Mechanisms underlying reduced responsiveness of neonatal neutrophils to distinct chemoattractants.

Potential mechanisms underlying impaired chemotactic responsiveness of neonatal neutrophils were investigated. Two distinct chemoattractants were compared: bacterially derived N-formyl-methionyl-leucyl-phenylalanine (fMLP) and a unique chemotactic monoclonal antibody, designated DL1.2, which binds to a neutrophil antigen with an apparent molecular mass of 120 kDa. Chemotaxis of neutrophils toward fMLP, as well as DL1.2, was reduced in neonates when compared with adult cells. This did not appear to be a result of decreased fMLP receptor or DL1.2 antigen expression by neonatal neutrophils. fMLP, but not DL1.2, induced a rapid increase in intracellular calcium in adult and neonatal cells, which reached a maximum within 30 s. The calcium response of cells from neonates to fMLP was reduced when compared with adult cells, and an unresponsive subpopulation of neonatal neutrophils was identified. NF-kappaB nuclear binding activity induced by fMLP and DL1.2, as well as expression of the p65 NF-kappaB subunit and IkappaB-alpha, was also significantly reduced in neonatal cells, when compared with adult cells. In contrast, although fMLP, but not DL1.2, activated p42/44 and p38 mitogen-activated protein (MAP) kinases in neutrophils, no differences were observed between adults and neonates. Chemotaxis of adult and neonatal neutrophils toward fMLP and DL1.2 was also blocked to a similar extent by inhibitors of phosphatidylinositol 3-kinase, as well as an inhibitor of NF-kappaB. These findings indicate that reduced chemotactic responsiveness in neonatal neutrophils is a result of, at least in part, aberrations in chemoattractant-induced signaling. However, the biochemical pathways mediating this defect appear to be related to the specific chemoattractant.

Nitric oxide synthase sequences in the marine fish Stenotomus chrysops and the sea urchin Arbacia punctulata, and phylogenetic analysis of nitric oxide synthase calmodulin-binding domains.

The phylogenetic distribution and structural diversity of the nitric oxide synthases (NOS) remain important and issues that are little understood. We present sequence information, as well as phylogenetic analysis, for three NOS cDNAs identified in two non-mammalian species: the vertebrate marine teleost fish Stenotomus chrysops (scup) and the invertebrate echinoderm Arbacia punctulata (sea urchin). Partial gene sequences containing the well-conserved calmodulin (CaM)-binding domain were amplified by RT-PCR. Identical 375-bp cDNAs were amplified from scup brain, heart, liver and spleen; this sequence shares 82% nucleic acid and 91% predicted amino acid identity with the corresponding region of human neuronal NOS. A 387-bp cDNA was amplified from sea urchin ovary and testes; this sequence shares 72% nucleic acid identity and 65% deduced amino acid identity with human neuronal NOS. A second cDNA of 381 bp was amplified from sea urchin ovary and it shares 66% nucleic acid and 57% deduced amino acid identity with the first sea urchin sequence. Together with earlier reports of neuronal and inducible NOS sequences in fish, these data indicate that multiple NOS isoforms exist in non-mammalian species. Phylogenetic analysis of these sequences confirms the conserved nature of NOS, particularly of the calmodulin-binding domains.

Proteins and lipids define the diffusional field of nitric oxide.

Nitric oxide (NO) fluxes released from the surface of individual activated macrophages or cells localized in small aggregates were measured with a novel polarographic self-referencing microsensor. NO fluxes could be detected at distances from the cells of 100-500 microm. The initial flux and the distance from the cells at which NO could be detected were directly related to the number of cells in the immediate vicinity of the probe releasing NO. Thus, whereas NO fluxes of approximately 1 pmol. cm(-2). s(-1) were measured from individual macrophages, aggregates composed of groups of cells varying in number from 18 to 48 cells produced NO fluxes of between approximately 4 and 10 pmol. cm(-2). s(-1). NO fluxes required the presence of L-arginine. Signals were significantly reduced by the addition of hemoglobin and by N-nitro-L-arginine methyl ester. NO fluxes were greatest when the sensor was placed immediately adjacent to cell membranes and declined as the distance from the cell increased. The NO signal was markedly reduced in the presence of the protein albumin but not by either oxidized or reduced glutathione. A reduction in the NO signal was also noted after the addition of lipid micelles to the culture medium. These results demonstrate that NO can be detected at significant distances from the cell of origin. In addition, both proteins and lipids strongly influence the net movement of free NO from macrophages. This suggests that these tissue components play an important role in regulating the biological activity of NO.

