Imad Y. Haddad

Quantitation of nitrotyrosine levels in lung sections of patients and animals with acute lung injury.

Activated alveolar macrophages and epithelial type II cells release both nitric oxide and superoxide which react at near diffusion-limited rate (6.7 x 10(9) M-1s-1) to form peroxynitrite, a potent oxidant capable of damaging the alveolar epithelium and pulmonary surfactant. Peroxynitrite, but not nitric oxide or superoxide, readily nitrates phenolic rings including tyrosine. We quantified the presence of nitrotyrosine in the lungs of patients with the adult respiratory distress syndrome (ARDS) and in the lungs of rats exposed to hyperoxia (100% O2 for 60 h) using quantitative immunofluorescence. Fresh frozen or paraffin-embedded lung sections were incubated with a polyclonal antibody to nitrotyrosine, followed by goat anti-rabbit IgG coupled to rhodamine. Sections from patients with ARDS (n = 5), or from rats exposed to hyperoxia (n = 4), exhibited a twofold increase of specific binding over controls. This binding was blocked by the addition of an excess amount of nitrotyrosine and was absent when the nitrotyrosine antibody was replaced with nonimmune IgG. In additional experiments we demonstrated nitrotyrosine formation in rat lung sections incubated in vitro with peroxynitrite, but not nitric oxide or reactive oxygen species. These data suggest that toxic levels of peroxynitrite may be formed in the lungs of patients with acute lung injury.

Safety of inhaled nitric oxide after lung transplantation.

BACKGROUND The present study tests the hypothesis that therapy with inhaled nitric oxide (iNO) at the time of lung transplantation in patients undergoing bilateral angle lung transplantation: (i) is safe; and (ii) does not increase either the duration of mechanical ventilation or the incidence of acute graft dysfunction. METHODS We conducted a prospective, non-randomized trial of iNO at 20 parts per million. The treatment group was comprised of 14 patients (10 females, 4 males) undergoing lung transplantation to address severe end-stage lung disease and pulmonary hypertension (mean pulmonary artery pressure > 30 mmHg). Clinical and histologic parameters were compared with 22 historical control subjects who were matched with the study population for age, diagnosis and disease severity (17 females, 5 males) and had undergone lung transplantation in the preceding 2-year time period. No significant differences were noted between the 2 study groups at baseline. RESULTS No toxic effect of iNO treatment was evident. Although the incidence of acute graft dysfunction was the same in both groups, the occurrence of acute graft rejection in the initial 4 weeks after transplant was less frequent in the iNO group than in the control group (7% vs 32%, p = 0.05). Fifty percent of the treatment group, as compared with 22% of the control group, were discharged from the hospital within 2 weeks of the procedure (p = 0.05). CONCLUSIONS Early initiation of iNO in lung transplant patients with pulmonary hypertension is safe and may decrease the incidence of acute graft rejection. We speculate that iNO may exert an immunomodulatory effect.

Interactions of keratinocyte growth factor with a nitrating species after marrow transplantation in mice

