From www.bloodjournal.org by guest on August 27, 2017. For personal use only.

IMMUNOBIOLOGY

IFN-stimulated neutrophils and monocytes release a soluble form

of TNF-related apoptosis-inducing ligand (TRAIL/Apo-2 ligand)
displaying apoptotic activity on leukemic cells
Cristina Tecchio, Veronica Huber, Patrizia Scapini, Federica Calzetti, Daniela Margotto, Giuseppe Todeschini,
Lorenzo Pilla, Giovanni Martinelli, Giovanni Pizzolo, Licia Rivoltini, and Marco A. Cassatella

Tumor necrosis factor (TNF)related apop- ties that were significantly reduced by leukocytes, in vivo, might represent an
tosis-inducing ligand (TRAIL) is a mem- either a combination of TRAIL-R1/Fc and important source of sTRAIL. In this re-
ber of the TNF superfamily exerting cyto- TRAIL-R2/Fc chimeras or neutralizing anti- gard, we show that sTRAIL serum levels
toxic activities toward tumor cells. Herein, TRAIL, antiTRAIL-R1, and antiTRAIL-R2 as well as leukocyte-associated TRAIL
we demonstrate that therapeutic concen- antibodies, suggesting that they were me- significantly increase in melanoma pa-
trations of interferon (IFN) stimulate diated by released sTRAIL acting on both tients following IFN administration. Col-
the expression of high levels of TRAIL TRAIL receptors. Since diseases such as lectively, these findings indicate that
mRNA and the release of elevated chronic myeloid leukemia (CML) and mela- sTRAIL released by IFN-activated neutro-
amounts of a soluble bioactive form of noma are effectively treated with IFN, we phils and monocytes contributes not only
TRAIL (sTRAIL) in both human neutro- also demonstrate that CML neutrophils to the immunoregulatory actions but also
phils and monocytes. Supernatants har- and peripheral blood mononuclear cells to the therapeutic activities of IFN.
vested from IFN-treated neutrophils/ (PBMCs) cultured with IFN at therapeu- (Blood. 2004;103:3837-3844)
monocytes elicited, on TRAIL-sensitive tic concentrations retain the capacity of
leukemic cell lines, proapoptotic activi- releasing sTRAIL, suggesting that CML 2004 by The American Society of Hematology

Introduction
Tumor necrosis factor (TNF)related apoptosis-inducing ligand fundamental role of this molecule in cancer immunosurveillance.19
(TRAIL/Apo-2 ligand) is a member of the TNF superfamily Moreover, in recent years, it has been suggested that the immuno-
identified on the basis of the homology of its extracellular domain modulatory activity of interferons (IFNs) in controlling cancer
with Fas/Apo-1 ligand.1,2 Although primarily expressed as a type II development might also be attributable to their capacity of
transmembrane protein, TRAIL may exist in a soluble form inducing membrane-bound TRAIL expression in different effector
(sTRAIL) either generated through enzymatic shedding or released cells.14,15,18,20-22 In the light of the tumor-selective proapoptotic
in association with microvesicles.3-6 TRAIL exerts its activity by activity of membrane-bound TRAIL, the potential use of recombi-
interacting with a complex system of 2 death receptors (DR4/ nant soluble forms of this molecule as cancer therapeutics is
TRAIL-R1 and DR5/TRAIL-R2) and 3 decoy receptors (DcR1/ presently being exploited in several preclinical and preliminary
TRAIL-R3, DcR2/TRAIL-R4, and osteoprotegerin/OPG).7,8 Al- clinical studies.23
though these receptors are characterized by a high sequence TRAIL is expressed on the surface of different activated cells of
homology in their extracellular domains, only DR4/TRAIL-R1 and the immune system such as IFN-, IFN-, or lipopolysaccharide
DR5/TRAIL-R2 contain a functionally active cytoplasmic death (LPS)stimulated monocytes5 and/or dendritic cells12,13; CD4 T
domain that allows an apoptotic response upon TRAIL stimula- lymphocytes upon specific T-cell receptor (TCR) engagement or
tion.9,10 The biologic significance of TRAIL as mediator of innate IFN stimulation14; and IFN-stimulated natural killer cells.15
and specific immunity against transformed and virus-infected cells More recently, induction of membrane-bound TRAIL has been also
has been clearly documented by several reports.11-18 In particular, it shown on measles virusstimulated dendritic cells16 and Newcastle
has been demonstrated that activated monocytes, dendritic cells, disease virusstimulated monocytes.17 Even though neutrophils are
natural killer cells, and T lymphocytes may exert tumoricidal well known to produce ligands of the TNF family, such as TNF,
activities through membrane-bound TRAIL.5,8,12-15 Evidence that Fas ligand (FasL), CD30L24, and B-lymphocyte stimulator (BLyS),25
TRAIL-deficient mice are more susceptible to experimental and it has not yet been investigated whether they eventually express
spontaneous tumor initiation and metastasis further support the membrane-bound TRAIL or release sTRAIL.

