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Originally published online as doi:10.1189/jlb.0704402 on October 21, 2004

Published online before print October 21, 2004
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(Journal of Leukocyte Biology. 2005;77:112-119.)
© 2005 by Society for Leukocyte Biology

Expression of the largest CD97 and EMR2 isoforms on leukocytes facilitates a specific interaction with chondroitin sulfate on B cells

Mark J. Kwakkenbos*, Walter Pouwels*, Mourad Matmati*, Martin Stacey{dagger}, Hsi-Hsien Lin{dagger}, Siamon Gordon{dagger}, René A. W. van Lier* and Jörg Hamann*,1

* Laboratory for Experimental Immunology, Academic Medical Centre, University of Amsterdam, The Netherlands; and
{dagger} Sir William Dunn School of Pathology, University of Oxford, United Kingdom

1 Correspondence: Laboratory for Experimental Immunology, G1-106, Academic Medical Centre, University of Amsterdam, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands. E-mail: j.hamann{at}amc.uva.nl


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ABSTRACT
 
The EGF-TM7 receptors CD97 and EMR2 are heptahelical molecules predominantly expressed on leukocytes. A characteristic of these receptors is their ability to interact with cellular ligands via the N-terminal epidermal growth factor (EGF)-like domains. The first two EGF domains of CD97 (but not EMR2) bind CD55 (decay-accelerating factor), while the fourth EGF domain of both CD97 and EMR2 interacts with the glycosaminoglycan chondroitin sulfate (CS). Using fluorescent beads coated with soluble recombinant CD97 and EMR2 protein, and isoform-specific monoclonal antibodies, we have determined the cellular and molecular characteristics of the interaction with CS. The fourth EGF domain of CD97 and EMR2 is expressed on activated lymphocytes and myeloid cells, whereas the ligand is specifically found on B cells within the peripheral blood. The interaction between CD97/EMR2 and CS may therefore play a role in the interaction of activated T cells, dendritic cells, and macrophages with B cells.

Key Words: B cell • EGF-like domain • ligand specificity • multivalent probe • proteoglycan


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INTRODUCTION
 
Membrane proteins that are involved in cell-cell interactions are critical for a normal immune response. These proteins can be divided into different categories, namely those involved in antigen recognition, costimulation, and cellular adhesion and migration [1 ]. A group of receptors, which have recently been recognized to have a function in cell adhesion and/or migration, is the epidermal growth factor-seven-span transmembrane (EGF-TM7) family [2 3 4 ], which belongs to the adhesion class (LNB-TM7 family) of TM7 receptors [5 6 7 ]. Five predominantly leukocyte-restricted human EGF-TM7 receptors have been identified: CD97 [8 , 9 ], EGF-like module-containing mucin-like receptor protein (EMR)1 [10 ], EMR2 [11 ], EMR3 [12 ], and EMR4 [13 ]. Whereas expression of EMR1-4 is restricted to myeloid cells [3 ], CD97 is more widely expressed on myeloid and lymphoid cells as well as smooth muscle cells [8 , 14 , 15 ].

An extended extracellular region comprising N-terminal EGF-like domains characterizes the EGF-TM7 receptors [3 ]. These EGF domains are coupled to the TM7 part via an extended spacer region. As a result of alternative RNA splicing, receptor isoforms possessing variable numbers of EGF domains are expressed.

The EGF domains of EGF-TM7 receptors have been shown to mediate binding to cellular ligands. Two ligands for CD97 have been identified so far. Whereas EGF domains 1 and 2 facilitate binding to CD55 [4 , 16 17 18 ], EGF domain 4 interacts with the glycosaminoglycan (GAG) chondroitin sulfate (CS) [19 ]. CD97 has three isoforms containing three (EGF1,2,5), four (EGF1,2,3,5), or five (EGF1,2,3,4,5) EGF domains [9 ]. Although all isoforms can bind CD55, only the largest isoform, containing EGF domain 4, interacts with CS. The affinity for CD55 differs between the different CD97 isoforms and is highest for the three EGF domain-containing isoform [17 , 18 ]. The largest isoform, possessing five EGF domains, has the lowest affinity for CD55.

Ligand specificity for CS is shared by EMR2, whose EGF domain region is highly similar to that of CD97. Only six out of 236 amino acids differ within the five EGF domains [11 ]. Despite this small difference, EMR2 does not bind to CD55 [11 , 18 ]. Four isoforms have been described for EMR2, containing two (EGF1,2), three (EGF1,2,5), four (EGF1,2,3,5), or five (EGF1,2,3,4,5) EGF domains. As for CD97, only the largest isoform binds CS [19 ].

