Modulatory Effects and Action Mechanisms of Tryptanthrin on Murine Myeloid Leukemia Cells

Leukemia is the disorder of hematopoietic cell development and is characterized by an uncoupling of cell proliferation and differentiation. There is a pressing need for the development of novel tactics for leukemia therapy as conventional treatments often have severe adverse side effects. Tryptanthrin （6,12-dihydro-6,12-dioxoindolo- （2,1-b）-quinazoline） is a naturally-occurring, weakly basic alkaloid isolated from the dried roots of medicinal indigo plants （Ban-Lan-Gen）. It has been reported to have various biological and pharmacological activities, including anti-microbial, anti-inflammatory, immunomodulatory and anti-tumor effects. However, its modulatory effects and action mechanisms on myeloid cells remain poorly understood. In this study, tryptanthrin was shown to suppress the proliferation of the murine myeloid leukemia WEHI-3B JCS cells in a dose- and time-dependent manner. It also significantly reduced the growth of WEHI-3B JCS cells in vivo in syngeneic BALB/c mice. However, it exhibited no significant direct cytotoxicity on normal murine peritoneal macrophages. Flow cytometric analysis showed an obvious cell cycle arrest of the tryptanthrin-treated WEHI-3B JCS cells at the G0/G1 phase. The expression of cyclin D2, D3, Cdk 2, 4 and 6 genes in WEHI-3B JCS cells was found to be down-regulated at 24 h as measured by RT-PCR. Morphological and functional studies revealed that tryptanthrin could induce differentiation in WEHI-3B JCS cells, as shown by the increases in vacuolation, cellular granularity and NBT-reducing activity in tryptanthrin-treated cells. Collectively, our findings suggest that tryptanthrin might exert its anti-tumor effect on the murine myelomonocytic leukemia WEHI-3B JCS cells by causing cell cycle arrest and by triggering cell differentiation.     

Histamine H2 receptor activity and histamine metabolism in human U-937 monocyte-like cells and human peripheral monocytes

Functional and specific histamine H₂ receptors were characterized in human peripheral monocytes and in U-937 cells, before and after retinoid acid-induced differentiation into monocyte/macrophage-like cells. The relative potencies of histamine and the H₁, H₂ receptor agonists and antagonists studied are remarkably similar in U-937 cells and U-937 monocytes. There is no change in histamine concentration and activity of the enzymes forming and degrading histamine during monocytic-like differentiation. The results raise the possibility that histamine H₂ receptors might be involved in pathophysiological regulations (proliferation/differentiation and biological function) of normal and leukemic monocytes.     

The death-associated protein kinase 2 is up-regulated during normal myeloid differentiation and enhances neutrophil maturation in myeloid leukemic cells

The death-associated protein kinase 2 (DAPK2) belongs to a family of Ca(2+)/calmodulin-regulated serine/threonine kinases involved in apoptosis. During investigation of candidate genes operative in granulopoiesis, we identified DAPK2 as highly expressed. Subsequent investigations demonstrated particularly high DAPK2 expression in normal granulocytes compared with monocytes/macrophages and CD34(+) progenitor cells. Moreover, significantly increased DAPK2 mRNA levels were seen when cord blood CD34(+) cells were induced to differentiate toward neutrophils in tissue culture. In addition, all-trans retinoic acid (ATRA)-induced neutrophil differentiation of two leukemic cell lines, NB4 and U937, revealed significantly higher DAPK2 mRNA expression paralleled by protein induction. In contrast, during differentiation of CD34(+) and U937 cells toward monocytes/macrophages, DAPK2 mRNA levels remained low. In primary leukemia, low expression of DAPK2 was seen in acute myeloid leukemia samples, whereas chronic myeloid leukemia samples in chronic phase showed intermediate expression levels. Lentiviral vector-mediated expression of DAPK2 in NB4 cells enhanced, whereas small interfering RNA-mediated DAPK2 knockdown reduced ATRA-induced granulocytic differentiation, as evidenced by morphology and neutrophil stage-specific maturation genes, such as CD11b, G-CSF receptor, C/EBPepsilon, and lactoferrin. In summary, our findings implicate a role for DAPK2 in granulocyte maturation.     

Nuclear factor-kappaB and caspases co-operatively regulate the activation and apoptosis of human macrophages

Accumulating evidence suggests that macrophages function as major effector cells in the pathological process of various human diseases. We examined here the role of nuclear factor-kappaB (NF-kappaB) and caspases in the regulation of activation and apoptosis of macrophages. Activation of the human monoblastic leukaemia cell line, U937, by phorbol 12-myristate 13-acetate (PMA) increased the expression of CD14/CD86, and cytokine production. PMA stimulation also increased the expression of both pro-caspase-8 and pro-caspase-3 in U937, but not apoptosis or intracellular caspase-3 activity. PMA also increased the expression of X-chromosome-linked inhibitor of apoptosis protein (XIAP) in U937, suggesting an inhibitory action for XIAP on the caspase cascade in PMA-stimulated U937. Electrophoretic mobility shift assay (EMSA) showed a significant increase of nuclear NF-kappaB activity in PMA-stimulated U937. When a potent NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC), was added to U937 cell culture in the presence of PMA, apoptosis was triggered by activation of caspase-3, which was induced by caspase-8 activation. XIAP expression was markedly suppressed in PMA-treated U937 in the presence of PDTC. The inhibitors of caspase-8 and caspase-3 mostly inhibited apoptosis of U937 treated with PMA in the presence of PDTC. Furthermore, a phenotype of U937 treated with PMA and PDTC in the presence of caspase inhibitor was almost identical to that of unstimulated U937. Our results suggest that the signalling pathways involved in the activation and apoptosis of human macrophages could be co-operatively regulated by the use of NF-kappaB and caspase inhibitors, thus enabling the control of macrophage function and number.     

Heme-induced heme oxygenase-1 (HO-1) in human monocytes inhibits apoptosis despite caspase-3 up-regulation

Monocyte activation, apoptosis and differentiation are hallmarks of most inflammatory vascular disorders. We studied the effects of heme oxygenase-1 (HO-1) induced by its substrate hemin on apoptosis, caspase-3 expression and the differentiation of freshly isolated human monocytes. Hemin induced HO-1 in a dose- and time-dependent fashion as measured by semi-quantitative RT-PCR and flow cytometry. Apoptosis was markedly suppressed by hemin in cells rendered apoptotic by serum deprivation or dexamethasone as determined by flow cytometric detection of annexin V binding or transmission electron microscopy (TEM). The specific HO-1 inhibitor zinc protoporphyrin (ZnPP) reversed the effects of hemin on monocyte apoptosis and diminished cell lifespan. Surprisingly, the cytoprotective effects of hemin were positively correlated with caspase-3 up-regulation. Hemin-induced apoptosis suppression was enhanced by the caspase-3 inhibitor DEVD-CHO, indicating that caspase-3 was active in a pro-apoptotic fashion. Hemin inhibited CD95 as a putative cytoprotective mechanism. Morphological studies and detection of CD86 showed that monocytes differentiated into macrophages in response to hemin after relatively long incubation times, a phenomenon that might be provoked by caspase-3-regulated pathways. Our results confirm a similar cytoprotective effect of hemin/HO-1 for monocytes as has been shown for other cells, despite caspase-3 up-regulation. The fact that HO-1 may adversely affect monocyte survival and differentiation could be of particular significance in future therapies for occlusive vascular diseases or transplant rejection.     

Porphyromonas gingivalis Fimbriae Inhibit Caspase-3-Mediated Apoptosis of Monocytic THP-1 Cells under Growth Factor Deprivation via Extracellular Signal-Regulated Kinase-Dependent Expression of p21 Cip/WAF1

Apoptotic regulation of monocytes/macrophages appears to be closely associated with chronic inflammatory reactions. Since it was demonstrated earlier that certain bacterial cell components are involved in apoptotic regulation of these cells, in the present study, we investigated whether the bacterial fimbria, an important cell structure involved in bacterial adherence to host cells, regulates apoptosis of human monocytic THP-1 cells induced under growth factor deprivation. To investigate this point, we used fimbriae of Porphyromonas gingivalis, a pathogen causing periodontal disease, which is a chronic inflammatory disease. The fimbriae inhibited apoptosis of the cells under growth factor deprivation. This inhibitory action of the fimbriae was completely neutralized by anti-fimbrial antibody. The fimbriae stimulated activation of extracellular signal-regulated kinase (ERK) and expression of cyclin-dependent kinase inhibitor p21 Cip/WAF1 (p21) in the cells. The stimulatory effect of the fimbriae on the expression of the p21 protein was inhibited by treatment with PD98059, a specific inhibitor of ERK. The cell apoptosis was inhibited by treatment with Ac-DEVD-CHO, an inhibitor of caspase-3. The fimbriae inhibited the serum withdrawal-induced cleavage of the caspase-3 proform and poly(ADP-ribose) polymerase, one of the caspase-3 substrates. Furthermore, PD98059 and antisense p21 oligonucleotide blocked the fimbrial inhibition of apoptosis and caspase-3 activation of the cells induced by serum withdrawal. These results show that the bacterial fimbriae inhibited apoptosis of THP-1 cells induced under growth factor deprivation via ERK-dependent expression of p21. The present study suggests that bacterial fimbriae act as potent regulators of chronic inflammatory disease, e.g., periodontal disease, through blocking apoptosis of monocytes/macrophages.     

