Acute ethanol consumption synergizes with trauma to increase monocyte tumor necrosis factor α production late postinjury

The hypothesis that acute ethanol uptake plus trauma can synergize to increase immunosuppression was tested. We found that, unlike non-alcohol-exposed patients, patients with acute alcohol use prior to trauma have a transient decrease in monocyte tumor necrosis factor (TNF) production during the very early postinjury (0–3 days) period. However, TNF production by these alcoholexposed patients' monocytes (MØ) became hyperelevated late postinjury (>9 days). Consequently, these massively elevated MØ TNF levels can contribute to posttrauma immunosuppression after acute alcohol use. We also demonstrate that normal monocyte activation with the superantigen,Staphylococcus enterotoxin B (SEB), results in a preferential induction of cellassociated MØ TNF production, described as characteristic of immunosuppressed trauma patients. Acutein vitro ethanol treatment down-regulated the elevated TNF production by trauma patients' MØ after either SEB, muramyl-dipeptide (MDP), interferon- plus MDP, or lipopolysaccharide (LPS) stimulation. Both SEB- and LPS-induced TNF mRNA induction was inhibited by acute alcohol treatment in normal MØ, indicating that ethanol can regulate cytokine gene expression. An additional immunosuppressive effect of acute ethanol's stimulation was suggested by its induction of elevated transforming growth factor production in trauma patients' activated MØ.     

Genetic characterization of an α-specific gene responsible for sexual agglutinability in Saccharomyces cerevisiae: mapping and gene dose effect

Summary A recessive ag1 mutation leads to specific defect in sexual agglutinability specifically in cells of the yeast Saccharomyces cerevisiae. The cryptopleurine resistance gene cry^R 1, closely linked to the mating type locus, was used to select / strains which emerged from / strains by mitotic nonreciprocal recombination, to genetically analyse ag1, since ag1 is expressed only in mating type. The ag1 gene was found to be linked to the centromere tightly, to met3 at 4.4 cM, and to ilv3 at 12 cM on chromosome X. Sexual agglutinability of cells was shown to be dependent on the dose of the AG1 gene, using / isogenic strains carrying AG1/AG1, AG1/ag1 or ag1/ag1. The sst2-1 mutation did not suppress the ag1 mutation. Based on these results, function of the AG1 gene is discussed.Abbreviations cM centimorgan - FDS first division segregation - NPD nonparental ditype - PD parental ditype - SDS second division segregation - TT tetratype     

Potential roles for tumour necrosis factorα during embryonic development

This paper reviews the evidence indicating possible roles for tumour necrosis factor-alpha (TNF) in development. It is proposed that TNF may have essentially three major roles during embryonic development, which may be analogous to its roles in the immune system and during inflammation: a role in programmed cell death; a role as a cellular growth and differentiation factor; and also a role in the remodelling of extracellular matrix, and the regulation of cell adhesion molecules and integrins. The concept of the existence of a cytokine array during embryogenesis, analogous to that occurring in inflammation, is discussed, as well as potential roles for TNF in the induction of ubiquitin; protective mechanisms embryonic cells may employ against TNF-mediated cytotoxicity; and a consideration of the role TNF may play in a free radical theory of development.     

Down-regulation of tumor necrosis factor α activity by acute ethanol treatment in human peripheral blood monocytes

As the most commonly used drug that can modulate both metabolic and immune pathways, ethanol is evaluated in this report as a regulator of tumor necrosis factor (TNF) production in human peripheral blood monocytes (M) in combination with a variety of stimuli. While acute ethanol treatment did not induce TNF in M, it was a potent down-regulator of M TNF production whether induced by the combination of interferon- plus muramyl dipeptide (MDP) (P<0.001), lipopolysaccharide (LPS) alone (P<0.01), or interferon- plus LPS. Down-regulation of M TNF by ethanol was dose dependent and statistically significant in the biologically relevant, 25–150 mM, ethanol concentration range. We also demonstrate that these ethanol concentrations did not affect M viability. TNF down-regulation by ethanol was most effective when ethanol was administered 4 hr prior to MDP stimulation; however, it was also effective—though to a lesser extent—if it was added at the time of MDP stimulation. Furthermore, ethanol also down-regulated TNF production of thein vivo preactivated M of trauma patients, which produce hyperelevated levels of TNF. We have previously shown that the majority of posttrauma elevated M TNF is produced by the M subpopulation expressing high-affinity type I Fc receptors (FcRI). When the FcRI cross-linking-stimulated M subpopulation was treated with acute ethanol, TNF production was suppressed again both inin vivo preactivated M of trauma patients and in M of normal controls. In experiments utilizing cyclooxygenase inhibitor, we also demonstrate that ethanol has a direct, prostaglandin E₂-independent, effect on M TNF production. These results demonstrate that acute ethanol exposure has the potential to down-regulate M production of TNF significantly regardless of the TNF-inducing stimulus. Decreased capacity of M to produce TNF might, therefore, contribute to the immunological and metabolic abnormalities described after ethanol uptake.     

