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Laboratoire de Recherche sur l’Arthrite et l’Inflammation, Department of Medicine, Faculty of Medicine, Centre Hospitalier de l’Université Laval, Sainte-Foy, Québec, Canada
Correspondence: Dr André Beaulieu, Laboratoire de Recherche sur l’Arthrite et l’Inflammation, Centre Hospitalier de l’Université Laval, 2705 Boul. Laurier, Sainte-Foy, Québec, Canada G1V 4G2. E-mail: andre.beaulieu{at}crchul.ulaval.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, IL-1, LPS, and Matrigel®. CLECSF6 bears the immunoreceptor tyrosine-based inhibition motif (ITIM) involved in signal transduction resulting in the inhibition of leukocyte activation. We propose that some neutrophil activators modulate the expression of CLECSF6 at the mRNA (GM-CSF, IL-3, IL-4, and IL-13) or protein (TNF-, IL-1, LPS, and Matrigel®) levels in ways that block ITIM-based transduction of anti-inflammatory signals and therefore promote inflammation.
Key Words: inhibition • receptor • mRNA • protein • cytokine
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-), interleukin (IL)-1, and other proinflammatory agents such as lipopolysaccharide (LPS) and extracellular matrix (ECM) proteins are activators of neutrophil functions [1
2
3
4
5
6
7
8
]. The signals for the control of the inflammatory response are transduced by cell receptors among which is a growing family of proteins bearing an immunoreceptor tyrosine-based motif involved in inhibition (ITIM) or in activation (ITAM) of immune activities of leukocytes. ITIM-bearing receptors transduce signals for the inhibition of leukocyte activation via binding to phosphatases such as SHP-1 (SH2-containing tyrosine phosphatase 1), SHP-2, and SHIP (SH2-containing inositol 5-phosphatase) [9 10 11 12 ]. These phosphatases can have an inhibitory effect on the JAK/STAT and Ras/MAP kinase transcription activation pathways [13 14 15 16 ]. Two types of receptors were shown to bear ITIMs: immunoglobulin-like receptors and C-type lectins. Lectins are sugar-binding proteins. The subfamily of type C is composed of calcium-dependent, carbohydrate-binding proteins that are involved in adhesion, endocytosis, and humoral defense [17 18 19 ].
In our search for new genes that are modulated in answer to proinflammatory stimuli in neutrophils, we used differential-display polymerase chain reaction (DDPCR) to analyze messenger RNA (mRNA) expression in neutrophils treated or not with GM-CSF. We have found a complementary DNA (cDNA) with an open-reading frame (ORF) bearing a C-type-lectin-like sequence and an inhibitory motif ITIM. The corresponding gene was called CLECSF6 (C-type lectin superfamily 6). We demonstrate that it produces two mRNA forms in neutrophils. The long version encodes a transmembrane protein that presents the features of a repressor of cell activation. The short version misses a region that is believed to be responsible for the oligomerization of its protein product and thus, for the formation of functional receptors. Proinflammatory agents such as TNF-, IL-1, LPS, and Matrigel® cause a down-regulation of the expression of the CLECSF6 protein at the surface of neutrophils. Other agonists of neutrophils, GM-CSF, IL-3, IL-4, and IL-13, induce the overproduction of the short mRNA version of CLECSF6. These results suggest that proinflammatory agents acting at the mRNA or the protein level down-regulate the expression of functional ITIM-bearing CLECSF6 surface receptors and that this could be a step toward proinflammatory activation of neutrophils. To our knowledge, this is the first detailed study of the expression and modulation of an ITIM-bearing lectin in neutrophils.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell isolation and culture
Human neutrophils were isolated from peripheral blood by centrifugation on Ficoll-Paque (endotoxin-tested levels lower than the sensitivity of the test; Amersham Pharmacia Biotech, Baie D’Urfé, Canada), as described previously [20
]. They were cultured at a concentration of 10 million cells per ml at 37°C and 5% CO2 in RPMI 1640 (contains the lowest levels of endotoxin attainable; Life Technologies) supplemented with 2 mM glutamine, 50 U/ml penicillin, and 50 mg/ml streptomycin in the presence of 1% autologous serum (Life Technologies). Cytokines were added when appropriate as described in the text.
