* Laboratory for Experimental Immunology and Department of Immunobiology, CLB, and
Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, The Netherlands; and
Sir William Dunn School of Pathology, University of Oxford, United Kingdom
Correspondence: Dr. Jörg Hamann, Laboratory for Experimental Immunology, G1-106, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. E-mail: j.hamann{at}amc.uva.nl.
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part and a TM7/cytoplasmic ß part. Analysis of EMR2 expression on primary blood leukocytes, on hematopoietic cells lines, and in situ revealed a myeloid-restricted profile. Highest expression levels were detected on the more mature CD16+ blood monocytes, on macrophages, and on BDCA-3+ myeloid DC, whereas little if any expression was found on granulocytes. Unlike CD97, no expression was observed on resting or activated lymphocytes. Different expression patterns and the inability of EMR2 to interact with the CD97 ligand CD55 indicate that the molecular twins EMR2 and CD97 likely have nonredundant functions.
Key Words: CD97 • myeloid lineage • proteolytic processing
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Heterodimeric expression of two EGF-TM7 family members has been shown recently. Translated as a single polypeptide, cleavage of CD97 proximal to the first transmembrane domain results in the formation of a heterodimer comprised of an extracellular subunit noncovalently associated with a TM7/cytoplasmic ß subunit [7
, 8
]. Likewise, for ETL, a heterodimeric structure has been described, and cleavage has been mapped to a conserved motif immediately N-terminal to the TM7 region. This motif, containing four invariant cysteines, is found in all LNB-TM7 receptors but also in several non-TM7 receptors [15
]. Based on the earlier observation that this motif is a proteolytic site in the LNB-TM7 receptor CIRL/latrophilin [16
], it has been termed GPCR proteolytic site (GPS) [17
]. Although a likely characteristic of all LNB-TM7 receptors, no functional consequence has yet been ascribed to the proteolytic cleavage.
Receptors of the EGF-TM7 family are expressed by cells of the immune system and by smooth muscle cells. Using monoclonal antibody (mAb), expression of human CD97 and mouse EMR1 has been analyzed in detail. CD97 is expressed on a broad array of hematopoietic cells including activated lymphocytes, granulocytes, monocytes, macrophages (except for microglia), and dendritic cells (DC) [6 , 18 19 20 ]. In addition, smooth muscle cells and malignant cells in various epithelial tumors express CD97 [20 , 21 ]. In contrast, expression of mouse EMR1 is restricted to myeloid cells including monocytes, mature macrophages, and Langerhans cells (LC; ref. [5 ], and references therein). Knowledge of the distribution of other EGF-TM7 family members so far is based solely on RNA analysis. Whereas EMR2 and EMR3 transcripts have been detected in granulocytes, monocytes, and macrophages [13 , 14 ], ETL transcripts were found in smooth muscle cells [15 ].
To date, the only EGF-TM7 family member with a ligand identified is CD97. CD97 binds CD55/decay accelerating factor [22 ], a molecule that regulates the complement cascade by inhibiting C3/C5 convertases [23 ]. Affinity of the CD55 binding site, which is formed by the EGF domain region, differs between CD97 isoforms and is highest for the three EGF domain-containing smallest isoform [24 ]. CD97(EGF1,2,5) binds CD55 with a Kd of 86 µM and an off rate of 0.6 s-1 [25 ]. Whether the CD97-CD55 interaction is involved in cell adhesion or has another physiological function still remains elusive.
Recent characterization of EMR2 has revealed that the five EGF domains of this novel EGF-TM7 receptor differ from those of CD97 by only six amino acids [13 ]. Surprisingly, despite the fact that only three amino acids are different in the three EGF domain-containing isoforms, the affinity of EMR2(EGF1,2,5) for CD55 is at least one order of magnitude weaker than that of CD97(EGF1,2,5) [25 ]. Consequently, EMR2 transfectants do not bind CD55-expressing cells in cell-adhesion assays.
Because of the similarity between CD97 and EMR2, previous mAb raised against EGF domains of CD97 are known to cross-react with those of EMR2 [13 ]. In this study, a new mAb specific for the stalk region of EMR2 has been used to investigate the structure and cellular distribution of EMR2. In addition, because most previous studies on the expression of CD97 were done with mAb against the first EGF domain, these data were evaluated using a specific mAb against the stalk region of CD97.
