Published online before print December 6, 2007
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,
,
* Departments of Urology and
Medicine and
Inflammation Program, University of Iowa, and
VA Medical Center, Iowa City, Iowa, USA
1Correspondence: Department of Urology, 3204 MERF, University of Iowa, 375 Newton Road, Iowa City, IA 52242-1089, USA. E-mail: thomas-griffith{at}uiowa.edu
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Key Words: granulopoiesis • BCG • myelopoiesis • PMN
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Neutrophils originate in the bone marrow as progenitor stem cells that develop into myeloblasts, the first stage committed to neutrophil maturation [1 , 5 ]. From this point, these primitive neutrophil precursors progress into the promyelocyte stage marked by azurophilic granule (AG) formation [6 ]. Next are the myelocyte and metamyelocyte stages, when the specific granules (SG) and gelatinase granules (GG) are formed, respectively. The metamyelocyte stage also marks the last point of mitotic activity [5 , 6 ]. The maturing myelocytes progress to the band cell stage and are then released into the circulation, where development is completed with the formation of secretory vesicles (SV), resulting in mature, terminally differentiated neutrophils.
The effector functions of neutrophils are mediated by stores of preformed proteins and enzymes synthesized during granulocytic maturation in the bone marrow. Many of these effector molecules are stored in the granules, providing a readily available source of these essential proteins through recruitment and/or exocytosis of granules, allowing neutrophils to rapidly respond to environmental signals. Neutrophils possess three types of granules: AG, SG, and GG [7 ]. In addition, neutrophils contain SV that can be mobilized in response to a variety of agonists. Each of these granule subtypes is formed during distinct periods of neutrophil development, and granule proteins are packaged according to the timing of their synthesis (reviewed in Faurschou and Borregaard [7 ] and Borregaard and Cowland [8 ]). For example, myeloperoxidase (MPO) is synthesized during the promyelocyte stage of development and is found only in AG. In contrast, lactoferrin (LF) synthesis occurs during the myelocyte stage and with subsequent packaging in SG. In many cases, this regulated synthesis and packaging of proteins allow molecules with similar effector functions to be stored in the same granules. Thus, mobilization of one type of granule subtype provides a source of proteins essential for a particular function to be released at the same time. SV contain surface receptors and adhesion molecules, providing neutrophils the ability to respond to signals and begin migration by adherence to tissues. GG are involved in degrading the extracellular matrix and allowing effective chemotaxis. SG and AG contain a variety of microbicidal proteins and enzymes essential in the killing and destruction of phagocytosed microorganisms. Interestingly, the granules are released in reverse order to the timing of their synthesis, with SV mobilized first, followed by GG, SG, and finally, AG [7 , 8 ].
TRAIL is a member of the TNF superfamily of cytokines that is cytotoxic against tumor cells and little to no effects on normal cells, giving it the potential to be used as a broad spectrum, anti-tumor molecule [9 10 11 12 ]. Our laboratory has shown that TRAIL is expressed on neutrophils present in the voided urine of bladder cancer patients undergoing immunotherapy with Mycobacterium bovis bacillus Calmette-Guérin (BCG) [13 ]. Further studies demonstrated that neutrophils contain a preformed, intracellular pool of TRAIL that is released from neutrophils stimulated in vitro with BCG or components of the mycobacterial cell wall [14 , 15 ]. These findings suggest that TRAIL may be localized in neutrophil granules allowing rapid release upon stimulation with BCG. In support of this, preliminary studies from our laboratory and others have found TRAIL in neutrophil granules, but the precise localization differed in these reports [14 , 16 ]. Therefore, the goal of this study was to further characterize the distribution of TRAIL in neutrophils. Our findings describe a distribution of TRAIL that is unique among characterized granule proteins. Furthermore, we demonstrate that TRAIL is actively produced in each stage of neutrophil development, consistent with its broad distribution among granule subtypes.
