Journal of Leukocyte Biology eBioscience full spectrum cell analysis
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.0805431 on October 21, 2005

Published online before print October 21, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0805431v1
79/1/123    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cassatella, M. A.
Right arrow Articles by Tecchio, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cassatella, M. A.
Right arrow Articles by Tecchio, C.
(Journal of Leukocyte Biology. 2006;79:123-132.)
© 2006 by Society for Leukocyte Biology

Interferon-activated neutrophils store a TNF-related apoptosis-inducing ligand (TRAIL/Apo-2 ligand) intracellular pool that is readily mobilizable following exposure to proinflammatory mediators

Marco A. Cassatella*,1, Veronica Huber{dagger}, Federica Calzetti*, Daniela Margotto*, Nicola Tamassia*, Giuseppe Peri{ddagger}, Alberto Mantovani{ddagger}, Licia Rivoltini{dagger} and Cristina Tecchio*,§

* Department of Pathology, Division of General Pathology, University of Verona, Italy;
{dagger} Unit of Immunotherapy of Human Tumors, Istituto Nazionale Tumori, Milano, Italy;
{ddagger} Istituto di Ricerche Farmacologiche Mario Negri, Milano and Istituto Clinico Humanitas, Rozzano, Italy; and
§ Division of Hematology, Ospedale Regionale Cà Foncello, Treviso, Italy

1 Correspondence: Department of Pathology, Section of General Pathology, University of Verona, Strada Le Grazie 4, 37134 Verona, Italy. E-mail: marco.cassatella{at}univr.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neutrophils are versatile cells, which play a role, not only in inflammatory processes but also in immune and antitumoral responses. Recently, we have reported that interferon (IFN)-activated neutrophils are able to release biologically active tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/APO2 ligand), a molecule exerting selective, apoptotic activities toward tumor and virus-infected cells, as well as immunoregulatory functions on activated T lymphocytes. Herein, we show that only a minor fraction of the total TRAIL, newly synthesized by IFN-activated neutrophils within 24 h, is released outside, the rest being retained intracellularly, mainly in secretory vesicles and light membrane fractions. We demonstrate that the intracellular pool of TRAIL present in IFN-pretreated neutrophils is rapidly mobilizable to the cell surface and can be secreted following exposure to proinflammatory mediators such as TNF-{alpha}, lipopolysaccharide, formyl-methionyl-leucyl-phenylalanine, CXC chemokine ligand 8/interleukin-8, insoluble immunocomplexes, and heat shock protein Gp96. These various proinflammatory agonists functioned as effective secretagogue molecules only, in that they failed to augment TRAIL mRNA expression or TRAIL de novo synthesis in freshly isolated neutrophils or cultured with or without IFN. In addition, supernatants from IFN-treated neutrophils stimulated with proinflammatory mediators induced the apoptosis of target cells more effectively than supernatants from neutrophils activated with IFNs alone. Collectively, our results uncover a novel mechanism, whereby the release of soluble TRAIL by neutrophils can be greatly amplified and further reinforce the notion that neutrophils are important cells in tumor surveillance and immunomodulation.

Key Words: T lymphocyte • LPS • fMLP • CXCL


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In recent years, it has been demonstrated that neutrophils, although classically viewed as critical cellular components in preprogrammed innate host defense, can contribute to the development of adaptive immune and antitumoral responses [1 ]. In this regard, the potential immunoregulatory role of neutrophils is currently the subject of a new wave of enthusiastic research, as it is now clear that under various circumstances, neutrophils interact in a continuous cross-talk with other leukocyte subsets and in turn, modulate their effector and antitumoral functions [1 2 3 ]. For instance, neutrophils are well known to produce a variety of cytokines and chemokines, including interleukin (IL)-12, macrophage inflammatory protein-1{alpha} (MIP-1{alpha})/CC chemokine ligand 3 (CCL3), MIP-1ß/CCL4, MIP-3ß/CCL19, MIP-3{alpha}/CCL20, interferon-{gamma} (IFN-{gamma})-inducible protein of 10 kDa/CXC chemokine ligand 10 (CXCL10), monokine induced by IFN-{gamma}/CXCL9, and IFN-inducible T cell {alpha} chemoattractant/CXCL11, which greatly influence leukocyte trafficking and activation [2 , 4 , 5 ]. Among the cytokines of great interest, which neutrophils produce, are the ligands belonging to the tumor necrosis factor (TNF) superfamily for their ability to activate signaling pathways involved in cell survival, death, and differentiation, which orchestrate the development, organization, and homeostasis of lymphoid and other tissues [6 ]. In this regard, evidence that neutrophils express TNF-{alpha}, Fas ligand, CD30 ligand, and B lymphocyte stimulator (BLyS) and that such production may have pathophysiological implications under specific settings in human diseases or other experimental models has been clearly provided [4 , 7 ].

A member of the TNF superfamily which displays selective apoptotic cell death in a variety of tumor cells by engaging the death receptors DR4 and DR5 and sparing most normal cells and that also exerts immunoregulatory functions toward activated T lymphocytes, is TNF-related apoptosis-inducing ligand (TRAIL) [8 9 10 ]. Preclinical studies in mice and nonhuman primates have shown the potential use of recombinant soluble (rs)TRAIL and agonistic anti-DR5 or -DR4 antibodies for cancer therapy [11 ]. Moreover, a vital role for endogenously expressed TRAIL in immunosurveillance of developing and metastatic tumors has been clearly revealed [11 ]. TRAIL is expressed primarily as a type II membrane protein by immune cells {activated monocytes, dendritic cells (DC), natural killer (NK) cells, and T lymphocytes [12 13 14 15 16 17 18 ]}, but increasing evidence points to the existence of a biologically active, soluble form generated through enzymatic shedding of the membrane-bound form or through the release in association with microvesicles [19 20 21 ]. It is interesting that recent reports have indicated the possible involvement of sTRAIL in the outcome of several pathologies by demonstrating that in vivo, significant amounts of this molecule can be detected in sera obtained from patients affected by neoplastic, autoimmune, and infectious diseases [22 23 24 ]. In particular, sTRAIL has been shown to serve as a potential response marker for bladder cancer patient outcomes after Bacillus Calmette-Guerin (BCG) instillations [25 ] and for IFN-ß treatment in multiple sclerosis [26 ]. In the former situation, high TRAIL expression on the neutrophils present in the urine of patients immediately after (BCG) instillation was reported to occur [25 ]. It is important that these latter data have corroborated previous findings made by us [22 ] and three more groups [27 28 29 ], showing that peripheral blood neutrophils, in response to IFN-{alpha} and IFN-{gamma}, accumulate high levels of TRAIL transcripts and release significant amounts of sTRAIL, which retains proapoptotic activities toward different leukemic cell lines. In addition, we have shown that sTRAIL serum levels as well as leukocyte-associated TRAIL significantly increase in melanoma patients following IFN-{alpha} administration [22 ], further highlighting the possible participation of neutrophils to TRAIL-mediated tumor immunosurveillance in vivo [30 ].

