title: Human eosinophil adhesion and degranulation stimulated with eotaxin and RANTES in vitro: lack of interaction with nitric oxide.
abstract: BACKGROUND
  Airway eosinophilia is considered a central event in the pathogenesis of asthma. The toxic components of eosinophils are thought to be important in inducing bronchial mucosal injury and dysfunction. Previous studies have suggested an interaction between nitric oxide (NO) and chemokines in modulating eosinophil functions, but this is still conflicting. In the present study, we have carried out functional assays (adhesion and degranulation) and flow cytometry analysis of adhesion molecules (VLA-4 and Mac-1 expression) to evaluate the interactions between NO and CC-chemokines (eotaxin and RANTES) in human eosinophils.
  
  
  METHODS
  Eosinophils were purified using a percoll gradient followed by immunomagnetic cell separator. Cell adhesion and degranulation were evaluated by measuring eosinophil peroxidase (EPO) activity, whereas expression of Mac-1 and VLA-4 was detected using flow cytometry.
  
  
  RESULTS
  At 4 h incubation, both eotaxin (100 ng/ml) and RANTES (1000 ng/ml) increased by 133% and 131% eosinophil adhesion, respectively. L-NAME alone (but not D-NAME) also increased the eosinophil adhesion, but the co-incubation of L-NAME with eotaxin or RANTES did not further affect the increased adhesion seen with chemokines alone. In addition, L-NAME alone (but not D-NAME) caused a significant cell degranulation, but it did not affect the CC-chemokine-induced cell degranulation. Incubation of eosinophils with eotaxin or RANTES, in absence or presence of L-NAME, did not affect the expression of VLA-4 and Mac-1 on eosinophil surface. Eotaxin and RANTES (100 ng/ml each) also failed to elevate the cyclic GMP levels above baseline in human eosinophils.
  
  
  CONCLUSIONS
  Eotaxin and RANTES increase the eosinophil adhesion to fibronectin-coated plates and promote cell degranulation by NO-independent mechanisms. The failure of CC-chemokines to affect VLA-4 and Mac-1 expression suggests that changes in integrin function (avidity or affinity) are rather involved in the enhanced adhesion.
authors.affiliation: Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas, Campinas (São Paulo), Brazil. lintomen2001@yahoo.com.br
authors.forename: Letícia
authors.initials: L
authors.lastname: Lintomen
authors.forename: Gilberto
authors.initials: G
authors.lastname: Franchi
authors.forename: Alexandre
authors.initials: A
authors.lastname: Nowill
authors.forename: Antonio
authors.initials: A
authors.lastname: Condino-Neto
authors.forename: Gilberto
authors.initials: G
authors.lastname: de Nucci
authors.forename: Angelina
authors.initials: A
authors.lastname: Zanesco
authors.forename: Edson
authors.initials: E
authors.lastname: Antunes
mesh_terms: D039481:CD11b Antigen / Q000378:metabolism; D002448:Cell Adhesion / Q000502:physiology; D015550:Cell Degranulation / Q000502:physiology; D018946:Chemokine CCL5 / Q000378:metabolism; D019742:Chemokines, CC / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004802:Eosinophilia / Q000378:metabolism*; D004804:Eosinophils / Q000166:cytology* / Q000378:metabolism* / Q000502:physiology; D005434:Flow Cytometry; D006801:Humans; D066298:In Vitro Techniques; D039441:Integrin alpha4 / Q000378:metabolism; D039041:Integrin alpha4beta1 / Q000096:biosynthesis / Q000378:metabolism; D016177:Macrophage-1 Antigen / Q000096:biosynthesis / Q000378:metabolism; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000502:physiology*
chemical_list: D039481:CD11b Antigen; D018946:Chemokine CCL5; D019742:Chemokines, CC; D004791:Enzyme Inhibitors; D039041:Integrin alpha4beta1; D016177:Macrophage-1 Antigen; D039441:Integrin alpha4; D009569:Nitric Oxide; D019331:NG-Nitroarginine Methyl Ester