Mature natural killer (NK) cells use Ca²⁺-dependent granule exocytosis and release of cytotoxic proteins, Fas ligand (FasL), and membrane-bound or secreted cytokines (tumor necrosis factor [TNF]-α) to induce target cell death. Fas belongs to the TNF receptor family of molecules, containing a conserved intracytoplasmic “death domain” that indirectly activates the caspase enzymatic cascade and ultimately apoptotic mechanisms in numerous cell types. Two additional members of this family, DR4 and DR5, transduce apoptotic signals upon binding soluble TNF-related apoptosis-inducing ligand (TRAIL) that, like FasL, belongs to the growing TNF family of molecules. Here, we report that TRAIL produced or expressed by different populations of primary human NK cells is functional, and represents a marker of differentiation or activation of these, and possibly other, cytotoxic leukocytes. During differentiation NK cells, sequentially and differentially, use distinct members of the TNF family or granule exocytosis to mediate target cell death. Phenotypically immature CD161⁺/CD56⁻ NK cells mediate TRAIL-dependent but not FasL- or granule release–dependent cytotoxicity, whereas mature CD56⁺ NK cells mediate the latter two.

Human natural killer (NK) cell differentiation from immature lineage negative (Lin-) umbilical cord blood cells was examined in vitro. Cells expressing differentiation antigens of mature NK cells (CD56, CD16, CD2, CD8, NKR-P1A) were generated from Lin- cells cultured with interleukin (IL)-2 and a murine bone marrow stromal cell line expressing the human membrane-bound form of stem cell factor. Two subsets of NK cells were identified in these cultures: one expressed both NKR-P1A and CD56 and, in variable proportions, all other NK cell differentiation antigens; the second subset expressed only NKR-P1A and, unlike the former, was not cytotoxic. Neither subset expressed interferon (IFN)-gamma mRNA even after stimulation with phorbol di-ester and Ca2+ ionophore, but both expressed tumor necrosis factor alpha mRNA and the cytotoxic granule-associated proteins TIA-1, perforin, and serine esterase-1. After 10-d culture with IL-2, IL-12, and irradiated B lymphoblastoid cells, approximately 45% of the NKR-P1A+/ CD56- cells became CD56+, and the same cultures contained cells capable of cytotoxicity and of IFN-gamma production. These results indicate that NKR-P1A expression in the absence of other NK cell markers defines an intermediate, functionally immature stage of NK cell differentiation, and that effector functions develop in these cells, concomitantly with CD56 expression, in the presence of IL-12. These cells likely represent the counterpart of a CD3-/NKR-P1A+/ CD56-/CD16- cell subset that, as shown here, is present both in adult and neonatal circulating lymphocytes.

Oxysterols are bioactive lipids that act as regulators of lipid metabolism, inflammation, cell viability and are involved in several diseases, including atherosclerosis. Mounting evidence linked the atherosclerosis to endothelium dysfunction; in fact, the endothelium regulates the vascular system with roles in processes such as hemostasis, cell cholesterol, hormone trafficking, signal transduction and inflammation. Several papers shed light the ability of oxysterols to induce apoptosis in different cell lines including endothelial cells. Apoptotic endothelial cell and endothelial denudation may constitute a critical step in the transition to plaque erosion and vessel thrombosis, so preventing the endothelial damaged has garnered considerable attention as a novel means of treating atherosclerosis. Endoplasmic reticulum (ER) is the site where the proteins are synthetized and folded and is necessary for most cellular activity; perturbations of ER homeostasis leads to a condition known as endoplasmic reticulum stress. This condition evokes the unfolded protein response (UPR) an adaptive pathway that aims to restore ER homeostasis. Mounting evidence suggests that chronic activation of UPR leads to cell dysfunction and death and recently has been implicated in pathogenesis of endothelial dysfunction. Autophagy is an essential catabolic mechanism that delivers misfolded proteins and damaged organelles to the lysosome for degradation, maintaining basal levels of autophagic activity it is critical for cell survival. Several evidence suggests that persistent ER stress often results in stimulation of autophagic activities, likely as a compensatory mechanism to relieve ER stress and consequently cell death. In this review, we summarize evidence for the effect of oxysterols on endothelial cells, especially focusing on oxysterols-mediated induction of endoplasmic reticulum stress.

Flow cytometry has recently become a choice technique for the quantitative analysis of apoptosis. Monoparametric DNA analysis usually allows identification of apoptotic cells as a “subdiploid” peak. Progression through apoptosis leads to chromatin condensation, nuclear fragmentation and eventually to cell disruption. Thus, a major problem for the flow cytometric analysis of apoptotic populations is discrimination between debris and apoptotic cells. Here we demonstrate that the best parameter on which to make such a distinction is the DNA content, no matter what type of cell is studied. In contrast, discrimination between apoptotic, non‐apoptotic cells, and debris is possible on the basis of scattering signals only in few selected cases, depending on the morphology of the intact cells.

To determine whether production of type 1 and type 2 cytokines defines discrete stages of natural killer (NK) cell differentiation, cytokine expression was analyzed in human NK cells generated in vitro in the presence of interleukin-15 (IL-15) and/or IL-2 from umbilical cord blood hematopoietic progenitors. Like peripheral NK cells, the CD161⁺/CD56⁺ NK cells from these cultures contained a tumor necrosis factor alpha (TNF-α)⁺/granulocyte macrophage–colony-stimulating factor (GM-CSF)⁺ subset, an interferon gamma (IFN-γ)⁺ subset, mostly included within the former, and very few IFN-γ⁻/IL-13⁺ cells. Instead, most immature CD161⁺/CD56⁻ NK cells, detectable only in the cultures with IL-2, produced IL-13, TNF-α, and GM-CSF, but not IFN-γ, and contained an IL-5⁺ subset. In short-term cultures with IL-12 and feeder cells, a proportion of the immature cells acquired the ability to produce IFN-γ. Part of these produced both IFN-γ and IL-13, irrespective of induced CD56 expression. These in vitro data indicate that ability to produce the type 2 cytokines IL-13 and IL-5 defines CD161⁺ NK cells at intermediate stages of differentiation, and is lost upon terminal functional differentiation, concomitant with acquired ability to produce IFN-γ.