Debra L. Laskin

Macrophages and Tissue Injury: Agents of Defense or Destruction?

The past several years have seen the accumulation of evidence demonstrating that tissue injury induced by diverse toxicants is due not only to their direct effects on target tissues but also indirectly to the actions of resident and infiltrating macrophages. These cells release an array of mediators with cytotoxic, pro- and anti-inflammatory, angiogenic, fibrogenic, and mitogenic activity, which function to fight infections, limit tissue injury, and promote wound healing. However, following exposure to toxicants, macrophages can become hyperresponsive, resulting in uncontrolled or dysregulated release of mediators that exacerbate acute tissue injury and/or promote the development of chronic diseases such as fibrosis and cancer. Evidence suggests that the diverse activity of macrophages is mediated by distinct subpopulations that develop in response to signals within their microenvironment. Understanding the precise roles of these different macrophage populations in the pathogenic response to toxicants is key to designing effective treatments for minimizing tissue damage and chronic disease and for facilitating wound repair.

Production of nitric oxide and peroxynitrite in the lung during acute endotoxemia.

Nitric oxide is a short‐lived cytotoxic mediator that has been implicated in the pathogenesis of endotoxin‐induced tissue injury and septic shock. In the present studies we determined whether this mediator is produced in the lung during acute endotoxemia. We found that intravenous injection of rats with bacterially derived lipopolysaccharide (LPS), a condition that induces acute endotoxemia, caused a time‐dependent increase in inducible nitric oxide synthase (iNOS) mRNA expression in the lung, which reached a maximum after 24 h. This was correlated with nitric oxide production in the lung as measured by electron paramagnetic spin trapping, which was detectable within 6 h. Alveolar macrophages (AMs) and interstitial macrophages (IMs) isolated from rats 6–12 h after induction of acute endotoxemia were also found to exhibit increased nitric oxide production in response to in vitro stimulation with interferon‐γ (IFN‐γ) and LPS measured by nitrite accumulation in the culture medium. The effects of acute endotoxemia on nitric oxide production by these cells were, however, transient and returned to control levels by 24 h in AMs and 36 h in IMs. Interestingly, although nitrite accumulation in the culture medium of IMs isolated 48 h after induction of acute endotoxemia and stimulated with low concentrations of IFN‐γ and LPS was reduced, when compared with cells from control animals, these cells, as well as AMs, continued to express high levels of iNOS protein and mRNA. This was correlated with increased peroxynitrite production by the cells. Peroxynitrite has been shown to act as a nitrating agent and can generate nitrotyrosine residues in proteins. Using a specific antibody and immunohistochemistry, we found evidence of nitrotyrosine residues in sections of lungs 48 h after treatment of rats with endotoxin. These data suggest that nitric oxide produced by IMs and AMs can react with superoxide anion to form peroxynitrite. Taken together, the present studies demonstrate that AMs and IMs are activated following acute endotoxemia to produce reactive nitrogen intermediates and that both cell types contribute to inflammatory responses in the lung. J. Leukoc. Biol. 56: 759–768; 1994.

Macrophages and Inflammatory Mediators in Chemical Toxicity: A Battle of Forces

Macrophages function as control switches of the immune system, providing a balance between pro- and anti-inflammatory responses. To accomplish this, they develop into different subsets: classically (M1) or alternatively (M2) activated macrophages. Whereas M1 macrophages display a cytotoxic, proinflammatory phenotype, much like the soldiers of The Dark Side of The Force in the Star Wars movies, M2 macrophages, like Jedi fighters, suppress immune and inflammatory responses and participate in wound repair and angiogenesis. Critical to the actions of these divergent or polarized macrophage subpopulations is the regulated release of inflammatory mediators. When properly controlled, M1 macrophages effectively destroy invading pathogens, tumor cells, and foreign materials. However, when M1 activation becomes excessive or uncontrolled, these cells can succumb to The Dark Side, releasing copious amounts of cytotoxic mediators that contribute to disease pathogenesis. The activity of M1 macrophages is countered by The Force of alternatively activated M2 macrophages, which release anti-inflammatory cytokines, growth factors, and mediators involved in extracellular matrix turnover and tissue repair. It is the balance in the production of mediators by these two macrophage subpopulations that ultimately determines the outcome of the tissue response to chemical toxicants.

