Diane E. Heck

Multifunctional role of nitric oxide in inflammation

In response to infection or tissue damage, an array of soluble and lipid mediators as well as cytokines and growth factors cause both immune and nonimmune cells to produce rather large amounts of nitric oxide. Nitric oxide and its oxidation products are toxic and can cause tissue injury. The endocrine system can protect against nitric-oxide-mediated tissue damage by producing corticosteroids, growth factors, and cytokines that are potent inhibitors of nitric oxide production. This review focuses on our current understanding of the role of nitric oxide in the inflammatory response. An emphasis has been placed on the potential for nitric oxide in tissue damage.

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.

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.

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.

Role of inflammatory cytokines and nitric oxide in hepatic and pulmonary toxicity

Exposure of humans and experimental animals to inhaled irritants such as ozone, induces an acute inflammatory response and lung injury. We hypothesize that macrophage-derived inflammatory cytokines and cytotoxic mediators contribute to the pathogenic process. Treatment of rats with ozone (2 ppm, 3 h) results in damage to the alveolar epithelium and increased protein in lung lavage fluid. This is associated with an increase in the number of macrophages in the lung. We found that these cells are activated to release the proinflammatory cytokine tumor necrosis factor-alpha (TNF alpha) which has been implicated in tissue injury. Following ozone inhalation, alveolar macrophages also produce increased amounts of the cytotoxic mediator, nitric oxide. This response is time-dependent and correlated with expression of inducible nitric oxide synthase (NOS II) protein and mRNA. Inhibition of macrophages with gadolinium chloride abrogates ozone-induced inflammation, mediator production and tissue injury. These data demonstrate, that macrophages and mediators they release contribute to irritant-induced lung injury. Ozone inhalation also caused alterations in the liver, including increased nitric oxide production and protein synthesis suggesting that ozone induces an acute phase response. We speculate that this is mediated by cytokines such as TNF alpha produced by alveolar macrophages. In this regard we noted increased expression of TNF alpha in both lung and liver tissue. Thus cytokines produced locally by macrophages following toxicant exposure may exert pathophysiologic effects outside the target organ.

Role of cytochrome P450 reductase in nitrofurantoin-induced redox cycling and cytotoxicity

The one-electron reduction of redox-active chemotherapeutic agents generates highly toxic radical anions and reactive oxygen intermediates (ROI). A major enzyme catalyzing this process is cytochrome P450 reductase. Because many tumor cells highly express this enzyme, redox cycling of chemotherapeutic agents in these cells may confer selective antitumor activity. Nitrofurantoin is a commonly used redox-active antibiotic that possesses antitumor activity. In the present studies we determined whether nitrofurantoin redox cycling is correlated with cytochrome P450 reductase activity and cytotoxicity in a variety of cell lines. Recombinant cytochrome P450 reductase was found to support redox cycling of nitrofurantoin and to generate superoxide anion, hydrogen peroxide, and, in the presence of redox-active iron, hydroxyl radicals. This activity was NADPH dependent and inhibitable by diphenyleneiodonium, indicating a requirement for the flavin cofactors in the reductase. Nitrofurantoin-induced redox cycling was next analyzed in different cell lines varying in cytochrome P450 reductase activity including Chinese hamster ovary cells (CHO-OR) constructed to overexpress the enzyme. Nitrofurantoin-induced hydrogen peroxide production was 16-fold greater in lysates from CHO-OR cells than from control CHO cells. A strong correlation between cytochrome P450 reductase activity and nitrofurantoin-induced redox cycling among the cell lines was found. Unexpectedly, no correlation between nitrofurantoin-induced ROI production and cytotoxicity was observed. These data indicate that nitrofurantoin-induced redox cycling and subsequent generation of ROI are not sufficient to mediate cytotoxicity and that cytochrome P450 reductase is not a determinant of sensitivity to redox-active chemotherapeutic agents.

UVB light upregulates prostaglandin synthases and prostaglandin receptors in mouse keratinocytes

Prostaglandins belong to a class of cyclic lipid-derived mediators synthesized from arachidonic acid via COX-1, COX-2 and various prostaglandin synthases. Members of this family include prostaglandins such as PGE(2), PGF(2alpha), PGD(2) and PGI(2) (prostacyclin) as well as thromboxane. In the present studies we analyzed the effects of UVB on prostaglandin production and prostaglandin synthase expression in primary cultures of undifferentiated and calcium-differentiated mouse keratinocytes. Both cell types were found to constitutively synthesize PGE(2), PGD(2) and the PGD(2) metabolite PGJ(2). Twenty-four hours after treatment with UVB (25 mJ/cm(2)), production of PGE(2) and PGJ(2) increased, while PGD(2) production decreased. This was associated with increased expression of COX-2 mRNA and protein. UVB (2.5-25 mJ/cm(2)) also caused marked increases in mRNA expression for the prostanoid synthases PGDS, mPGES-1, mPGES-2, PGFS and PGIS, as well as expression of receptors for PGE(2) (EP1 and EP2), PGD(2) (DP and CRTH2) and prostacyclin (IP). UVB was more effective in inducing COX-2 and DP in differentiated cells and EP1 and IP in undifferentiated cells. UVB readily activated keratinocyte PI-3-kinase (PI3K)/Akt, JNK and p38 MAP signaling pathways which are known to regulate COX-2 expression. While inhibition of PI3K suppressed UVB-induced mPGES-1 and CRTH2 expression, JNK inhibition suppressed mPGES-1, PGIS, EP2 and CRTH2, and p38 kinase inhibition only suppressed EP1 and EP2. These data indicate that UVB modulates expression of prostaglandin synthases and receptors by distinct mechanisms. Moreover, both the capacity of keratinocytes to generate prostaglandins and their ability to respond to these lipid mediators are stimulated by exposure to UVB.

