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Munc13-4 Regulates Granule Secretion in Human Neutrophils¹

Christophe Pivot-Pajot^*, Frederique Varoqueaux, Geneviève de Saint Basile and Sylvain G. Bourgoin^2,*,

^* Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du Centre Hospitalier de l’Université Laval and Département d’Anatomie-Physiologie, Université Laval, Québec City, Québec, Canada; Department of Neurobiology, Max Planck Institute of Experimental Medecine, Goettingen, Germany; and Institut National de la Santé et de la Recherche Médicale U429, Hôpital Necker-Enfants Malades, Paris, France

The neutrophil plays a central role in the innate host immune defense. Regulated exocytosis of its granules and release of antimicrobial and cytotoxic substances are key events to limit the spread of pathogens. However, the molecular mechanisms that control exocytosis of neutrophil granules are ill-defined. Recently, it was shown that Munc13-4 is essential for the priming of granules in several hematopoietic cells. In this study, we show that Munc13-4 is expressed in human neutrophils, and that its expression is increased during granulocytic differentiation of HL-60 and PLB-985 cells. Cell fractionation analysis reveals that Munc13-4 is mainly cytosolic and is recruited rapidly to membranes following stimulation with fMLF (N-formyl-methionyl-leucyl-phenylalanine). Moreover, a pool of Munc13-4 associated with mobilizable secondary and tertiary granules is relocalized to the plasma membrane after stimulation with fMLF. The fMLF-induced translocation of Munc13-4 is strictly dependent on calcium in neutrophils. C2 domains of Munc13-4 are essential for binding to phospholipid vesicles in a Ca²⁺-independent manner. Finally, down-regulation of Munc13-4 using small interfering RNA decreases exocytosis of tertiary granules in PLB-985 cells, whereas overexpression of Munc13-4 enhances secretion of MMP-9 (matrix metalloproteinase-9) from tertiary granules. Our findings suggest a role for Munc13-4 as a component of the secretory machinery in neutrophils.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by grants from the Canadian Institutes of Health Research and the Arthritis Society of Canada (TAS04/0061 to S.G.B). C.P.P was the recipient of a fellowship from La Fondation pour la Recherche Médicale.

² Address correspondence and reprint requests to Dr. Sylvain G. Bourgoin, Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du Centre Hospitalier de l’Université Laval, 2705 Boulevard Laurier, Room T1-49, Sainte-Foy, Québec, Canada, G1V 4G2. E-mail address: sylvain.bourgoin{at}crchul.ulaval.ca

³ Abbreviations used in this paper: fMLF, N-formyl-methionyl-leucyl-phenylalanine; ADA, adenosine deaminase; CB, cytochalasin B; dbcAMP, dibutyryl cyclic AMP; ([Ca²⁺]_c), cytosolic calcium; DFP, diisopropylfluorophosphate; LDH, lactate dehydrogenase; LF, lactoferrin; MHD, Munc13 homology domain; MMP-9, matrix metalloproteinase-9; MPO, myeloperoxidase; PMA, phorbol myristate acetate; siRNA, small interfering RNA; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor.