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* Department of Morphology and Embryology, Human Anatomy Section, University of Ferrara, Italy;
Department of Drug Sciences, "G. D’Annunzio" University of Chieti, Italy;
Department of Human Normal Morphology, University of Trieste, Italy; and
Hemostasis Research Center on Physiopathology of Hemostasis, Department of Histology, Catholic University of Rome, Italy
Correspondence: Giorgio Zauli, M.D., Ph.D., Department of Human Normal Morphology, University of Trieste, Via Manzoni 16, 34138 Trieste, Italy. E-mail: zauli{at}univ.trieste.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: hematopoiesis • signal transduction • death receptor • arachidonic acid
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Prostanoids synthesized from COX-1 and COX-2 pathways can function as extracellular or intracellular messengers [1 ]. Classical prostanoids, such as PGE2, are secreted extracellularly and mediate their effects via plasma membrane G-protein-coupled receptors [4 ]. Circulating blood cells are known to diversely express active COX isoforms: For instance, granulocytes and monocytes constitutively express COX-1 and upon endotoxin, cytokine, or phagocytic stimuli, up-regulate COX-2, which contributes to the local inflammatory/immunologic responses [3 ]. Aside from the presence and effects in terminally differentiated blood cells, the expression and the function of COX isoenzymes in hematopoietic progenitors and precursors remain largely unknown.
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), also known as Apo-2 ligand, is a member of the structurally related TNF family of cytokines [5
, 6
]. A subset of these cytokines, including TNF, CD95L, death receptor (DR)-3 ligand (TWEAK), and TRAIL, is known to activate the cell death program [7
]. These cytokines, also called death ligands, induce apoptosis by activating their cognate surface receptors. These DRs are members of the TNF receptor superfamily and include TNFR1, CD95 (Fas/Apo-1), DR-3, DR-4, DR-5, and DR-6. In particular, TRAIL, which exists as a type II membrane protein or as a soluble protein [8
], specifically binds and induces apoptosis via DR-4 (also known as TRAIL-receptor R1) and DR-5 (also known as TRAIL-R2) [9
]. The unique feature of TRAIL, with respect to CD95L and TNF-
, is considered its ability to induce apoptosis in a variety of malignant cells, including several of hematopoietic origin [10
11
12
13
14
15
], displaying minimal toxicity on normal cells and tissues [9
]. In spite of its potential as an anticancer therapeutic, the physiologic role of TRAIL is presumably more complex than merely activating caspase-dependent cell death of cancer cells. Although little is currently known about possible nonapoptotic functions induced by TRAIL, previous data reported by other investigators and our group suggest that TRAIL might play a role in the negative regulation of human erythropoiesis [16
17
18
].
This study was designed to investigate the biological activity of TRAIL on cells of the myeloid lineage in terms of ability to modulate COX expression and function. As an experimental system, we have chosen the HL-60 myeloid leukemia cell line. Parallel experiments were carried out using primary-cultured myeloid cells, derived from CD34+ hematopoietic progenitors, as well as primary CD14+ monocytes, isolated from peripheral blood, to also investigate in normal myeloid cells whether TRAIL modulates the production of prostanoids.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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was purchased from Sigma Chemical Co. (St. Louis, MO). The broad caspase inhibitor Cbz-Val-Ala-Asp (Ome)-fluoromethyl ketone (z-VAD-fmk), the control peptide Cbz-Phe-Ala-fluoromethyl ketone, and the selective caspase 8 Cbz-Ile-Glu(Ome)-Thr-Asp(Ome)-CH2F (z-IETD-fmk) and caspase 9 Cbz-Leu-Glu(Ome)-His-Asp(Ome)-CH2F (z-LEHD-fmk) inhibitors were from Calbiochem. Although z-VAD-fmk blocks the activity of most effector caspases [3 , 4 , 7 ] as well as of caspase 1, z-IETD-fmk and z-LEHD-fmk specifically block the activity of caspases 8 and 9, respectively. All caspase inhibitors were dissolved in dimethyl sulfoxide, stocked in aliquots at -20°C, and used at the final concentration of 10 µM.
AA and PGE2 were purchased from Cayman Chemical (Ann Arbor, MI). Concentrated stock solutions of valeryl salicylate (COX-1 inhibitor; Cayman Chemical), NS-398 (COX-2 inhibitor; Biomol, Plymouth Meeting, PA), and indomethacin (nonselective COX inhibitor; Sigma Chemical Co.) were prepared in ethanol. The IC50 values for valeryl salicylate are 0.8 mM (for inhibition of COX-1) and 15 mM (for inhibition of COX-2). The IC50 values for NS-398 are 1.8 µM (for inhibition of COX-2) and 75 µM (for inhibition of COX-1).
Cells
HL-60 cell line is an M2/M3-type acute myeloid leukemia, and Jurkat is a CD4+ lymphoblastoid T cell line. All cell lines were grown in RPMI (Gibco-Life Technologies, Grand Island, NY) supplemented with 10% fetal calf serum (Gibco-Life Technologies) at an optimal cell density of 3 x 105–1.5 x 106 cells/ml. Cell stocks were routinely monitored for mycoplasma contamination by the gene probe method (Gentile Probe Inc., San Diego, CA).
Cord blood (CB) specimens and leukapheresis units were collected, according to institutional guidelines, during normal full-term deliveries and from peripheral blood (PB) normal donors, respectively. CB and PB mononuclear cells were isolated by density gradient (Ficoll/Histopaque-1077; Sigma Chemical Co.). CD34+ cells were isolated from CB mononuclear cells by using a magnetic cell-sorting program Mini-MACS and the CD34 isolation kit (Miltenyi Biotech, Auburn, CA) in accordance with the manufacturer’s instructions. CD34+-derived myeloid cultures were carried out by seeding CD34+ cells in X-vivo medium (BioWittaker), supplemented with nucleosides (10 µg/ml each), 0.5% bovine serum albumin (BSA; fraction V of Chon), 10-4 M BSA-adsorbed cholesterol, 10 µg/ml insulin, 200 µg/ml iron-saturated transferrin, 5 x 10-5 M 2-ß-mercaptoethanol (all purchased from Sigma Chemical Co.), stem-cell factor (SCF; 50 ng/ml), interleukin (IL)-3 (10 ng/ml), and granulocyte-colony stimulating factor (G-CSF; 10 ng/ml) or macrophage-CSF (M-CSF), as previously described [17 ]. All cytokines were purchased from Genzyme (Cambridge, MA). Every 3–4 days, cultures were demi-populated by removing half-volume of the medium, which was substituted with fresh medium supplemented with cytokines and analyzed for phenotypic expression of surface myeloid maturation markers. Treatment of the cells with control His. peptide or TRAIL was typically performed between days 12 and 14 of culture.
CD14+ cells were isolated from PB mononuclear cells using a magnetic cell-sorting program Mini-MACS and the CD14+ isolation kit (Miltenyi Biotech) in accordance with the manufacturer’s instructions. The purity of CD14+-selected cells was determined after each purification by flow cytometry using a monoclonal antibody (mAb), which recognizes a separate epitope of the CD14 molecule directly conjugated to fluorescein isothiocyanate (FITC; Becton-Dickinson, San Jose, CA). CD14+ cell purity was at least 95%.
Cell treatments
HL-60 and primary cells were treated with TRAIL (0.5 µg/ml) or His. control peptide in the presence or absence of pharmacological inhibitors. All experiments were performed on exponentially growing cells, showing a viability of at least 95%. The cytotoxic effects were evaluated at each time point after treatment by counting viable cells by trypan blue dye exclusion and evaluating apoptosis and cell cycle by propidium iodide (PI; Sigma Chemical Co.) staining as previously detailed [17
]. For Western blot analyses, 24–36 h after TRAIL treatment, dead HL-60 cells were removed from the cultures by using the Dead Cell Removal kit (Miltenyi Biotech).
Phenotypic analyses
Expression of surface antigens was analyzed by FACScan (Lysis II program, Becton-Dickinson). Surface expression of TRAIL-R1, TRAIL-R2, TRAIL-R3, and TRAIL-R4 was analyzed by indirect staining with primary goat anti-human TRAIL-R1, TRAIL-R2, TRAIL-R3, and TRAIL-R4 immunoglobulin G (IgG; all from R&D Systems, Oxon, UK) followed by phycoerythrin (PE)-conjugated rabbit anti-goat IgG secondary Ab (Sigma Chemical Co.). Background fluorescence was assessed using normal goat IgG followed by a second layer as above.
The phenotype of CD34-derived cells in liquid culture was analyzed by flow cytometry after staining using CD11b, CD15, and CD14 PE- and/or FITC-conjugated mAb (Becton-Dickinson). Nonspecific fluorescence was assessed using isotype-matched control mAb. Samples were assayed in duplicate, and gates containing viable cells were used to collect 10,000 events.
Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis
The presence of COX mRNA transcripts was examined by RT-PCR. RNA purification was performed using the RNA easy isolation system (Promega, Madison, WI) following the manufacturer’s protocol. Synthesis of first-strand cDNA and amplification was performed using the Access RT-PCR system (Promega) and specific primer sets, following the manufacturer’s protocol. As a control for DNA contamination, equal amounts of RNA were used for PCR without template retro-transcription. RT-PCR reactions were performed using the following primer sets: COX-1 (forward: 5'-TTCTTGCTGTTCCTGCTCCTG-3', reverse: 5'-GCATTGACAAACTCCCAGAAC-3'); COX-2 (forward: 5'-TCCTGGCGCTCAGCCATACAG-3', reverse: 5'-GTAGCCATAGCTAGCATTGTA-3'); and ß-actin (forward: 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3', reverse: 5'-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3'). The resulting PCR products from RT-PCR were resolved on 2% agarose gels and visualized with ethidium bromide.
Western blot
For the analysis of COX-1 and COX-2 protein expression by Western blot, cells were harvested in lysis buffer containing 1% Triton X-100 and sonicated. Protein determination was performed by Bradford assay (Bio-Rad, Richmond, CA). Equal amounts of protein lysates (40–50 µg) for each sample were migrated in 10% acrylamide gels and blotted onto nitrocellulose filters. For some experiments, positive controls, constituted by cell lysates, commercially provided (Cayman Chemical) or derived from TNF--stimulated endothelial cells, were included in the gels. Blotted filters were blocked for 2 h in a 5% suspension of dried skimmed milk in phosphate-buffered saline and were further incubated for 1 h at room temperature with specific Ab for COX-1 and COX-2 (Cayman Chemical), as previously described [19
, 20
], and for tubulin (Sigma Chemical Co.). After incubation with peroxidase-conjugated antirabbit or antimouse IgG (Sigma Chemical Co.), specific reactions were revealed with the enchanced chemiluminescence Western blotting detection reagent (Amersham, Arlington Heights, IL).
Densitometric analysis
Quantitative evaluation of the relative immunoblotting patterns of expression for COX-1 and tubulin proteins was performed considering the mean density of pixels in selected areas. Densitometric values, expressed by arbitrary units, were estimated by the Optimas Corporation Analyzing Program, version 6.1, and were normalized to the corresponding tubulin densitometric values to avoid variation as a result of unequal loading of the sample before statistical comparison.
Multiple film exposures were used to verify the linearity of the samples analyzed and to avoid saturation of the film.
Measurement of prostanoids
PGE2 were determined in the supernatants of cell cultures using previously validated radioimmunoassays [21
]. The anti-PGE2 serum used was previously described [21
].
To evaluate COX activity, cells were harvested from cultures, and viable cells were scored by trypan blue dye exclusion, washed twice with Hanks’ balanced saline solution with 1 mg/ml BSA, and incubated for 40 min in 1 ml of the same buffer with 10 µM AA. Supernatants were collected and stored at -80°C until measurement of prostanoid. Experiments were always performed in duplicate.
Statistical analysis
The results were evaluated by using analysis of variance with subsequent comparisons by Student’s t-test for paired or nonpaired data as appropriate. Statistical significance was defined as P < 0.05. Values are reported as means ± SD.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The dose response for inhibition of prostanoid production was next determined by preincubating HL-60 cells with COX-1 and COX-2 pharmacological inhibitors, followed by exposure to TRAIL for 6 h (Fig. 3C) . Valeryl salicylate (a COX-1 inhibitor) and NS-398 (a COX-2 inhibitor) were used at concentration ranges comprising their IC50 values (0.8 mM for valeryl salicylate on COX-1 and 1.8 µM for NS-398 on COX-2). Valeryl salicylate and NS-398 showed a comparable, dose-dependent inhibitory effect on the basal PGE2 production (Fig. 3C) , suggesting that in spite of the difficulty to detect COX-2 protein by Western blot, both COX isoenzymes contribute to prostanoid production in unstimulated HL-60 cells. In sharp contrast, in TRAIL-treated cultures, valeryl salicylate was significantly more potent (P<0.05) than NS-398 in suppressing the increase of PGE2 production induced by TRAIL at all the concentrations of pharmacological inhibitors examined (Fig. 3C) . These data confirmed that COX-1 plays a predominant role in the induction of PGE2 production mediated by TRAIL.
TRAIL-induced COX activity of myeloid cell lines via caspase-dependent and -independent pathways
Having previously shown that TRAIL activates the caspase pathway in a panel of myeloid cell lines, including HL-60 [23
], we have next investigated whether this pathway might play a role in the up-regulation of PGE2 induced by TRAIL by using selective pharmacological inhibitors. For this purpose, cells were preincubated with each inhibitor, followed by exposure to TRAIL for an additional 6 h. In control HL-60 cultures, cell viability was not affected by any of the inhibitors used at indicated concentrations (data not shown). Incubation of HL-60 cells with the broad inhibitor of effector caspases, z-VAD-fmk, which completely abrogated the TRAIL-induced apoptosis (data not shown), significantly (P<0.01), although not completely, decreased the TRAIL-mediated increase of PGE2 production (Fig. 4A
). The ability of TRAIL to induce PGE2 production was also evaluated in the presence of selective inhibitors for caspase 8 and caspase 9 [24
]. As shown in Figure 4A
, z-IETD-fmk, a selective caspase 8 inhibitor, was as effective as z-VAD-fmk in decreasing (P<0.01) PGE2 induction by TRAIL. Conversely, z-LEHD-fmk, a selective caspase 9 inhibitor, was unable to affect TRAIL-mediated PGE2 production (Fig. 4A)
. In additional experiments, we have therefore analyzed whether the caspase pathway modulated PGE2 production by affecting COX-1 protein expression. As shown in Figure 4B
, when HL-60 cells were incubated with z-VAD-fmk, the ability of TRAIL to up-regulate COX-1 protein expression was not affected, indicating that the caspase pathway likely interfered with TRAIL-induced COX activity but not with protein level.
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(Fig. 5A
and 5B)
, the prototype of the TNF family of cytokines, which has well-characterized, inflammatory properties. It is also particularly noteworthy that at variance to what was observed when added to myeloid HL-60 leukemic cells, TRAIL did not induce any significant cytotoxicity on primary, normal myeloid cells and in CD14+ monocytes, as evaluated in terms of viable cell counts and quantification of apoptosis (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Here, we have shown for the first time that besides inducing a marked cytotoxicity on the HL-60 myeloid cell line, TRAIL enhances the release of PGE2 by the subset of cells that survived to TRAIL cytotoxicity. Of note, similar biological effects were also observed in primary CD34-derived myeloid precursors and primary CD14+ monocytes. As demonstrated by using relative, selective COX-1 (valeryl salicylate) and COX-2 (NS-398) pharmacological inhibitors, the enhanced PGE2 release induced by TRAIL in myeloid cells was apparently a result of increased activity of the COX-1 protein, the predominant isoform expressed in HL-60, and COX-2 was not involved in TRAIL signaling. In agreement with our findings, other authors have shown that THP1 and U937 monoblastic cell lines and circulating monocytes show a constitutive expression of COX-1, and COX-2 is up-regulated in these cells only upon incubation with LPS or inflammatory cytokines such as IL-1 [30 31 32 33 34 ]. It is interesting that these studies have also reported an approximately twofold increase in prostanoid production following COX-1 activation [30 31 32 33 34 ]. Thus, the degree of COX-1 activation observed in this study is similar to that observed in previous studies and clearly distinguishes the changes occurring upon COX-1 activity from those of COX-2, which results in a several-fold increase of prostanoids. The potential relevance of this twofold increase in prostanoid production by TRAIL is underscored by the fact that the PGE2 concentrations reached in TRAIL-treated cultures are approximately in the same range of those able to promote HL-60 cell survival.
TRAIL has garnered the most interest therapeutically, as several studies have demonstrated cytotoxicity toward tumoral cells in vitro and tumoricidal activity in vivo in animal models, without inducing significant toxicity in mice and nonhuman primates [9 , 35 36 37 ]. However, the actual biological function of the TRAIL/TRAIL receptors system is still not clear. In this contest, previous studies from other investigators and our group have demonstrated that TRAIL acts as a negative regulator of erythropoiesis in physiological and pathological conditions by directly killing glycophorin Aintermediate normal erythroblasts [16 17 18 ]. On the contrary, here, we have demonstrated that TRAIL does not show any cytotoxic effect on primary, normal CD34-derived cells committed toward the myeloid lineage or on freshly isolated CD14+ monocytes, and it enhances the PGE2 release by these cells. As mature monocytic/macrophages represent a main cell type of the bone marrow microenvironment, the ability of TRAIL to up-regulate PGE2 release by monocytes is particularly noteworthy. In fact, PGE2 are known to inhibit the cell-cycle progression of myeloid colony-forming units-GM progenitors, while expanding the life span of mature myeloid cells [25 26 27 , 29 ]. Consistenly, we have found that the addition of exogenous PGE2 was able to partially counteract the TRAIL-mediated apoptosis in HL-60 cells, also exhibiting a protective effect on these cells. The ability of TRAIL to modulate COX-1 activity and to up-regulate PGE2 production by primary, normal myeloid cells in the absence of cytototoxicity envisions a novel, physiological role of TRAIL in the regulation of normal myelopoiesis. The concept that the TRAIL/TRAIL-receptor system plays a physiological role in the regulation of normal hematopoiesis is strengthened by the previous demonstration that TRAIL protein is expressed at the bone marrow level [17 ].
In the attempt to clarify the mechanisms underlying the ability of TRAIL to up-regulate COX-1 activity, we have demonstrated that the caspase cascade plays a role in this process, without affecting the expression levels of COX-1 protein. These data are in agreement with previous studies that have shown how the caspase activation in intact cells does not necessarily lead to cell death and argue for a checkpoint in the apoptotic pathway downstream of caspases. Several reports have recently suggested that caspases may have a function outside of apoptosis [38 ]. In particular, it has been recently demonstrated that the caspase family of proteases mediates the activation/proliferation of quiescent T lymphocytes [39 ] and the expression of inflammatory cytokines such as IL-8 [40 ].
Recently, two distinct caspase-dependent pathways have been described in response to the CD95 ligand [24
]. The type I pathway triggers the activation of large amounts of caspase-8 followed by the rapid cleavage of caspase-3. In the type II pathway, low concentrations of caspase 8, insufficient to allow an effective downstream activation of caspase 3, induce loss of mitochondria transmembrane potential (
m). This, in turn, induces the release of cytocrome C and procaspase 9 from mitochondria, its activation in the cytosol, and the activation of caspase 3 [24
]. We could demonstrate that in HL-60 cells, z-IETD-fmk, a selective pharmacological inhibitor of caspase 8, reproduced the ability of z-VAD-fmk to significantly decrease the TRAIL-mediated PGE2 production. Conversely, z-LEHD-fmk, a selective pharmacological inhibitor of caspase 9, was unable to interfere with TRAIL biological activities, indicating that the type II intrinsic pathway, which has been mainly involved in the induction of apoptosis [24
], does not play a role in the TRAIL-induced PGE2 up-regulation. Moreover, the presence of residual PGE2 not inhibible by z-VAD-fmk or z-IETD-fmk suggests that additional pathways might be involved in mediating TRAIL-induced COX-1 up-regulation in myeloid cells. In this respect, a recent study has demonstrated that agonists able to activate nuclear factor (NF)-
B subsequently induce COX-1 activity and increase PGE2 synthesis in the U937 monoblastic cell line [41
]. It has to be underlined that the interaction of TRAIL with its cognate DRs TRAIL-R1 and TRAIL-R2 also results in the activation of the NF-B pathway in various cell types (reviewed in ref. [9
]). Therefore, it is possible that the NF-B pathway participates to the TRAIL-mediated increase of PGE2 synthesis, accounting for the z-VAD-insensitive increase of PGE2 biosynthesis.
The data derived from this study indicate that COX activation is not involved in TRAIL-induced cytotoxicity in leukemic cells. Indeed, the COX inhibitor indomethacin, used at concentrations that completely abrogated the TRAIL-mediated PGE2 increase, did not block the TRAIL-mediated apoptosis in HL-60 cells and exogenous PGE2 but rather counteracted TRAIL cytotoxicity. Thus, although TRAIL induces two functional outcomes in myeloid leukemic cells, apoptosis and PGE2 production, in primary normal myeloid cells, it enhances PGE2 production without inducing apoptosis.
In conclusion, the findings derived from this study might have implications in understanding the biology of the TRAIL/TRAIL-receptor system in hematopoiesis by disclosing a novel, physiological role for TRAIL as an activator of the prostanoid production in cells committed toward the myeloid lineage.
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ACKNOWLEDGEMENTS |
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Received May 24, 2002; accepted August 7, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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