Granulocyte-macrophage colony-stimulating factor modulates tapasin ...

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Granulocyte-macrophage colony-stimulating factor modulates tapasin expression in human neutrophils. Souad El Ouakfaoui, Dominique Heitz, Robert Paquin, ...
Granulocyte-macrophage colony-stimulating factor modulates tapasin expression in human neutrophils Souad El Ouakfaoui, Dominique Heitz, Robert Paquin, and Andre´ D. Beaulieu Laboratoire de Recherche sur l’Arthrite, Centre de Recherche du Centre Hospitalier de l’Universite´ Laval, Que´bec, Canada

Abstract: Differential display-polymerase chain reaction (DD-PCR) was used to evaluate changes in mRNA expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) treated human neutrophils to better understand how this cytokine affects the functions of neutrophils at the molecular level. Although a variety of cDNA fragments were identified as modulated by GM-CSF with the use of DD-PCR, one fragment in particular, NGS-17 (neutrophil GM-CSF-stimulated fragment #17), was characterized. The NGS-17 fragment hybridized to a 3.8-kb mRNA that encodes for a protein of a predicted molecular mass of 47.6 kDa. After cloning and sequencing, this gene was found to code for the recently sequenced tapasin or TAP-A protein. Immunoprecipitation and immunoblotting studies using anti-tapasin antibodies showed that tapasin is expressed in neutrophils and is associated with the MHC class I-TAP complex. Moreover, tapasin expression was found to be induced by dimethyl sulfoxide and by retinoic acid in HL-60 cells. This is the first report on the expression of tapasin in human neutrophils. It provides novel information, at the molecular level, on how GM-CSF enhances the functions of these cells. J. Leukoc. Biol. 65: 205–210; 1999. Key Words: MHC class-I · HL-60 cells · polymerase chain reaction

INTRODUCTION Neutrophils are mobile cells that can ingest and kill invading microorganisms. They can influence the afferent as well as the efferent arms of the immune response [1]. Moreover, they are often the most abundant cellular component of inflammatory lesions. This is the case in rheumatoid arthritis, where neutrophils accumulate abundantly in the synovial fluid of patients. The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is a 22-kDa monomeric glycoprotein that controls growth, differentiation, death, and functions of multipotential hematopoietic progenitor cells. It also enhances the functional activities of mature cells involved in host defense. Pathologically, it might be involved in inflammatory reactions and autoimmunity. Furthermore, the evidence is growing that

GM-CSF has potential clinical utility in enhancing the rate of neutrophil recovery after cancer chemotherapy and in enhancing the immune response [2, 3]. Future clinical applications may include a role for GM-CSF in the treatment of HIVassociated neutropenia and defects in neutrophil functions [4]. In neutrophils, GM-CSF acts as a powerful activating factor [5, 6]. It increases not only phagocytosis and antibody celldependant cytotoxicity but also stimulates secretion of cytokines and soluble inflammatory mediators, as well as expression of important cell surface proteins [7–9]. Recently, it was shown that activated neutrophils undergo significant de novo synthesis of many proteins, particularly in response to GM-CSF, tumor necrosis factor a (TNF-a), and N-formyl-methionylleucyl-phenylalanine (fLMP) [5, 10]. The cytokines interleukin (IL)-2, IL-13, and IL-4 were found to have similar effects on neutrophils [11–13]. However, the nature of all the proteins modulated by the above proinflammatory cytokines remain to be fully characterized. We used the recently described method of differential display-polymerase chain reaction (DD-PCR) [14] to analyze gene expression in activated neutrophils. More specifically, we characterized cDNA fragments corresponding to mRNA molecules that are modulated by GM-CSF. Here, we report the characterization of one of these cDNA fragments. It was found to be part of the recently cloned and sequenced gene coding for the protein tapasin or TAP-A. Immunoblotting and immunoprecipitation experiments allowed us to demonstrate that tapasin, in neutrophils, is assembled in the multimeric MHC class I-TAP complex. In addition, we observed that tapasin expression is induced in undifferentialted HL-60 cells by the differentiating agents dimethyl sulfoxide (DMSO) and retinoic acid (RA), with tapasin expression being maximal in fully differentiated cells. These findings demonstrate that tapasin is likely to play an active role in MHC class I restricted antigen processing in neutrophils.

