology.

The molecular cloning of a group of proteins, collectively referred to as cytokines, and including interleukins, chemokines, growth factors, colony stimulating factors, and tumor necrosis factors, has allowed for the increased understanding of the mechanisms for many disease processes as well as provided strategies for the development of novel therapies. Conceptually similar to hormones and peptides, this group of phylogenetically related molecules are also involved in various toxicological processes, including apoptosis, cell repair, and in particular inflammation. In this review, we offer a description of what many believe represents the primary regulatory cytokine, tumor necrosis factor (TNF)alpha and its role in toxicological processes. For over a decade it has been suspected that this molecule helps mediate the shock state induced by bacterial endotoxin and the wasting diathesis that typifies chronic diseases. Advances in molecular biology that have provided tools to modulate TNFalpha regulation and synthesis have allowed for the identification of additional roles for TNFalpha in homeostasis, cellular damage, and repair. This review provides a brief summary of our understanding of TNFalpha biology followed by a discussion of its role in toxicological responses. This is followed by specific examples of organ-specific and tissue-specific responses to chemical damage where TNFalpha has been implicated. The review concludes with a review of its implication in human risk assessment, particularly as it relates to genetic polymorphisms of TNFalpha expression and disease susceptibility.

Autoimmunity and risk assessment.

Among the issues dealing with identifying potential adverse immunologic effects (i.e., suppression, hypersensitivity, or autoimmunity) associated with xenobiotic exposure, general agreement exists among the regulatory and pharmaceutical communities that predictive tests for autoimmunity are in most need of development in order to improve risk assessment. The estimation of risk (i.e., the probability of a deleterious effect resulting from exposure) involves both the qualitative evaluation of whether a hazard exists and the quantitative evaluation for determining an acceptable level of exposure in humans. Unless adequate human data are available, which is uncommon, this is based on animal studies. Although animal models exist to study autoimmune processes, these models do not readily lend themselves to interpretation in the risk assessment process due, for the most part, to the complexity of autoimmune disease(s), as they are multifactorial and exhibit genetic heterogeneity in humans. To improve the risk assessment process, researchers must develop and validate animal models that not only incorporate mechanistic information into the assessment process but also allow for consideration of potent genetic, physiologic, and environmental influences.

Redox-dependent regulation of interleukin-8 by tumor necrosis factor-alpha in lung epithelial cells.

Increasing evidence supports a major role for interleukin-8 (IL-8), a potent neutrophil chemoattractant, in the chronic progression of inflammatory lung diseases. The present studies were designed to characterize the molecular events involved in IL-8 induction in pulmonary epithelial cells in response to tumor necrosis factor-alpha (TNF-alpha). IL-8 induction by TNF-alpha was redox sensitive, as indicated by electron spin resonance analysis and inhibition with membrane permeable hydroxyl scavengers. Furthermore using cell transfection and mobility shift assays, it was found that transcriptional activation of the IL-8 gene required TNF-alpha-induced activation and binding of nuclear factor-kappaB (NF-kappaB)- and NF-IL-6, nuclear transcription factors to regulatory elements in the IL-8 promoter. Activation of the IL-8 promoter by these transcription factors was also redox-sensitive. This response was mediated through the TNF-R1 receptor (p55), and not the TNF-R2 (p75) receptor, although both receptors can be found on pulmonary epithelial cells. Taken together these studies indicate that TNF-alpha-induced redox changes in lung epithelial cells are responsible for the transcriptional activation of IL-8 and that coordinate activation of NF-kappaB and NF-IL-6 mediate the response.

Arsenic enhancement of skin neoplasia by chronic stimulation of growth factors.

Although numerous epidemiological studies have shown that inorganic arsenicals cause skin cancers and hyperkeratoses in humans, there are currently no established mechanisms for their action or animal models. Previous studies in our laboratory using primary human keratinocyte cultures demonstrated that micromolar concentrations of inorganic arsenite increased cell proliferation via the production of keratinocyte-derived growth factors. As recent reports demonstrate that overexpression of keratinocyte-derived growth factors, such as transforming growth factor (TGF)-alpha, promote the formation of skin tumors, we hypothesized that similar events may be responsible for those associated with arsenic skin diseases. Thus, the influence of arsenic in humans with arsenic skin disease and on mouse skin tumor development in transgenic mice was studied. After low-dose application of tetradecanoyl phorbol acetate (TPA), a marked increase in the number of skin papillomas occurred in Tg.AC mice, which carry the v-Ha-ras oncogene, that received arsenic in the drinking water as compared with control drinking water, whereas no papillomas developed in arsenic-treated transgenic mice that did not receive TPA or arsenic/TPA-treated wild-type FVB/N mice. Consistent with earlier in vitro findings, increases in granulocyte/macrophage colony-stimulating factor (GM-CSF) and TGF-alpha mRNA transcripts were found in the epidermis at clinically normal sites within 10 weeks after arsenic treatment. Immunohistochemical staining localized TGF-alpha overexpression to the hair follicles. Injection of neutralizing antibodies to GM-CSF after TPA application reduced the number of papillomas in Tg.AC mice. Analysis of gene expression in samples of skin lesions obtained from humans chronically exposed to arsenic via their drinking water also showed similar alterations in growth factor expression. Although confirmation will be required in nontransgenic mice, these results suggest that arsenic enhances development of skin neoplasias via the chronic stimulation of keratinocyte-derived growth factors and may be a rare example of a chemical carcinogen that acts as a co-promoter.