From the Department of Pathology, University of Verona, Verona, Italy; Unit of Cancro, CARIVERONA-2001 and Fondazione del Monte di Bologna e
Immunotherapy of Human Tumors, Istituto Nazionale Tumori, Milano, Italy; Ravenna.
Department of Clinical and Experimental Medicine, University of Verona,
Verona, Italy; Institute of Hematology and Medical Oncology L. & A. Seragnoli Reprints: Marco A. Cassatella, Department Pathology, Section of General
University of Bologna, Bologna, Italy. Pathology, Strada Le Grazie 4, 37134 Verona, Italy; e-mail: marco.
cassatella@univr.it.
Submitted August 14, 2003; accepted January 5, 2004. Prepublished online
as Blood First Edition Paper, January 15, 2004; DOI 10.1182/blood-2003- The publication costs of this article were defrayed in part by page charge
08-2806. payment. Therefore, and solely to indicate this fact, this article is hereby
marked advertisement in accordance with 18 U.S.C. section 1734.
Supported by grants from Ministero dellIstruzione, dellUniversita e della
Ricerca (COFIN, FIRB, and 60%), Associazione Italiana per la Ricerca sul 2004 by The American Society of Hematology

BLOOD, 15 MAY 2004 VOLUME 103, NUMBER 10 3837

 From www.bloodjournal.org by guest on August 27, 2017. For personal use only.

3838 TECCHIO et al BLOOD, 15 MAY 2004 VOLUME 103, NUMBER 10

In this work, we show that peripheral blood neutrophils and MN), human TRAIL-R1, -R2, -R3, -R4 (Apotech Corporation, Epalinges,
mononuclear cells up-regulate TRAIL mRNA and release signifi- Switzerland), or control mouse immunoglobulin G1 (IgG1; Sigma), fol-
cant amounts of functionally active sTRAIL in response to IFN. lowed by biotinylated secondary sheep antimouse IgG (Sigma). Immunola-
Given the use of IFN in the treatment of diseases such as chronic beling was revealed using phycoerythrin-conjugated streptavidin (Becton
Dickinson, Mountain View, CA). Analysis was performed with FACScan
myeloid leukemia (CML)26 and melanoma, we also demonstrate
using CellQuest software (Becton Dickinson). For each sample, the mean
that neutrophils and mononuclear cells isolated from CML patients fluorescence intensity (MFI) was calculated by subtracting the MFI of the
efficiently release sTRAIL after treatment with IFN. Furthermore, corresponding immunolabeled isotype control.25
we report that administration of low-dose IFN (3 million units) to
patients with stage IV metastatic melanoma results in a dramatic Western blot analysis
increase of sTRAIL serum levels and leukocyte-associated TRAIL.
Supernatants from IFN-activated neutrophils and PBMCs were analyzed
Taken together, our data suggest that the release of sTRAIL by
by Western blot under reducing, nonreducing, and native conditions.
IFN-treated peripheral blood cells represents a novel mechanism
Supernatants were first concentrated by a Centricon Plus 20 device
whereby therapeutic concentrations of IFN might exert immuno- (Amicon, Beverly, MA) and then analyzed for total protein concentration
modulatory and antitumor activities. by Lowry assay (Bio-Rad Laboratories, Hercules, CA). Under native
conditions, equal amounts of protein with additional 10% of glycerol were
separated on a 6% phenoxyacetic acid (PAA) gel. Under nonreducing and
Patients, materials, and methods reducing conditions, equal amounts of protein for each sample were
separated on a 4% to 12% Bis-Tris gel (Invitrogen SRL, Milan, Italy).
Cell purification and culture Before loading, samples were denatured in NuPage lithium dodecyl sulfate
(LDS) sample buffer (Invitrogen SRL) at 60C (nonreducing conditions) or
Hghly purified granulocytes (neutrophils 96.5%, eosinophils 3%), at 95C (reducing conditions, containing NuPAGE Sample Reducing
peripheral blood mononuclear cells (PBMCs), monocytes, and lymphocytes Agent; Invitrogen) for 15 minutes. After electrophoresis, proteins were
were isolated under endotoxin-free conditions.27 Immediately after purifica- electroblotted onto a polyvinylidene fluoride (PVDF) membrane (Amer-
tion, cells were suspended in RPMI 1640 supplemented with 10% sham Pharmacia Biotech Italia, Milan, Italy). Membranes were stained
low-endotoxin fetal calf serum (FCS; 0.5 endotoxin units (EU)/mL; using 1 g/mL of antihuman TRAIL mAb (clone B35-1; BD Biosciences,
Biowhittaker Europe, Verviers, Belgium) and cultured at 5 106/mL in the Calderara, Italy), developed with a secondary mouse antihuman IgG
absence or in the presence of 100 to 1000 U/mL IFN (Roche Laboratories, conjugated to horseradish peroxidase (BD Biosciences), and detected by
Nutley, NJ), 100 ng/mL LPS (Sigma, St Louis, MO), 10 ng/mL granulocyte- SuperSignal detection system (Pierce Chemical, Rockford, IL). In all
