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Originally published online as doi:10.1189/jlb.0406276 on November 15, 2006

Published online before print November 15, 2006
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(Journal of Leukocyte Biology. 2007;81:440-448.)
© 2007 by Society for Leukocyte Biology

The human EGF-TM7 receptor EMR3 is a marker for mature granulocytes

Mourad Matmati*, Walter Pouwels*, Robin van Bruggen{dagger}, Machiel Jansen{dagger}, Robert M. Hoek*, Arthur J. Verhoeven{dagger} and Jörg Hamann*,1

* Department of Experimental Immunology and
{dagger} Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

1 Correspondence: Department of Experimental Immunology, K0-144, Academic Medical Center, University of Amsterdam, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands. E-mail: j.hamann{at}amc.uva.nl


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
EMR3 is a member of the epidermal growth factor-seven-transmembrane (EGF-TM7) family of adhesion class TM7 receptors. This family also comprises CD97, EMR1, EMR2, and EMR4. To characterize human EMR3 at the protein level, we generated Armenian hamster mAb. Using the mAb 3D7, we here demonstrate that EMR3, like other EGF-TM7 receptors, is expressed at the cell surface as a heterodimeric molecule consisting of a long extracellular {alpha}-chain, which possesses at its N-terminus EGF-like domains and a membrane-spanning ß-chain. Flow cytometric analysis revealed that all types of myeloid cells express EMR3. In peripheral blood, the highest expression of EMR3 was found on granulocytes. More mature CD16+ monocytes express high levels of EMR3, and CD16– monocytes and myeloid dendritic cells (DC) are EMR3dim/low. Lymphocytes and plasmacytoid DC are EMR3–. It is interesting that in contrast with CD97 and EMR2, CD34+CD33–/CD38– committed hematopoietic stem cells and CD34+CD33+/CD38+ progenitors in bone marrow do not express EMR3. In vitro differentiation of HL-60 cells and CD34+ progenitor cells revealed that EMR3 is only up-regulated during late granulopoiesis. These results demonstrate that the expression of EGF-TM7 receptors on myeloid cells is differentially regulated. EMR3 is the first family member found mainly on granulocytes.

Key Words: expression profiling • granulopoiesis • monoclonal antibody


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The five members of the epidermal growth factor-seven-transmembrane (EGF-TM7) family, CD97 and EGF module-containing, mucin-like hormone receptor (EMR)1–4, are adhesion class TM7 molecules with a long extracellular region [1 ]. Proximal to the first transmembrane segment, a G-protein-coupled receptor-proteolytic site (GPS) gives rise to autocatalytic processing of the polypeptide into an extracellular {alpha}- and a membrane-spanning ß-chain, which noncovalently associate at the cell surface [2 ]. At the membrane-distal terminus of the {alpha}-subunit, variable numbers of EGF domains facilitate the binding of cellular ligands. The first two EGF domains of CD97 bind CD55 (decay-accelerating factor), whereas the fourth EGF domain of CD97 and EMR2 interacts with the glycosaminoglycan chondroitin sulfate [3 4 5 ]. Ligands for the other EGF-TM7 family members are unknown to date.

EGF-TM7 receptors are expressed predominantly by cells of the immune system. Whereas CD97 is found on almost all leukocytes, expression of the EMR molecules is restricted to myeloid cells [1 ]. Increased expression of CD97 and EMR2 has been observed at various sites of inflammation [6 7 8 9 ]. Recent functional studies indicate a role for CD97 in leukocyte trafficking and angiogenesis [10 , 11 ] and of EMR1 (also known as F4/80 in the mouse) in the generation of peripheral tolerance [12 ]. The molecular mechanism(s) behind these different functions remain to be unraveled.

EMR3 is a still poorly characterized EGF-TM7 receptor. EMR3 possesses two EGF domains and has been suggested to bind a ligand at the surface of monocyte-derived macrophages and activated neutrophils [13 ]. It is remarkable that the TM7 region of EMR3 is highly similar with that of EMR2, and in fact, EMR3 was discovered unintentionally as a PCR coamplification product during the cDNA cloning of EMR2 [13 ]. Transcript analysis indicated a predominantly leukocyte-restricted expression with highest levels in polymorphonuclear (PMN) cells.