Abbreviations: DD-PCR, differential display polymerase chain reaction; GM-CSF, granulocyte-macrophage colony-stimulating factor; TNF-a, tumor necrosis factor a; fMLP, N-formyl-methionyl-leucyl-phenylalanine; IL, interleukin; DMSO, dimethyl sulfoxide; RA, retinoic acid; FCS, fetal calf serum; PBS, phosphate-buffered saline; ECL, enhanced chemiluminescence. Correspondence: Andre´ D. Beaulieu, Laboratoire de Recherche sur l’Arthrite, Centre de Recherche du Centre Hospitalier de l’Universite´ Laval, Que´bec, Canada G1V 4G2. E-mail: [email protected] Received July 15, 1998; revised October 23, 1998; accepted October 25, 1998.

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Heidelberg, Germany). Each screening [15] was performed on 5 3 105 colonies.

MATERIALS AND METHODS Cell isolation and culture conditions Human neutrophils were freshly isolated from venous blood of different healthy donors by centrifugation over Ficoll-Hypaque (Pharmacia Biotech, Quebec, Canada) as previously described [5]. Cell viability of neutrophils was 95% as determined by trypan blue staining. Isolated neutrophils were resuspended at a density of 10 3 106/mL in RPMI supplemented with 100 U/mL penicillin/ streptomycin and 100 U/mL L-glutamine (GIBCO-BRL) and 1% human autologous serum. They were incubated at 37°C, 5% CO2 for 4 h in either the absence or presence of GM-CSF at 3 nM. Recombinant human GM-CSF (sp. act. 9 3 106 units/mg) was a gift from the Genetics Institute (Boston, MA). HL-60 cells were maintained in RPMI-1640 (GIBCO-BRL) supplemented with 100 U/mL penicillin/streptomycin, 100 U/mL L-glutamine (GIBCO-BRL), and 10% fetal calf serum (FCS, Hyclone, Logan, UT). HL-60 cells were differentiated to neutrophils by treatment with 1.25% DMSO or 1 µM RA for 7 days. The terminally differentiated cells were then subjected to GM-CSF treatment as described above.

RNA extraction and Northern blotting analysis Cells were washed in RPMI and prepared for immediate RNA extraction using Trizol reagent (Life Technologies, GIBCO-BRL) according to the suggested protocol. Northern blots were performed using 10 µg of extracted RNA. Equal loading and RNA integrity were verified by ethidium bromide staining of the 28S and 18S ribosomal RNA bands. After migration on 1% agarose denaturing gel, RNA was transferred on Hybond N membrane (Amersham International, Little Chalfont, UK) during 3 h with the VacuGene TM Xl vacuum blotting system according to the manufacturer’s instructions (Pharmacia). Membranes were then UV cross-linked for 3 min. Prehybridizations and hybridizations were performed in 50% formamide at 42°C [15]. After washing, membranes were exposed to Kodak X-Omat films (Eastman-Kodak, Rochester, NY) at 280°C with an intensifying screen. All the fragments used as probes were purified on low-melting-point agarose gels. Probes were labeled by random priming using the Prime-A-Gene labeling system (Promega Corp., Madison, WI) and a-[32P]dCTP (Amersham International). GAPDH (579 bp), a probe used to test equal loading of RNA, was obtained by PCR using the primers 5’-ACCAGCGCTGCTTTTAACTCT-3’ and 5’-CAGTAGAGGCAGGGATGATGTTCT-3’ based on the sequence published in GenBank under the accession number M17851.

PCR differential display of mRNA

Rapid amplification of 5’-cDNA ends (RACE) Marathon RACE-ready human spleen cDNA (Clontech Laboratories, Heidelberg, Germany) was used to clone the 5’ end of NGS-17. This was performed by using the NGS-17 specific primer 5’-AGGCAGCCAGAAGGTCCC-3’ and the AP1 anchor primer provided in the kit. One microliter of this reaction served as template in a nested PCR reaction using the same gene-specific primer and the primer AP2 from Clontech Laboratories. PCR reaction was run on low-meltingpoint 1% agarose gel and the band of interest that hybridized to NGS-17 probe was eluted from the gel and cloned according to the TA cloning procedure (Invitrogen Corp.). The ligation product was transformed in E. coli DH5-a strain according to the Hanahan procedure [16] and positive clones were sequenced.