macrophage colony-stimulating factor (GM-CSF; Schering-Plough, Ken- Western blot experiments soluble nondisulfide-linked trimeric recombi-
ilworth, NJ), 10 nM fMLP (formyl-methionyl-leucyl-phenylalanine; Sigma), nant human soluble TRAIL (rhsTRAIL; R&D Systems) was used as a
or 1000 U/mL G-CSF (Chugai Pharmaceutical, London, United Kingdom). positive control.
After the desired incubation period, cells were collected and spun at 350g
for 5 minutes. The resulting supernatants were immediately frozen in liquid
ELISA
nitrogen and stored at 70C. The corresponding pellets were either
extracted for total RNA or thawed in phosphate-buffered saline (PBS) Concentrations of sTRAIL and sFas ligand in cell-free supernatants,
containing 0.5% nonidet P-40 (NP40), 5 mM EDTA (ethylenediaminetet- cells pellets, and sera obtained from healthy donors and melanoma
raacetic acid), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 5 g/mL patients were measured by using commercial enzyme-linked immunosor-
leupeptin/pepstatin A and then spun (14 000g, 5 min) to remove cell debris. bent assay (ELISA) kits (soluble TRAIL/Apo2L and soluble FasL
Peripheral blood leukocytes and bone marrow samples from patients ELISA KITs; Diaclone Research, Besancon, France), according to the
affected by CML were obtained, after signed informed consent, during manufacturers instructions.
routine diagnosis at the Hematology Unit of the University of Verona.
Approval was obtained by the University of Verona institutional review Apoptosis assessment
board. Bone marrow samples from CML patients in chronic phase
underwent the same procedures used to purify peripheral blood neutrophils, Apoptotic rate of neutrophils was determined by propidium iodide staining,
allowing us to obtain a mixed cell population composed of myeloblasts, as previously described,29 whereas apoptosis of leukemic cell lines was
promyelocytes, myelocytes, and metamyelocytes, along with some band measured by annexin-VFLUOS Staining kit (Roche Diagnostic, Mann-
and segmented neutrophils. MEG-01 and KU-812 cell lines, derived from heim, Germany). Neutrophils were preincubated with or without 100 U/mL
CML patients in megakaryocytic and myeloid blast crisis, respectively IFN for 4 hours and then cultured in the absence or in the presence of 1 to
(obtained from DSMZ, Braunschweig, Germany), and Fas/DR4 Jurkat 20 ng/mL of SuperKillerTRAIL (Apotech Corporation) for additional 20
cells (J32 clone; kindly provided by Dr L. Zamai, Institute of Morphologi- hours. In other experiments, normal or CML neutrophils were cultured with
cal Sciences, University of Urbino, Italy)28 were cultured in RPMI 1640 or without 100 U/mL IFN in the presence of up to 500 nM imatinib
supplemented with 10% FCS. mesylate (Novartis Pharma AG, Basel, Switzerland) and then harvested
after 24 hours. MEG-01, Jurkat J32 (both TRAIL sensitive), and KU-812
RNA isolation and Northern blot analysis (TRAIL insensitive) cell lines were seeded at a density of 1 106 cells/mL
in the presence of media conditioned by IFN alone or by neutrophils/
Total RNA from neutrophils and PBMCs was extracted by the guani- monocytes cultured with IFN. Supernatants from neutrophils were
dinium isothiocyanate method and analyzed by Northern blot for concentrated by Centricon Plus 20 device (Amicon) as described29 and then
TRAIL, Fas ligand, interleukin 1 receptor antagonist (IL-1ra), and actin used at a final concentration of 5 ng/mL sTRAIL (as measured by ELISA).
mRNA expression, as already described.25,27 The following reagents were used to neutralize TRAIL-induced apoptosis
of leukemic cell lines: Fc chimera TRAIL-R1 and Fc chimera TRAIL-R2
Flow cytometry for membrane-bound TRAIL (R&D Systems) at 100 ng/mL; mAb 2E5 (anti-TRAIL; Alexis, San Diego,
and TRAIL receptors CA) at 3 g/mL; mAb HS101 and mAb HS201 (antiTRAIL-R1 and
antiTRAIL-R2, respectively; Alexis) at 5 g/mL.
Briefly, freshly isolated or cultured cells were centrifuged, suspended in 100
L of PBS containing 0.2% albumin and 0.2% sodium azide, and then Patients
incubated for 15 minutes with 10% complement-inactivated human serum.
Cells were then incubated with 10 g/mL monoclonal antibodies (mAbs) After signed informed consent was obtained, blood was collected from 6
raised against human TRAIL (TNFSF10; R&D Systems, Minneapolis, healthy subjects and 6 patients with stage IV metastatic melanoma treated at
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BLOOD, 15 MAY 2004 VOLUME 103, NUMBER 10 SOLUBLE TRAIL RELEASE BY HUMAN PHAGOCYTES 3839

