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FDF03, a Novel Inhibitory Receptor of the Immunoglobulin Superfamily, Is Expressed by Human Dendritic and Myeloid Cells¹

Nathalie Fournier^*, Lionel Chalus^*, Isabelle Durand^*, Eric Garcia^*, Jean-Jacques Pin^*, Tatyana Churakova, Segal Patel, Constance Zlot, Dan Gorman, Sandra Zurawski, John Abrams, Elizabeth E. M. Bates^* and Pierre Garrone^2,*

^* Laboratory for Immunological Research, Schering-Plough, Dardilly, France; DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA 94304

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, we describe human FDF03, a novel member of the Ig superfamily expressed as a monomeric 44-kDa transmembrane glycoprotein and containing a single extracellular V-set Ig-like domain. Two potential secreted isoforms were also identified. The gene encoding FDF03 mapped to chromosome 7q22. FDF03 was mostly detected in hemopoietic tissues and was expressed by monocytes, macrophages, and granulocytes, but not by lymphocytes (B, T, and NK cells), indicating an expression restricted to cells of the myelomonocytic lineage. FDF03 was also strongly expressed by monocyte-derived dendritic cells (DC) and preferentially by CD14⁺/CD1a^- DC derived from CD34⁺ progenitors. Moreover, flow cytometric analysis showed FDF03 expression by CD11c⁺ blood and tonsil DC, but not by CD11c^- DC precursors. The FDF03 cytoplasmic tail contained two immunoreceptor tyrosine-based inhibitory motif (ITIM)-like sequences. When overexpressed in pervanadate-treated U937 cells, FDF03 was tyrosine-phosphorylated and recruited Src homology-2 (SH2) domain-containing protein tyrosine phosphatase (SHP)-2 and to a lesser extent SHP-1. Like engagement of the ITIM-bearing receptor LAIR-1/p40, cross-linking of FDF03 inhibited calcium mobilization in response to CD32/FcRII aggregation in transfected U937 cells, thus demonstrating that FDF03 can function as an inhibitory receptor. However, in contrast to LAIR-1/p40, cross-linking of FDF03 did not inhibit GM-CSF-induced monocyte differentiation into DC. Thus, FDF03 is a novel ITIM-bearing receptor selectively expressed by cells of myeloid origin, including DC, that may regulate functions other than that of the broadly distributed LAIR-1/p40 molecule.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Inhibitory receptors containing one or several cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIM)>³> are members of the C-type lectin family or Ig superfamily (Ig-SF). The ITIM is generally defined by the consensus sequence L/V/IxYxxL/V that, following phosphorylation on tyrosine, recruits Src homology-2 (SH2) domain-containing protein tyrosine phosphatases SHP-1 and/or SHP-2, as well as the SH2 domain-bearing inositol phosphatase (SHIP) in the case of FcRIIB (1, 2, 3, 4, 5). ITIM-bearing receptors negatively regulate cellular functions when coaggregated with stimulatory receptors that signal through an immunoreceptor tyrosine-based activation motif (ITAM). The growing number of novel ITIM-bearing receptors identified thus far and their expression by virtually all leukocyte populations suggest that inhibitory receptors may regulate various aspects of immune responses (6, 7), as well as the fate of nonhemopoietic cells, as described for the SHPS-1/SIRP molecules (8, 9).

Among APCs, dendritic cells (DC) are unique leukocyte populations using their role as sentinels to capture Ag at the periphery of an organism and by their capacity to present processed Ag to both CD4⁺ and CD8⁺ naive T cells, thus initiating primary immune responses (for review, see Refs. 10 and 11). In our effort to identify receptors involved in Ag capture and presentation by human DC, we previously described an ITIM-containing C-type lectin, designated DCIR, that displayed features intermediate between NK cell receptors and typical type II lectins involved in ligand internalization (12). While the function of DCIR is not yet determined, its restricted expression on APCs, and particularly on DC, has lead to questions on the potential role(s) of inhibitory receptors in DC function. Different groups have previously reported the presence on APCs of the ITIM-bearing molecules ILT3/LIR-5, ILT2/LIR-1, and ILT4/LIR-2 belonging to the ILT/LIR/MIR family (13, 14, 15, 16). It has been shown that these Ig-SF members can function as negative regulators of monocyte and DC activation, most probably through recruitment of SHP-1 (15, 17, 18, 19, 20). Both ILT2/LIR-1 and ILT4/LIR-2, but not ILT3/LIR-5, bind to HLA class I molecules (14, 17, 19). While the physiological role of inhibitory receptors for HLA class I on monocytes and DC is not yet understood, the demonstration that ILT2/LIR-1 is also a receptor for the UL18 molecule, an homologue of human MHC class I encoded by CMV (19, 21), suggests that pathogens may use inhibitory receptors of APCs to down-regulate immune responses. In addition to its inhibitory activity, ILT3/LIR-5 is also internalized following cross-linking on monocytes and may be involved in Ag capture and loading for presentation into MHC class II (15). As reported for the FcRIIB (22, 23), internalization of ILT3/LIR-5 might be mediated through its cytoplasmic ITIMs, because the ITIM sequence contains the tyrosine-based internalization motif Yxx, where represents any hydrophobic residue (24). Finally, the recent demonstration that the broadly expressed inhibitory receptor LAIR-1/p40 (25) can inhibit the differentiation of monocytes into DC in response to GM-CSF (26) suggests that ITIM-bearing receptors may also play an important regulatory role during the early commitment of DC precursors by interfering with signaling mediated through growth factor receptors.

In the present study, we report the cloning and characterization of FDF03, a novel member of the Ig-SF that has a restricted expression in myelomonocytic cells including in vitro-derived DC and in vivo CD11c-positive DC. The FDF03 cytoplasmic tail contains two ITIM-like sequences and preferentially associates with SHP-2 in pervanadate-activated monocytic U937 cells. Moreover, we show that coaggregation of FDF03 with CD32/FcRII inhibits intracellular Ca²⁺ mobilization in U937 cells, as does LAIR-1/p40, thus indicating that FDF03 possesses an inhibitory activity. However, in contrast to LAIR-1/p40, FDF03 failed to inhibit monocyte differentiation into DC in response to GM-CSF, suggesting that FDF03 may be involved in the regulation of different pathways or may use different inhibitory signals to that of LAIR-1/p40.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Hemopoietic factors, cytokines, and reagents

All cultures were performed in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine (all from Life Technologies, Gaithersburg, MD), and 160 µg/ml gentamicin (Schering-Plough, Levallois-Perret, France). The source and sp. act. of recombinant human cytokines used in this study have been previously described (12). The following factors were used at optimal concentration: GM-CSF (100 ng/ml), TNF- (2.5 ng/ml), stem cell factor (SCF), and M-CSF (25 ng/ml), G-CSF (25 ng/ml), and IL-4 (50 U/ml). In some experiments, cells were activated with 1 ng/ml PMA (Sigma, St. Louis, MO) and 1 µg/ml ionomycin (Calbiochem, La Jolla, CA) or with 25 ng/ml LPS (Sigma).

Cell preparations

Umbilical cord blood samples, peripheral blood samples, and tonsils were obtained according to institutional guidelines. PBMC were purified from human peripheral blood by Ficoll-Hypaque centrifugation. Monocytes were purified from PBMC by centrifugation over a 50% Percoll gradient followed by immunomagnetic depletion of contaminating T, B, and NK cells as described elsewhere (12). The isolated cells were >95% CD14⁺ as judged by staining with anti-CD14 mAb and flow cytometric analysis. Granulocytes were purified from whole blood, T lymphocytes (>95% CD3⁺) were purified from PBMC by immunomagnetic depletion, and B cells (>98% CD19⁺) were isolated from tonsils essentially as previously described (12, 27). CD34⁺ hemopoietic progenitors were purified from umbilical cord blood as previously described (28). In all experiments, the isolated cells were 80–95% CD34⁺ as judged by staining with anti-CD34 mAb.