Functional heterogeneity in liver and lung macrophages.

Although initially considered merely "scavenger cells" that participate in immunologic responses only after B and T lymphocytes have performed their biological tasks, more recent evidence suggests that macrophages play a key role in host defense as well as in the maintenance of normal tissue structure and function. For macrophages to perform their biological functions, they must be activated. This involves up-regulation of an array of signaling pathways resulting in altered gene expression and increased biochemical and functional activity. Macrophages have been identified in almost all tissues of the body. However, the basal activity of these cells, as well as their ability to respond to inflammatory mediators, varies considerably with their location. In addition, even within a particular tissue, there is evidence of macrophage heterogeneity. The largest populations of macrophages in the body are located in the liver and lung. Because of the unique attributes of these tissues, hepatic and pulmonary macrophages play essential roles not only in nonspecific host defense but also in the homeostatic responses of these tissues. In this review, the functional and biochemical activities of macrophages localized in the liver and lungs are compared. Evidence suggests that these represent distinct cell populations with unique functions and responsiveness to inflammatory agents.

Prooxidant and antioxidant functions of nitric oxide in liver toxicity.

In response to tissue damage and inflammation induced by a variety of xenobiotics including acetaminophen, carbon tetrachloride, ethanol, galactosamine, and endotoxin, as well as disease states such as viral hepatitis, and postischemic and regenerative injury, the liver produces large quantities of nitric oxide. Indeed, nearly all cell types in the liver including hepatocytes, Kupffer cells, stellate cells, and endothelial cells have the capacity to generate nitric oxide. Thus, these cells, as well as infiltrating leukocytes, may indirectly augment tissue injury. In many models of liver damage, nitric oxide and its oxidation products such as peroxynitrite contribute to the injury process by directly damaging the tissue or by initiating additional immunologic reactions that result in damage. In some models, nitric oxide donors or peroxynitrite can mimic the cytotoxic actions of liver toxins. Moreover, agents that prevent the generation of nitric oxide or antioxidants that bind reactive nitrogen intermediates, or knockout mice with reduced capacity to produce nitric oxide, are protected from xenobiotic-induced tissue injury. In contrast, there have been reports that blocking nitric oxide production enhances xenobiotic-induced tissue injury. This has led to the concept that nitric oxide either inactivates proteins critical for xenobiotic-induced tissue injury or acts as an antioxidant, reducing cellular levels of cytotoxic reactive oxygen intermediates. Whether or not nitric oxide or secondary oxidants generated from nitric oxide act as mediators of tissue injury or protect against toxicity is likely to depend on the precise targets of these reactive nitrogen intermediates, as well as levels of superoxide anion present and the extent to which tissue injury is mediated by reactive oxygen intermediates. In addition, as toxicity is a complex process involving a variety of cell types and many soluble mediators, the contribution of each of these factors must be taken into account when considering the role of nitric oxide as a determinant of tissue injury.

Minimal amidine structure for inhibition of nitric oxide biosynthesis.

