Free radicals, chemokines, and cell injury in HIV-1 and SIV infections and alcoholic hepatitis

Oxidative stress has been observed in HIV-1 infection and alcoholic liver disease. The formation of reactive oxygen species (ROS) contributes to cell injury through apoptosis and/or necrosis and secretion of proinflammatory cytokines and chemokines. The major sources of ROS and chemokines are the Kupffer cells. During chronic ethanol consumption they are primed and activated for enhanced formation of pro-inflammatory factors, probably as a result of ethanol-induced translocation of gut-derived endotoxin into the circulation. Pro-inflammatory factors produced in the liver stimulate neutrophilic and/or lymphocytic infiltration to this organ. The presence of inflammatory cells in the liver may compromise the hepatocytes to injury by releasing cytotoxic factors, i.e., ROS, cytolytic proteases. Kupffer cells also interact with the glycoprotein 120 of SIV and HIV-1, which can induce oxidative stress and chemokine release. CD4+ lymphocytes that are infected with the virus generate intracellular ROS, which in turn leads to apoptosis and cell death. Downregulation of CD4+ cells contributes to immunodeficiency, while enhanced sequestration of inflammatory cells in the liver during chronic ethanol use with or without HIV-1/SIV may result in hepatocyte injury and exacerbation of alcoholic liver disease.     

Differential effects of apoptotic versus lysed cells on macrophage production of cytokines: role of proteases

Granulocytes undergoing apoptosis are recognized and removed by phagocytes before their lysis. The release of their formidable arsenal of proteases and other toxic intracellular contents into tissues can create significant damage, prolonging the inflammatory response. Binding and/or uptake of apoptotic cells by macrophages inhibits release of proinflammatory cytokines by mechanisms that involve anti-inflammatory mediators, including TGF-beta. To model the direct effects of necrotic cells on macrophage cytokine production, we added lysed or apoptotic neutrophils and lymphocytes to mouse and human macrophages in the absence of serum to avoid complement activation. The results confirmed the ability of lysed neutrophils, but not lymphocytes, to significantly stimulate production of macrophage-inflammatory protein 2 or IL-8, TNF-alpha, and IL-10. Concomitantly, induction of TGF-beta1 by lysed neutrophils was significantly lower than that observed for apoptotic cells. The addition of selected serine protease inhibitors and anti-human elastase Ab markedly reduced the proinflammatory effects, the lysed neutrophils then behaving as an anti-inflammatory stimulus similar to intact apoptotic cells. Separation of lysed neutrophils into membrane and soluble fractions showed that the neutrophil membranes behaved like apoptotic cells. Thus, the cytokine response seen when macrophages were exposed to lysed neutrophils was largely due to liberated proteases. Therefore, we suggest that anti-inflammatory signals can be given by PtdSer-containing cell membranes, whether from early apoptotic, late apoptotic, or lysed cells, but can be overcome by proteases liberated during lysis. Therefore, the outcome of an inflammatory reaction and the potential immunogenicity of Ags within the damaged cell will be determined by which signals predominate.     

Reactive oxygen and ischemia/reperfusion injury of the liver

Pharmacological experiments suggested that reactive oxygen species contribute to ischemia-reperfusion injury of the liver. Since there is no evidence that quantitatively sufficient amounts of reactive oxygen are generated intracellularly to overwhelm the strong antioxidant defense mechanisms in the liver and cause parenchymal cell injury, the role of reactive oxygen in the pathogenesis remains controversial. This paper reviews the data and conclusions obtained with pharmacological intervention studies in vivo, the sources of reactive oxygen in the liver as well as the growing evidence for the importance of liver macrophages (Kupffer cells) and infiltrating neutrophils in the pathogenesis. A comprehensive hypothesis is presented that focuses on the extracellular generation of reactive oxygen in the hepatic sinusoids, where Kupffer cell-derived reactive oxygen species seem to be involved in the initial vascular and parenchymal cell injury and indirectly also in the recruitment of neutrophils into the liver. Reactive oxygen species may also contribute to the subsequent neutrophil-dependent injury phase as one of the toxic mediators released by these inflammatory cells.     