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Flow cytometry
Flow cytometry was performed by standard procedures on a FACScan (Becton Dickinson, Mountain View, CA). Apart from the EMR2 mAb 2A1, the CD97 mAb CLB-CD97/1 (binds to EGF domain 1) and CLB-CD97/3 (binds to stalk region) [26
] directed toward the following antigens were used: CD11b (CLB-B2.12; CLB, Amsterdam, The Netherlands), CD14 [Leu-M3, phycoerythrin (PE)-labeled; Becton Dickinson, San Jose, CA], CD14 [fluorescein isothiocyanate (FITC)-labeled; Becton Dickinson, San Jose, CA], CD16 (CLB-FcRgran/1, FITC-labeled; CLB), CD25 (CLB-IL2R/1, FITC-labeled; CLB), CD63 (CLB-gran/12; CLB), and CD69 (TP1.55.3, PE-labeled; CLB). An IgG1 mouse anti-dog mAb (6E9; CLB) was used as control Ig (cIg). Prior to incubation with primary mAb, Fc receptors were blocked with phosphate-buffered saline (PBS)/0.5% bovine serum albumin (BSA) containing 10–20% human pooled serum. PE (Immunotech, Marseille, France)- or FITC-conjugated (CLB) goat anti-mouse Ig were used as secondary reagents.
To characterize monocyte-derived cell subsets, three-color flow cytometry was performed on a FACS-Calibur (Becton Dickinson, Mountain View, CA). Human whole blood samples were incubated in a first step with biotinylated 2A1 or CLB-CD97/3, followed by a second step with mAb to CD14 (PE-labeled) and CD16 (FITC-labeled), and streptavidin-APC (PharMingen, San Diego, CA). Prior to cytometry, erythrocytes were shocked using FACS lysing solution (Becton Dickinson, San Jose, CA).
Western blot analysis and immunoprecipitation
To generate expression constructs containing a C-terminal V5/histidine tag, CD97(EGF1,2,5) and EMR2(EGF1,2,5) cDNA were cloned into pcDNA3.1/V5/His-TOPO (Invitrogen). Using Lipofectamine Plus, COS cells were transfected with these constructs or, as mock controls, with pcDNA3.1/V5/His-TOPO containing CD97(EGF1,2,5) cDNA in reverse orientation (immunoprecipitation) or pcDNA3.1/Zeo(+)-CD97(EGF1,2,5) (Western blot). Three days post-transfection, cells were harvested, and expression was tested by flow cytometry using 2A1 and CLB-CD97/3. For immunoprecipitation, cells were biotinylated with NHS-LC-biotin (Pierce, Rockfort, IL) 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 ethylenediaminetetraacetate, and 1% Nonidet P-40, supplemented with protease inhibitors. After centrifugation, cell extracts were incubated with an irrelevant mouse mAb (5 µg/ml), precleared with protein A-sepharose (Sigma Chemical Co., St. Louis, MO), and incubated with 2A1 or CLB-CD97/3 at 4°C for 1 h. Immune complexes were adsorbed onto protein A-sepharose, washed extensively, eluted under reducing conditions, separated electrophoretically by 8.75% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to Hybond-C extra membrane (Amersham Life Science, Piscataway, NJ). The membrane was incubated overnight (o/n) at 4°C in 3% Protivar (protein-rich milk) in Tris-buffered saline supplemented with 0.05% Tween 20 (TBST), followed by incubation for 1 h with polystreptavidin-horseradish peroxidase (HRP; CLB) in 1% Protivar in TBST. After washing, precipitated protein was visualized by enhanced chemiluminescence (ECL; Amersham Life Sciences).
For Western blot analysis, transfected COS cells were lysed as described. Cell extracts were separated electrophoretically by 12.5% SDS-PAGE and transferred to Hybond-C extra membrane. The membrane was incubated o/n at 4°C in 3% Protivar in TBST followed by staining for 1 h with an HRP-labeled V5 mAb (Invitrogen) in 1% Protivar in TBST. After washing, binding was visualized by ECL.
Lymphocyte activation
Human peripheral blood mononuclear cells (PBMC) were cultured at 2 x 106 cells/ml in Iscove’s modified Dulbecco’s medium/10% fetal calf serum without addition or with 1 µg/ml phytohemagglutinin (PHA), 10 ng/ml phorbol 12-myristate 13-acetate (PMA), or 1:1000 CD3 ascites (CLB-T3/3; CLB). After 0, 4, and 24 h, cells were harvested, washed, and analyzed by flow cytometry.