In the present study, we report a first characterization of EMR3 at the protein level. We demonstrate that EMR3 is a heterodimeric molecule expressed on mature myeloid cells and found primarily on granulocytes. Although the cellular distribution of EMR3 partially overlaps with that of CD97 and EMR2, it is absent on hematopoietic stem and progenitor cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Generation and stable expression of a hybrid EMR3 construct
A hybrid EMR3 expression construct in which the first EGF domain has been replaced by the first EGF domain of CD97 was generated by overlap extension-PCR as described earlier [10 , 14 ]. In a first step, two overlapping PCR fragments were generated in separate PCR reactions. The N-terminal part of CD97, including EGF domain 1, was amplified from human CD97 cDNA [15 ] using a standard T7 primer (sense) and the specific primer 5'-CATTAATGTCGTCACAAGTCTCCGTCGGGGTG-3' (antisense; the sequence overlapping with EGF domain 2 of EMR3 is underlined). EGF domain 2 and part of the stalk region of EMR3 was amplified from human EMR3 cDNA [13 ], kindly provided by Dr. Martin Stacey (Sir William Dunn School of Pathology, Oxford, UK), using the specific primers 5'-GACTTGTGACGACATTAATGAATGTACACCACC-3' (sense; the sequence overlapping with EGF domain 1 of CD97 is underlined) and 5'-GGTGCTGGTGTTCTGGATGGC-3' (antisense). The PCR fragments were purified and mixed together to serve as template for the second step of the overlap extension-PCR performed with a standard T7 primer and the specific primer 5'-GGTGCTGGTGTTCTGGATGGC-3'. A hybrid construct was obtained by replacing the N-terminal part of EMR3 cDNA in pcDNA3 (Invitrogen-Gibco, Paisley, UK) with the PCR amplicon, thereby using a HindIII site in the multiple cloning site of the vector and a BamHI site in the EMR3 sequence.

Using Lipofectamine Plus reagent (Life Technologies, Gaithersburg, MD), the Armenian hamster fibroblast line ARHO12 [16 ], kindly provided by Professor Jannie Borst (NKI, Amsterdam, The Netherlands), was transfected with the pcDNA3-EMR3-CD97(EGF1) hybrid construct. After selection with G418 (Invitrogen-Gibco), stable, transfected clones were tested for expression by flow cytometry with the mAb CLB-CD97/1, which binds to the first EGF domain of CD97. Stable transfectants expressing wild-type CD97(EGF1,2,5) and EMR2(EGF1,2,5) were generated in a similar way.

Generation of mAb
EMR3 mAb were generated in an Armenian hamster (Cricetulus migratorius; Cytogen, West Roxbury, MA) as described previously [10 , 14 ]. Immunization was performed with permission of our institute’s Animal Ethics Committee (University of Amsterdam, The Netherlands). One animal was immunized four times i.p. with 10 x 106-irradiated (50 Gray) ARHO-EMR3-CD97(EGF1) transfectants in PBS. Three days after the final boost, spleen cells were fused with mouse SP2/0 cells by standard hybridoma technology. Binding of hybridoma supernatants to ARHO transfectants was tested by flow cytometry. From 33 hybridomas, which were initially found to bind EMR3-CD97(EGF1) but not CD97(EGF1,2,5), one clone (3D7) was selected and subcloned until it was monoclonal and stable. The hybridoma was grown in large amounts, and Ig was purified using protein A bound to Sepharose CL-4B (Pharmacia, Piscataway, NJ). Applying anti-Armenian hamster Ig mAb (PharMingen, San Diego, CA), 3D7 was determined to be an IgG2. F(ab')2 fragments were generated by pepsin digestion with the ImmunoPure F(ab')2 preparation kit (Pierce, Rockland, IL), and 3D7 Ig and F(ab')2 fragments were biotinylated using ImmunoPure NHS-LC-biotin (Pierce).