Characterization of a novel myeloid antigen regulated during differentiation of monocytic cells

The monoclonal antibody HC1/6 generated against phorbol 12-myristate 13-acetate-treated U-937 cells recognizes a new cell surface antigen with a broad relative molecular mass ranging from 100 to 150 kDa. This antigen is also present on monocytes, platelets and endothelial cells and is weakly expressed by granulocytes. In contrast, it is absent from T, B and erythroblastoid cells. The antigen HC1/6 is also expressed by normal tissue macrophages in tonsil, lung and kidney, as well as in skin biopsies from pathologies such as sarcoidosis and lepromatous leprosy. The expression of the HC1/6 antigen is increased up to 5-fold when U-937 (promonocytic) and HL-60 (myelomonocytic) cell lines are stimulated with phorbol 12-myristate 13-acetate. Conversely, the expression of the HC1/6 antigen is down-regulated in monocytes upon treatment with interferon-gamma. These findings are discussed in relation with other myeloid cell surface markers.     

Resveratrol, a natural stilbene in grapes and wine, enhances intraphagocytosis in human promonocytes: a co-factor in antiinflammatory and anticancer chemopreventive activity

Trans-resveratrol, a natural stilbene present in wine and grapes, has been studied mainly for its antiinflammatory and anticancer activities. In this study the activity of resveratrol on proliferative immunological parameters (differentiation, apoptosis, phagocytosis and intracellular killing) was studied using a U937 human promonocytic cell line in comparison with another polyphenol, quercetin. After incubation of the pathogen, Candida albicans, intracellular killing by macrophage-like cells was decreased by quercetin and resveratrol 10 microM but was enhanced by resveratrol 1 microM after 20 h of treatment. Phagocytosis rate, expressed as phagocytosis frequency, (i.e., percentage number of phagocytosing cells/total cells) at 20 h was highest with resveratrol 10 microM and was higher with quercetin 10 microM than with resveratrol 1 microM. The phagocytosis index exhibited the same trend. While both polyphenols demonstrated cytostatic activity on U937 growth, a prointraphagocytic effect for resveratrol 10 microM-treated cells at 10 min, resveratrol 1 microM-treated cells at 20 h and resveratrol 10 microM-treated cells at 48 h was observed. Morphological examination with optic microscopy demonstrated both apoptotic and differentiating cells, even after 10 min treatment. Resveratrol-induced apoptosis (following 4 h treatment) was confirmed by flow cytometry at concentrations as low as 1 microM and 100 nM in the assay for detection of membrane phosphatidylserine. Resveratrol- or quercetin-treated, but unstimulated cells, did not produce tumor necrosis factor-alpha protein. As phosphatidylserine externalization triggers specific recognition by monocytes and macrophages, removal of intact apoptotic cells is important a) in cell population selection and differentiation for antiblastic therapy, and b) in preventing the release of toxic inflammatory substances such as reactive oxygen substances and proteolytic enzymes by dying cells. This observation suggests that wine polyphenols, at the same concentrations as those found in plasma after moderate wine consumption, are important cofactors in antiinfective, antiinflammatory and anticancer nonspecific immune reactions.     

Resistance to apoptosis in <Emphasis Type="Italic">Leishmania infantum</Emphasis>-infected human macrophages: a critical role for anti-apoptotic Bcl-2 protein and cellular IAP1/2