In the present study, we demonstrate that the capacity of IFN-treated neutrophils to release sTRAIL can be greatly amplified by proinflammatory mediators of different origin, including those potentially present in the tumor microenvironment. We show that only a minor fraction of the total TRAIL synthesized by neutrophils upon IFN type I (IFN-{alpha}, IFN-ß) or IFN type II (IFN-{gamma}) treatment is released extracellularly, as sTRAIL is the major fraction remaining, in fact, inside the cytoplasm of neutrophils themselves. However, we also show that most of the intracellular TRAIL pool retained in IFN-activated neutrophils can be either mobilized rapidly onto the surface membrane or secreted as sTRAIL, if the cells are stimulated with TNF-{alpha}, lipopolysaccharide (LPS), formyl-methionyl-leucyl-phenylalanine (fMLP), CXCL8/IL-8, insoluble immunocomplexes (IC), or heat shock protein (hsp) Gp96. Moreover, this rapidly secreted sTRAIL upon stimulation with proinflammatory cytokines proved to be bioactive, as apoptosis of Jurkat T cells was potentiated as compared with cell death induced by sTRAIL released by neutrophils, activated by IFN only. These observations not only uncover a novel mechanism that dramatically exacerbates the release of sTRAIL by neutrophils during pathological inflammatory and neoplastic responses but also substantiate the role of IFN-activated neutrophils as a major potential source of sTRAIL.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell purification and culture
Highly purified granulocytes (neutrophils>96.5%, eosinophils<3%), peripheral blood mononuclear cells (PBMC), and Percoll-purified monocytes were isolated under endotoxin-free conditions from buffy coats of healthy donors as described previously [31 ]. Immediately after purification, neutrophils were suspended at 5 x 106/ml in RPMI-1640 medium supplemented with 10% low endotoxin fetal bovine serum [<0.06 EU/ml by Limulus amoebocyte lysate (LAL) assay, BioWhittaker, Verviers, Belgium], treated with or without 1000 U/ml IFN-{alpha} (Roferon, Roche Laboratories, Nutley, NJ), 1000 U/ml IFN-ß (Betaferon®, Schering AG, Berlin, Germany), or 200 U/ml IFN-{gamma} (R&D Systems, Minneapolis, MN), unless otherwise specified, and then, usually plated in 24-well tissue-culture wells (Orange, Trasadingen, Switzerland). After 20 h of incubation, cultures were usually stimulated for an additional 4 h (unless otherwise indicated) with the following substances: TNF-{alpha} (0.5–5 ng/ml; Peprotech, Rocky Hill, NJ), LPS (0.1–100 ng/ml, from Escherichia coli, serotype 026:B6, Sigma Chemical Co., St. Louis, MO), fMLP (10–1000 nM, Sigma Chemical Co.), 60 µg/ml insoluble IC prepared as described previously [32 ], and hsp Gp96 (1–50 µg/ml, Immatics Biotechnologies GmbH, Tubingen, Germany). The levels of contaminating endotoxin in the Gp96 preparations (declared by Immatics Biotechnologies GmbH as <0.026 EU/µg by LAL assay and equivalent to <0.1 ng/ml LPS) had no effect on TRAIL release. In selected experiments, IFN-preincubated neutrophils were treated with or without 6 mM pentoxifylline (PTF), 3.5 µM monensin (Alexis, San Diego, CA), 10 µM jasplakinolide (JK), or 20 µg/ml cycloheximide (Sigma Chemical Co.) prior to further stimulation with proinflammatory mediators. After the desired incubation period, cells were collected and spun at 350 g for 5 min. The resulting supernatants were immediately frozen in liquid nitrogen and stored at –80°C. The corresponding pellets were extracted for total RNA or thawed in phosphate-buffered saline containing 0.5% Nonidet P-40 (NP-40), 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 5 µg/ml leupeptin/pepstatin A and then spun (14,000 g, 5 min) to remove cell debris.

Enzyme-linked immunosorbent assay (ELISA)
Concentrations of sTRAIL ligand in cell-free supernatants and cell pellets were measured by using a commercial ELISA kit from Diaclone Research (Besancon, France), according to the manufacturer’s instructions. The presence of NP-40 did not interfere with the measurement of sTRAIL.

Flow cytometry for membrane-bound TRAIL (mTRAIL)
This was performed according to the same protocol recently described by us [22 ].

Quantitative polymerase chain reaction (Q-PCR)
Total RNA was extracted from 107 neutrophils using the RNeasy mini kit followed by DNase I treatment, according to the manufacturer’s protocol (Qiagen, Crawley, UK). Total RNA (1 µg) from each sample was used as a template for the reverse transcription (RT) reaction, using random hexamers and SuperScript II RT (Invitrogen, Carlsbad, CA). All samples were reverse-transcribed under the same conditions and from the same RT master mix to minimize differences in RT efficiency [33 ]. Oligonucleotide primers (purchased from Invitrogen) were: TRAIL forward, CAG AGG AAG AAG CAA CAC ATT, and TRAIL reverse, GGT TGA TGA TTC CCA GGA GTT TAT TTT G; CXCL8 forward, CTG GCC GTG GCT CTC TTG, and CXCL8 reverse, CCT TGG CAA AAC TGC ACC TT; ß2-microglobulin forward, CTC CGT GGC CTT AGC TGT G, and ß2-microglobulin reverse, TTT GGA GTA CGC TGG ATA GCC T. Triplicate Q-PCR reactions for each sample were performed in 20 µL containing 20 ng cDNA, 10 µL 2x Platinum SYBR Green qPCR SuperMix UDG (Invitrogen), and forward/reverse primers at a final concentration of 200 nM. The Q-PCR reactions were performed in 96-well plates with optical caps using the DNA Engine Opticon 2 System (MJ Research, Waltham, MA). The reaction conditions were identical for all primer sets as follows: 50°C for 2 min, 95°C for 2 min, and then, 40 cycles of 95°C for 15 s and 60°C for 1 min. Control wells containing no template were used to exclude the presence of contaminating template molecules and to identify potential primer-dimer products from the dissociation curve analysis. Amplification plots were analyzed using Opticon Monitor software Version 2.02 (MJ Research), and data were calculated with Q-Gene software (www.BioTechniques.com). mRNA expression levels are reported as the number of gene copies per copies of the control mRNA. ß2-microglobulin was selected as a normalizing gene according to its stable expression levels in leukocytes [33 ].

Western blot analysis
Supernatants from neutrophils activated as described in "Cell purification and culture" were analyzed under reducing conditions by Western blot analysis, as described previously [22 ]. Nondisulfide-linked, trimeric human rsTRAIL (rhsTRAIL; R&D Systems) was used as a positive control. Equal loading was checked by Ponceau Red S (Sigma Chemical Co.) staining.

Apoptosis assessment
Jurkat 32 cells were seeded at a density of 106/ml in 96-well plates and cultured in the presence of media conditioned by neutrophils treated overnight with IFN-{alpha} and subsequently stimulated with or without 100 ng/ml or 10 nM fMLP for 4 h. Concentrated supernatants were prepared as described previously [22 ], added to Jurkat cells at a dilution of 1:10, and cultured for 6 h or overnight at 37°C, 5% CO2. Apoptosis was assessed by Annexin V/propidium iodide staining (rh Annexin V/FITC kit, Bender MedSystems, Vienna, Austria) and analyzed by FACSCalibur and CELLQuest software. To assess specificity of TRAIL-mediated killing activities, the supernatants were preincubated with 1 µg/ml anti-TRAIL monoclonal antibodies (mAb; RIK2, BD Biosciences, San Jose, CA) for 4 h at 4°C.

Subcellular fractionation
Subcellular fractionation of neutrophils was performed exactly as described by Kjeldsen et al. [34 ]. Briefly, neutrophils cultured with IFN-{alpha} for 20 h were disrupted by nitrogen cavitation (Parr Instruments Co., Moline, IL), and the resulting cavitate then centrifuged to eliminate pellet nuclei and the remaining intact cells [35 ]. The postnuclear supernatant containing cytosol, granules, and light membranes was separated immediately by centrifugation on a three-layer Percoll density gradient [34 ]. Gradient fractions of 3 ml were collected and analyzed for localization of subcellular organelles by marker assays as follows: An {alpha}-mannosidase functional assay was used for azurophil granules [36 ]; vascular endothelial growth factor (VEGF) and lactoferrin-specific ELISA were used for specific granules [37 ]; a gelatinase ELISA for gelatinase granules; and an albumin ELISA for secretory vesicles and light membranes [38 ]. Aliquots of all fractions were also assayed for sTRAIL content. Preliminary experiments confirmed that the presence of Percoll does not interfere with the various methods used to determine the different markers [34 ].

