Involvement of diacylglycerol produced by phospholipase D activation in Aß-induced reduction of sAPPα secretion in SH-SY5Y neuroblastoma cells.

We previously reported that the thiol proteinase inhibitor, E-64-d, ameliorated amyloid ß (Aß)-induced reduction of soluble amyloid precursor protein α (sAPPα) secretion by reversing ceramide-induced protein kinase C down-regulation in SH-SY5Y neuroblastoma cells. In the present study, we showed that Aß (1-42) peptide enhanced diacylglycerol (DAG) production by phospholipase D (PLD) activation in these cells. We subsequently examined whether PLD was involved in Aß-induced reduction of sAPPα secretion and showed that 2 µM CAY10593, which selectively inhibits PLD2, ameliorated reduction of sAPPα secretion, whereas 50 nM CAY10593, which selectively inhibits PLD1, did not. Moreover, 50 µM propranolol, a phosphatidic acid phosphohydrolase inhibitor, also ameliorated Aß-induced reduction of sAPPα secretion, suggesting that DAG may be responsible for Aß-induced reduction of sAPPα. We subsequently examined whether DAG affects sAPPα secretion and showed that a DAG analog reduced sAPPα secretion in SH-SY5Y cells. In addition, DAG enhanced ceramide production by stimulating neutral sphingomyelinase (N-SMase) activity. We previously demonstrated that Aß stimulates N-SMase activity in SH-SY5Y cells. Here, we showed that inhibition of PLD2 by 2 µM CAY10593 suppressed Aß-induced N-SMase activation. Taken together, the results suggest that DAG produced through the PLD pathway is involved in Aß-induced reduction of sAPPα secretion in SH-SY5Y cells.

The thiol proteinase inhibitor E-64-d ameliorates amyloid-ß-induced reduction of sAPPα secretion by reversing ceramide-induced protein kinase C down-regulation in SH-SY5Y neuroblastoma cells.

In Alzheimer's disease (AD), enhancing α-secretase processing of amyloid precursor protein (APP) is an important pathway to decrease neurotoxic amyloid ß (Aß) secretion. The α-secretase is reported to be regulated by protein kinase C (PKC) and various endogenous proteins or cell surface receptors. In this report, we first examined whether Aß reduces α-secretase activity, and showed that Aß peptide 1-40 (0.001 and 0.01 µM) reduced the secretion of soluble amyloid precursor protein α (sAPPα) in carbachol-stimulated SH-SY5Y neuroblastoma cells. E-64-d (3 µM), which is a potent calpain inhibitor that prevents PKC degradation, ameliorated the Aß-induced reduction of sAPPα secretion. In addition, we observed that Aß significantly enhanced ceramide production by activating neutral sphingomyelinase. The cell-permeable ceramide analog, C2-ceramide (1 µg/mL), also reduced sAPPα secretion, and in addition, E-64-d eliminated the observed decrease of sAPPα secretion. C2-ceramide induced down-regulation of PKC-α, -ß1, and -ß2 isozymes in SH-SY5Y cells. These findings suggest that ceramide may play an important role in sAPPα processing by modulating PKC activity.

Improvement of deficient natural killer activity and delayed bactericidal activity by a thiol proteinase inhibitor, E-64-d, in leukocytes from Chediak-Higashi syndrome patients in vitro.