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PLB-985 cells
PLB-985 cells are a promyelocytic leukemic cell line with the capacity to progress into later stages of neutrophil development [18
]. PLB cells were cultured in RPMI-1640 medium with 10% FCS and incubated at 37°C in 5% CO2, passaging every 3–5 days depending on cell density. For experiments in which cells were induced to differentiate, culture medium was supplemented with 0.5% dimethylformamide (DMF; Sigma Chemical Co., St. Louis, MO, USA), and aliquots of cells were collected each day over a 4-day period. Harvested cells were placed in lysis buffer [PBS+1% Nonidet P-40 (NP-40) and Complete Mini protease inhibitors (Roche, Mannheim, Germany)] for protein analysis or TRIzol reagent (Invitrogen, Carlsbad, CA, USA) for RNA isolation. Untreated, Day 0 cells were used as controls and for normalization of RNA levels.
CD34-positive stem cells
Stem cells were obtained from the DeGowan Blood Center at the University of Iowa Hospitals and Clinics. Blood was drawn from healthy donors treated with G-CSF to promote the transient mobilization of stem cells into the peripheral blood. Cells were isolated by leukapheresis followed by MACS purification for CD34-positive cells using the human CD34 microbead kit (Miltenyi Biotech, Auburn, CA, USA), according to the manufacturer’s protocol. Purified stem cells were stored in liquid nitrogen. CD34-positive stem cells were thawed and cultured in StemSpan medium supplemented with the stem cell expansion cocktail [fetal liver tyrosine kinase 3 ligand, stem cell factor, thrombopoietin (Stem Cell Technologies, Vancouver, BC, Canada)] to allow for cell expansion. After 14 days, cells were separated by positive selection for CD34 using MACS. The purified, CD34-positive cells isolated after the 14-day expansion were true progenitors, as all lineage-committed cells eventually lose CD34 expression during maturation, whereas the true progenitor stem cells expand by mitosis and retain CD34 expression and pluripotent capacity. The pure progenitor stem cells were cultured in StemSpan supplemented with recombinant human G-CSF (50 ng/ml, Peprotech, Rocky Hill, NJ, USA) for 14 days with samples taken every 3 days for microscopic and RNA analysis. Harvested cells were placed in lysis buffer PBS + 1% NP-40 and Complete Mini protease inhibitors (Roche) for protein analysis or TRIzol reagent (Invitrogen) for RNA isolation. Purified, CD34-positive progenitors that had not been treated with G-CSF were used for normalization of RNA levels.
Subcellular fractionation of neutrophils
Neutrophil granule fractions were obtained using subcellular fractionation techniques as described in previous studies [14
]. Briefly, neutrophils were cavitated in a nitrogen bomb after treatment with the serine protease inhibitor diisopropylfluorophosphate (1 mM). Unbroken cells and nuclei were pelleted by centrifugation at 200 g. Postnuclear supernatants were placed on top of two-layer Percoll gradients made as described previously [19
] and centrifuged at 48,400 g for 15 min at 4°C to separate the subcellular fractions. Isolated granule fractions were carefully removed from the gradient, and Percoll was removed from each fraction by centrifugation at 110,000 g for 1 h at 4°C. Fractions were then washed and resuspended in relaxation buffer (10 mM PIPES, 100 mM KCl, 3 mM NaCl, 3.5 mM MgCl2) with added ATP [1 mM ATP(Na)2] and PMSF (1 mM) and kept on ice.
Free-flow electrophoresis
Free-flow electrophoresis was performed as described [20
] to separate SV from plasma membrane (PM) using the 
-fraction isolated by two-layer Percoll density centrifugation as starting material. Briefly, the fraction was treated with neuraminidase (10 units/ml) to reduce the surface charge on PM vesicles and then resuspended in 1 ml buffer (6 mM triethanolamine, pH 7.4, sucrose 270 mM, with conductivity adjusted to 420 µs/cm). Free-flow electrophoresis was performed on an Elphor VaP 22 (Bender and Hobein, Munich, Germany) with electrode buffer (50 mM triethanolamine and 50 mM acetic acid, pH 7.4) at 5°C using a flow rate of 3.12 ml/h/fraction and current of 120 mA, giving a voltage of 1120 V. Latent alkaline phosphatase activity assay was used to discriminate fractions containing SV from PM. Fractions were pooled, taking only the centermost fractions with peak activity to avoid cross-contamination as described previously [21
], sacrificing total yield for purity. Because of this, the amount of protein from the sum of pooled SV and PM fractions does not equal the original, unseparated SV/PM.