We reported that allogeneic T cells given to irradiated mice at the time of marrow transplantation stimulated tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and nitric oxide (⋅ NO) production in the lung, and the addition of cyclophosphamide (known to stimulate superoxide production) favored the generation of a nitrating species. Although keratinocyte growth factor (KGF) prevents experimental lung injury by promoting epithelial repair, its effects on the production of inflammatory mediators has not been studied. KGF given before transplantation inhibited the T cell-induced increase in bronchoalveolar lavage fluid protein, TNF-α, IFN-γ, and nitrite levels measured on day 7 after transplantation without modifying cellular infiltration or proinflammatory cytokines and inducible ⋅ NO synthase mRNA. KGF also suppressed ⋅ NO production by alveolar macrophages obtained from mice injected with T cells. In contrast, the same schedule of KGF failed to prevent permeability edema or suppress TNF-α, IFN-γ, and ⋅ NO production in mice injected with both T cells and cyclophosphamide. Because only epithelial cells respond to KGF, these data are consistent with the production of an epithelial cell-derived mediator capable of downregulating macrophage function. However, the presence of a nitrating agent impairs KGF-derived responses.We reported that allogeneic T cells given to irradiated mice at the time of marrow transplantation stimulated tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and nitric oxide (. NO) production in the lung, and the addition of cyclophosphamide (known to stimulate superoxide production) favored the generation of a nitrating species. Although keratinocyte growth factor (KGF) prevents experimental lung injury by promoting epithelial repair, its effects on the production of inflammatory mediators has not been studied. KGF given before transplantation inhibited the T cell-induced increase in bronchoalveolar lavage fluid protein, TNF-alpha, IFN-gamma, and nitrite levels measured on day 7 after transplantation without modifying cellular infiltration or proinflammatory cytokines and inducible. NO synthase mRNA. KGF also suppressed. NO production by alveolar macrophages obtained from mice injected with T cells. In contrast, the same schedule of KGF failed to prevent permeability edema or suppress TNF-alpha, IFN-gamma, and. NO production in mice injected with both T cells and cyclophosphamide. Because only epithelial cells respond to KGF, these data are consistent with the production of an epithelial cell-derived mediator capable of downregulating macrophage function. However, the presence of a nitrating agent impairs KGF-derived responses.

Nitric oxide inhibits heterologous CFTR expression in polarized epithelial cells

Nitric oxide (⋅ NO) has been implicated in a wide range of autocrine and paracrine signaling mechanisms. Herein, we assessed the role of exogenous ⋅ NO in the modulation of heterologous gene expression in polarized kidney epithelial cells (LLC-PK1) that were stably transduced with a cDNA encoding human wild-type cystic fibrosis transmembrane conductance regulator (CFTR) under the control of a heavy metal-sensitive metallothionein promoter (LLC-PK1-WTCFTR). Exposure of these cells to 125 μM DETA NONOate at 37°C for 24 h (a chemical ⋅ NO donor) diminished Zn2+-induced and uninduced CFTR protein levels by 43.3 ± 5.1 and 34.4 ± 17.1% from their corresponding control values, respectively. These changes did not occur if red blood cells, effective scavengers of ⋅ NO, were added to the medium. Exposure to ⋅ NO did not alter lactate dehydrogenase release in the medium or the extent of apoptosis. Coculturing LLC-PK1-WTCFTR cells with murine fibroblasts that were stably transduced with the human inducible ⋅ NO synthase cDNA gene also inhibited CFTR protein expression in a manner that was antagonized by 1 mM N G-monomethyl-l-arginine in the medium. Pretreatment of LLC-PK1-WTCFTR with ODQ, an inhibitor of guanylyl cyclase, did not affect the ability of ⋅ NO to inhibit heterologous CFTR expression; furthermore, 8-bromo-cGMP had no effect on heterologous CFTR expression. These data indicate that ⋅ NO impairs the heterologous expression of CFTR in epithelial cells at the protein level via cGMP-independent mechanisms.Nitric oxide (. NO) has been implicated in a wide range of autocrine and paracrine signaling mechanisms. Herein, we assessed the role of exogenous. NO in the modulation of heterologous gene expression in polarized kidney epithelial cells (LLC-PK(1)) that were stably transduced with a cDNA encoding human wild-type cystic fibrosis transmembrane conductance regulator (CFTR) under the control of a heavy metal-sensitive metallothionein promoter (LLC-PK(1)-WTCFTR). Exposure of these cells to 125 microM DETA NONOate at 37 degrees C for 24 h (a chemical. NO donor) diminished Zn(2+)-induced and uninduced CFTR protein levels by 43.3 +/- 5.1 and 34.4 +/- 17.1% from their corresponding control values, respectively. These changes did not occur if red blood cells, effective scavengers of. NO, were added to the medium. Exposure to. NO did not alter lactate dehydrogenase release in the medium or the extent of apoptosis. Coculturing LLC-PK(1)-WTCFTR cells with murine fibroblasts that were stably transduced with the human inducible. NO synthase cDNA gene also inhibited CFTR protein expression in a manner that was antagonized by 1 mM N(G)-monomethyl-L-arginine in the medium. Pretreatment of LLC-PK(1)-WTCFTR with ODQ, an inhibitor of guanylyl cyclase, did not affect the ability of. NO to inhibit heterologous CFTR expression; furthermore, 8-bromo-cGMP had no effect on heterologous CFTR expression. These data indicate that. NO impairs the heterologous expression of CFTR in epithelial cells at the protein level via cGMP-independent mechanisms.

