Apoptosis Of Human Neutrophils Research Articles

Effects of endotoxin/lipopolysaccharide on early apoptosis of human neutrophil through PIM3

Objective: To explore the effects of endotoxin/lipopolysaccharide (LPS) on early apoptosis of human neutrophil through PIM3. Methods: Venous blood samples were collected from a healthy adult volunteer to isolate neutrophils. The neutrophils were divided into control group, LPS group, and LPS+ PIM447 group according to the random number table. No treatment was given to the cells in control group. The cells in LPS group underwent LPS stimulation (1 μL, 1 μg/mL). The cells in LPS+ PIM447 group underwent PIM447 (1 μL, final amount-of-substance concentration of 5 μmol/L) intervention 30 min before having the same LPS stimulation as that in LPS group. After conventional culture for 1 h, the early cell apoptosis rate was determined with flow cytometer; the generation level of reactive oxygen species (ROS) was assessed with dihydrogenrhodamine 123 fluorescent probe staining method; and the level of PIM3 was detected by Western blotting. After conventional culture for 2 h, the cell chemotaxis distance was measured by agarose chemotaxis cell model. The sample numbers of each group in the 4 experiments were all 5. Data were processed with one-way analysis of variance and Student-Newman-Keuls test. Results: (1) The early apoptosis rate of cells in LPS group [(0.891±0.012)%] was close to that in control group [(1.351±0.183)%, P>0.05)]. The early apoptosis rate of cells in LPS+ PIM447 group [(82.057±0.121)%] was higher than that in LPS group (P<0.01). (2) The cell chemotaxis distance of cells in LPS group [(984±5) μm] was significantly shorter than that in control group [(2 241±7) μm, P<0.01]. The cell chemotaxis distance of cells in LPS+ PIM447 group [(1 785±11) μm]was significantly longer than that in LPS group (P<0.05). (3) The generation level of ROS in cells of LPS group was significantly higher than that in control group (P<0.05). The generation level of ROS in cells of LPS+ PIM447 group was significantly lower than that in LPS group (P<0.05). (4) The expression level of PIM3 in cells of LPS group (1.297±0.015) was significantly higher than that in control group (0.789±0.021, P<0.05). The expression level of PIM3 in cells of LPS+ PIM447 group (0.731±0.011) was significantly lower than that in LPS group (P<0.05). Conclusions: LPS stimulation can reduce the early apoptosis of human neutrophils. Pre-intervention with PIM447 can significantly increase the early apoptosis of neutrophils after LPS stimulation, recover the chemotaxis, and inhibit the production of ROS. The mechanism may be related to LPS promoting the expression of PIM3.

Distinct Trypanosoma cruzi isolates induce activation and apoptosis of human neutrophils.

Neutrophils are critical players in the first line of defense against pathogens and in the activation of subsequent cellular responses. We aimed to determine the effects of the interaction of Trypanosoma cruzi with human neutrophils, using isolates of the two major discrete type units (DTUs) associated with Chagas’ disease in Latin America (clone Col1.7G2 and Y strain, DTU I and II, respectively). Thus, we used CFSE-stained trypomastigotes to measure neutrophil-T. cruzi interaction, neutrophil activation, cytokine expression and death, after infection with Col1.7G2 and Y strain. Our results show that the frequency of CFSE+ neutrophils, indicative of interaction, and CFSE intensity on a cell-per-cell basis were similar when comparing Col1.7G2 and Y strains. Interaction with T. cruzi increased neutrophil activation, as measured by CD282, CD284, TNF and IL-12 expression, although at different levels between the two strains. No change in IL-10 expression was observed after interaction of neutrophils with either strain. We observed that exposure to Y and Col1.7G2 caused marked neutrophil death. This was specific to neutrophils, since interaction of either strain with monocytes did not cause death. Our further analysis showed that neutrophil death was a result of apoptosis, which was associated with an upregulation of TNF-receptor, TNF and FasLigand, but not of Fas. Induction of TNF-associated neutrophil apoptosis by the different T. cruzi isolates may act as an effective common mechanism to decrease the host’s immune response and favor parasite survival.

