Sestina Falcone

Macropinocytosis: regulated coordination of endocytic and exocytic membrane traffic events

Macropinocytosis, a form of bulk uptake of fluid and solid cargo into cytoplasmic vacuoles, called macropinosomes, has been studied mostly in relation to antigen presentation. Early membrane traffic events occurring in this process are, however, largely unknown. Using human dendritic cells we show that a marked increase in the rate of macropinocytosis occurs a few minutes after application of two markers (small latex beads or dextran), depends on a slow intracellular Ca2+ concentration ([Ca2+]i) rise that precedes the PI3K-dependent step, and is preceded and accompanied by exocytosis of enlargeosomes compensating in part for the macropinocytic plasma membrane internalization. Unexpectedly, macropinosomes themselves, which share markers with endosomes, undergo Ca2+-dependent exocytosis so that, after ∼20 minutes of continuous bead or dextran uptake, an equilibrium is reached preventing cells from overloading themselves with the organelles. Large [Ca2+]i increases induced by ionomycin trigger rapid (<1 minute) exocytic regurgitation of all macropinosomes, whereas endosomes remain apparently unaffected. We conclude that, in dendritic cells, the rate of macropinocytosis is not constant but increases in a regulated fashion, as previously shown in other cell types. Moreover, macropinosomes are not simple containers that funnel cargo to an endocytic pathway, but unique organelles, distinct from endosomes by their competence for regulated exocytosis and other membrane properties.

Activation of Acid Sphingomyelinase and Its Inhibition by the Nitric Oxide/Cyclic Guanosine 3′,5′-Monophosphate Pathway: Key Events in Escherichia coli-Elicited Apoptosis of Dendritic Cells

Depletion of dendritic cells (DCs) via apoptosis contributes to sepsis-induced immune suppression. The mechanisms leading to DC apoptosis during sepsis are not known. In this study we report that immature DCs undergo apoptosis when treated with high numbers of Escherichia coli. This effect was mimicked by high concentrations of LPS. Apoptosis was accompanied by generation of ceramide through activation of acid sphingomyelinase (A-SMase), was prevented by inhibitors of this enzyme, and was restored by exogenous ceramide. Compared with immature DCs, mature DCs expressed significantly reduced levels of A-SMase, did not generate ceramide in response to E. coli or LPS, and were insensitive to E. coli- and LPS-triggered apoptosis. However, sensitivity to apoptosis was restored by addition of exogenous A-SMase or ceramide. Furthermore, inhibition of A-SMase activation and ceramide generation was found to be the mechanism through which the immune-modulating messenger NO protects immature DCs from the apoptogenic effects of E. coli and LPS. NO acted through formation of cGMP and stimulation of the cGMP-dependent protein kinase. The relevance of A-SMase and its inhibition by NO/cGMP were confirmed in a mouse model of LPS-induced sepsis. DC apoptosis was significantly higher in inducible NO synthase-deficient mice than in wild-type animals and was significantly reduced by treatment ex vivo with NO, cGMP, or the A-SMase inhibitor imipramine. Thus, A-SMase plays a central role in E. coli/LPS-induced DC apoptosis and its inhibition by NO, and it might be a target of new therapeutic approaches to sepsis.

Nitric Oxide Boosts Chemoimmunotherapy via Inhibition of Acid Sphingomyelinase in a Mouse Model of Melanoma

Cisplatin is one of the most effective anticancer drugs, but its severe toxic effects, including depletion of immune-competent cells, limit its efficacy. We combined the systemic treatment with cisplatin with intratumor delivery of dendritic cells (DC) previously treated ex vivo with a pulse of nitric oxide (NO) released by the NO donors (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]-diazen-1-ium-1,2-diolate or isosorbide dinitrate. We found that this chemoimmunotherapy, tested in the B16 mouse model of melanoma, was significantly more efficacious than cisplatin alone, leading to tumor regression and animal survival at low doses of cisplatin that alone had no effect. Tumor cure was not observed when combining cisplatin with DCs not exposed to NO donors, indicating the key role of the pretreatment with NO. We investigated the mechanisms responsible for the synergic effect of NO-treated DCs and cisplatin and found that NO-treated DCs were protected both in vitro and in vivo from cisplatin-induced cytotoxicity. Cisplatin triggered DC apoptosis through increased expression and activation of acid sphingomyelinase; pretreatment of DCs with NO donors prevented such activation and inhibited activation of the downstream proapoptotic events, including generation of ceramide, activation of caspases 3 and 9, and mitochondrial depolarization. The effects of NO were mediated through generation of its physiologic messenger, cyclic GMP. We conclude that NO and NO generating drugs represent promising tools to increase the efficacy of chemoimmunotherapies in vivo, promoting the survival and increasing the function of injected cells by targeting a key pathway in cisplatin-induced cytotoxicity.