Pharmacological modulation of nitric oxide synthase activity has been achieved using structural analogs of arginine. In the present studies, we demonstrated that the minimal amidine structure required for enzymatic inhibition is formamidine. We found that the production of nitric oxide by primary cultures of rat hepatocytes and several mouse and human cell lines, including RAW 264.7 macrophages, PAM 212 keratinocytes, G8 myoblasts, S180 sarcoma, CX-1 human colon cells, and GH3 rat pituitary cells, was inhibited in a concentration- and time-dependent manner by formamidine. Formamidine was 2- to 6-fold more effective in inhibiting nitric oxide production in cells expressing inducible nitric oxide synthase (NOS2) than in a cell line expressing calcium-dependent neuronal nitric oxide synthase (NOS1). Whereas formamidine had no effect on gamma-interferon-induced expression of nitric oxide synthase protein, its enzymatic activity was blocked. Kinetic analysis revealed that formamidine acts as a simple competitive inhibitor with respect to arginine (K(i) formamidine approximately 800 microM). Using a polarographic microsensor to measure real-time flux of nitric oxide release from RAW 264.7 macrophages, formamidine was found to require 30-90 min to inhibit enzyme activity, suggesting that cellular uptake of the drug may limit its biological activity. Our data indicate that formamidine is an effective inhibitor of nitric oxide production. Furthermore, its low toxicity may make it useful as a potential therapeutic agent in diseases associated with the increased production of nitric oxide.

Pharmacologic therapy of persistent pulmonary hypertension of the newborn.

Persistent pulmonary hypertension of the newborn (PPHN) is a potentially life-threatening condition characterized by a failure of pulmonary vascular resistance to decrease adequately during the transition to extrauterine life. Inhaled nitric oxide, a vasodilator that acts selectively on the pulmonary circulation, has revolutionized the treatment of this condition. However, inhaled nitric oxide has not proven effective in all patients, particularly those with congenital diaphragmatic hernias or meconium aspiration syndrome. Furthermore, large clinical trials of inhaled nitric oxide have failed to demonstrate significant differences in mortality between nitric oxide-treated and control infants with PPHN. Other therapeutic approaches to PPHN have been limited by a relative lack of specificity for the pulmonary circulation, and have received much less attention. Pharmacologic approaches, including pulmonary surfactants, prostacyclin, endothelin antagonists, Ca(2+)-channel blockers, magnesium sulfate, and tolazoline, have exhibited varying degrees of efficacy in lowering pulmonary vascular pressures in humans and/or animals. A number of these agents are also effective when used in combination. For example, phosphodiesterase inhibitors have been reported to act synergistically with inhaled nitric oxide. Surfactants also appear to be useful in PPHN, particularly in patients with congenital diaphragmatic hernia, when used in combination with other therapies. Surfactant lavage and other novel therapies may also be effective in combination therapy of meconium aspiration syndrome. Further studies should be directed at defining the optimal therapies in specific clinical settings. Validation of multiple therapeutic modalities for PPHN, including inhaled nitric oxide, will allow for rational, combined vasodilator strategies that are specific for the underlying pathophysiology in each patient.

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.

The toxicology of inhaled nitric oxide.

Inhaled nitric oxide is a targeted pulmonary vasodilator that improves clinical outcomes for newborn patients with persistent pulmonary hypertension of the newborn, and may be effective in treating some premature patients with acute respiratory distress syndrome or lung disease of prematurity. Nitric oxide is now recognized as playing an important role in the regulation of diverse physiological processes. However, the pharmacological properties of inhaled nitric oxide are not easy to separate from its toxicological effects. For example, the intended effect of inhaled nitric oxide, vasodilation in the lung, is mediated, in part, by increased cellular cyclic GMP (cGMP). However, increased cGMP can also interfere with normal cellular proliferation. Nitric oxide has also been shown to cause DNA strand breaks and/or base alterations that are potentially mutagenic. Inhaled nitric oxide can rapidly react with oxygen in the lung to form nitrogen dioxide, which is a potent pulmonary irritant. Nitric oxide also reacts with superoxide anion to form peroxynitrite, a cytotoxic oxidant that can interfere with surfactant functioning. The overall effect of inhaled nitric oxide in potentiating or attenuating inflammation and oxidative damage in diseased lung is dependent on the dose administered. Furthermore, despite rapid inactivation by circulating hemoglobin, inhaled nitric oxide exerts effects outside the lung, including blocking platelet aggregation, causing methemoglobinemia, and possibly inducing extrapulmonary vasodilation. The toxicology of inhaled nitric oxide is not completely understood and must be considered in the design of protocols for its safe and effective clinical use.

Nitric oxide and peroxynitrite in ozone-induced lung injury.