Bacterial lipoproteins and other factors released byFrancisella tularensismodulate human neutrophil lifespan: Effects of aTLR1SNP on apoptosis inhibition

Francisella tularensis infects several cell types including neutrophils, and aberrant neutrophil accumulation contributes to tissue destruction during tularaemia. We demonstrated previously that F. tularensis strains Schu S4 and live vaccine strain markedly delay human neutrophil apoptosis and thereby prolong cell lifespan, but the bacterial factors that mediate this aspect of virulence are undefined. Herein, we demonstrate that bacterial conditioned medium (CM) can delay apoptosis in the absence of direct infection. Biochemical analyses show that CM contained F. tularensis surface factors as well as outer membrane components. Our previous studies excluded roles for lipopolysaccharide and capsule in apoptosis inhibition, and current studies of [14C] acetate‐labelled bacteria argue against a role for other bacterial lipids in this process. At the same time, studies of isogenic mutants indicate that TolC and virulence factors whose expression requires FevR or MglA were also dispensable, demonstrating that apoptosis inhibition does not require Type I or Type VI secretion. Instead, we identified bacterial lipoproteins (BLPs) as active factors in CM. Additional studies of isolated BLPs demonstrated dose‐dependent neutrophil apoptosis inhibition via a TLR2‐dependent mechanism that is significantly influenced by a common polymorphism, rs5743618, in human TLR1. These data provide fundamental new insight into pathogen manipulation of neutrophil lifespan and BLP function.

Angiotensin-(1-7) Promotes Resolution of Neutrophilic Inflammation in a Model of Antigen-Induced Arthritis in Mice.

Defective resolution of inflammation may be crucial for the initiation and development of chronic inflammatory diseases, such as arthritis. Therefore, it has been suggested that therapeutic strategies based on molecules that facilitate inflammation resolution present great potential for the treatment of chronic inflammatory diseases. In this study, we investigated the effects and role of angiotensin-(1-7) [Ang-(1-7)] in driving resolution of neutrophilic inflammation in a model of arthritis. For this purpose, male C57BL/6 mice were subjected to antigen-induced arthritis and treated with Ang-(1-7) at the peak of the inflammatory process. Analysis of the number of inflammatory cells, apoptosis, and immunofluorescence for NF-κB was performed in the exudate collected from the knee cavity. Neutrophil accumulation in periarticular tissue was measured by assaying myeloperoxidase activity. Apoptosis of human neutrophil after treatment with Ang-(1-7) was evaluated morphologically and by flow cytometry, and NF-κB phosphorylation by immunofluorescence. Efferocytosis was evaluated in vivo. Therapeutic treatment with Ang-(1-7) at the peak of inflammation promoted resolution, an effect associated with caspase-dependent neutrophils apoptosis and NF-κB inhibition. Importantly, Ang-(1-7) was also able to induce apoptosis of human neutrophils, an effect associated with NF-κB inhibition. The pro-resolving effects of Ang-(1-7) were inhibited by the Mas receptor antagonist A779. Finally, we showed that Ang-(1-7) increased the efferocytic ability of murine macrophages. Our results clearly demonstrate that Ang-(1-7) resolves neutrophilic inflammation in vivo acting in two key step of resolution: apoptosis of neutrophils and their removal by efferocytosis. Ang-(1-7) is a novel mediator of resolution of inflammation.

Human Cytomegalovirus Delays Neutrophil Apoptosis and Stimulates the Release of a Prosurvival Secretome.

