Soluble glucomannan isolated from Candida utilis primes blood phagocytes Veronika Hájková a , Aneta Svobodová b , Daniela Krejc ˇová b , Milan C ˇ íz ˇ b , Vladimír Velebny ´ a , Antonín Lojek b , Jamel El-Benna c,d , Lukáš Kubala b, * a CPN spol. s.r.o., Dolní Dobrouc ˇ 401, 562 01 Dolní Dobrouc ˇ, Czech Republic b Institute of Biophysics of the Academy of Sciences of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno, Czech Republic c INSERM, U773, Centre de Recherche Biomédicale Bichat Beaujon CRB3, Paris F-75018, France d Universite Paris 7 Denis Diderot, Faculte de Medecine, site Bichat, Paris F-75018, France article info Article history: Received 9 April 2009 Received in revised form 11 June 2009 Accepted 23 June 2009 Available online 27 June 2009 Keywords: Phagocyte Oxidative burst Degranulation Polysaccharide Glucans abstract It is well documented that the polysaccharide glucomannan (GM), an abundant constituent of the fungal cell wall, in the form of particulate induces strong activation of phagocytes, however, the effects of sol- uble GM are not known. Activation of phagocyte anti-microbial mechanisms is a crucial part of the innate host defense against invading pathogens. However, under uncontrolled inflammatory conditions they contribute to damage of surrounding tissues. Thus, to prevent these deleterious effects, the activation of phagocytes is a tightly regulated process. Therefore, in this study we analyzed the effect of soluble GM on some neutrophil functions such as reactive oxygen species production, degranulation, and recep- tor mobilization at the plasma membrane. Soluble GM at the tested concentrations did not stimulate oxi- dative burst of phagocytes directly but significantly potentiated oxidative burst in response to opsonized zymosan particles. GM induced significant phosphorylation of p47phox subunit of NADPH oxidase on Ser345. This priming effect of GM was accompanied by time and concentration dependent degranulation characterized by increased surface expression of receptors stored in neutrophil granules (CD10, CD11b, CD14, CD35, and CD66b). Degranulation was further confirmed by increase of elastase activity in media. Thus, it could be suggested that soluble GM induces priming of phagocytes connected with their degran- ulation, the increase of surface receptor expression, and potentiation of oxidative burst response to opso- nized particles through the activation of NADPH oxidase. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Polysaccharides such as glucans and glucomannans (GMs), which are major structural components of fungal cell walls, are known to be potent activators of leukocytes including human blood phagocytes. 1–4 These polysaccharides have been shown to stimulate various facets of immune responsiveness in humans, including anti-infective activities against fungal, bacterial, viral, and protozoal infections. Many studies suggest that the activity and immunostimulatory potential of microbial polysaccharides de- pend on structural parameters such as primary structure, degree of branching, solution conformation, solubility, and molecular weight. 1–5 Thus, polysaccharides obtained from various microbial sources and by diverse isolation procedures significantly differ in their biological activity. The widely exploited insoluble particles isolated from Saccharomyces cerevisiae called zymosan, composed of b-glucans and GM, induce intensive oxidative burst of phago- cytes accompanied by intensive degranulation. 1–5 Interestingly, in contrast to numerous studies describing effects of particular GM (e.g., zymosan particles) on leukocytes, only limited informa- tion exists about the effects of soluble forms of b-glucans and par- ticularly GM on phagocyte activation, and specifically induction of respiratory burst of neutrophils. To the best of our knowledge there are no reports about the effects of soluble GM on the physi- ological response of human blood phagocytes. Phagocytes, including polymorphonuclear leukocytes (PMNLs) and monocytes, play a key role in host defense against invading pathogens and play a crucial role in inflammatory processes. 6,7 In response to a variety of stimuli, phagocytes degranulate their secretory vesicles and release large quantities of reactive oxygen species (ROS) in a phenomenon described as the respiratory burst. 6,8 Oxidative burst is primarily characterized by the produc- tion of superoxide anion, which gives rise to other forms of ROS. 6 The production of superoxide anion during respiratory burst is 0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2009.06.034 Abbreviations: CL, luminol-enhanced chemiluminescence; GM, glucomannan; GM-CSF, granulocyte–macrophage colony-stimulating factor; HBSS, Hank’s buffered salt solution; NADPH, nicotinamide adenine dinucleotide phosphate; OD, optical density; OZP, opsonized zymosan particles; PGG-glucan, poly-b1-glucotriosyl-b1-3- pyranose glucan; PMNL, polymorphonuclear leukocytes; PBS, phosphate buffer solution; RLU, relative light units; RFU, relative fluorescence unit; ROS, reactive oxygen species; SEM, standard error of mean; TNF-a, tumor necrosis factor a. * Corresponding author. Tel.: +420 541 517 117; fax: +420 541 211 293. E-mail address: kubalal@ibp.cz (L. Kubala). Carbohydrate Research 344 (2009) 2036–2041 Contents lists available at ScienceDirect Carbohydrate Research journal homepage: www.elsevier.com/locate/carres