Increased expression of inducible nitric oxide synthase and cyclo-oxygenase-2 in the airway epithelium of asthmatic subjects and regulation by corticosteroid treatment.

BACKGROUND: Nitric oxide (NO) and prostanoids are mediators of vascular and bronchial tone that are postulated to be involved in asthma. Increased levels of both are found in asthmatic subjects and are synthesised by enzymes that have cytokine inducible forms: inducible NO synthase (iNOS) and cyclo-oxygenase-2 (COX-2), respectively. We hypothesised that the in vivo expression of iNOS and COX-2 in the airways would be increased in asthma, and that these cytokine inducible enzymes may represent targets for regulation by corticosteroid treatment. METHODS: Bronchial biopsy specimens were obtained from three groups of subjects: atopic asthmatics treated with beta(2) agonists alone (n=7), atopic asthmatics additionally receiving regular treatment with corticosteroids (n=8), and non-asthmatic control subjects (n=10). Expression of iNOS and COX-2 mRNA and immunoreactive protein was studied using in situ hybridisation and quantitative immunohistochemistry. RESULTS: Immunoreactivity and the hybridisation signal for iNOS and COX-2 were mainly localised in the airway epithelium. The proportion of epithelium immunostained was significantly greater in the non-steroid treated asthmatic subjects (iNOS 8.6 (1.8)%; COX-2 26.3 (4.6)%) than either the steroid treated asthmatics (iNOS 3.4 (1.0)%, p=0.009; COX-2 13.0 (0.6)%, p=0.0015) or the non-asthmatic controls (iNOS 4.2 (0.9)%, p=0.018; COX-2 11.6 (0.6)%, p=0.0003). Similarly, the hybridisation signal was stronger in the non-steroid treated group of asthmatic subjects than in the other two groups. CONCLUSIONS: These findings highlight the potential role of the airway epithelium both as a contributor to the inflammatory process in asthma and as a target for inhaled corticosteroid treatment in this disease.

Validation of endobronchial biopsy specimens for nerve quantitation by computer-assisted image analysis.

The aim of this study was to assess the validity of endoscopic bronchial biopsy specimens for the quantitation of nerves. To this end, endobronchial biopsy was simulated ex vivo on surgically resected lung specimens and nerve densities were compared in airway smooth muscle of biopsy and surrounding tissue. Specimens were stained immunohistochemically for the general neural marker protein gene product 9.5 (PGP 9.5) and for vasoactive intestinal peptide (VIP), and nerve densities were quantitated using computer-assisted image analysis. Nerve density for total (PGP 9.5-immunoreactive) nerves was slightly higher in biopsies than in corresponding lung tissue, but this difference did not reach statistical significance (p=0.08). There was also no significant difference in the density of VIP-immunoreactive nerves (p=0.60). These findings support the use of endobronchial biopsy specimens to quantitate nerves in asthma and other airway diseases.

Airway endothelin levels in asthma: influence of endobronchial hypertonic saline challenge.

BACKGROUND: The pathophysiology of exercise-induced asthma is not well understood. Hypertonicity of the airway lining fluid resulting from loss of water due to hyperventilation is considered to play a role, but the precise mechanism by which hypertonicity can induce bronchoconstriction is unknown. Peptides of the endothelin (ET) family have potent smooth muscle contractile properties, and have been linked to airway narrowing in stable asthma. We postulated that ET release may contribute to the acute bronchoconstrictor response induced by a hypertonic stimulus. METHODS: Seven male asthmatic subjects underwent local endobronchial challenge with hypertonic (3.6%) saline and, as a control, isotonic (0.9%) saline aerosols in separate bronchopulmonary segments. Bronchoalveolar lavage (BAL) was performed at both sites during the phase of immediate bronchoconstriction. Concentrations of immunoreactive ET and of the mast cell products, histamine, tryptase and prostaglandin D2, in BAL fluid were measured. RESULTS: Concentrations of ET in BAL fluid from the hypertonic saline-challenged sites were significantly lower than those in BAL fluid from sites exposed to isotonic saline (0.19 [0.11-1.24] fmol/mL vs. 0.40 [0.20-2.36] fmol/mL, P<0.05). Concentrations of histamine, tryptase, and prostaglandin D2 did not differ significantly between the two sites. CONCLUSIONS: These findings do not support the hypothesis that ET release within the airway lumen is involved in the bronchoconstrictor response induced by hypertonic saline.