Human cytomegalovirus (HCMV) is a major cause of viral disease in the young and the immune-suppressed. At sites of infection, HCMV recruits the neutrophil, a cell with a key role in orchestrating the initial immune response. Herein, we report a profound survival response in human neutrophils exposed to the clinical HCMV isolate Merlin, but not evident with the attenuated strain AD169, through suppression of apoptosis. The initial survival event, which is independent of viral gene expression and involves activation of the ERK/MAPK and NF-κB pathways, is augmented by HCMV-stimulated release of a secretory cytokine profile that further prolongs neutrophil lifespan. As aberrant neutrophil survival contributes to tissue damage, we predict that this may be relevant to the immune pathology of HCMV, and the presence of this effect in clinical HCMV strains and its absence in attenuated strains implies a beneficial effect to the virus in pathogenesis and/or dissemination. In addition, we show that HCMV-exposed neutrophils release factors that enhance monocyte recruitment and drive monocyte differentiation to a HCMV-permissive phenotype in an IL-6-dependent manner, thus providing an ideal vehicle for viral dissemination. This study increases understanding of HCMV–neutrophil interactions, highlighting the potential role of neutrophil recruitment as a virulence mechanism to promote HCMV pathology in the host and influence the dissemination of HCMV infection. Targeting these mechanisms may lead to new antiviral strategies aimed at limiting host damage and inhibiting viral spread.

Serum α-1 Antitrypsin (AAT) antagonizes intrinsic apoptosis induction in neutrophils from patients with systemic inflammatory response syndrome

Excessive neutrophil activation accompanied by delayed apoptotic cell death in inflammatory conditions causes progressive damage of cells and tissues, leading to life-threatening multiple organ dysfunction syndrome. Previous work suggested that circulating serum factors during inflammation are critically involved in the suppression of neutrophil cell death although the identity of these antiapoptotic mediators remained elusive. In this study, we identified the acute phase protein α-1 Antitrypsin (AAT) as a potent suppressor of staurosporine (STS)-induced apoptosis in human neutrophils through a mechanism implicating caspases-independent pathways. We show here that serum levels of AAT, potentially in part released by stimulated neutrophils, are markedly elevated in major trauma patients suffering from systemic inflammatory response syndrome (SIRS). Notably, AAT depletion from serum increased sensitivity of human neutrophils for STS-induced cell death. In fact, AAT was demonstrated to confer intrinsic apoptosis resistance by preventing PKC/Akt inactivation and subsequent proteasomal degradation of antiapoptotic Mcl-1 protein in response to STS treatment. Neither MAP kinase ERK1/2 nor caspases were found to be involved in AAT-triggered antiapoptotic pathways in neutrophils. In summary, these results establish a novel pivotal role of circulating AAT in mediating survival by antagonizing the proapoptotic action of the PKC inhibitor STS and should be considered for AAT augmentation therapies in future.

Effect of Propofol on Human Neutrophil Apoptosis in vitro

Effect of Propofol on Human Neutrophil Apoptosis in vitro

Non-canonical PI3K-Cdc42-Pak-Mek-Erk Signaling Promotes Immune-Complex-Induced Apoptosis in Human Neutrophils

SummaryNeutrophils are peripheral blood leukocytes that represent the first line of immune cell defense against bacterial and fungal infections but are also crucial players in the generation of the inflammatory response. Many neutrophil cell surface receptors regulate important cellular processes via activation of agonist-activated PI3Ks. We show here that activation of human neutrophils with insoluble immune complexes drives a previously uncharacterized, PI3K-dependent, non-canonical, pro-apoptotic signaling pathway, FcγR-PI3Kβ/δ-Cdc42-Pak-Mek-Erk. This is a rare demonstration of Ras/Raf-independent activation of Erk and of PI3K-mediated activation of Cdc42. In addition, comparative analysis of immune-complex- and fMLF-induced signaling uncovers key differences in pathways used by human and murine neutrophils. The non-canonical pathway we identify in this study may be important for the resolution of inflammation in chronic inflammatory diseases that rely on immune-complex-driven neutrophil activation.

Effects of ghrelin on the apoptosis of human neutrophils invitro.