Granulocyte stimulation
Neutrophilic granulocytes were stimulated according to the protocol of Kuipers et al. [27
]. In short, neutrophils purified by density-gradient centrifugation and erythrocyte lysis were resolved at 2 x 106 cells/ml in HEPES buffer supplemented with 0.5% human serum albumin, 1 mM CaCl2, and 1 g/l glucose. Stimulation was performed at 37°C, while cells were shaken gently. After 5 min of preincubation, cells were stimulated by addition of platelet-activating factor (PAF; 1 µmol/l, 2 min) or PAF (1 µmol/l, 2 min), followed by formyl-Met-Leu-Phe (fMLP; 1 µmol/l, 15 min) or cytochalasin B (5 mg/ml, 5 min), followed by fMLP (1 µmol/l, 15 min). Control reactions were performed without additions (2 min). Reactions were stopped with excess ice-cold HEPES buffer. After centrifugation, cells were fixed with PBS/1% paraformaldehyde (10 min), washed, and analyzed by flow cytometry.
Isolation of DC subpopulations
Blood DC of the myeloid and plasmacytoid lineage were isolated from peripheral blood according to the protocol of Dzionek et al. [28
]. In short, after depletion of CD19+ B cells using anti-CD19 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany), cells were labeled with PE-conjugated mAb directed against blood dendritic cell antigen (BDCA)-1, BDCA-3, or BDCA-4 for 15 min at 4°C in FcR-blocking reagents (Miltenyi Biotec). Next, anti-PE microbeads were added and incubated for another 15 min at 4°C. BDCA-1-, BDCA-3-, or BDCA-4-expressing cells were subsequently isolated using immunomagnetic cell sorting (MACS, Milteyi Biotec). Enriched cell fractions were washed, stained with mAb, and analyzed by flow cytometry.
Preparation of tissue sections and immunohistochemistry
Human tonsil was obtained postoperatively from the ENT Department of Oxford Radcliffe Trust (Radcliffe Infirmary, Oxford, UK). Other normal human tissues were obtained from the Clinical Laboratory Sciences Department of the John Radcliffe Hospital (Oxford, UK). Tissues were snap-frozen in liquid nitrogen. Cryostat sections (10 µm) were cut and stored at -70°C. For immunohistochemistry, the tissue sections were fixed with cold acetone. Fixed sections were washed in PBS and blocked with PBS/0.5% BSA containing 2% of normal serum of the species in which the secondary Ab was raised. Subsequently, 2A1, CLB-CD97/3, and mAb to CD68 (EBM11; DAKO, Glostrup, Denmark) and CD1a (NA1/34; DAKO) diluted in the blocking buffer were applied. Sections were washed in PBS, and endogenous peroxidase activity was then depleted with 1% of H2O2 in methanol. After another round of washes, sections were incubated with biotinylated secondary Ab and subsequently incubated with an avidin-biotin-peroxidase complex (ABC Elite, Vector Laboratories, Burlingame, CA). Peroxidase activity was detected with 0.5 mg/ml 3,3'-diaminobenzidine tetrahydrochloride and 0.024% H2O2 in PBS containing 10 mM imidazole. Sections were counterstained with methyl green. Photos of stained sections were taken using a Zeiss Axioplan 2 microscope with SPOT program (Diagnostic Instruments, Inc., Sterling Heights, MI).
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Figure 1. Generation of an EMR2-specific mAb. (A) Schematic structure of the largest isoform of EMR2 possessing one classical and four calcium-binding (shaded) EGF domains. 3T3-Cells stably expressing this isoform were used for immunization of a Balb/c mouse. (B) Flow cytometric analysis of the specificity of existing CD97 mAb and the newly produced EMR2 mAb 2A1. 3T3-Cells expressing EMR2(EGF1–5) or CD97(EGF1–5) were stained with 2A1, CLB-CD97/1, CLB-CD97/3, or cIg. Goat anti-mouse Ig-PE was used as secondary Ab.
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part of 65 kD (isoform with three EGF domains) linked to a TM7/cytoplasmic ß part of 28 kD. The 100-kD band observed in Figure 2A
is likely to represent the /ß heterodimer of each protein.
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Figure 2. Biochemical characterization of EMR2 and CD97. (A) Immunoprecipitation of EMR2 and CD97 from biotinylated COS cells expressing EMR2(EGF1,2,5)-V5/His or CD97(EGF1,2,5)-V5/His using 2A1 and CLB-CD97/3. As a mock control, COS cells transfected with a CD97(EGF1,2,5) construct in inverse orientation were used. The second-step reagent polystreptavidin-HRP was visualized by ECL. (B) Western blot detection of the C-terminal tag from the above-described recombinants using an HRP-labeled V5 mAb followed by ECL visualization. As mock control, COS cells expressing nontagged CD97(EGF1,2,5) were used.