Immunoprecipitation
ARHO cells, untransfected or stably expressing EMR3-CD97(EGF1), were biotinylated with NHS-LC-biotin (Pierce) for 30 min, washed three times with PBS, and lysed in buffer containing 150 mM NaCl, 10 mM triethanolamine-HCl (pH 7.8), 5 mM EDTA, and 1% Nonident P-40, supplemented with protease inhibitors. After centrifugation, cell extracts were incubated with an irrelevant mouse mAb, precleared with protein A-Sepharose (Sigma Chemical Co., St. Louis, MO), and incubated with 3D7 (5 µg/ml) for 30 min on ice. Immune complexes were adsorbed onto protein A-Sepharose, washed extensively, and eluted under reducing conditions. Release of N-linked oligosaccharides was performed with peptide-N-glycosidase F (PNGase F; Sigma Chemical Co.), according to the manufacturer’s protocol. The samples were subjected to 8.75% SDS-PAGE and transferred to Hybond-C extra membrane (Amersham Life Science, Piscataway, NJ). The membrane was incubated overnight at 4°C in 3% Protivar (protein-rich milk) in TBST, followed by incubation for 1 h with poly-streptavidin-HRP (Sanquin, Amsterdam, The Netherlands) in 1% Protivar in TBST. After washing, precipitated protein was visualized by ECL (Amersham Life Science).

Flow cytometry
Flow cytometry was performed by standard procedures on a FACSCalibur with CellQuest Pro software (BD Biosciences, San Jose, CA). The following mAb directed to EGF-TM7 receptors were used: CD97 (Clone CLB-CD97/1, binds to EGF domain 1 and CLB-CD97/3, binds to stalk region [17 ], EMR2 Clone 2A1 [18 ]), and EMR3 (Clone 3D7). In addition, we used mAb to the following molecules: CD11b [Clone CLB-B2.12, Sanquin; and Clone ICRF44, allophycocyanin (APC)-labeled, BD Biosciences], CD14 (Clone Leu-M3, PE- and FITC-labeled, BD Biosciences), CD16 (Clone Leu 11c, PE-labeled, BD Biosciences), CD33 (Clone P67.6, PerCP-Cy5.5-labeled, BD Biosciences), CD34 (Clone 8G12, FITC-labeled, BD Biosciences), CD38 (Clone HB7, PE-labeled, BD Biosciences), CD45 (Clone 2D1, PerCP-Cy5.5- and APC-labeled, BD Biosciences), CD63 (Clone CLB-gran/12, Sanquin), and IgE (Clone Hu-901, FITC-labeled, Kirkegaard and Perry, Gaithersburg, MD). As control Ig, we used mouse IgG1 (Clone 6E9, Sanquin), mouse IgG1K (biotinylated, BD Biosciences), and hamster IgG (biotinylated, Southern Biotech, Birmingham, AL). PE-conjugated goat antihamster-Ig (BD Biosciences), streptavidin-PE (Molecular Probes, Eugene, OR), and streptavidin-APC (BD Biosciences) were used as a second-step reagent.

Prior to incubation of cells with mAb, FcRs were blocked for 20 min with 10–20% human pooled serum. Incubations with first- and second-step reagents, diluted in PBS/0.5% BSA, were performed for 45 min on ice.

To analyze expression on peripheral blood leukocytes (PBL), whole blood or buffy coat cells were incubated in a first step with biotinylated 3D7. In most experiments, biotinylated mAb to CD97 and EMR2 were applied in parallel. The second step included mAb to subset markers along with streptavidin-APC. The main leukocyte populations were divided with mAb to CD14 (PE-labeled), CD16 (PE-labeled), and CD33 (PerCP-Cy5.5-labeled) [19 20 21 ]. In contrast with Bakker et al. [21 ], we designated the CD14–CD16–CD33dim population, based on the high presence of Fc{epsilon}RI-attached IgE, as basophils. Monocyte subsets were separated with mAb to CD14 (FITC-labeled) and CD16 (PE-labeled) [22 ]. Prior to cytometry, erythrocytes were removed with FACS lysing solution (BD Biosciences).

To analyze expression on hematopoietic stem and progenitor cells, normal bone marrow, kindly provided by the Department of Hematology (AMC, The Netherlands), was incubated in a first step with biotinylated EGF-TM7 receptor mAb, followed by a second step with mAb to CD45 (APC- or PerCP-Cy5.5-labeled), CD34 (FITC-labeled), and CD33 (PerCP-Cy5.5-labeled) or CD38 (PE-labeled), along with streptavidin-PE or -APC [21 , 23 , 24 ].