High expression of endothelial nitric oxide synthase in plexiform lesions of pulmonary hypertension.

The pathogenesis of pulmonary hypertension (PH) remains poorly understood. Vasoconstriction, although likely to be a major factor in the disease, varies between patients and studies of a variety of vasoactive substances have sometimes yielded conflicting results. Amongst these substances, alteration of the nitric oxide (NO) system has been cited as a possible pathogenic factor but both reduction and elevation of the expression of endothelial NO-synthase (eNOS) have been reported in pulmonary vessels. The present study has used immunocytochemistry with well-characterized antibodies to eNOS to investigate its expression in lung tissue taken at transplantation from 44 patients with PH (22 primary, 22 secondary) and 12 non-hypertensive controls. Semi-quantitative assessment showed that although the levels of eNOS expression in pulmonary vessels were variable within both hypertensives and controls, a statistically significant (P < 0.01) reduction of immunoreactivity was found in small arterioles from hypertensives compared with controls. In contrast, consistently strong expression of eNOS was seen in the endothelium of plexiform lesions in both the primary and the secondary PH patients. Although a decrease in the NO system of patients with PH has been reported, these findings show a distinct regional distribution of the enzyme with particularly high levels in plexiform lesions, a previously unreported observation, and offer a new perspective on the disease and on the evaluation of possible novel therapeutic approaches.

Induction of nitric oxide synthase in human vascular smooth muscle: interactions between proinflammatory cytokines.

OBJECTIVE: We have attempted to demonstrate the induction of inducible nitric oxide synthase in human vascular tissue and define the capacity of different cytokines to induce this enzyme. METHODS: Segments of human arteries were stimulated with lipopolysaccharide (10 micrograms/ml), interleukin-1 beta (5 U/ml), tumor necrosis factor-alpha (10 U/ml), and interferon-gamma (200 U/ml). Cytokines were either used alone or in certain combinations, as well as in the presence of L-NG-monomethyl-arginine (100 mumol/l) or cycloheximide (1 mumol/l). Induction was assessed by measurement of mRNA expression, immunocytochemical localisation of the expressed protein, nitric oxide synthase activity and levels of nitrite, a product of nitric oxide formation. RESULTS: PCR analysis showed the presence of mRNA for iNOS in stimulated samples which could be inhibited by cycloheximide. There was positive staining with an antibody against human iNOS in the media of stimulated vessel segments. Stimulated segments were also shown to contain Ca(2+)-independent nitric oxide synthase activity. The cytokines and lipopolysaccharide together gave a significant rise in levels of nitrite in the medium after 36 and 48 h, which was inhibited by L-NG-monomethyl-arginine and cycloheximide. Only interferon-gamma incubated alone was capable of increasing nitrite levels. This effect was enhanced by co-incubation with either interleukin-1 beta, tumor necrosis factor-alpha or lipopolysaccharide. CONCLUSION: We have shown that increased production of nitrite by human vascular tissue in response to cytokines is associated with induction of iNOS as shown at the molecular and protein levels, and further supported by the presence of increased Ca(2+)-independent nitric oxide synthase activity following cytokine stimulation.

Expression of inducible nitric oxide synthase and formation of peroxynitrite in posttransplant obliterative bronchiolitis.

BACKGROUND: Obliterative bronchiolitis is characterized histologically by inflammation, epithelial cell damage and loss, fibrosis, and eventual obliteration of airways. Production of high levels of the potential cytotoxin nitric oxide by inducible nitric oxide synthase has been implicated in several inflammatory diseases. The damaging effects of nitric oxide are mediated by peroxynitrite, are formed from nitric oxide and superoxide, and can be demonstrated by the detection of nitrotyrosine. Our previous finding of high inducible nitric oxide synthase expression in inflamed airway epithelium led us to hypothesize that release of nitric oxide in obliterative bronchiolitis mediates the characteristic epithelial damage. METHODS: Immunocytochemistry was carried out to seek expression of inducible nitric oxide synthase and nitrotyrosine in transplant samples from patients with obliterative bronchiolitis (n=10) and, as controls, unused donor lungs (n=5). RESULTS: Inducible nitric oxide synthase was strongly expressed in the damaged airway epithelium in obliterative bronchiolitis and in inflammatory cells, where its distribution was matched by that of nitrotyrosine. Normal controls showed little or no immunoreactivity for any of the antigens studied. CONCLUSIONS: Our findings suggest that nitric oxide may play a role in the pathogenesis of obliterative bronchiolitis and indicate that further work is essential to fully understand the processes and mechanisms involved.