Immunoprecipitation, protein electrophoresis, and immunoblotting
TRAIL was immunoprecipitated from whole cell lysates or isolated granule fractions as described previously [13
]. Immunoprecipitation was necessary to obtain enough TRAIL for immunoblot analysis, as loading pure fractions on SDS-PAGE resulted in inadequate amounts of protein for analysis. Briefly, anti-TRAIL mAb M181 (10 µg/ml, Amgen, Seattle, WA, USA) was added to samples for 2 h at 4°C, followed by the addition of protein G-Sepharose beads (Sigma Chemical Co.), and incubated overnight at 4°C. The beads were pelleted by centrifugation, washed with PBS/0.1% Triton X-100, and resuspended in nonreducing SDS-PAGE sample buffer. Samples were boiled for 5 min to release precipitates from the protein G beads, separated by SDS-PAGE, and transferred to nitrocellulose membranes, which were blocked overnight at 4°C in 5% nonfat dry milk, reconstituted in PBS-Tween-20 (0.05% v/v). The membrane was then incubated with an anti-TRAIL polyclonal antiserum (0.1 µg/ml, Peprotech). After washes, membranes were treated with a HRP-conjugated goat anti-rabbit mAb (Jackson ImmunoResearch, West Grove, PA, USA; 1:5000 dilution) and developed by chemiluminescence (SuperSignal West Pico chemiluminescence substrate, Pierce, Rockford, IL, USA).
Immunofluorescence confocal microscopy
Immunofluorescence microscopy was performed using a combination of methods described previously [22
, 23
]. Glass coverslips coated with bovine tendon collagen were added to 24-well tissue-culture plates and incubated in autologous normal human serum for 30 min at 37°C prior to adherence of neutrophils. Approximately 106 neutrophils were added to each well of the 24-well tissue-culture plate centrifuged at 350 g and allowed to adhere for 30 min at 37°C. Monolayers were washed with 0.9% NaCl to remove nonadherent neutrophils. Coverslips containing cell monolayers were processed as described [23
] with fixation in 10% formalin for 15 min, followed by permeabilization with acetone at –20°C for 5 min. Monolayers were rehydrated with PBS and stored overnight in blocking buffer with PBS supplemented with 5 mg/ml BSA, 0.5 mg/ml NaN3. Cells were stained using combinations of antibodies for TRAIL (biotinylated mouse anti-human mAb, RIK-2, a gift from Dr. Hideo Yagita, Juntendo University, Tokyo, Japan), MPO (mouse anti-human mAb, FITC conjugate, Fitzgerald Industries International, Concord, MA, USA), LF (rabbit anti-human LF antiserum, Fitzgerald Industries International), and albumin (mouse anti-human mAb, Sigma Chemical Co.). Secondary antibodies included anti-rabbit or anti-mouse Alexa-647 (Molecular Probes/Invitrogen) and streptavidin-Texas Red conjugates (Caltag/Invitrogen). Coverslips were mounted onto glass slides and viewed using a Zeiss 510 confocal microscope. Images were taken as optical sections from apical to basolateral cell surfaces at 
0.5 µm intervals. Zeiss LSM software was used to compile optical sections into a single image.