Statistical analysis
Data are expressed as means ± SD, unless otherwise indicated. Statistical evaluation was performed using Student’s t-test. P values less that 0.05 were regarded as significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TRAIL synthesized by human neutrophils in response to type I and type II IFN is mainly accumulated in intracellular stores
We have recently reported that human neutrophils express TRAIL mRNA and release sTRAIL following exposure to IFN-{alpha} or to a lower extent, IFN-{gamma} [22 ]. As a number of cytokines newly synthesized by activated neutrophils, including CXCL8/IL-8, CXCL20/MIP-3{alpha}, IL-1 receptor antagonist [39 40 41 ], and even members of the TNF family such as BLyS [7 ], are, in large part, stored in intracellular compartments, we investigated whether TRAIL too, once manufactured in response to IFNs, accumulates in the cytoplasm or instead, is fully released outside the cell. As shown in Figure 1 , measurement of total TRAIL production, i.e., released sTRAIL in parallel with cell-associated TRAIL, demonstrated that a 24-h treatment of neutrophils with 1000 U/ml IFN-{alpha}, 1000 U/ml IFN-ß, and 200 U/ml IFN-{gamma} induces a remarkable de novo synthesis of TRAIL, which relative to cells treated with medium only, increases approximately four- to sevenfold, depending on the IFN type. In this regard, a significant, higher TRAIL synthesis over control was induced by concentrations of IFN-{alpha}, IFN-ß, and IFN-{gamma}, equal to 1 U/ml (data not shown). However, only a minor fraction of the total TRAIL synthesized by IFN-treated neutrophils was detectable in their extracellular medium (14±3% for IFN-{alpha}, n=9; 18±2% for IFN-ß, n=6; 7±1% for IFN-{gamma}, n=4), indicating that type I and type II IFNs, although efficiently triggering a remarkable, novel synthesis of TRAIL, only activate a limited release of sTRAIL.



View larger version (16K):
[in this window]
[in a new window]
  		Figure 1. Total TRAIL synthesis in neutrophils cultured with type I and type II IFNs. Neutrophils (5x106/ml) were incubated for 24 h at 37°C with 1000 U/ml IFN-{alpha}, 1000 U/ml IFN-ß, and 200 U/ml IFN-{gamma}. Cell-free supernatants and the corresponding pellets were harvested, and the levels of TRAIL were determined by ELISA. The mean values ± SD of the total production of TRAIL, depicted as cell-associated and released from four to nine independent experiments, are shown.

 
Proinflammatory mediators promote the release of sTRAIL in type I and type II IFN-treated neutrophils
As the ELISA used to measure TRAIL in cell-associated samples does not discriminate between the full-length and the sTRAIL protein forms and as mTRAIL is expressed poorly in neutrophils and upon IFN-stimulation, slightly increases in some donors only [22 , 28 ], we assumed initially that cell-associated TRAIL mostly consisted of preformed sTRAIL. Therefore, we investigated whether intracellular TRAIL could be mobilized eventually into the extracellular environment. To test such a hypothesis, we measured released TRAIL in parallel with cell-associated TRAIL in neutrophils, which were preincubated with type I and type II IFNs for 20 h and then treated with optimal concentrations of classical, proinflammatory agonists, such as TNF-{alpha} (5 ng/ml), fMLP (10 nM), LPS (100 ng/ml), insoluble IC (60 µg/ml), and CXCL8/IL-8 (100 ng/ml; Fig. 2 ). IFN-treated neutrophils were also stimulated with the hsp 96 (Gp96), a danger signal present in the tumor microenvironment, which has been shown recently to bind to granulocytes [42 ]. All these stimuli, added alone for up to 24 h to freshly purified or to 20 h cultured neutrophils, proved to be completely unable to stimulate TRAIL synthesis/release (data not shown), confirming and extending previous findings made by us [22 ] and other groups [27 , 28 ]. In contrast, addition of the various proinflammatory agonists to IFN-preincubated neutrophils triggered a remarkable release of sTRAIL (Fig. 2) , without, however, further increasing the effect of IFN on TRAIL de novo synthesis (Fig. 2A 2B 2C) or TRAIL gene expression (Fig. 3 , upper). In these latter experiments, accumulation of IL-8 transcripts was instead up-regulated by TNF-{alpha}, fMLP, and LPS (Fig. 3 , lower), illustrating not only the effectiveness of these proinflammatory mediators in increasing the IL-8 gene as expected [41 ] but also, their selective inability to up-regulate TRAIL mRNA expression.



View larger version (39K):
[in this window]
[in a new window]
  		Figure 2. Characterization of TRAIL synthesis and secretion by IFN-treated neutrophils stimulated with proinflammatory mediators. Neutrophils were preincubated as follows: (A) with 1000 U/ml IFN-{alpha} for 20 h prior to the addition of 5 ng/ml TNF-{alpha}, 10 nM fMLP, and 100 ng/ml LPS for the times indicated; (B) with 1000 U/ml IFN-ß for 20 h prior to a 4-h addition of TNF-{alpha}, fMLP, and LPS at the doses indicated; and (C) with or without 1000 U/ml IFN-{alpha} for 20 h prior to a 4-h addition of various concentrations of Gp96. Cell-free supernatants and the corresponding cell pellets were then harvested for determination of antigenic TRAIL. (A and B) The mean values ± SEM of the total production or released fraction of TRAIL obtained from three experiments [*, a significant increase of sTRAIL release (P<0.01)]. (C) The mean values of the total production of TRAIL (depicted as cell-associated and released) of a single experiment representative of two with similar results. (D) Neutrophils were incubated with or without IFN-{alpha}, IFN-ß, or IFN-{gamma} for 20 h prior to addition of TNF-{alpha} (5 ng/ml), fMLP (10 nM), LPS (100 ng/ml), IC (60 mg/ml), or Gp96 (50 mg/ml). After an additional 4 h, cell-free supernatants and the corresponding cell pellets were harvested for determination of antigenic TRAIL. The percentages of secreted sTRAIL were calculated from the total TRAIL levels. Data report the mean values ± SD of the percentages of sTRAIL release calculated from four to 11 independent experiments. *, A significant increase of sTRAIL release (P<0.01).

 


View larger version (20K):
[in this window]
[in a new window]
  		Figure 3. Effect of TNF-{alpha}, fMLP, and LPS on TRAIL mRNA expression in neutrophils incubated with or without IFN-{alpha}. Neutrophils incubated with or without 1000 U/ml IFN-{alpha} for 20 h were stimulated with 5 ng/ml TNF-{alpha}, 10 nM fMLP, and 100 ng/ml LPS for 45 and 120 min, before total RNA extraction and analysis of TRAIL, CXCL8, and ß2-microglobulin mRNA expression by real-time PCR. TRAIL (upper) and CXCL8 (lower) mRNA levels are depicted as the number of copies of the gene per copies of the control mRNA [ß2-microglobulin (ß2m)]. Data show one representative experiment out of three performed with similar results.