the Istituto Nazionale Tumori of Milano. Patients were enrolled in clinical selectively expressed FasL mRNA in response to IFN.30,31 Finally,
protocols of vaccine therapy that scheduled subcutaneous administration of TNF gene was expressed neither in resting nor in IFN-treated
IFN. Blood samples were collected prior to and 24 hours after the first leukocytes (Figure 1B).
administration of 3 million units (MU) IFN (IFN2b, Schering-Plough
Segrate, Milan, Italy; IFN, Alfa Wasserman Alanno, Pescara, Italy). Serum was Soluble TRAIL release by IFN-stimulated leukocytes
immediately separated from blood by centrifugation at 860g for 10 minutes
at 4C and stored at 80C. PBMCs were also isolated from these blood To determine whether up-regulation of TRAIL mRNA corre-
samples and lysed for TRAIL measurement in cell-associated pellets. lated with an increased accumulation of the related membrane-
bound protein, expression of surface TRAIL was analyzed by
Statistical analysis flow cytometry in either freshly isolated or cultured neutrophils
Data are expressed as means SEM. Statistical evaluation was performed
and PBMCs. As demonstrated by the representative experiment
by the Student t test for paired data. shown in Figure 2, low levels of constitutive TRAIL expression
were found in freshly isolated neutrophils (38 11 MFI, n 5)
but not on freshly isolated monocytes or lymphocytes from the
same donors. As previously reported,11 expression of membrane-
Results bound TRAIL increases in 24-hour cultured monocytes (35 13
Human neutrophils incubated with IFN up-regulate TRAIL MFI, n 5) and is further augmented by IFN (66 11 MFI),
mRNA expression whereas in lymphocytes remains negative, regardless of the
presence of IFN in the incubation medium20 (Figure 2).
To investigate TRAIL mRNA expression in neutrophils, these cells Surprisingly, while the levels of surface TRAIL did not change
were cultured with a variety of mediators and then processed for in 24-hour cultured neutrophils (25 7 MFI) compared with
Northern blot analysis. Figure 1 shows that resting neutrophils freshly isolated cells, they did not increase at all upon treatment
express constitutive levels of TRAIL mRNA that persisted for up to with IFN (24 8 MFI; Figure 2), even if the cytokine was
24 hours of culture. TRAIL mRNA expression was not substan- used at 1000 U/mL (data not shown).
tially increased by the addition of GM-CSF, LPS, fMLP (Figure To explain such discrepancy, we investigated whether, at
1A), or G-CSF (not shown) to the cultures of neutrophils, even least in neutrophils, TRAIL was released as a soluble protein
though these stimuli up-regulated the IL-1ra gene expression (sTRAIL) instead of being expressed on the cell surface. For this
(Figure 1A).24 By contrast, IFN-treated neutrophils exhibited a purpose, neutrophils, PBMCs, or, alternatively, Percoll-purified
considerably high accumulation of TRAIL mRNA (Figure 1A), monocytes were cultured for 24 hours in the absence or the
with maximum expression levels reached after 5 hours of incuba- presence of 100 U/mL IFN to analyze their capacity to release
tion (Figure 1B). The genuine ability of neutrophils to express sTRAIL into the cell-free supernatants. Figure 3A shows that
TRAIL mRNA was further confirmed by the Northern blot while untreated leukocytes constitutively released small but
experiments performed with PBMCs (Figure 1B), which displayed detectable amounts of sTRAIL, both neutrophils and PBMCs
a different time course of TRAIL mRNA accumulation and which cultured with IFN secreted significant amounts of sTRAIL. On
a per cell basis, PBMCs resulted consistently more effective
than neutrophils (Figure 3A), with monocytes contributing for
85% 6% (n 5) of the total sTRAIL release by PBMCs. Time-
course experiments revealed that sTRAIL was released as early as after
3 hours of incubation with IFN, progressively accumulating into the
supernatants for up to 48 hours (data not shown). The genuine
release of sTRAIL by neutrophils was substantiated by the findings
that sFasL was present only in supernatants harvested from
cultured PBMCs but not from activated neutrophils (Figure 3B), in
line with the Northern blot data (Figure 1) and with earlier
studies.31 Importantly, concentrations of IFN ranging from 100 to
1000 U/mL were equally effective in inducing sTRAIL release by
either neutrophils or PBMCs (data not shown). Measurement of
total TRAIL production by leukocytes (ie, released sTRAIL in
parallel with cell-associated TRAIL) demonstrated that treatment
with IFN for 24 hours induced approximately a 5-fold increase of
total TRAIL synthesis relative to cells treated with medium only in
both neutrophils and PBMCs (data not shown).
To further confirm the release of sTRAIL by IFN-activated
neutrophils/PBMCs as detected by ELISA, we initially performed
Western blot analysis of concentrated supernatants under reducing
conditions. As reported in Figure 4A, sTRAIL was detectable as 2
Figure 1. Expression of TRAIL mRNA in IFN-stimulated neutrophils and major bands of approximately 24 kDa and 48 kDa in both
PBMCs. (A) Neutrophils were stimulated for 3 hours with 100 U/mL IFN, 10 ng/mL
GM-CSF, 100 ng/mL LPS, and 10 nM fMLP before total RNA extraction and analysis
neutrophils and PBMC supernatants, likely representing the mono-
of TRAIL, IL-1ra, and actin mRNA expression by Northern blotting. (B) Neutrophils meric and dimeric forms of the protein, respectively. A recombinant
and PBMCs, purified from the same donor, were cultured for the times indicated with bioactive soluble TRAIL (rhsTRAIL), produced as a nondisulfide-
or without 100 U/mL IFN and then subjected to Northern blot analysis for TRAIL,
linked homotrimer, showed a similar band pattern with an addi-
FasL, IL-1ra, and actin mRNA expression. Time 0 indicates RNA extraction in freshly
isolated leukocytes. Data are representative of at least 2 independent experiments tional band at about 64 kDa, representing the trimeric conformation
for each panel. of the protein. In the attempt to investigate the aggregation state of
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3840 TECCHIO et al BLOOD, 15 MAY 2004 VOLUME 103, NUMBER 10