One of the hallmarks of the inflammatory response associated with tissue injury is the accumulation of macrophages at sites of damage. These cell types release proinflammatory cytokines and cytotoxic mediators to destroy invading pathogens and initiate wound repair. However, when produced in excessive amounts, these macrophage-derived mediators may actually contribute to tissue injury. This process involves both direct damage to target tissues and amplification of the inflammatory response. One group of macrophage-derived mediators of particular interest are reactive nitrogen intermediates including nitric oxide and peroxynitrite which have been implicated in tissue injury induced by a variety oftoxicants. Our laboratory has been investigating the role of reactive nitrogen intermediates in lung injury induced by oxidants such as ozone. Inhalation of ozone causes epithelial cell damage and Type II cell hyperplasia. This is associated with an accumulation of activated macrophages in the lower lungs which we have demonstrated contribute to toxicity. To analyze the role of macrophage-derived reactive nitrogen intermediates in ozone toxicity, we used transgenic mice lacking the gene for inducible nitric oxide synthase (NOSII). Treatment of wild type control animals with ozone (0.8 ppm) for 3 hr resulted in an increase in bronchoalveolar lavage (BAL) fluid protein reaching a maximum 24-48 hr after exposure. This was correlated with increased expression of NOSII protein and mRNA by alveolar macrophages and increased production of nitric oxide as well as peroxynitrite. Ozone inhalation also resulted in the appearance of nitrotyrosine residues in the lungs, an in vivo marker of peroxynitrite-induced damage. In contrast, in NOSII knockout mice, BAL protein was not increased demonstrating that these mice were protected from ozone-induced epithelial injury. Moreover, alveolar macrophages from the transgenic mice did not produce nitric oxide or peroxynitrite even after ozone inhalation. There was also no evidence for the formation of nitrotyrosine in lung tissue. These data indicate that ozone-induced lung injury is mediated by reactive nitrogen intermediates.

Nucleic acid microarray technology for toxicology: promise and practicalities.

Much ongoing research in toxicology focuses on a hypothesis-driven mechanism of action approach aimed at understanding the molecular events mediating the actions of the chemicals of interest. Using this approach, investigators develop hypotheses based on observations, which may be derived from a host of resources but most frequently have been made within their own laboratories, or uncovered by others and reported in the scientific literature. Although the bulk of current understanding ofbiochemical toxicology emerged using studies based on observations derived in this way, this process, which is essentially based on existing information, may often limit the expansion knowledge. More simply expressed, one only finds that which one seeks. Without a clear understanding of the processes targeted by a specific toxin the problem of making observations that globally and accurately reflect the events mediating pathology which have been induced by the toxic agent is challenging. Recently, the development of high-throughput technologies for biochemical analysis of gene expression has led to innovative approaches in addressing the problem of making broad-based observations that more accurately reflect the entire spectrum of molecular lesions induced by specific toxins. These strategies include the use of new techniques in analysis of gene expression to convey information on alterations in mRNA levels, one of the earliest cellular signs initiated in response to a potential toxin. Prior to this time studies on toxicant-induced altered gene expression were limited to single, or small numbers of identified genes chosen by an investigator who reasoned, based on an existing observations, that levels of the proteins encoded by these genes were likely to be altered during toxic injury. Now, using cDNA or oligonucleotide genome-wide arrays, toxin-induced alterations in gene expression of thousands of genes can be examined simultaneously. Using these tools, molecular toxicologists can for the first time employ reasoned strategies to make observations, and then formulate hypotheses based on these observations.

Nitric oxide in the lung: therapeutic and cellular mechanisms of action.