 
Time-course and dose-response studies demonstrated that the ability of proinflammatory substances to activate the release of sTRAIL in type I and type II IFN-treated neutrophils occurs rapidly (being clearly evident within 45 min, Fig. 2A ) and is triggered by concentrations of TNF-{alpha}, fMLP, LPS, and Gp96, as low as 0.5 ng/ml, 1 nM, 1 ng/ml (Fig. 2B) , and 20 µg/ml (Fig. 2C) , respectively. As depicted in Figure 2D , which summarizes the stimulatory effect of all proinflammatory mediators used, insoluble IC, other than TNF-{alpha}, fMLP, LPS, and CXCL8 (data not shown), also potentiated the release of sTRAIL in IFN-{alpha}- or IFN-{gamma}-pretreated neutrophils. Furthermore, concentrations of IFN-{alpha}/ß or IFN-{gamma} as low as 10 U/ml proved effective in preparing neutrophils to synthesize and accumulate an intracellular TRAIL pool ready to be released upon appropriate stimulation (data not shown). Finally, the contemporaneous addition of more than one stimulus to the cultures of IFN-treated neutrophils (i.e., fMLP plus LPS, fMLP plus TNF-{alpha}, TNF-{alpha} plus LPS, or LPS plus fMLP and TNF-{alpha}) did not result in any additive effect on sTRAIL release (data not shown), indicating that similar intracellular pathways are used by these stimuli or that they act on the same mobilizable pool of intracellular TRAIL.

Bioactivity and Western blot analysis of sTRAIL protein released by activated neutrophils
We have previously shown that sTRAIL, released by IFN-activated neutrophils, is bioactive and induces TRAIL-mediated, specific apoptosis in various leukemic cell lines [22 ]. To verify whether the increased release of sTRAIL by IFN-{alpha}-treated neutrophils stimulated with proinflammatory factors was associated with an enhanced bioactivity, we evaluated the proapoptic effect of our supernatants on the TRAIL-sensitive T cell line Jurkat 32 [8 , 43 ]. By using the Annexin V/propidium iodide staining methods [22 ], we could uncover a significant potentiation of specific TRAIL-mediated apoptosis in Jurkat cells exerted by supernatants from IFN-activated neutrophils treated with 10 nM fMLP or 100 ng/ml LPS over that exerted by supernatants harvested from cells activated with IFN-{alpha} alone (Fig. 4A ). Under these conditions, apoptosis was mediated by sTRAIL, as it was almost blocked completely by anti-TRAIL mAb and was already detectable after 6 h (Fig. 4) but substantially maintained for up to 24 h of Jurkat cell culture (not shown). We could not use supernatants from IFN-{alpha}-treated neutrophils stimulated with TNF-{alpha} as a result of the well-known, direct, proapoptotic effect of TNF-{alpha} on Jurkat cells.



View larger version (32K):
[in this window]
[in a new window]
  		Figure 4. Biological activity and Western blot analysis of sTRAIL released by IFN-{alpha}-treated neutrophils stimulated with proinflammatory agonists. (A) The apoptosis rate of the Jurkat 32 leukemic cell line was assessed by Annexin-V-FLUOS staining after a 6-h culture in the presence of conditioned medium, previously prepared from neutrophils, incubated for 24 h with IFN-{alpha} alone or with IFN-{alpha} (for 20 h) followed by 10 nM fMLP or 100 ng/ml LPS (for an additional 4 h). Jurkat cell incubation was carried out in the absence or the presence of 1 µg/ml anti-TRAIL neutralizing antibodies (RIK2). The histograms depict the mean values ± SEM of the percentage of apoptosis enhancement exerted by supernatants derived from IFN-{alpha}-treated neutrophils stimulated with fMLP or LPS compared with that observed with supernatants derived from neutrophils treated with IFN-{alpha} only. (B) Supernatants harvested from neutrophils cultured for 20 h with 1000 U/ml IFN-{alpha} and then stimulated with 5 ng/ml TNF-{alpha} or 10 nM fMLP were concentrated and analyzed for the presence of sTRAIL by Western blotting. Concentrated supernatants (50 µg/sample) were run under reducing conditions and then stained with anti-TRAIL mAb. rhsTRAIL (nondisulfide-linked homotrimer) at 80 ng/sample was included as a positive control. Loading control was performed by Ponceau Red S staining. Data are representative of analyses performed in two healthy donors.

 
We also performed Western blot analysis of concentrated supernatants under reducing conditions. As shown in Figure 4B , sTRAIL was mostly detectable in its monomeric (24 kDa) and trimeric (64 kDa) aggregation status in concentrated supernatants. Although untreated neutrophils release little amounts of sTRAIL, an overnight incubation with IFN greatly enhances their sTRAIL release [22 ]. It is remarkable that a further increase of sTRAIL is detectable in supernatants after the addition to IFN-{alpha}-pretreated neutrophils of TNF-{alpha} or in this particular experiment, fMLP (Fig. 4B) . Nondisulfide-linked, trimeric rhsTRAIL, used as a positive control, appeared as a band of 64 kDa at the same height of the trimeric sTRAIL detected in supernatants of neutrophils. Similar results were obtained by Western blot analysis of samples harvested from IFN-ß-treated neutrophils (data not shown). Collectively, these data not only support the results obtained by ELISA but also confirm that in IFN-treated neutrophils, proinflammatory mediators trigger the release of a sTRAIL fraction, which retains a biologic activity.

Proinflammatory mediators induce the expression of mTRAIL in type I and type II IFN-treated neutrophils
To clarify whether IFN-treated neutrophils, stimulated with proinflammatory mediators, could also express greater levels of mTRAIL, we performed studies by flow cytometry analysis. Confirming and extending our recent observations [22 ], Figure 5 shows that cultured neutrophils display low levels of mTRAIL [18±14 mean fluorescence intensity (MFI), n=5], which only minimally change after an overnight incubation in the presence of IFN-{alpha} (48±27 MFI, n=5) or IFN-ß (32±14 MFI, n=3; data not shown). In contrast, surface expression of mTRAIL rapidly increases in IFN-{alpha}-treated neutrophils (Fig. 5) or IFN-ß-treated neutrophils (not shown) exposed to proinflammatory agonists. Up-regulation of mTRAIL proved to be, in fact, maximally evident after 45 min of stimulation with TNF-{alpha} (82±5 MFI, n=3; Fig. 5A ), LPS (62±20 MFI, n=3; Fig. 5B ), and fMLP (131±37 MFI, n=4; Fig. 5C ), slowly declining thereafter (Fig. 5) . Similar patterns of mTRAIL expression were observed in neutrophils cultured overnight with IFN-ß or IFN-{gamma} and then activated with fMLP (data not shown). Under these conditions, the protein synthesis inhibitor cycloheximide did not influence at all the up-regulatory effect of fMLP and TNF-{alpha} on mTRAIL expression (data not shown), again consistent with the gene expression data (Fig. 3) . These results demonstrate that in the context of inflammatory settings, the intracellular TRAIL pool, which accumulates in IFN-treated neutrophils, can be mobilized rapidly onto the surface membrane, other than being exocytosed in the extracellular environment.



View larger version (37K):
[in this window]
[in a new window]
  		Figure 5. Effect of TNF-{alpha}, LPS, and fMLP on mTRAIL expression in neutrophils incubated with or without IFN-{alpha}. Neutrophils incubated with or without 1000 U/ml IFN-{alpha} for 20 h were stimulated for 45 min, 2 h, and 4 h with 5 ng/ml TNF-{alpha} (A), 100 ng/ml LPS (B), and 10 nM fMLP (C) before flow cytometry analysis for mTRAIL. These profiles are representative of analyses performed in three healthy donors. PMN, Polymorphonuclear neutrophils; IgG1, immunoglobulin G1.