Acute respiratory distress syndrome (ARDS) is characterized by lung inflammation and the diffuse infiltration of neutrophils into the alveolar space. Neutrophils are abundant, short-lived leukocytes that play a key role in immune defense against microbial infections. These cells die via apoptosis following the activation and uptake of microbes, and will also enter apoptosis spontaneously at the end of their lifespan if they do not encounter pathogens. Apoptosis is essential for the removal of neutrophils from inflamed tissues and for the timely resolution of neutrophilic inflammation. Ghrelin is an endogenous ligand for the growth hormone (GH) secretagogue receptor, produced and secreted mainly from the stomach. Previous studies have reported that ghrelin exerts anti-inflammatory effects in lung injury through the regulation of the apoptosis of different cell types; however, the ability of ghrelin to regulate alveolar neutrophil apoptosis remains largely undefined. We hypothesized that ghrelin may have the ability to modulate neutrophil apoptosis. In this study, to examine this hypothesis, we investigated the effects of ghrelin on freshly isolated neutrophils in vitro. Our findings demonstrated a decrease in the apoptotic ratio (as shown by flow cytometry), as well as in the percentage of cells with decreased mitochondrial membrane potential (ΔΨm) and in the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling-positive rate, accompanied by an increased B-cell lymphoma 2/Bax ratio and the downregulation of cleaved caspase-3 in neutrophils following exposure to lipopolysaccharide (100 ng/ml). However, pre-treatment with ghrelin at a physiological level (100 nM) did not have a notable influence on the neutrophils in all the aforementioned tests. Our findings suggest that ghrelin may not possess the ability to modulate the neutrophil lifespan in vitro.

Effects of phosphodiester and phosphorothioate ODN2216 on leukotriene synthesis in human neutrophils and neutrophil apoptosis.

Polymorphonuclear leukocytes (PMNLs, neutrophils) play a major role in the initiation and resolution of the inflammatory response, and neutrophil apoptosis is a critical step in resolving inflammation. We examined the effects of oligodeoxynucleotide (ODN) species with different numbers of phosphodiester and phosphorothioate bonds on leukotriene synthesis in PMNLs and on neutrophil apoptosis. Our modifications were based on the well-known ODN2216 molecule (Krug etal., 2001). Treatment of cultured human neutrophils with ODN2216 accelerated apoptosis except in the case of a species with only phosphodiester bonds. The ODNs with poly(g) (phosphorothioate) sequences at both ends and a phosphodiester inner core had maximal effects on leukotriene synthesis in neutrophils and inhibited formation of 5-lipoxygenase metabolites. Addition of phosphodiester and phosphorothioate ODNs to PMNLs produced distinct effects on superoxide and nitric oxide formation: phosphorothioate-containing ODNs concomitantly stimulated production of nitric oxide and superoxide, which may rapidly combine to generate peroxynitrite. Altogether, our results describe strong activation of neutrophil's cellular responses by phosphorothioate ODN2216. We propose that phosphorothioate modification of ODNs represents a potential mechanism of PMNL activation.

Ptdglc-Enriched Lipid Rafts Are a Novel Platform for Apoptosis Signaling in Human Neutrophilic Lineage Cells