Nitric Oxide Confers Therapeutic Activity to Dendritic Cells in a Mouse Model of Melanoma

Susceptibility of dendritic cells (DCs) to tumor-induced apoptosis reduces their efficacy in cancer therapy. Here we show that delivery within exponentially growing B16 melanomas of DCs treated ex vivo with nitric oxide (NO), released by the NO donor (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO), significantly reduced tumor growth, with cure of 37% of animals. DETA-NO-treated DCs became resistant to tumor-induced apoptosis because DETA-NO prevented tumor-induced changes in the expression of Bcl-2, Bax, and Bcl-xL; activation of caspase-9; and a reduction in the mitochondrial membrane potential. DETA-NO also increased DC cytotoxic activity against tumor cells and DC ability to trigger T-lymphocyte proliferation. All of the effects of DETA-NO were mediated through cGMP generation. NO and NO-generating drugs may therefore be used to increase the anticancer efficacy of DCs.

Synergism of nitric oxide and maturation signals on human dendritic cells occurs through a cyclic GMP-dependent pathway

Nitric oxide (NO), generated by phagocytes at inflammation sites, contributes to regulate immune responses through autocrine and paracrine actions on bystander cells. Among the latter are dendritic cells (DCs). Little is known about regulation of DC function by NO, especially in the human system. We exposed human monocyte‐derived DCs to the NO donor (z)‐1‐[2‐(2‐aminoethyl)‐N‐(2‐ammonioethyl)amino] diazen‐1‐ium‐1,2 diolate (DETA‐NO) during their maturation process induced by treatment with tumor necrosis factor α or lipopolysaccharide or by CD40 activation. We report here that after exposure to DETA‐NO, DCs exhibit a significantly increased ability to activate T lymphocytes stimulated by mycobacterial antigens, Staphylococcus aureus Cowen strain B, allo‐antigens, or cross‐linking of the CD3–T cell receptor complex. This effect persists after removal of DETA‐NO, depends on the generation of cyclic guanosine 5′‐monophosphate, and is a result of enhanced release by DCs of soluble factors, in particular interleukin (IL)‐12. This modulation of DC function is a result of a synergism between NO and the various maturation stimuli, as neither enhanced T cell activation nor IL‐12 release was observed after DC exposure to DETA‐NO only. These results provide the first evidence that NO acts as a cosignaling molecule regulating human DC response to maturation stimuli.

Characterization of two novel SETX mutations in AOA2 patients reveals aspects of the pathophysiological role of senataxin

Ataxia with oculomotor apraxia (AOA) type 2 (AOA2 MIM 606002) is a recessive subtype of AOA characterized by cerebellar atrophy, oculomotor apraxia, early loss of reflexes, and peripheral neuropathy. Various mutations either in homozygous or compound heterozygous condition were so far identified in the associated gene SETX (MIM 608465). SETX encodes a large protein called senataxin with a DNA-RNA helicase domain and a putative N-terminus protein interaction domain. Here, we report the identification of two novel homozygous mutations in SETX gene, c.340_342delCTT (p.L114Del) and c.1669C > T (p.R557X), in two AOA2 families. The characterization of the mutant lymphoblastoid cell lines for sensitivity to oxidative DNA-damaging agents indicates that the p.L114Del deletion confers an increased sensitivity to H2O2, camptothecin, and mitomycin C, previously found to induce death in lymphoblasts harbouring other SETX mutations; the cells carrying the nonsense mutation display instead values within the normal range. Further analysis of a neuronal cell model SKNBE, transfected with the mutant senataxin proteins, reveals increased sensitivity also to staurosporine and excitotoxicity associated with the p.L114Del mutant only. We also demonstrate that the sensitizing effect of p.L114Del on apoptosis can be reversed by senataxin silencing. The ability of a single amino acid deletion to sensitize cells to death by different agents, compared to the lack of effect of a whole protein deletion, seems to exclude a protective role played by the native protein while suggesting that a specific mutation confers to the protein the ability to enhance the toxic effect of various cell damaging agents.