Immunization and challenge with toluene diisocyanate decrease tachykinin and calcitonin gene-related peptide immunoreactivity in guinea pig central airways.

Toluene diisocyanate (TDI) is a potent sensitizer that causes occupational asthma in a significant proportion of subjects exposed. We used an animal model to investigate whether neuropeptide changes occur in the airways of immunized and TDI-challenged guinea pigs. Animals were immunized by weekly intradermal injections, challenged with TDI (5 to 20 ppb) after the third injection, and killed 6 h after exposure. Control guinea pigs received injections of saline. Lung tissue was processed immediately and analyzed for nerves using the streptavidin-biotin complex peroxidase method with antisera to the neural marker protein gene product 9.5 (PGP 9.5), substance P (SP), and calcitonin gene- related peptide (CGRP). We also quantified the inflammatory infiltrate in the submucosa of central airways, and we measured the serum level of specific IgG and IgG1. Specific antibodies against TDI were present only in immunized animals. Immunized as compared with nonimmunized animals had a significant increase in eosinophils in the submucosa of central airways, and a further increase was observed 6 h after TDI challenge. Immunization and TDI challenge did not modify the number of mononuclear cells in the submucosa of central airways in both nonimmunized and immunized animals. TDI exposure did not change the overall innervation in both nonimmunized and immunized animals, but the density of PGP 9.5-positive nerves was significantly different between nonimmunized and immunized TDI-challenged animals. The density of SP-, and CGRP-immunostained nerves was significantly lower in immunized TDI-challenged than in nonimmunized animals. TDI exposure significantly decreased the density of SP-positive nerves in nonimmunized animals. A negative relationship was found between the presence of airway inflammation, as indexed by eosinophil cell infiltration, and the density of PGP 9.5-, SP-, and CGRP-immunostained nerves. In conclusion, TDI produces airway inflammation and neuropeptides changes in the central airways of immunized guinea pigs 6 h after TDI challenge. These findings support an interaction between tachykinins, inflammatory (i.e., eosinophils) and possibly immune cells.

Structural, biochemical and functional effects of distending pressure in the human saphenous vein: implications for bypass grafting.

BACKGROUND: Distension of the saphenous vein before and after coronary artery bypass grafting results in damage to mechanisms that regulate vascular tone. We have investigated the relationship between the magnitude of distending pressure and the degree of structural, biochemical and functional damage to the vessel wall. METHODS: Vessel segments that had been distended to either 100 or 300 mmHg were set up in isolated organ baths and the function of the smooth muscle and endothelial cells examined. All segments examined were then fixed for assessment of structural damage by scanning electron microscopy and for immunocytochemical localisation of endothelial nitric oxide synthase. RESULTS: Segments of saphenous vein distended to 100 mmHg retained their responsiveness to KCl (90 mmol/l) and phenylephrine (10(-6) mol/l), but those pressurised to 300 mmHg had significantly reduced responses to both agents. There was also a significant reduction in response to the endothelium-dependent dilators, acetylcholine (10(-10)-10(-6) mol/l) and bradykinin (10(-10)-10(-6) mol/l) in those segments distended to 300 mmHg. Quantitative studies of structural endothelial damage showed a significant loss of endothelium at 300 mmHg distension pressure. Remaining endothelial cells retained strong positive staining for endothelial nitric oxide synthase. By electron microscopic examination, those vessels distended to 100 mmHg showed lifting and rounding of individual cells, whereas segments distended to 300 mmHg revealed major areas of denuded endothelium. CONCLUSIONS: Distension of saphenous veins to pressures equivalent to those in the systemic circulation result in structural and biochemical changes in the endothelium that are not paralleled by immediate functional vasomotor changes.

Lack of inducible nitric oxide synthase in bronchial epithelium: a possible mechanism of susceptibility to infection in cystic fibrosis.