Transmission electron microscopy (TEM)
Freshly isolated neutrophils were fixed with a solution of 2% paraformaldyde and 0.05% glutaraldehyde in PBS, dehydrated with increasing concentrations of ethanol, and embedded with LR-white resin. Thin sections were cut using a Reichert Austria Ultracut E ultramicrotome (Vienna, Austria) and placed onto formvar grids. Sections were blocked with normal goat serum and immunolabeled with different combinations of antibodies for TRAIL (biotinylated mouse anti-human mAb, RIK-2), MPO (mouse anti-human mAb), LF (rabbit anti-human LF antiserum), and albumin (mouse anti-human mAb, Sigma Chemical Co.). Secondary antibodies included anti-mouse ultra-small gold conjugate, anti-rabbit ultra-small gold conjugate, and streptavidin 10 nm gold conjugate (Electron Microscopy Sciences, Hatfield, PA, USA). Sections that were immunolabeled for TRAIL and MPO were stained for MPO with primary and secondary antibodies prior to labeling with biotinylated anti-TRAIL and streptavidin gold to avoid cross-reactivity. Gold labeling was enhanced with silver using the Aurion R-Gent silver enhancement reagent for electron microscopy (SE-EM) kit, according to the manufacturer’s protocol (Electron Microscopy Sciences). Grids were examined using a Hitachi H-7000 TEM.
Quantitative RT-PCR
RNA was isolated from cells using Trizol reagent (Invitrogen) and quantified using a spectrophotometer at 260 nm. Samples (1 µg each) were reverse-transcribed with Superscript II (Roche) using random hexamers. Resulting cDNA products were examined by quantitative PCR using Taqman (TRAIL, MPO, p47phox) or Sybr green (LF). Taqman primers for TRAIL, MPO, and p47phox were purchased from Applied Biosystems (Foster City, CA, USA). Primers for human LF were designed and purchased from IDT (Coralville, IA, USA): 5'-AGGCACAGGAAAAGTTTGGAAAGG-3', 5'-AGTTCTGGATGGCAGTGAAGTAGC-3'. Quantitative PCR with individual primer sets was performed using IQ Supermix (Taqman) or IQ SYBR Green Supermix (Sybr Green, BioRad, Hercules, CA, USA), and reactions were performed using a MJ PTC-200 Chromo4 thermocycler (BioRad). RNA levels in each sample were normalized to controls, with data expressed as the mean of relative mRNA levels with accompanying SEM values calculated from at least three independent experiments.
Statistical analysis
Analysis of data was performed using SigmaStat 3.5 and SigmaPlot 10 (Systat Software, San Jose CA, USA). Mean values were compared by one-way ANOVA and Dunnett or Bonferroni post-tests with statistical significance defined as P < 0.05.
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Figure 1. Presence of TRAIL in all subcellular fractions from naïve neutrophils. (A) Membrane-bound intracellular vesicles and granules were obtained from freshly isolated neutrophils after subcellular fractionation and free-flow electrophoresis as described in Materials and Methods. Fractions were analyzed by ELISA for TRAIL. (B) TRAIL was immunoprecipitated from fractions obtained after subcellular fractionation and free-flow electrophoresis and analyzed by immunoblot under nonreducing conditions to visualize TRAIL aggregates. Full-length TRAIL (FL TRAIL) and soluble TRAIL (S TRAIL) were used as standards, migrating as monomers, dimers, and trimers. Data in each panel are representative of more than five experiments with different donors, and each panel is from a different experiment and donor.
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To assess the distribution of TRAIL in neutrophils using a different analytical approach, we used immunofluorescence confocal microscopy to visualize TRAIL localization in intact neutrophils (Fig. 2A ). TRAIL had a punctuate distribution in intact neutrophils, consistent with its localization in small granules, supporting the results of our subcellular fractionation experiments. It is important to note that there was neither diffuse (cytoplasmic) nor peripheral (PM) localization of TRAIL, suggesting that TRAIL is found only in the granules and not in the cytoplasm or PM in intact neutrophils. Next, we determined the localization of TRAIL relative to defined granule markers. TRAIL colocalized independently with MPO and LF, respective markers of AG and SG (Fig. 2B and 2C) . As a control, we stained neutrophils for MPO and LF (Fig. 2D) . MPO and LF did not colocalize in our experiments as expected, as the two proteins are segregated into different granule populations [6 ]. In cells that were stained for all three proteins (TRAIL/MPO/LF; Fig. 2E ), TRAIL colocalized with MPO, with LF, or with neither marker, consistent with location in AG, SG, or SV, respectively. To directly assess SV localization, we stained neutrophils for TRAIL and albumin, a marker of SV [27 ], demonstrating that TRAIL and albumin did colocalize (Fig. 2F) .