Phosphatidylglucoside (PtdGlc) is a recently discovered type of cell surface glycophospholipid that was originally detected in human cord red cells. PtdGlc was also detected in early erythroblastic leukemia cells, astroglial cells from fetal rat brains and the HL-60 acute promyelocytic leukemia (APL) cell line, and thought to be associated with their differentiation (Biochim Biophys Acta. 1851: 90, 2015). Indeed, the recombinant anti-PtdGlc Fab antibody rGL-7 induced HL-60 cells to differentiate into neutrophilic cells along with inducing the phosphorylation of Lyn and Hck (PNAS 100: 7454, 2003). These activities were suppressed by methyl-β-cyclodextrin, which reduced endogenous cholesterol, suggesting that PtdGlc forms lipid raft-like domains on HL-60 cells. Another anti-PtdGlc monoclonal antibody DIM21 induced the formation of FAS-colocalized clusters of PtdGlc on plasma membranes and the apoptosis of human neutrophils[RJ1]. However, it is still unclear about the molecular connection of PtdGlc, inserted into the external leaflet of the membrane bilayer, with signal transduction molecules located at the cytoplasmic side. In this study, we investigated the differentiation effect of DIM21 by comparing the ones of dimethyl sulfoxide (DMSO) and all-trans retinoic acid (ATRA), and identified the extracellular proteins associate with PtdGlc-enriched domains on plasma membranes during the activation of apoptosis signaling in neutrophilic lineage cells.HL60 cells or chronic neutrophilic leukemia (MOLM-20) cells were treated with DMSO or ATRA in the absence or presence of DIM21, and the expression of differentiation markers was determined. DIM21 induced the neutrophilic differentiation marker CD11b by itself, and synergistically enhanced DMSO-induced CD11b expression in HL-60 and MOLM-20 cells. DIM21 significantly enhanced annexin V positive DMSO-treated neutrophilic differentiated HL-60 (D-HL60) cells, but not MOLM-20 cells. FITC BrdU Flow analysis showed that the both ATRA and DIM21 induced the cell cycle accumulation in G0/G1 phase and reduced the percentage in S phase, suggesting that not only ATRA but also DIM21 is capable of reducing cell proliferation. Treatment with ATRA or DMSO increased the expression of PtdGlc and FAS in HL60, while DIM21 in itself is not capable of inducing the PtdGlc expression. To isolate the extracellular proteins associate with PtdGlc-enriched domains on plasma membranes, PtdGlc associated molecules on plasma membranes were labeled with FITC by the enzyme-mediated activation of radical sources (EMARS) method. Silver staining and western blotting analyses showed that the treatment with DIM21 enhanced the expression of several proteins in HL60, including FAS, integrin αM/β2 (CD11b/CD18). Furthermore, about five FITC-labeled proteins were immunoprecipitated with anti-FITC antibody and detected by sequenced SDS-PAGE. The shot-gun proteomics analysis detected an approximately equal number of FITC-labeled CD18 peptides in the resting and DIM21-stimulated HL60 cells. Other FITC-labeled proteins detected by proteomics analysis were included insulin receptor and Insulin like growth factor-1-receptor which is responsible in cell growth and survival control. These findings indicate that PtdGlc might have an intimate involvement in cell survival through the direct interaction with integrins and other transmembrane receptors. The novel strategies targeting PtdGlc by monoclonal antibody DIM21 warrant further exploration in patients with leukemia including APL.[RJ1] DisclosuresEkyalongo:ONO PHARMACEUTICAL CO., LTD: Research Funding. Iwabuchi:ONO PHARMACEUTICAL CO., LTD: Research Funding.

Secondary necrotic neutrophils release interleukin-16C and macrophage migration inhibitory factor from stores in the cytosol.

Neutrophils harbor a number of preformed effector proteins that allow for immediate antimicrobial functions without the need for time-consuming de novo synthesis. Evidence indicates that neutrophils also contain preformed cytokines, including interleukin (IL)-1ra, CXCL8 and CXCL2. In the search for additional preformed cytokines, a cytokine array analysis identified IL-16 and macrophage migration inhibitory factor (MIF) as preformed cytokines in lysates from human primary neutrophils. Both IL-16 and MIF are unconventional cytokines because they lack a signal sequence. Using confocal immunofluorescence microscopy as well as western blot analysis of subcellular fractions, IL-16 and MIF were found to be stored in the cytosol rather than in the granules of human neutrophils, which implies an unconventional secretion mechanism for both cytokines. IL-16 is synthesized and stored as a precursor (pre-IL-16). We present evidence that the processing of pre-IL-16 to the biologically active IL-16C is mediated by caspase-3 and occurs during both spontaneous and UV-induced apoptosis of human neutrophils. Although IL-16 processing occurs during apoptosis, IL-16C and MIF release was observed only during secondary necrosis of neutrophils. Screening a panel of microbial substances and proinflammatory cytokines did not identify a stimulus that induced the release of IL-16C and MIF independent of secondary necrosis. The data presented here suggest that IL-16 and MIF are neutrophil-derived inflammatory mediators released under conditions of insufficient clearance of apoptotic neutrophils, as typically occurs at sites of infection and autoimmunity.

