Priming of neutrophils and differentiated PLB-985 cells by pathophysiological concentrations of TNF-α is partially oxygen dependent.

Activation of polymorphonuclear leukocytes (PMN) can be modulated to intermediate 'primed' states characterized by enhanced responsiveness to subsequent stimuli. We studied priming in response to TNF-α in human PMN and PLB-985 cells, a myeloid cell line differentiated to a neutrophilic phenotype (PLB-D). PMN generated reactive oxygen species (ROS) in response to TNF-α alone, and NADPH oxidase activity increased in response to stimulation with formyl-Met-Leu-Phe after priming. PLB-D cells also demonstrated priming of NADPH oxidase activity. Similar to priming by endotoxin, priming of the respiratory burst by TNF-α was predominantly oxygen dependent, with marked attenuation of ROS generation if primed anaerobically. Both PMN and PLB-D cells displayed significant increases in cell surface CD11b and gp91(phox) expression after TNF-α priming and PMN displayed activation of MAPK. In response to TNF-α priming, neither mobilization of intracellular proteins nor activation of MAPK pathways was NADPH oxidase dependent. Priming of PMN and PLB-D cells by low TNF-α concentrations enhanced chemotaxis. These data demonstrate that pathophysiological concentrations of TNF-α elicit NADPH oxidase-derived ROS and prime cells for enhanced surface protein expression, activation of p38 and ERK1/2 MAPK pathways, and increased chemotaxis. Furthermore, PLB-D cells undergo TNF-α priming and provide a genetically modifiable model to study priming mechanisms.

ClC-3 and IClswell are required for normal neutrophil chemotaxis and shape change.

Polymorphonuclear leukocytes undergo directed movement to sites of infection, a complex process known as chemotaxis. Extension of the polymorphonuclear leukocyte (PMN) leading edge toward a chemoattractant in association with uropod retraction must involve a coordinated increase/decrease in membrane, redistribution of cell volume, or both. Deficits in PMN phagocytosis and trans-endothelial migration, both highly motile PMN functions, suggested that the anion transporters, ClC-3 and ICl(swell), are involved in cell motility and shape change ( Moreland, J. G., Davis, A. P., Bailey, G., Nauseef, W. M., and Lamb, F. S. (2006) J. Biol. Chem. 281, 12277-12288 ). We hypothesized that ClC-3 and ICl(swell) are required for normal PMN chemotaxis through regulation of cell volume and shape change. Using complementary chemotaxis assays, EZ-TAXIScantrade mark and dynamic imaging analysis software, we analyzed the directed cell movement and morphology of PMNs lacking normal anion transporter function. Murine Clcn3(-/-) PMNs and human PMNs treated with anion transporter inhibitors demonstrated impaired chemotaxis in response to formyl peptide. This included decreased cell velocity and failure to undergo normal cycles of elongation and retraction. Impaired chemotaxis was not due to a diminished number of formyl peptide receptors in either murine or human PMNs, as measured by flow cytometry. Murine Clcn3(-/-) and Clcn3(+/+) PMNs demonstrated a similar regulatory volume decrease, indicating that the ICl(swell) response to hypotonic challenge was intact in these cells. We further demonstrated that ICl(swell) is essential for shape change during human PMN chemotaxis. We speculate that ClC-3 and ICl(swell) have unique roles in regulation of PMN chemotaxis; ICl(swell) through direct effects on PMN volume and ClC-3 through regulation of ICl(swell).