Apoptosis is essential for maintaining tissue homoeostasis in multi-cellular organisms, also occurring as a defence mechanism against a number of infectious agents, such as parasites. Among intracellular protozoan parasites reported to interfere with the apoptotic machinery of the host cell, Leishmania (L.) sp. have been described, although the various species might activate different pathways in their host cells. Since until now it is not yet well clarified the signalling pathway involved in the apoptosis modulation by L. infantum, the aim of this work was to investigate the role of the anti-apoptotic protein, Bcl-2, and the inhibitors of apoptosis IAP1/2 (cIAP1/2) in cell death resistance showed in L. infantum-infected human macrophages. We observed that actinomycin D-induced apoptosis in U-937 cells, evaluated by Annexin V-CY3, DNA fragmentation and caspase-3, caspase-8, caspase-9 activation assays, was inhibited in the presence of L. infantum promastigotes and that, in these conditions, Bcl-2 protein expression resulted significantly upregulated. Interestingly, L. infantum infection in combination with the Bcl-2 inhibitor, ABT-737, significantly increased the apoptotic process in actinomycin D-treated cells, suggesting a role for Bcl-2 in the anti-apoptotic regulation of human macrophages induced by L. infantum infection. Moreover, Western blotting analysis demonstrated not only a significantly upregulation of cIAP1/2 in infected U-937 cells, but also that the inhibition of cIAPs, employing specific siRNAs, restored the apoptotic effect of actinomycin in infected macrophages. These results clearly support the hypothesis that Bcl-2 and cIAPs are strongly involved in the anti-apoptotic action played by L. infantum in human macrophages.     

Gamma-interferon (IFN-gamma) augments apoptotic response to mistletoe lectin-II via upregulation of Fas/Fas L expression and caspase activation in human myeloid U937 cells

Mistletoe lectin-II, a major composition of Korean mistletoe (Viscum album coloratum), is known as a potent apoptosis inducer. The previous research has demonstrated that Korean mistletoe lectin-II induces apoptosis via c-Jun N terminal kinase (JNK) activation in human myeloid U937 cells. The purpose of this research is to prove the synergistic action of mistletoe lectin-II and interferon-γ (IFN-γ) in the apoptotic cytotoxicity of U937. When U937 cells were treated with mistletoe lectin-II after being differentiated by IFN-γ, the proteolytic activity of caspase-3 and 9 was markedly elevated and that of caspase-8 was prolonged for 18 hr. The activation of caspase-3-like protease requires the earlier cleavage of poly(ADP-ribose) polymerase(PARP). Caspase-1 was, however, not activated during the resting phase and nor in IFN-γ-differentiated U937 cells. Western blot analysis revealed that, in IFN-γ-differentiated U937 cells, the expression of Fas (CD95/APO-1) & Fas ligand(FasL) increases the apoptotic sensitivity against Mistletoe lectin-II. Fas (CD95/APO-1) & FasL were not significantly induced solely by mistletoe lectin-II. Furthermore the activity of JNK1 in U937 cells was also markedly increased with IFN-γ-differentiation, compared to that of the control. These results suggest that the IFN-γ-differentiation of U937 cells increases the susceptibility to mistletoe lectin-II-induced apoptosis.     

Phosphatidylserine expression and phagocytosis of apoptotic thymocytes during differentiation of monocytic cells

Expression of phosphatidylserine (PS) on the surface of both macrophages and their apoptotic targets is required for efficient phagocytosis. Monocytes, the precursors of macrophages, do not express PS on their surface and do not efficiently phagocytose apoptotic cells. We report here that PS appears on the surface of both human monocytic U937 cells and primary human monocytes as they differentiate in culture and acquire the ability to phagocytose apoptotic thymocytes. Phagocytosis was blocked by pretreating either the apoptotic target or the phagocyte with annexin V to mask PS and was CD14-dependent. Expression of PS, like other events characteristic of differentiating monocytes such as Mac-1 expression, was independent of the agent used to induce differentiation and was insensitive to the addition of caspase inhibitors. These results demonstrate that PS is expressed on monocytes as part of their differentiation program and is independent of apoptosis.     

Recombinant human granulocyte-macrophage colony-stimulating factor induces secretion of autoinhibitory monokines by U-937 cells