Figure 2. Expression of membrane-bound TRAIL in freshly isolated and

IFN-stimulated leukocytes. Neutrophils and PBMCs, purified from the same
donor, were analyzed by flow cytometry immediately after isolation or after incubation
for 24 hours in the absence or in the presence of 100 U/mL IFN. Monocytes and
lymphocytes were identified in the total PBMC population according to forward light Figure 4. Western blot analysis of sTRAIL released by IFN-stimulated neutro-
scatter and side scatter parameters. Staining with anti-TRAIL mAb are indicated with phils and PBMCs. Supernatants harvested from neutrophils and PBMCs, cultured
normal and bold histograms for cells cultured in the absence or in the presence of for 24 hours with 100 U/mL IFN, were concentrated and analyzed for the presence
IFN, respectively. Dotted histograms represent staining with isotype control IgG1 of sTRAIL by Western blot, as described in Patients, materials, and methods. Data
mAbs. These profiles are representative of analyses performed in 5 healthy donors. are representative of analyses performed in 2 healthy donors. (A) Concentrated
supernatants (50 g/sample) from neutrophils (lane 1) and PBMC (lane 2) were run
under reducing conditions and stained with anti-TRAIL mAb. The rhsTRAIL (nondisul-
fide-linked homotrimer) at 100 ng/sample (lane 3) was included as positive control.
(B) The rhsTRAIL (lane 1), supernatants of activated neutrophils (lane 2), and
PBMCs (lane 3) were analyzed by Western blot under nonreducing conditions;
samples were heated at 60C for 15 minutes before loading. (C) Freshly concen-
trated supernatants of activated PBMCs (lanes 1-2) and neutrophils (lane 3-4) were
analyzed under native conditions in comparison with rhsTRAIL (lane 5).

the bioactive sTRAIL released by activated cells, Western blot

analysis was performed under nonreducing and native conditions.
As shown in Figure 4B (nonreducing), a band of approximately 66
kDa was detectable in supernatants from both activated neutrophils
and PBMCs and rhsTRAIL preparations. This band likely repre-
sents the trimeric form of the protein, which maintains aggregation
stability in the experimental setting used (denaturation at 60C).
Moreover, higher aggregated forms (multimers) of sTRAIL, as
well as dimers and monomers, were detectable. Western blot
analysis under native conditions (Figure 4C) further confirms the
similarity of the migration patterns displayed by the bioactive
rhsTRAIL and the sTRAIL present in cell supernatants. Altogether,
these results suggest that sTRAIL released by IFN-activated
leukocytes is characterized by a similar aggregation state as the one
observed with the proapoptotic homotrimeric rhsTRAIL.

IFN protects neutrophils from TRAIL-induced apoptosis

Recently, a recombinant TRAIL preparation that has been

engineered to self-associate spontaneously into stable structures
with high biologic activity, called leucine zipper (LZ)TRAIL,
was demonstrated to accelerate the constitutive apoptotic rate of
human neutrophils.32 In our hands, increasing concentrations
(1-20 ng/mL) of rhsTRAIL His-tag CC-mutant (a multimeric
Figure 3. Release of sTRAIL by IFN-stimulated neutrophils and PBMCs of soluble recombinant form of TRAIL characterized by a biologic
healthy donors and CML patients. Neutrophils and PBMCs isolated from healthy activity higher than LZ-TRAIL) confirmed that soluble forms of
donors or CML patients were incubated for 24 hours in the absence or in the presence
recombinant TRAIL significantly increase the spontaneous
of 100 U/mL IFN. The yields of sTRAIL (A) and sFasL (B) were determined in the
corresponding cell-free supernatants by ELISA. The mean values SEM of cytokine neutrophil apoptosis in a 24-hour culture period (from 30% 6%
release from 3 to 4 independent experiments are shown. to 52% 6% with 20 ng/mL rhsTRAIL; P .01, n 5). In
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BLOOD, 15 MAY 2004 VOLUME 103, NUMBER 10 SOLUBLE TRAIL RELEASE BY HUMAN PHAGOCYTES 3841