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Figure 3. Expression of EMR2 and CD97 on PBL. Cells were analyzed by flow cytometry with 2A1 (bold, solid line), CLB-CD97/1 (solid line), CLB-CD97/3 (dashed line), and cIg (dotted line). Goat anti-mouse Ig-PE was used as a secondary Ab. Cell types were determined on the basis of forward- and side-scatter.
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Figure 4. Expression of EMR2 and CD97 on leukemia cell lines. Lymphoid (Ramos and Jurkat) and myeloid (KG1a, HL60, THP1, K562, U937, and MonoMac6) cell lines were analyzed by flow cytometry with 2A1 (bold, solid line), CLB-CD97/1 (solid line), CLB-CD97/3 (dashed line), and cIg (dotted line). Goat anti-mouse Ig-PE was used as a secondary Ab.
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EMR2 is not up-regulated during stimulation of lymphocytes and granulocytes
To test whether EMR2 is up-regulated during lymphocyte activation, as has been demonstrated for CD97 [6
, 18
, 19
, 24
], PBMC were stimulated with PHA, PMA, or CD3 mAb and analyzed for expression of EMR2 and CD97 after 4 and 24 h (Fig. 5
). In contrast to CD97, no up-regulation of EMR2 was observed, confirming previous reverse transcriptase-polymerase chain reaction (RT-PCR) data [13
].
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Figure 5. Expression of EMR2 and CD97 on activated PBMC. PBMC were cultured with PHA, PMA, CD3 mAb, or without addition. Expression of EMR2 and CD97 was analyzed at different time points by flow cytometry with 2A1, CLB-CD97/1, CLB-CD97/3, and cIg. Goat anti-mouse Ig-PE was used as secondary Ab. Staining with conjugated mAb for CD69 and CD25 was used as positive control for cellular activation.
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Table 1. EMR2 and CD97 Expression on Activated, Purified, Neutrophilic Granulocytes
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Figure 6. Expression of EMR2 and CD97 on monocyte subsets. Blood monocytes were analyzed by three-color flow cytometry for expression of EMR2 and CD97 on typical blood monocytes (CD14++CD16-), macrophage-like monocytes (CD14++CD16+), and DC-like monocytes (CD14+CD16+). CD14 and CD16 were stained with conjugated mAb, EMR2, and CD97 with biotinylated 2A1 and CLB-CD97/3. Streptavidin-PE was used as second-step reagent. Monocytes were determined on the basis of forward- and side-scatter. Expression of EMR2 and CD97 is given as mean fluorescence intensity (MFI) ± SEM of five experiments.
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Figure 7. Expression of EMR2 and CD97 on blood DC. Blood DC expressing BDCA-1 (myeloid DC), BDCA-3 (myeloid DC), and BDCA-4 (plasmacytoid DC) were purified by MACS. Expression of EMR2 and CD97 was analyzed by flow cytometry with biotinylated 2A1 (bold, solid line), CLB-CD97/1 (solid line), and CLB-CD97/3 (dashed line). Streptavidin-FITC was used as second-step reagent. As reference, expression of CD14 is shown (dotted line). Whereas BDCA-3+ and BDCA-4+ DC are CD14-, some BDCA-1+ cells express CD14 [28
].
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Table 2. Tissue Distribution of EMR2 and CD97 in Various Tissues
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Figure 8. Immunohistochemical localization of EMR2 and CD97 in human tissues. Skin (A–D), lung (E–H), tonsil (I–L), and liver (M–P) were stained with 2A1 (A, E, I, M), CLB-CD97/3 (B, F, J, N), mAb for CD68 (C, G, K, O), and CD1a (D, H, L, P). (A) Staining of skin with 2A1 showed weak labeling of few macrophages. (B) In an adjacent section, CLB-CD97/3 labeled macrophages and smooth muscle cells (arrowhead). (C, D) Although the CD68 mAb stained those macrophages in the dermis, the CD1a mAb showed strong staining in the epidermis (arrow). A few macrophages/histiocytes in the dermis were also stained for CD1a. (E) In the lung, interstitial macrophages (arrow) but not alveolar macrophages were weakly labeled by 2A1. (F) In an adjacent section, CLB-CD97/3 strongly labeled smooth muscle cells (arrowheads), underlining the secondary bronchus as well as in the lung tissues. Interstitial macrophages were weakly stained. (G) The CD68 mAb labeled interstitial macrophages as well as alveolar macrophages (arrow). (H) Staining of lung with the CD1a mAb showed little reactivity. (I) In addition to macrophages/histiocytes present in the connective tissues of a tonsil, 2A1 strongly labeled interfollicular macrophages/histiocytes (arrow), whereas no labeling was detected on the tingible body macrophages of the germinal centers. (J) CLB-CD97/3 labeled macrophages/histiocytes as well as lymphocytes in a tonsil section. (K) The CD68 mAb stained interfollicular macrophages and tingible body macrophages (arrow) of the germinal centers. (L) The CD1a mAb labeled only a few histiocytes in the interfollicular area and in the connective tissues of a tonsil. (M) Staining of liver with 2A1 showed no reactivity. (N) A small subpopulation of Kupffer cells was labeled with CLB-CD97/3 (arrow). (O, P) Kupffer cells were strongly stained by the CD68 Ab, whereas no labeling was seen with the CD1a Ab. Control staining using isotype-matched mouse IgG1 showed no reactivity in those tissue sections described above (not shown). The black bar in P represents 50 µm.