Expression ofβ1 integrins in non-neoplastic mammary epithelium,fibroadenoma and carcinoma of the breast

1 Integrins were examined immunohisto-chemically in normal and mastopathic mammary glands, 12 benign tumours and 90 carcinomas of the breast using monoclonal antibodies against1 and1 to6 subunits. When compared with epithelial cells of non-neoplastic mammary glands and of benign tumours, carcinoma cells showed considerable quantitative changes in the pattern of2,3 and6 subunit expression. In contrast, the distribution pattern of1,1,4 and5 antigens corresponded to the situation observed in non-neoplastic mammary gland epithelium in most instances. An abnormal expression of2 was found in 71.0% of the carcinomas ranging from a remarkably low number of2-positive tumour cells in 27.5% of the cases to a complete absence of the2 molecule in 43.5% of the carcinomas. Of the carcinomas 39.9% exhibited quantitative changes in3 expression with an abnormally low content of3-positive neoplastic cells in 15.4% and a complete absence of this molecule in 24.5% of the cases. Expression of6 was abnormal in 73.2% of the carcinomas, consisting in a greater number of6-negative tumour cells in 31.9% and in a complete absence of6 in 41.3% of the tumours. The abnormally low expression/absence of2 and3 subunits correlated with oestrogen receptor negativity (P<0.033 andP<0.04, respectively). In addition, abnormally low expression/absence of2 correlated with poor differentiation of the tumours (P< 0.014). The quantitative changes in the expression pattern of1-associated subunits in breast carcinomas may cause a disturbed cell-cell and/or cell-matrix interaction that increases the invasive and migratory property of the tumour cells.     

The role ofγδ T cells in the normal and disordered immune system

Summary A small population of T cells does not express the conventional T cell receptor characterized by the and polypeptide chains (TCR) but instead, two polypeptides termed and (TCR). This alternative receptor is able to recognize antigen. It appears early in T cell ontogeny, but its role in the thymus prior to the availability of TCR remains unclear. In selected sites such as skin or gut TCR predominates in mice which might suggest a role of T cells in the first line of defense against infection, T cells secrete lymphokines and display cytotoxic activity. However, their activation requirements may differ from what is known for T cells since MHC-nonrestricted and also CD4 and CD8 negative T cells have been described. Preferential activation by mycobacterial antigens possibly indicates a special repertoire of the T cells. In various diseases slightly increased numbers of T cells were found, but these preliminary studies have not yet provided evidence for a major pathogenetic role of T cells.List of abbreviations C constant region (immunoglobulin or TCR gene segment) - CD4 cluster of differentiation 4 (mainly on helper cells) - CD8 cluster of differentiation 8 (mainly on cytotoxic cells) - D diversity region (immunoglobulin or TCR gene segment) - DNA desoxyribonucleic acid - IL2 interleukin 2 - J joining region (immunoglobulin or TCR gene segment) - kD kiloDalton - MHC major histocompatibility complex - NK natural killer (cells) - RA rheumatoid arthritis - TCR T cell receptor - V variable region (immunoglobulin or TCR gene segment)     

Altered IL-1 Expression and Compartmentalization in Monocytes from Patients with AIDS Stimulated with Mycobacterium avium Complex

The pathophysiologic basis for the exuberant intracellular growth of Mycobacterium avium complex (MAC) in AIDS patients is unclear but may relate to altered expression of modulatory cytokines. Interleukin (IL)-1, IL-6, and TNF- expression by monocytes from AIDS patients and healthy subjects (HS) stimulated with isogeneic MAC strains (SmT, smooth-transparent, virulent; SmD, smooth-domed, avirulent) was examined. Spontaneous cytokine production was not observed in patients with AIDS. MAC strains induced less IL-1 and IL-1 release in AIDS patients than HS (P < 0.05). The ratio of cell-associated to supernatant IL-1 also was increased in AIDS patients (P = 0.03). IL-1 mRNA expression paralleled protein release in either group of subjects. In both HS and AIDS patients, stimulation with SmD induced more IL-1 and TNF- release by monocytes compared to SmT. In AIDS patients, SmD also induced greater IL-6 release than SmT (P < 0.01). Alterations in monocyte expression and compartmentalization of the regulatory cytokines IL-1 and IL-6 may enhance bacterial replication and contribute to the patho-genesis of MAC infection in AIDS.     