Sequencing of DNA PCR fragments, library, and RACE clones were sequenced using the T7 polymerase sequencing kit (Pharmacia Biotech AB) based on the dideoxymediated chain termination method [17]. Vector primers and various NGS-17specific oligonucleotides were used. Sequence reactions were run on 6% polyacrylamide gels [15] and exposed overnight at room temperature to Kodak Biomax film (Eastman-Kodak).

DNA and protein sequence searches Homology searches were performed in EMBL/GenBank/DDBJ databases using DNA or protein sequences. DNA Strider program was helpful to find possible open reading-frames, to analyze the protein sequence composition, as well as to calculate the pI and to produce hydropathy curves. Comparisons of DNA and protein sequences were carried out using the FASTA and the TFASTA programs from the GCG package version 8.1 (Genetic Computer Group, 1994). The MOTIF program was used to find protein consensus regions.

Metabolic labeling, immunoprecipitation, and immunoblotting

Seeking for the entire cDNA of NGS-17 was performed by screening of a homemade neutrophil cDNA library. Briefly, mRNA from GM-CSF-stimulated human neutrophils were isolated and reverse transcribed using both oligo(dT) and random primers. The cDNA fragments obtained were cloned in pBluescript SK(2) vector and transformed [16] in Escherichia coli Sure strain (Stratagene,

The anti-tapasin antibodies for immunoprecipitation (Rgp48C) and for Western blotting (Rgp48N) were kindly provided by Dr. P. Cresswell (Yale University). The mouse monoclonal antibody w6/32 anti-HC class I was a gift from Dr. R. Roy (Laval University). Neutrophils freshly isolated from different donors as described above were incubated with or without 3 nM GM-CSF at 37°C for 4 h in the presence of 0.2 mCi/mL [35S]cysteine and [35S]methionine (Amersham). Cells were harvested, washed with ice-cold phosphate-buffered saline (PBS), and lysed in 1% digitonin (Boehringer Mannheim) lysis buffer in TBS [10 mM Tris, 150 mM NaCl (pH 7.4)] supplemented with 1.5 µg/mL iodoacetamide and protease inhibitors (2 mM phenylmethylsulfonyl fluoride, 10 µg/mL leupeptin, 30 µg/mL aprotinin, 10 µg/mL pepstatin; Boehringer Mannheim) [18]. For immunoprecipitation studies, the lysates were cleared and added to a rabbit antibody to tapasin (Rgp48C), previously bound to protein A Sepharose beads (Pharmacia), for 1 h at 4°C [18]. Immunoprecipitates were washed twice with 0.1% digitonin and once with distilled water before being separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), treated by 1 M sodium salicylate, dried, and exposed to Kodak film overnight or longer at 280°C. For Western blotting, total or immunoprecipitated digitonin lysates were resolved by 10% SDS-PAGE and proteins were electrophoretically transferred to PVDF membranes (Millipore) at 100 mA for 1 h. The membranes were blocked by incubation in 1% BSA (Sigma) in TBS and probed with antibody to tapasin (Rgp48N) at a 1:3000 dilution for 1 h at 25°C, washed, and incubated in a 1:50,000 dilution of goat anti-rabbit horseradish peroxidase secondary reagent (Jackson Immunoresearch Lab). Reactive bands were detected by chemiluminescence (ECL, Amersham) [18].

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Differential mRNA display [14] was performed using RNAmap kits A and B (GenHunter Co., Brookline, MA) according to the manufacturer’s instructions. Briefly, 0.2 µg of DNase-treated total RNA (using Message Clean kit; GeneHunter Corp., Nashville, TN) from neutrophils incubated or not with GM-CSF were reverse transcribed with the following specific anchored oligo(dT) primers: T12MA, T12MC, T12MG (M may be dA, dC, or dG; 5 min at 65°C, 60 min at 37°C, and 5 min at 95°C). The first strand cDNAs were then amplified in the presence of 35S-dATP (Amersham International) using the corresponding T12MN and an arbitrary 10-mer (AP1–20) as primers. After an initial 5-min denaturation period at 94°C, amplification was performed in 40-µL reaction for 40 cycles (30 s at 94°C, 2 min at 40°C, 30 s at 72°C), followed by 5 min extension at 72°C. The amplified fragments were resolved by electrophoresis on a 6% denaturing polyacrylamide gel, which was dried and exposed overnight to a Kodak X-OMAT film (Eastman-Kodak). Bands of interest were cut from the gel, eluted, and reamplified with the same sets of primer pairs. They then served as probes to hybridize Northern blots with mRNA from neutrophils treated or not with GM-CSF. This was done to confirm their modulation by this cytokine. Modulated fragments were cloned in pCRII vector with the use of TA cloning kit (Invitrogen Corp., San Diego, CA).