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Biochemical analysis of EMR2 revealed the existence of two receptor subunits, an extracellular part and a TM7/cytoplasmic ß part. Likely, as has been demonstrated for CD97 [7
], the EMR2 polypeptide is cleaved in the endoplasmic reticulum or early Golgi into these two subunits, which associate noncovalently and are expressed on the cell surface as a heterodimer (see Fig. 1A
for the schematic structure). Apart from CD97, CIRL/latrophilin [16
], and ETL [15
], EMR2 is the fourth LNB-TM7 receptor that is shown to undergo proteolytic processing. Based on the size of the receptor subunits, N-terminal amino acid sequencing of the CIRL/latrophilin ß subunit, and mutation studies on ETL, the processing site has been mapped to a cysteine box immediately N-terminal to the TM7 region. This site, recently termed GPS motif [17
], is found in all LNB-TM7 receptors, suggesting that all these receptors are potential heterodimers. The functional relevance of the proteolytic processing still remains elusive.
For many GPCR with an extended extracellular region, ligand binding is a two-step process [31
]. First, a complex is formed between the N-terminal receptor region and the ligand. Next, this complex interacts with the TM7 region to trigger signals via G proteins. A heterodimeric structure as found in EGF-TM7 receptors could facilitate signal transduction in different ways. Noncovalent association with the ß part may directly increase the structural flexibility of the part to make contact with the TM7 exoloops. Alternatively, the subunit could be released after ligand binding and subsequently, an additional, second ligand might activate the receptor. However, as yet, the coupling of G proteins to EGF-TM7 receptors has not been formally proven.
In contrast to the structural similarity, EMR2 and CD97 have distinct expression profiles. Whereas CD97 is distributed broadly and is found on all types of hematopoietic cells as well as on smooth muscle cells [6 , 18 19 20 ], expression of EMR2 is restricted to myeloid cells. Flow cytometry analysis and RT-PCR (unpublished results) indicate that expression levels of EMR2 are generally lower compared with CD97. Highest levels of EMR2 are found on macrophages and myeloid DC, pointing to a function of EMR2 in those cells. In contrast to previous RNA blot data [13 ], only low EMR2 expression was found on granulocytes even after cellular activation. The nature of this discrepancy is uncertain and will be investigated further. In part, expression of EMR2 resembles that of F4/80, the mouse homologue of EMR1, which is expressed on mature tissue macrophages [5 ]. Detailed comparison of the expression pattern of EMR1, EMR2, and the most recently identified EMR3 [14 ] will await the generation of mAb to human EMR1 and EMR3 receptors. Questions that remain regarding the expression of EMR2 include the regulation of expression during myeloid differentiation, the up-regulation at sites of inflammation, and the presence on malignant cells.
Because current knowledge of the distribution of CD97 is mainly based on mAb against the first EGF domain, which have been shown recently to also recognize EMR2 [13 ], we used this study to evaluate previous data about the expression of CD97. No significant differences in binding between CLB-CD97/1 and CLB-CD97/3, which recognize the first EGF domain and the stalk region of CD97, respectively, were found. This confirms similar observations from a recent immunohistochemical study [20 ]. Obviously, because of the more restricted expression pattern of EMR2 and the much higher expression levels of CD97, mAb against the EGF domain region primarily reflect expression of CD97.
The differences found for EMR2 and CD97 with regard to cellular distribution, activation-dependent up-regulation, and ligand binding strongly imply that both molecules have a different function. From the structural similarity between EMR2 and CD97, a cellular ligand for EMR2 is to be expected. Its identification and a further characterization of the expression of EMR2 should shed more light on the function of EMR2 and other EGF-TM7 family members as well as on the biology of macrophages.
Received September 12, 2001; revised January 23, 2002; accepted January 23, 2002.
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