Scientific Report 2007

Molecular and Experimental Medicine

Division Of Biochemistry

Neutrophil Exocytosis and Extracellular Killing of Bacteria

S.D. Catz, D.B. Munafo, A.A. Brzezinska, B.A. Ellis, J.L. Johnson

Role of Rab27 Family Members in Exocytosis of Neutrophil Granules

Neutrophils kill microorganisms via microbicidal products released into the phagosome or into the extracellular space. Neutrophils contain 4 types of excytosable storage organelles with different tendencies to undergo exocytosis: azurophilic, specific, and gelatinase granules and secretory vesicles. We are characterizing the secretory machinery that regulates the exocytosis of these organelles.

We recently showed that the small GTPase Rab27a and its effector JFC1 localize in a minor subpopulation of myeloperoxidase-containing granules (azurophilic) and that interfering with the Rab27a-JFC1 secretory machinery inhibits myeloperoxidase secretion in granulocytes. We also showed that Rab27a-deficient mice have impaired secretion of myeloperoxidase in vivo after intraperitoneal injection of lipopolysaccharide. Contrarily, mobilization of CD11b from intracellular granules (gelatinase granules and secretory vesicles) in response to the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine or lipopolysaccharide was not affected in Rab27a-deficient neutrophils.

Rab27b, a GTPase that shares 72% identity with Rab27a, is expressed in granulocytes and is upregulated in the absence of Rab27a. Granulocytes from mice deficient in Rab27b and from mice in which the genes for both Rab27a and Rab27b are inoperable mobilized CD11b in response to stimuli as efficiently as did wild-type controls. However, Rab27b deficiency impaired the mobilization of a subpopulation of specific granules expressing the IL-10 receptor. This finding suggests that Rab27b is involved in the mobilization of specific granules and that CD11b-containing vesicles are secreted independently of the Rab27 family members.

Localization and Activity of the NADPH Oxidase on Neutrophil Extracellular Traps

Neutrophils engulf microorganisms in a process known as phagocytosis. The bactericidal ability of these leukocytes relies on the antimicrobial peptides released into the phagosome and on the capacity of the neutrophils to generate reactive oxygen species. Many microorganisms escape phagocytosis-dependent killing. To overcome these escape tactics, neutrophils have developed alternative tools to kill bacteria. It has recently become apparent that extracellular neutrophil microbicidal components can be regulated by a novel mechanism: the formation of organized extracellular DNA fibers containing histones and other proteins with bactericidal ability. The composition of these neutrophil extracellular traps (NETs) and the bactericidal mechanism used are incompletely understood.

We tested the hypothesis that the NADPH oxidase is present and active on NETs. We showed that the oxidase is assembled on punctate structures distributed on NETs. Detection of NET-associated oxidase subunits was abolished by treatment with deoxyribonuclease and depended on cell stimulation. Using a superoxide-specific DNA-binding fluorescent probe, we found that superoxide anion is produced on NETs. Interfering with extracellular oxidase subunits by means of specific antibodies impaired extracellular bacterial killing. Our data support a role for NET-associated NADPH oxidase in the microbicidal mechanism used to combat nonphagocytosed microorganisms.

Publications

Johnson, J.L., Ellis, B.A., Munafo, D.B., Brzezinska, A.A., Catz, S.D. Gene transfer and expression in human neutrophils: the phox homology domain of p47^phox translocates to the plasma membrane but not to the membrane of mature phagosomes. BMC Immunol. 7:28, 2006.

Munafo, D.B., Johnson, J.L., Ellis, B.A., Rutschmann, S., Beutler, B., Catz, S.D. Rab27a is a key component of the secretory machinery of azurophilic granules in granulocytes. Biochem. J. 402:229, 2007.

Pacquelet, S., Johnson, J.L., Ellis, B.A., Brzezinska, A.A., Lane, W.S., Munafo, D.B., Catz, S.D. Cross-talk between IRAK-4 and the NADPH oxidase. Biochem. J. 403:451, 2007.