Nuclear imaging to support anti-inflammatory drug discovery and development.

Nuclear medicine contributes important tools to support anti-inflammatory drug discovery and development. The support provided is manifold: new molecular entities (NME, either small molecules or biologics) labeled with radioisotopes can be applied in animal models and humans to measure biodistribution, target engagement, and pharmacokinetics. In addition, nuclear imaging techniques can be used to select or enrich the patient populations in clinical trials, to assess disease activity, target status and distribution and to quantify response to therapeutic interventions. In the first part of this review we will outline how nuclear imaging techniques can be applied to support informed decision making in drug development. In the second part, we will briefly high-light the use of nuclear imaging of inflammation in drug development in selected diseases, specifically rheumatoid arthritis (RA), inflammatory bowel diseases (IBD), atherosclerosis and - as an emerging topic - cancer.

Nuclear imaging to support anti-inflammatory drug discovery and development.

Nuclear medicine contributes important tools to support antiinflammatory drug discovery and development in many ways. The support provided is manifold: new molecular entities (NME, either small molecules or biologics) labeled with radioisotopes can be applied in animal models and humans to measure biodistribution, target engagement, and pharmacokinetics. In addition, nuclear imaging techniques can be used to select or enrich the patient populations in clinical trials, to assess disease activity, target status and distribution and to quantify response to therapeutic interventions. In the first part of this review we will outline how nuclear imaging techniques can be applied to support informed decision making in drug development. In the second part, we will briefly highlight the use of nuclear imaging of inflammation in drug development in selected diseases, specifically rheumatoid arthritis (RA), inflammatory bowel diseases (IBD), atherosclerosis (ATS) and as an emerging topic cancer.

Nitric oxide-induced F-actin disassembly is mediated via cGMP, cAMP, and protein kinase A activation in rat mesangial cells.

Glomerular mesangial cells contain actin and myosin, and in analogy to vascular smooth muscle cells, they can contract and relax to regulate the glomerular filtration rate. A key molecule that determines hemodynamic properties is nitric oxide, which is produced by nitric oxide synthase isoenzymes located in individual cells of the kidney. The contractility of mesangial cells is based on the interaction of actin microfilament bundles (F-actin) with myosin. We had the notion that nitric oxide influences the shape change of mesangial cells, so we analyzed the signal transduction involved. Chemically unrelated nitric oxide donors induced F-actin dissolution, which was mediated by cGMP but was unrelated to protein kinase G activation. Actin disassembly was achieved with inhibitors of phosphodiesterase-3 and -4 or forskolin-evoked cAMP generation. We assumed that signal transmission involves activation of protein kinase A, and we went on to attenuate F-actin disassembly by protein kinase A inhibition. In conclusion, we found evidence that nitric oxide triggered F-actin dissolution via cGMP generation, inhibition of cAMP-hydrolyzing phosphodiesterase-3, and subsequent protein kinase A activation.

Depletion of hepatic glutathione prevents death receptor-dependent apoptotic and necrotic liver injury in mice.

The activation of the death receptors, tumor necrosis factor-receptor-1 (TNF-R1) or CD95, is a hallmark of inflammatory or viral liver disease. In different murine in vivo models, we found that livers depleted of gamma-glutamyl-cysteinyl-glycine (GSH) by endogenous enzymatic conjugation after phorone treatment were resistant against death receptor-elicited injury as assessed by transaminase release and histopathology. In apoptotic models initiated by engagement of CD95, or by injection of TNF or lipopolysaccharide into galactosamine-sensitized mice, hepatic caspase-3-like proteases were not activated in the GSH-depleted state. Under GSH depletion, also caspase-independent, TNF-R1-mediated injury (high-dose actinomycin D or alpha-amanitin), as well as necrotic hepatotoxicity (high-dose lipopolysaccharide) were entirely blocked. In the T-cell-dependent model of concanavalin A-induced hepatotoxicity, GSH depletion resulted in a suppression of interferon-gamma release, delay of systemic TNF release, hepatic nuclear factor-kappaB activation, and an abrogation of sinusoidal endothelial cell detachment as assessed by electron microscopy. When GSH depletion was initiated 3 hours after concanavalin A injection, ie, after the peak of early pro-inflammatory cytokines, livers were still protected. We conclude that sufficient hepatic GSH levels are a prerequisite for the execution of death receptor-mediated hepatocyte demise.