Antitumor effects of liposomal IL1α and TNFα against the pulmonary metastases of the B16F10 murine melanoma in syngeneic mice

Interleukin 1 alpha (IL1) and tumor necrosis factor alpha (TNF) have been successfully incorporated into specific phosphatidylcholine (PC) and phosphatidylserine (PS) multilamellar vesicle (MLV) liposomes by modifying the concentration of calcium ion and pH of the encapsulation buffer. Under these conditions, some of the cytokines may attach to the exterior surface of the MLV and therefore be readily accessible to target cells for receptor binding and signal transduction. These cytokine-associated liposomes are stable for up to 2 weeks in serum-free buffer, and leakage of cytokines into medium containing 10% fetal bovine serum was about 50% at the end of a 3-day incubation period at 37°C. The biological activities mediated by liposomal IL1 and TNF were specific: the stimulation of thymidine uptake in T-helper D10 lymphocytes and the cytolysis of TNF-sensitive L929 target cells could be blocked by specific neutralizing antibodies in a dose-dependent fashion. When administered intravenously into C57BL/6 mice bearing the syngeneic B16F10 murine melanoma cells, dual entrapment of liposomal IL1 and TNF significantly reduced the number of metastatic tumor nodules in the lungs and prolonged the life span of the animals. Thus, liposomal IL1 and TNF displayed significant in vivo antitumor activity against the IL1- and TNF-resistant B16F10 metastatic murine melanoma.     

Flow Cytometric Analysis of In Vitro Proinflammatory Cytokine Secretion in Peripheral Blood from Multiple Sclerosis Patients

The cytokines, interferon- (IFN-), tumor necrosis factor- (TNF-rpar;, and interleukin-2 (IL-2) are important endogenous proinflammatory proteins and have been linked to disease activity in multiple sclerosis. In this study, we use flow cytometric methodology to compare the secretion of IFN-, IL-2, and TNF- from peripheral blood-derived T cells of multiple sclerosis patients to the secretion in healthy controls. The percentages of IFN-, IL-2, and TNF- secreting cells are not significantly different between multiple sclerosis patients and controls. However, the TNF- secreting CDS cell percentage is correlated with the IFN- and IL-2 secreting CD3 cell percentages in multiple sclerosis patients. In the controls, only the TNF- secreting CD3 cell percentage is correlated with IFN-. These findings show that correlated secretion of cytokines occurs in multiple sclerosis and suggest that concerted intercytokine interactions may play an important role in the disease.     

alpha1-acid glycoprotein possesses in vitro pro- and antiinflammatory activities

We studied the effects of ₁-acid glycoprotein on tumor necrosis factor- (TNF-) and interleukin-10 (IL-10) production and lymphocyte response to phytohemagglutinin in cultured peripheral blood mononuclear leukocytes from 6 healthy donors. We observed 2 opposite responses to ₁-acid glycoprotein: first, stimulation of TNF- and IL-10 production and inhibition of lymphocyte proliferation, and second, suppression of cytokine production and stimulation of lymphocyte proliferation. In cell cultures isolated from 4 of 6 donors, the TNF-/IL-10 ratio remained unchanged after addition of native ₁-acid glycoprotein, but some fractions isolated by chromatography on concanavalin A-Sepharose changed this parameter. These changes were most pronounced after treatment with fraction C enriched with molecules with incomplete (biantennary) carbohydrate chains. The mechanisms of ₁-acid glycoprotein-induced effects on peripheral blood mononuclear leukocytes are discussed.     