HL-60 cells (cat. no. CCL-240, American Type Culture Collection, Manassas, VA) were cultured at 37°C and 5% CO2 in RPMI 1640 supplemented with 10% fetal bovine serum and antibiotics. Cells at passage 12 or less were used. For differentiation into neutrophils, they were treated with 1.25% DMSO or 1 µM retinoic acid for up to 7 days as described [21 , 22 ].
For flow cytometry, whole blood was diluted 1/10 in RPMI 1640 with supplements listed above, but no serum was added. Dilute blood was cultured at 37°C and 5% CO2 for times between 1 and 24 h, as described in Results. For the combination of dexamethasone with LPS, cells were pretreated for 0, 1, or 2 h with dexamethasone before the addition of LPS.
RNA extraction
Neutrophils were isolated as described above and cultured for various times with or without cytokines as described in figure legends. Total RNA was prepared from 25 to 30 million cells using 1 ml TRIzol reagent (Life Technologies) following the recommended protocol.
DDPCR
DDPCR was performed as recommended by Liang and Pardee [23
]. Total RNA from neutrophils, untreated or stimulated with 30 ng/ml GM-CSF for 2 h, was used. Briefly, RNA was treated with DNase using the MessageClean kit (GenHunter Corp., Nashville, TN). Total RNA was then reverse-transcribed to cDNA and amplified. The PCR amplifications were performed with the RNAimage kit (GenHunter Corp.) using Amplitaq DNA polymerase (Perkin Elmer, Foster City, CA) and [35S] dATP. Amplification conditions were 94°C for 1 min; 40 repeats of 94°C for 30 s; 40°C for 2 min and 72°C for 30 s; and 72°C for 5 min. Products of the DDPCR were migrated on a 6% polyacrylamide gel, dried, and exposed overnight. Bands corresponding to modulated fragments were cut out from the dried gel, eluted, and reamplified using the same primer pairs. Reamplified PCR products were hybridized to Northern blots of stimulated and untreated neutrophils from at least three donors to confirm that they corresponded to modulated RNAs.
Northern blots
For Northern blots, 10 µg total RNA from untreated and stimulated cells was migrated on 1.4% agarose gels containing formaldehyde [24
] and was vacuum-transferred [24
] on Hybond-N membranes (Amersham Pharmacia Biotech).
Probes and hybridizations
Probe DDb27 is a 437 bp PCR fragment obtained by DDPCR, which was reamplified directly using the primers AP20 and T11MC (GenHunter Corp.) or after cloning in pCR2.1 using universal primers. Probe glyceraldehyde 3-phosphate (GAPD) is a 579 bp PCR product obtained with the primers 5'-ACCAGCGCTGCTTTTAACTCT-3' and 5'-CAGTAGAGGCAGGGATGATGTTCT-3' (DDBJ/EMBL/GenBank accession number M17851). Amplification conditions were 94°C for 5 min; 35 repeats of 94°C for 30 s; 60°C for 30 s and 72°C for 30 s; and 72°C for 10 min. All the probes were cut from a low melting point agarose 1% gel using a standard protocol [24
] and were labeled without further purification. Probes were labeled by random priming with [32P] dCTP using the Prime-A-Gene labeling system (Promega Corp., Madison, WI) and were hybridized to the membranes using a standard protocol [24
].
Cloning and sequencing of PCR fragments
The PCR fragments were ligated to the vector pCR2.1 using the TA cloning kit from Invitrogen Corp. (San Diego, CA), according to the recommended protocol. Competent Escherichia coli cells, strain DH5, were prepared according to the protocol of Hanahan [25
] and were transformed with the ligation products by heat shock. Bacterial colonies were screened for positive clones by hybridization [24
]. Plasmid DNA extractions were performed using a standard protocol [26
]. Cloned cDNA fragments were sequenced by the Service d’Analyse et de Synthèse de l’Université Laval (Québec, Canada).