Granulocytes and macrophages were also generated in vitro from CD34⁺ hemopoietic progenitors in the presence of G-CSF and SCF for 12 days and M-CSF and SCF for 12 days, respectively. Aliquots of cells were further treated with 1 ng/ml PMA and 1 µg/ml ionomycin for 1 and 6 h and then pooled. Activated and nonactivated cells were lysed for RNA extraction.

Generation of DC from CD34⁺ progenitors and from monocytes

Cultures of CD34⁺ cells were established in the presence of SCF, GM-CSF, TNF-, and 5% AB⁺ pooled human serum, as described (28, 29). By day 6, human serum was removed and cells were further cultured in the presence of GM-CSF and TNF- until day 12. At this time point, aliquots of cells were activated with PMA and ionomycin for 1 and 6 h, then pooled and lysed for RNA extraction. For analysis of FDF03 expression by flow cytometry, cells were collected at the time points indicated within the text. In some experiments, CD1a⁺ and CD14⁺ DC precursor subsets were sorted at day 6 by flow cytometry and further cultured until day 12 in the presence of GM-CSF and TNF-. Monocyte-derived DC were produced by culturing purified blood monocytes for 6 days in the presence of GM-CSF and IL-4 (30). In some experiments, 5 x 10⁵ monocyte-derived DC per well (24-well culture plate) were further activated with LPS (25 ng/ml) for 72 h or by coculture with 4 x 10⁴ irradiated (7500 rad) murine fibroblastic L cells untransfected or transfected with the cDNA for CD40 ligand (27).

Northern blot analysis

Human mRNA adult tissue blots (Clontech, Palo Alto, CA) were hybridized with a 377-bp DNA probe from the 3'-end of FDF03 cDNA, produced by PCR amplification of a region defined by the oligonucleotides 5'-CAGCAGCGGACTAAAGCCAC (forward primer) and 5'-GCATCTCGCTCCATTATCAA (reverse primer). This fragment was labeled with ³²P-dCTP using the High Prime kit (Boehringer Mannheim, Meylan, France). Membranes were prehybridized and hybridized under standard conditions (31). Low and high stringency washes were 2x SSC/0.2% SDS and 0.2x SSC/0.2% SDS, respectively, each done twice for 30 min. The membranes were incubated with Biomax MR film (Kodak, Rochester, NY) for 7 days.

RNA, DNA, and RT-PCR analysis

Cells were lysed, total RNA was extracted (32), and first-strand cDNAs were prepared after DNase I treatment (in the presence of RNase inhibitor) of 5 µg of total RNA using oligo(dT) primers (Pharmacia, Uppsala, Sweden) and the Superscript kit (Life Technologies). Synthesis of cDNAs was controlled by performing RT-PCR using ß-actin primers. RT-PCR with the primers 5'-CCCCTTCTCCTTCTATTACC (forward primer) and 5'-TTTAGTCCGCTGCTGACCTT (reverse primer) specific for human FDF03 cDNA (1 ng/ml) was performed using the AmpliTaq enzyme and buffer (Perkin-Elmer, Paris, France), dNTPs at 0.8 mM, and DMSO at 5% final concentration. Cycle conditions were 94°C, 40 s; 55°C, 1 min; 72°C, 2 min, for 35 cycles.

Cloning of FDF03-TM and FDF-M14 isoforms was performed by RT-PCR using a pair of primers designed in the 5'-untranslated region (UTR) and 3'-UTR of FDF03 cDNA (forward 5'-ACAGCCCTCTTCGGAGCCTCA and reverse 5'-AAGCTGGCCCTGAACTCCTGG). PCR products were cloned using the pCRII vector (TA cloning kit; Invitrogen, San Diego, CA). Double-stranded plasmid DNA was sequenced on an ABI 373A sequencer (Applied Biosystems, Foster City, CA) using dye terminator technology. Sequencher (Gene Codes Corporation, Ann Arbor, MI) and Lasergene (DNASTAR, London, U.K.) software were used to analyze sequences. Comparisons against the GenBank databases were performed using the BLAST algorithm.

Identification of a mouse homologue of FDF03

tBlastn searches against the mouse expressed sequence tag (EST) database identified different ESTs (gb, AI606524, AI595493, and AV021745) with homology to human FDF03 cDNA and allowed to build a contig coding for a mouse protein homologous to human FDF03. PCR amplification of cDNAs from mouse spleen using oligonucleotides designed in the 5'- and 3'-ends of this sequence (forward 5'-CCTGAGCACCCCAGTGTCCC and reverse 5'-GTGAATTTCTGTGTCTGCCT) permitted the amplification of the entire open reading frame (ORF) of mouse FDF03 (mFDF03).

Chromosomal localization of the FDF03 gene

Chromosomal localization was performed with the Stanford G3 radiation hybrid medium resolution panel (Research Genetics, Huntsville, AL). PCR was as described above using oligonucleotides that amplify a 505-bp fragment specific to the human gene (forward 5'-ACAGCCCTCTTCGGAGCCTCA, reverse 5'-GGCAGAAATACACAGACTGG). The results were scored manually, and analysis was performed with the radiation hybrid mapper program (http//shgc-www.stanford.edu).

Generation of anti-FDF03 mAb 36H2

Female Lewis rats were primed using DNA immunization via intradermal injection into the tail of 50 µg of a pCDM8 expression plasmid (Invitrogen) encoding the FDF03 molecule. Plasmid immunizations were repeated thereafter on day 7 (40 µg) and day 29 (10 µg). Animals were boosted with FDF03-Ig fusion protein on day 67 (10 µg i.p. in CFA) and on day 82 (2.5 µg i.p. in IFA). On day 127, a combined immunizing dose (2 µg i.v. in saline; 2 µg i.p. in IFA) was administered, and splenocytes were harvested 4 days later and fused with the mouse myeloma P3X63-AG8.653. Hybridomas were initially selected that recognized FDF03-Ig (but not control-Ig) fusion protein in indirect ELISA. Hybridomas were then further selected based on their ability to immunoprecipitate an FDF03-AP fusion protein, as previously described (33), and to recognize PBMC via FACS staining. The rat IgG2a mAb TC17-36H2 (mAb 36H2) was selected for use in this study.

Surface biotinylation, immunoprecipitation, and Western blot analysis

For surface-biotinylation, purified blood monocytes were washed three times with ice-cold PBS, pH 8, then resuspended in freshly prepared solution of 0.5 mg/ml sulfo-N-hydroxy succinimidester of biotin (Pierce, Rockford, IL) in PBS and agitated at room temperature for 30 min. Cells were washed three times with ice-cold PBS, pH 8, and then treated in a lysis buffer containing 50 mM Tris-HCl, pH 8, 1% Nonidet P-40, 150 mM NaCl, and protease inhibitors (Boehringer Mannheim). Lysates were then incubated at 4°C for 20 min, and the insoluble material was pelleted by centrifugation at 12,000 x g for 10 min at 4°C. Soluble extracts were precleared three times with control mAb and protein G-Agarose (Boehringer Mannheim). The extract was then incubated with the mAb of interest for 1 h before adding protein G-Agarose for at least 3 h. Beads were washed three times in lysis buffer, resuspended in SDS-PAGE sample buffer with or without 5% 2-ME, boiled for 3 min, and centrifuged. Immunoprecipitates were separated by SDS-PAGE using 12% polyacrylamide gel and then transferred to a polyvinylidene difluoride membrane (Immobilon P; Millipore, Bedford, MA). Blots were blocked with 1% BSA, 0.1% Tween-20 in PBS, then incubated with HRP-conjugated avidin (ExtrAvidin peroxidase; Sigma) for 30 min. Proteins were detected by enhanced chemiluminescence (Boehringer Mannheim). To analyze FDF03 glycosylations, precipitates were untreated or digested overnight at 37°C with N-glycosidase F (0.2 U/50 µl), or with O-glycosidase (2 mU/50 µl) with or without neuraminidase (5 mU/50 µl) before SDS-PAGE in reducing conditions. All enzymes were from Boehringer Mannheim, and digestions were performed in 0.1 M potassium/phosphate buffer, pH 7, 0.05% SDS, 1% 2-ME, 1% Nonidet P-40, and 50 mM EDTA.