 
Proinflammatory mediators inducing sTRAIL, released by IFN-treated neutrophils, act as secretagogue-like molecules
To better elucidate the mechanisms whereby TNF-{alpha}, fMLP, LPS, and IC induce the extracellular mobilization of sTRAIL, neutrophils incubated with IFN-{alpha} or IFN-{gamma} for 20 h were treated for 1 h with PTF, a general inhibitor of neutrophil degranulation [37 ], or monensin, a specific inhibitor of post-Golgi secretion [44 ], prior to stimulation with proinflammatory agonists for 3 h. Cell-free supernatants and cell pellets were then harvested, and TRAIL was measured by ELISA in the two fractions. As shown in Figure 6A , PTF, but not monensin, significantly reduced the release of sTRAIL induced by TNF-{alpha} (57±7% inhibition), fMLP (54±5% inhibition), LPS (47% inhibition), and IC (35±2% inhibition) in IFN-{alpha}-treated neutrophils. JK, another inhibitor of degranulation [45 ], diminished sTRAIL release-induced proinflammatory mediators similarly to PTF (data not shown). Flow cytometry analysis performed under identical experimental conditions revealed that fMLP- and TNF-{alpha}-dependent up-regulation of mTRAIL was also reduced significantly by PTF (60±5% inhibition for fMLP and 42±4% inhibition for TNF-{alpha}, n=3) but not by monensin (Fig. 6B and 6C) . Similar results were observed in IFN-{gamma}- or IFN-ß-treated neutrophils (data not shown). At the same concentrations, monensin inhibited the release of BLyS triggered by LPS, fMLP, and TNF-{alpha} in granulocyte-colony stimulating factor (G-CSF)-treated neutrophils [38 ]. Taken together, with the time-course experiments (Fig. 2A) , these findings indicate that a secretory pathway underlies sTRAIL exocytosis in IFN-treated neutrophils stimulated by proinflammatory agonists.



View larger version (23K):
[in this window]
[in a new window]
  		Figure 6. Effect of PTF and monensin on TRAIL secretion. (A) IFN-{alpha}-treated neutrophils were incubated for 1 h with or without 6 mM PTF or 3.5 µM monensin prior to TNF-{alpha} (5 ng/ml), fMLP (10 nM), LPS (100 ng/ml), and IC (60 µg/ml) addition. After 3 h, cell-free supernatants and cell pellets were harvested for ELISA determination of antigenic TRAIL. Data report the mean values ± SD of the percentages of inhibition on TNF-{alpha}-, fMLP-, LPS-, and IC-induced release of TRAIL provoked by PTF and monensin over IFN-{alpha}-treated cells (calculated from three to four independent experiments). *, A significant inhibition of sTRAIL release by PTF (P<0.01). (B and C) IFN-{alpha}-treated neutrophils were incubated for 1 h with or without 6 mM PTF or 3.5 µM monensin (MON) prior to addition of fMLP (B) or TNF-{alpha} (C). After a further 60 min, flow cytometry analysis for mTRAIL was performed. The profiles shown are representative of analyses performed in three independent experiments.

 
Intracellular localization of TRAIL in IFN-{alpha}-treated neutrophils
To identify the intracellular localization of TRAIL in IFN-{alpha}-treated neutrophils, we performed subcellular fractionation experiments of neutrophil-derived nitrogen cavitates [35 ] centrifuged on a three-layer, discontinuous Percoll density gradient [34 ]. As depicted in Figure 7 , this methodology permits a clear separation and identification of the various intracellular compartments of neutrophils, including the azurophil granules (measured as {alpha}-mannosidase content), the specific granules (measured as VEGF content or lactoferrin as well), the gelatinase granules (measured as gelatinase content), and the secretory vesicles/light membranes (measured as albumin content). In these sets of experiments, TRAIL was found to clearly localize mainly to the band corresponding to the secretory vesicles/light membranes fraction (enriched for albumin) but not in primary, secondary, and gelatinase granules (Fig. 7) . As secretory vesicles are notoriously, easily degranulated [46 ], a TRAIL localization in these organelles is consistent with its rapid secretion in response to proinflammatory mediators, as described above.



View larger version (24K):
[in this window]
[in a new window]
  		Figure 7. De novo synthesized TRAIL is localized to the secretory vesicle/light membrane fraction in IFN-{alpha}-treated neutrophils, which when treated with 1000 U/ml IFN-{alpha} for 20 h, were disrupted by nitrogen cavitation and then fractionated on a three-layer Percoll density gradient. Localization of neutrophil organelles in the gradient was shown by marker analysis as follows: {alpha}-mannosidase activity for azurophil granules; VEGF ELISA for specific granules; gelatinase ELISA for gelatinase granules; and albumin ELISA for the secretory vesicles/light membranes. TRAIL accumulation in all gradient fractions was determined by ELISA.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The capacity of IFN to induce TRAIL synthesis and release in various leukocyte types, including neutrophils, has been widely demonstrated in several laboratories [12 , 18 , 20 , 22 , 27 , 28 ]. In particular, we [22 ] and others [27 ] have recently demonstrated that human neutrophils, upon incubation with IFN-{alpha} or IFN-{gamma}, release in a time-dependent manner a sTRAIL, which fully retains proapoptotic activity against leukemic cell lines [22 ]. Similar findings have been reported by Koga and coworkers [28 ], who confirmed that IFN-{gamma} and IFN-{alpha} are able to induce TRAIL expression and release in neutrophils and PBMC, and other cytokines such as TNF-{alpha}, G-CSF, granulocyte macrophage-CSF, and transforming growth factor-ß proved ineffective. In addition, the same authors validated the cytotoxic effect of neutrophil-derived TRAIL toward leukemic cells [28 ], thus confirming and further highlighting the participation of neutrophils to TRAIL-mediated tumor immunosurveillance [3 , 30 ]. More recently, Ludwig et al. [25 ], by studying the mechanisms mediating the immunostimulatory effect of BCG in bladder cancer, provided further data supporting a TRAIL-mediated antitumoral activity of human neutrophils. These authors demonstrated that intravesical administration of BCG, which induces an early granulocytic influx and a T helper cell type 1 response, is followed by an increase of urinary levels of IFN-{gamma} and subsequently, of a functionally active sTRAIL. Of note, only neutrophils, but not other leukocytes present in voided urine after BCG instillation, expressed mTRAIL, thus demonstrating that neutrophils contribute strongly to TRAIL production/release and supporting a relevant TRAIL-mediated role of these cells in tumor surveillance [25 ].

In this work, we extend to IFN-ß the capacity to activate the production of bioactive sTRAIL in neutrophils, in agreement with similar effects exerted on monocytes/PBMC [47 ]. It is surprising that we also report that sTRAIL is only minimally released by neutrophils incubated with IFN-{alpha} or IFN-ß. This effect does not seem to be limited to type I IFNs, as similar findings were reported to occur in neutrophils treated with IFN-{gamma} for 4 h [28 ] and herein confirmed over a longer time course. In addition, we have uncovered that most of the newly synthesized TRAIL remains stored within intracellular compartments, which include the highly mobilizable secretory vesicles. Accordingly, we further show that such an intracellular pool of TRAIL can be mobilized rapidly onto the membrane or mostly (but not completely), secreted into the extracellular medium, following exposure of IFN-treated neutrophils to proinflammatory mediators, including TNF-{alpha}, fMLP, LPS, insoluble IC, Gp96, and CXCL8/IL-8. Of note, these latter agents were able to trigger the release of sTRAIL, without inducing TRAIL gene expression or TRAIL de novo synthesis. Taken together with previous findings [22 , 27 , 28 ], our data suggest that the release of sTRAIL may derive not only from an IFN-dependent transcriptional activation and de novo synthesis [22 ] but also from the mobilization of TRAIL intracellular stores. This latter event, however, occurs only in cells that have accumulated a pool of stored TRAIL and that are able to respond rapidly to proinflammatory or tumor-derived stimuli, such as in the case of IFN-treated neutrophils. It is worth mentioning here that the release of sBLyS (another TNF-related member) by human neutrophils has also been shown recently to be controlled by transcriptional and post-transcriptional events [7 , 38 ], highlighting the peculiarity of neutrophils as cells committed to produce, accumulate, and release members of the TNF family in a finely regulated manner.