Cystic fibrosis (CF) is an inherited disorder associated with severe inflammation and repeated bacterial infection and colonization in the lung. Airway epithelium is involved in defence against bacteria, but this system may be defective in CF. Pro-inflammatory cytokines can stimulate the expression of inducible nitric oxide synthase (iNOS), an enzyme generating nitric oxide, which functions as an important mediator in host defence mechanisms. To understand better the poor resistance to infections in the CF lung, the expression of the iNOS gene was investigated in explanted lungs from patients with cystic fibrosis (n = 13), bronchiectasis (n = 3), emphysema (n = 14), and in normal lungs (n = 8). In addition, bronchial epithelial cell lines were examined to study iNOS gene expression in vitro. Strong immunoreactivity for iNOS was seen in inflammatory cells and bronchial epithelium in all the diseased lungs, except for bronchial epithelium in CF. Quantitative analysis showed a significant reduction in the area of epithelium immunostained in CF [CF 6.8 +/- 1.6 (% +/- SEM); emphysema 18.2 +/- 2.8; normal 9.6 +/- 0.8, P < 0.01], regardless of steroid treatment. These results were supported by in situ hybridization of iNOS mRNA, which showed a pattern of gene expression in CF, emphysema, and normal lung which paralleled that of protein immunoreactivity. Stimulation with cytokines (IL-1 beta, TNF-alpha, and IFN-gamma) increased the expression of iNOS mRNA detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in cultures of normal (16HBE14o-), but not CF (CFBE41o-, with delta F508 CFTR mutation) epithelial cells. Expression of iNOS in inflammatory cells suggests that the gene is normal in CF. Absence of iNOS from bronchial epithelium may be due to low expression of the gene resulting from abnormalities in the signalling system that normally causes induction, such as cytokine receptors, second messengers or transcription factors. The resulting deficiency of the nitric oxide defence system may be relevant to the susceptibility of CF patients to pulmonary bacterial colonization.

Increased VIP-positive nerve fibers in the mucous glands of subjects with chronic bronchitis.

The presence and distribution of neuropeptide-containing nerves within bronchial surgical specimens has been investigated in bronchitic (n = 12) and in nonbronchitic subjects (n = 7). Lung tissue, obtained from patients undergoing thoracotomy for limited lung lesions, was processed immediately and analyzed for nerves using the streptavidin-biotin complex peroxidase method with antisera to the neural marker protein gene product 9.5 (PGP 9.5) and the neuropeptides vasoactive intestinal peptide (VIP), substance P (SP), calcitonin-gene related peptide (CGRP). There were no significant differences between the two groups with respect to the density of PGP 9.5-, SP-, or CGRP-positive nerves in both the locations assessed (smooth muscle layer and glands). The density of VIP-positive nerves was significantly higher in the glands of bronchitic than in nonbronchitic subjects. A negative relationship was found between the presence of airway inflammation, as indexed by mononuclear cell tissue infiltration, and the density of PGP 9.5-positive nerves in both smooth muscle and glands. Likewise, a relationship was found between the smoking history (packs/yr and age of onset of smoking) and the density of VIP-positive nerves in glands. These findings support a role for VIP in the hallmark of chronic bronchitis, i.e., sputum production.

Immunoreactive endothelin in bronchial biopsy specimens: increased expression in asthma and modulation by corticosteroid therapy.

BACKGROUND: The human endothelin (ET) family comprises three 21-amino-acid peptides, which are potent bronchoconstrictors and have a number of other biologic properties relevant to the pathophysiology of asthma. OBJECTIVE: We sought to compare the expression of immunoreactive ET in bronchial biopsy specimens from subjects with asthma treated only with inhaled beta2-agonists, subjects with asthma treated with beta2-agonists and corticosteroids, and control subjects without asthma. METHODS: Biopsy specimens were obtained by fiberoptic bronchoscopy and stained immunohistochemically with a specific ET antiserum. Epithelial ET expression was quantitated by using a computer-assisted system of image analysis. Numbers of inflammatory cells and depth of subepithelial collagen deposition were also determined. RESULTS: Immunoreactive ET was principally localized in the airway epithelium. The proportion of epithelium immunostained was significantly increased in the subjects with asthma not treated with steroids (35.4% +/- 3.8%) compared with that of both the control subjects (16.2% +/- 1.9%, p < 0.0001) and the subjects with asthma treated with steroids (14.3% +/- 2.0%, p < 0.0001). The last two groups did not differ significantly from one another. There were no significant correlations between ET expression and either physiologic parameters or indexes of airway inflammation and remodeling. CONCLUSION: Bronchial epithelial expression of immunoreactive ET is increased in subjects with asthma receiving treatment only with beta2-agonists but not in subjects with asthma also receiving corticosteroid therapy. These findings are consistent with the hypothesis that ET is implicated in the pathophysiology of asthma.

