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* Respiratory Medicine Unit, Section of Functional Genomics, Division of Genomic Medicine, University of Sheffield, Royal Hallamshire Hospital, Sheffield, United Kingdom; and
Respiratory Medicine Division, Department of Medicine, University of Cambridge School of Medicine, Addenbrooke’s and Papworth Hospitals, Cambridge, United Kingdom
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also has a transient proapoptotic effect.
We have studied the role of the death receptor ligand TRAIL in human
neutrophils. We identified the presence of mRNAs for TRAIL, TRAIL-R2,
and TRAIL-R3, and cell surface expression of TRAIL-R2 and -R3 in
neutrophil populations. Neutrophil apoptosis is specifically
accelerated by exposure to a leucine zipper-tagged form of TRAIL, which
mimics cell surface TRAIL. Using blocking Abs to TRAIL receptors,
specifically TRAIL-R2, and a TRAIL-R1:FcR fusion protein, we have
excluded a role for TRAIL in regulating constitutive neutrophil
apoptosis. No additional proapoptotic effect of leucine zipper TRAIL
was identified following TRAIL treatment of neutrophils in the presence
of gliotoxin, an inhibitor of NF-
B, suggesting TRAIL does not
activate NF-B in human neutrophils. TRAIL treatment of human
neutrophils did not induce a chemotactic response. The susceptibility
of neutrophils to TRAIL-mediated apoptosis suggests a role for TRAIL in
the regulation of inflammation and may provide a mechanism for
clearance of neutrophils from sites of
inflammation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, via binding to the type 1 TNFR
(TNF-R1),3 is also
able to accelerate neutrophil apoptosis at early time points
(10). However, the proapoptotic effects of death receptor
ligation can be suppressed in both peripheral blood (9)
and inflammatory neutrophils (11) by inflammatory
mediators present at sites of inflammation.
Death receptors, in addition to acceleration of apoptosis, can
transduce additional functions. TNF- has a biphasic effect upon
neutrophil apoptosis, with early induction followed by later (12 h
onward in vitro) inhibition of apoptosis (10). Ligation of
TNF-R1 by TNF- not only leads to formation of a death-inducing
signaling complex, that signals induction of apoptosis, but also
activates the transcription factor NF-B (12). In the
neutrophil, inhibition of NF-B signaling concomitant with the
addition of TNF- results in rapid induction of apoptosis, implying
the later inhibition of apoptosis by TNF- is NF-B-dependent
(13, 14). Fas, in contrast, is not generally thought to
activate NF-B. Soluble forms of Fas ligand (FasL) are able to induce
a pronounced chemotactic response in neutrophils (15, 16),
without detectable intracellular Ca2+
mobilization.
A further proapoptotic member of the TNF superfamily, TRAIL, has been implicated in the regulation of immune function (17). Two of the known receptors for TRAIL, TRAIL-R1 (DR-4), and TRAIL-R2 (DR-5), have been identified as death receptors (17). The remaining receptors, TRAIL-R3 (DcR1), TRAIL-R4 (DcR2), and a soluble receptor osteoprotegerin, have been termed decoy receptors for their ability to inhibit TRAIL-induced apoptosis when overexpressed (17).
We have investigated whether human neutrophils may respond to TRAIL via the presence of appropriate receptors on the neutrophil cell surface. We have examined the effects of both TRAIL and leucine zipper (LZ)-tagged TRAIL (a fusion of the extracellular portion of human (hu) TRAIL with a LZ self-association domain) upon neutrophil apoptosis and upon nonapoptotic functions described for other death receptor ligands.
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Materials and Methods |
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All chemicals were of analytical reagent grade and were
purchased from Sigma-Aldrich (Poole, U.K.) unless stated otherwise.
