Neutrophil granulocytes are rapidly recruited from the bloodstream to the site of acute inflammation where they die in large numbers. Because release of toxic substances from dead neutrophils can propagate the inflammatory response leading to tissue destruction, clearance of dying inflammatory neutrophils has a critical function in the resolution of the inflammatory response. Apoptotic neutrophils are phagocytosed primarily by macrophages, provided these cells are present in adequate numbers. However, macrophages are rare at sites of acute inflammation, whereas the number of neutrophils can be extremely high. In the current study, in vitro experiments with human neutrophils were carried out to investigate whether neutrophils can ingest apoptotic neutrophils. We show that naïve granulocytes isolated from venous blood have a limited capacity to phagocytose apoptotic cells. However, exposure to activating stimuli such as LPS, GM-CSF and/or IFN-γ results in enhanced phagocytosis of apoptotic cells. The efficient uptake of apoptotic cells by neutrophils was found to depend on the presence of heat labile serum factors. Importantly, the contact to or uptake of apoptotic cells inhibited neutrophil functions such as respiratory burst and the release of the proinflammatory cytokines TNF-α and interferon-inducible protein-10. Contact to apoptotic cells, however, induced the secretion of IL-8 and growth-related oncogene-α, which was independent of NF-κB and p38 MAPK but involved C5a and the ERK1/2 pathway. The data suggest that activated neutrophils participate in the clearance of apoptotic cells. In addition, because apoptotic cells inhibit proinflammatory functions of neutrophils, uptake of apoptotic cells by neutrophils contributes to the resolution of inflammation.

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Neutrophil granulocytes provide the first line of defense against bacterial, fungal, and parasitic infections. They phagocytose and kill many invading pathogens. Certain pathogenic microorganisms such as the intracellular protozoan parasite Leishmania major (L. major) can survive inside neutrophils. Mature neutrophils have a very short life span due to spontaneous apoptosis. Previously, we have reported that infections with L. major are able to delay spontaneous apoptosis. In the present study, we addressed the underlying mechanisms of regulation of both extrinsic and intrinsic apoptosis. We show that interaction with L. major transiently activates ERK1/2 phosphorylation. Pharmacological inhibition of ERK1/2 phosphorylation reversed the apoptosis delay. Moreover, infection leads to the enhanced and sustainable expression of the anti-apoptotic proteins Bcl-2 and Bfl-1, respectively. As downstream events, the release of cytochrome c from mitochondria and processing of caspase-6 were inhibited. We also confirm that infection with L. major results in reduced FAS expression on the surface of neutrophils. The presented data indicate that infection with L. major affects both intrinsic as well as extrinsic pathways of neutrophil apoptosis. Enhanced life span of host neutrophils enables the parasite to survive within neutrophils.

Upon LPS binding, TLR4 activates a MyD88-dependent pathway leading to the transcriptional activation of proinflammatory genes, as well as a MyD88-independent/TRIF-dependent pathway, responsible for the transcriptional induction of IFN-β. Previous findings delineated that human neutrophils are unable to induce the transcription of IFN-β in response to TLR4 stimulation. Because neutrophils do not express protein kinase C ε, a molecule recently reported as essential for initiating the MyD88-independent/TRIF-dependent pathway, we optimized an electroporation method to transfect PKCε into neutrophils with very high efficiency. By doing so, a significant IFN-β mRNA expression was induced, in the absence of LPS stimulation, not only in PKCε-overexpressing neutrophils but also in cells transfected with a series of empty DNA plasmids; however, LPS further upregulated the IFN-β transcript levels in plasmid-transfected neutrophils, regardless of PKCε overexpression. Phosphoimmunoblotting studies, as well as chromatin immunoprecipitation assays targeting the IFN-β promoter, revealed that IFN-β mRNA induction occurred through the cooperative action of IRF3, activated by transfected DNA, and NF-κB, activated by LPS. Additional immunoblotting and coimmunoprecipitation studies revealed that neutrophils constitutively express various cytosolic DNA sensors, including IFN-inducible protein 16, leucine-rich repeat (in Flightless I) interacting protein-1, and DDX41, as well as that IFN-inducible protein 16 is the intracellular receptor recognizing transfected DNA. Consistently, infection of neutrophils with intracellular pathogens, such as Bartonella henselae, Listeria monocytogenes, Legionella pneumophila, or adenovirus type 5, promoted a marked induction of IFN-β mRNA expression. Taken together, these data raise questions about the role of PKCε in driving the MyD88-independent/TRIF-dependent response and indicate that human neutrophils are able to recognize and respond to microbial cytosolic DNA.