To our knowledge, the demonstration of preformed, intracellular stores of TRAIL in activated neutrophils, which are readily mobilizable upon appropriate stimulation, is here demonstrated for the first time. However, the presence of similar TRAIL molecules has been observed already in the cytoplasm of the human Jurkat T cell line and T cell blasts, in which the rapid TRAIL release, following CD59 triggering or phytohemagglutinin (PHA) stimulation, was linked to the activation-induced cell death process [20 ]. In this context, Mariani and Krammer [19 ], using an in vitro cleavage assay, showed that the generation of recombinant sTRAIL appears to be dependent on the functional activity of cysteine proteases, pointing to a sTRAIL shedding regulated by membrane-bound proteases. However, further studies have better characterized the physiological metabolism of sTRAIL secretion in the same cells and shown the mobilization of TRAIL-containing microvesicles toward plasma membrane [20 ]. More recently, preformed cytotoxic sTRAIL stores have been observed in the human melanoma cell line MelJuSo, in which sTRAIL secretion appears to be associated with the release of microvesicles upon activation with PHA or with an {alpha}-melanocyte-stimulating hormone [21 ]. Concerning human neutrophils, the data provided in the present study seem to correlate with a vesicle mobilization, as revealed by the experiments performed by using PTF and JK, which unlike monensin, significantly inhibited TNF-{alpha}-, fMLP-, LPS-, and IC-induced mTRAIL expression or sTRAIL release, and by the subcellular fractionation experiments, which localized intracellular TRAIL to the highly mobilizable, secretory vesicles.

The physiological significance of preformed sTRAIL stores may be of the utmost importance for IFN-activated neutrophils, which are recruited to tumor or inflammatory sites, as in this way, they can rely on a potent, cytotoxic weapon. Upon exposure to a proinflammatory cytokine milieu, containing, for instance, TNF-{alpha} or other factors produced by exogenous elements or cancer cells infiltrating the tissue, significant amounts of biologically active sTRAIL could be in fact released and concentrated rapidly within a delimited area, in this way, avoiding unwanted or potentially toxic, systemic effects. In this context, we demonstrate that the hsp Gp96, an endoplasmic reticulum-resident chaperone, which can be overexpressed and released by neoplastic cells (including melanoma) undergoing damage or necrotic death [48 49 50 51 ], in turn, acting as a proinflammatory molecule with potent stimulatory activity on innate and adaptive immunity [48 , 52 ], is also able to induce sTRAIL release by IFN-activated neutrophils. This observation acquires a particular significance in light of our previous findings about the marked increase of sTRAIL serum levels and leukocyte-associated TRAIL in melanoma patients following IFN-{alpha} administration [22 ].

The functional significance of infiltrating leukocytes in neoplastic tissues is still unclear, as neutrophils have been considered either as promoting a cellular element of malignant growth and progression, or as important effectors of tumor immunosurveillance [3 ]. Of note, these contrasting findings have been associated with the expression of the interplay between the type and amount of cytokines and chemotactic factors released by tumor or tumor-associated cells and the degree of recruitment and activation of the intermingled granulocytes [3 ]. In this regard, the sTRAIL secretory induction by Gp96 on neutrophils appears as utmost importance for two reasons: It includes neutrophils among the elements (DC, macrophages), which are activated by this protein, independently of bound peptides [52 ]; the release of sTRAIL by IFN-activated neutrophils in response to a tumor-derived protein, along with the demonstration of polymorphonuclear cell infiltration in some tumors, further supports a direct involvement of these cells in tumor immunosurveillance. Furthermore, the capacity of neutrophils to transiently express surface TRAIL, which for most of the immune effector cells (i.e., activated monocytes, lymphocytes, DC, and NK cells) represents a potent effector mechanism, whereby they exert TRAIL-mediated cytotoxic activities, provides neutrophils with an additional weapon usable for killing activities toward neoplastic elements. In summary, the identification of an intracellular pool of TRAIL, which can be secreted or moved rapidly onto the membrane following appropriate stimulation, sheds light on a previously unknown tool possessed by IFN-activated human neutrophils in inflammatory, infectious, and neoplastic settings. Taken together, the findings presented in this work strongly support the prominent role played by neutrophils in innate and adaptive immunity [4 ] and further outline the importance of TRAIL as one of their most important effector molecules.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from Ministero dell’Istruzione, dell’Università e della Ricerca (PRIN 2003, FIRB, and 60%), Fondazione Cassa di Risparmio di Verona-Vicenza-Ancona e Belluno, and from Associazione Italiana per la Ricerca sul Cancro (AIRC).