light of the ability of IFN to induce the release of an similar to those of healthy donors (data not shown). In contrast,
endogenous sTRAIL, we investigated whether type I IFN could CML neutrophils displayed a constitutive apoptotic rate lower than
eventually promote neutrophil apoptosis. However, this ap- normal neutrophils, remaining, however, susceptible to the
peared not to be the case, since 100 U/mL IFN not only proapoptotic effect of rhsTRAIL His-tag CC-mutant (by
reduced the constitutive apoptosis of neutrophils (to 20% 5%; 10.5% 4%; n 2). Collectively, these data demonstrate that
P .01) but also prevented that induced by 20 ng/ml rhsTRAIL the capacity of leukemic leukocytes to release sTRAIL in
(24% 10%; P .01). Similarly to neutrophils, PBMCs re- response to IFN is unaltered.
mained more viable (by 10% 5%, n 3) when cultured in the
presence of IFN. In agreement with the data reported by
Neutrophil- and monocyte-derived supernatants induce
Renshaw et al,32 flow cytometry analysis of TRAIL receptors in
apoptosis of TRAIL-sensitive leukemia cells
freshly isolated neutrophils confirmed a low expression of
TRAIL-R2 (38.7 9.5 MFI, n 5), a high expression of To ascertain the biologic activity of sTRAIL released by IFN-
TRAIL-R3/DcR1 (332.1 8.3 MFI), and the absence of both treated phagocytes, we tested the ability of supernatants from
TRAIL-R1 and TRAIL-R4. IFN-treated neutrophils and monocytes to induce apoptosis of
TRAIL-sensitive cell lines. For this purpose, we used Fas/DR4
Neutrophils and PBMCs isolated from CML patients
Jurkat (J32 clone), a well-characterized TRAIL-sensitive T-acute
release sTRAIL upon stimulation with therapeutic
lymphoblastic leukemia cell line,28 and MEG-01, a Philadelphia
concentrations of IFN
chromosomepositive (Ph) CML cell line displaying susceptibil-
Having observed that leukocytes from healthy donors release ity to recombinant TRAIL. We also used KU-812, another Ph
significant amounts of sTRAIL upon treatment with concentrations CML cell line, which, unlike MEG-01, was resistant to rhsTRAIL
of IFN (100 U/mL), which were therapeutically reached in His-tag CC-mutant. Flow cytometric analysis of TRAIL receptor
plasma of CML patients,33 we investigated the release of sTRAIL expression revealed a positive staining for TRAIL-R1 and
by leukocytes isolated from these subjects. For this purpose, TRAIL-R2 in MEG-01 and KU-812 cells and for only TRAIL-R2
peripheral blood neutrophils and PBMCs isolated from chronic- in J32 cells, the latter finding in agreement with previous observa-
phase CML patients at the diagnosis were cultured for 24 hours in tions.34,35 Preliminary experiments revealed that viability of J32,
the absence or presence of 100 U/mL IFN before harvesting their MEG-01, and KU-812 cells was unaffected by doses of IFN of up
supernatants for sTRAIL measurement. As depicted in Figure 3A, to 10 000 U/mL for 72 hours. Basal apoptosis rates of J32,
IFN-stimulated neutrophils and PBMCs isolated from CML MEG-01, and KU-812 cells detected in a 40-hour culture period
patients release sTRAIL at levels similar to those of normal were 9% 1%, 20% 1%, and 22% 5% (n 5), respectively,
leukocytes. Strikingly, elevated levels of sTRAIL were also as measured by annexin-V binding. These percentages variably
measured in supernatants harvested from cultures of a mixture of increased if the cells were cultured in the presence of medium
immature myeloid cells (myeloblasts, promyelocytes, myelocytes, conditioned by supernatants harvested from resting monocytes
and metamyelocytes) containing some bands and segmented neutro- (Figure 5) or neutrophils. Remarkably, apoptosis of MEG-01 and
phils isolated from the bone marrow of CML patients. In fact, when J32, but not of KU-812, greatly augmented if leukemia cells were
incubated in the presence of IFN, these cells released increased cultured in the presence of medium containing supernatants
amounts of sTRAIL (268 53 pg/mL versus 136 142 pg/mL, harvested from monocytes stimulated with IFN (Figure 5). That
respectively; n 2) indicating that even immature CML myeloid the latter enhancement was specifically provoked by endogenous
elements are responsive to IFN and contribute to sTRAIL release sTRAIL was demonstrated by the observations that it was greatly
in their microenvironment. Similarly to what was observed in reduced either by a combination of TRAIL-R1/Fc and TRAIL-
healthy donors, sFasL was detected only in supernatants of CML R2/Fc chimeras (Figure 5) or by neutralizing anti-TRAIL mAb 25E
PBMCs treated with IFN (Figure 3B). However, the yields of (data not shown), whereas it was not influenced by neutralizing
sFasL recovered in these samples were lower than those recovered anti-IFN mAb (data not shown). Supernatants of IFN-treated
from normal PBMCs. Additional experiments demonstrated that neutrophils also increased the apoptosis rate of MEG-01 cells
the patterns of membrane-bound TRAIL and TRAIL receptor (from 52% 5% to 67% 6%; n 3), such effect being partially
expression in freshly isolated CML neutrophils were substantially neutralized (by 59% 4%) by TRAIL inhibitors. Anti-TRAIL

Figure 5. The sTRAIL present in supernatants of IFN-treated monocytes enhances apoptosis in human leukemic cell lines. Apoptosis rate of J32, MEG-01, and
KU-812 leukemic cell lines was assessed by annexin-VFLUOS staining after a 2-day culture in the presence of conditioned medium prepared from resting () and
IFN-stimulated (f) monocytes. Cell incubation was carried out in the absence or the presence of a combination of 100 ng/mL TRAIL-R1/Fc and TRAIL-R2/Fc chimeras. The
experiment depicted is representative of 3.
 From www.bloodjournal.org by guest on August 27, 2017. For personal use only.

3842 TECCHIO et al BLOOD, 15 MAY 2004 VOLUME 103, NUMBER 10

mAb 25E or TRAIL-R1/Fc and TRAIL-R2/Fc chimeras com-

pletely suppressed the proapoptotic effect of 20 ng/mL rhsTRAIL
of 82% 5%, 42% 3%, and 24% 5% in J32, MEG-01, and
KU-812 cells, respectively. By using specific TRAIL-R1 and
TRAIL-R2neutralizing antibodies, we could also demonstrate that
the proapoptotic activities of supernatants from IFN-treated
neutrophils and monocytes on MEG-01 cells are initiated through
the involvement of both receptors, TRAIL-R2 appearing to signal
more efficiently (Figure 6). The rhsTRAIL-induced apoptosis of
MEG-01 cells was also mediated by the engagement of both
TRAIL receptors, but, in this case, TRAIL-R1 appeared to play a
more important role than TRAIL-R2 (Figure 6). Interestingly, the
inhibitory action of TRAIL-R1 and TRAIL-R2 antibodies on the
proapoptotic effects exerted by either leukocyte-derived superna-
tants or rhsTRAIL was only partial (Figure 6) and did not augment
if the 2 reagents were used in combination instead of singularly
(data not shown).