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Figure 2. TRAIL colocalization with multiple markers of neutrophil granules and vesicles. Isolated neutrophils were stained using antibodies for TRAIL (A), TRAIL and MPO (B), TRAIL and LF (C), MPO and LF (D), TRAIL, MPO, and LF (E), or TRAIL and albumin (F). Images were acquired at x1000 original maganification and zoomed x2 original magnification to visualize granules. TRAIL appears as red fluorescence in A–F, MPO as green and LF as blue in B–E, and albumin as green in F. Colocalization of red–green results in yellow, red–blue in pink, green–blue in aqua/teal, and red–green–blue in white.
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Figure 3. Ultrastructural localization of TRAIL in intact, naïve neutrophils. Freshly isolated neutrophils were examined by TEM after staining for TRAIL (A and B), TRAIL and MPO (C and D), or TRAIL and albumin (E and F). TRAIL was labeled with 10 nm gold, whereas MPO and albumin were stained with ultra-small gold. Original magnification for each panel was as follows: x30,000 (A), x20,000 (B), x30,000 (C), x60,000 (D), x30,000 (E), x70,000 (F). Original scale bars represent 500 nm in each image. The PM and nucleus (N) are labeled. Examples of regions where TRAIL colocalized with MPO or albumin are indicated (white arrows). Regions where TRAIL did not colocalize with either marker are also indicated (black arrows).
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PLB-985 cells are myelomonoblasts that can be induced into granulocyte differentiation by treatment with DMSO or DMF [18 , 28 ]. Induced PLB cells progress into the metamyelocyte stage after 4 days in culture. To assess TRAIL expression, PLB cells were induced with 0.5% DMF, and cells were collected daily for 4 days. Changes in nuclear morphology and the presence of cytoplasmic granules were seen after Wright-Giemsa stain, consistent with progression through the early stages of neutrophil maturation (data not shown). RNA was isolated from PLB cells at each day of differentiation and analyzed by quantitative RT-PCR. DMF treatment induced TRAIL expression that remained constant throughout the 4 days of differentiation (Fig. 4A ). In contrast, MPO expression was markedly reduced after DMF treatment, and LF expression was low from Days 1 to 2 and peaked on Day 3 (Fig. 4B and 4C) . The cytosolic protein p47phox was constant throughout the 4 days (Fig. 4D) , consistent with previous observations [29 ]. We analyzed cell lysates taken at each day for TRAIL protein by immunoprecipitation and immunoblot (Fig. 4E) , similar to the experiments performed on mature peripheral blood neutrophils. TRAIL was present in PLB cells at Day 0 (noninduced) and after 4 days of differentiation. Similar to mature neutrophils, only the truncated S TRAIL was detected in PLB cells, suggesting that neutrophil-like cells synthesize only the truncated form of TRAIL.
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Figure 4. TRAIL is consistently expressed in DMF-treated PLB-985 cells, which were induced to differentiate with DMF as described in Materials and Methods, and cells were collected daily for 4 days. RNA levels were determined by quantitative RT-PCR for TRAIL (A), MPO (B), LF (C), and p47phox (D). (E) TRAIL protein was immunoprecipitated from PLB lysates obtained at Days 0 and 4 and analyzed by immunoblot under nonreducing conditions to visualize TRAIL aggregates. FL TRAIL and S TRAIL were used as standards, migrating as monomers, dimers, and trimers.