Phagocytosis of nonapoptotic cells dying by caspase-independent mechanisms.

Caspase activation, exposure of phosphatidylserine (PS) on the outer surface of the plasma membrane, and rapid phagocytic removal of dying cells are key features of apoptosis. Nonapoptotic/necrotic modes of death occur independent of caspase activation, but the role of phagocytosis is largely unknown. To address this issue, we studied phagocytosis by human monocyte-derived macrophages (HMDM) and rat microglial cells. Target cells (Jurkat) were stimulated by several different methods that all caused caspase-independent death. First, we induced necrosis by combining toxins with ATP-depleting agents. Under these conditions, neither PS was exposed nor were such cells phagocytosed before their death. However, once the plasma membrane integrity was lost, the dead cells were rapidly and efficiently engulfed by HMDM. Next, we triggered Jurkat cell death with staurosporine in the presence of the pan-caspase inhibitor zVAD-fmk. Under these conditions, death occurred by delayed necrosis and without exposure of PS. Nevertheless, such lethally challenged cells were phagocytosed before the loss of membrane integrity. Finally, we triggered Ca2+ influx in Jurkat cells with an ionophore, or in neurons by glutamate receptor stimulation, respectively. In both models, PS was exposed on the cell surface. Ca2+-stressed cells were phagocytosed starting at 30 min after stimulation. Protein kinase C inhibitors prevented Ca2+-mediated PS exposure and phagocytosis. Essentially, similar phagocytosis data were obtained for all models with HMDM and microglia. We conclude that also cells dying nonapoptotically and independent of caspase activation may be recognized and removed before, or very quickly after, membrane lysis.

Human monocytes express functional receptors for granulocyte colony-stimulating factor that mediate suppression of monokines and interferon-gamma.

In a double-blind, placebo-controlled, randomized study, 10 healthy men received either a single dose of 480 microg granulocyte colony-stimulating factor (G-CSF) or saline. Blood taken from the volunteers was stimulated with 10 microg/mL endotoxin and released cytokines were measured by enzyme-linked immunosorbent assay. Expression of G-CSF receptors on leukocytes was examined by flow cytometry and reverse transcriptase-polymerase chain reaction. Functional activity of these receptors was tested by challenging isolated leukocyte populations to release cytokines with endotoxin in the presence of G-CSF. The G-CSF treatment attenuated the release of the proinflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-12, IL-1beta, and interferon (IFN)-gamma in ex vivo lipopolysaccharide (LPS)-stimulated whole blood. In blood from untreated volunteers the presence of G-CSF in vitro also attenuated the LPS-stimulated release of these cytokines. G-CSF in vitro also attenuated TNF-alpha release from elutriation-purified monocytes. In the presence of 10 ng/mL recombinant TNF-alpha, the attenuation of LPS-inducible IFN-gamma release by G-CSF was blunted in whole blood. However, G-CSF had no such effect on IFN-gamma release from isolated lymphocytes stimulated with anti-CD3 or a combination of TNF-alpha and IL-12. G-CSF receptor expression was detected in human neutrophils and monocytes but not in lymphocytes by means of RT-PCR as well as flow cytometry. These results indicate that G-CSF receptors expressed on monocytes are functional in modulating monokine release. We conclude that the attenuation of IFN-gamma release from lymphocytes is not a direct effect of G-CSF on these cells but is rather due to the inhibition of monocytic IL-12 and TNF-alpha release by G-CSF. (Blood. 2000;95:270-276)

Characterization of the phosphodiesterase (PDE) pattern of in vitro-generated human dendritic cells (DC) and the influence of PDE inhibitors on DC function.