Nitric oxide is produced by many cell types in the lung and plays an important physiologic role in the regulation of pulmonary vasomotor tone by several known mechanisms. Nitric oxide stimulates soluble guanylyl cyclase, resulting in increased levels of cyclic GMP in lung smooth muscle cells. The gating of K+ and Ca2+ channels by cyclic GMP binding is thought to play a role in nitric oxide-mediated vasodilation. Nitric oxide may also regulate pulmonary vasodilation by direct activation of K+ channels or by modulating the expression and activity of angiotensin II receptors. Administration of nitric oxide by inhalation has been shown to acutely improve hypoxemia associated with pulmonary hypertension in humans and animals. This is presumably due to its ability to induce pulmonary vasodilation. Inhaled nitric oxide improves oxygenation and reduces the need for extracorporeal membrane oxygenation in term and near-term infants with persistent pulmonary hypertension. However, long-term benefits to these infants have been difficult to demonstrate. In other pathologic conditions, such as prematurity and acute respiratory distress syndrome, short-term benefits have not been shown conclusively to outweigh potential toxicities. For example, high-dose inhaled nitric oxide decreases surfactant function in the lung. Inhaled nitric oxide also acts as a pulmonary irritant, causing priming of lung macrophages and oxidative damage to lung epithelial cells. Conversely, protective effects of nitric oxide have been described in a number of pathological states, including hyperoxic and ischemia/reperfusion injury. Nitric oxide has also been reported to protect against oxidative damage induced by other reactive intermediates, including superoxide anion and hydroxyl radical. The dose and timing of nitric oxide administration needs to be ascertained in clinical trials before recommendations can be made regarding its optimal use in patients.

Increased nitric oxide synthase in the lung after ozone inhalation is associated with activation of NF-kappa B.

Acute inhalation of ozone is associated with a inflammatory response characterized by the accumulation of macrophages at sites of tissue injury. These cells, along with resident alveolar epithelial cells, become activated and release cytotoxic and proinflammatory mediators, such as nitric oxide (.NO), that we speculate contribute to toxicity. In these studies we analyzed mechanisms regulating increased .NO synthase activity in lung macrophages and type II cells after ozone inhalation. Brief exposure of rats to ozone (2 ppm for 3 hr) resulted in an increase in .NO production by alveolar macrophages as well as type II cells in response to the inflammatory mediators lipopolysaccharide and interferon gamma. These effects were apparently due to increased expression of inducible .NO synthase (iNOS) protein and mRNA, which were evident in vitro and in situ in histologic sections. .NO production and iNOS protein expression by both macrophages and epithelial cells were blocked by pyrrolidine dithiocarbamate (PDTC), an agent that inhibits activity of nuclear transcription factor kappa B (NF-kappa B). Cells from ozone-treated animals were less sensitive to the effects of PDTC than cells from control animals. Using electrophoretic mobility shift assays, we measured NF-kappa B binding activity in nuclear extracts of cells from control and ozone-exposed animals. Treatment of rats with ozone resulted in a time-dependent increase in NF-kappa B binding activity in both cell types, reaching a maximum in cells isolated 12 to 24 hr after ozone inhalation. Taken together, these data suggest that changes in the activity of NF-kappa B signaling are important in the response of lung macrophages and type II epithelial cells to cytokines after ozone inhalation.

Inhaled nitric oxide primes lung macrophages to produce reactive oxygen and nitrogen intermediates.

Inhaled nitric oxide is a selective pulmonary vasodilator used for the treatment of pulmonary hypertension. The potential adverse effects of inhaled nitric oxide are unknown and represent the focus of the present studies. Whereas inhalation of nitric oxide (10 to 100 ppm, 5 h) by Balb/c mice had no effect on the number or type of cells recovered from the lung, a dose-related increase in bronchoalveolar lavage protein was observed, suggesting that nitric oxide induces alveolar epithelial injury. To determine if this was associated with altered alveolar macrophage activity, we quantified production of reactive oxygen and nitrogen intermediates by these cells. Interferon-gamma, alone or in combination with lipopolysaccharide (LPS), induced expression of inducible nitric oxide synthase (iNOS) protein and nitric oxide production by alveolar macrophages. Cells from mice exposed to 20 to 100 ppm nitric oxide produced significantly more nitric oxide and expressed greater quantities of iNOS than cells from control animals. Superoxide anion production and peroxynitrite generation by alveolar macrophages were also increased after exposure of mice to nitric oxide. This was correlated with increased antinitrotyrosine antibody binding to macrophages in histologic sections. Taken together, these data demonstrate that inhaled nitric oxide primes lung macrophages to release reactive oxygen and nitrogen intermediates. Increased production of these mediators by macrophages following inhalation of nitric oxide may contribute to tissue injury.