Isolation of DC subsets
Myeloid dendritic cells [DC; blood DC antigen (BDCA)-1+] and plasmacytoid DC (BDCA-4+) [25 ] were isolated from PBMC by positive MACS in the mini-MACS system (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. In short, after depletion of CD19+ B cells with anti-CD19 microbeads, cells were labeled with PE-conjugated mAb directed against BDCA-1 or BDCA-4, followed by incubation with anti-PE microbeads and separation over a magnetic column. Enriched cells (>90% pure), kindly provided by the Department of Cell Biology and Histology (AMC), were processed for flow cytometric analysis.

Differentiation of HL-60 cells
The human promyeloid tumor cell line HL-60 has been shown to differentiate into neutrophil-like cells in the presence of DMSO [26 ]. We stimulated HL-60 cells in IMDM supplemented with 10% FCS with 1.25% DMSO for 1–5 days prior to flow cytometric analysis. The morphology of the cells was examined by microscopy of cytospins stained with Diff-Quick (Dade Behring, Düdingen, Switzerland).

Differentiation of hematopoietic progenitor cells toward neutrophils
CD34+ cells were isolated from cord blood and differentiated toward neutrophils as described previously by Buitenhuis et al. [27 ]. In short, CD34+ cells were sorted by MACS (Miltenyi Biotec) from the mononuclear fraction of umbilical cord blood. 1–2x105 cells/ml were cultured in IMDM supplemented with 10% FCS in the presence of SCF (2.5 nM), FLT-3 (2.5 nM), GM-CSF (0.1 nM), IL-3 (0.1 nM), and G-CSF (1.5 nM). After 6 days of culture, only G-CSF was added to the cells. On Days 13 and 17, cells were analyzed by flow cytometry on a LSRII flow cytometer with FACSDiva software (BD Biosciences). On Day 17, cells were sorted based on EMR3 expression on a MoFlow cell sorter (Cytomation, Fort Collins, CO). Cytospins of the EMR3– and EMR3+ were stained using the May-Grünwald-Giemsa staining.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of an EMR3-specific mAb
To generate an EMR3-specific mAb, an Armenian hamster was immunized with ARHO cells stably expressing a hybrid EMR3 receptor, in which the first EGF domain was switched for the one of CD97 (Fig. 1A ). Screening of hybridoma supernatants by flow cytometry revealed a panel of clones to be specific for the hybrid EMR3 receptor. To exclude that the mAb are reacting with the first EGF domain of CD97 or with the TM7 region of EMR2, which is highly similar to that of EMR3 [13 ], supernatants were tested on ARHO cells expressing CD97 and EMR2. Approximately 30 potentially EMR3-specific hybridoma were selected, and the mAb 3D7 (Fig. 1B) was purified and used in this study to characterize EMR3.


Figure 1
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  		Figure 1. Generation of an EMR3-specific mAb. (A) Schematic structure of EMR3, which possesses two EGF domains, the second one containing a calcium-binding site [13 ]. ARHO fibroblasts, stably expressing a hybrid EMR3 receptor, in which the first EGF domain has been replaced by EGF Domain 1 of CD97, were used for immunization of an Armenian hamster. (B) The mAb 3D7 is specific for EMR3. Binding of 3D7 to ARHO cells expressing the hybrid EMR3-CD97(EGF1) receptor or wild-type CD97 and EMR2 was determined by flow cytometry. Along with 3D7, cells were stained with CLB-CD97/1 (anti-CD97) and 2A1 (anti-EMR2). Control Ig stainings are shown in gray. (C) Biochemical characterization of EMR3, which was immunoprecipitated with 3D7 from biotinylated ARHO cells, untransfected or stably expressing EMR3-CD97(EGF1). To remove N-linked glycans, precipitated EMR3 was treated with PNGase F (right lane).