During differentiation of human monocytes (CD14(+)/CD1a(-)) to CD14(-)/CD1a(+)dendritic cells (DC), a drastic decrease in PDE4 activity was observed, while activities of PDE1 and PDE3 substantially increased. DC released tumour necrosis factor-alpha (TNF) in response to lipopolysaccharide (LPS) challenge, which was abolished both by dexamethasone and the cyclic AMP-elevating drugs db-cAMP and PGE(2). In addition, rolipram, at PDE4-selective concentrations, blocked TNF release by 37 +/- 5% (P<0.05 vs. control). The PDE3 inhibitor motapizone only marginally influenced TNF synthesis, but a synergistic inhibitory effect was noted in combination with rolipram. Qualitatively, similar inhibitory effects were observed in DC-stimulated T cell responses. Motapizone, lacking efficacy when used alone, increased the effect of rolipram in blocking CD4(+)T lymphocyte proliferation in response to antigen (Ag) (tetanus toxoid, TT; keyhole limpet hemocyanin, KLH) presented by DC and in allogeneic mixed leukocyte reactions (MLR). However, in these coculture systems the T cells rather than the DC seem to be the major target cells of PDE-inhibitor action. In summary, PDE inhibitors can affect DC function directly as demonstrated by blocking TNF release and their efficacy reflects the changes in the PDE activity profile during differentiation from their monocyte precursors. These results together with the known efficacy of PDE3/4 inhibitors in T cells support the concept of combined PDE3/4 inhibitors for asthma therapy.

Effect of filgrastim treatment on inflammatory cytokines and lymphocyte functions.

Twenty-four healthy male volunteers received either placebo or 75, 150, or 300 microg filgrastim (recombinant methionyl human granulocyte colony-stimulating factor) for 12 days to study effects on monocytes and lymphocytes. In all filgrastim-treated groups, tumor necrosis factor alpha (TNF-alpha), interleukin-12 (IL-12), and interferon gamma (IFN-gamma) release by whole blood in response to endotoxin (lipopolysaccharide) was reduced. IL-12 added in vitro to lipopolysaccharide-stimulated blood of filgrastim-treated donors restored IFN-gamma and TNF-alpha release, suggesting that the anti-inflammatory effect of granulocyte colony-stimulating factor is exercised through IL-12 suppression. Phytohemagglutinin- or anti-CD3 antibody-induced lymphocyte proliferation ex vivo was reduced by 60% from day 5 to day 15, after a 50% increase at day 2 with concomitant doubled IL-2 release. In vivo, filgrastim induced doubling of all T-cell populations by day 8. Filgrastim decreased proinflammatory cytokine production and lymphocyte proliferation ex vivo throughout prolonged treatment at all doses. This indicates that endogenous granulocyte colony-stimulating factor may counterregulate the inflammatory cytokine cascade and implies a potential indication for filgrastim in chronic inflammatory conditions.

Phosphodiesterase profile of human B lymphocytes from normal and atopic donors and the effects of PDE inhibition on B cell proliferation.

1. CD19+ B lymphocytes were purified from the peripheral blood of normal and atopic subjects to analyse and compare the phosphodiesterase (PDE) activity profile, PDE mRNA expression and the importance of PDE activity for the regulation of B cell function. 2. The majority of cyclic AMP hydrolyzing activity of human B cells was cytosolic PDE4, followed by cytosolic PDE7-like activity; marginal PDE3 activity was found only in the particulate B cell fraction. PDE1, PDE2 and PDE5 activities were not detected. 3. By cDNA-PCR analysis mRNA of the PDE4 subtypes A, B (splice variant PDE4B2) and D were detected. In addition, a weak signal for PDE3A was found. 4. No differences in PDE activities or mRNA expression of PDE subtypes were found in B cells from either normal or atopic subjects. 5. Stimulation of B lymphocytes with the polyclonal stimulus lipopolysaccharide (LPS) induced a proliferative response in a time- and concentration-dependent manner, which was increased in the presence of interleukin-4 (IL-4). PDE4 inhibitors (rolipram, piclamilast) led to an increase in the cellular cyclic AMP concentration and to an augmentation of proliferation, whereas a PDE3 inhibitor (motapizone) was ineffective, which is in accordance with the PDE profile found. The proliferation enhancing effect of the PDE4 inhibitors was partly mimicked by the cyclic AMP analogues dibutyryl (db) cyclic AMP and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3',5'-cyclic monophosphorothioate, Sp-isomer (dcl-cBIMPS), respectively. However, at concentrations exceeding 100 microM db-cyclic AMP suppressed B lymphocyte proliferation, probably as a result of cytotoxicity. Prostaglandin E2 (PGE2, 1 microM) and forskolin (10 microM) did not affect B cell proliferation, even when given in combination with rolipram. 6. Inhibition of protein kinase A (PKA) by differentially acting selective inhibitors (KT 5720, Rp-8-Br-cyclic AMPS) decreased the proliferative response of control cells and reversed the proliferation enhancing effects of rolipram. 7. Importantly, PDE4 activity in LPS/IL-4-activated B lymphocytes decreased by about 50% compared to unstimulated control values. 8. We conclude that an increase in cyclic AMP, mediated by down-regulation of PDE4 activity, is involved in the stimulation of B cell proliferation in response to LPS/IL-4. B cell proliferation in response to a mitogenic stimulus can be further enhanced by pharmacological elevation of cyclic AMP.