cDNA library screening


Journal of Leukocyte Biology


NGS-17 expression in HL-60 cells

Differentially expressed mRNAs in GM-CSF stimulated human neutrophils identified by PCR differential display analysis

We investigated the expression of NGS-17 mRNA during differentiation of the promyelocytic cell line HL-60. HL-60 cells achieved the terminal differentiation stage to neutrophillike cells after 7 days of incubation with either DMSO or RA. Figure 2 shows that NGS-17 mRNA is expressed in undifferentiated HL-60 cells at a lower level than that observed in fully differentiated cells (Fig. 2, A and B). In addition, in RAdifferentiated HL-60 cells, the expression of NGS-17 mRNA was observed to be increased by GM-CSF treatment, whereas in undifferentiated cells this effect was not as apparent (Fig. 2C).

Total RNA extracts from neutrophils, cultured in either the absence or presence of GM-CSF at 3 nM for 4 h, were used to identify genes that are responsive to proinflammatory stimuli. This concentration of the cytokine and time of stimulation were chosen based on the results of previous experiments on neutrophils performed in our laboratory using GM-CSF [5, 10]. We tested 20 out of the 30 possible combinations of primer sets between three degenerated anchored oligo(dT) primers (T12MA, T12MG, and T12MC) and 10 short arbitrary 10-mers (AP1–10) [14]. Many cDNA fragments were differentially expressed when comparing untreated and GM-CSF-treated neutrophils. These results were reproducible using neutrophils from several different donors. One 300-bp cDNA fragment that we termed NGS-17 was selected for further characterization (Fig. 1A). This fragment was reamplified by PCR using the same set of primers used in differential display (i.e. T12MG and AP8: 5’-GGATTGTGCG-3’) and subcloned by the TA cloning procedure in pCRII vector. The cloned cDNA fragment was used as a probe for Northern blot analysis to confirm that the expression of this cDNA is indeed modulated by GM-CSF. In Northern blot, NGS-17 hybridized to an mRNA of approximately 3.8 kb. Basal levels of the mRNA were present in non-activated neutrophils but expression was markedly stimulated after GM-CSF treatment of the cells (Fig. 1B). The stimulatory effect was detectable after 2 h of GM-CSF treatment, optimal at 4 h, and maintained up to 6 h (not shown).

NGS-17 is identical to tapasin A search in the EMBL/GenBank/DDBJ database showed that the NGS-17 predicted protein was identical to a protein the sequence of which had just recently been entered in the database by two other groups and called tapasin (accession number: AF009510) [19] or TAP-A (accession number: Y13582) [20]. However, our approach allowed us to sequence the entire 3’ untranslated region. This information was entered in the database and is available under accession number AF029750.

Tapasin expression in human neutrophils Tapasin protein expression was examined in human neutrophils using an antibody raised to tapasin [18]. Immunoblot analysis using digitonin lysates of neutrophils showed tapasin as a band of approximately 48 kDa (Fig. 3). Tapasin was expressed in unstimulated neutrophils at basal levels and its expression was increased in neutrophils treated 4 h with GM-CSF. Results on Western blots were reproducible using three different donors. To confirm the association of tapasin with MHC class I,

Fig. 1. Identification of NGS-17 gene by differential display of mRNA by PCR. (A) Total RNA was extracted using neutrophils freshly isolated from two different donors (1 and 2). Unstimulated control (C) cells were compared to cells stimulated with 3 nM GM-CSF (GM) for 4 h. The RNA was then reverse transcribed using T12MG as primer. PCR was carried out using T12MG and AP8. PCR fragments were displayed on a 6% DNA sequencing gel. The GM-CSF-stimulated fragment NGS-17 is indicated by an arrow at the right of the gel. (B) Northern blot analysis of NGS-17. Ten micrograms of total RNA extracted from control neutrophils and neutrophils stimulated with GM-CSF were run on 1% agarose denaturing gel, blotted, and probed with the NGS-17 fragment. NGS-17 mRNA position is shown by an arrow at the right. Equal loading was assessed by hybridization with a GAPDH cDNA probe. This result is representative of six separate experiments.