Detection of increased sTRAIL levels in sera of patients

treated with IFN

To assess whether release of sTRAIL takes place in vivo, we

analyzed the levels of sTRAIL in sera of 6 stage IV metastatic
melanoma patients undergoing treatment with low-dose (3 MU)
IFN. Serum samples were collected prior to and 24 hours after
IFN administration. The amounts of sTRAIL present in these sera Figure 7. Detection of increased sTRAIL levels in sera of IFN-treated patients.
(A) Serum samples obtained from 6 healthy donors and 6 stage IV metastatic
before treatment with IFN (1460 203 pg/mL) were substan- melanoma patients were collected either prior to or 24 hours after IFN
tially comparable to those found in healthy donors (1239 153 administration and then analyzed for sTRAIL content by ELISA. The figure shows
pg/mL; Figure 7A). Remarkably, sTRAIL serum levels dramati- the mean values of averaged duplicate determinations. (B) PBMCs obtained from
cally increased in all patients after IFN treatment (3133 273 2 different patients (collected either prior to or 24 hours after IFN infusion) were
lysed as described in Patients, materials, and methods and the levels of
pg/mL; P .0006; Figure 7A), whereas those of FasL did not intracellular TRAIL determined by ELISA. The mean values of TRAIL levels
significantly change (39 11 vs 54 14 pg/mL; P .1). Analy- associated to PBMC pellets are shown.
sis of the intracellular levels of TRAIL demonstrated that the
accumulation of leukocyte-associated TRAIL dramatically aug-
mented following IFN exposure (Figure 7B), consistent with a TRAIL (sTRAIL) that fully retains proapoptotic activities toward
TRAIL de novo synthesis. Collectively, these data demonstrate that TRAIL-sensitive leukemic cell lines. Other agonists such as fMLP,
serum levels of sTRAIL are increased following systemic IFN LPS, G-CSF, and GM-CSF failed to up-regulate TRAIL gene
administration and that peripheral blood leukocytes greatly contrib- expression and soluble protein release. Since additional experi-
ute to this sTRAIL release in vivo. ments proved that IFN, at lower levels than IFN, also promotes
sTRAIL release by neutrophils (C.T., M.A.C., unpublished observa-
tions, December 2003), we could conclude that IFNs act as specific
Discussion stimuli for sTRAIL production by neutrophils. Membrane-bound
TRAIL was expressed on the surface of freshly isolated neutrophils
In this study, we demonstrate that upon incubation with therapeutic at very low levels, without, however, increasing in response to
concentrations of IFN, human neutrophils and monocytes are either IFN or IFN (C.T., M.A.C., unpublished observations,
induced to accumulate TRAIL mRNA and release a soluble form of December 2003). In contrast, surface TRAIL was induced by IFN

Figure 6. Effect of antiTRAIL-R1 and antiTRAIL-R2 neutralizing antibodies on MEG-01 apoptosis-induced by supernatants harvested from IFN-treated
leukocytes. MEG-01 cells were pretreated for 30 minutes with antiTRAIL-R1 or antiTRAIL-R2 neutralizing antibodies (5 g/mL for each one) before addition of
conditioned medium prepared from resting and IFN-stimulated monocytes and neutrophils or 20 ng/mL rhsTRAIL. Apoptosis rate of MEG-01 cells was then assessed
after a 2-day culture. Means SEM of the percentage of apoptotic inhibition exerted by antiTRAIL-R1 or antiTRAIL-R2 neutralizing antibodies calculated from 3 to 5
independent experiments are shown.
 From www.bloodjournal.org by guest on August 27, 2017. For personal use only.