Phosphodiesterase profiles of highly purified human peripheral blood leukocyte populations from normal and atopic individuals: a comparative study.

BACKGROUND: Several previous reports have suggested an increased activity of cAMP phosphodiesterases (PDEs) and a higher sensitivity of these enzymes toward PDE inhibitors in leukocytes of patients suffering from atopic dermatitis. OBJECTIVE: The purpose of the present study was to comprehensively analyze and compare the PDE expression and activity profile of highly purified populations of leukocytes from normal and atopic blood donors. In addition, the influence of PDE inhibitors on function of leukocytes from normal and atopic individuals was investigated. METHODS: Density gradient centrifugation, elutriation, and magnetic cell sorting techniques were used to purify eosinophils, monocytes, and B and T lymphocytes from peripheral human blood. Complementary DNA-polymerase chain reaction was used to analyze PDE4 subtype messenger RNA (mRNA) expression levels in addition to PDE isoenzyme activities. PDE4 inhibitor sensitivity was determined in monocyte homogenates from both groups. Functionally, suppression of lipopolysaccharide-induced synthesis of tumor necrosis factor-alpha in monocytes as well as phytohemagglutinin-induced T cell proliferation in peripheral blood mononuclear cell fractions by PDE4 and PDE3/4 inhibitors was compared. RESULTS: Identical PDE activities and mRNA expression profiles were found in all cells from normal and atopic donors except that there was an increase in the mRNA levels of PDE4A and PDE4B2 in atopic T cells, which was, however, not reflected in overall PDE4A activity. In addition, no differences in sensitivity of the functional responses to PDE inhibitors were noted. The mixed PDE3/4 inhibitor zardaverine was a more potent inhibitor of T cell proliferation than rolipram, a selective PDE4 inhibitor. CONCLUSION: No evidence for alterations of PDE activities in atopy is provided by our findings.

In vitro differentiation of human monocytes to macrophages: change of PDE profile and its relationship to suppression of tumour necrosis factor-alpha release by PDE inhibitors.