El Ouakfaoui et al.

Tapasin expression in stimulated neutrophils


Fig. 2. Induction of NGS-17 mRNA expression during differentiation of HL-60 cells by DMSO and RA. Total RNA was extracted from HL-60 cells incubated with (1) or without (2) DMSO (A) or RA (B) for 7 days to induce full differentiation to mature neutrophils. (C) Terminally RA-differentiated (1) and undifferentiated (2) HL-60 cells were treated either in the absence (C) or presence (GM) of 3 nM GM-CSF for 4 h. Northern blots were performed with extracted RNA and probed with a NGS-17 probe. Comparable RNA loading was confirmed by ethidium bromide staining of 28S rRNA. This result is representative of three separate experiments.

immunoprecipitation studies were undertaken using either the anti-tapasin antibody (Rgp48C) or the mouse monomorphic anti-class I antibody (w6/32). We used digitonin lysates from neutrophils 35S-metabolically labeled and stimulated with GM-CSF for 4 h. Both antibodies coprecipitated the same complex of bands (Fig. 4A): a band at 43 kDa corresponding to HC class I molecule; a band at 48 kDa identified as tapasin in an experiment to be described in Figure 4B; a band at approximately 58 kDa that could represent calreticulin; two bands at approximately 68 and 69 kDa that could represent TAP1 and TAP2 molecules. As expected, the HC class I band was more abundant in the immunoprecipitate when using the

anti-HC antibody (w6/32) than when using the anti-tapasin (Rgp48C) antibody. The position of the tapasin band in this complex was confirmed by Western blotting. The total digitonin lysate and

Fig. 3. Modulation of tapasin expression in human neutrophils. Neutrophils from three donors were isolated and incubated either in the absence (C) or presence (GM) of 3 nM GM-CSF for 4 h at 37°C. Cleared lysates, obtained from 1% digitonin total neutrophil extracts, were resolved by 10% SDS-PAGE and blotted to a PVDF membrane. Tapasin was detected with the anti-tapasin antibody (Rgp48N) and revealed by enhanced chemiluminescence. The molecular mass markers (kDa) are indicated at the left and the tapasin band is indicated at the right.

Fig. 4. Tapasin is associated with the MHC class I complex in human neutrophils. Digitonin extracts were prepared from [35S]cysteine and methionine-labeled neutrophils and stimulated with GM-CSF as previously described. (A) Cleared extracts were immunoprecipitated with either the rabbit anti-tapasin (Rgp48C) antibody or the anti-HC class I mouse monoclonal antibody (w6/32). Immunoprecipitates were run on 10% SDS-PAGE and the film was exposed for 48 h. The co-precipitated complex is composed of HC, tapasin, calreticulin, and the heterodimer TAP. (B) Western blotting of total (T) and w6/32-immunoprecipitated (I) digitonin extracts. Tapasin was detected using the anti-tapasin (Rgp48N) antibody and revealed by enhanced chemiluminescence. The molecular mass markers (kDa) are indicated at the left of the gel and the tapasin band is indicated at the right. The other visible band above the tapasin band corresponds to the mouse immunoglobulin that cross-reacts with the secondary antibody used in detection by enhanced chemiluminescence. These results are representative of four separate experiments.

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Journal of Leukocyte Biology

the w6/32-immunoprecipitate were reacted with the antitapasin antibody (Rgp48N). This revealed tapasin to be present in the total lysate (T in Fig. 4B) as well as in the w6/32immunoprecipitate (I in Fig. 4B).