Synergistic effects of tumour necrosis factor-α and interferon-γ on feline haemopoietic progenitors

Pathogenic mechanisms that underlie feline leukaemia virus subgroup-C (FeLV-C) induced erythroid aplasia are unknown. FeLV-C infection is associated with higher serum levels of interferon- (IFN-) and tumour necrosis factor- (TNF-), which may act synergistically to cause haemopoietic suppression. In the present studies, the synergistic effects of TNF- and IFN- on feline bone marrow progenitors in vitro were evaluated. Bone marrow mononuclear cells from specific-pathogen-free cats were exposed to TNF- (100 and 200 pg/ml) and IFN- (100 or 200 units/ml), alone or in combination, for 2 h before plating for clonal assays of colony forming units. Our results show that TNF- and IFN- in combination caused marked suppression of feline colony forming units-erythroid (CFU-E), burst forming units-erythroid (BFU-E), and colony forming units-fibroblasts (CFU-F), whereas colony forming units-granulocyte/macrophage (CFU-GM) were minimally affected. The same concentrations of TNF- and IFN- alone had minimal effects on CFU-E, BFU-E and CFU-F. These results suggest that TNF- and IFN- may play a significant role in regulating haemopoiesis in cats and may be involved in the pathogenesis of erythroid aplasia in cats infected with feline leukaemia virus.     

Regional location of α1-antichymotrypsin and α1-antitrypsin genes on human chromosome 14

The human protease inhibitor genes ₁ antitrypsin (₁-PI) and ₁-antichymotrypsin (₁-ACT) are acute-phase proteins which are induced in response to inflammation. These inhibitors function to limit the activity of serine proteases in vivo. ₁-PI acts as an inhibitor of neutrophil elastase to protect the elastin fibers of the lung. Genetic deficiencies of ₁-PI result in development of chronic pulmonary emphysema. The physiologic role of ₁-ACT has not been clearly defined, but it also appears to function in the maintenance of protease-protease inhibitor equilibrium in the lung. Nucleic acid and protein sequence homologies detected between ₁-PI and ₁ t-ACT suggested an evolutionary relationship. Gene mapping experiments were performed to determine if these protease inhibitor genes reside at the same chromosomal locus in man. In situ hybridization data demonstrate that both ₁-PI and ₁-ACT map to the same region, q31–q32.3, on chromosome 14.     

Expression of transforming growth factor-alpha and its receptor during human liver development and maturation

We investigated the expression of transforming growth factor-alpha (TGF-) and its receptor during human liver development and maturation, using immunohistochemistry. In the fetal liver, strong immunoreactivity for TGF- and its receptor was noted in intrahepatic bile duct cells of various developmental stages; moderate immunoreactivity for TGF- and mild immunoreactivity for TGF- receptor were found in immature hepatocytes. In the postnatal liver, reactivity for TGF- in hepatocytes decreased gradually and was negative or only weakly positive in the adult liver, while reactivity for TGF- receptor in hepatocytes increased gradually and was strongly positive in the adult liver. In contrast, immunoreactivity of TGF- and its receptor in intrahepatic bile duct cells persisted in the postnatal liver and was positive in the adult liver. These data suggest that the system of TGF- and its receptor has an important role in the proliferation and differentiation of intrahepatic biliary cells and hepatocytes in the fetal liver. The decreasing expression of TGF- in hepatocytes in the postnatal liver may indicate that proliferative activity of hepatocytes gradually decreases with liver maturation. The presence of TGF- and its receptor in intrahepatic bile ducts in the postnatal liver suggests that the system of TGF- and TGF- receptor is operative postnatally.     

Synoviocytes in chronic synovitis in situ and cytokine stimulated synovial cells in vitro neo-express α1, α3 and α5 chains of β1 integrins

The expression of the 1 integrins was examined immunohistochemically in synoviocytes from normal synovial membrane and from chronic synovitis of different aetiology and intensity. Normal synoviocytes were 61-positive but lacked 1 through 5. In mild inflammation type A synoviocytes neo-expressed 1, 3, and 5 chains. In severe inflammation both type A and B synoviocytes expressed 3, 4, 5, and 6 chains. The effects of inflammatory cytokines, as single agents or in combination, on the 1 integrin expression in cultured normal synoviocytes was determined by immunocytochemistry and flow cytometry. The 1 chain, while absent in unstimulated synoviocytes, was induced by interleukin-1 (IL-1), tumour necrosis factor- (TNF-), and interferon- (INF-). This effect was enhanced by combining IL-1 and TNF-. Expression of the 3 chain was up-regulated by IL-1 and, more intensely, by IFN-. Transforming growth factor (TGF-) inhibited the up-regulating effect of IL-1 and antagonized the effect of IFN- on 3 chain expression. Expression of the 5 chain was up-regulated significantly by co-stimulation through IL-1 together with TGF- or TNF-. Thus, the 1 integrin profile of cytokine activated synoviocytes in vitro resembled that of synoviocytes in synovitis in situ. These data suggest that IL-1, TNF-, IFN-, and TGF- are likely to be among the effectors regulating 1 integrin expression in synoviocytes in vivo.     