Endothelial nitric oxide synthase in hypoxic newborn porcine pulmonary vessels.

AIMS: To determine if the failure of neonatal pulmonary arteries to dilate is due to a lack of nitric oxide synthase (NOS). METHODS: A monoclonal antibody to endothelial NOS was used to demonstrate the distribution and density of NOS in the developing porcine lung after a period in hypobaric hypoxia. Newborn piglets were made hypertensive by exposure to hypobaric hypoxia (50.8 kPa) from < 5 minutes of age to 2.5 days of age, 3-6 days of age or 14-17 days of age. A semiquantitative scoring system was used to assess the distribution of endothelial NOS by light microscopy. RESULTS: NOS was present in the arteries in all hypoxic animals. However, hypoxia from birth caused a reduction in NOS compared with those lungs normal at birth and those normal at 3 days. Hypoxia from 3-6 days led to a high density of NOS compared with normal lungs at 6 days. Hypoxia from 14-17 days had little effect on the amount of NOS. On recovery in room air after exposure to hypoxia from birth there was a transient increase in endothelial NOS after three days of recovery, mirroring that seen at three days in normal animals. CONCLUSIONS: Suppression of NOS production in the first few days of life may contribute to pulmonary hypertension in neonates.

Development of an in vitro model to study the response of saphenous vein endothelium to pulsatile arterial flow and circumferential deformation.

OBJECTIVES: To develop an in vitro model of human saphenous vein bypass to facilitate study of the early adaptive responses of venous endothelium to arterial flow conditions. DESIGN MATERIAL AND METHODS: Segments of human saphenous vein (with or without external polytetrafluoroethylene (PTFE) stents to limit circumferential and radial deformation) were mounted in a bypass circuit and subjected to pulsatile flow with oxygenated Krebs solution to simulate arterial or venous flow conditions for a period of 90 min. The viability of the vein was assessed by the tissue ATP concentration and vasomotor responses to phenylephrine, sodium nitroprusside and bradykinin (endothelium-dependent). Immunohistochemistry was used to assess both endothelial preservation (CD31) and the expression of proteins involved in leukocyte adhesion: E-selectin, P-selectin and ICAM-1. Freshly excised veins were used as controls. RESULTS: The concentration of ATP was 320 +/- 11 nmol/g in freshly excised vein (n = 8) and following exposure to the arterial flow circuit increased to 566 +/- 60 nmol/g (n = 8, paired t-test, p = 0.003) in unstented veins and to 421 +/- 49 nmol/g (n = 8, paired t-test, p = 0.002) in externally stented veins (with PTFE). Both endothelium-dependent and sodium nitroprusside-induced vasodilatation responses were preserved after veins were exposed to the arterial flow circuit, but the sensitivity to phenylephrine was increased: EC50 decreasing from 9 microM, p = 0.008. There was a 5-10% decrease in staining area for CD31 after veins, stented or unstented, were exposed to the arterial flow circuit. However, after exposure to the arterial flow circuit, the staining area ratio for ICAM-1/CD31, which remained unchanged in externally stented veins, increased two-fold in unstented veins, p > 0.01: there were no changes in the staining area ratio P-selectin/CD31 and no staining for E-selectin was observed. CONCLUSION: Vasomotor responses and tissue ATP concentration indicate that the viability of saphenous vein can be maintained for up to 90 min in an ex vivo flow circuit and the CD31 staining indicated endothelial preservation. This opens up the possibility of investigating the early changes in saphenous vein endothelium following exposure to arterial pressure, as at bypass surgery. First results suggest that there is rapid upregulation of the leukocyte adhesion molecule ICAM-1, which can be prevented by limiting the circumferential deformation of the vein with an external PTFE stent.

Circumferential deformation and shear stress induce differential responses in saphenous vein endothelium exposed to arterial flow.

