No abstract

Neutrophils are the first line of defense against bacteria and fungi and help combat parasites and viruses. They are necessary for mammalian life, and their failure to recover after myeloablation is fatal. Neutrophils are short-lived, effective killing machines. Their life span is significantly extended under infectious and inflammatory conditions. Neutrophils take their cues directly from the infectious organism, from tissue macrophages and other elements of the immune system. Here, we review how neutrophils traffic to sites of infection or tissue injury, how they trap and kill bacteria, how they shape innate and adaptive immune responses, and the pathophysiology of monogenic neutrophil disorders.

Gasdermin-D (GSDMD) in inflammasome-activated macrophages is cleaved by caspase-1 to generate N-GSDMD fragments. N-GSDMD then oligomerizes in the plasma membrane (PM) to form pores that increase membrane permeability, leading to pyroptosis and IL-1β release. In contrast, we report that although N-GSDMD is required for IL-1β secretion in NLRP3activated human and murine neutrophils, N-GSDMD does not localize to the PM or increase PM permeability or pyroptosis. Instead, biochemical and microscopy studies reveal that N-GSDMD in neutrophils predominantly associates with azurophilic granules and LC3 + autophagosomes. N-GSDMD trafficking to azurophilic granules causes leakage of neutrophil elastase into the cytosol, resulting in secondary cleavage of GSDMD to an alternatively cleaved N-GSDMD product. Genetic analyses using ATG7-deficient cells indicate that neutrophils secrete IL-1β via an autophagy-dependent mechanism. These findings reveal fundamental differences in GSDMD trafficking between neutrophils and macrophages that underlie neutrophil-specific functions during inflammasome activation.

This work presents direct evidence that the bcl-2 gene is transcriptionally regulated by nuclear factor-kB (NF-kB) and directly links the TNF-a/NF-kB signaling pathway with Bcl-2 expression and its pro-survival response in human prostate carcinoma cells. DNase I footprinting, gel retardation and supershift analysis identi®ed a NFkB site in the bcl-2 p2 promoter. In the context of a minimal promoter, this bcl-2 p2 site 1 increased transcription 10-fold in the presence of the p50/p65 expression vectors, comparable to the increment observed with the consensus NF-kB site, while for the full p2 promoter region transcriptional activity was increased sixfold by over-expression of NF-kB, an eect eliminated by mutating the bcl-2 p2 site 1. The expression of Bcl-2 has been linked to the hormone-resistant phenotype of advanced prostate cancer. Here we show that an increase in the level of expression of Bcl-2 in the human prostate carcinoma cell line LNCaP observed in response to hormone withdrawal is further augmented by TNF-a treatment, and this eect is abated by inhibitors of NFkB. Concomitantly, bcl-2 p2 promoter studies in LNCaP cells show a 40-fold increase in promoter activity after stimulation with TNF-a in the absence of hormone. Oncogene (2001) 20, 7342 ± 7351.

Nitric oxide ('NO) release, oxygen uptake and hydrogen peroxide (H202) production elicited by increasing phorbol12-myristate 13-acetate (PMA) concentrations were measured in human neutrophils. Half-maximal activities were sequentially elicited at about 0.0001-0.001 pg PMA/ml CNO) and O.OOl~.Ol pg PMA/ml (H,OJ. At saturated PMA concentrations, 'NO production, oxygen uptake and H,O, release were 0.56 ? 0.04, 3.32 f 0.52 and 1.19 f 0.17 nmol min-' 1 O6 cells-'. 'NO production accounts for about 30% of the total oxygen uptake. Luminol-dependent chemiluminescence, reported to detect NO reactions in other inflammatory cells, was also half-maximally activated at about 0.00-0.01 pg PMA/ml. Preincubation with A'"-monomethyl-L-arginine (L-NMMA) decreased 0, uptake and 'NO release but increased H,Oz production, while superoxide dismutase (SOD) increased 'NO detection by 30%. Chemiluminescence was also reduced by preincubation with L-NMMA and/or SOD. The results indicate that 'NO release is part of the integrated response of stimulated human neutrophils and that, in these cells, kinetics of-NO and O;-release favour the formation of other oxidants like peroxynitrite.