Regulated overproduction of α-amylase by transformation of the amylolytic yeast Schwanniomyces occidentalis

Summary High frequency transformation of a Schwanniomyces occidentalis mutant defective in the last step of tryptophan synthesis was achieved with plasmids containing the tryptophan synthetase gene (TRP5) of Saccharomyces cerevisiae and an autonomous replication sequence from S. occidentalis, which we called SwARS1. The SwARS1 fragment is also functional in S. cerevisiae. The average copy number of the plasmids in both yeast species was 5–10 per cell under selective conditions. S. occidentalis cells that were transformed with an autonomously replicating plasmid carrying the cloned -amylase gene from S. occidentalis secreted about five times more -amylase than cells without additional copies of the -amylase gene. Both the chromosomal copy and the plasmid-carried copies of the -amylase gene were repressed in the presence of glucose. This transformation system provides a possibility to improve starch degradation by S. occidentalis.     

Interferon-α activates cytotoxic function but inhibits interleukin-2-mediated proliferation and tumor necrosis factor-α secretion by immature human natural killer cells

Natural killer (NK) cells play an important role in host defense mechanisms against infection and neoplasia. Interferon- (IFN-) has been shown to activate NK cells and to augment their cytotoxic activity, albeit its role in the maturation pathway of NK cells has not been elucidated. The present study examined whether IFN- activates the immature NK subset (Free cells) to become cytotoxic and also ascertained whether IFN- uses the same pathway of activation as that mediated by interleukin-2 (IL-2). Incubation of sorted Free cells overnight with IFN- resulted in augmentation of their cytotoxic function against NK sensitive target cells. The enhanced cytotoxic activity was not accompanied by a new recruitment of NK-target binder cells but by an increase in the frequency of killer cells in the conjugate fraction. Activation of the Free subset by IFN- resulted in upregulation of CD69, CD11b, and CD2 surface expression and stimulated secretion of IFN-. Unlike IL-2, IFN- did not stimulate the Free cells to proliferate or secrete TNF- and activation of cytotoxicity and modulation of surface antigens by IFN- were independent of TNF-. The failure of IFN- to stimulate secretion and proliferation by Free cells appeared to be mediated by negative signals. This was corroborated in experiments demonstrating that when Free cells were cultured with both IFN- and IL-2, a significant inhibition was observed for both the IL-2 dependent secretion of TNF- and proliferation. These results demonstrate that IFN- serves as both an activator and a regulator of NK function. Further, activation of the immature Free NK cells by IL-2 and IFN- proceeds by TNF--dependent and independent pathways, respectively. The findings also support our contention that the mechanism of activation of the cytotoxic machinery of NK cells is not linked to the mechanism of activation of cytokine secretion and/or proliferation.Abbreviations used IFN interferon - IL interleukin - PBL peripheral blood leukocytes - PE phycoerythrin - PE-GAM PE-conjugated Fab2 goat anti-mouse IgG - NK natural killer - NRS normal rabbit serum - TNF tumor necrosis factor - FCS fetal calf serum - FITC fluorescein isothiocyanate - PBS phosphate-buffered saline - MACS magnetic cell sorting - ELISA enzyme-linked immunosorbent assay - BSA bovine serum albumin - PKC protein kinase C - mAb monoclonal antibody - PBMC peripheral blood mononuclear cells - BCLL B-chronic lymphocytic leukemia - E effector - T target     

Epidermal growth factor,transforming growth factor-α, and epidermal growth factor receptor content in normal and carcinomatous gastric and colonic tissue

Summary Epidermal growth factor (EGF) and transforming growth factor- (TGF-) are polypeptides which bind to the EGF receptor (EGFr) and may play a role in cell growth and carcinogenesis. Our study investigated the content of EGF, TGF-, and EGFr in tumors of the stomach and the colon in comparison with the sourrounding mucosa. EGF was detected in half of the stomach specimens with concentrations between 1 and 9 ng/g weight irrespective of histology. In the colon no EGF was found in the tumor or normal mucosa. In the stomach normal mucosa contained higher TGF- concentrations (mean 22.4 ng/g) than the tumors (mean 11.8 ng/g), but the difference was not statistically significant because of a wide variation in mucosal values. By contrast, the colon mucosa displayed significantly higher TGF- concentrations than the tumor tissues (33 ng/g versus 12 ng/g; P < 0.01). EGFr content in the gastric mucosa was lower compared to gastric carcinoma (48 fmol/g versus 75 fmol/g) yet not significantly different. In contrast, colorectal tumor specimens disclosed significantly higher concentrations than the mucosal tissues (mean of 155 fmol/g versus 80 fmol/g; P < 0.01). In conclusion, TGF- should not be considered a tumorigenic but a physiological growth factor in the stomach and colon. An elevated EGFr content in colorectal tumors in comparison with the normal mucosa could lead to a growth advantage by an autostimulating mechanism.Abbreviations EGF epidermal growth factor - EGFr epidermal growth factor receptor - TGF- transforming growth factor - ROC receiver operating characteristic Dedicated to Prof. Dr. G. Paumgartner on the occasion of his 60th birthday     

