The authors thank Professor Sir John Vane for his stimulating advice and help. 1 Redington AE ... 23 Springall DR, Riveros-Moreno V, Buttery L, et al. Immuno-.
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Increased expression of inducible nitric oxide synthase and cyclo-oxygenase-2 in the airway epithelium of asthmatic subjects and regulation by corticosteroid treatment A E Redington, Q-H Meng, D R Springall, T J Evans, C Créminon, J Maclouf, S T Holgate, P H Howarth, J M Polak
University Medicine, Southampton General Hospital, Tremona Road, Southampton SO16 6YD, UK A E Redington S T Holgate P H Howarth Department of Histochemistry, Imperial College School of Medicine, Hammersmith Hospital, London W12 0NN, UK Q-H Meng D R Springall J M Polak Department of Infectious Diseases, Imperial College School of Medicine T J Evans CEA, SPI/DRM, Bâtiment 136, CEA-Saclay, 91191 Gif-sur-Yvette, Cedex, France C Créminon U348 INSERM, Hôpital Lariboisière, 8 rue Patin, 75475 Paris Cedex 10, France J Maclouf Correspondence to: Dr A E Redington, Academic Department of Medicine, Castle Hill Hospital, Castle Road, Cottingham, East Yorkshire HU16 5JQ, UK a.e.redington@ medschool.hull.ac.uk Received 14 September 2000 Returned to authors 2 December 2000 Revised version received 19 January 2001 Accepted for publication 22 January 2001
Abstract 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 â2 agonists alone (n=7), atopic asthmatics additionally receiving regular treatment with corticosteroids (n=8), and nonasthmatic 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. (Thorax 2001;56:351–357) Keywords: asthma; corticosteroids; immunohistochemistry; in situ hybridisation; nitric oxide synthase; cyclo-oxygenase (COX)
Mucosal inflammation in asthma is characterised by infiltration and activation of immune
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cells—principally mast cells, eosinophils, T lymphocytes, and monocytes/macrophages— and activation of structural cells such as airway epithelial cells and subepithelial myofibroblasts.1 These various cell populations are believed to release biochemical mediators whose biological properties determine the clinical and pathophysiological expression of the disease. Prominent among these putative mediators are nitric oxide (NO) and prostanoids. NO plays an important role in a wide variety of pulmonary functions including regulation of vascular and airway smooth muscle tone. Endogenous NO is normally present in the exhaled air of animals and humans.2 Levels of exhaled NO are increased in asthma3–5 and are reduced by corticosteroid treatment.6 7 NO is generated by the enzyme NO synthase (NOS) which catalyses the conversion of L-arginine to L-citrulline. Three major forms of NOS have been recognised, all of which are present in human airways.8 9 Endothelial NOS (eNOS) and neuronal NOS (nNOS) are constitutively expressed and produce small (picomolar) amounts of NO that are probably involved in normal pulmonary homeostasis. In contrast, inducible NOS (iNOS) is transcriptionally regulated by cytokines and produces much larger (nanomolar) quantities of NO. Prostanoids are also centrally involved in pulmonary physiology and have been implicated in the pathophysiology of diseases such as asthma.10 Of particular relevance is their ability to regulate bronchial tone: prostaglandin(PG)D2, PGF2á, and 9á11â-PGF2á are potent bronchoconstrictors11 12 whereas PGE2 is capable of causing either contraction or relaxation of airway smooth muscle.12 13 Levels of prostanoids in bronchoalveolar lavage (BAL) fluid are increased in asthma,14 as is release of PGE2 by cultured bronchial epithelial cells derived from asthmatic subjects.15 The initial rate limiting step in prostanoid biosynthesis is the conversion of arachidonic acid to PGH2 by the enzyme cyclo-oxygenase (COX), also known as prostaglandin endoperoxide synthase. In a variety of species two distinct but homologous COX isoenzymes, denoted COX-1 and COX-2, that are encoded by separate genes have been identified and cloned.16–18 Under most conditions COX-1 is constantly expressed and is believed to produce low levels of prostanoids which serve a physiological housekeeping role. In contrast, COX-2 is readily
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Redington, Meng, Springall, et al Table 1
Clinical and physiological characteristics of the subjects studied
Controls 1 2 3 4 5 6 7 8 9 10 Mean SE Geometric mean
Age
Sex
Atopy
24 20 22 19 23 22 19 35 21 22 22.7 1.5
M F F F M F F F M F
Y N Y N Y Y Y N Y Y
700 17 30 32 >32 >32 >32 >32 >32 >32 >32
Non-steroid treated asthmatics 1 21 2 19 3 21 4 20 5 20 6 21 7 23 Mean 20.7 SE 0.5 Geometric mean Steroid treated asthmatics 1 21 2 25 3 41 4 20 5 21 6 21 7 55 8 Mean SE Geometric mean
IgE (IU/ml)
33 29.6 4.5
F F F F F F M
Y Y Y Y Y Y Y
NA 442 229 727 266 275 1230
122.3 100.3 77.4 99.0 91.3 131.4 105.9 103.9 6.9
3.85 6.92 3.49 0.59 0.29 2.21 0.32
S S S S S S S
1.41 M M F M F F F
Y Y Y Y Y Y Y
182 267
