The immunogold-silver staining technique is shown to be of great value in the detection of regulatory peptide-containing nerves and endocrine cells in routinely fixed, paraffin-wax-embedded tissues. The method appears to be better for this system than peroxidase anti-peroxidase (PAP) which can yield poor or variable results.

Small cell carcinoma of the lung is a highly malignant tumour. Its known biological products which include bombesin, do not allow the prediction of tumour behaviour. Molecular biology has revealed the amino acid sequence of human pro-bombesin, which consists of a signal peptide, the bioactive bombesin molecule and a C-terminal peptide. We have raised a rabbit antiserum to the first (N-terminal) 21 amino acids of the predicted C-terminal peptide. A total of 505 (361 neuroendocrine) surgically resected pulmonary tumours were evaluated for the presence of immunoreactive bombesin and C-terminal peptide. Strong immunostaining was obtained with the antiserum to the C-terminal peptide of human probombesin in 70% of the small cell carcinomas (175/250), in 63% of atypical (aggressive) carcinoids (31/49) but only in 16% of benign carcinoids (10/62). In contrast, bombesin immunostaining was focal and only moderately strong and the relative proportion of positive cases was quite evenly distributed amongst the neuroendocrine tumours: 35% of carcinoids (22/62), 22% of atypical carcinoids (11/49) and 25% of small cell carcinoma (62/250). None of the squamous, adeno, or large cell undifferentiated carcinomas were immunoreactive for bombesin or the C-terminal peptide. Radioimmunoassay and chromatography of extracts of tumours recovered from wax blocks revealed high concentrations of C-terminal peptide immunoreactivity (241±66 pmol/g of tissue) in all 12 small cell carcinomas studied, moderate concentrations in carcinoid tumours (50±7 pmol/g) and none in non-small cell carcinomas. Patients with tumours showing immunoreactivity to the C-terminal peptide of human pro-bombesin had a significantly shorter survival time than those without immunoreactive peptide (185±16.49 days, mean± SEM, and with 1128±226 days, respectivelyP> 0.02). The apparent presence of the C-terminal peptide of human pro-bombesin in higher concentrations than bombesin in the more malignant class of endocrine tumours, mainly small cell carcinomas associated with the poorest prognosis, suggests that the antiserum to this C-terminal peptide is not only a useful pathological marker but may prove to be of value in investigating the biological behaviour of small cell carcinomas and predicting the clinical course of the disease.

We have previously shown depletion of nerves and neuropeptides in skin biopsies of diabetic patients, even in the absence of clinical signs and symptoms of sensory and autonomic neuropathy, but were unable to examine the changes occurring at an early stage of the disease. Therefore, the distribution and relative density of peptide-containing nerves was studied in streptozotocin-treated rats in order to assess the progression of neural changes in the initial stages of diabetes. Skin samples dissected from the lip and footpad of diabetic rats, 2, 4, 8 and 12 weeks after streptozotocin injection and age matched controls were sectioned and were immunostained with antisera to the neuropeptides substance P, calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY), and to a general neural marker, protein gene product 9.5 (PGP 9.5). No change was apparent in the distribution or relative density of immunoreactive cutaneous nerve fibres 2, 4 and 8 weeks after streptozotocin treatment. By 12 weeks there was a marked increase in the number of CGRP-immunoreactive fibres present in epidermis and dermis, and of VIP-immunoreactive fibres around sweat glands and blood vessels. A parallel increase was seen in nerves displaying PGP 9.5 immunoreactivity. No differences were detected in nerves immunoreactive for either substance P in the epidermis and dermis, and NPY around blood vessels. The alterations in the peptide immunoreactivities may be similar in the initial stages of human diabetes.

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.

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. Copyright (C) 2000 John Wiley & Sons, Ltd.

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.

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 connflicting 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 mere 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. (C) 1998 John Wiley & Sons, Ltd.

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 immunocyto‐chemical localisation of endothelial nitric oxide synthase. Results Segments of saphenous vein distended to 100 mmHg retained their responsiveness to KCI (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.

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 mu g/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-N-G-monomethyl-arginine (100 mu mol/l) or cycloheximide (1 mu mol/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 Ca2+-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-N-G-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 Ca2+-independent nitric oxide synthase activity following cytokine stimulation. (C) 1998 Elsevier Science B.V. All rights reserved.

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.

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.

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.

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.

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.

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 mu mol/L; cytomix plus aprotinin, 2.7 +/- 0.6 mu mol/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.

Although the production of NO within rodent phagocytes is well-characterized, its production and function within human phagocytes are less clear. We show here that neutrophils within human buffy coat preparations stimulated with a mixture of interleukin 1, tumor necrosis factor alpha, and interferon gamma contain inducible NO synthase mRNA and protein, one of the enzymes responsible for NO production. The protein colocalizes with myeloperoxidase within neutrophil primary granules. Using an inhibitor of NO synthase, L-N-monomethyl arginine, we show that activity of this enzyme is required for the formation of nitrotyrosine around phagocytosed bacteria, most likely through the intermediate production of peroxynitrite, a reaction product of NO and superoxide anions.

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.

BACKGROUND AND AIM:Endothelin has bronchoconstrictive, vasoactive, and inflammatory properties and may be involved in the pathogenesis of asthma. We have studied the involvement of endothelin in asthma by examining its expression and release by mononuclear phagocytes obtained from 56 patients with asthma and 32 control subjects and the activation of mononuclear phagocytes by endothelin. METHODS:Endothelin immunoreactivity was studied by using immunocytochemistry on monocytes and alveolar macrophages. Spontaneous and lipopolysaccharide-induced endothelin release from monocytes and alveolar macrophages was studied by radioimmunoassay. The proportion of intracellular endothelin was assessed after cell disruption by Triton (Union Carbide Corp., Bound Brook, N.J.). The release of fibronectin and tumor necrosis factor-alpha induced by endothelin was studied in alveolar macrophages by enzyme immunoassay. RESULTS:Endothelin immunoreactivity was significantly increased in cells from patients with asthma in comparison with those from the control group, but its release by alveolar macrophages was similar in both groups. Levels in the cell lysates and supernatants were similar for patients with asthma and normal subjects. Endothelin significantly increased the release of tumor necrosis factor-alpha and fibronectin by alveolar macrophages from normal subjects and patients with stable asthma, but it significantly decreased their release in patients with unstable asthma. CONCLUSION:This study suggests a role for endothelin in airway inflammation in asthma. Endothelin may act in a different fashion on alveolar macrophages, depending on the degree of stability of the disease.