Flow cytometric analysis and cell sorting

Cell-surface expression of FDF03 was determined by immunofluorescence staining and flow cytometric analysis with a FACScalibur (Becton Dickinson, Mountain View, CA). For single staining, cells were incubated for 30 min at 4°C with 5 µg/ml purified rat anti-FDF03 mAb 36H2, then washed twice in PBS, 1% BSA, 0.1% NaN₃, and labeled with PE-conjugated F(ab')₂ goat anti-rat IgG (H+L) or with biotin-conjugated F(ab')₂ goat anti-rat IgG (H+L) (Biosource International, Camarillo, CA) followed by PE-conjugated streptavidin (Becton Dickinson). Nonspecific staining was determined by using control rat IgG mAb. For double staining, cells were first labeled with anti-FDF03 mAb 36H2 or isotype control mAb and further with biotin-conjugated F(ab')₂ goat anti-rat IgG, then washed and incubated for 15 min in 5% normal mouse serum (Dako, Glostrup, Denmark) for saturation. Cells were then labeled with PE-conjugated streptavidin and with FITC-conjugated anti-CD3, anti-CD19, anti-CD16, anti-CD56, anti-CD14, or anti-CD15 mAbs (all from Becton Dickinson). To analyze expression of FDF03 on CD34⁺ progenitor-derived CD1a⁺ and CD14⁺ DC, cells were labeled as described above but FDF03 was revealed with tricolor-conjugated streptavidin (Caltag, Burlingame, CA). Separation of CD1a⁺ and CD14⁺ DC subsets from cultured CD34⁺ cord blood cells was performed by sorting on a FACStar^Plus (Becton Dickinson) as previously described (12, 29).

To analyze FDF03 expression on blood and tonsil DC, blood mononuclear cells (obtained after centrifugation over Ficoll gradient) and tonsil mononuclear cells (obtained by digestion with collagenase) were first depleted of T and B cells by using a cocktail of anti-CD3 and anti-CD19 mAbs and goat anti-mouse IgG-coated magnetic beads essentially as previously described (34). The resulting cell population was labeled with a cocktail of FITC-conjugated anti-CD3, anti-CD14, anti-CD15, anti-CD16, anti-CD20, and anti-CD57 mAbs (lineage-FITC), APC-conjugated anti-CD11c mAb, peridinin chlorophyl protein-conjugated anti-HLA-DR mAb (all mAbs from Becton Dickinson) or PE-Cy5-conjugated anti-CD4 mAb (Immunotech, Marseille, France) and with biotin-conjugated anti-FDF03 mAb 36H2 followed by PE-conjugated streptavidin. Simultaneous acquisition of the four fluorescence parameters was performed on a FACScalibur.

Transfection of FDF03 cDNA in U937 cells

A synthetic Sal-CD8-myc-Bam fragment was first generated by annealing two single-stranded oligonucleotides corresponding to the sequence of the human CD8 signal peptide followed by the c-myc epitope (EQKLISEEDL), introducing SalI and BamHI restriction sites on 5' and 3', respectively. FDF03 deleted of the leader sequence (FDF03delSP) was amplified by PCR from a full-length FDF03 cDNA clone using a forward primer 5'-GGATCCACAGGATCTGGTCCAAGCTACCTTTATGGG introducing a BamHI site (underlined) and a reverse primer 5'-ATAGCGGCCGCCTTAGGCCTTTAAGACAGAGTACAGGGTC introducing a NotI site (underlined) 3' to the FDF03 stop codon. Following cloning in pCRII vector and enzymatic digestions, the fragments Sal-CD8-myc-Bam and Bam-FDF03delSP-Not were ligated together into the SalI-NotI site of pMET7. The resulting CD8-myc-FDF03 construction was transfected into U937 cells (20 µg cDNA for 10 x 10⁶ cells) by electroporation in a GenePulser at 250 V with 975 µF capacitance (Bio-Rad, Richmond, CA). After 48 h of culture, cells expressing FDF03 were positively selected by staining with anti-FDF03 mAb 36H2 and sorting on a FACStar^Plus (Becton Dickinson). After four successive rounds of culture and sorting, a cell line stably expressing FDF03 was obtained.

Tyrosine phosphorylation and phosphatase recruitment

To analyze tyrosine phosphorylation and association with SHP-1 and SHP-2 of FDF03 and LAIR-1, FDF03-transfected U937 cells were stimulated or not with 100 mM sodium pervanadate for 10 min at 37°C and cells were lysed in 1% Nonidet P-40 in the presence of protease and phosphatase inhibitors essentially as previously described (35). Lysates were immunoprecipitated by anti-FDF03 mAb 36H2 or anti-LAIR-1 mAb DX26 (25) and protein G-Agarose. Immunoprecipitates were separated by SDS-PAGE on 12% polyacrylamide gels and transferred to Immobilon-P membranes. The membranes were blocked and then incubated with HRP-anti-phosphotyrosine mAb 4G10 (Upstate Biotechnology, Lake Placid, NY) or with polyclonal rabbit Abs against SHP-1 (C19) or SHP-2 (C18) (Santa Cruz Laboratories, Santa Cruz, CA) followed by HRP-conjugated anti-rabbit Igs (Amersham, Arlington Heights, IL). Proteins were detected by enhanced chemiluminescence.

Analysis of intracellular calcium mobilization

FDF03-transfected U937 cells were loaded with 5 µM Indo-1 AM and 5 µM Pluronic F-27 (Molecular Probes, Eugene, OR) for 45 min at 37°C. Cells were washed and kept at room temperature in the dark in RPMI 1640 supplemented with 10% FCS (complete medium) until analysis. For each experiment, an aliquot of Indo-1-loaded cells (10⁶ cells) was resuspended in 1 ml complete medium at 37°C, and the ratio of violet/blue fluorescent emissions at 405 and 530 nm, respectively, was analyzed on a FACStar^Plus flow cytometer (Becton Dickinson) equipped with an argon laser tuned at 350–364 nm (Spectra-Physics, Mountain View, CA) used for UV excitation. When the 405/530 nm ratio baseline was stable, 10 µg/ml of the Abs to be tested were added and the fluorescent emissions were recorded for 2 min. Cells were then kept at 37°C for 2 min, and cross-linking of Abs was achieved by adding 20 µg/ml of a goat F(ab')₂ anti-mouse Ig (Fc fragment-specific) that cross-reacts with rat IgG (Jackson ImmunoResearch, West Grove, PA). Fluorescent emissions were then recorded for the remaining time (5 min). The ratio of Indo-1 violet to blue fluorescence (405/530 nm ratio) was displayed as a function of the elapsed time. The anti-CD32 mAb IV.3 used to induce Ca²⁺ mobilization was obtained from Medarex (Lebanon, NH).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cloning of FDF03 cDNA

By random sequencing of a cDNA library from human activated monocytes (500 sequences), we selected a clone containing a putative leader sequence and representative of a cell-surface receptor, designated FDF03. Fig. 1A shows the nucleotide and deduced amino acid sequences of FDF03. The nucleotide sequence was 1249 bp in length and contained an ORF of 912 nt with the first start codon ATG contained in a consensus Kozak sequence and preceded by a stop codon. The 3'-untranslated sequence of 184 nt contained a classical AATAAA polyadenylation signal. The deduced polypeptide conformed to a type I transmembrane protein composed of 303 aa including a 21-aa signal peptide, a 175-aa extracellular domain, a hydrophobic sequence of 22 aa characteristic of a transmembrane segment, and a 85-aa cytoplasmic tail. The predicted molecular mass of the FDF03 polypeptide was 34 kDa.

