Lack of eosinophil extracellular trap formation due to failure of plasma membrane breakdown in the absence of elastase.

Activated eosinophils are described to release eosinophil extracellular traps (EETs), which consist of the cell's DNA covered with granule-derived antimicrobial peptides. Upon stimulation of eosinophils with the known EET-inducers phorbol 12-myristate 13-acetate, monosodium urate crystals, or Candida albicans, we observed that their plasma membrane became compromised, resulting in accessibility of the nuclear DNA for staining with the impermeable DNA dye Sytox Green. However, we did not observe any DNA decondensation or plasma membrane rupture by eosinophils, which sharply contrasts with neutrophil extracellular trap (NET) formation and the subsequent cell death known as NETosis. Neutrophil elastase (NE) activity is thought to be essential for the cleavage of histones and chromatin decondensation during NETosis. We observed that the neutrophils of a patient with a mutation in ELANE, leading to congenital neutropenia and NE deficiency, were unable to undergo NETosis. Taken together, we may suggest that the natural absence of any NE-like proteolytic activity in human eosinophils explains why EET formation is not observed, even when eosinophils become positive for an impermeable DNA dye in response to stimuli that induce NETosis in neutrophils.

Efficient complement-mediated clearance of immunosuppressed T cells by macrophages.

Cancer is one of the leading causes of death worldwide. Treatment outcome is largely dictated by the tumor type, disease stage, and treatment success rates, but also by the variation among patients in endogenous anti-tumor responses. Studies indicate that the presence of neutrophils in the tumor microenvironment is associated with a worse patient outcome due to their ability to suppress local anti-tumor T cell activity. Our previous studies investigated the mechanisms by which neutrophils suppress and damage T cells to become smaller in size (small T cells), debilitating their effector activities. Several studies indicate a role for tumor-associated macrophages in scavenging damaged or dead cells. We hypothesized that the observed lack of small T cells in the TME by confocal microscopy is due to immediate uptake by macrophages. In this study, we confirmed that indeed only the smaller, damaged T cells are taken up by macrophages, once serum-opsonized. Damaged T cells opsonized with complement factor C3 fragments were phagocytosed by macrophages, resulting in almost instantaneous and highly efficient uptake of these small T cells. Inhibition of the complement receptors CR1, CR3 and CR4 expressed by macrophages completely blocked phagocytosis. By contrast, actively proliferating T cells (large T cells) were neither impaired in neutrophil-MDSC activity nor opsonized for phagocytosis by macrophages. Rapid removal of damaged T cells suggests a role of complement and macrophages within the tumor microenvironment to clear suppressed T cells in cancer patients.

Analysis of centrosomal area actin reorganization and centrosome polarization upon lymphocyte activation at the immunological synapse.

T cell receptor (TCR) and B cell receptor (BCR) stimulation of T and B lymphocytes, by antigen presented on an antigen-presenting cell (APC) induces the formation of the immunological synapse (IS). IS formation is associated with an initial increase in cortical filamentous actin (F-actin) at the IS, followed by a decrease in F-actin density at the central region of the IS, which contains the secretory domain. This is followed by the convergence of secretion vesicles towards the centrosome, and the polarization of the centrosome to the IS. These reversible, cortical actin cytoskeleton reorganization processes occur during lytic granule secretion in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells, proteolytic granules secretion in B lymphocytes and during cytokine-containing vesicle secretion in T-helper (Th) lymphocytes. In addition, several findings obtained in T and B lymphocytes forming IS show that actin cytoskeleton reorganization also occurs at the centrosomal area. F-actin reduction at the centrosomal area appears to be associated with centrosome polarization. In this chapter we deal with the analysis of centrosomal area F-actin reorganization, as well as the centrosome polarization analysis toward the IS.