Exuberant Inflammation in Nicotinamide Adenine Dinucleotide Phosphate-Oxidase-Deficient Mice After Allogeneic Marrow Transplantation

We have shown that NO and superoxide ()contribute to donor T cell-dependent lung dysfunction after bone marrow transplantation (BMT) in mice. We hypothesized that inhibiting production during inducible NO synthase induction would suppress oxidative/nitrative stress and result in less severe lung injury. Irradiated mice lacking the phagocytic NADPH-oxidase (phox−/−), a contributor to generation, were conditioned with cyclophosphamide and given donor bone marrow in the presence or absence of inflammation-inducing allogeneic spleen T cells. On day 7 after allogeneic BMT, survival, weight loss, and indices of lung injury between phox−/− and wild-type mice were not different. However, the majority of macrophages/monocytes from phox−/− mice given donor T cells produced fewer oxidants and contained less nitrotyrosine than cells obtained from T cell-recipient wild-type mice. Importantly, suppressed oxidative stress was associated with marked infiltration of the lungs with inflammatory cells and was accompanied by increased bronchoalveolar lavage fluid levels of the chemoattractants monocyte chemoattractant protein-1 and macrophage-inflammatory protein-1α and impaired clearance of recombinant mouse macrophage-inflammatory protein-1β from the circulation. Furthermore, cultured macrophages/monocytes from NADPH-deficient mice produced 3-fold more TNF-α compared with equal number of cells from NADPH-sufficient mice. The high NO production was not modified during NADPH-oxidase deficiency. We conclude that phox−/− mice exhibit enhanced pulmonary influx of inflammatory cells after BMT. Although NO may contribute to increased production of TNF-α in phox−/− mice, the data suggest that NADPH-oxidase-derived oxidants have a role in limiting inflammation and preventing lung cellular infiltration after allogeneic transplantation.

Simultaneous absence of surfactant proteins A and D increases lung inflammation and injury after allogeneic HSCT in mice.

The relative contributions of the hydrophilic surfactant proteins (SP)-A and -D to early inflammatory responses associated with lung dysfunction after experimental allogeneic hematopoietic stem cell transplantation (HSCT) were investigated. We hypothesized that the absence of SP-A and SP-D would exaggerate allogeneic T cell-dependent inflammation and exacerbate lung injury. Wild-type, SP-D-deficient (SP-D(-/-)), and SP-A and -D double knockout (SP-A/D(-/-)) C57BL/6 mice were lethally conditioned with cyclophosphamide and total body irradiation and given allogeneic bone marrow plus donor spleen T cells, simulating clinical HSCT regimens. On day 7, after HSCT, permeability edema progressively increased in SP-D(-/-) and SP-A/D(-/-) mice. Allogeneic T cell-dependent inflammatory responses were also increased in SP-D(-/-) and SP-A/D(-/-) mice, but the altered mediators of inflammation were not identical. Compared with wild-type, bronchoalveolar lavage fluid (BALF) levels of nitrite plus nitrate, GM-CSF, and MCP-1, but not TNF-alpha and IFN-gamma, were higher in SP-D-deficient mice before and after HSCT. In SP-A/D(-/-) mice, day 7 post-HSCT BALF levels of TNF-alpha and IFN-gamma, in addition to nitrite plus nitrate and MCP-1, were higher compared with mice lacking SP-D alone. After HSCT, both SP-A and SP-D exhibited anti-inflammatory lung-protective functions that were not completely redundant in vivo.