Received August 2, 2005; accepted September 5, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Cassatella, M. A. eds. The neutrophil. An emerging regulator of inflammatory and immune response 2003 Karger Basel.
  2. Scapini, P., Lapinet-Vera, J. A., Gasperini, S., Calzetti, F., Bazzoni, F., Cassatella, M. A. (2000) The neutrophil as a cellular source of chemokines Immunol. Rev. 177,195-203[CrossRef][Medline]
  3. Di Carlo, E., Forni, G., Lollini, P., Colombo, M. P., Modesti, A., Musiani, P. (2001) The intriguing role of polymorphonuclear neutrophils in antitumor reactions Blood 97,339-345[Free Full Text]
  4. Cassatella, M. A. (1999) Neutrophil-derived proteins: selling cytokines by the pound Adv. Immunol. 73,369-509[Medline]
  5. Scapini, P., Laudanna, C., Pinardi, C., Allavena, P., Mantovani, A., Sozzani, S., Cassatella, M. A. (2001) Neutrophils produce biologically active macrophage inflammatory protein-3{alpha} (MIP-3{alpha})/CCL20 and MIP-3ß/CCL19 Eur. J. Immunol. 31,1981-1988[CrossRef][Medline]
  6. Ware, C. F. (2003) The TNF superfamily Cytokine Growth Factor Rev. 14,181-184[CrossRef][Medline]
  7. Scapini, P., Nardelli, B., Nadali, G., Calzetti, F., Pizzolo, G., Montecucco, C., Cassatella, M. A. (2003) G-CSF-stimulated neutrophils are a prominent source of functional BLyS J. Exp. Med. 197,297-302[Abstract/Free Full Text]
  8. Pitti, R. M., Marsters, S. A., Ruppert, S., Donahue, C. J., Moore, A., Ashkenazi, A. (1996) Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine family J. Biol. Chem. 271,12687-12690[Abstract/Free Full Text]
  9. Wiley, S. R., Schooley, K., Smolak, P. J., Din, W. S., Huang, C. P., Nicholl, J. K., Sutherland, G. R., Smith, T. D., Rauch, C., Smith, C. A. (1995) Identification and characterization of a new member of the TNF family that induces apoptosis Immunity 3,673-682[CrossRef][Medline]
  10. Wajant, H., Pfizenmaier, K., Scheurich, P. (2002) TNF-related apoptosis inducing ligand (TRAIL) and its receptors in tumor surveillance and cancer therapy Apoptosis 7,449-459[CrossRef][Medline]
  11. Yagita, H., Takeda, K., Hayakawa, Y., Smyth, M. J., Okumura, K. (2004) TRAIL and its receptors as targets for cancer therapy Cancer Sci. 95,777-783[CrossRef][Medline]
  12. Halaas, O., Vik, R., Ashkenazi, A., Espevik, T. (2000) Lipopolysaccharide induces expression of APO2 ligand/TRAIL in human monocytes and macrophages Scand. J. Immunol. 51,244-250[CrossRef][Medline]
  13. Griffith, T. S., Wiley, S. R., Kubin, M. Z., Sedger, L. M., Maliszewski, C. R., Fanger, N. A. (1999) Monocyte-mediated tumoricidal activity via the tumor necrosis factor-related cytokine, TRAIL J. Exp. Med. 189,1343-1354[Abstract/Free Full Text]
  14. Fanger, N. A., Maliszewski, C. R., Schooley, K., Griffith, T. S. (1999) Human dendritic cells mediate cellular apoptosis via tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) J. Exp. Med. 190,1155-1164[Abstract/Free Full Text]
  15. Yu, Y., Liu, S., Wang, W., Song, W., Zhang, M., Zhang, W., Qin, Z., Cao, X. (2002) Involvement of tumor necrosis factor-{alpha}-related apoptosis-inducing ligand in enhanced cytotoxicity of lipopolysaccharide-stimulated dendritic cells to activated T cells Immunology 106,308-315[CrossRef][Medline]
  16. Dorothee, G., Vergnon, I., Menez, J., Echchakir, H., Grunenwald, D., Kubin, M., Chouaib, S., Mami-Chouaib, F. (2002) Tumor-infiltrating CD4+ T lymphocytes express APO2 ligand (APO2L)/TRAIL upon specific stimulation with autologous lung carcinoma cells: role of IFN-{alpha} on APO2L/TRAIL expression and -mediated cytotoxicity J. Immunol. 169,809-817[Abstract/Free Full Text]
  17. Smyth, M. J., Cretney, E., Takeda, K., Wiltrout, R. H., Sedger, L. M., Kayagaki, N., Yagita, H., Okumura, K. (2001) Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) contributes to interferon {gamma}-dependent natural killer cell protection from tumor metastasis J. Exp. Med. 193,661-670[Abstract/Free Full Text]
  18. Zamai, L., Ahmad, M., Bennett, I. M., Azzoni, L., Alnemri, E. S., Perussia, B. (1998) Natural killer (NK) cell-mediated cytotoxicity: differential use of TRAIL and Fas ligand by immature and mature primary human NK cells J. Exp. Med. 188,2375-2380[Abstract/Free Full Text]
  19. Mariani, S. M., Krammer, P. H. (1998) Surface expression of TRAIL/Apo-2 ligand in activated mouse T and B cells Eur. J. Immunol. 28,1492-1498[CrossRef][Medline]
  20. Martinez-Lorenzo, M. J., Alava, M. A., Gamen, S., Kim, K. J., Chuntharapai, A., Pineiro, A., Naval, J., Anel, A. (1998) Involvement of APO2 ligand/TRAIL in activation-induced death of Jurkat and human peripheral blood T cells Eur. J. Immunol. 28,2714-2725[CrossRef][Medline]
  21. Martinez-Lorenzo, M. J., Anel, A., Alava, M. A., Pineiro, A., Naval, J., Lasierra, P., Larrad, L. (2004) The human melanoma cell line MelJuSo secretes bioactive FasL and APO2L/TRAIL on the surface of microvesicles. Possible contribution to tumor counterattack Exp. Cell Res. 295,315-329[CrossRef][Medline]
  22. Tecchio, C., Huber, V., Scapini, P., Calzetti, F., Margotto, D., Todeschini, G., Pilla, L., Martinelli, G., Pizzolo, G., Rivoltini, L., Cassatella, M. A. (2004) IFN{alpha}-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[Abstract/Free Full Text]
  23. Han, L. H., Sun, W. S., Ma, C. H., Zhang, L. N., Liu, S. X., Zhang, Q., Gao, L. F., Chen, Y. H. (2002) Detection of soluble TRAIL in HBV infected patients and its clinical implications World J. Gastroenterol. 8,1077-1080[Medline]
  24. Matsuyama, W., Yamamoto, M., Higashimoto, I., Oonakahara, K., Watanabe, M., Machida, K., Yoshimura, T., Eiraku, N., Kawabata, M., Osame, M., Ariumura, K. (2004) TNF-related apoptosis-inducing ligand is involved in neutropenia of systemic lupus erythematosus Blood 104,184-191[Abstract/Free Full Text]
  25. Ludwig, A. T., Moore, J. M., Luo, Y., Chen, X., Saltsgaver, N. A., O’Donnell, M. A., Griffith, T. S. (2004) Tumor necrosis factor-related apoptosis-inducing ligand: a novel mechanism for Bacillus Calmette-Guerin-induced antitumor activity Cancer Res. 64,3386-3390[Abstract/Free Full Text]
  26. Wandinger, K. P., Lunemann, J. D., Wengert, O., Bellmann-Strobl, J., Aktas, O., Weber, A., Grundstrom, E., Ehrlich, S., Wernecke, K. D., Volk, H. D., Zipp, F. (2003) TNF-related apoptosis inducing ligand (TRAIL) as a potential response marker for interferon-ß treatment in multiple sclerosis Lancet 361,2036-2043[CrossRef][Medline]
  27. Kamohara, H., Matsuyama, W., Shimozato, O., Abe, K., Galligan, C., Hashimoto, S., Matsushima, K., Yoshimura, T. (2004) Regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and TRAIL receptor expression in human neutrophils Immunology 111,186-194[CrossRef][Medline]
  28. Koga, Y., Matsuzaki, A., Suminoe, A., Hattori, H., Hara, T. (2004) Neutrophil-derived TNF-related apoptosis-inducing ligand (TRAIL): a novel mechanism of antitumor effect by neutrophils Cancer Res. 64,1037-1043[Abstract/Free Full Text]
  29. Liu, L. G., Tanaka, H., Ito, K., Ito, T., Sultana, T. A., Kyo, T., Kimura, A. (2005) Absence of gene mutation in TRAIL receptor 1 (TRAIL-R1) and TRAIL receptor 2 (TRAIL-R2) in chronic myelogenous leukemia and myelodysplastic syndrome, and analysis of mRNA expressions of TRAIL and TRAIL-related genes in chronic myelogenous leukemia Acta Haematol. 113,113-123[CrossRef][Medline]