View larger version (61K): [in this window] [in a new window]   FIGURE 1. FDF03 belongs to the Ig-SF. A, Nucleotide and predicted amino acid sequences of human FDF03. The nucleotide sequence of the 1249-bp cDNA clone and the derived amino acid sequence are numbered starting from the start codon ATG and methionine, respectively. The predicted signal peptide (dotted) and the transmembrane domain are underlined. The potential N-linked glycosylation site in extracellular domain is underlined twice, and the three tyrosine-based motifs within the cytoplamic tail are boxed. A consensus polyadenylation signal (AATAAA) in the 3'-UTR is underlined. Arrows above the nucleotide sequence indicate sequences deleted in FDF03-TM and FDF-M14 isoforms. This sequence has been submitted to the GenBank, EMBL, and DDBJ databases under accession no. AJ400841. B, Alignment of the putative Ig-like domain of FDF03 with the variable (V) domain of Ig and Ig-like proteins. Amino acids conserved between FDF03 (aa 32–151) and V-set domains of human Ig (P01713), Ig (P01608), and TCRß (P01733) are shaded. Residues characteristic of the V-set and generally conserved among Ig-like proteins are boxed. Asterisks indicate the position of the conserved cysteines in the Ig fold. The positions of the predicted ß-strands are indicated above the sequences. Sequences were analyzed using the MegAlign function of Lasergene (DNASTAR). C, Putative soluble forms of human FDF03 (hFDF03) and sequence of a mouse homologue (mFDF03) of hFDF03. Amino acid sequences of hFDF03-TM, hFDF-M14, and mFDF03 (GenBank, EMBL, and DDBJ accession nos. AJ400842, AJ400843, and AJ400844, respectively) are aligned with that of hFDF03. Only amino acids that differ from that of hFDF03 are noted, and residues identical with hFDF03 are indicated by dashes. Gaps introduced to optimize the sequence alignment are indicated by points.

Searching databases of known polypeptide sequences indicated that the extracellular domain of FDF03 shared significant homology only with the variable (V) domain of human and mouse Igs. Further alignment studies (exemplified in Fig. 1B) confirmed that FDF03 was a novel member of the Ig-SF with a single Ig-related domain of the V-set (36, 37). While most Ig domains are characterized by the presence of a pair of cysteines forming a disulfide bridge that stabilizes the Ig fold, the FDF03 extracellular domain contained only one cysteine at position 125 (asterisks in Fig. 1B). However, typical residues conserved among Ig-like proteins and contained in strands forming the two ß-sheets of the Ig domain were also present within the FDF03 extracellular region (boxes in Fig. 1B). Moreover, a long spacing between the predicted ß-strands C and D identified the two additional strands C' and C'' that are characteristics of the V-set of Ig domain (36, 37). A single potential N-linked glycosylation site (NWT) was present in the Ig domain at position 100. Finally, the membrane-proximal region of the FDF03 extracellular domain contained a high proportion of threonine and serine that represent potential sites for O-linked glycosylation. By analogy with other Ig-SF members, this region is predicted to display an extended open conformation typical of hinge-like sequences.

The 85-aa intracellular domain of FDF03 possessed two putative protein kinase C phosphorylation sites (on serine residues 279 and 285 within the motifs SPR and SHR, respectively) and three tyrosine residues at position 246, 269, and 298 in sequences YENI, YASL, and YSVL, respectively. Following tyrosine phosphorylation, these motifs may represent potential binding sites for SH2 domain-containing signaling molecules (38). In particular, tyrosine 269 is centered within the sequence IVYASL that perfectly matches the consensus L/V/IxYxxL/V of the ITIM (2, 5, 39), and is spaced by 28 aa from the more distal tyrosine that is also contained within an ITIM-like sequence TLYSVL (Fig. 1A).

Identification of a mouse homologue of human FDF03

Searching mouse cDNA databases, we identified ESTs with homology to human FDF03 cDNA and we used them to build a contig coding for a protein homologous to human FDF03 and to design oligonucleotides in the 5'-end and 3'-end of this sequence (see Materials and Methods). PCR amplification of cDNAs from mouse spleen further permitted the amplification of a 1.2-kb nucleotide sequence containing an entire ORF encoding a predicted protein (mFDF03) of 289 aa in length (Fig. 1C). The mFDF03 extracellular region also contained a single Ig-like domain of the V-type and shared the amino acid residues characteristics of human FDF03. In particular, the IPFSFY sequence (at position 45–50 of mFDF03) in the predicted strand B is conserved within both proteins. However, mFDF03 contained an YFGRV sequence instead of the YFCRV sequence in strand F of human FDF03 (aa 117 to 121 in mFDF03), thus indicating that the mouse protein possessed none of the two cysteines typical of the Ig domains. Of importance, the intracytoplasmic tail of mFDF03 contained two tyrosines in tandem sequence IVYASI-x23-TVYSIV that is homologous to the tandem IVYASL-x23-TLYSVL of ITIM-like sequences in human FDF03 (Fig. 1C).

Soluble forms of FDF03 can be produced by alternative splicing

During the analysis of FDF03 expression by RT-PCR (see below), we identified a second PCR product shorter than the expected size. This product, amplified by RT-PCR from activated PBMC cDNA using primers designed in the 5'-UTR and 3'-UTR of FDF03 cDNA, was purified, cloned, and sequenced. All cDNA clones but one had an identical insert of 943 bp that matched with FDF03 cDNA sequence but contained a deletion of 219 nt coding for the extracellular threonine-rich region and the transmembrane domain of FDF03 (FDF03 nucleotides 455–673, arrows in Fig. 1A). The resulting nucleotide sequence coded for a putative protein designated FDF03-TM of 230 aa, composed of the extracellular Ig like-domain directly linked to the entire intracytoplasmic domain of FDF03 (Fig. 1C). The other cDNA clone identified was designated FDF-M14. It contained a 900-bp insert that matched the FDF03 nucleotide sequence but with a 253-bp deletion (FDF03 nucleotides 455–707, arrows in Fig. 1A) that also resulted in deletion of the region coding for the hinge region and transmembrane domain of FDF03. In this case, a change in reading frame introduced premature stop codons and the deduced FDF-M14 protein was a short 175-aa polypeptide composed of the extracellular FDF03 Ig-like domain linked to 24 aa at the carboxyl terminus that are not related to the full-length FDF03 amino acid sequence (Fig. 1C).

These two molecules lacking the transmembrane domain may thus represent soluble isoforms of FDF03 generated by alternative splicing of FDF03 mRNA (see Discussion).

FDF03 is expressed as a monomeric glycoprotein

The anti-FDF03 mAb 36H2, produced by immunizing rats against human FDF03 extracellular domain, was used throughout this study. Immunoprecipitation from lysates of biotin-labeled blood monocytes using this mAb revealed a 44-kDa protein under both nonreducing and reducing conditions (Fig. 2A). Incubation of the precipitate with N-glycosidase F resulted in a shift of the molecular mass to 41 kDa, while treatment with neuraminidase and O-glycosidase did not change the electrophoretic migration of the molecule (Fig. 2B). Taken together, these data indicate that FDF03 is expressed as a monomer and that N-linked glycosylations at the single site (N100) within the FDF03 Ig domain account for the observed molecular mass (44 kDa) of the FDF03 polypeptide.