Percoll was obtained from Pharmacia Biotech (St. Albans, U.K.). Culture
media (HBSS, RPMI 1640), penicillin, and streptomycin were obtained
from Life Technologies (Paisley, U.K.). Recombinant human (rh)
TRAIL (His-tagged,
Thr95-Gly281) was obtained
from R&D Systems (Oxford, U.K.) and recombinant human untagged TRAIL
was obtained from Upstate Biotechnology (Lake Placid, NY).
huLZ-TRAIL (18) and mAbs against TRAIL-R1 (IgG2a
huTRAIL-M271), TRAIL-R2 (IgG1 huTRAIL-M413), TRAIL-R3 (IgG1
huTRAIL-M430), TRAIL-R4 (IgG1 huTRAIL-M444), and TRAIL (IgG1
huTRAIL-M181) have been described previously
(19, 20, 21) and were the kind gifts of Amgen (Seattle,
WA). Isotype control Abs (IgG1 and IgG2a) were obtained from R&D
Systems and the Alexa 488-labeled antimouse secondary Ab was obtained
from Molecular Probes (Eugene, OR). rhGM-CSF was obtained from Roche
Molecular Biochemicals (Lewes, U.K.) and rhTNF- was obtained from
ImmunoKontact (Frankfurt, Germany). TRAIL-R1:Fc, Fas:Fc, and TNFR1:Fc
were kind gifts from Dr. S. Farrow (Glaxo Smith Kline, Stevenage,
U.K.).
Cell preparation and culture
Human peripheral blood neutrophils were isolated from venous blood of healthy volunteers by dextran sedimentation and centrifugation through a discontinuous plasma-Percoll gradient as previously described (2, 22). Inflammatory neutrophils were purified from the joint aspirates of patients with rheumatoid arthritis, using a very similar protocol as previously described (11). Purity was assessed by counting >500 cells on duplicate cytospin preparations, and was always >95%. The contaminating cells were almost exclusively eosinophils. Neutrophils were incubated at 37°C in RPMI containing 1% penicillin and streptomycin and supplemented with 10% FCS, in 96-well nontissue culture-treated Falcon "Flexiwell" plates (BD Biosciences, Oxford, U.K.). The studies were approved by the South Sheffield Research Ethics Committee and all patients gave fully informed, written consent.
Assessment of apoptosis
Apoptosis was quantified by morphology on Giemsa-stained cytospins, by blind counting of >300 cells per slide on duplicate cytospins. This method has been shown to correlate closely with other measurements of neutrophil apoptosis, including annexin V binding (23) and shedding of CD16 (24). In addition, necrosis was assessed at all time points by exclusion of the vital dye trypan blue and was <2% in all cases.
RNase protection assay (RPA)
Neutrophil RNA was isolated using RNeasy columns (Qiagen, Valencia, CA), using 5 µg of RNA in each RPA sample. RPAs were conducted using the RiboQuant protocol following the manufacturer’s instructions. The probes for RPA were the RiboQuant APO-3c probe set and a custom probe set including probes for TRAIL-R1, -R2, and -R4 (BD PharMingen, San Diego, CA).
Flow cytometry
Analysis was conducted using a BD Biosciences FACSCalibur flow cytometer. Cells were gently resuspended and removed from culture, then washed in FACS buffer (PBS, 2.5% FCS, and 0.1% sodium azide). Appropriate saturating concentrations of Ab were added to cells in wash buffer and incubated at room temperature for 15 min. Cells were washed and incubated with a 1/40 dilution of Alexa 488 antimouse secondary in wash buffer and incubated for 15 min at room temperature, after which they were washed twice and resuspended in 200 µl of PBS. A minimum of 104 cells were analyzed per sample.
Chemotaxis assay
Cell migration was assessed using a modified Boyden chamber (Receptor Technologies, Adderbury, U.K.) (25). The upper and lower wells were separated by a nitrocellulose filter containing 5-µm diameter pores at a density of 4000 per cm2 (NeuroProbe, Gaithersburg, MD). Neutrophils were suspended at 3 x 106 cells/ml in PBS (with divalent cations) and 225 µl were placed into the upper wells, with 30 µl of test substance or vehicle control in the lower wells. C5a (maximally effective concentration, 100 nM) and FasL (0.1 nM) were included as positive controls together with PBS with 1% BSA (control vehicle for TRAIL) as a negative control. The chamber was incubated at 37°C in a 5% CO2 atmosphere for 90 min. Cells passing into the lower wells were counted using a hemocytometer. Each experiment was performed in triplicate at least three times using independent donors.