  30. Cummins, J. M., Kohli, M., Rago, C., Kinzler, K. W., Vogelstein, B., Bunz, F. (2004) X-linked inhibitor of apoptosis protein (XIAP) is a nonredundant modulator of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in human cancer cells Cancer Res. 64,3006-3008[Abstract/Free Full Text]
  31. Cassatella, M. A., Bazzoni, F., Flynn, R. M., Dusi, S., Trinchieri, G., Rossi, F. (1990) Molecular basis of interferon-{gamma} and lipopolysaccharide enhancement of phagocyte respiratory burst capability. Studies on the gene expression of several NADPH oxidase components J. Biol. Chem. 265,20241-20246[Abstract/Free Full Text]
  32. Brunkhorst, B. A., Lazzari, K. G., Strohmeier, G., Weil, G., Simons, E. R. (1991) Calcium changes in immune complex-stimulated human neutrophils. Simultaneous measurement of receptor occupancy and activation reveals full population stimulus binding but subpopulation activation J. Biol. Chem. 266,13035-13043[Abstract/Free Full Text]
  33. Hayashi, F., Means, T. K., Luster, A. D. (2003) Toll-like receptors stimulate human neutrophil function Blood 102,2660-2669[Abstract/Free Full Text]
  34. Kjeldsen, L., Sengelov, H., Borregaard, N. (1999) Subcellular fractionation of human neutrophils on Percoll density gradients J. Immunol. Methods 232,131-143[CrossRef][Medline]
  35. McDonald, P. P., Bovolenta, C., Cassatella, M. A. (1998) Activation of distinct transcription factors in neutrophils by bacterial LPS, interferon-{gamma}, and GM-CSF and the necessity to overcome the action of endogenous proteases Biochemistry 37,13165-13173[CrossRef][Medline]
  36. De Togni, P., Cabrini, G., Di Virgilio, F. (1984) Cyclic AMP inhibition of fMet-Leu-Phe-dependent metabolic responses in human neutrophils is not due to its effects on cytosolic Ca2+ Biochem. J. 224,629-635[Medline]
  37. Gaudry, M., Bregerie, O., Andrieu, V., El Benna, J., Pocidalo, M. A., Hakim, J. (1997) Intracellular pool of vascular endothelial growth factor in human neutrophils Blood 90,4153-4161[Abstract/Free Full Text]
  38. Scapini, P., Carletto, A., Nardelli, B., Calzetti, F., Roschke, V., Merigo, F., Tamassia, N., Pieropan, S., Biasi, D., Sbarbati, A., Sozzani, S., Bambara, L., Cassatella, M. A. (2005) Proinflammatory mediators elicit the secretion of the intracellular B-lymphocyte stimulator (BLyS) pool that is stored in activated neutrophils: implications for inflammatory diseases Blood 105,830-837[Abstract/Free Full Text]
  39. Scapini, P., Crepaldi, L., Pinardi, C., Calzetti, F., Cassatella, M. A. (2002) CCL20/macrophage inflammatory protein-3{alpha} production in LPS-stimulated neutrophils is enhanced by the chemoattractant formyl-methionyl-leucyl-phenylalanine and IFN-{gamma} through independent mechanisms Eur. J. Immunol. 32,3515-3524[CrossRef][Medline]
  40. McDonald, P. P., Gasperini, S., Calzetti, F., Cassatella, M. A. (1998) Modulation by interferon-{gamma} of the production and gene expression of IL-1 receptor antagonist in human neutrophils Cell. Immunol. 184,45-50[CrossRef][Medline]
  41. Meda, L., Gasperini, S., Ceska, M., Cassatella, M. A. (1994) Modulation of proinflammatory cytokine release from human polymorphonuclear leukocytes by {gamma} interferon Cell. Immunol. 157,448-461[CrossRef][Medline]
  42. Radsak, M. P., Hilf, N., Singh-Jasuja, H., Braedel, S., Brossart, P., Rammensee, H. G., Schild, H. (2003) The heat shock protein Gp96 binds to human neutrophils and monocytes and stimulates effector functions Blood 101,2810-2815[Abstract/Free Full Text]
  43. Cha, S. S., Kim, M. S., Choi, Y. H., Sung, B. J., Shin, N. K., Shin, H. C., Sung, Y. C., Oh, B. H. (1999) 2.8 A resolution crystal structure of human TRAIL, a cytokine with selective antitumor activity Immunity 11,253-261[CrossRef][Medline]
  44. Tartakoff, A. M. (1983) Perturbation of vesicular traffic with the carboxylic ionophore monensin Cell 32,1026-1028[CrossRef][Medline]
  45. Rizoli, S. B., Rotstein, O. D., Parodo, J., Phillips, M. J., Kapus, A. (2000) Hypertonic inhibition of exocytosis in neutrophils: central role for osmotic actin skeleton remodeling Am. J. Physiol. Cell Physiol. 279,C619-C633[Abstract/Free Full Text]
  46. Faurschou, M., Borregaard, N. (2003) Neutrophil granules and secretory vesicles in inflammation Microbes Infect. 5,1317-1327[CrossRef][Medline]
  47. Ehrlich, S., Infante-Duarte, C., Seeger, B., Zipp, F. (2003) Regulation of soluble and surface-bound TRAIL in human T cells, B cells, and monocytes Cytokine 24,244-253[CrossRef][Medline]
  48. Rivoltini, L., Castelli, C., Carrabba, M., Mazzaferro, V., Pilla, L., Huber, V., Coppa, J., Gallino, G., Scheibenbogen, C., Squarcina, P., Cova, A., Camerini, R., Lewis, J. J., Srivastava, P. K., Parmiani, G. (2003) Human tumor-derived heat shock protein 96 mediates in vitro activation and in vivo expansion of melanoma- and colon carcinoma-specific T cells J. Immunol. 171,3467-3474[Abstract/Free Full Text]
  49. Castelli, C., Rivoltini, L., Rini, F., Belli, F., Testori, A., Maio, M., Mazzaferro, V., Coppa, J., Srivastava, P. K., Parmiani, G. (2004) Heat shock proteins: biological functions and clinical application as personalized vaccines for human cancer Cancer Immunol. Immunother. 53,227-233[CrossRef][Medline]
  50. Missotten, G. S., Journee-de Korver, J. G., de Wolff-Rouendaal, D., Keunen, J. E., Schlingemann, R. O., Jager, M. J. (2003) Heat shock protein expression in the eye and in uveal melanoma Invest. Ophthalmol. Vis. Sci. 44,3059-3065[Abstract/Free Full Text]
  51. Heike, M., Frenzel, C., Meier, D., Galle, P. R. (2000) Expression of stress protein gp96, a tumor rejection antigen, in human colorectal cancer Int. J. Cancer 86,489-493[CrossRef][Medline]
  52. Nicchitta, C. V. (2003) Re-evaluating the role of heat-shock protein-peptide interactions in tumor immunity Nat. Rev. Immunol. 3,427-432[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Leukoc. Biol.Home page
M. P. Simons, K. G. Leidal, W. M. Nauseef, and T. S. Griffith
TNF-related apoptosis-inducing ligand (TRAIL) is expressed throughout myeloid development, resulting in a broad distribution among neutrophil granules
J. Leukoc. Biol., March 1, 2008; 83(3): 621 - 629.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
M. Mosca, N. Polentarutti, G. Mangano, C. Apicella, A. Doni, F. Mancini, M. De Bortoli, I. Coletta, L. Polenzani, G. Santoni, et al.
Regulation of the microsomal prostaglandin E synthase-1 in polarized mononuclear phagocytes and its constitutive expression in neutrophils
J. Leukoc. Biol., August 1, 2007; 82(2): 320 - 326.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
N. Tamassia, V. Le Moigne, F. Calzetti, M. Donini, S. Gasperini, T. Ear, A. Cloutier, F. O. Martinez, M. Fabbri, M. Locati, et al.
The MYD88-Independent Pathway Is Not Mobilized in Human Neutrophils Stimulated via TLR4
J. Immunol., June 1, 2007; 178(11): 7344 - 7356.
[Abstract] [Full Text] [PDF]

Home page
 J. Exp. Med.Home page
S. Jaillon, G. Peri, Y. Delneste, I. Fremaux, A. Doni, F. Moalli, C. Garlanda, L. Romani, H. Gascan, S. Bellocchio, et al.
The humoral pattern recognition receptor PTX3 is stored in neutrophil granules and localizes in extracellular traps
J. Exp. Med., April 16, 2007; 204(4): 793 - 804.
[Abstract] [Full Text] [PDF]