Cytokine generation, promoter activation, and oxidant-independent NF-kappaB activation in a transfectable human neutrophilic cellular model.
BACKGROUND
Human neutrophils are key players of innate immunity, and influence inflammatory and immune reactions through the production of numerous cytokines and chemokines. Despite major advances in our understanding of this important functional response of neutrophils, the short lifespan of these cells and their resistance to transfection have always been an obstacle to the detailed dissection of signaling pathways and effector responses that is often possible in other cell types.


RESULTS
Here, we report that granulocytic differentiation of human PLB-985 cells with DMSO yields cells that are neutrophil-like with respect to surface markers, acquisition of responsiveness to physiological neutrophil stimuli (fMLP, LPS), cytokine expression and production profile, and transcription factor activation profile (NF-kappaB, C/EBP, AP-1, STAT). We also show that granulocytic PLB-985 cells can be reliably tranfected by nucleofection in a rapid and efficient manner. Indeed, we overexpressed several proteins and luciferase constructs into these cells. In particular, overexpression of a dominant negative IkappaB-alpha confirmed the central role of NF-kappaB in the production of cytokines by granulocytes. Moreover, the use of PLB-985 granulocytes in which the NADPH oxidase is inactive due to the targeted disruption of a key component (gp91phox) revealed that NF-kappaB activation and kappaB-dependent responses are independent of endogenous reactive oxygen intermediates in these cells. Antioxidant studies performed in primary human neutrophils support this conclusion.


CONCLUSIONS
Our results unveil a new facet of the NF-kappaB system of human granulocytes, and pave the way for deciphering signal transduction pathways and promoter activation in these cells.
Ear
Thornin
T
Pulmonary Division, Faculty of Medicine, Université de Sherbrooke and Centre de recherche du CHUS, Sherbrooke, Qc, Canada. thornin.ear@USherbrooke.ca
McDonald
Patrick P
PP
D002454:Cell Differentiation; D016207:Cytokines / Q000096:biosynthesis*; D005434:Flow Cytometry; D006098:Granulocytes / Q000166:cytology*; D006801:Humans; D008954:Models, Biological; D016328:NF-kappa B / Q000378:metabolism*; D009504:Neutrophils / Q000166:cytology* / Q000378:metabolism; D016877:Oxidants; D011401:Promoter Regions, Genetic; D014162:Transfection
D016207:Cytokines; D016328:NF-kappa B; D016877:Oxidants