Cloning of neutrophil ORFs using a homemade library
We used a cDNA pool from an available homemade neutrophil library to clone the ORFs of the new gene. The library was prepared with cDNA of neutrophils stimulated with GM-CSF and cloned in pBluescript as described elsewhere [27
, 28
]. DNA was extracted from a pool of library clones by standard methods [26
] and used as a template for the PCR amplification of CLECSF6-related fragments. Primers corresponding to the 5' and 3' ends of the ORF and containing a BamHI restriction site (lowercase letters below; 5' end, 5'-GCggatccATGACTTCGGAAATCACTTATGCT-3'; 3' end, 5'-GCggatccGAGAATGTTCAGTTCATAAGTGGA-3') were used to amplify the cDNA. Amplification conditions were as described above for probe preparation. PCR products were cloned and sequenced as described above.
Preparation of anti-CLECSF6 antibodies
The extracellular portion of CLECSF6 was amplified by PCR using primers containing a BamHI restriction site (lowercase letters): 5' end, 5'-GCggatccCAAAAATATTCTCAGCTTCTTGAA-3'; 3' end, 5'-GCggatccGAGAATGTTCAGTTCATAAGTGGA-3'. It was cloned in the BamHI site of pGEX-2T (Amersham Pharmacia Biotech) and expressed in E. coli strain BL21(DE3) as a fusion with glutathione-S-transferase (GST). The polypeptide obtained, GST-DDb27CRD, was used to immunize rabbits to obtain a polyclonal antiserum. Briefly, it was isolated by separation on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel [29
], followed by cutting and drying the acrylamide band containing the polypeptide. The dried gel was hydrated in phosphate-buffered saline (PBS) and was mixed with an equal volume of complete Freund’s adjuvant to form an emulsion. The emulsion was injected into rabbits (150 µg protein/rabbit; four sites of injection). This was followed by three boosts at 3-week intervals with the same dose of polypeptide. Sera were evaluated for polypeptide recognition by enzyme-linked immunosorbent assay (ELISA) and by Western blotting.
A peptide with the sequence LGPQRSVCEMMKIHL (mapping at positions 223–237 in Fig. 3 and called 27P4) was synthesized by the Service de Séquence de Peptides de l’Est du Québec (Canada) using the multiple antigenic-peptide system [30 , 31 ]. Anti-27P4 antibodies were raised in rabbits as described above except that the peptides were resuspended directly in PBS and that 500 µg peptide was used for each immunization (five sites of injection per rabbit).
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Western blots and immunodetection
HL-60 cells were cultured and differentiated as described above, and cell samples were harvested every day. Cells were lysed in Laemli’s sample buffer [29
] containing 100 mM dithiothreitol and protease inhibitors (CompleteTM cocktail tablets, Roche Diagnostics Corp., Indianapolis, IN) and were boiled for 7 min. Lysates of 2 x 106 cells were separated by SDS-polyacrylamide gel electrophoresis [29
] on a 10% polyacrylamide gel and were transfered to ImmobilonTM-P membranes (Millipore Corp., Mississauga, Canada). Blots were blocked in TBST (20 mM Tris, 500 mM NaCl, pH 7.4, 1% Tween 20) containing 2% milk powder. They were incubated with a mouse monoclonal anti-SHP-1 (diluted 1/50; Santa Cruz Biotechnology, Santa Cruz, CA), washed, and incubated with a goat anti-mouse peroxydase conjugate (diluted 1/5000; Sigma Chemical Co., St. Louis, MO). All incubations and washes were done in TBST with 0.2% milk powder. Blots were revealed with the Western blot chemiluminescence reagent Plus (NEN Life Sciences Products, Boston, MA) using X-OMATTM AR films (Eastman Kodak Co., Rochester, NY).