Statistical analysis
The results are expressed as mean ± SE of the number (n) of independent experiments, each using cells from separate donors and with each experiment performed in duplicate. Statistical analysis was performed by use of the paired Student’s t test and results were considered to be statistically significant where p < 0.05.
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Results |
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We first examined the effect of incubation with TRAIL upon human neutrophil apoptosis. rhHis-tagged monomeric TRAIL (100 ng/ml), at time points up to 22 h, did not influence rates of constitutive neutrophil apoptosis (Fig. 1a). The same was true for concentrations of TRAIL from 1 ng/ml to 1 mg/ml, and for a nontagged form of TRAIL (data not shown). However, signaling by related death receptor ligands is more efficient if they retain the stable cross-linked structure of the membrane-bound ligand. For example, the cytotoxicity of FasL can be increased >1000-fold by Ab cross-linking, to levels comparable to the cytotoxic potency of cell membrane-bound FasL (26, 27). A recent study has shown similar potentiation of the cytotoxic effects of TRAIL on tumor cell lines by cross-linking (28). LZ-TRAIL have been engineered which self-associate spontaneously into stable structures with high biological activity (18). Therefore, we tested the response of neutrophils to LZ-TRAIL. The addition of LZ-TRAIL to human neutrophils significantly accelerated apoptosis at 6 h (Fig. 1a) and this effect was concentration-dependent (Fig. 1b). Recent reports have suggested that the proapoptotic effects of cross-linked forms of TRAIL may not be specific (29, 30). To confirm that the effect seen in this study was mediated by TRAIL receptor ligation, blocking Abs to the TRAIL death receptors, TRAIL-R1 and TRAIL-R2, were added at concentrations known to block interaction with TRAIL (19). The presence of blocking Ab to TRAIL-R2 completely abrogated the response to LZ-TRAIL, whereas Ab to TRAIL-R1 was without effect (Fig. 2). Use of isotype-matched control Abs did not alter rates of neutrophil apoptosis (data not shown). TRAIL-mediated apoptosis of Jurkat cells, which are known to express TRAIL-R2 (31, 32), was similarly inhibited by Ab to TRAIL-R2 at a concentration (5 µg/ml) previously shown to prevent TRAIL binding (19) (data not shown). These data confirm acceleration of neutrophil apoptosis following TRAIL ligation occurs via TRAIL-R2-dependent mechanisms.
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Expression of TRAIL and its receptors at RNA and protein levels was examined in human neutrophils. Multiprobe RPA analysis revealed protection of mRNA species corresponding to TRAIL-R2 and TRAIL-R3 and also TRAIL in mRNA samples prepared from freshly isolated human peripheral blood neutrophils (Fig. 3a). mRNAs for TRAIL-R1 and TRAIL-R4 (custom probe set; BD Biosciences) were not detected in an additional three independent experiments using mRNA from different donors (examples shown in Fig. 3). However, we were able to detect mRNA fragments of appropriate size for TRAIL-R1 and TRAIL-R4 in other cell types, e.g., human umbilical vein endothelial cells (S. J. Rowe, unpublished observations). To extend the RPA findings, cell surface expression of TRAIL and TRAIL receptors was examined by flow cytometry. There was increased cell surface binding of Abs to TRAIL-R2 (Fig. 4c) and TRAIL-R3 (Fig. 4d) compared with isotype control Abs, consistent with low level expression of TRAIL-R2 and higher level expression of TRAIL-R3. Freshly isolated human neutrophils did not show specific binding of Abs to TRAIL (Fig. 4a), TRAIL-R1 (Fig. 4b), or TRAIL-R4 (Fig. 4e), consistent with lack of expression of these proteins on the neutrophil surface. Detection of cell surface protein for TRAIL, TRAIL-R1 and TRAIL-R4 has been confirmed in other cell types, e.g., HUVECs (Ref. 20 and S. J. Rowe, unpublished observations). Neutrophils thus express a TRAIL death receptor, TRAIL-R2, and a presumed decoy receptor, TRAIL-R3, at both mRNA and protein levels, with no evidence of expression of TRAIL-R1 or TRAIL-R4. TRAIL itself was detected at the RNA level but not as a cell surface protein. TRAIL and TRAIL receptor expression was also studied in neutrophils purified from joint aspirates of patients with rheumatoid arthritis, as previously described (11). No differences were observed in expression either of TRAIL or its receptors compared with peripheral blood neutrophils (data not shown).
