Cigarette smoke condensate attenuates phorbol ester-mediated neutrophil  extracellular trap formation.

Refilwe Philadelphia Bokaba, Ronald Anderson, Annette Johanna Theron, Gregory Ronald Tintinger
1. Department of Immunology, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa
2. Institute for Cellular and Molecular Medicine, SAMRC Extramural Unit for Stem Cell Research and Therapy, Department of Immunology, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa
3. Tshwane Academic Division of the National Health Laboratory Service, Pretoria South Africa
4. Department of Internal Medicine, University of Pretoria and Steve Biko Academic Hospital, Pretoria, South Africa

Abstract
Background:Neutrophil extracellular traps (NETs) constitute a network of chromatin fibres containing histone and antimicrobial peptides that are released by activated neutrophils. NETs protect the host against infection by trapping and facilitating  phagocytosis of potentially harmful pathogens.

Objectives:The aim of the current study was to investigate the effects of cigarette smoke condensate (CSC) on phorbol-ester  (PMA)-mediated NETosis in vitro.

Methods: Isolated human blood neutrophils were exposed to PMA (6.25 ng/ml) in the presence or absence of CSC (40-80 µg/ ml) for 90 min at 37oC. NET formation was measured using a spectrofluorimetric procedure to detect extracellular DNA and  fluorescence microscopy was used to visualize nets. Oxygen consumption by PMA-activated neutrophils was measured using an  oxygen sensitive electrode.

Results: Activation of neutrophils with PMA was associated with induction of NETosis that was significantly attenuated in the  presence of CSC (40 and 80 µg/ml), with mean fluorescence intensities of 65% and 66% of that observed with untreated cells,  respectively, and confirmed by fluorescence microscopy. The rate and magnitude of oxygen consumption by activated neutrophils pre-treated with CSC (80 µg/ml) was significantly less than that observed with untreated cells (73% of the control system),  indicative of decreased production of reactive oxidants in the presence of CSC.

Conclusion:The inhibition of NETosis observed in the presence of CSC correlated with attenuation of oxygen consumption  by PMA-activated neutrophils suggesting a mechanistic relationship between these events. If operative in vivo, smoking-related  attenuation of NETosis may impair host immune responses and increase the risk of respiratory infections.

Keywords: Neutrophils, reactive oxygen species, respiratory infection, smoking.