View larger version (26K): [in this window] [in a new window]   FIGURE 2. FDF03 is a cell-surface monomeric glycoprotein. A, Lysates of surface biotinylated monocytes were immunoprecipitated with anti-FDF03 mAb 36H2 or control rat IgG with protein G-Agarose, boiled in nonreducing or reducing conditions, and analyzed by SDS-PAGE on 12% gels. Following transfer on membranes, blots were incubated with HRP-streptavidin and proteins were revealed by enhanced chemiluminescence. B, Precipitates of biotin-labeled monocytes with mAb 36H2 were treated with N-glycosidase F or with neuraminidase and/or O-glycosidase before SDS-PAGE in reducing conditions and Western blot analysis as above.

Using human radiation hybrid mapping panels and PCR analysis, the gene coding for FDF03 was mapped to chromosome 7q22, within the interval D7S479–D7S2545 of microsatellite anchor markers AFM036xg5 and AFMa052ya5 (data not shown). The closest marker was SGC33905 localizing the gene encoding human fetal liver cytochrome P450 subfamily IIIA polypeptide 7, CYP3A7.

Northern blot analysis on the Clontech immune system blots, performed with a 377-bp probe that hybridized the 3'-end ORF and 3'-UTR of FDF03 mRNA, showed the highest levels of expression in PBL, strong levels of expression in spleen and bone marrow, and lower expression in lymph node (Fig. 3). FDF03 mRNA was not detected in thymus, appendix, and fetal liver (Fig. 3). On total RNA tissue blots, detectable expression was seen in spinal cord, placenta, and lung, but no expression was seen in heart, brain, liver, skeletal muscle, kidney, pancreas, prostate, testis, ovary, small intestine, and colon (Fig. 3 and data not shown). This analysis generally showed the presence of two bands of 1.4 and 1.3 kb that may represent mRNA for FDF03 and for the transmembrane-deleted isoforms FDF03-TM and FDF-M14, respectively, because the probe used in these experiments could hybridize with all three transcripts.

View larger version (89K): [in this window] [in a new window]   FIGURE 3. FDF03 is mostly expressed in immune tissues. Northern blots of human tissues were analyzed with a probe corresponding to 277 bp overlapping the 3' of the ORF and 3'-UTR of FDF03 cDNA labeled with [32P]dCTP. Two bands were obtained, with the major band at 1.4 kb corresponding to the expected size for FDF03 mRNA and both the FDF03-TM and/or FDF-M14 isoforms.

The cellular distribution of FDF03 was first determined by RT-PCR analysis on various isolated cells and cell lines using a pair of primers able to amplify both FDF03 and FDF03-TM cDNAs. As shown in Fig. 4A, FDF03 and FDF03-TM cDNAs were strongly detected in PBMC, blood monocytes, and granulocytes, while no signal was amplified from purified blood T cells, NK cells, and tonsillar B cells. FDF03 and FDF03-TM cDNAs were also present in macrophages (M in Fig. 4A) derived from CD34⁺ cells in the presence of M-CSF, as well as in DC generated either from CD34⁺ cord blood progenitors with GM-CSF and TNF- (CD34⁺-DC in Fig. 4A) or from blood monocytes with GM-CSF and IL-4 (mono-DC in Fig. 4A). Moreover, FDF03 was not expressed by the cell lines JY, RAMOS, DAUDI, BL2 (B cell lines), JURKAT, and MOLT4 (T cell lines), TF1 (erythro-leukemia), CHA (kidney carcinoma), MRC5 (fetal lung fibroblasts), and SW620 and HT29 (colon carcinoma cell lines) (data not shown).

View larger version (63K): [in this window] [in a new window]   FIGURE 4. FDF03 is expressed by cells of myelo-monocytic origin. A, RT-PCR analysis of FDF03 and FDF03-TM mRNA expression in various cell populations. Pools of cDNA from different samples were prepared as described in Materials and Methods. cDNA of macrophages (M) and granulocytes were derived from cord blood CD34+ progenitors cultured in the presence of M-CSF or G-CSF, respectively. DC were generated from cord blood CD34+ cells with GM-CSF and TNF- for 12 days (CD34+ DC) or from blood monocytes with GM-CSF and IL-4 for 7 days (mono-DC). Freshly isolated cells were PBMC, monocytes, T cells, NK cells, and tonsillar B cells. RT-PCR was performed with primers that amplified both FDF03 and FDF03-TM, but not FDF-M14, as described in Materials and Methods. Amplification of plasmids containing FDF03 or FDF03-TM cDNA (pFDF03 and pFDF03-TM) was used as control of specificity, and amplification of ß-actin was used as control for cDNA quantity and quality. B, Expression of FDF03 on peripheral blood leukocytes. FDF03 expression was analyzed by flow cytometry after staining of circulating mononuclear or polynuclear cells with anti-FDF03 mAb 36H2 followed by PE-conjugated goat anti-rat Ig. After saturation with mouse serum, cells were incubated with FITC-conjugated anti-CD20, anti-CD3, anti-CD16, anti-CD56 (for lymphocytes), anti-CD14 (for monocytes), or with anti-CD15 mAb for granulocytes. Acquisition was performed by gating on the different cell populations according to their forward and right angle scatter parameters.

Taken together, these data indicate that FDF03 is mostly expressed by cells of myelo-monocytic origin and by in vitro-derived DC. However, it should be noted that FDF03 mRNA was not detected in a number of myeloid cell lines including U937, HL60, and THP-1 (data not shown).

FDF03 is preferentially expressed by monocytic/CD14⁺-derived DC and by CD11c⁺ DC

DC represent heterogeneous populations of cells according to their origin and stage of activation/maturation (10, 40, 41). Because FDF03 mRNA was detected in bulk preparations of in vitro-derived DC (Fig. 4A), we further analyzed whether cell-surface expression of the FDF03 receptor could be differently regulated during DC differentiation as well as on DC subpopulations. As shown in Fig. 5A, FDF03 was not expressed on the surface of CD34⁺ cord blood progenitor cells, but was induced during their culture with a combination of SCF, GM-CSF, and TNF-, with the strongest expression observed on immature DC on days 5–7 of culture. On day 6, three-color flow cytometric analysis demonstrated that FDF03 was mostly expressed by the CD14⁺/CD1a^- subset of cells, when compared with the CD14^-/CD1a⁺ subset (Fig. 5B). These two populations were sorted on day 6 according to their CD14 and CD1a expression and recultured for 6 days in the presence of GM-CSF and TNF-. As shown in Fig. 5B, on day 12, CD14⁺-derived DC still expressed detectable levels of surface FDF03, and both FDF03 mRNA and FDF03-TM mRNA were amplified by RT-PCR in those DC (day 12 DC in Fig. 5B). In contrast, the DC derived from the CD1a⁺ subset of cells no longer expressed FDF03 at day 12, both at the cell surface and mRNA levels (Fig. 5B). In contrast, DC generated from peripheral blood monocytes in the presence of GM-CSF and IL-4 expressed high levels of FDF03 both at their immature stage (day 7 in Fig. 5C) and after further activation by signals inducing DC maturation (day 9 in Fig. 5C), such as LPS and CD40 ligand (42). Taken together, these results indicate a preferential expression of FDF03 by monocyte- and CD14⁺/CD1a^--derived DC and suggest that FDF03 can be expressed by both immature and mature DC.