Mechanisms of cadmium-mediated acute hepatotoxicity

The mechanism of cadmium-mediated acute hepatotoxicity has been the subject of numerous investigations and although some uncertainties persist, sufficient evidence has emerged to provide a reasonable account of the toxic process. Acute hepatotoxicity involves two pathways, one for the initial injury produced by direct effects of cadmium and the other for the subsequent injury produced by inflammation. Primary injury appears to be caused by the binding of Cd2+ to sulfhydryl groups on critical molecules in mitochondria. Thiol group inactivation causes oxidative stress, the mitochondrial permeability transition, and mitochondrial dysfunction. Although cadmium may injure hepatocytes directly, there are compelling reasons to believe that hepatocellular injury is produced in vivo as the result of ischemia caused by damage to endothelial cells. Secondary injury from acute cadmium exposure is thought to occur from the activation of Kupffer cells and a cascade of events involving several types of liver cells and a large number of inflammatory and cytotoxic mediators. In this regard, it is clear that Kupffer cell activation and neutrophil infiltration are important events in the toxic process, and the involvement of proinflammatory cytokines and chemokines has also been implicated. The precise roles of the soluble mediators of inflammation warrant further investigation.     

Intracellular signaling in neutrophil priming and activation

In order for neutrophils to function effectively in host defense, they have evolved specific attributes including the ability to migrate to the site of inflammation and release an array of toxic products including proteolytic enzymes, reactive oxygen species, and cationic proteins. While these compounds are intended for killing invading pathogens, if released inappropriately, they may also contribute to tissue damage. Such inflammatory tissue injury may be important in the pathogenesis of a variety of clinical disorders including arthritis, ischemia-reperfusion tissue injury, the systemic inflammatory response syndrome (SIRS), and the acute respiratory distress syndrome (ARDS). Despite the importance of neutrophil function in host defense and dysfunction in disease states, much remains unknown about the intracellular signaling pathways regulating neutrophil activity. This review will focus on the signaling molecules regulating leukocyte ‘effector’ functions including receptors, GTP-binding proteins, phospholipases, polyphosphoinositide metabolism, and protein kinases and phosphatases.     

Recent insights into the role of the innate immune system in the development of alcoholic liver disease

The innate immune system is responsible for the rapid, initial response of the organism to potentially dangerous stresses, including pathogens, tissue injury, and malignancy. Pattern-recognition receptors of the toll-like receptor (TLR) family expressed by macrophages provide a first line of defense against microbial invasion. Activation of these receptors results in a stimulus-specific expression of genes required to control the infection, including the production of inflammatory cytokines and chemokines, followed by the recruitment of neutrophils to the site of infection. The early stages in the development of alcoholic liver disease (ALD) follow a pattern characteristic of an innate immune response. Kupffer cells, the resident macrophages in the liver, are activated in response to bacterial endotoxins (lipopolysaccharide, LPS), leading to the production of inflammatory and fibrogenic cytokines, reactive oxygen species, as well as the recruitment of neutrophils to the liver. One mechanism by which chronic ethanol can turn the highly regulated innate immune response into a pathway of disease is by disrupting the signal transduction cascades mediating the innate immune response. Recent studies have identified specific modules in the TLR-4 signaling cascade that are disrupted after chronic ethanol exposure, including CD14 and the mitogen-activated protein kinase family members, ERK1/2 and p38. Enhanced activation of these TLR-4 dependent signaling pathways after chronic ethanol likely contributes to the development of alcoholic liver disease.     

Nuclear localization of TRK-A in liver cells

The liver represents a site of expression of neurotrophins and their receptors. We have characterized the expression and intracellular localization of the nerve growth factor (NGF) receptor, Trk-A, in liver cells in vivo and in vitro. In both normal and fibrotic liver tissue, Trk-A immunostaining was present in different cell types, including parenchymal cells and cells of the inflammatory infiltrate. In hepatocytes and activated stellate cells (HSC), Trk-A showed a predominant nuclear localization, both in the presence and absence of injury. In cultured HSC, Trk-A was found to be functional, because exposure of the cells to recombinant NGF resulted in stimulation of cell migration and activation of intracellular signaling pathways, including Ras-ERK and PI3K/Akt. Remarkably, in cultured HSC, Trk-A staining was found constitutively in the nucleus. In these cells, Trk-A could be stained only by antibodies directed against the intracellular domain but not by those recognizing the extracellular portion of Trk-A suggesting that the intracellular portion of the receptor is the major determinant of nuclear Trk-A staining. In contrast to HSC, freshly isolated hepatocytes did not show any nuclear localization of the intracellular portion of Trk-A. In pheocromocytoma cells, nuclear staining for Trk-A was not present in conditions of serum deprivation, but could be induced by exposure to NGF or to a mixture of soluble mediators. We conclude that nuclear localization of the intracellular domain of Trk-A is observed constitutively in liver cells such as HSC, while in other cell types it could be induced in response to soluble factors.     

