Apoptosis of human neutrophils induced by protein phosphatase 1/2A inhibition is caspase-independent and serine protease-dependent.

Protein phosphatase (PP) activity is associated with the regulation of apoptosis in neutrophils. However, the underlying regulatory mechanism(s) in apoptosis remain unclear. The type of cell death induced by okadaic acid (OA), the inhibitor of PP1 and PP2A, is characterized by apoptotic morphological changes of the cells and annexin V-positive staining without DNA fragmentation. The apoptotic effects of OA and calyculin A on neutrophils were observed at concentrations ranging from 50 to 200 nM, or 10 to 50 nM, respectively. Cyclosporine A (a PP2B specific inhibitor), however, did not exhibit any pro-apoptotic effects. OA and calyculin A, but not cyclosporine A, exhibited significant effects on protein levels and on the electrophoretic mobility of Mcl-1. zVAD-fmk, a pancaspase inhibitor, failed to inhibit the effect of OA on the caspase-3 activity, procaspase-3 processing, and the apoptotic rate of neutrophils. However, 4-(2-aminoethyl) benzenesulfonylfluoride (AEBSF), a general serine protease inhibitor, significantly abrogated the OA-induced mobility shift in procaspase-3, caspase-3 activation, and the apoptotic morphological changes in neutrophils. Moreover, OA enhanced the serine protease activity of the neutrophils. The addition of the proteinase-3 protein increased the rate of neutrophil apoptosis, which was also blocked by AEBSF but not by zVAD-fmk. These results suggest that OA induces procaspase-3 processing but that OA-induced apoptosis is caspase-independent and serine protease-dependent.

Phosphatidic acid induces the differentiation of human acute promyelocytic leukemic cells into dendritic cell-like.

We investigated whether phosphatidic acid (PA) can differentiate the promyelocytic leukemia (PML)-retinoic acid receptor alpha (RAR alpha)-expressing acute promyelocytic leukemic cell line, NB4, to dendritic cell (DC)-like cells. Dioctanoyl-PA alone upregulated the expression of DC markers. The expression of DC markers on NB4 cells was potentiated by the overexpression of phospholipase D and upregulation was blocked by the addition of n-butanol, an inhibitor of PA production. The expression of CD11c, CD83, and CCR7 in PA-treated NB4 cells was further increased by tumor necrosis factor (TNF)-alpha treatment. Increased functional capacities were also found in PA-differentiated and TNF-alpha-activated NB4 cells with respect to changes in T-cell proliferation, cytokine production, endocytic activity, and cytolytic capacity against undifferentiated NB4 cells. PA alone increased the phosphorylation of extracellular signal-regulated kinase (ERK)-1/2. The expression of DC markers was downregulated by PD98059, a specific inhibitor of ERK kinase or transient transfection of mutant-ERK. The level of PML-RAR alpha fusion protein was decreased by PA treatment and PD98059 blocked the decrease of PML-RAR alpha. These results suggest that PA induces differentiation of NB4 cells into DC-like cells and that the upregulation of antigen presenting cell markers is mediated by the activation of ERK and the downregulation of PML-RAR alpha levels.

Delayed apoptosis and modulation of phospholipase D activity by plasmid containing mammalian cDNA in human neutrophils.

Phospholipase D (PLD) has been reported to have an anti-apoptotic role in neutrophils. This study examined the effects of plasmids containing the cDNA of PLD on the apoptosis of neutrophils. The apoptotic rate of neutrophils treated with the pCDNA3.1 plasmid was similar to that of the untreated cells after 24 h culture. However, the addition of pCDNA3.1 containing the cDNA of either human PLD1 (pCDNA3.1-PLD1) or -PLD2 (pCDNA3.1-PLD2) to the culture media with or without transfection reagent significantly decreased the rate of spontaneous apoptosis but not Fas-stimulated apoptosis and the decreased apoptosis was blocked by 1-butanol. pCDNA3.1-PLD blocked the cleavage of procaspase-3 and -8. The phorbol myristate acetate stimulated the PLD activities of pCDNA3.1-PLD-treated neutrophils but did not stimulate the activities of untreated or pCDNA3.1-treated neutrophils. The level of the PLD1 protein was higher in the cultured neutrophils with pCDNA3.1-PLD than with the media or pCDNA3.1. The spontaneous apoptosis of neutrophils was inhibited and the PLD1 expression level was increased by the linearized or promoterless forms of pCDNA3.1-PLD1 and the plasmids containing the cDNA of the enhanced green fluorescent protein (pEGFP) and EGFP-PLD1. These results suggest that the plasmids containing mammalian cDNA inhibit the spontaneous apoptosis of neutrophils and modulate PLD.