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Although we were unable to detect expression of TRAIL protein,
TRAIL was expressed at the mRNA level. The lower limit of detection of
this molecule by flow cytometry has not been determined, so low level
or transient expression of cell surface TRAIL could not be excluded.
Because biologically active human TRAIL has been demonstrated to
accelerate neutrophil apoptosis and because constitutive neutrophil
apoptosis appears to use the same caspase-dependent cell death pathways
as TRAIL-induced apoptosis (33, 34), it was conceivable
that low level expression of TRAIL at the cell surface might influence
constitutive neutrophil apoptosis. We confirmed this was not the case
by two different strategies. First, neutrophils were incubated with
blocking concentrations of Ab to TRAIL-R2, the only signaling TRAIL
receptor present on human neutrophils. This Ab has been shown to
abolish binding of TRAIL to TRAIL-R2 at concentrations above 3.3
µg/ml (19), and to block TRAIL-induced neutrophil
apoptosis (Fig. 2), but was without effect upon constitutive neutrophil
apoptosis (Fig. 5a). Secondly,
we used a soluble TRAIL-R1:Fc fusion protein to disrupt interactions
between TRAIL and its receptors, using verified reagents and
concentrations (35). Incubation of neutrophils with
Fc-fusion proteins to TRAIL-R1, Fas, or TNF-R1 did not modulate rates
of constitutive apoptosis (Fig. 5b). Because neutrophils in
our culture system neither express FasL (11, 36) nor
secrete TNF- (10), the Fas:Fc and TNFR1:Fc fusion
proteins are included as negative controls. Higher concentrations of
fusion proteins had no effect on rates of constitutive neutrophil
apoptosis (data not shown). The lack of effect of either TRAIL-R1:Fc
fusion protein or receptor blockade argues against a role for TRAIL
signaling in the regulation of constitutive neutrophil apoptosis.
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Acceleration of neutrophil apoptosis by Fas ligation can be inhibited by inflammatory mediators, such as GM-CSF, that are present at sites of inflammation (9, 11). To test whether TRAIL-induced apoptosis could be similarly modulated, neutrophils were cultured with LZ-TRAIL in the presence or absence of GM-CSF. Suppression of apoptosis by GM-CSF was observed both in untreated cells and in those treated with LZ-TRAIL (Fig. 6).
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treatment of human neutrophils reveals a biphasic effect upon
neutrophil lifespan, with acceleration of apoptosis at early time
points and delay at later time points (10). The later
delay of apoptosis is largely due to activation of the NF-B complex,
leading to transcription of a cassette of antiapoptotic factors
(38). When neutrophils are treated with TNF- in the
presence of gliotoxin there is a dramatic acceleration of apoptosis,
ascribed to the NF-B-blocking actions of gliotoxin
(14). To assess the potential for TRAIL to induce
accelerated apoptosis under similar conditions, neutrophils were
incubated in the presence or absence of LZ-TRAIL at 100 ng/ml, with or
without gliotoxin at 100 ng/ml (Fig. 8).
There was no additional effect on apoptosis when TRAIL receptors were
cross-linked in the presence of gliotoxin, other than the additive
cytotoxic effects of LZ-TRAIL and gliotoxin alone. This is in contrast
to the action of TNF- which, in combination with gliotoxin, caused
almost 100% apoptosis at this time point.