Lipid peroxidation as molecular mechanism of liver cell injury during reperfusion after ischemia

The pathophysiological importance of reactive oxygen species has been extensively documented in the pathogenesis of hepatic ischema-reperfusion injury. Kupffer cells and neutrophils were identified as the dominant sources of the postischemic oxidant stress. To test the hypothesis that a direct free radical-mediated injury mechanism (lipid peroxidation; LPO) may be involved in the pathogenesis, highly sensitive and specific parameters of LPO, i.e., hydroxy-eicosatetraenoic acids (HETES), and F₂-isoprostanes, were determined by gas chromatographic-mass spectrometric analysis in liver tissue and plasma during 45 min of hepatic ischemia and up to 24 h of reperfusion. A significant 60–250% increase of F₂-isoprostane levels in plasma was found at all times during reperfusion; the HETE content increased only significantly at 1 h of reperfusion and in severely necrotic liver tissue at 24 h with increases between 90–320%. On the other hand, in a model of LPO-induced liver injury (infusion of 0.8 μmol tert-butylhydroperoxide/min/g liver), the hepatic HETE content increased two to fourfold over baseline values at 45 min, i.e., before liver injury. A further increase to 12- to 30-fold of baseline was observed during moderate liver injury. Based on these quantitative comparisons of LPO and liver injury, it seems highly unlikely that LPO is the primary mechanism of parenchymal cell injury during reperfusion, although it cannot be excluded that LPO may be important as a damaging mechanism in a limited compartment of the liver, e.g., endothelial cells, close to the sources of reactive oxygen, e.g., Kupffer cells and neutrophils.     

Anti-inflammatory role of fetuin-A in injury and infection

Infection and injury are two seemingly unrelated processes that often converge on common innate inflammatory responses mediated by pathogen- or damage-associated molecular patterns (PAMPs or DAMPs). If dysregulated, an excessive inflammation manifested by the overproduction and release of proinflammatory mediators (e.g., TNF, IFN-γ, and HMGB1) may adversely lead to many pathogenic consequences. As a counter-regulatory mechanism, the liver strategically re-prioritizes the synthesis and systemic release of acute phase proteins (APP) including the fetuin-A (also termed alpha-2-HS-glycoprotein for the human homologue). Fetuin-A is divergently regulated by different proinflammatory mediators, and functions as a positive or negative APP in injury and infection. It not only facilitates anti-inflammatory actions of cationic polyamines (e.g., spermine), but also directly inhibits PAMP-induced HMGB1 release by innate immune cells. Peripheral administration of fetuin-A promotes a short-term reduction of cerebral ischemic injury, but confers a long-lasting protection against lethal endotoxemia. Furthermore, delayed administration of fetuin-A rescues mice from lethal sepsis even when the first dose is given 24 hours post the onset of disease. Collectively, these findings have reinforced an essential role for fetuin-A in counter-regulating injury- or infection-elicited inflammatory responses.     

Hydrogen peroxide and redox modulation sensitize primary mouse hepatocytes to TNF-induced apoptosis

Tumor necrosis factor alpha (TNF) plays an important role in mediating hepatocyte injury in various liver pathologies. TNF treatment alone does not cause the death of primary cultured hepatocytes, suggesting other factors are necessary to mediate TNF-induced injury. In this work the question of whether reactive oxygen species can sensitize primary cultured hepatocytes to TNF-induced apoptosis and necrosis was investigated. Sublethal levels of H(2)O(2), either as bolus doses or steady-state levels generated by glucose oxidase, were found to sensitize cultured hepatocytes to TNF-induced apoptosis. High levels of H(2)O(2) also triggered necrosis in hepatocytes regardless of whether TNF was present. Similarly, antimycin, a complex III inhibitor that increases reactive oxygen species generation from mitochondria, sensitized hepatocytes to TNF-induced apoptosis at low doses but caused necrosis at high doses. Redox changes seem to be important in sensitizing primary hepatocytes, because diamide, a thiol-oxidizing agent, and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of GSSG reductase, also increased TNF-induced apoptosis in cultured primary hepatocytes at sublethal doses. High doses of diamide and BCNU predominantly triggered necrotic cell death. Agents that sensitized hepatocytes to TNF-induced apoptosis -- H(2)O(2), antimycin, diamide, BCNU -- all caused a dramatic fall in the GSH/GSSG ratio. These redox alterations were found to inhibit TNF-induced IkappaB-alpha phosphorylation and NF-kappaB translocation to the nucleus, thus presumably inhibiting expression of genes necessary to inhibit the cytotoxic effects of TNF. Taken together, these results suggest that oxidation of the intracellular environment of hepatocytes by reactive oxygen species or redox-modulating agents interferes with NF-kappaB signaling pathways to sensitize hepatocytes to TNF-induced apoptosis. The TNF-induced apoptosis seems to occur only in a certain redox range -- in which redox changes can inhibit NF-kappaB activity but not completely inhibit caspase activity. The implication for liver disease is that concomitant TNF exposure and reactive oxygen species, either extrinsically generated (e.g., nonparenchymal or inflammatory cells) or intrinsically generated in hepatocytes (e.g., mitochondria), may act in concert to promote apoptosis and liver injury.     