1. During in vitro culture in 10% human AB serum, human peripheral blood monocytes acquire a macrophage-like phenotype. The underlying differentiation was characterized by increased activities of the macrophage marker enzymes unspecific esterase (NaF-insensitive form) and acid phosphatase, as well as by a down-regulation in surface CD14 expression. 2. In parallel, a dramatic change in the phosphodiesterase (PDE) profile became evident within a few days that strongly resembled that previously described for human alveolar macrophages. Whereas PDE1 and PDE3 activities were augmented, PDE4 activity, which represented the major cyclic AMP-hydrolysing activity of peripheral blood monocytes, rapidly declined. 3. Monocytes and monocyte-derived macrophages responded to lipopolysaccharide (LPS) with the release of tumour necrosis factor-alpha (TNF). In line with the change in CD14 expression, the EC50 value of LPS for induction of TNF release increased from approximately 0.1 ng ml-1 in peripheral blood monocytes to about 2 ng ml-1 in macrophages. 4. Both populations of cells were equally susceptible towards inhibition of TNF release by cyclic AMP elevating agents such as dibutyryl cyclic AMP, prostaglandin E2 (PGE2) or forskolin, which all led to a complete abrogation of TNF production in a concentration-dependent manner and which were more efficient than the glucocorticoid dexamethasone. 5. In monocytes, PDE4 selective inhibitors (rolipram, RP73401) suppressed TNF formation by 80%, whereas motapizone, a PDE3 selective compound, exerted a comparatively weak effect (10-15% inhibition). Combined use of PDE3 plus PDE4 inhibitors resulted in an additive effect and fully abrogated LPS-induced TNF release as did the mixed PDE3/4 inhibitor tolafentrine. 6. In monocyte-derived macrophages, neither PDE3- nor PDE4-selective drugs markedly affected TNF generation when used alone (< 15% inhibition), whereas in combination, they led to a maximal inhibition of TNF formation by about 40-50%. However, in the presence of PGE2 (10 nM), motapizone and rolipram or RP73401 were equally effective and blocked TNF release by 40%. Tolafentrine or motapizone in the presence of either PDE4 inhibitor, completely abrogated TNF formation in the presence of PGE2. Thus, an additional cyclic AMP trigger is necessary for PDE inhibitors to become effective in macrophages. 7. Finally, the putative regulatory role for PDE1 in the regulation of TNF production in macrophages was investigated. Zaprinast, at a concentration showing 80% inhibition of PDE1 activity (100 micromol l-1), did not influence TNF release. At higher concentrations (1 mmol l-1), zaprinast became effective, but this inhibition of TNF release can be attributed to a significant inhibitory action of this drug on PDE3 and PDE4 isoenzymes. 8. In summary, the in vitro differentiation of human peripheral blood monocytes to macrophages is characterized by a profound change in the PDE isoenzyme pattern. The change in the PDE4 to PDE3 ratio is functionally reflected by an altered susceptibility towards selective PDE inhibitors under appropriate stimulating conditions.

Tumor necrosis factor-induced apoptosis during the poisoning of mice with hepatotoxins.

BACKGROUND & AIMS: Treatment with tumor necrosis factor (TNF) induces murine hepatocyte apoptosis in vitro and in vivo when sensitizing concentrations of toxins are present. The aim of this study was to investigate whether endogenously formed TNF contributes to liver failure caused by hepatotoxins. METHODS: The extent of liver damage, induced by alpha-amanitin or actinomycin D (ActD), was examined under various experimental conditions, preventing the action of TNF on hepatocytes. RESULTS: TNF induced apoptosis of murine hepatocytes or human hepatoma cells in the presence of alpha-amanitin or ActD. TNF and alpha-amanitin induced such hepatotoxicity also in vivo in a synergistic way. After in vivo administration of high doses of ActD or alpha-amanitin alone, hepatic TNF-messenger RNA was increased and hepatocytes underwent apoptosis. A neutralizing antiserum against TNF-alpha prevented the liver injury. Hepatotoxicity of ActD or alpha-amanitin also was prevented by pretreatment of mice with low doses of the tolerizing cytokine interleukin 1. Mice deficient for the 55-kilodalton TNF receptor were protected from ActD- or alpha-amanitin-induced toxicity. Endotoxin-unresponsive C3H/HeJ mice also had liver failure after ActD treatment, and this damage was prevented by treatment with anti-TNF antiserum. CONCLUSIONS: Hepatotoxins such as alpha-amanitin may induce liver failure by an indirect mechanism involving sensitization of parenchymal cells toward endogenously produced TNF.

Protection from T cell-mediated murine liver failure by phosphodiesterase inhibitors.