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Discussion |
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Appropriately controlled flow cytometric studies showed that TRAIL
itself was absent from the neutrophil cell surface, although it appears
to be abundantly expressed at the mRNA level. It is possible that TRAIL
is expressed and then cleaved from the cell surface by the action of
tissue proteases, as described for other death receptor ligands
(40, 41). This is likely in view of its structure, and a
protease activity has been described that may act to cleave TRAIL from
the cell surface (42). Experiments using TRAIL-R2 blocking
Ab or a TRAIL-R1:Fc fusion protein, however, excluded an autocrine or
paracrine effect of TRAIL produced in culture upon constitutive
neutrophil apoptosis. Nonetheless, neutrophils have the potential to
express TRAIL protein, particularly because significant levels of the
mRNA are expressed and neutrophils typically contain a relatively
limited repertoire of mRNAs (43). We attempted
to up-regulate surface TRAIL by treatment with IFN-
, which has been
shown to increase TRAIL expression on other primary myeloid cells
(monocytes) (21), but neither IFN-, LPS, nor TNF-
up-regulated TRAIL expression (data not shown). The most striking
feature of TRAIL receptor expression on neutrophils was the high levels
of anti-TRAIL-R3 Ab binding, consistent with high-level expression
of TRAIL-R3, a GPI-linked extracellular protein with no functional
signaling domains yet identified (44, 45). That
neutrophils remain sensitive to TRAIL despite these levels of TRAIL-R3
expression suggests that TRAIL-R3 is an inefficient decoy in this
context. This may be explained by the observation that, at
physiological temperatures, TRAIL-R2 binds TRAIL with higher affinity
than other TRAIL receptors (46). Attempts to address this
specifically in neutrophils have been hampered by the lack of a
specific blocking Ab to TRAIL-R3. Phosphatidylinositol phospholipase C
has been shown to cleave TRAIL-R3 from the cell surface, markedly
enhancing sensitivity to TRAIL (47). In our hands,
phosphatidylinositol phospholipase-C treatment of neutrophils did not
alter TRAIL sensitivity, despite 50% reductions in anti-TRAIL-R3
Ab binding (data not shown). It may be that TRAIL-R3 has other roles,
for example cooperating with another death receptor to transduce a
proapoptotic signal. A precedent exists in neutrophils for this type of
signaling: type 2 TNFR, which does not itself contain a death domain,
can cooperate with TNF-R1 to transmit its proapoptotic signal
(10).
His-tagged, monomeric TRAIL was without effect upon neutrophil
apoptosis, in agreement with the data of Daigle and Simon
(39). Recombinant sFasL is similarly without a
proapoptotic effect when added to human neutrophils (16),
whereas TNF- can exert its proapoptotic action without modification
(10). Acceleration of human neutrophil apoptosis was seen
only with LZ-TRAIL, which is thought to closely mimic membrane-bound
TRAIL, and cause association and activation of TRAIL receptors. This
effect was specific, because it was inhibited by an Ab shown to block
binding of TRAIL to TRAIL-R2 but not by an Ab to TRAIL-R1. Other groups
have shown nonspecific effects with some preparations of TRAIL
(29, 30), but we believe we have excluded such effects in
these studies.
Both Fas and TNF-R1 are known to transduce signals that regulate
cellular functions other than survival. We sought a specific
chemotactic activity of soluble TRAIL, analogous to that seen for FasL
(15, 16). The data excluded a chemotactic or chemokinetic
effect of TRAIL upon human neutrophils, a finding that was fully
supported by the inability of TRAIL to induce neutrophil shape change
(polarization) under nongradient conditions. This latter assay is a
particularly sensitive index of neutrophil priming and activation and
is invariably positive with all known chemotactic agents
(37). The possibility that TRAIL could activate NF-B
was also considered. The addition of gliotoxin to TRAIL-treated
neutrophils did not lead to rapid induction of apoptosis, as is the
case with TNF-. These data, combined with evidence that TRAIL does
not activate NF-B in TRAIL-sensitive cell types (48, 49), makes TRAIL-mediated NF-B activation in neutrophils
unlikely. Therefore, signaling via TRAIL receptors results in neither
chemotaxis nor NF-B activation. However, the numerous receptors for
TRAIL, and the lack of a clearly defined function for all the receptors
identified, suggest that the full extent of the cellular effects of
TRAIL in human neutrophils have yet to be fully elucidated.