 
EMR3 possesses a GPS, indicating that the molecule is proteolytically cleaved into an extracellular {alpha}-subunit and membrane-spanning ß-subunit. We examined the molecular structure of cell surface-expressed EMR3 by immunoprecipitation from a lysate of ARHO transfectants. As shown in Figure 1C , 3D7 precipitated a specific product of ~56 kDa, which was reduced in size by PNGase F treatment to ~35 kDa. This corresponds well with the predicted molecular mass of the mature {alpha}-chain of 35.6 kDa. These results imply that EMR3, like other EGF-TM7 receptors, is expressed at the cell surface as a heterodimeric molecule.

Granulocytes express high levels of EMR3
Using flow cytometry of human whole blood, we assessed the expression of EMR3 on different subtypes of PBL. Analysis based on scatter characteristics and the coexpression of CD14, CD16, and CD33 [20 ] revealed that EMR3 is expressed broadly on myeloid cells (Fig. 2 ). Highest expression levels were found on granulocytes. PMN, in addition to a majority of neutrophilic granulocytes, comprise a smaller subset of eosinophilic granulocytes, which can be dissected based on a specific, high-depolarization signal [28 ] or the dim expression of CD16 [29 ]. As shown in Figure 3 , eosinophils are EMR3high and express consistently higher levels of EMR3 compared with neutrophils.


Figure 2
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  		Figure 2. Expression of EMR3 on main leukocyte populations. PBL were analyzed by flow cytometry, such that cells were divided in subsets based on forward-scatter (FSC) and side-scatter (SSC) analysis. Further analysis of PMN (R1), which consist of neutrophils and a small fraction of eosinophils, is shown in Fig. 3 . The gated mononuclear cells (R2) were separated on the basis of CD33 and CD14/CD16 expression. In the panels displaying the selected subsets, EMR3 expression was analyzed and compared with control Ig staining (gray histograms). Geometric means are provided with the upper numbers presenting the control. The results are representative of six independent donors.

 

Figure 3
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  		Figure 3. Expression of EMR3 on eosinophils. PBL were analyzed by flow cytometry. PMN were gated based on FSC and SSC analysis (see Fig. 2 , R1). Subsequently, eosinophils were selected on the basis of a specific high-depolarization signal [28 ]. EMR3 expression was determined compared with control Ig staining (gray histograms). Geometric means are provided with the upper numbers presenting the control. Data are representative of two independent donors. Analysis based on CD16 expression (eosinophils are CD16dim [29 ]) revealed similar results (four independent donors; data not shown).

 
Monocytes and DC express EMR3, albeit at lower amounts compared with granulocytes. Lymphocytes do no express EMR3, even after stimulation with PHA, PMA, or CD3 mAb for 4 h and 24 h (data not shown). A small EMR3high population of 1–3%, which we reproducibly found within the lymphocyte gate and which was negative for CD3, CD14, CD16, CD19, and CD56 but dim for CD33 (Fig. 2) , represents basophils [20 ], as confirmed by the high presence of Fc{epsilon}RI (data not shown).

Monocytes and myeloid DC express different levels of EMR3
Blood monocytes can be divided on the basis of CD14 and CD16 expression into cells, which vary in their maturity and migratory properties [22 , 30 31 32 ]. Analysis of EMR3 in these subpopulations revealed a fivefold higher expression level on CD16+ cells compared with CD16– monocytes (Fig. 4 ). Thus, monocytes with a high capacity for transendothelial migration express substantially increased levels of EMR3.


Figure 4
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  		Figure 4. Expression of EMR3 on monocyte subsets. PBL were analyzed by flow cytometry. Monocytes, gated by FSC and SSC analysis, were divided on the basis of CD14 and CD16 expression. In the selected subsets, EMR3 expression was measured and compared with control Ig staining (gray histograms). Geometric means are provided with the upper numbers presenting the control. In five independent donors, relative expression on the CD14+CD16+ cells was 534 ± 83% (geometric mean fluorescence intensity±SEM) compared with the CD14++CD16– subset, set to 100%.

 
The main DC populations in blood are premature myeloid and plasmacytoid DC [33 ]. Staining of PBL subtypes (Fig. 2) indicated low levels of EMR3 on blood DC. To confirm these data and to assign EMR3 expression to DC lineages, we stained DC subsets purified on the basis of BDCA marker expression [25 ]. As shown in Figure 5 , EMR3 was found exclusively on myeloid DC, and EMR2, extending our previous findings [18 ], is present on myeloid and plasmacytoid DC.