Computer analyses
The search for ORFs and the translation of cDNA sequences into polypeptide sequences were performed using the DNA Strider program version 1.2 (C. Marck, Centre d’Etudes de Sarclay, Gif-sur-Yvette, France) [32
]. The MOTIFS program of the GCG Wisconsin package version 10.0 (Genetic Computer Group, Madison, WI) was used to find protein consensus regions. The PSORT program [33
] was used to predict the cellular localization of CLECSF6. The Winmdi program version 2.8 was used to analyze the flow cytometry results (http://facs.scripps.edu/software.html). Quantifications were made with an Image Laser Scanner Storm 860 (Molecular Dynamics, Sunnyvale, CA) with Image Quant software.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DDb27 is a fragment of a C-type lectin
A comparison with the DDBJ/EMBL/GenBank database showed that fragment DDb27 is homologous to an expressed sequence tag (EST), which was obtained from Research Genetics Inc. (Huntsville, AL) and studied further (accession number AA677149). It is a 1124 bp cDNA that contains one large ORF encoding a polypeptide of 237 amino acids with a calculated molecular weight of 27.5 kDa. Another EST (accession number AA380065) displayed a sequence identical to the 5' end of EST AA677149 plus a 145 bp stretch at the 5' end, suggesting that the complete cDNA for the sequenced gene was 1269 bp in length aside from the poly-A tail (accession number AF109146).
The analysis of the deduced amino acid sequence of the cDNA suggested that the ORF codes for a transmembrane protein (Fig. 1 ; transmembrane domain is underlined) with an N-terminal cytoplasmic domain and a C-terminal extracellular domain [33 , 34 ]. A comparison of its sequence with public databases revealed that the extracellular domain presents a high degree of similarity with the carbohydrate recognition domain (CRD) of group II C-type lectins. The HUGO/GDB Nomenclature Committee approved the name CLECSF6 for the gene that corresponds to this cDNA. The comparison with the DDBJ/EMBL/GenBank database also showed that it is homologous to clone BAC RPCI11-69M1, assigned to chromosome position 12p13.1. Many C-type lectins have been mapped near this position.
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Neutrophils produce two ORFs related to CLECSF6
On Northern blots of neutrophil RNA, the DDb27 probe detected two mRNAs of 1.1 kb and 1.4 kb repeatedly (Figs. 2A
and 3A
). To elucidate the origin of the double mRNA hybridization signal, the cloning of CLECSF6 ORFs expressed in neutrophils was undertaken. CLECSF6-related sequences were amplified by PCR using a template made of neutrophil total cDNA. Primers used contained the sequences of the 5' and 3' ends of the ORF. The PCR products were cloned and hybridized with the DDb27 probe. Two types of clones were obtained. The first type (long version) was identical to the cDNA sequence described above. The second one (short version) was missing 99 nucleotides, producing an ORF shorter by 33 codons with a predicted weight of 23.5 kDa (accession number AF200738). Figure 1
shows in bold the 34 missing residues that are replaced by a single lysine in the short version. Taken together, our results strongly suggest that the long version corresponds to the 1.4 kb mRNA and the short version, to the 1.1 kb signal. It is interesting that the missing residues correspond to the region of the neck that is believed to be responsible for the oligomerization of C-type lectin receptors [37
, 38
].
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, IL-1ß, IL-8, G-CSF, Matrigel®, LPS, and TNF-, did not modulate the accumulation of CLECSF6 mRNAs significantly (Fig. 2A)
nor did IL-6 and fMLP (N-formyl-met-leu-phe; not shown). The Northern blot shown in Figure 2A
was also hybridized with GAPD to compare RNA loadings (Fig. 2B)
. The effect of four anti-inflammatory agents on the expression of the DDb27-related mRNAs was also studied. Among them, IL-4 and IL-13 triggered the accumulation of CLECSF6 mRNAs in a pattern similar to the one observed with GM-CSF and IL-3 (Fig. 3A and 3B) . The observation that IL-4 and IL-13 mimic the effect of GM-CSF and IL-3 on the mRNA expression of CLECSF6 supports previous studies that these so-called anti-inflammatory agents can activate neutrophils paradoxically [39 40 41 42 43 ]. IL-10 and dexamethasone did not influence the CLECSF6 mRNA accumulation.