Mechanisms of viral hepatitis induced liver injury

Among seven human hepatitis viruses (A to E, G and TT virus), hepatitis B (HBV) and C (HCV) viruses are able to persist in the host for years and principally contribute to the establishment of chronic hepatitis. During the course of persistent infection, continuous intrahepatic inflammation maintains a cycle of liver cell destruction and regeneration that often terminates in hepatocellular carcinoma (HCC). While the expression and retention of viral proteins in hepatocytes may influence the severity and progression of liver disease, the mechanisms of liver injury in viral hepatistis are defined to be due not to the direct cytopathic effects of viruses, but to the host immune response to viral proteins expressed by infected hepatocytes. In the process of liver injury, hepatocellular death (apoptosis) induced by the proapoptotic molecules of T cells activated following antigen recognition triggers a cascade of antigen nonspecific effector systems and causes necroinflammatory disease. Accordingly, the regulation of the immune response, e.g., via the cell death pathways, in chronically infected patients should prevent the development of HCC.     

Acute lung injury and cell death: how many ways can cells die?

Apoptosis has been considered as an underlying mechanism in acute lung injury/acute respiratory distress syndrome and multiorgan dysfunction syndrome. Recently, several alternative pathways for cell death (such as caspase-independent cell death, oncosis, and autophagy) have been discovered. Evidence of these pathways in the pathogenesis of acute lung injury has also come into light. In this article, we briefly introduce cell death pathways and then focus on studies related to lung injury. The different types of cell death that occur and the underlying mechanisms utilized depend on both experimental and clinical conditions. Lipopolysaccharide-induced acute lung injury is associated with apoptosis via Fas/Fas ligand mechanisms. Hyperoxia and ischemia-reperfusion injury generate reactive oxidative species, which induce complex cell death patterns composed of apoptosis, oncosis, and necrosis. Prolonged overexpression of inflammatory mediators results in increased production and activation of proteases, especially cathepsins. Activation and resistance to death of neutrophils also plays an important role in promoting parenchymal cell death. Knowledge of the coexisting multiple cell death pathways and awareness of the pharmacological inhibitors targeting different proteases critical to cell death may lead to the development of novel therapies for acute lung injury.     

Apoptotic Neutrophils Augment the Inflammatory Response to Mycobacterium tuberculosis Infection in Human Macrophages

Macrophages in the lung are the primary cells being infected by Mycobacterium tuberculosis (Mtb) during the initial manifestation of tuberculosis. Since the adaptive immune response to Mtb is delayed, innate immune cells such as macrophages and neutrophils mount the early immune protection against this intracellular pathogen. Neutrophils are short-lived cells and removal of apoptotic cells by resident macrophages is a key event in the resolution of inflammation and tissue repair. Since anti-inflammatory activity is not compatible with effective immunity to intracellular pathogens, we therefore investigated how uptake of apoptotic neutrophils modulates the function of Mtb-activated human macrophages. We show that Mtb infection exerts a potent proinflammatory activation of human macrophages with enhanced gene activation and release of proinflammatory cytokines and that this response was augmented by apoptotic neutrophils. The enhanced macrophage response is linked to apoptotic neutrophil-driven activation of the NLRP3 inflammasome and subsequent IL-1β signalling. We also demonstrate that apoptotic neutrophils not only modulate the inflammatory response, but also enhance the capacity of infected macrophages to control intracellular growth of virulent Mtb. Taken together, these results suggest a novel role for apoptotic neutrophils in the modulation of the macrophage-dependent inflammatory response contributing to the early control of Mtb infection.     

Inflammation in the course of early myocardial ischemia

Experimental models of acute ischemic myocardial injury indicate that the inflammatory response after the ischemic event contributes to tissue damage. This is especially apparent with reperfusion of the ischemic tissue. In such models some therapeutic strategies designed to reduce neutrophil accumulation or function have resulted in apparently beneficial effects. Although such findings are encouraging, interventions into these pathological processes using specific molecular targets will require greater understanding of specific mechanisms. Current evidence indicates that potential sites of therapeutic intervention will be found in pathways leading to complement activation, generation of lipid-derived mediators, adhesion of neutrophils to endothelial cells and cardiac myocytes, and activation of neutrophil secretory processes releasing, for example, proteolytic enzymes and reactive oxygen. Understanding the dynamic interplay between the mediators, adhesion pathways, and secretory processes that results in myocardial damage will allow a rational approach to controlling the detrimental inflammatory consequences of ischemia and reperfusion.