J. A. Nadel

Cigarette smoke induces bronchoconstrictor hyperresponsiveness to substance P and inactivates airway neutral endopeptidase in the guinea pig. Possible role of free radicals.

We examined the effects of acute exposure to cigarette smoke on the airway responses to substance P in anesthetized guinea pigs and on the activity of airway neutral endopeptidase (NEP). After exposure to air or to cigarette smoke we measured the change in total pulmonary resistance (RL) induced by increasing concentrations of aerosolized substance P in the absence or presence of the NEP inhibitor phosphoramidon. In the absence of phosphramidon the bronchoconstrictor responses to substance P were greater in cigarette smoke-exposed guinea pigs than in air-exposed animals. Phosphoramidon did not further potentiate the responses to substance P in smoke-exposed guinea pigs, whereas it did so in air-exposed animals. In the presence of phosphoramidon, bronchoconstrictor responses to substance P in animals exposed to air or to cigarette smoke were not different. Aerosols of SOD delivered before cigarette smoke exposures dramatically reduced smoke-induced hyperresponsiveness to substance P, whereas heat-inactivated SOD had no effect on smoke-induced hyper-responsiveness to substance P. Cigarette smoke solution inhibited NEP activity from tracheal homogenate in a concentration-dependent fashion, an inhibitory effect that was mostly due to the gas phase of the smoke, but not to nicotine. The mild chemical oxidant N-chlorosuccinimide mimicked the concentration-dependent inhibitory effect of smoke solution on airway NEP activity. We conclude that cigarette smoke causes enhanced airway responsiveness to substance P in vivo by inactivating airway NEP. We suggest that cigarette smoke-induced inhibition of airway NEP is due to effects of free radicals.

Novel Pseudomonas product stimulates interleukin-8 production in airway epithelial cells in vitro.

Because high concentrations of IL-8 are found in the sputum of cystic fibrosis patients, we hypothesized that Pseudomonas aeruginosa (PA) induces the production of IL-8 in airway epithelial cells and in monocytes. Therefore, we incubated the supernatant from PA culture with human transformed bronchial epithelial cells (16-HBE) or with monocytes. The culture medium of 16-HBE cells that had been incubated with PA supernatant for 6 h had chemotactic activity that was inhibited by an antibody to human IL-8. The PA supernatant induced IL-8 production by primary bronchial epithelial cells, by 16-HBE cells, and by monocytes. After incubation with PA supernatant, 16-HBE cells showed a marked increase in the levels of IL-8 gene expression. The PA product responsible for IL-8 production resisted freezing, boiling, and proteolysis. This product was not lipid extractable and was present in a 1-kD filtrate. We conclude that a small molecular mass product of PA stimulates IL-8 production by 16-HBE cells and by monocytes, and that the chemotactic activity produced by 16-HBE cells after exposure to PA is due principally to IL-8.

Hypertonic saline increases vascular permeability in the rat trachea by producing neurogenic inflammation.

In this study, we examined whether inhalation of hypertonic saline aerosols increases vascular permeability in the rat trachea, and we examined the role of neurogenic inflammation in this response. Stereological point counting was performed to measure the percent area occupied by Monastral blue-labeled blood vessels as a means of quantifying the increase in vascular permeability in tracheal whole mounts. Hypertonic saline aerosols (3.6-14.4% NaCl) increased vascular permeability in a dose-dependent fashion compared with 0.9% NaCl. Thus, the area density of Monastral blue-labeled vessels after inhalation of 3.6% NaCl was greater (21.2 +/- 3.5% mean +/- SEM, n = 5) than after 0.9% NaCl aerosol (3.3 +/- 0.9%, n = 5, P less than 0.5). The neutral endopeptidase inhibitor phosphoramidon (2.5 mg/kg, i.v.) significantly potentiated the increase of vascular permeability caused by 3.6% NaCl. Desensitization of sensory nerve endings by pretreatment with capsaicin markedly reduced the usual increase in vascular permeability caused by 3.6% NaCl, but the increase in vascular permeability induced by aerosolized substance P (10(-4) M) was unchanged. These findings suggest that hypertonic saline increases vascular permeability in the rat trachea by stimulating the release of neuropeptides from sensory nerves.

Toluene diisocyanate increases airway responsiveness to substance P and decreases airway neutral endopeptidase.