Arginine metabolism in keratinocytes and macrophages during nitric oxide biosynthesis: Multiple modes of action of nitric oxide synthase inhibitors

Nitric oxide is an important cellular mediator produced in keratinocytes and macrophages from arginine by the enzyme nitric oxide synthase during inflammatory reactions in the skin. We found that gamma-interferon stimulated nitric oxide production and the expression of inducible nitric oxide synthase in both cell types. However, macrophages produced more nitric oxide and nitric oxide synthase protein, and at earlier times than keratinocytes. Keratinocytes treated with gamma-interferon took up more arginine than macrophages; however, they were less efficient in metabolizing this amino acid and exhibited reduced nitric oxide synthase enzyme activity. In both cell types, the nitric oxide synthase inhibitors, N(G)-monomethyl-L-arginine (NMMA), L-N5-(iminoethyl)ornithine, L-canavanine, and N(omega)-nitro-L-arginine, as well as lysine, ornithine, and homoarginine markedly reduced arginine uptake. In contrast, N(omega)-nitro-L-arginine methyl ester and N(omega)-nitro-L-arginine benzyl ester were poor inhibitors of arginine uptake, while aminoguanidine had no effect on uptake of arginine by the cells. Moreover, NMMA was found to inhibit simultaneously arginine uptake and nitric oxide synthase enzyme activity in both cell types, whereas aminoguanidine only affected nitric oxide synthase activity. No major differences were observed between keratinocytes and macrophages. Taken together, these data demonstrate that, although keratinocytes and macrophages both synthesize nitric oxide, its production is regulated distinctly in these two cell types. Furthermore, in these cells, nitric oxide synthase inhibitors such as NMMA exhibit at least two sites of action: inhibition of nitric oxide synthase and cellular uptake of arginine.

UVB light suppresses nitric oxide production by murine keratinocytes and macrophages.

Nitric oxide is an important mediator of excessive cell growth and inflammation associated with many epidermal proliferative disorders. It is a highly reactive oxidant generated in keratinocytes and macrophages via the inducible form of the enzyme nitric oxide synthase (NOS2). In the present studies, we examined the effects of ultraviolet light (UVB, 2.5-25mJ/cm(2)) on interferon-gamma (IFN-gamma)-induced expression of NOS2 in these cells. Transient transfection assays using wild-type and mutant NOS2 promoter/luciferase reporter constructs showed that DNA binding of the transcription factors Stat1 and NF-kappaB was essential for optimal expression of the NOS2 gene. Whereas NF-kappaB was constitutively expressed in both cell types, Stat1 phosphorylation and nuclear binding activity were dependent upon IFN-gamma. UVB light, which is used therapeutically to treat inflammatory dermatosis, was found to suppress IFN-gamma-induced expression of NOS2 mRNA and protein, and nitric oxide production in both keratinocytes and macrophages. In macrophages, this was associated with complete inhibition of NF-kappaB nuclear binding activity and partial (approximately 20-25%) reduction of Stat1 activity. In keratinocytes, both responses were partially reduced at the highest doses of UVB light (15-25mJ/cm(2)). Whereas in macrophages UVB light suppressed NOS2 wild-type promoter-luciferase reporter activity, this activity was stimulated in keratinocytes. These data suggest that UVB light functions to suppress NOS2 gene expression in macrophages by inhibiting the activity of key regulatory transcription factors. In contrast, in keratinocytes, inhibition occurs downstream of NOS2 promoter activity.

Production of hydrogen peroxide by cutaneous T-cell lymphoma following photopheresis with psoralens and ultraviolet light

SummaryTreatment of peripheral blood mononuclear cells with 8-methoxypsoralen (8-MOP) and ultraviolet light, a procedure known as PUVA, has been found to be useful in the management of systemically disseminated cutaneous T-cell lymphoma (CTCL). In the present study we used a highly sensitive flow cytometric assay in conjunction with the hydroperoxide-sensitive dye 2′,7′-dichlorofluorescein diacetate to measure intracellular hydrogen peroxide in normal lymphocytes and CTCL following PUVA treatment. Based on their laser light-scattering properties, lymphocytes were separated into three major subpopulations. We found that ultraviolet light alone caused an increase in the hydrogen peroxide content of each of the subpopulations, a response that was augmented when the cells were pretreated with 8-MOP (50 ng/ml). Cells from CTCL patients were more sensitive to the effects of 8-MOP than were normal lymphocytes. In both cell types, the production of hydrogen peroxide was found to be inhibitable by catalase. We noted an increase in hydrogen peroxide production following photopheresis; however, this was observed only 24 h after treatment. In addition, a further increase in hydrogen peroxide production was observed in lymphocytes isolated from peripheral blood that had been obtained from patients at 15 min after a second photopheresis treatment. Hydrogen peroxide is known to modulate the action of cytokines as well as the immunological responses of leukocytes. Our data suggest that the production of hydrogen peroxide by lymphocytes may be important in the action of PUVA in CTCL.