Role of macrophages and inflammatory mediators in chemically induced toxicity

Macrophages are critical cellular effectors of nonspecific host defense. They are also potent secretory cells releasing an array of mediators including proinflammatory and cytotoxic cytokines and growth factors, bioactive lipids, hydrolytic enzymes and reactive oxygen and nitrogen intermediates, each of which has been implicated in tissue injury. The research in our laboratories has focused on analyzing the role of macrophages in chemically induced injury in the lung and the liver. In both these tissues, a localized accumulation of macrophages is observed following toxicant exposure. This is directly correlated with the generation of cytotoxic inflammatory mediators at these sites. Moreover, when macrophage functioning is blocked, pulmonary and hepatic injury induced by toxicants such as ozone or acetaminophen is prevented. These findings provide direct support for our hypothesis that macrophages contribute to tissue injury. Approaches using pharmacologic inhibitors and transgenic animals are currently being used to evaluate the specific macrophage-derived products involved in the pathogenic process. Our results suggest that the extent to which a particular mediator contributes to injury depends on the nature of the toxicant, the target tissue, and quantities of the mediator produced.

Mechanisms Mediating the Vesicant Actions of Sulfur Mustard after Cutaneous Exposure

Sulfur mustard (SM), a chemical weapon first employed during World War I, targets the skin, eyes, and lung. It remains a significant military and civilian threat. The characteristic response of human skin to SM involves erythema of delayed onset, followed by edema with inflammatory cell infiltration, the appearance of large blisters in the affected area, and a prolonged healing period. Several in vivo and in vitro models have been established to understand the pathology and investigate the mechanism of action of this vesicating agent in the skin. SM is a bifunctional alkylating agent which reacts with many targets including lipids, proteins, and DNA, forming both intra- and intermolecular cross-links. Despite the relatively nonselective chemical reactivity of this agent, basal keratinocytes are more sensitive, and blistering involves detachment of these cells from their basement membrane adherence zones. The sequence and manner in which these cells die and detach is still unresolved. Much has been discovered over the past two decades with respect to the mechanisms of SM-induced cytotoxicity and the intracellular and extracellular targets of this vesicant. In this review, the effects of SM exposure on the skin are described, as well as potential mechanisms mediating its actions. Successful therapy for SM poisoning will depend on following new mechanistic leads to develop drugs that target one or more of its sites of action.

Oxidants and antioxidants in sulfur mustard–induced injury

Sulfur mustard (SM) is a chemical weapon that targets the skin, eyes, and lung. It was first employed during World War I and it remains a significant military and civilian threat. As a bifunctional alkylating agent, SM reacts with a variety of macromolecules in target tissues including nucleic acids, proteins and lipids, as well as small molecular weight metabolites such as glutathione. By alkylating subcellular components, SM disrupts metabolism, a process that can lead to oxidative stress. Evidence for oxidative stress in tissues exposed to SM or its analogs include increased formation of reactive oxygen species, the presence of lipid peroxidation products and oxidized proteins, and increases in antioxidant enzymes such as superoxide dismutase, catalase, and glutathione‐S‐transferase. Inhibition of antioxidant enzymes including thioredoxin reductase by SM can also disrupt cellular redox homeostasis. Consistent with these findings, SM‐induced toxicity has been shown to be reduced by antioxidants in both in vitro and in vivo models. These data indicate that drugs that target oxidative stress pathways may represent important candidates for reducing SM‐induced tissue injury.

Regulation of cyclooxygenase-2 by nitric oxide in activated hepatic macrophages during acute endotoxemia

Eicosanoids generated via cyclooxygenase‐2 (COX‐2) and nitric oxide produced from inducible nitric oxide synthase (NOSII) have been implicated in endotoxin‐induced tissue injury. In the present studies, we characterized COX‐2 and NOSII activity in rat hepatic macrophages and their interaction during acute endotoxemia. Kupffer cells from control animals were found to constitutively express COX‐2 and NOSII mRNA and protein. Whereas treatment of the cells with lipopolysaccharide (LPS) and/or interferon‐γ (IFN‐γ) had no major effect on COX‐2, NOSII expression increased. Induction of acute endotoxemia resulted in a rapid and transient increase in constitutive COX‐2 expression and prostaglandin E2 (PGE2) production by liver macrophages as well as NOSII expression and nitric oxide release. Cells from endotoxin‐treated rats were also sensitized to generate more nitric oxide and express increased NOSII in response to LPS and IFN‐γ. Inhibition of NOSII with aminoguanidine reduced COX‐2 mRNA and protein expression as well as PGE2 production by activated macrophages from endotoxemic, but not control animals. In contrast, SC236, a specific COX‐2 inhibitor, had no effect on NOSII mRNA or protein levels or on nitric oxide production by hepatic macrophages, even after endotoxin administration. These data suggest that activation of COX‐2 may be important in the pathophysiological response of hepatic macrophages to endotoxin. Moreover, nitric oxide is involved in regulating COX‐2 in activated liver macrophages during acute endotoxemia.