Figure 5
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  		Figure 5. Expression of EMR3 and EMR2 blood DC. Myeloid and plasmacytoid DC were isolated from PBMC by positive immunomagnetic bead selection on the basis of BDCA expression [25 ]. Expression of EMR3 and EMR2 was analyzed by flow cytometry. Gray histograms show control Ig staining. Geometric means are provided with the upper numbers presenting the control. Data are representative of six independent donors.

 
EMR3 is up-regulated during late neutrophilic development
Myeloid cells develop from hematopoietic stem and downstream progenitor cells, which can be defined in humans based on the expression of CD34 in combination with CD33 or CD38 [23 , 24 ]. Analysis of these cells from bone marrow revealed a striking difference between EMR3 and its relatives EMR2 and CD97 (Fig. 6 ). Although EMR3 was completely absent from CD34+ cells, EMR2 and CD97 were detected on CD34+CD33–/CD38– hematopoietic stems as well as on CD34+CD33+/CD38+ progenitor cells.


Figure 6
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  		Figure 6. Expression of EMR3, EMR2, and CD97 on bone marrow (BM) stem and progenitor cells. Normal bone marrow was analyzed by flow cytometry. CD34+ cells were gated based on SSC and the expression of CD45 and CD34. Stem and progenitor cells were divided on the basis of CD33 or CD38 staining. EMR3, EMR2, and CD97 expression was determined and compared with control Ig (cIg) staining. Quadrant cell percentages are provided. The results are representative of three independent donors.

 
Absence of EMR3 on hematopoietic stem and progenitor cells indicated that the molecule is up-regulated during the development of myeloid cells. To prove this assumption, we first analayzed expression on promyeloid HL-60 cells, which differentiate in the presence of DMSO into neutrophil-like cells [26 ]. Whereas undifferentiated HL-60 cells are EMR3–, DMSO-induced differentiation resulted in a strong up-regulation of EMR3 on almost all cells on Day 5 (Fig. 7 ). EMR3 expression, following that of CD11b, increased further during longer differentiation, resulting in a uniformly positive cell population after 7 days of culture (data not shown). Expression levels of EMR2 and CD97, in contrast, increased only slightly (twofold) or remained stable, respectively.


Figure 7
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  		Figure 7. Expression of EMR3, EMR2, and CD97 on DMSO-differentiated HL-60 cells, which were grown in the presence of 1.25% DMSO for 5 days, were analyzed at the indicated days by flow cytometry. Staining for CD11b was included as a marker for granulocyte differentiation. Gray histograms show control Ig staining. Geometric means are provided, and the upper numbers present the control. Data are representative of three independent experiments.

 
To confirm the up-regulation of EMR3 during granulopoiesis with a more physiological approach, we used a protocol developed by Coffer and colleagues [27 ], which allows differentiation of CD34+ progenitor cells in vitro in the presence of G-CSF toward neutrophils. EMR3 was detectable after 17 days of differentiation, and thus appeared later than the maturation markers CD11b and CD16, which were detectable already at Day 13 (Fig. 8A ). Microscopic examination of the EMR3+ fraction, sorted on Day 17, revealed banded nuclei in ~15% and segmented nuclei in ~80% of the cells (Fig. 8B) . The late appearance of EMR3 on a small number of neutrophils with banded or segmented nuclei implies that EMR3 is a marker for terminally differentiated cells.