We observed repeatedly that the shorter signal corresponds to the most abundant DDb27-related mRNA species expressed in neutrophils stimulated with GM-CSF, IL-3, IL-4, and IL-13. In other cases, both forms are expressed at similar levels. Quantitative representations of the data are presented in Figures 2C and 3C . In most cases, the short and long mRNA versions of CLECSF6 were evaluated separately and are shown to be comparable. However, in the cases of GM-CSF, IL-3, IL-4, and IL-13, the relative expression of the 1.4-kb mRNA is too low to be evaluated independently because like most of the mRNA bands, it does not appear on short expositions (not shown). This means that it is expressed at much lower levels than the 1.1-kb mRNA upon treatment with these four agents and that the strong signal observed on Northern blots is caused by the accumulation of the short, 1.1-kb mRNA.
Myeloid cell maturation is required for CLECSF6 expression
Although neutrophils and other myeloid cells express the CLECSF6 mRNA, we as well as Bates et al. [36
] observed that the myeloid precursor cell line HL-60 does not (Fig. 4A
, lane 1). We performed the differentiation of HL-60 cells into neutrophil-like cells by the use of retinoic acid and DMSO. The expression of the CLECSF6 mRNAs was monitored throughout the differentiation process. Figure 4
(A–C), shows that nontreated HL-60 cells do not produce CLECSF6 mRNAs. However, the mRNA expression rises during differentiation with either agent to reach low-to-medium levels after 7 days. This suggests that the expression of the CLECSF6 mRNAs is a feature of mature neutrophils. To confirm that HL-60 cells did differentiate in our hands, we monitored the expression of the SHP-1 protein by immunoblotting. This phosphatase has been shown to be up-regulated in HL-60 cells during differentiation by DMSO or retinoic acid [44
]. Our results were consistent with the ones of Uesugi et al. [44
] showing a low expression of SHP-1 in resting cells and a rapid raise upon differentiation (Fig. 4D
and 4E)
. We also observed repeatedly the size reduction expected for differentiated cells when comparing resting cells and 7-day-treated cells by microscopic observation and by the variation of forward scatter in flow cytometry experiments (not shown).
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Fresh whole blood was labeled with anti-GST-DDb27CRD antibodies, and the expression of CLECSF6 at the surface of neutrophils was analyzed by flow cytometry. Neutrophils were found to express CLECSF6 at their surface. Whole blood was diluted in RPMI medium and cultured for various lengths of time in the presence and absence of cytokines. The time course of the expression of CLECSF6 on the surface of cultured neutrophils was monitored. When no cytokine was added, levels of surface expression of CLECSF6 were stable for many hours and slightly reduced after 24 h (Fig. 5A ). The CLECSF6 surface expression was not modulated by changes in the pH of the medium in a range varying from 6.2 to 8.7 (not shown).
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Immunoblotting analyses were undertaken to study the total expression of CLECSF6 in neutrophils. No signal was obtained, even when gels were overloaded with neutrophil extracts, suggesting that the total expression of CLECSF6 is lower than the sensitivity threshold of anti-GST-DDb27CRD antibodies for solubilized proteins. Similar results have been obtained in a system studying the P-glycoprotein (PGP) in the ACHN renal adenocarcinoma cell line [46 ]. Although the mRNA expression of PGP has been demonstrated, and its protein expression has been observed by flow cytometry, no protein was detected by immunoblotting using an antibody known to work in these conditions. This has been attributed to the low protein expression level in the cells studied [46 ].
Neutrophil surface expression of the CLECSF6 protein is down-modulated by proinflammatory stimuli
The effect of proinflammatory agents on the surface expression of CLECSF6 was studied. None of them enhanced the expression of the protein. However, TNF-, IL-1, and LPS down-modulated the surface expression of CLECSF6 (Fig. 5B)
in a dose-dependant manner (not shown). A dose of 10 ng/ml (TNF-, IL-1) or 0.1 ng/ml (LPS) was enough to induce the down-modulation, although doses up to 100 ng/ml (TNF-, IL-1) or 10 ng/ml (LPS) had not significantly influenced the RNA accumulation caused by these agents. Typically, the minimal expression levels were reached after 4 h of treatment. In the case of IL-1, only half of the donors reacted to IL-1 treatments by the down-modulation of CLECSF6 surface expression. Other proinflammatory agents such as GM-CSF, IL-ß, IL-3, IL-6, IL-8, G-CSF, and fMLP did not modulate the expression of CLECSF6. In the cases of GM-CSF and IL-3, although an important CLECSF6 mRNA accumulation had been observed in cells treated with a dose of 1 ng/ml (GM-CSF) to 10 ng/ml (IL-3), no modulation of the surface protein accumulation could be observed at concentrations up to 1 µg/ml.