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Figure 5. Consistent expression of TRAIL in G-CSF-treated progenitor stem cells. CD34-positive stem cells were expanded, purified as described in Materials and Methods, and induced to differentiate into neutrophils with G-CSF. (A) Cellular morphology was examined at several days during the 14-day differentiation period by light microscopy after Wright-Giemsa staining (original magnification, 1000x). (B) Cells were collected at several days during the 14-day differentiation period, and RNA levels were analyzed by quantitative RT-PCR for TRAIL (B), MPO (C), LF (D), and p47phox (E).
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for 20 h. IFN- treatment induces de novo synthesis of TRAIL in mature neutrophils, resulting in an accumulation of intracellular stores that can augment the total amount of TRAIL released after stimulation with an agonist, such as BCG [15
, 16
]. Thus, different experimental conditions may have contributed to the slightly contrasting localization of TRAIL reported in these studies. In this study, we examined freshly isolated, resting peripheral blood neutrophils with the combination of subcellular fractionation and free-flow electrophoresis as a rigorous and thorough approach to assess subcellular distribution. TRAIL was indeed found in all defined compartments (AG, SG, and SV), and in agreement with the findings reported by Cassatella et al. [16 ], the SV fraction had high amounts of TRAIL. It is plausible that prolonged treatment with high doses of IFN augments the TRAIL present within the SV population to levels that dwarf the amounts of TRAIL in the other granule populations. Thus, neutrophils may potentially possess two distinguishable populations of TRAIL: preformed TRAIL synthesized during maturation and stored in many granules and TRAIL produced de novo in response to IFN and contained primarily within a SV-like compartment. Consistent with the targeting-by-timing hypothesis, TRAIL synthesized in mature neutrophils would be targeted to the SV, as azurophilic and secondary granule biogenesis had occurred earlier in myeloid development. We speculate that these two modes of storage likely have functional implications. Stores of preformed TRAIL would be available for rapid and immediate release in response to stimulation, whereas IFN-triggered, de novo production would enable neutrophils to continually secrete TRAIL over prolonged periods. This is especially relevant in the context of BCG immunotherapy for bladder cancer, in which high levels of IFN accumulate in the bladder environment during therapy and may augment the total amount of TRAIL released by neutrophils in this setting [32 33 34 ]. This is an area of focus for future studies that will compare the kinetics of TRAIL released from unstimulated and IFN-treated neutrophils.
In addition to the unique distribution of TRAIL in neutrophils, an interesting aspect of our findings was that neutrophils contained only the S TRAIL protein, as compared with the full-length, membrane-bound form expressed on the surface of PBMCs [35 36 37 38 39 40 ]. Similarly, neutrophil precursors that were actively synthesizing TRAIL only produced the truncated form of TRAIL. We found no evidence of a transitional, full-length form, suggesting that neutrophils produce only the truncated S TRAIL. Production of the S TRAIL may allow for more rapid release into the extracellular environment as compared with the kinetics of proteolytic cleavage required to release surface-expressed TRAIL from the membrane. In support of this, previous work from our laboratory demonstrated that neutrophils stimulated with M. bovis BCG release the S TRAIL [14 , 15 ]. Furthermore, S TRAIL is found at high levels in the urine of bladder cancer patients after treatment with BCG [13 ]. Synthesis of the truncated form of TRAIL in neutrophils could occur through alternative splicing TRAIL mRNA transcripts or by rapid, post-translational processing of the full-length protein. Splice variants of TRAIL have been identified in other cells types [41 ], so future studies will examine the size of TRAIL mRNAs for evidence of alternatively spliced transcripts. We are also pursuing protein biosynthesis studies in neutrophils after IFN priming, which will allow us to follow potential post-translational modifications and cleavage of the TRAIL protein.