Adaptation of saphenous vein to the hemodynamic stresses of the arterial circulation is critical to the maturation of vein bypass grafts. We have investigated early adaptive responses of venous endothelium by placing excised human saphenous vein in a bypass circuit with either venous or arterial flow conditions, using external stenting to resolve the effects of longitudinal (shear) from circumferential stress. Endothelial protein concentrations were assessed by immunostaining area (ratio of protein/CD31) and Western blotting of endothelial cell lysates (staining ratio protein/vWf). In both unstented and stented veins nitric oxide synthase increased after 90 min of arterial flow: twofold increase of immunostaining area (P = 0.001), four- to fivefold increase by Western blotting (P = 0.02), and increased A23187mediated maximum endothelium-dependent relaxation of vein rings (P = 0.01). In unstented veins, ICAM-1 concentration was increased after 45 min of arterial flow: twofold increase by immunostaining (P = 0.001) and Western blotting (P = 0.038), with maximum fibrinogen-mediated endothelium-dependent relaxation increasing from 55.9+/-4.9 to 97+/-2.1% (P = 0.01). In contrast, in unstented veins there was a threefold decrease of VCAM-1 and no change in P-selectin after arterial flow for 45 and 90 min, respectively. However, no changes in ICAM-1 and VCAM-1 were observed in stented veins. The flow-induced alterations in nitric oxide synthase, ICAM-1, and VCAM-1 were abolished when 3 mM tetraethylammonium ion (K+ channel blocker) was included in the vein perfusate. The very rapid changes in ICAM-1 and VCAM-1 expression are a response to circumferential stress, whereas the slower upregulation of nitric oxide synthase is a response to longitudinal (shear) stress. Similar changes could influence the adhesiveness of endothelium in newly implanted saphenous vein bypass grafts.

Airway endothelin levels in asthma: influence of endobronchial allergen challenge and maintenance corticosteroid therapy.

Endothelins (ETs) are 21 amino acid peptides which, in addition to their other properties, are potent bronchoconstrictors. Whilst there is evidence of the involvement of ET in the pathophysiology of chronic asthma, its contribution to the acute allergic response is undefined. To examine this, we have undertaken segmental bronchoprovocation with allergen and saline at separate sites in six atopic asthmatics receiving treatment with bronchodilators only and six atopic asthmatics additionally receiving treatment with inhaled corticosteroids. Each challenged segment was lavaged 10 min after bronchoprovocation and concentrations of immunoreactive ET were measured in bronchoalveolar lavage fluid. In the non-steroid-treated subjects, there were significantly lower ET levels at the allergen-challenged sites compared to the saline-challenged sites (p<0.05). In the steroid-treated subjects, on the other hand, there was no significant difference between the two sites. Levels of ET at the saline-challenged sites were significantly lower in the steroid-treated subjects compared to the non-steroid-treated subjects (p<0.04). These findings do not support the hypothesis that allergen exposure in asthma results in immediate release of endothelin. However, release at later time-points and a role for endothelin in late-phase bronchoconstriction are not excluded.

Aprotinin is associated with a decrease in nitric oxide production during cardiopulmonary bypass.

BACKGROUND: Cardiopulmonary bypass (CPB) is associated with an increase in airway nitric oxide (NO), plasma levels of tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 beta. Cytokine induction of the inducible form of nitric oxide synthase (iNOS) has been implicated in organ injury. In addition, serine protease inhibitors reduce cytokine-induced iNOS expression. Aprotinin, a serine protease inhibitor, has been demonstrated to exhibit significant antiinflammatory effects. We hypothesized that aprotinin administration during CPB would significantly reduce endogenous airway NO production. METHODS: Airway NO was measured during CPB in 10 patients receiving aprotinin and in 10 control subjects. In vitro, aprotinin was added to cultures of a murine lung epithelial cell line and was stimulated with cytomix, a combination of TNF, interleukin-1, and interferon-gamma. RESULTS: Airway NO concentration was increased after 50 minutes of CPB duration compared with that measured at 5 minutes in control subjects (53 +/- 5 versus 19 +/- 3 parts per billion, p < 0.05) but not in the aprotinin group (21 +/- 6 versus 15 +/- 3 parts per billion). Aprotinin reduced nitrite concentrations in the cell culture supernatant fluids after 24 hours (cytomix, 21.5 +/- 2.1 mumol/L; cytomix plus aprotinin, 2.7 +/- 0.6 mumol/L, p < 0.05). Immunohistochemistry showed a reduction in cytokine-induced iNOS expression and Northern blot analysis showed a decrease in iNOS mRNA. CONCLUSIONS: These data demonstrate that aprotinin reduces NO production in vivo and reduces cytokine-induced iNOS expression in vitro.