Injection of the T cell mitogens concanavalin A (Con A) into nonsensitized or of staphylococcal enterotoxin B (SEB) into D-galactosamine (GalN)-sensitized mice is known to cause fulminant liver failure via a cytokine response syndrome with tumor necrosis factor-alpha (TNF) as the plvotal mediator. We examined in vivo whether the phosphodiesterase (PDE) inhibitors motapizone (PDE3-selective) and rolipram (PDE4-selective) affected cytokine release and hepatic injury after T cell activation. Both motapizone as well as rolipram dose-dependently (0.1-10 mg/kg) attenuated the systemic release of TNF and interferon-gamma as initiated by injection of Con A (25 mg/kg) or SEB (2 mg/kg). Although interleukin-4 production was not affected by motapizone or decreased by rolipram, circulating levels of interleukin-10, however, were significantly increased in PDE inhibitor-treated mice compared with controls. Associated with the suppression of the central mediator TNF, motapizone and rolipram protected mice from liver injury in the Con A as well as in the SEB model. Moreover, the combined administration of motapizone plus rolipram at doses which were ineffective when given alone completely protected mice from GalN/SEB toxicity. These data demonstrate that PDE inhibitors effectively attenuate an inflammatory T cell response in vivo and strongly suggest a therapeutic potential as anti-inflammatory drugs in T cell-related disorders. We conclude that cAMP-elevating drugs shift the balance of T cell-derived cytokines from a proinflammatory to an enhanced anti-inflammatory factor release, thus protecting mice from TNF-mediated hepatic failure.

T cell stimulus-induced crosstalk between lymphocytes and liver macrophages results in augmented cytokine release.

Polyclonal T cell stimulation in humans leads to a cytokine burst syndrome that may result in organ failure or lethality. Mechanisms of such cytokine-dependent morbidity can be studied in mice challenged with the T cell mitogen concanavalin A (Con A). In this model tumor necrosis factor (TNF)-dependent toxicity is characterized by a relatively selective liver failure. We examined here whether a crosstalk between liver macrophages and lymphocytes may be the underlying cause for the overshooting TNF response. Lymphocytes from lymph nodes, thymus, or the spleen were cocultured with Kupffer cells and stimulated with the polyclonal T cell stimuli Con A, anti-CD3 mAb, or staphylococcal enterotoxin B. We observed a rapid and synergistically augmented release of TNF, and also of IL-1, IL-2, IL-4, IL-6, and IFN-gamma, compared to stimulation of the individual cell types alone. This dramatically upregulated cytokine response did not require direct cell contact, but was mediated by a soluble factor. In order to find out whether TNF upregulation would require additional cell types in the liver, we used cocultures of T cells and a macrophage cell line and confirmed our previous results. In this model system an increase in TNF mRNA was observed in macrophages, but not in T cells. We conclude that the T cell-macrophage crosstalk following polyclonal T cell stimulation may be responsible for an overshooting TNF release from macrophages. This mechanism finally may lead to organ damage such as liver injury upon Con A injection into mice.

Role of sinusoidal endothelial cells of the liver in concanavalin A-induced hepatic injury in mice.

CD4+ T lymphocytes have been identified as being responsible for organ damage in the murine model of experimental liver injury induced by intravenous injection of concanavalin A (Con A). Liver sinusoidal endothelial cells (SEC) and Kupffer's cells (KC) are among the first cells that come into contact with lymphocytes in the liver sinusoid. We aimed to investigate the respective role of these cell populations in the initial steps of T-cell-mediated liver injury in Con A-induced hepatitis. By electron microscopy, we could show that intravenously applied Con A bound predominantly to SEC but not to KC. KC depletion by gadolinium chloride treatment of mice did not result in protection from liver injury, indicating that KCs are not primarily involved in the generation of liver injury. We could show that a CD4+ T-cell line (LNC.2) displayed selective cytotoxicity toward SEC (>50%) but not KC (12%) or fibroblasts (5%) in the presence of Con A in vitro. Microscopic observation revealed that the SEC monolayer was rapidly destroyed by LnC2 in the presence of Con A. Specificity of the Con A-induced cytotoxicity was shown by the ability of a competitive ligand, methyl-alpha-D-mannopyranoside, to reduce T-cell-mediated cytotoxicity to SEC by more than 50%. Tumor necrosis factor alpha (TNF-alpha) was produced by LnC2 in high amounts after Con A stimulation (>6 ng/mL), but antiserum to TNF-alpha did not reduce LnC2-mediated cytotoxicity toward SEC. In conclusion, we could show for the first time that liver SECs have accessory function and are selectively destroyed by CD4+ T lymphocytes in the presence of Con A. We speculate that SEC damage is an early event in T-cell-mediated liver injury recruiting T lymphocytes from the sinusoidal circulation. Loss of the SEC barrier function then exposes underlying hepatocytes to further attack by activated T lymphocytes. These results offer a model of initiating events in T-cell-mediated liver diseases, such as viral or autoimmune hepatitis, and suggest an important role for sinusoidal endothelial cells.