Substance P and related tachykinins contribute to the airway hyperresponsiveness caused by toluene diisocyanate (TDI) in guinea pigs. Neutral endopeptidase (NEP) is an important modulator of substance P-induced responses. To test the hypothesis that exposure to TDI would increase responsiveness to substance P by inhibiting activity of this enzyme, we determined the dose of substance P required to increase pulmonary resistance by 200% above baseline (PD200) before and after administration of the pharmacologic inhibitor phosphoramidon in guinea pigs studied 1 h after a 1-h exposure to air or 3 ppm TDI. TDI exposure increased responsiveness to substance P significantly. However, phosphoramidon caused a significantly greater leftward shift of the substance P dose-response curve in air-exposed animals than it did in TDI-exposed animals, so that after phosphoramidon, mean values of PD200 in animals exposed to air or TDI did not differ. Tracheal NEP activity was significantly less after exposure to TDI than after exposure to air, whereas activity in the esophagus was the same in both groups. These results suggest that TDI exposure increases the bronchoconstrictor responsiveness of guinea pigs to substance P, in large part through inhibition of airway NEP.

Immunocytochemical localization of arachidonate 15-lipoxygenase in erythrocytes, leukocytes, and airway cells.

In reticulocytes, the enzyme 15-lipoxygenase (15-LO) is believed to contribute to cellular differentiation, and in leukocytes and airway cells 15-LO generates inflammatory mediators. The recent availability of antibodies to 15-LO now allows us to determine which specific cells contain the enzyme, to characterize its subcellular localization, and to determine its expression at the translational level. A polyclonal antibody to recombinant human reticulocyte 15-LO was used with a standard immunofluorescent technique. In rabbit red blood cells, fluorescence appeared during the course of anemia. Early reticulocytes did not fluoresce, but more mature reticulocytes showed increased fluorescent intensity. Late reticulocytes contained little fluorescence. Among human leukocytes, only eosinophils fluoresced. In human trachea, 15-LO immunofluorescence was localized to epithelial cells, and both basal and ciliated cells fluoresced. In all cells studied, fluorescence was localized to the cytoplasm and was variable in degree among cells in each preparation. We conclude that the 15-LO of airway cells and eosinophils is immunologically related to the reticulocyte 15-LO. Furthermore, the variable fluorescence among cells (e.g., in epithelium) and during development (e.g., reticulocytes) suggests a role of 15-LO in cell growth and development.

Glucocorticoids inhibit neurogenic plasma extravasation and prevent virus-potentiated extravasation in the rat trachea.

Capsaicin increases the permeability of blood vessels in the rat tracheal mucosa through a mechanism involving the release of tachykinins from sensory nerves. This capsaicin-induced increase in vascular permeability is potentiated by viral infections of the respiratory tract. The present study was done to determine whether this neurogenic plasma extravasation can be inhibited by glucocorticoids, to learn the time course of this inhibition, and to determine whether glucocorticoids can prevent the potentiating effect of viral respiratory infections on neurogenic plasma extravasation. Groups of pathogen-free F344 rats were treated with dexamethasone for 2 or 8 h (4 mg/kg i.p.) or 48 or 120 h (0.5-4 mg/kg per d i.p.). Another group of rats was treated with dexamethasone for 120 h following the intranasal inoculation of Sendai virus. The magnitude of plasma extravasation produced by capsaicin or substance P was assessed after this treatment by using Monastral blue pigment and Evans blue dye as intravascular tracers. We found that dexamethasone reduced, in a dose-dependent fashion, the magnitude of plasma extravasation produced in the rat trachea by capsaicin and substance P. Significant inhibition was produced by a dose of dexamethasone as small as 0.5 mg/kg i.p. The effect of dexamethasone had a latency of several hours and reached a maximum after 2 d of treatment. Furthermore, dexamethasone prevented the potentiation of neurogenic plasma extravasation usually present after 5 d of Sendai virus respiratory infection.

Neutral endopeptidase and kininase II mediate glucocorticoid inhibition of neurogenic inflammation in the rat trachea.

Glucocorticoids inhibit plasma extravasation induced in the rat tracheal mucosa by substance P and other tachykinins released from sensory nerves. This study was performed to determine whether this antiinflammatory effect of glucocorticoids is mediated by the tachykinin-degrading enzymes neutral endopeptidase (NEP) and kininase II (angiotensin converting enzyme, ACE). In addition, we studied the effect of dexamethasone on a nonpeptide inflammatory mediator, platelet-activating factor (PAF), which is not degraded by NEP or ACE. Adult male pathogen-free F344 rats were treated for 2 d with dexamethasone (0.5 mg/kg per d i.p.), or with the vehicle used to dissolve the steroid. The magnitude of plasma extravasation produced by an intravenous injection of substance P (5 micrograms/kg) or PAF (10 micrograms/kg) was then assessed by using Monastral blue pigment as an intravascular tracer. The role of NEP and ACE activities in the changes produced by dexamethasone was investigated by examining the effect of the selective inhibitors of these enzymes, phosphoramidon and captopril. Dexamethasone reduced the substance P-induced extravasation by 57% but did not affect the PAF-induced extravasation. The suppressive effect of dexamethasone on substance P-induced extravasation was completely reversed by simultaneously inhibiting NEP and ACE activities, but the inhibition of these enzymes had no effect on PAF-induced extravasation, regardless of whether the rats were pretreated with dexamethasone or not. These results suggest that NEP and ACE mediate a selective inhibitory effect of glucocorticoids on neurogenic plasma extravasation.