Mechanisms of Nitric Oxide Induced Injury to the Alveolar Epithelium

The major function of the lung is gas exchange. The movement of both oxygen and carbon dioxide across the blood-gas barrier is by simple diffusion. This process is optimized by the large alveolar surface area, the close proximity of the alveolar and pulmonary capillary membranes, and the lack of any significant amount of fluid in the alveolar space. The relative dryness of the alveolar space is thought to be the result of: (1) the low permeability of the alveolar epithelium to both electrolytes and plasma proteins; (2) the presence of pulmonary surfactant, which lowers the surface tension of the blood-gas interface; and (3) the ability of alveolar epithelial cells to actively transport sodium ions from the alveolar to the basolateral spaces1.

Pneumonia and Empyema

In humans, the lung represents the largest epithelial surface of the body exposed to the external environment. This area is 40-fold larger than the skin. As a consequence, the upper airways and lower lung are continuously exposed to a variety of airborne particles and microbial agents. Despite this constant attack, sterility of the conducting airways, bronchioles, and alveoli is maintained by a complex pulmonary host defense system. Throughout the upper (nasopharynx) and lower (conducting airways and alveolar spaces) respiratory tracts, the innate and adaptive immune systems work synchronously to identify and eliminate foreign non-self particles, including microbes. In invertebrates, the innate system is the sole mechanism of host defense against pathogens, but in higher vertebrates it constitutes the fi rst line of defense. The innate defenses are constitutive, rapid, and nonspecifi c. The innate system is based on pattern recognition of repetitive molecular patterns shared by microorganisms. Major advances in innate immunity have focused on the discovery of a series of cell-surface receptors called toll-like receptors (TLRs), fi rst described in Drosophila, but now at least 11 homologues have been discovered in humans [1] and 13 homologues in mice. Individual TLRs differ in their ligand specifi cities (Figure 17.1). The interaction between a TLR and a microbial component triggers adaptor proteins and signal molecules, leading to transcription factors activation, production of proinfl ammatory cytokines, and expression of host defense peptides [2]. Importantly, the innate system and TLR activation also induce co-stimulatory molecules that stimulate and drive the inducible and slower specifi c adaptive immune system such that antigen-presenting cells present antigen to T helper (Th) cells that differentiate along two pathways: the Th1 pathway, important in cell-mediated immunity, and Th2 pathway involved in humoral responses [3]. Mechanical defenses also play a major role in respiratory host defense. Aerodynamic fi ltration in the nose and nasopharynx prevents particles that are >10 μm from passing to the lower respiratory tract. Particles from 5 to 10 μm are fi ltered by impaction in the conducting airways. Material deposited along the airways is removed by the mucociliary system, which starts in the nasopharynx and ends in the terminal bronchioles. Ciliary beating occurs in a precise and well-orchestrated fashion, propelling mucus and deposited organisms toward the oropharynx. A fi nal constituent of the mechanical defense of the respiratory tract is cough. This Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 Pulmonary Host Defense . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 Pneumonia Types in the Pediatric Intensive Care Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206 General Treatment Principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210

Pulmonary Vascular Voltage-Sensitive K+ Channel Expression is Developmentally Regulated † 1630

Pulmonary Vascular Voltage-Sensitive K+ Channel Expression is Developmentally Regulated † 1630

NITRIC OXIDE MODULATION OF GENE EXPRESSION

The free radical nitric oxide (·NO) is the major form of the endothelial-derived relaxing factor that is enzymatically synthesized from arginine oxidation by an NADPH-dependent nitric oxide synthase. ·NO causes smooth muscle relaxation by activating soluble guanylate cyclase, and inhibits platelet aggregation and adhesion to endothelium by increasing cGMP41. Because of the vasorelaxant properties of ·NO and its rapid inactivation in the blood by its reaction with hemoglobin, ·NO inhalation has been advocated as a means of selectively reducing pulmonary hypertension and improving ventilation/perfusion mismatching in a variety of clinical situations9,40.