DNA fragmentation in mouse organs during endotoxic shock.

The systemic inflammatory response syndrome has still an unpredictable outcome, and patients often die of multiple organ failure despite circulatory stabilization therapy. The still incompletely understood pathophysiological mechanisms include organ damage due to direct toxic actions of cytokines elicited by overactivation of the host response. To study this process of organ failure in experimental septic shock, we injected mice with a lethal dose of endotoxin and examined apoptotic and necrotic tissue damage biochemically, histologically, and ultrastructurally. Endotoxin administration caused oligonucleosomal as well as random DNA fragmentation in liver, lung, kidney, and intestine. In the liver, DNA fragmentation was not restricted to hepatocytes but also occurred in nonparenchymal cells. The DNA fragmentation was mediated by tumor necrosis factor and attenuated by endogenous nitric oxide release. Unlike the situation in D-galactosamine-sensitized mice, in which injection or release of tumor necrosis factor causes massive hepatocyte apoptosis, liver failure due to high doses of endotoxin was characterized by single-cell necrosis, a low incidence of apoptosis, and simultaneous damage to nonparenchymal cells. We conclude that, even though endotoxin causes cytokine-mediated DNA fragmentation in several organs including the liver, hepatocyte apoptosis itself seems to be a minor phenomenon in high-dose endotoxic shock in mice.

Interferon gamma plays a critical role in T cell-dependent liver injury in mice initiated by concanavalin A.

BACKGROUND & AIMS: T cell-dependent liver injury involving endogenous tumor necrosis factor (TNF) alpha can be induced by either concanavalin A in naive mice or by activating anti-CD3 antibody or staphylococcal enterotoxin B in D-galactosamine-sensitized mice. In this study, the role of interferon gamma (IFN-gamma) in these T-cell models was addressed. METHODS: Mice were pretreated with a neutralizing anti-mouse IFN-gamma antiserum before injection of T cell-activating agents. Plasma cytokine and transaminase levels were determined. Apoptotic cell death was assessed by hepatic DNA fragmentation. RESULTS: Anti-IFN-gamma antiserum significantly protected mice from concanavalin A-induced liver injury. Circulating IFN-gamma was completely suppressed, and TNF was reduced by 50%. Recombinant TNF-alpha administered to mice treated with concanavalin A and anti-IFN-gamma antiserum failed to initiate liver injury. Similar results were obtained with recombinant IFN-gamma in concanavalin A-challenged mice under the condition of TNF neutralization. Neither hepatic DNA fragmentation nor release of transaminases was inhibited by anti-IFN-gamma antiserum when liver injury was induced by staphylococcal enterotoxin B or anti-CD3 antibody in D-galactosamine-sensitized mice. CONCLUSIONS: Both TNF as well as IFN-gamma are critical mediators of liver injury in concanavalin A-treated mice, whereas hepatic DNA fragmentation and liver failure in the D-galactosamine models depend only on TNF.

Immunotoxicology of T cell-dependent experimental liver injury.

Microbial toxins as well as certain drugs and xenobiotics exert their toxic potential towards mammalian organisms by either activation or suppression of the immune system. We have investigated the immune stimulatory effect of either the bacterial superantigen staphylococcal enterotoxin B (SEB) or of the T cell activating anti-CD3 monoclonal antibody (alpha CD3-mAb) or of the T cell mitogenic plant lectin concanavalin A (Con A) in murine in vivo and in vitro systems. Any of these agents evoked a strong cytokine response in vitro and in vivo. Tumor necrosis factor-alpha (TNF) was identified as a common cytotoxic mediator which induced hepatocyte apoptosis as characterized by histological examination and internucleosomal DNA fragmentation, that preceded the release of liver specific enzymes into plasma and the histological appearance of necrosis. These mechanisms of acute liver failure observed under experimental conditions are discussed to account also for liver injury during acute episodes of autoimmune or viral hepatitis or rejection of liver grafts.