To complement our subcellular fractionation analysis of TRAIL distribution, we used microscopy to examine the localization of TRAIL in intact neutrophils. Immunofluorescence microscopy and TEM demonstrated that TRAIL was within granules, and there was no evidence of TRAIL localized in the cytoplasm (diffuse staining) or the PM (perimeter staining). Based on these results, the cytoplasmic distribution in the subcellular fractionation studies may result from lysis of the ER, Golgi, and disruption of a small percentage of granules during the cavitation process. Furthermore, the difference in the PM localization between subcellular fractionation and microscopy studies is also perplexing. Neutrophils express TRAIL receptors in the membrane [42 ], so it is possible that in disrupted neutrophils, small amounts of TRAIL could bind to these exposed receptors. However, previous reports have shown TRAIL surface expression on neutrophils [16 , 42 43 44 45 ], so it is also possible that there are low levels of TRAIL in the PM.
Given the implications of our subcellular localization for the timing of TRAIL biosynthesis during myeloid development, we examined TRAIL gene expression in neutrophil precursors to test the hypothesis that TRAIL is expressed throughout maturation to achieve the broad localization among neutrophil granules and vesicles. TRAIL expression was examined in DMF-induced PLB cells and CD34-positive progenitors that were induced toward granulocyte differentiation with G-CSF. Consistent with our hypothesis, TRAIL was expressed constitutively in induced PLB and CD34-positive progenitors, whereas other granule markers (MPO, LF) were only expressed at specific intervals, congruent with their finite granular localization. Various transcription factors, such as C/EBP isoforms, GATA-1, and PU.1, have been identified to regulate the expression of neutrophil proteins during specific intervals of development [46 ], allowing targeting to the proper granule subtype [7 , 8 ]. The expression pattern of TRAIL during neutrophil development suggests the involvement of multiple transcription factors, allowing continuous expression and packaging into different granule subtypes. Sequence analysis of the TRAIL promoter region revealed the presence of putative response elements for several transcription factors involved in gene expression during neutrophil development (C/EBP, GATA, PU.1, Ets, and others), as well as sites for IFN-regulatory factors. This sequence analysis will provide a framework for future studies to identify the transcription factors that regulate TRAIL expression in neutrophils.
In summary, the results of this study describe an expression and localization pattern of TRAIL that is unique in comparison with other described neutrophil proteins. Our findings suggest many potential avenues to be explored by future studies, such as the role of various transcription factors during neutrophil maturation, the biosynthesis and packaging of TRAIL in maturing and mature IFN-primed neutrophils, and the kinetics and mechanisms involved in agonist-dependent release. Furthermore, the biological role of TRAIL in neutrophils remains unclear. Previous studies from our laboratory have suggested neutrophils release large amounts of TRAIL in the bladder in response to BCG instillation, resulting in apoptosis of bladder tumor cells [13 , 14 ]. These findings, along with work from other laboratories [43 , 45 ], suggest that neutrophils may contribute to immune surveillance against cancer cells, a function not conventionally attributed to these cells. In addition, TRAIL is involved in the apoptosis of senescent neutrophils, allowing proper turnover of the circulating population [44 , 47 ]. Understanding the functions of neutrophil-derived TRAIL has developed into an exciting new area of research. The findings presented in this study provide a foundation for future studies aimed to further elucidate the biology of TRAIL in neutrophils.
Received July 10, 2007; revised November 2, 2007; accepted November 5, 2007.
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B and protects against TRAIL-mediated apoptosis, yet retains an incomplete death domain Immunity 7,813-820[CrossRef][Medline]-dependent suppression of subcutaneous tumor growth Cell. Immunol. 214,194-200[CrossRef][Medline]: two novel splice variants of the human TNF-related apoptosis-inducing ligand (TRAIL) without apoptotic potential Br. J. Cancer 88,918-927[CrossRef][Medline]-stimulated neutrophils and monocytes release a soluble form of TNF-related apoptosis-inducing ligand (TRAIL/Apo-2 ligand) displaying apoptotic activity on leukemic cells Blood 103,3837-3844
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