Another important stimulus of the activation of neutrophils is the contact with ECM proteins during transendothelial migration [6 7 8 ]. To evaluate the effect of ECM proteins on the expression of CLECSF6, we cultured diluted blood on a layer of Matrigel®, an ECM of murine origin. The culture of neutrophils on Matrigel® caused a down-modulation of the CLECSF6 expression (Fig. 5B) .
We studied the effect of some anti-inflammatory agents, namely IL-4, IL-10, IL-13 (up to 1 µg/ml), and dexamethasone (up to 4 µg/ml) on the CLECSF6 protein surface expression. None of these modulated the CLECSF6 surface expression. In the cases of IL-4 and IL-13, a concentration of 10 ng/ml had been sufficient to induce the accumulation of CLECSF6 mRNA. When used in combination with LPS or TNF-, none of the anti-inflammatory agents were able to prevent the two proinflammatory agents from down-modulating the CLECSF6 surface expression (not shown).
The surface expression and down-modulation of CLECSF6 are confirmed by anti-peptide antibodies
To confirm the results obtained with the anti-GST-DDb27CRD antibodies, we repeated some of the flow cytometry analyses described above using anti-peptide antibodies. A 15-mer peptide with the sequence corresponding to residues 223–237 in Figure 1
and called 27P4 was synthesized and used to prepare polyclonal antibodies in rabbits. Although less sensitive than anti-GST-DDb27CRD antibodies, anti-27P4 antibodies revealed the same down-modulation effect of TNF- and LPS on the CLECSF6 surface expression in neutrophils (Fig. 5C)
. This confirmed that the protein analyzed with the anti-GST-DDb27CRD antibodies is indeed CLECSF6.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cells that express CLECSF6 mRNAs produce a short and a long version. The long version was found simultaneously by us and by another group, who called it DCIR [36 ]. Here, we describe for the first time the sequence of the short mRNA version. It is identical to the long version except for a gap of 99 nucleotides and thus appears to be a product of alternate splicing rather than another gene of the same family. The short version lacks 33 amino acids in a region called the neck. Another C-type lectin, Dectin-2, was shown to exist in multiple forms including one devoid of the neck region [47 ]. The neck was proposed to be responsible for the multimerization of lectins. This region contains a cysteine residue (number 91 in Fig. 1 ). In another C-type lectin, CD69, a cysteine at a similar position was shown to be involved in intermolecular interactions [37 , 38 ]. This suggests that two versions of the CLECSF6 protein could be produced: one with a full-length neck, which would oligomerize, and one lacking the neck, which would remain in a monomeric form. Evidence accumulated in the study of ASGRP, a C-type lectin similar to CLECSF6, reveals that it is expressed as an oligomer at the cell surface and that this structure is required for efficient ligand binding. The oligomers are formed of nonidentical subunits encoded by separate but related genes [48 , 49 ]. No information on the oligomerization state or putative partners of CLECSF6 is available to date. However, data obtained with CD69 and ASGRP suggest that the ratio of long and short CLECSF6 proteins produced in a cell could affect the formation of putative CLECSF6 oligomers and ultimately regulate its affinity for ligands. We observed that GM-CSF and IL-3 stimulate the accumulation of the short CLECSF6 mRNA. Therefore, in vivo, these cytokines could contribute to the reduction of the CLECSF6 activity by promoting the synthesis of the CLECSF6 version that cannot oligomerize. It is interesting that in addition to GM-CSF and IL-3, IL-4 and IL-13 also trigger the short mRNA of CLECSF6 to accumulate, and IL-10 does not. IL-4 and IL-13, although usually referred to as anti-inflammatory cytokines, were shown to have cell-activation features [39 40 41 42 43 ]. To our knowledge, no such activity was shown for IL-10. Therefore, the cell-activating capacity of IL-4 and IL-13 could necessitate the inactivation of CLECSF6, and this would be achieved by the increase of the ratio of the short CLECSF6 version to the long one.