Quantitative microscopical methods for the identification and localisation of nerves and neuroendocrine cell markers in mammalian lung.

The lung contains a dense innervation and a population of endocrinelike cells both of which are believed to have a role in pulmonary function and to be involved in disease processes. They contain a number of regulatory peptides that affect vascular and bronchial tone, growth and repair. They can be detected and localised by immunocytochemistry, thereby allowing investigation of the normal distribution and changes in disease processes. The application of image analysis has added greatly to the amount of information that can be obtained from such morphological studies. Data can be obtained on either the overall distribution and amount of the antigen in a tissue, thereby allowing comparisons between normal and disease states, or following experimental manipulation. Furthermore, the actual intracellular level can be assessed, which adds the previously unattained dimension of comparisons between cells. Thus the density of innervation in the specific regions of the lung tissue, either total nerves or specific peptide-containing cells, may be estimated and used to show release of a peptide or to determine changes in the nerve density in disease. Image processing and image analysis have reduced the labour-intensive manual input required to perform such studies. The continuing development of digital image processing and computer technology will increase the application of these methods in lung research of normal and pathological material.

Stereological estimation of blood vessel surface and volume densities in human normal and rhinitic nasal mucosa.

A technique is proposed for applying well-established stereological methods to study fixed nasal biopsy material to obtain an unbiased estimate of blood vessel surface and volume densities. Biopsies of the nasal mucosa from the anterior 10 mm of an inferior turbinate were obtained from 18 subjects (15 males, 3 females with a mean age of 28.5 years [range: 17-54 years]), ten of whom had perennial allergic rhinitis, and eight control subjects. The mucosal tissue volumes were estimated by water displacement. Zamboni's-fixed cryostat sections (10 microns thick), stained with haematoxylin and eosin, were examined histologically. Computerised images of randomly selected tissue sections were analysed with point-counting and intercept-counting techniques to determine large blood vessel volume and surface densities, respectively. There were no significant differences between the volumes of tissue analysed from the control and rhinitic subjects (p = 0.35). The average volume density of the vessels was similar in the control group (6.17 +/- 1.41%) and the rhinitic group (7.8 +/- 5.59%; p = 0.38), but with a greater variability in the rhinitic group. Surface density estimations were 3.14 +/- 0.74 mm-1 in the control group and 3.10 +/- 1.41 mm-1 in the rhinitic group. Therefore, on average, the volume and surface densities of the cavernous blood vessels in rhinitis were unaltered and there was no evidence of vascular remodelling.

Human lung mononuclear cells induce nitric oxide synthase in murine airway epithelial cells in vitro: role of TNFalpha and IL-1beta.

Nitric oxide (NO) is a gas released by the airway epithelium, but the mechanism regulating NO release is unclear. We hypothesized that lung mononuclear cell release of tumor necrosis factor alpha (TNF) and interleukin-1beta (IL-1) would induce epithelial cells to release NO. Lung mononuclear cells were obtained from seven normal volunteers by bronchoalveolar lavage and cultured with Escherichia coli lipopolysaccharide for 24 h. The mononuclear cell culture-conditioned media (M-CM) were then applied to cultures of the murine lung epithelial cell line, LA-4. Nitrite and nitrite + nitrate concentrations were 0.9 +/- 0.1 and 11.8 +/- 2.4 microM in the M-CM. Culturing LA-4 cells line with the M-CM (1:10 dilution) resulted in a marked and time-dependent increase in nitrite or nitrite + nitrate compared with LA-4 cells cultured in media alone (2.4 +/- 0.5 versus 0.9 +/- 0.1 microm and 16.6 +/- 0.6 versus 11.8 +/- 2.4 microM after 24 h). Antibodies to TNF and/or IL-1 significantly reduced the nitrite or nitrite + nitrate concentrations and the concentrations of TNF and IL-1 in the M-CM correlated with nitrite concentrations in the LA-4 culture supernatant fluids (r2 = 0.848 and 0.956). Inducible nitric oxide synthase (iNOS) protein and mRNA examined by immunohistochemistry and Northern blot analysis revealed a marked elevation in the cells cultured with the M-CM which was significantly reduced by TNF and IL-1 antibodies. These data demonstrate that mononuclear cells can stimulate LA-4 cells to express iNOS by releasing TNF and IL-1.