Expression of nitric oxide synthases and in vitro migration of eosinophils from allergic rhinitis subjects Heloisa H.A. Ferreira a, * , Mo ˆnia L.S. Lodo a , Antonio R. Martins b , Ludmyla Kandratavicius b , Antonio F. Salaroli c , Nicola Conran d , Edson Antunes d , Gilberto De Nucci d a Clinical Pharmacology and Gastroenterology Unit (USF), USF, Braganc ßa Paulista (SP), Brazil b Department of Pharmacology, Ribeira ˜o Preto Medical School, USP, Ribeira ˜o Preto (SP), Brazil c Sa ˜o Francisco University (USF) Hospital, USF, Braganc ßa Paulista, (SP), Brazil d Department of Pharmacology, Faculty of Medical Sciences, UNICAMP, Campinas (SP), Brazil Received 19 December 2001; received in revised form 5 March 2002; accepted 8 March 2002 Abstract The expression of nitric oxide (NO) synthases and the role of the NO cyclic GMP pathway on the migration of eosinophils from untreated patients with allergic rhinitis were investigated. Inducible NO synthase was strongly expressed in eosinophils from healthy individuals, but not in eosinophils from allergic rhinitis patients. The neuronal isoform was observed in eosinophils from each group studied, whereas no staining for the endothelial isoform was detected in either group. The chemotaxis to N-formyl-methionyl-leucyl-phenylalanine (fMLP, 5 Â 10 À 7 M) and eotaxin (100 ng/ml) was significantly potentiated in allergic rhinitis eosinophils. In both groups, N N -nitro-L-arginine methyl ester (L-NAME, 1.0 mM) or 1H(1,2,4)-oxadiazolo(4,3,-a)quinoxalin-1-one (ODQ, 0.2 mM) markedly reduced the chemotaxis. The selective iNOS inhibitor N-(3-(aminomethyl)benzyl)acetamidine (1400 W, 0.1 –1.0 mM) significantly reduced the chemotaxis of eosinophils from healthy but not from allergic rhinitis subjects. The inhibition by L-NAME was restored by 3-morpholinosydnonimine (SIN-1) and S- nitroso-N-acetyl-penicillamine, whereas the inhibition by ODQ was restored by dibutyryl cyclic GMP. In conclusion, both endothelial and inducible NO synthase isoforms are absent in allergic rhinitis eosinophils, suggesting that the NO cyclic GMP pathway in this cell type is maintained through the activity of a neuronal isoform. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Allergic rhinitis; Eosinophil; Nitric oxide (NO) synthase; 1400 W; Eotaxin; Chemotaxis 1. Introduction Allergic rhinitis is characterized by the accumulation of inflammatory cells, particularly eosinophils, in tissues (Var- ney et al., 1992). The participation of eosinophils in this process, however, depends upon their migration from the circulation to sites of inflammation (Gleich and Adolphson, 1986). The mechanisms responsible for the recruitment of these cells are not clearly understood, but it is likely that various mechanisms, which involve cytokine, chemokine and adhesion molecules, operate in order to attain a selective recruitment of eosinophils in these diseases (Lundahl et al., 1998). Eosinophils from atopic subjects (allergic asthma and/or rhinitis) may have been preexposed to cytokines in vivo (Sehmi et al., 1992), and primed eosinophils are reported to present an increased migratory response to different chemotactic agents (Bruijnzeel et al., 1993). Nitric oxide (NO) is produced in mammalian cells from L-arginine and oxygen by a family of enzymes known as NO synthases (NOS). The three NOS isoforms are the neuronal (nNOS or type I), endothelial (eNOS or type III) and inducible (iNOS or type II) types. The nNOS and eNOS are constitutively expressed, whereas the iNOS can be induced by bacterial lipopolysaccharide or certain cytokines such as tumor necrosis factor-a, interleukin-1 and interferon g (Moncada et al., 1991). These three NOS isoforms have been observed in rat peritoneal eosinophils (Zanardo et al., 1997), human peripheral blood eosinophils (Del Pozo et al., 1997) and in dermal eosinophilic pustular folliculitis (Maruo et al., 1999). Nitric oxide is thought to be an important inflammatory mediator in several atopic diseases, and relatively large amounts of NO are continuously produced in the nasal 0014-2999/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII:S0014-2999(02)01507-8 * Corresponding author. Sa ˜o Francisco University, Avenida Sa ˜o Francisco de Assis, 218, 12916-900, Braganc ßa Paulista (SP), Brazil. Tel.: +55-11-4034-8393; fax: +55-11-4034-1825. E-mail address: heloisaferreira@saofrancisco.edu.br (H.H.A. Ferreira). www.elsevier.com/locate/ejphar European Journal of Pharmacology 442 (2002) 155 – 162