Modulation of neutrophil apoptosis by beta-amyloid proteins.

This study examined the effect of amyloid beta peptide (Abeta) and the secretase inhibitors of amyloid precursor proteins (APP) on the spontaneous apoptosis of neutrophils. Abeta(1-40) decreased the apoptotic rate of neutrophils. The delayed apoptosis by Abeta was not blocked by pertussis toxin and N-formyl peptide receptor-like 1 antagonistic peptide, WRWWWW. The inhibitors of phoshoinositide 3-kinase (LY294002), phospholipase C (U73122), or Ca++-dependent protein kinase C (Go6976) abrogated the anti-apoptotic effect of Abeta on neutrophils. Moreover, the Abeta-induced delay of apoptosis was inhibited by a calcium chelator, BAPTA/AM. The amount of the APP protein was reduced in the cultured neutrophils and the APP level in the Abeta or pancaspase-treated neutrophils was lower than that in the cultured neutrophils. However, the reduction in APP level was recovered after treating them with the secretase inhibitors or anti-Fas antibody. The exogenous addition of cell permeable beta- and gamma-secretase inhibitors resulted in an increase in the rate of the apoptosis. The regulation of neutrophil apoptosis by the addition of Abeta and secretase inhibitors occurred via the caspase -8, -9, -3, and mitochondrial-dependent pathways. This suggests that the intracellular beta-amyloid proteins play a role as regulating factor of neutrophil survival and that Abeta-induced delay of apoptosis is mediated by other receptors rather than a seven-transmembrane G protein-coupled receptor(s).

IL-10-dependent down-regulation of MHC class II expression level on monocytes by peritoneal fluid from endometriosis patients.

Endometriosis is a gynecologic disorder characterized by the ectopic growth of misplaced endometrial cells. Moreover, immunological abnormalities of cell-mediated and humoral immunity may be associated with the pathogenesis of endometriosis. The effects of peritoneal fluid (PF) from endometriosis patients on the expression levels of MHC class II and costimulatory molecules on the cell surfaces of monocytes were investigated. Compared to the PF of controls, the addition of 10% PF (n=10) from patients with endometriosis to culture medium significantly reduced the percentage of MHC class II-positive cells in cultures of a THP-1, monocytic cell line at 48 h. The effect of endometriosis patient PF (EPF) was dose-dependent, and similar effect was observed in peripheral blood monocytes. An inverse correlation was found between MHC class II expression level and IL-10 concentration in EPF (r=-0.518; p=0.019) and in the supernatant of peripheral blood monocyte cultured in EPF (r=-0.459; p=0.042) (n=20). The expression levels of costimulatory molecules (CD80 and CD86), but not of CD54 and B7-H1, were down-regulated by EPF. The mRNA level of HLA-DR was unaffected by EPF but protein level was reduced by EPF. Neutralizing IL-10 antibody abrogated MHC class II down-regulation on monocytes, which had been induced by EPF. However, in a functional assay, monocytes treated with EPF failed to stimulate T cell in mixed leukocyte reaction, although T cell proliferation was increased with EPF-treated monocytes and Staphylococcus enterotoxin B. These results suggest that MHC class II expression level on monocytes is down-regulated by EPF, but the cell stimulatory ability of monocytes does not coincide with MHC class II expression level.

Phosphatidic acid and tumor necrosis factor-alpha induce the expression of CD83 through mitogen activated protein kinase pathway in a CD34+ hematopoietic progenitor cell line, KG1.