Mycobacterium tuberculosis strains modify granular enzyme secretion and apoptosis of human neutrophils.

Mycobacterium tuberculosis has evolved to employ multiple strategies to avoid an efficient host immune response. Accordingly, enzymes are important antimycobacterial elements and apoptosis decides the fate of any cell. Hence, we carried out this study to discern the amplitude of two clinical stains (S7 & S10) and a laboratory strain (H37Rv) of M. tuberculosis in modifying the release of lytic enzymes and apoptosis of neutrophils from healthy volunteers and pulmonary tuberculosis patients. We detected reduced levels of elastase in neutrophils from pulmonary tuberculosis patients. The laboratory strain H37Rv is found to increase the release of elastase and myeloperoxidase in neutrophils from both the groups. This strain is more efficient compared to clinical strains in inducing late apoptosis/necrosis in neutrophils. Our results proclaim the susceptibility of neutrophils in responding to infection with H37Rv. Also, at the functional level, neutrophils undergo changes related to release of enzymes after acquiring tuberculosis.

Glucocorticoid-Induced Leucine Zipper Is Expressed in Human Neutrophils and Promotes Apoptosis through Mcl-1 Down-Regulation

Glucocorticoid-induced leucine zipper (GILZ) is a potent anti-inflammatory protein, the expression of which is mainly induced by glucocorticoids (GCs) in haematopoietic cells. GILZ regulates signal transduction pathways of inflammation and plays a role in cell survival. The objective of this study was to evaluate the expression and mechanisms of action of GILZ in the apoptosis of human neutrophils. GILZ expression was induced by GCs in human neutrophils, enhanced upon phosphatidylinositol 3-kinase inhibition and resulted in apoptosis amplification. We then stably transfected PLB-985 cells with the human gilz gene and differentiated both control and GILZ-overexpressing clones in neutrophil-like cells. GILZ overexpression in PLB-985 cells led to an exacerbated apoptosis, associated with caspase-3, caspase-9 and caspase-8 activations, and a loss of mitochondrial potential, suggesting that GILZ-induced apoptosis used the mitochondrial pathway. The expression of BH3 interacting domain death agonist, Bcl-2 interacting mediator of cell death, annexin-A1 and Bcl-2-associated X was not affected in PLB-985-GILZ clones, but phosphorylation and subsequent proteasomal degradation of myeloid cell leukemia-1 (Mcl-1) were observed. Noteworthy, Mcl-1 phosphorylation was related to a significant and sustained activation of c-Jun N-terminal kinase (JNK) in PLB-985-GILZ clones. These results reveal GILZ to be a new actor in apoptosis regulation in neutrophil-like cells involving JNK and Mcl-1.

Effect of tannic acid, resveratrol and its derivatives, on oxidative damage and apoptosis in human neutrophils

In this study we compared the antioxidant and DNA protective activity of tannic acid and stilbene derivatives, resveratrol, 3,5,4′-trimethoxystilbene (TMS) and pterostilbene in human neutrophils stimulated to oxidative burst by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in relation to apoptosis induction.All polyphenols within the concentration range 1–100 μM reduced the intracellular ROS and H2O2 production in the TPA-stimulated cells. Tannic acid was the most effective polyphenol in protection against DNA damage induced by TPA. In the resting neutrophils resveratrol and to lesser extent other polyphenols increased DNA damage and increased the level of p53. Pretreatment of the TPA-stimulated cells with tannic acid or stilbenes led to the induction of apoptosis. The most significant effect was observed as a result of treatment with TMS and resveratrol. These compounds appeared the most effective inducers of p53 in the TPA-challenged neutrophils, what may suggest that pro-apoptotic activity of these stilbenes might be related to p53 activation.Overall, the results of our present study demonstrate that tannic acid and stilbenes modulate the ROS production, ultimately leading to cell apoptosis in human neutrophils stimulated to oxidative burst. In resting neutrophils they exhibit pro-oxidant activity, which is accompanied by p53 induction.