Combined effect of hydrogen peroxide induced oxidative stress and IL-1 alpha on IL-8 production in CaCo-2 cells (a human colon carcinoma cell line) and normal intestinal epithelial cells

Oxidative stress, IL-1, and IL-8 are known to contribute to mucosal inflammation of the gastrointestinal tract. We examined the IL-8 response after brief exposure to hydrogen peroxide induced oxidative stress in CaCo-2 cells (a human colon carcinoma cell line) and in human intestinal epithelial cells. In addition, we examined whether exposure to oxidative stress, followed by IL-1, could modulate IL-8 production. A transient up-regulation of IL-8 mRNA expression was observed after hydrogen peroxide treatment. Hydrogen peroxide induced oxidative stress was also observed to promote IL-8 secretion. Exposure to hydrogen peroxide, followed by IL-1, enhanced IL-8 production over that achieved with IL-1 alone. Thus, oxidative stress and IL-1 were observed to cooperatively enhance IL-8 production.     

Molecular cloning and characterization of a Candida tsukubaensis α-glucosidase gene in the yeast Saccharomyces cerevisiae

Summary The molecular cloning of an -glucosidase gene isolated from a Candida tsukubaensis (CBS 6389) genomic library in Saccharomyces cerevisiae is reported. The cloned gene is contained within a 6.2 kb Sau3A DNA fragment and directs the synthesis and secretion of an amylolytic enzyme into the extracellular medium of the recombinant host, S. cerevisiae. The cloned enzyme was found to have an unusually broad substrate specificity and is capable of hydrolysing -1,2, -1,3, -1,4 and 1,6 linked, as well as aryl and alkyl, d-glucosides. On the basis of its substrate specificity profile, the cloned enzyme was classified as an -glucosidase (E.C. 3.2.1.20). It has a pH optimum in the range 4.2–4.6, a temperature optimum of 58°C and is readily inactivated at pasteurization temperature (60°C). Southern blot analysis failed to reveal any homology between the cloned gene and genomic DNA isolated from other well characterized amylolytic yeasts. A rapid plate-assay, based on the utilization of a chromogenic substrate X--d-glucoside to detect the expression of the cloned -glucosidase in S. cerevisiae transformants, was developed.     

A murine model of NKT cell-mediated liver injury induced by alpha-galactosylceramide/d-galactosamine

Natural killer-T (NKT) cells are rich in the liver. However, their involvement in liver injury is not fully understood. We developed here a new murine model of NKT-cell-activation-associated liver injury, and investigated a role of tumor necrosis factor alpha (TNF-) and Fas in pathogenesis. We injected intraperitoneally alpha-galactosylceramide (-GalCer), an NKT-cell stimulant, into d-galactosamine (GalN)-sensitized mice. Survival rate, pathological changes of the liver, and plasma concentrations of cytokines were studied. Alpha-GalCer/GalN administration gave a lethal effect within 7 h, making pathological changes such as massive parenchymal hemorrhage, hepatocyte apoptosis, sinusoidal endothelial cell injury, and close apposition of lymphocytes to apoptotic hepatocytes. Anti-NK1.1 mAb-pretreated mice and V14NKT knock out (KO) mice did not develop liver injury. Tumor necrosis factor-alpha (TNF-) and interferon-gamma (IFN-) were elevated at 4 h in the plasma. These cytokines were produced by hepatic lymphocytes as demonstrated by in vitro stimulation with -GalCer. The lethal effect was suppressed in TNF- KO mice, TNF receptor-1 KO mice, and lpr/lpr (Fas deficient) mice, whereas it was not in IFN- KO mice. These results indicate that the present liver injury is characterized by parenchymal hemorrhage and hepatocyte apoptosis, and mediated by TNF- secretion and direct cytotoxicity of -GalCer-activated NKT cells.