Exaggerated hepatotoxicity of acetaminophen in mice lacking tumor necrosis factor receptor-1. Potential role of inflammatory mediators.

Transgenic mice with a targeted disruption of the tumor necrosis factor receptor 1 (TNFR1) gene were used to analyze the role of TNF-alpha in pro- and anti-inflammatory mediator production and liver injury induced by acetaminophen. Treatment of wild-type mice with acetaminophen (300 mg/kg) resulted in centrilobular hepatic necrosis. This was correlated with expression of inducible nitric oxide synthase (NOS II) and nitrotyrosine staining of the liver. Expression of macrophage chemotactic protein-1 (MCP-1), KC/gro, interleukin-1beta (IL-1beta), matrix metalloproteinase-9 (MMP-9), and connective tissue growth factor (CTGF), inflammatory mediators known to participate in tissue repair, as well as the anti-inflammatory cytokine, interleukin-10 (IL-10), also increased in the liver following acetaminophen administration. TNFR1(-/-) mice were found to be significantly more sensitive to the hepatotoxic effects of acetaminophen than wild-type mice. This was correlated with more rapid and prolonged induction of NOS II in the liver and changes in the pattern of nitrotyrosine staining. Acetaminophen-induced expression of MCP-1, IL-1beta, CTGF, and MMP-9 mRNA was also delayed or reduced in TNFR1(-/-) mice relative to wild-type mice. In contrast, increases in IL-10 were more rapid and more pronounced. These data demonstrate that signaling through TNFR1 is important in inflammatory mediator production and toxicity induced by acetaminophen.

Inflammatory effects of inhaled sulfur mustard in rat lung

Inhalation of sulfur mustard (SM), a bifunctional alkylating agent that causes severe lung damage, is a significant threat to both military and civilian populations. The mechanisms mediating its cytotoxic effects are unknown and were investigated in the present studies. Male rats Crl:CD(SD) were anesthetized, and then intratracheally intubated and exposed to 0.7-1.4mg/kg SM by vapor inhalation. Animals were euthanized 6, 24, 48h or 7days post-exposure and bronchoalveolar lavage fluid (BAL) and lung tissue collected. Exposure of rats to SM resulted in rapid pulmonary toxicity, including focal ulceration and detachment of the trachea and bronchial epithelia from underlying mucosa, thickening of alveolar septal walls and increased numbers of inflammatory cells in the tissue. There was also evidence of autophagy and apoptosis in the tissue. This was correlated with increased BAL protein content, a marker of injury to the alveolar epithelial lining. SM exposure also resulted in increased expression of markers of inflammation including cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNFα), inducible nitric oxide synthase (iNOS), and matrix metalloproteinase-9 (MMP-9), each of which has been implicated in pulmonary toxicity. Whereas COX-2, TNFα and iNOS were mainly localized in alveolar regions, MMP-9 was prominent in bronchial epithelium. In contrast, expression of the anti-oxidant hemeoxygenase, and the anti-inflammatory collectin, surfactant protein-D, decreased in the lung after SM exposure. These data demonstrate that SM-induced oxidative stress and injury are associated with the generation of cytotoxic inflammatory proteins which may contribute to the pathogenic response to this vesicant.

Application of the Amplex Red/Horseradish Peroxidase Assay to Measure Hydrogen Peroxide Generation by Recombinant Microsomal Enzymes

The formation of reactive oxygen species by the cytochrome P450 monooxygenase system is thought to be due to autoxidation of NADPH-cytochrome P450 reductase and the nonproductive decay of oxygen-bound cytochrome P450 intermediates. To characterize this process in recombinant microsomal enzymes, we used a highly sensitive hydrogen peroxide assay based on Amplex red oxidation. This assay is 20 times more sensitive (LLD=5.0pmol/assay and LLQ=30pmol/assay) than the standard ferrous thiocyanate assay for detection of hydrogen peroxide. We found low, but detectable, spontaneous generation of hydrogen peroxide by recombinant human NADPH-cytochrome P450 reductase complexes (0.09nmol hydrogen peroxide/min/100Units of NADPH-cytochrome P450 reductase). Significantly higher rates of hydrogen peroxide production were observed when recombinant cytochrome P450 enzymes were coexpressed with NADPH-cytochrome P450 reductase (0.31nmol of hydrogen peroxide/min/100Units of NADPH-cytochrome P450 reductase). This was independent of the addition of any exogenous cytochrome P450 substrates. These data demonstrate that cytochrome P450s are a major source of hydrogen peroxide in the recombinant cytochrome P450 monooxygenase system. Moreover, substrate binding is not required for the cytochrome P450s to generate reactive oxygen species.