Propofol Treatment Inhibits Constitutive Apoptosis in Human Primary Neutrophils and Granulocyte-Differentiated Human HL60 Cells.

Apoptosis regulation is essential for neutrophil homeostasis. We previously demonstrated that a process involving glycogen synthase kinase (GSK)-3β determines neutrophil apoptosis. As for this apoptotic process, an overdose of propofol (2,6-Diisopropylphenol; 25 μg/ml or 140 μM) also causes GSK-3β-mediated macrophage apoptosis; however, the early deactivation of GSK-3β with low-dose propofol has been shown. Therefore, we hypothesize that low-dose propofol may induce neutrophil survival via GSK-3β inactivation. Following in vitro culture, the therapeutic concentration of propofol (10 μg/ml or 56 μM) treatment decreased constitutive apoptosis in isolated human primary neutrophils and in granulocyte-differentiated HL60 cells after all-trans retinoic acid (1 μM) treatment. The inactivation of phosphatidylinositol 3-kinase (PI3-kinase)/AKT and the activation of GSK-3β results in myeloid cell leukemia 1 (Mcl-1) down-regulation, the loss of the mitochondrial transmembrane potential, and caspase-3 activation in these cells, which is accompanied by apoptosis. Notably, propofol treatment attenuates these effects in a PI3-kinase-regulated manner. We found that propofol initiates PI3-kinase/AKT-mediated GSK-3β inactivation and Mcl-1 stabilization, rescuing the constitutive apoptosis in primary neutrophils and granulocyte-differentiated acute promyelocytic leukemia HL60 cells.

Thiotaurine protects mouse cerebellar granule neurons from potassium deprivation-induced apoptosis by inhibiting the activation of caspase-3.

Taurine (2-aminoethanesulphonic acid) is an endogenous amino acid that has a number of protective roles in most mammalian cells, including modulation of cytoplasmic calcium levels, antioxidant effects and protection against mitochondrial dysfunction and endoplasmic reticulum stress associated with neurological disorders. Thiotaurine (2-aminoethane thiosulfonate), a molecule structurally related to hypotaurine and taurine, counteracts the damaging effect of oxidants and prevents apoptosis of human neutrophils. In this study we have compared the effect of taurine and thiotaurine in protecting cerebellar granule neurons (CGNs) from apoptotic death. Two experimental paradigms were exploited to induce apoptosis: i) CGNs were continuously cultured in 5 mM K+-containing medium up to 6 days in the presence or absence of 1 mM either taurine or thiotaurine (chronic paradigm); ii) CGNs were cultured in 25 mM K+-containing medium for 6 days and then shifted to a 5 mM K+-containing medium in the presence or absence of 1 mM either taurine or thiotaurine (acute paradigm). In the first condition, CGNs survive up to 5 days, and then start to die; in the second condition, CGNs nicely differentiate elongating neurites but enter apoptosis within 30 min when shifted to the 5 mM K+-containing medium. These assays showed that taurine and thiotaurine counteracted the apoptotic death induced by low potassium in both the acute and chronic paradigms. However, while they displayed a similar efficacy in the chronic paradigm, the thiotaurine showed a significantly higher (20 %) efficacy compared to taurine in the acute paradigm. This finding pinpoints the thiotaurine as a powerful anti-apoptotic molecule in neurons that are fully differentiated and have established synaptic connections.