Bronchodilation by pituitary adenylate cyclase-activating peptide and related peptides

Pituitary adenylate cyclase-activating peptide (PACAP) is present in nerves in the vicinity of bronchial and vascular smooth muscle in the airways. At least one endogenous form of PACAP, PACAP 1-27, has high affinity binding sites in the lung, probably including cholinergic nerve terminals, bronchial smooth muscle, epithelial and mononuclear inflammatory cells. The mechanism of action for PACAP 1-27 and 1-38 in vivo involves endogenous catecholamines, peptidases and nitric oxide, depending on tissue type. Intracellularly, cyclic adenosine monophosphate (cAMP) as well as calcium and sodium mobilization is probably involved. PACAP 1-27 and 1-38 inhibit airway smooth muscle tone in vitro and in vivo. The inhibitory effect of PACAP 1-38 is more sustained than that of PACAP 1-27, in vitro as well as in vivo. PACAP 1-38 also causes more sustained inhibition of bronchoconstriction after inhalation in vivo, than does vasoactive intestinal peptide (VIP). PACAP 1-27 given intravenously virtually abolishes allergen-induced bronchoconstriction in vivo. Novel synthetic analogues of PACAP 1-27 cause more sustained inhibition of airway smooth muscle tone in vitro and in vivo than do PACAP 1-27 or 1-38. Both PACAP 1-27 and 1-38 inhibit arterial smooth muscle tone but, administration of PACAP 1-27, 1-38 or a structural analogue of PACAP 1-27 in the airways, induces no cardiovascular side effects at doses inhibiting bronchoconstriction. PACAP 1-38 enhances phagocytosis in macrophages and inhibits the release of the pro-inflammatory cytokine interleukin-2 in lymphocytes, suggesting antiinflammatory effects. It is concluded that pituitary adenylate cyclase-activating peptide 1-27 and 1-38, or structurally related molecules, may be useful as bronchodilators but their effect on human bronchial smooth muscle and on human inflammatory cells is in need of evaluation.

Cystic fibrosis sputum induces a secretory response from airway gland serous cells that can be prevented by neutrophil protease inhibitors

High activities of the neutrophil proteases, elastase and cathepsin G, are found in the sputum of patients with cystic fibrosis (CF). Because both proteases have been shown to be potent secretagogues for airway submucosal glands, and because hypersecretion is a characteristic feature of CF, the objective of the present study was to examine whether there is secretagogue activity in CF sputum, and to determine the contribution of neutrophil proteases to the secretagogue activity. Confluent monolayers of cultured bovine tracheal serous cells were pulse-labelled with Na2(35)SO4, incubated with diluted CF sputum supernatants in the presence or absence of different protease inhibitors, and the subsequent release of the radio-labelled macromolecules was measured. CF sputum potently induced secretion concentration-dependently. Addition of the selective neutrophil elastase inhibitor ICI 200,355 inhibited the secretory response to CF sputum supernatant by 89%. Addition of a cathepsin G-inhibitor resulted in further inhibition of the secretory response. Addition of phosphoramidon, a drug known to inhibit Pseudomonas aeruginosa elastase, had no effect. We conclude that CF sputum potently stimulates airway submucosal gland cell secretion. These studies with protease inhibitors suggest that neutrophil proteases account substantially for the secretagogue activity present in CF sputum.

Recombinant Neutral Endopeptidase Attenuates Substance P-Induced Plasma Extravasation in the Guinea Pig Skin

To determine whether exogenously administered neutral endopeptidase (NEP; enkephalinase, EC 3.4.24.11) inhibits the substance P-induced increase in vascular permeability in the skin, we examined the effects of recombinant human NEP on plasma extravasation induced by intradermal injection of substance P in guinea pig skin. Injection of substance P (2.5 X 10(-8) M) induced significant plasma extravasation in the skin (53 +/- 4 mm2 of Evans blue extravasation; mean +/- 1 SEM). In vitro incubation of substance P with recombinant human NEP prior to injection prevented the substance P-induced plasma extravasation in the skin in a dose-dependent fashion. Intradermal preinjection of recombinant human NEP partially inhibited plasma extravasation induced by subsequent injection of substance P (52 +/- 9% of the control without NEP). The H1 and H2 histamine antagonists pyrilamine and cimetidine, and a muscarinic antagonist, atropine, had no effects on substance P-induced responses. Two products of substance P degradation by NEP containing the carboxy-terminal portion, substance P7-11 and substance P8-11, were also without effect. These findings suggest that recombinant human NEP can attenuate substance P-induced increases in vascular permeability in guinea pig skin and, therefore, may be useful in treating dermatologic disorders in which abnormal responses to substance P or other neuropeptides cleaved by NEP may occur.