We previously reported that administration of a potent calpain inhibitor, E-64-d, which protects protein kinase C (PKC) from proteolysis, in a mouse model of Chediak-Higashi syndrome (CHS) (beige mice), decreases its susceptibility to Staphylococcus aureus infection. In the present study, we examined the in vitro effect of E-64-d on both deficient natural killer (NK) and delayed bactericidal activities of leukocytes from six CHS patients. Our results showed that pretreatment of peripheral blood mononuclear cells (PBMCs) obtained from CHS patients with E-64-d (1 microg/ml) significantly enhanced NK activity against K562 cells. The delayed bactericidal activity of polymorphonuclear cells (PMNs) against S. aureus also showed marked improvement. This was recovered to almost normal levels when PMNs were pretreated with E-64-d (1 microg/ml). On the other hand, the same concentration of E-64-d did not affect either the NK or bactericidal activity of normal controls. In addition, we confirmed that following E-64-d treatment, the abnormal down-regulation of PKC activity after concanavalin A (Con A) stimulation was eliminated in PBMCs obtained from CHS patients. To examine whether PKC is involved in the NK cell-mediated cytolysis and bactericidal activity of PMNs, two potent PKC inhibitors, chelerythrin and GO6976, were used. We found that chelerythrin inhibits NK activity of normal PBMCs in a dose-dependent manner, and GO6976 inhibits NK activity at doses that inhibit Ca(2+)-dependent PKC isozymes. These inhibitors also suppressed the bactericidal activity of PMNs against S. aureus. Taken together, our findings suggested that E-64-d improved the compromised NK and bactericidal activity of leukocytes from CHS patients by reversing the down-regulation of PKC activity.

Effect of a thiol proteinase inhibitor, E-64-d, on susceptibility to infection with Staphylococcus aureus in Chediak-Higashi syndrome (beige) mice.

We previously reported that abnormally down-regulated protein kinase C (PKC) activity is responsible for the impaired cellular function of natural killer cells and polymorphonuclear cells (PMNs), and the giant granule formation in fibroblasts in the beige mouse, an animal model of Chediak-Higashi syndrome. Here, we examine the effect of oral or intraperitoneal administration of E-64-d, which protects PKC from calpain-mediated proteolysis, on the impaired cellular function in PMNs from beige mice. We found that oral administration of E-64-d (12.5 mg/kg body weight per day) for three consecutive days, significantly improved the abnormally increased concanavalin A (Con A) cap formation and the decreased lysosomal enzyme activity in beige PMNs. In addition, E-64-d significantly improved the delayed bactericidal activity against Staphylococcus aureus. In contrast, E-64-d at the same dose did not affect these cellular functions in PMNs from C57BL/6J mice. We confirmed that the abnormal down-regulation of PKC after Con A stimulation was eliminated in PMNs from E-64-d-treated beige PMNs. We then examined whether the administration of E-64-d to beige mice improved the susceptibility to experimental infection with S. aureus (2x10(8)/mouse). Both intraperitoneal and oral administration of E-64-d to beige mice resulted in a significant increase in survival, whereas E-64-d at the same dose did not alter the survival rate in normal mice. These results suggest that the administration of E-64-d may be effective against severe bacterial infection in Chediak-Higashi syndrome.

Antithetical effects of hemicellulase-treated Agaricus blazei on the maturation of murine bone-marrow-derived dendritic cells.

We report the effects of hemicellulase-treated Agaricus blazei (ABH) on the maturation of bone-marrow-derived dendritic cells (BMDCs). ABH activated immature BMDCs, inducing up-regulation of surface molecules, such as CD40, CD80 and major histocompatibility complex class I antigens, as well as inducing allogeneic T-cell proliferation and T helper type 1 cell development. However, unlike lipopolysaccharide (LPS), ABH did not stimulate the BMDCs to produce proinflammatory cytokines, such as interleukin-12 (IL-12) p40, tumour necrosis factor-alpha, or IL-1beta. In addition, ABH suppressed LPS-induced DC responses. Pretreatment of DCs with ABH markedly reduced the levels of LPS-induced cytokine secretion, while only slightly decreasing up-regulation of the surface molecules involved in maturation. ABH also had a significant impact on peptidoglycan-induced or CpG oligodeoxynucleotide-induced IL-12p40 production in DCs. The inhibition of LPS-induced responses was not associated with a cytotoxic effect of ABH nor with an anti-inflammatory effect of IL-10. However, ABH decreased NF-kappaB-induced reporter gene expression in LPS-stimulated J774.1 cells. Interestingly, DCs preincubated with ABH and then stimulated with LPS augmented T helper type 1 responses in culture with allogeneic T cells as compared to LPS-stimulated but non-ABH-pretreated DCs. These observations suggest that ABH regulates DC-mediated responses.