Figure 8
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  		Figure 8. Up-regulation of EMR3 during in vitro granulopoiesis. (A) CD34+ bone marrow progenitor cells, differentiated in the presence of G-CSF toward neutrophils [27 ], were analyzed at the indicated days by flow cytometry. Staining for CD11b and CD16 was included as a marker for granulocyte differentiation. Gray histograms show control Ig staining. Data are representative of three independent experiments. (B) EMR3– and EMR3+ cells were sorted on Day 17 of differentiation. Representative photographs of cytospins, stained with May-Grünwald-Giemsa, are shown.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The EGF-TM7 family comprises the five receptors CD97 and EMR1–4 [1 ]. Whereas EMR2 and EMR3 have been characterized in humans (ref. [18 ] and this study), EMR1 and EMR4 have been studied in mice [34 , 35 ]. This dichotomy is caused by the highly active evolution that EGF-TM7 receptor genes underwent since their appearance in early mammals. The genes encoding EMR2 and EMR3 got lost in murids [36 , 37 ], the EMR4 gene became inactive in humans as a result of a frame-shift mutation [38 ]. CD97 is the only EGF-TM7 receptor that on current evidence, is confirmed to be expressed in primates and rodents [6 , 14 , 15 ]. In this study, EMR3, the last remaining member of the EGF-TM7 family that was not studied at the protein level so far, is described.

EGF-TM7 receptors are expressed on leukocytes. Previous studies indicated that expression of the EMR molecules is restricted to myeloid cells [1 ]. We here show that EMR3 is also found exclusively on cells of the myeloid lineages with highest expression levels on granulocytes and CD16+ monocytes. Expression of EMR3 is dim on the majority of monocytes and low on myeloid DC. Although the cellular distribution of EMR3 partly overlaps with that of EMR2 [18 ], expression levels can differ tremendously in a specific cell population. EMR2 is found high on monocytes and macrophages but only dim on granulocytes and DC. CD97, in contrast to EMR2 and EMR3, is found on essentially all types of leukocyte, including the different lymphoid lineages. Table 1 summarizes the expression of the human EGF-TM7 receptors CD97, EMR2, and EMR3. It is interesting that EMR3 is the only EGF-TM7 receptor that is present mainly on granulocytes.


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  		Table 1. Cellular Distribution of the Human EGF-TM7 Receptors CD97, EMR2, and EMR3 on Hematopoietic Cells

 
An intriguing finding of this study is the late up-regulation of EMR3 during granulopoiesis. In contrast with CD97 and EMR2, EMR3 is absent on hematopoietic stem and progenitor cells. We show that in vitro differentiation of promyeloid HL-60 cells (with DMSO) or CD34+ bone marrow progenitor cells (with G-CSF) results in up-regulation of EMR3. Markedly, EMR3 appears late during neutrophil differentiation. Comparison with CD11b and CD16, which are expressed late during granulopoiesis [42 ], and morphological analysis of EMR3+ cells indicate that EMR3 does not emerge at the cell surface prior to the transition of metamyelocytes toward mature neutrophils. As EMR3 is a late-stage marker of neutrophil maturation, it will be interesting to investigate how EMR3 expression and localization are regulated.

The expression of EMR3 on mature neutrophils and monocytes implies that EMR3 might contribute to the core function of phagocytic cells in fighting pathogens in a direct way or by affecting the capacity to sense pathogens, to migrate, or to communicate with other cell types. The recently reported ability of CD97 mAb to block neutrophil migration [10 , 11 ] might provide a clue for further functional investigations into the EGF-TM7 family. So far, the function of EMR3 is elusive, and its elucidation will be delayed by the absence of EMR3 in rodents and all premammalian animals. Consequently, in vivo studies based on genetic ablation or blockade with antibodies in the animal models most commonly used are impossible. In addition and in contrast with many other families of transmembrane molecules, TM7 receptors only occasionally respond to mAb cross-linking. In agreement, C5a-stimulated migration of neutrophils through a layer of HUVEC was not affected by the presence of 3D7 (data not shown). Thus, understanding the biological role of EMR3 will essentially depend on knowledge gained from research of other EGF-TM7 family members. A vital and direct goal will be the identification of the ligand(s) of EMR3 and the investigation of ligation-induced molecular changes.


   ACKNOWLEDGEMENTS
 
This work was supported by the Landsteiner Foundation for Bloodtransfusion Research. We thank Dr. Martin Stacey for providing us with the EMR3 cDNA, Dr. Edward F. Knol and Dr. Linde Meyaard for experimental advice, Professor René van Lier for helpful discussions, and Professor Dirk Roos for critical reading of the manuscript.

Received April 15, 2006; revised October 18, 2006; accepted October 20, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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