View larger version (33K): [in this window] [in a new window]   FIGURE 5. Expression of FDF03 by in vitro-derived DC. A and B, Expression of FDF03 on CD34+-derived DC. A, Purified cord blood CD34+ progenitor cells were cultured with SCF, GM-CSF, and TNF- from day 0 to day 6, then washed and recultured with GM-CSF and TNF- until day 12. At the time indicated, FDF03 expression was analyzed by flow cytometry after staining of cells with anti-FDF03 mAb 36H2 (histograms in solid line) or control rat IgG (histograms in dotted line) followed by PE-conjugated goat anti-rat Ig. B, Cord blood CD34+ cells cultured for 6 days as described above were labeled with PE-conjugated anti-CD14 and FITC-conjugated anti-CD1a mAbs and then FACS sorted according to CD14 and CD1a expression. CD14+/CD1a- cells and CD14-/CD1a+ cells were further cultured with GM-CSF and TNF-. FDF03 expression was determined just after cell sorting (day 6) and after 6 days of the second culture (day 12), after staining with anti-FDF03 mAb 36H2 (histograms in solid line) or control rat IgG (histograms in dotted line) followed by biotin-labeled anti-rat Ig and tricolor (TC)-conjugated streptavidin. Data presented in A and B are representative of at least three independent experiments. FDF03 expression was also analyzed by RT-PCR as described in Fig. 4. C, Expression of FDF03 on monocyte-derived DC. Purified blood monocytes were cultured with GM-CSF and IL-4. On day 7, cells were washed and recultured for 2 days with GM-CSF alone (medium), GM-CSF plus LPS (25 ng/ml), or with GM-CSF plus irradiated untransfected (control L cells) or CD40 ligand-transfected L cells. FDF03 expression was determined by flow cytometry after staining with anti-FDF03 mAb 36H2 (histograms in solid line) followed by PE-conjugated goat anti-rat Ig. Histograms in dotted line represent staining obtained with a rat IgG control mAb. Results presented are representative of five independent experiments.

View larger version (57K): [in this window] [in a new window]   FIGURE 6. Blood and tonsil CD11c+ DC, but not CD11c- DC, express FDF03. A, Blood mononuclear cells were first depleted with anti-CD3 and anti-CD19 mAbs and magnetic beads, then stained with FITC-conjugated mAbs against CD3, CD14, CD15, CD16, CD20, and CD57 (lineage-FITC), peridinin chlorophyl protein-conjugated anti-HLA-DR mAb, APC-conjugated CD11c mAb, and biotin-labeled anti-FDF03 mAb 36H2 revealed by PE-conjugated streptavidin. Following total cell acquisition, the expression of FDF03 vs CD11c was analyzed on HLA-DRhigh/lineage-FITC- DC (gate in left dot plot). B, Total tonsil cells were first depleted with anti-CD3 and anti-CD19 mAbs and magnetic beads, then stained with FITC-conjugated mAbs against CD3, CD14, CD15, CD16, CD20, and CD57 (lineage-FITC), PE-Cy5-conjugated anti-CD4 mAb, APC-conjugated CD11c mAb, and biotin-labeled anti-FDF03 mAb 36H2 revealed by PE-conjugated streptavidin. Following acquisition, the expression of FDF03 vs CD11c was analyzed on CD4+/lineage-FITC- DC (gate in left dot plot). Quadrant locations in FDF03 vs CD11c dot plots were determined according to the nonspecific fluorescence intensity obtained with APC-conjugated mouse and biotinylated rat control mAbs on HLA-DRhigh/lineage-FITC- (in A) or CD4+/lineage-FITC- (in B) cells. Data presented are representative of three experiments.

Following tyrosine phosphorylation, ITIM-bearing receptors can recruit protein tyrosine phosphatases SHP-1 and/or SHP-2 (5, 47). To determine whether the two ITIM-like sequences contained in the FDF03 intracytoplasmic tail could associate with SHP-1 and/or SHP-2, we transfected FDF03 cDNA in the monocytic U937 cell line and positively selected FDF03-expressing cells by using staining with mAb 36H2 and FACS sorting. This strategy was chosen because we did not find any cell line, including U937 (Fig. 7A, left histogram), that spontaneously expressed FDF03. After four rounds of successive sorting and culture, >90% of the cells expressed relatively high levels of FDF03 (Fig. 7A, right panel). This cell line (FDF03-U937 cells) was stimulated or not with sodium pervanadate (an inhibitor of protein tyrosine phosphatases that induces tyrosine phosphorylation) then lysed, and lysates were immunoprecipitated with anti-FDF03 mAb 36H2 or rat control IgG. Because FDF03-U937 cells also expressed the inhibitory receptor LAIR-1/p40 (25) (Fig. 7A), lysates were precipitated in parallel with anti-LAIR-1 mAb DX26 used as a positive control for ITIM-bearing molecule. After SDS-PAGE, Western blot analysis with the anti-phosphotyrosine mAb 4G10 revealed the presence of a major band of 44 kDa, corresponding to FDF03 molecular mass, in mAb 36H2 precipitates of pervanadate-activated, but not untreated, cells (Fig. 7B). A 40-kDa tyrosine-phosphorylated protein was specifically detected in mAb DX26 precipitates of pervanadate-treated FDF03-U937 cells (Fig. 7B), which corresponds to the previously reported molecular mass of LAIR-1/p40 on NK cells, monocytes, and DC (25, 26). When blots were probed with specific anti-SHP-1 Abs, a faint band of 73 kDa was specifically revealed in mAb 36H2 precipitates of pervanadate-treated FDF03-U937 cells (Fig. 7C), while a strong signal was observed in mAb DX26 precipitates, thus suggesting that FDF03 was less efficient than LAIR-1 in association with SHP-1. In contrast, anti-SHP-2 Abs demonstrated that FDF03 recruited SHP-2 as efficiently as LAIR-1 (Fig. 7D). Taken together, these results indicate that FDF03 is tyrosine-phosphorylated and can associate with SHP-2, and to a lesser extent with SHP-1, in pervanadate-activated U937 cells.

View larger version (56K): [in this window] [in a new window]   FIGURE 7. FDF03 and LAIR-1 are tyrosine-phosphorylated and recruit SHP-1 and SHP-2 in pervanadate-activated U937 cells. A, Expression of FDF03 and LAIR-1 on U937 cells. U937 cells were transfected with FDF03 cDNA, then positively selected for FDF03 expression by flow cytometry after staining with mAb 36H2. Filled histograms represent surface expression of FDF03 or LAIR-1 (staining with mAb DX26) on transfected cells after four successive rounds of FACS sorting and culture. Unfilled histograms show staining obtained with a rat (for FDF03) or a mouse (for LAIR-1) IgG control mAb. B–D, FDF03-transfected U937 cells were unstimulated (-) or stimulated (+) for 5 min with sodium pervanadate and then lysed in 1% Nonidet P-40 lysis buffer. Lysates were immunoprecipitated with anti-FDF03 mAb 36H2 or control rat IgG, or with anti-LAIR-1 mAb or control mouse IgG. After SDS-PAGE and Western blots (precipitate from lysate of 30 x 106 cells/line), precipitated proteins were analyzed using Abs specific for (B) proteins phosphorylated on tyrosines (anti-pTyr); (C) SHP-1 (anti-SHP-1); and (D) SHP-2 (anti-SHP-2).