BLOOD, 15 MAY 2004 VOLUME 103, NUMBER 10 SOLUBLE TRAIL RELEASE BY HUMAN PHAGOCYTES 3843

in monocytes, confirming the observations made by Griffith and suggested that, in Ph leukemias, the immunomodulatory
colleagues.11 These findings suggest that in neutrophils and mono- activity of IFN might be mediated by membrane-bound TRAIL
cytes, TRAIL surface expression and release are probably regulated expressed on activated T lymphocytes.41 In this paper, we show
by different mechanisms. It will be interesting to verify if that CML neutrophils and mononuclear cells, upon incubation
neutrophils are exclusively committed to release TRAIL, similarly with therapeutic dosages of IFN, release sTRAIL into the
to what was previously observed in the case of BlyS.25 extracellular environment as efficiently as normal leukocytes. In
Release of endogenous sTRAIL by activated neutrophils did contrast, sFasL was not expressed or released by normal or CML
not correlate with an increased apoptosis of the same cells, as neutrophils. Regretfully, we could not obtain sera of CML
IFN resulted in significantly prolong neutrophil survival. patients subjected to IFN administration due to the current use
These data apparently contradict those reported by Renshaw and of imatinib mesylate as treatment of choice in CML, but we had
colleagues,32 who have recently shown that a recombinant access to sera of melanoma patients undergoing IFN therapy.
soluble version of TRAIL strongly resembling the membrane- In these samples, we detected serum levels of sTRAIL that were
bound form (LZ-TRAIL) accelerates neutrophil apoptosis in the dramatically more elevated than those measured prior to cyto-
absence of additional proinflammatory stimuli. Indeed, even in kine injection or than those found in healthy donors. The sFasL
our hands, rhsTRAIL His-tag CC-mutant slightly augmented was instead only marginally increased in sera of IFN-treated
neutrophil apoptosis. It must be, however, specified that all patients. To our knowledge, this is the first evidence showing
commercial recombinant TRAIL preparations are specifically that IFN, in vivo, provokes the release of sTRAIL when
designed to exert potent proapoptotic activities and, in the administered systemically. The latter is further supported by
studies mentioned previously,32 such preparations have been very recent findings on increased sTRAIL levels in sera of
used at concentrations much higher than those released by patients affected by multiple sclerosis who have been given
activated neutrophils. Nonetheless, sTRAIL present as homotri- systemic IFN1 administration.42 Although we could not prove
meric or multimeric aggregates in supernatants conditioned by it for the reasons explained previously, it seems reasonable to
IFN-stimulated neutrophils or monocytes was biologically hypothesize that, in chronic-phase CML, which is a disease
functional, since it exerted remarkable cytotoxic activities clinically characterized by an high number of circulating
toward TRAIL-sensitive leukemic cell lines, including Jurkat neutrophils belonging to the CML clone, administration of IFN
(J32 clone) and MEG-01 cells. These proapoptotic effects were produces a massive release of sTRAIL into the serum. Accord-
mediated through TRAIL-R1 and TRAIL-R2, indicating that the ing to our data and to those published by Plasilova et al,43 who
soluble form of TRAIL released by IFN-activated leukocytes used a recombinant His-tag form of TRAIL, leukocyte-derived
can signal via both receptors. Even though the issue of a sTRAIL would preferentially induce apoptosis of CD34 Ph
possible differential activation of TRAIL-R1 and TRAIL-R2 by leukemic blasts rather than of leukemic neutrophils themselves.
natural or recombinant sTRAIL has been poorly addressed,36,37 This is further supported by the fact that CD34 cells isolated
our results are in agreement with those of Clarke et al.36 from CML patients, but not from healthy donors, express
Collectively, our data demonstrate that endogenous TRAIL can TRAIL-R1 and TRAIL-R2 death receptors (C.T., M.A.C.,
be biologically effective not only as an integral membrane unpublished observations, December 2003). Consistent with the
protein12-15 but also as a soluble released cytokine. To our absence of TRAIL-R1 and TRAIL-R2 on normal CD34 cells, it
knowledge, detection of TRAIL in a soluble form has been has been reported that, following exposure to His-TRAIL,
previously demonstrated only in supernatants derived from CD34 cells from healthy donors retain the capacity to repopu-
LPS-activated monocytes/macrophages,5 from reovirus-infected late human hematopoiesis when transplanted in sublethally
neoplastic cells, 36 and from phytohemagglutinin (PHA) irradiated severe combined immunodeficiency/nonobese dia-
activated and/or CD59-triggered Jurkat cells and normal T-cells betic (SCID/NOD) mice.43 Interestingly, preliminary experi-
blast.3,4,6 According to the latter reports, sTRAIL secretion by ments revealed that concentrations of imatinib mesylate up to
lymphocytes represents one of the mechanisms contributing to 500 nM do not interfere with the ability of IFN to trigger the
the activation-induced T-cell death (AICD) process by inducing release of sTRAIL by either normal or CML leukocytes (C.T.,
the apoptosis of the activated lymphocytes themselves.4,6 On the M.A.C., unpublished observations, January 2004), thus provid-
basis of our findings, and given the role of IFNs in innate ing a further rationale for the concurrent use of IFN and
immunity, it is tempting to speculate that recombinant as well as imatinib mesylate in CML.44 Our data also exclude sFasL as a
endogenous IFNs employ, other than membrane-bound TRAIL, potential molecule mediating the effects of IFN for the
sTRAIL derived from peripheral blood neutrophils and mono- following reasons: (1) it was not expressed and released by CML
nuclear cells as a potential cytotoxic mediator against trans- neutrophils, and (2) it was minimally increased in sera of IFN-
formed and virus-infected cells. Studies are ongoing in our treated patients. In line with this notion, Uno et al41 recently
laboratory to understand whether the cytotoxic potential of reported that recombinant TRAIL, but not FasL, effectively
sTRAIL could be enhanced by its interaction with extracellular induces apoptotic cell death in most of the CML and Ph acute
matrix, as recently demonstrated for sFasL.38 leukemia cell lines that they examined.
The observations made in this work have additional clinical In summary, our data indicate that IFN can exert its
implications. IFN has been proven to be an effective antiviral immunomodulatory activities in neoplastic and viral diseases by
and antineoplastic drug and its systemic administration is promoting the release of soluble TRAIL (other than by inducing
currently employed in the treatment of several malignancies the expression of the corresponding membrane-bound form) by
other than in viral diseases.39 For instance, CML is a clonal effector cells of the immune system. Our findings also support
myeloproliferative expansion of transformed hematopoietic the idea that, due to the lack of systemic toxic effects and to its
cells in which therapy with IFN was found to suppress the synergistic activity with other chemotherapeutic agents,23,44,45
leukemic clone.40 Interestingly, although the mechanisms rhsTRAIL may be an ideal candidate to substitute IFN in
whereby IFN functions have not been elucidated, it has been combination regimens.
 From www.bloodjournal.org by guest on August 27, 2017. For personal use only.

3844 TECCHIO et al BLOOD, 15 MAY 2004 VOLUME 103, NUMBER 10

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 From www.bloodjournal.org by guest on August 27, 2017. For personal use only.

2004 103: 3837-3844

doi:10.1182/blood-2003-08-2806 originally published online
January 15, 2004

IFN-stimulated neutrophils and monocytes release a soluble form of

TNF-related apoptosis-inducing ligand (TRAIL/Apo-2 ligand) displaying
apoptotic activity on leukemic cells
Cristina Tecchio, Veronica Huber, Patrizia Scapini, Federica Calzetti, Daniela Margotto, Giuseppe
Todeschini, Lorenzo Pilla, Giovanni Martinelli, Giovanni Pizzolo, Licia Rivoltini and Marco A.
Cassatella

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