We observed that Matrigel® down-modulates the CLECSF6 surface protein expression. Neutrophils are activated during transendothelial migration by the effect of ECM proteins [6
7
8
]. LPS, TNF-, and IL-1 also modulate the surface expression of CLECSF6 in neutrophils. Our results suggest that the down-modulation of surface-expressed CLECSF6 is related to the promotion of neutrophil activation and/or inflammation. LPS is a stimulator of TNF- expression [50
]. However, dexamethasone, an inhibitor of LPS-triggered TNF- expression [51
], did not impair LPS modulation of CLECSF6 expression. Therefore, LPS is able to modulate CLECSF6 independently of its stimulation of TNF- production in neutrophils. TNF- also triggers cells to produce cytokines such as IL-1 and ß, IL-6, and IL-8. Most of these cytokines were unable to modulate the CLECSF6 expression. The effects of IL-1 on CLECSF6 were variable. This means that the TNF--driven down-modulation of CLECSF6 does not require the production of these cytokines.
It is interesting that the effect of many agents on the CLECSF6 mRNA expression did not correlate with their effect on the CLECSF6 protein expression. Many cases are known where mRNA and proteins are not coregulated. Many examples have been shown in neutrophils [52 53 54 55 ]. Furthermore, a comparative study of the mRNA and protein expression in liver cells showed that the most expressed mRNA do not correlate with the most expressed proteins [56 ]. Our results suggest that the CLECSF6 expression can be modulated at many levels.
We propose that cellular activation toward inflammation could be actively repressed by a process in which CLECSF6 would be involved. The inhibition of this active repression by various proinflammatory agents could be a critical step toward inflammatory reactions. Mechanisms that could be used by proinflammatory agents include the down-modulation of the surface expression of CLECSF6. It is interesting that we observed down-modulation caused by TNF-, IL-1, LPS, and Matrigel®. Second, the balance between the long and short forms of CLECSF6 could produce an effect on its activity. The up-regulation of the expression of the short form of CLECSF6 mRNA by GM-CSF and IL-3 could serve this purpose. IL-4 and IL-13, which have been shown to possess cell-activating properties and to modulate the mRNA expression in the same way as GM-CSF and IL-3, could activate neutrophils by this putative mechanism. Last, the phosphorylation state of the ITIM bore by CLECSF6 could play an important role in its activity. Our inability to detect the CLECSF6 protein by immunoblotting impaired us from verifying these last two hypotheses.
ITIM-bearing receptors were found to bind phosphatases such as SHP-1, SHP-2, and SHIP. SHP-1 and SHIP were found to be involved in the negative regulation of leukocyte activities [10 , 11 ]. SHIP was shown to augment the rate of neutrophil apoptosis [57 ]. The overexpression of SHP-1 and SHP-2 proteins was found in neutrophils of patients with severe neutropenia [58 ]. Therefore, these mediators of ITIM-related down-activation are active in neutrophils. Work is in progress to assess the interaction between these phosphatases and CLECSF6 in neutrophils.
Our analysis of the CLECSF6 expression in neutrophils is in good agreement with the analysis of the DCIR expression in dendritic cells published by Bates et al. [36 ]. Both groups have observed that it is expressed only in immune cells and have found that its surface expression can be modulated by some proinflammatory agents. Therefore, CLECSF6 could play a similar role in neutrophils and in dendritic cells.
The development of chronic inflammatory diseases such as rheumatoid arthritis is believed to be a result of the disruption of the balance between pro- and anti-inflammatory signals that control the activities of leukocytes [1 2 3 4 5 ]. The down-modulation of the expression of CLECSF6 in neutrophils could be an important step of activation toward inflammation. This could be particularly significant in neutrophils because they are the first leukocytes to accumulate at inflammatory sites, and they participate in the recruitment of other cell types [50 , 59 ]. The study of CLECSF6 and its ligands will provide a better understanding of the process of inflammation and could also lead to the development of a mean to control the activities of neutrophils.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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Received June 7, 2001; revised December 12, 2001; accepted January 9, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-stimulated synoviocytes Genomics 53,137-145[Medline]
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