Our studies demonstrate acceleration of neutrophil apoptosis following
exposure to LZ-TRAIL in vitro and suggest that neutrophils may be
susceptible to membrane bound TRAIL in vivo. TRAIL is expressed on a
number of cells involved in regulation of immune function, including
certain subsets of T cells (42), particularly NK cells
(50, 51), and also macrophages (21). TRAIL
surface expression can be increased by treatment with IFNs (21, 52, 53). Stimulated T cells have been shown to release
microvesicles containing functional membrane-bound forms of both FasL
and TRAIL (54). Therefore, inflammatory neutrophils are
likely to be exposed to membrane bound TRAIL, and this may limit their
lifespan in vivo. Neutrophil lifespan may also be influenced by
exposure to TNF- or FasL during inflammation, with these different
death receptor ligands modulating cell survival in different contexts,
and in response to different stimuli. We have also shown that TRAIL,
unlike sFasL, does not induce a chemotactic response in neutrophils.
Therefore, TRAIL-based strategies may permit acceleration of neutrophil
apoptosis without inducing further neutrophil recruitment. The
restricted tissue sensitivities to TRAIL may also favor its use in the
treatment of inflammation. In the lung, for example, Fas ligation may
be harmful as a result of unwanted effects upon resident cell
populations (55, 56). In summary, these studies identify
possible roles for TRAIL both in regulation of neutrophilic
inflammation in vivo and in strategies for treatment of inflammatory
disease.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Moira K. B. Whyte, Respiratory Medicine Unit, Division of Genomic Medicine, University of Sheffield, Royal Hallamshire Hospital, Sheffield S10 2JF, U.K. E-mail address: m.k.whyte{at}sheffield.ac.uk
3 Abbreviations used in this paper: TNF-R1, type 1 TNFR; FasL, Fas ligand; LZ, leucine zipper; hu, human; rh, recombinant human; RPA, RNase protection assay; sFasL, soluble FasL.
Received for publication June 6, 2002. Accepted for publication November 6, 2002.
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S. A. Renshaw, C. E. Dempsey, F. A. Barnes, S. M. Bagstaff, S. K. Dower, C. D. Bingle, and M. K. B. Whyte Three Novel Bid Proteins Generated by Alternative Splicing of the Human Bid Gene J. Biol. Chem., January 23, 2004; 279(4): 2846 - 2855. [Abstract] [Full Text] [PDF]
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) and TRAIL-treated cells (
) for at least three independent
experiments, each performed in duplicate. *, Statistically
significant difference between control and LZ-TRAIL-treated populations
after 6 h in culture (p = 0.003).
b, Freshly isolated neutrophils were cultured with
LZ-tagged TRAIL at the concentrations shown, and sampled after 6 h
in culture. Mean ± SEM of the increase in percent-apoptotic cells
in excess of control cells is shown for five independent experiments,
each performed in duplicate.
), performed in duplicate. For all treatments,
p > 0.2. In separate experiments, appropriate
isotype control Abs were also without effect upon apoptosis (data not
shown). b, Incubation of neutrophils with Fc fusion
proteins to Fas, TNF-R1, and TRAIL-R1 does not modulate constitutive
neutrophil apoptosis. Neutrophils were incubated with each fusion
protein at concentrations of 10, 100, and 1000 ng/ml. The data shown
represent mean ± SEM percent-apoptosis of four independent
experiments performed in duplicate. The results shown are at
t = 22, but similar results were seen at
t = 6 (not shown). For all treatments,
p > 0.2.