To elucidate the signaling pathways involved in the expression of CD83, which is linked to the differentiation and maturation states of dendritic cells, we examined the effect of phosphatidic acid (PA) on the expression of CD83 in KG1, a CD34(+) hematopoietic progenitor cell. In the presence of tumor necrosis factor (TNF)-alpha, PA but not lyso-PA up-regulated CD83 on KG1 cells. Moreover, PA and TNF-alpha-induced expression of CD83 was slightly increased by propranolol, an inhibitor of PA phosphohydrolase but was unaffected by phospholipase A2 inhibitor. PA and TNF-alpha increased the phosphorylation of extracellular signal-regulated kinase (ERK)-1/2, p38-kinase, and c-Jun N-terminal kinase (JNK) by Western blotting. However, the up-regulation of CD83 by PA/TNF-alpha on KG1 was significantly abrogated by PD98059, a specific inhibitor of ERK kinase, but was enhanced by SP600125, a JNK inhibitor. Bis-indolylmaleimide, an inhibitor of protein kinase C, partially blocked the up-regulation of CD83 and ERK phosphorylation induced by PA and TNF-alpha. Moreover, the incubation of KG1 cells with phorbol ester and TNF-alpha for 5 days increased the protein level of phospholipase D. These results suggest that PA and TNF-alpha induce the up-regulation of CD83 and that their action is regulated by ERK and JNK.

Anti-apoptotic mechanism and reduced expression of phospholipase D in spontaneous and Fas-stimulated apoptosis of human neutrophils.

Neutrophil apoptosis is a constitutive process that can be enhanced or delayed by signals triggered by various stimuli. In this work, we investigated the action mechanism of phospholipase D (PLD) and its expression in the inhibition of spontaneous and Fas-mediated apoptosis. Anti-Fas antibody-stimulated apoptosis of neutrophils was significantly blocked by the exogenous addition of bacterial PLD from Streptomyces chromofuscus (scPLD), and neutrophils cultured for 24 h in the presence of anti-Fas antibody showed lower agonist-stimulated PLD activity compared to untreated cells. The amount of PLD1a protein reduced time-dependently in cultured neutrophils, but was recovered by treating with LPS or GM-CSF. The reduction in PLD1a protein level was blocked by caspase inhibitors. The exogenous addition of scPLD blocked the up-regulation of Fas-associated death domain expression, mitochondrial permeability, and the cleavages of procaspase-8, procaspase-3, and protein kinase C-delta. We also found that the protein level of apoptosis-inducing factor was increased in cultured neutrophils but its expression was reduced by scPLD. However, sulfasalazine-induced apoptosis and change of protein expression were not blocked by scPLD. Taken together, the activity and protein levels of PLD play a role as an anti-apoptotic factor by acting at multiple levels of the apoptotic cascade in neutrophils.

Differential expression of dendritic cell markers by all-trans retinoic acid on human acute promyelocytic leukemic cell line.

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) for naive T cells and play an important role in cancer immunology. All-trans retinoic acid (ATRA) is known to be a differentiating agent in the treatment of acute promyelocytic leukemia (APL). In this study, we investigated whether ATRA can differentiate the retinoic acid (RA)-sensitive promyelocytic leukemic cell line, NB4, to DC-like cells and whether these differentiated cells can activate T cells. NB4 cells were differentiated to myeloid cells by 4, 6, and 8 days of ATRA treatment. NB4 cells up-regulated markers found in DCs, including HLA-DR, costimulatory molecules (CD80 and CD86), adhesion molecules (CD40), and chemokine receptors (CCR6) when cultured for 8 days in the presence of 1 microM ATRA. Upregulation of CD83 was also detected on the surface of ATRA-treated NB4 cells versus untreated cells. The addition of cytokines alone, such as GM-CSF or CD40 ligand, did not affect the expression of CD83 in untreated NB4 cells but they up-regulated CD83 in ATRA-treated cells. CD11b was coexpressed with CD80, CD83, and CD86 in ATRA-treated NB4 cells. In a functional assay, ATRA-treated NB4 cells stimulated T cell proliferation when challenged with Staphylococcus enterotoxin B. These results suggest that the differentiation of NB4 cells by ATRA causes the cells to express DC markers, and that ATRA-differentiated NB4 cells are able to present antigens to T cells.