FDF03 inhibits CD32/FcRII-induced calcium mobilization in U937 cells

ITIM-bearing receptors inhibit activation signals, such as intracellular calcium mobilization, when coaggregated with ITAM-bearing receptors (4). Therefore, we analyzed whether FDF03 could also antagonize signaling of stimulatory receptors in FDF03-U937 cells. Efficient cross-linking of the different molecules was performed with a goat F(ab')₂ anti-mouse Ig (Fc-specific) that also reacts with rat IgG, so that the same reagents could be used regardless of the origin of the mAbs. As shown in Fig. 8, cross-linking of mAb 36H2 did not increase intracellular Ca²⁺ concentration in Indo-1-loaded FDF03-U937 cells. In contrast, aggregation of CD32/FcRII by cross-linking of mAb IV.3, which preferentially recognizes the activating isoforms of CD32 (48, 49), induced intracellular Ca²⁺ mobilization. Of interest, the coaggregation of CD32 with FDF03, but not with a control mAb, resulted in strong inhibition of calcium mobilization (Fig. 8). Similar inhibition of Ca²⁺ flux was observed when CD32 was coaggregated with LAIR-1 (Fig. 8). Altogether, these results indicate that FDF03 can function as an inhibitory receptor in monocytic U937 cells, similarly to LAIR-1/p40.

View larger version (57K): [in this window] [in a new window]   FIGURE 8. Cross-linking of FDF03 inhibits CD32/FcRII-induced calcium mobilization in U937 cells. FDF03-transfected U937 cells were loaded with Indo-1 AM then stored at room temperature before analysis on FACStarPlus flow cytometer using an argon laser for UV excitation. For each experiment, aliquots of cells were incubated at 37°C until the baseline of violet vs blue fluorescent emissions (405/530 nm ratio) was stable. Then 10 µg of Abs to be tested were added (arrowhead in each figure) and the 405/530 nm ratio was recorded for 120 s. After a further incubation of 120 s, 20 µg/ml of cross-linker were added (goat F(ab')2 anti-mouse/rat IgG, arrow in each figure), and the 405/530 nm fluorescence ratio was recorded for the remaining time. Data showed are representative of three experiments.

Because it has been reported that engagement of LAIR-1/p40 inhibited differentiation of monocyte into DC by interfering with GM-CSF activities (26), we wondered whether FDF03 could also regulate monocyte differentiation. To do this, blood monocytes were cultured with GM-CSF alone or GM-CSF plus IL-4, in the presence of anti-FDF03 mAb 36H2 or anti-LAIR-1 mAb DX26, together with goat F(ab')₂ anti-mouse/rat IgG used as cross-linker. As previously described (30, 50), GM-CSF rapidly down-regulated the expression of CD14 on monocytes, but induced expression of CD1a, as determined by flow cytometry at day 2 of culture (control mAb in Fig. 9A). Engagement of FDF03 by mAb 36H2 only weakly decreased CD1a expression and did not inhibit CD14 down-regulation whether the cells were cultured with GM-CSF alone (Fig. 9A, left panel) or in the presence of IL-4 (Fig. 9A, right panel). In contrast, and as expected, down-regulation of CD14 and induction of CD1a expression by GM-CSF was almost completely blocked by engagement of LAIR-1 with mAb DX26, even in the presence of IL-4 (Fig. 9A). Similar results (lack of inhibition by FDF03 but blockade by LAIR-1 cross-linking) were obtained after 6 days of culture with GM-CSF and were also observed for GM-CSF-induced up-regulation of CD1b and MHC class II molecules (data not shown). Moreover, cross-linking of FDF03 did not modify the percentage of viable cells recovered after 6 days of culture in the presence of GM-CSF with or without IL-4, while cross-linking of LAIR-1 resulted in a decrease in cell viability (Fig. 9B). Thus, engagement of LAIR-1, but not of FDF03, inhibits differentiation of monocytes into DC, most likely by interfering with the GM-CSF receptor signaling pathway (26).

View larger version (41K): [in this window] [in a new window]   FIGURE 9. Cross-linking of FDF03 does not inhibit the effects of GM-CSF on monocytes. Blood monocytes were cultured with GM-CSF alone or GM-CSF plus IL-4, in the presence of anti-FDF03 mAb 36H2 or anti-LAIR-1 mAb DX26 or with rat or mouse control mAb, respectively. Abs (final concentration of 10 µg/ml) were added at the onset of the culture together with 10 µg/ml of goat F(ab')2 anti-mouse IgG that cross-reacts with rat IgG and that was used as cross-linker. A, On day 2, cells were collected and expression of CD1a and CD14 was determined by flow cytometry following staining with FITC-conjugated anti-CD1a or anti-CD14 mAb. Filled histograms represent CD14 or CD1a expression on cells treated with control IgG, anti-FDF03, or anti-LAIR-1 mAb. Expression of CD1a and CD14 was equivalent on cells cultured with mouse or rat control IgG, and only results with rat IgG are shown (control mAb). Unfilled histograms represent nonspecific staining obtained with a FITC-conjugated negative control mAb. B, On day 6 of culture, cell viability was determined by flow cytometry after staining with propidium iodide. Results are expressed as percentage of viable cells (propidium iodide-negative cells) and are representative of one experiment of three.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We have shown that FDF03 is preferentially expressed in immune tissues and has a restricted expression in cells of the myelomonocytic lineage including monocytes, macrophages, DC, and granulocytes. Flow cytometric analysis demonstrated that FDF03 was expressed by the majority of the CD11c⁺ DC both in blood and tonsils, but not by the CD11c^- DC precursors. This preferential expression of FDF03 was confirmed at the mRNA levels because neither FDF03 nor FDF03-TM messengers could be amplified by RT-PCR in purified blood CD11c^- DC, while both cDNAs were detected in CD11c⁺ DC (data not shown). This is in agreement with the restricted expression of FDF03 in cells of the myeloid origin, because blood and tonsil CD11c^- DC precursors, corresponding to the previously so called plasmacytoid T cells or plasmacytoid monocytes, do not express myelomonocytic markers such as CD14, CD13, and CD33 (43, 44, 46, 61) and have been proposed to be of lymphoid origin. On in vitro-generated DC, FDF03 was preferentially expressed by monocyte-derived DC and by DC derived from the CD14⁺/CD1a^- precursors that display features of the interstitial/dermal-type DC, rather than by the CD1a⁺ subsets of cells that may represent precursors of epidermal/Langerhans cells (29, 40). In keeping with this, anti-FDF03 mAb did not stain Langerhans cells in skin epithelium nor immature CD1a⁺ Langerhans-like DC in tonsil epithelium, and we failed to detect FDF03 mRNA in purified skin Langerhans cells (data not shown). Moreover, FDF03 expression on DC was not down-regulated by signals inducing DC maturation such as LPS or CD40 ligand, suggesting that FDF03 can be expressed by both immature and mature DC. However, immunohistochemical analysis performed on frozen tonsil sections indicated that mAb 36H2 stained neither the CD11c⁺ germinal center DC in B cell follicles (34) nor the mature interdigitating DC in T cell areas (data not shown). This suggests that, in situ, these DC express very low, if any, levels of FDF03, but we cannot exclude that a low sensitivity of our immunostaining procedures may decrease FDF03 detection on tissue sections. However, FDF03 was strongly expressed by cells localized in close contact to the epithelial crypts of the tonsils, some of them expressing the CD11c marker (data not shown). We are currently isolating and characterizing the FDF03-expressing cell populations from tonsils.