Polymorphonuclear leukocyte apoptosis is accelerated by sulfatides or sulfatides-treated Salmonella Typhimurium bacteria.

Neutrophils die by apoptosis following activation and uptake of microbes or enter apoptosis spontaneously at the end of their lifespan if they do not encounter a pathogen. Here we report that sulfatides or sulfatides-treated Salmonella Typhimurium bacteria accelerated human neutrophil apoptosis. Neutrophil apoptosis was examined by flow cytometry. Sulfatides caused prominent increase in percentage of apoptotic cells after 2.5 hrs of incubation. Salmonella Typhimurium bacteria by themselves did not affect the basal level of apoptosis in neutrophil population. When neutrophils were added to S. Typhimurium “opsonized” by sulfatides, apoptotic index significantly increased, whereas the number of phagocyting cells was not influenced. Sulfatides' proapoptotic effect was strongly dependent on the activity of β-galactosidase; inhibition of this enzyme impaired its potency to accelerate apoptosis. These data support the mechanism of neutrophil apoptosis triggering based on sulfatides' ability to accumulate in intracellular compartments and mediate successive increase in ceramide content resulting from β-galactosidase activity.

Fucoidan delays apoptosis and induces pro-inflammatory cytokine production in human neutrophils

Although some immune modulatory effects of fucoidan have been elucidated, the effects of fucoidan on the apoptosis and activation of human neutrophils have not been investigated. In this study, we demonstrated that fucoidan purified from the brown seaweed Undaria pinnatifilda delays spontaneous apoptosis of human neutrophils and induces their activation. Fucoidan treatment inhibited apoptotic nuclei changes and phosphatidyl serine (PS) exposure on neutrophils cultured in vitro for 24h. The delay in neutrophil apoptosis mediated by fucoidan was associated with increased levels of the anti-apoptotic protein Mcl-1 and decreased levels of activated caspase-3. Screening of the signaling pathways by specific inhibitors indicated that fucoidan-induced delay in neutrophil apoptosis was dependent on the activation of PI3K/AKT signaling pathway, whereas MAPK signaling pathway was not critical. In addition, fucoidan enhanced the production of IL-6, IL-8 and TNF-α from neutrophils in an AKT-dependent manner. Taken together, these results demonstrated that fucoidan delays human neutrophil apoptosis and induces their production of pro-inflammatory cytokines. This knowledge could facilitate the development of novel therapeutic strategies for infectious diseases and neutropenia by controlling neutrophil homeostasis and function with fucoidan.

IL-32γ Delays Spontaneous Apoptosis of Human Neutrophils through MCL-1, Regulated Primarily by the p38 MAPK Pathway

IL-32γ is a multifunctional cytokine involved in various inflammatory and auto-immune diseases in which neutrophils can affect the evolution of these diseases. To persist at inflammatory sites, neutrophils require inhibition of their rapid and constitutive apoptosis, an inhibitory effect that phlogogenic cytokines support. To date, the effects of IL-32γ on neutrophils remain unknown. We demonstrate that IL-32γ delays, in a dose-dependent manner, the spontaneous apoptosis of human blood neutrophils by activating mainly p38 MAPK through rapid p38 phosphorylation. PI3-K and ERK1/2 MAPK are also involved, but to a lesser extent. Most of cytokines that induce retardation of neutrophil apoptosis activate the expression of MCL-1 at both mRNA and protein levels. IL-32γ added to human blood neutrophils in vitro is associated with sustained levels of MCL-1 protein. This effect in neutrophils corresponds to a decrease of MCL-1 protein degradation without any effect on MCL-1 mRNA levels. The sustained levels of MCL-1 induced by IL-32γ are only abrogated by the p38β MAPK inhibitor SB202190. Additionally, IL-32γ induces a reduction in caspase 3 activity in neutrophils. In conclusion, IL-32γ affects human blood neutrophils in vitro by increasing their survival, suggesting that this cytokine could have profound effects on the deleterious functions of neutrophils in several diseases.