Nitric oxide-induced membrane tubulovesicular extensions (cytonemes) of human neutrophils catch and holdSalmonella entericaserovar Typhimurium at a distance from the cell surface

Abstract

Nitric oxide (NO) plays an important role in host defense against bacterial infections such as salmonellosis. NO and 4-bromophenacyl bromide (BPB) induce the formation of long tubulovesicular extensions (TVE, cytonemes, membrane tethers) from human neutrophils. These TVE serve as cellular sensory and adhesive organelles. In the present study, we demonstrated that in the presence of the NO donor, diethylamine NONOate or BPB human neutrophils bound and aggregated Salmonella enterica serovar Typhimurium bacteria extracellularly by TVE. In contrast, inhibition of NO-synthase activity by N(omega)-nitro-L-arginine methyl ester stimulated neutrophil phagocytosis (ingestion) of bacteria. Neutrophil TVE consisted of membrane-covered cytoplasm as was shown by the fluorescent cytoplasmic dye 2',7'-bis(2carboxyethyl)-5,(6)-carboxyfluorescein, and the fluorescent lipid, BODIPY-labeled sulfatide. Disruption and shedding of TVE were accompanied by the appearance of specific invaginations (porosomes) on neutrophil cell bodies. These invaginations corresponded to the variations in diameter of TVE (160-240 nm). We hypothesized that TVE represented protrusions of neutrophil exocytotic trafficking through special structures on the neutrophil surface. In conclusion, we propose a novel mechanism by which NO-induced TVE formation enables neutrophils to bind and aggregate bacteria at a distance.

Microbial alkaloid staurosporine induces formation of nanometer-wide membrane tubular extensions (cytonemes, membrane tethers) in human neutrophils

“…Similar invaginations we observed on the neutrophil surface after shedding of NO-induced cytonemes as a result of interaction with bacteria. 11 Assembly of neutrophil cytoskeleton undergoes strong reorganization upon adhesion to substrata and depends on substrata. Human neutrophils adherent to a polystyrene plastic surface are vigorously activated and exhibited an increase of cytoskeleton-accociated actin (F-actin) and a decrease of monomeric (G-actin) concentration when compared to suspended cells before plating.…”

“…9 Nitric oxide (NO), the physiological regulator of leukocyte adhesion to endothelium, appears to be a natural causative factor for TVE formation. 10,11 NO-induced neutrophil tubulovesicular extensions connected neutrophils to substrata and to the other cells, and bound and aggregated pathogenic bacteria over a distance of several cell diameters.…”

Cytonemes as specialized signaling filopodia

“…In transformed mammalian cell lines, filopodia are associated with EGF (Lidke et al, 2005) and FGF (Koizumi et al, 2012) signal transduction. They have been observed extending from Xenopus XTC cells (Holzer et al, 2012), from B cells (Gupta and DeFranco, 2003), from mast cells induced by chemokines (Fifadara et al, 2010), and from neutrophils induced by nitric oxide and 4-bromophenacyl bromide (Galkina et al, 2009) or aggregation of the A3-adenosine receptor (Corriden et al, 2013). In developmental contexts, filopodia are associated with patterning of pigment cells in zebrafish (Inaba et al, 2012) and with transport of Hh in the chick limb bud (Sanders et al, 2013).…”

Membrane tubulovesicular extensions (cytonemes)

“…Extracellular binding of bacteria by neutrophil TVEs represents an alternative phagocytotic mechanism to bind and kill pathogens. [26][27][28][29] Notably, there are principle differences between TVEs and so-called "neutrophil extracellular traps," or NETs. According to Zychlinsky and colleagues, neutrophils exposed to uncoated coverslips in the presence of phorbol ether for 4-6 h "extrude" chromatin and granule proteins that form NETs for binding and killing pathogens.…”