DISCUSSION In this report, we show for the first time that neutrophils produce tapasin. This protein was first characterized in Swei cells [19] and in a B lymphoblastoid cell line [20]. It is associated with the MHC class I-TAP complex. Tapasin is considered to be an obligatory mediator of the assemblage of calreticulin/H chain/b2 microglobulin with TAP. Moreover, its expression was shown to correct the defective recognition of virus-infected 220 cells by class I-restricted cytotoxic T cells, establishing a critical functional role for tapasin in MHC class I-restricted antigen processing [19]. MHC class I molecules are expressed on the cell surface of almost all nucleated mammalian cells. Their main function is to transport and present peptides, derived from intracellularly degraded proteins, to cytotoxic T cells (CTL). They are also directly involved in the process leading to maturation and selection of a functional CD81 T cell repertoire [21]. The association of peptide fragments with MHC class I molecules is believed to be a requirement for the assembly of the MHC class I molecules in the endoplasmic reticulum (ER) and transportation of MHCTAP complex from ER to the cell surface [22]. In light of recent studies, TAP and tapasin are considered to be highly specific chaperones for class I molecules [23]. The precise role of tapasin in the loading of peptides by MHC class I molecules is not yet definitively assessed. However, several hypotheses are suggested: tapasin could stabilize empty class I molecules, function as the invariant chain does in the class II complex, or simply interact with TAP to ensure the proximity to the source of translocated peptides [19]. Our findings on the modulation of tapasin expression by GM-CSF in neutrophils are consistent with previous observations showing that this cytokine induces a 4.5-fold increase in the synthesis of MHC class I proteins by neutrophils [7, 24]. Moreover, GM-CSF-activated neutrophils are able to modulate and to maintain a constant plasma membrane expression of MHC class I molecules via posttranscriptional regulation of protein synthesis, as well as by release and internalization of MHC class I proteins [8]. Our study did not assess whether the increase in tapasin mRNA after GM-CSF treatment is due to increased transcription or decreased mRNA turnover. Experiments addressing this remain to be performed. However, of greater interest is the biological significance of our findings. Although the expression of MHC class I molecules on neutrophils has been known for some time, no evidence suggests that these cells are antigen-presenting cells to the same extent that monocyte/macrophages have been shown to be [24]. However, it is of interest that new roles for MHC class I molecules besides antigen presentation are being discovered. Recently, it has been shown that NK cells have two classes of receptors regulating NK cell activity, one exerting a positive effect, and another with a repressive function. The latter were shown to mediate their effect through MHC class I molecules [25, 26]. It

could therefore make physiological sense that GM-CSF, a potent agonist that increases the survival of neutrophils, would use the MHC class I system to protect these cells from NK cell activity. In contrast to this hypothesis, however, MHC class I molecules have been shown to serve as receptors for mediating apoptosis [27]. Although GM-CSF is a potent agent that retards apoptosis in neutrophils, one could speculate that the increased expression of MHC class I molecules on neutrophils is a mechanism used by these cells to ultimately regulate their lifespan once they have fully served their roles as GM-CSFactivated cells. All of this of course is speculative and will remain to be shown through studies specifically addressing these points. Finally, we have shown that RA and DMSO induce the synthesis of tapasin in HL-60 cells. These agents are known to induce the full differentiation of HL-60 cells toward mature neutrophils. Our findings provide new information on the events associated with the differentiation of neutrophils by RA and DMSO. Because it is known that the functional expression of the MHC class I system is dependent on tapasin, it makes physiological sense that tapasin expression is associated with the development of mature neutrophils. Furthermore, it is noteworthy that, in a different cell system, RA was shown to increase the expression of the transcription factor H-2RIIBP, a member of the nuclear hormone receptor superfamily, and responsible for the enhancement of MHC class I genes [28]. Whether or not this is the mechanism used in neutrophils remains to be determined. In conclusion, we show for the first time that human neutrophils constitutively produce tapasin in association with a fully assembled multimeric MHC class I-TAP complex, and that its expression is up-regulated on GM-CSF treatment. We also demonstrate that tapasin mRNA expression increases during RA- or DMSO-driven neutrophilic differentiation of HL-60 cells. These findings shed additional light, at the molecular level, on how GM-CSF enhances the functions of neutrophils. Further studies will be necessary, however, to establish the role of tapasin in neutrophilic development and survival.

ACKNOWLEDGMENTS We would like to thank Dr. Peter Cresswell for generously supplying antibodies and for his review of the manuscript. The authors are also grateful to Barbara Leclerc for DNA sequencing, Claude Potvin for the preparation of the cDNA library, and Dr. Manon Richard and Dr. Fatiha Chandad for their helpful scientific discussion and support during the course of this work.

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