The presence of ITIM-like sequences in the FDF03 cytoplasmic tail suggested that FDF03 might principally function as an inhibitory receptor of cell function and activation, as generally described for ITIM-bearing molecules (1, 3, 5, 6). This was confirmed by demonstrating that FDF03 blocked intracellular Ca²⁺ mobilization induced by CD32/FcRII aggregation in FDF03-transfected U937 cells, as did the ITIM-bearing receptor LAIR-1/p40. However, while cross-linking of LAIR-1 strongly inhibited the effects of GM-CSF and IL-4 on monocyte differentiation and survival, cross-linking of FDF03 only weakly decreased expression of CD1a and did not block down-regulation of CD14, nor affected survival of monocytes. While we cannot exclude that engagement of FDF03 by mAb 36H2 was not optimal in our experimental protocols, these results suggest that FDF03 may have function(s) different to that of the broadly expressed LAIR-1/p40 molecule (25, 26). Moreover, this discrepancy between FDF03 and LAIR-1 inhibitory activities might be explained by the stronger association of LAIR-1 with SHP-1 (as observed in U937 cells), because SHP-1 has been implicated in the down-regulation of signaling by receptors for erythropoietin, IL-3, and GM-CSF (62, 63, 64). Equally, we were unable to demonstrate that engagement of FDF03 negatively regulated functions of FDF03-expressing cells (monocytes and DC) using different assays including proliferation of CD34⁺ progenitor cells in response to SCF and GM-CSF or IL-3, or DC activation and maturation induced by CD40 ligand or LPS as measured by phenotypic parameters (increase expression of CD80, CD86, CD83, MHC class II, and CD40) and cytokine secretion (IL-12, IL-8, and IL-6) (data not shown). Moreover, anti-FDF03 mAb 36H2 did not affect T cell proliferation induced in allo-reactions with in vitro-derived DC (data not shown). Finally, unlike some ITIM-bearing receptors such as ILT3 and FcRIIB (15, 22, 23), FDF03 was not internalized in monocytes and in vitro-derived DC, suggesting that FDF03 does not function as an endocytic receptor for Ag capture and presentation (data not shown).

The cytoplasmic tail of FDF03 contains three tyrosine-based motifs that, following phosphorylation, may represent binding sites for SH2 domain-containing signaling molecules (38). However, the membrane proximal tyrosine in YENI motif is not present within the cytoplasmic region of the mouse homologue of FDF03, while both human and mouse proteins display an ITIM-like tandem IVYASL-x23-TLYSVL and IVYASI-x23-TVYSIV, respectively, which are closest to the motif allowing high-affinity binding to the tandem SH2 domains of SHP-2 (65). We have shown that FDF03 was tyrosine phosphorylated in pervanadate-treated transfected U937 cells and associated with SHP-2. We have also observed association of FDF03 with SHP-1, but apparently with a weaker efficiency when compared with SHP-1 recruitment by the LAIR-1 receptor in the same cells. In contrast to FDF03, but similarly to SHP-1 recruiting inhibitory receptors such as the KIRs (3, 4), the LAIR-1 cytoplasmic tail contains two typical ITIMs in the tandem VTYAQL-x24-ITYAAV (25). Interestingly, it has been shown that the hydrophobic residue (I/V/L) at position -2 upstream of phosphorylated tyrosine in ITIM is critical for binding to and activation of SHP-1 by peptides as well as the cytoplasmic tail of the KIR (47, 66). Moreover, as recently shown for the KIR molecules (67), the N-terminal and C-terminal ITIMs may have different efficiencies to associate with phosphatases in vivo, because the N-terminal ITIM was found to be sufficient to recruit SHP-2 but not SHP-1, while both ITIMs were required to recruit SHP-1. In keeping with this, the recently described T cell transmembrane adaptor protein SIT, which contains only one I/VxYxxV ITIM among its four cytoplasmic YxxL/V tyrosine motifs, associates in vivo with SHP-2, but not with SHP-1 and SHIP (68). Thus, this suggests that the presence of a threonine (T) instead of a hydrophobic residue at position -2 of the C-terminal tyrosine-based motif of FDF03 (TLYSVL), may decrease its potential association with SHP-1 but not with SHP-2, as seen in our study. It should be noted that a similar motif (TVYSIV) is also present at the C-terminal end of mouse FDF03. Interestingly, a threonine is also present at position -2 of the second tandem tyrosine motif YxxV/L of the molecules SHPS-1/SIRP, CD31/PECAM-1, and CD33 that have been primarily found to associate with SHP-2 as well as, in some studies, with SHP-1 (8, 9, 69, 70, 71, 72). Moreover, the TxYxxV/I sequence is present in the cytoplasmic tail of the two CD33-related Siglecs, OB-BP-1/Siglec-6 and OB-BP-2/Siglec-5 (59, 60), and in human and mouse PD-1 molecules (51, 73), but it has not been yet demonstrated whether these molecules can recruit SHP-1 and/or SHP-2. Finally, four TxYxxV/I motifs are present in the intracellular domain of human and mouse 2B4 molecules (74, 75), and three are in the signaling lymphocytic activation molecule SLAM (76), both receptors preferentially recruiting SHP-2. In both SLAM and 2B4 molecules, these motifs also represent docking sites for the SLAM-associated protein SAP (75, 77), an SH2-containing adaptor protein that competes with SHP-2 for recruitment to SLAM and 2B4, and is defective in the inherited X-linked lymphoproliferative syndrome XLP (77, 78, 79). Thus, although SAP does not appear to be expressed in myeloid cells, it would be of interest to analyze whether FDF03 may also associate with SAP or with homologous protein such as EAT-2 that recognizes a similar TxYxxV/I motif (80).

In conclusion, FDF03 represents a novel member of the Ig-SF selectively expressed in cells of the myelomonocytic lineage, including monocytes/macrophages, granulocytes, and DC that express the CD11c marker. Tyrosine-phosphorylated FDF03 preferentially recruits SHP-2 and can function as an inhibitory receptor, at least when overexpressed in the monocytic U937 cell line. However, we cannot exclude that FDF03 may have other regulatory functions because recruitment of SHP-2 may also mediate cellular activation (81, 82, 83). Of note, by screening molecules associating with SHP-1, Mousseau et al. (84) recently identified a protein designated PIRL (for paired Ig-like receptor, accession no. AF161080) whose amino acid and cDNA sequences are identical with that of FDF03. The description of the genomic organization of PIRL/FDF03 (84) clearly confirms that FDF03-TM and FDF-M14 are produced by alternative splicing of FDF03/PIRL cDNA. It should be noted that all the primers and probes used in our study are specific for FDF03 and cannot hybridize with the nucleotide sequence of PIRLß (accession no. AF161081), a putative activating counterpart of PIRL/FDF03 described by Mousseau et al. (84), the gene of which being also localized on chromosome 7q22. Of interest, this localization is close to a region associated with several chromosomal abnormalities relating to tumors and particularly with deletion in myelodysplastic syndrome and acute myeloid leukemia (85, 86, 87). We are currently analyzing whether abnormalities in the FDF03 gene locus may be associated with such malignancies.

	Acknowledgments

 
We thank S. Aït-Yahia, C. Massacrier, B. Vanbervliet, B. Salinas, and C. Péronne for technical help in cell and cDNA preparation, biochemistry, and DNA sequencing. We thank Drs. C. Caux, S. Lebecque, L. L. Lanier, S. Tangye, and T. Hauser for helpful discussions and reading of the manuscript and M. Vatan, C. Alexandre, and S. Bourdarel for editorial assistance.

	Footnotes

 
¹ This work was partially supported by Fondation Marcel Mérieux, Lyon, France (to N.F.).

² Address correspondence and reprint requests to Dr. Pierre Garrone, Laboratory for Immunological Research, Schering-Plough, 27 chemin des Peupliers, BP11, 69571 Dardilly cedex, France.

³ Abbreviations used in this paper: ITIM, immunoreceptor tyrosine-based inhibitory motif; DC, dendritic cells; EST, expressed sequence tag; Ig-SF, Ig superfamily; ITAM, immunoreceptor tyrosine-based activation motif; ORF, open reading frame; SH2, Src homology-2; SHIP, SH2 domain-containing inositol phosphatase; SHP, SH2 domain-containing protein tyrosine phosphatase; UTR, untranslated region; SCF, stem cell factor.

Received for publication February 1, 2000. Accepted for publication May 9, 2000.

	References

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