Cytokine expression in hepatocytes: role of oxidant stress.

Inflammatory mediators, including cytokines and chemokines, are associated with the pathology of chronic liver disease. Interleukin-8 (IL-8) in humans and macrophage inflammatory protein-2 (MIP-2) in rodents, both members of the C-X-C family of chemokines, are particularly potent neutrophil attractants and have been implicated in chronic liver diseases. In the liver, cytokine secretion is usually associated with non-parenchymal cells, particularly Kupffer cells. In the present studies, chemokine gene expression and secretion were investigated in hepatocytes treated with various stimulators. Using human Hep G2 cells, it was demonstrated that, in contrast to lipopolysaccharides (LPS), both tumor necrosis factor-alpha (TNF-beta) and H2O2 are potent inducers of IL-8, presumably acting via protein kinase C (PKC)-dependent pathways. MIP-2 expression occurred in freshly isolated rat hepatocytes following treatment with TNF-alpha, LPS, and to a lesser degree, H2O2. Both IL-8 and MIP-2 secretion were inhibited, although to varying degrees, by such antioxidants as TMTU, DMSO, catalase, and N-acetylcysteine. Furthermore, in vitro TNF-alpha neutralization experiments and transfection of Hep G2 cells with an IL-8 construct confirmed that TNF-alpha and H2O2 directly stimulate IL-8 secretion. RT-PCR analyses indicated that chemokine secretion induced by these agents operates via increased gene expression. Furthermore, a variety of cytokine genes were found to be expressed by hepatocytes, including MCP-1, cytokine-induced neutrophil chemoattractant (CINC), and IL-6. Taken together, these studies indicate that hepatocytes respond to biologically relevant levels of common activators, including H2O2, to produce cytokines and chemokines that contribute to pathophysiologic and repair processes in the liver.

Toxic metals stimulate inflammatory cytokines in hepatocytes through oxidative stress mechanisms.

Hepatocytes, as well as nonparenchymal cells, secrete proinflammatory cytokines and chemokines that are involved in the pathology of many liver diseases. In particular, tumor necrosis factor-alpha (TNFalpha), as well as members of the CXC family of chemokines, including interleukin (IL)-8 in humans and macrophage inflammatory protein (MIP)-2 in rodents, have been implicated in both damage and repair processes associated with various hepatotoxins. In the liver, cytokine secretion is usually associated with nonparenchymal cells, particularly Kupffer cells. In the present studies, cytokine gene expression and secretion were investigated in hepatocytes treated with cadmium chloride (CdCl2) or vanadium pentoxide (V2O5). Using human Hep G2 cells and freshly isolated rodent hepatocytes, it was demonstrated that metals increase gene expression and secretion of CXC chemokines and TNFalpha. IL-8 and MIP-2 secretion induced either by the metals or H2O2 were inhibited by antioxidants such as tetramethyl-thiourea and N-acetyl-cysteine. In vitro neutralization experiments with TNFalpha and in vivo studies with TNFalpha receptor knockout mice indicated that the metals directly stimulate CXC chemokine secretion without the need for TNFalpha. Taken together these studies indicate that, in addition to other inflammatory mediators and acute phase proteins, cytokines and chemokines are produced by hepatocytes, which may participate in hepatotoxic responses. The events responsible for their expression involve cellular redox changes.

Asbestos induces inflammatory cytokines in the lung through redox sensitive transcription factors.

Studies are summarized demonstrating that the inflammatory cytokines, interleukin IL-6 and IL-8, play a direct role in asbestos lung diseases and are produced by lung epithelial cells in direct response to the fibers. This response is controlled by changes in the cellular oxidative/state induced by iron present in the fiber through Fenton-type chemistry. As a result of this oxidative stress, the redox sensitive transcription factors, NF-kappaB and NF-IL-6, which help regulate cytokine gene expression, are activated.

Dietary restriction mitigates ozone-induced lung inflammation in rats: a role for endogenous antioxidants.

Studies were undertaken to determine whether dietary restriction protects against acute pulmonary oxidant challenge. Male F344 rats were fed NIH-31 diet either ad libitum or at restricted levels equal to 75% that of ad libitum intake. After 3 wk of dietary adaptation, animals were exposed by inhalation to 2.0 ppm ozone (O3) for 2 h or chamber air and evaluated for cellular and biochemical indices of pulmonary toxicity. Compared to air controls, bronchoalveolar lavage fluid (BALF) from O3 exposed ad libitum fed rats contained increased protein (145 versus 380 microg/ml), PMN infiltration (0 versus 11%) and fibronectin (45 versus 607 U/ml). Diet restriction abrogated these indicators of pulmonary inflammation induced by ozone. Binding of 18O3 to BALF protein and cells was significantly decreased in diet restricted rats while BALF ascorbate and glutathione levels, but not alpha-tocopherol or urate, were elevated compared to ad libitum fed rats. Taken together, these results indicate that dietary restriction affords protection against O3-induced oxidant toxicity. Protection is mediated partially by increases in ascorbate in the fluid bathing the lung surface, thereby providing an antioxidant sink which minimizes the ability of O3 to reach biological targets.