Colony-stimulating factors are required for survival proliferation, differentiation and functional activation of granulocytes, macrophages and their precursor cells. In the present report, however, we demonstrate antiproliferative activity of recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF) on monoblast cell line U-937 and provide evidence for the involvement of tumor necrosis factor alpha TNF-alpha and interleukin 1 beta (IL 1 beta) in its growth inhibitory action. GM-CSF (but not granulocyte CSF, G-CSF or macrophage CSF, M-CSF) suppressed DNA synthesis and self renewal of U-937 cells. Similarly, medium conditioned by U-937 cells in response to GM-CSF (GM-CSF U-937-CM) was able to reduce clonogenicity and [3H]thymidine uptake by U-937 cells. Since neutralization of GM-CSF present in GM-CSF U-937-CM by monoclonal antibody to GM-CSF did not abrogate the autoinhibitory activity present in GM-CSF U-937-CM, we considered the possibility that other soluble molecules are released by U-937 cells upon GM-CSF stimulation. Neutralization by antibodies to IL 1 beta and TNF-alpha suggested that both monokines could be the antiproliferative principle operating in GM-CSF U-937-CM. Moreover, employing IL 1 beta-specific enzyme-linked immunosorbent assay, TNF-alpha specific radioimmunoassay, Northern analysis using a cloned TNF-alpha-specific cDNA and an oligonucleotide probe for IL 1 beta, we demonstrate GM-CSF-inducible IL 1 beta and TNF-alpha gene expression by U-937 cells at the mRNA and protein level. Although M-CSF expression was induced under similar conditions, M-CSF failed to inhibit growth of U-937 cells.     

The functional activity of human monocytes passively sensitized with monoclonal anti-D suggests a novel role for Fc gamma RI in the immune destruction of blood cells.

The role of Fc gamma RI in the immune destruction of blood cells is uncertain as serum IgG levels are sufficient to competitively inhibit interactions between this high-affinity receptor and sensitized red cells. In the current study, it is proposed that, rather than functioning as a receptor for opsonized red cells, Fc gamma RI might, under appropriate conditions, mediate the passive sensitization (or 'arming') of human macrophages with IgG antibodies resulting in the in vivo destruction of unsensitized cells expressing the corresponding antigen. To examine this hypothesis, Fc gamma RI-bearing human monocytes and U937 cells were first passively sensitized by incubation in vitro with human monoclonal anti-D, and then incubated with D-positive red cells. The uptake of monoclonal anti-D by U937 cells was rapid and, in the presence of 2.5 micrograms/ml IgG1 or IgG3 anti-D, was almost complete after 5 min at 37 degrees. Subsequent incubation of passively sensitized U937 cells in an IgG-free medium for 1 hr at 37 degrees resulted in the loss from the cell surface of approximately 50% cell-bound IgG; the remaining cell-bound IgG was lost more slowly despite repeated washing. In functional assays, passively sensitized monocytes (M-IgG) mediated adherent, phagocytic and chemiluminescent (CL) responses to D-positive red cells. After incubation of M-IgG in 50% v/v fresh normal human serum (FNHS) for 2 hr, sufficient anti-D remained bound to monocytes to promote the adherence of red cells. The adherence and phagocytosis of red cells by M-IgG was enhanced by the simultaneous addition of 50% FNHS, probably owing to the binding of low levels of C3bi to red cells. In contrast, phagocytic and CL responses of unsensitized monocytes to anti-D-sensitized red cells (E-IgG) were abrogated in the presence of 0.25% v/v FNHS, presumably owing to blocking of Fc gamma RI by IgG. It is considered that in vivo, Fc gamma RI may mediate the passive sensitization of macrophages in close proximity with antibody-secreting cells in the reticular network of the splenic cords. Once 'armed' in this way, macrophages may destroy cells expressing the appropriate antigen.     

The roles of endogenous reactive oxygen species and nitric oxide in triptolide-induced apoptotic cell death in macrophages

Triptolide, a major active component extracted from the root of Tripterygium wilfordii Hook f, has been shown to possess potent immunosuppressive and anti-inflammatory properties. In the present report, we reported that triptolide increased the generation of reactive oxygen species (ROS) and nitric oxide (NO) and induced apoptosis of RAW 264.7 cells in a dose-dependent manner (5-25 ng/ml). The antioxidant, reduced glutathione (GSH), significantly inhibited triptolide-induced apoptosis and inhibited the degradation of Bcl-2 protein, disruption of mitochondrial membrane potential, release of cytochrome c from mitochondria into the cytosol, activation of caspase-3, and cleavage of poly-(ADP-ribose)-polymerase. The inducible nitric oxide synthase-specific inhibitor 1400w blocked triptolide-induced apoptosis, but did not alter mitochondria disruption and caspase-3 activation. These results, for the first time, implicated that the increased endogenous ROS and NO co-mediated triptolide-induced apoptosis in macrophages. ROS initiated triptolide-induced apoptosis by the mitochondria signal pathway, while the apoptotic cell death mediated by NO was not via mitochondria collapse and caspase-3 activation. In addition, combining mathematical calculation and computer simulation based on our conventional experimental results, we set and validated the apoptotic model and provided more dynamic processes of triptolide-induced apoptotic cascade in macrophages.