In a recent in vivo study, we demonstrated an essential role of CD97 in the migration of neutrophilic granulocytes [4 ]. In experimental colitis, homing of adoptively transferred neutrophils to the colon was significantly delayed when cells were preincubated with CD97 monoclonal antibody (mAb). The consequences of this defect in neutrophil migration for host defense became apparent in a model of pneumococcal pneumonia. Application of CD97 mAb impaired the recruitment of neutrophils to the lungs, thereby significantly reducing bacterial clearance and survival.

As yet, it is not clear how the molecular interactions engaged by CD97 contribute to cell migration. Whereas CD55 is a glycosylphosphatidylinositol-linked molecule that protects cells from complement-mediated damage by accelerating the decay of C3/C5 convertases [20 ], CS is a GAG, implicated in a wide range of physiological processes [21 , 22 ].

Following the recent identification of CS as a ligand of the largest isoform of CD97 and EMR2 [19 ], we here investigated the cellular and molecular characteristics of this interaction. Soluble recombinant CD97 and EMR2 isoforms were generated and coupled to fluorescent beads to generate multivalent probes. Using these tools in combination with isoform-specific mAb, we analyzed whether the CD55 binding EGF domains 1 and 2 and the CS binding EGF domain 4 cooperate in cellular contacts. In addition, we studied whether physiological interactions between CS and EGF-TM7 receptors can take place in the immune system.


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MATERIALS AND METHODS
 
Cell culture
Cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 50 IU/ml penicillin, and 50 µg/ml streptomycin. All cells were incubated at 37°C in 5% CO2 and 95% humidity.

Leukocyte isolation
Peripheral blood lymphocytes and peripheral blood mononuclear cells (PBMC) were isolated from heparinized peripheral blood samples of healthy volunteers using standard procedures. Surplus splenocytes, used for human leukocyte antigen typing of potential organ donors, were isolated from small parts of spleen by grinding the tissues through nylon sieves and density gradient centrifugation. According to paragraph 13 from the Dutch Law for Organ Donation, these specimens could be used for scientific research. Flow cytometric analysis using CD3, CD14, CD19, and CD56 mAb (see below) showed the cell mixture to contain mostly lymphocytes and monocytes. Typically, 50% of these cells were B cells (data not shown).

Production of biotinylated recombinant Fc proteins
CD97 isoform sequences, encoding three (EGF1,2,5), four (EGF1,2,3,5), or five (EGF1,2,3,4,5) EGF domains and 48 amino acids of the stalk region, were amplified by polymerase chain reaction (PCR) using primers KE5 (5'-GCTGGTACCATGGGAGGCCGCGTCTTTCTCG) and CD97BIO-3 (5'-ACGAATTCGATGACATTCTGGATGGT) and a pcDNA3.1(+) vector containing the CD97 isoforms as template [17 ]. PCR products were ligated into pGEM-T easy vector (Promega, Madison, WI) and after digestion with KpnI and EcoRI (sites introduced by primers, underlined), were subcloned into a pcDNA3.1(+) vector (Invitrogen, Leek, The Netherlands). These vectors contained C-terminal the CH3-CH2-hinge region sequence of mouse immunoglobulin G (IgG)2b linked to a peptide recognition sequence (DPNSGSLHHILDAQKMVWNHR) for the Escherichia coli biotin holoenzyme synthetase BirA. After translation, resulting soluble proteins comprised the CD97 EGF domains, part of the stalk region, truncated mouse IgG2b, and the BirA recognition peptide sequence. Generation of EMR2 constructs has been reported recently [19 ]. Soluble biotinylated Fc proteins were produced as described earlier [23 ]. Briefly, conditioned OptiMem 1 medium (Life Technologies Ltd., Paisley, Scotland), containing soluble recombinant protein collected from transfected human embryo kidney (HEK)293 cells was purified using a Protein A (Sigma Chemical Co., St. Louis, MO) column. After purification, recombinant protein derived from four transfected 225 cm2 cell-culture flasks was concentrated to ~0.5 ml using a 30-kDa molecular weight cut-off filter (Millipore, Bedford, MA) dialyzed against 10 mM Tris-HCl (pH 8) buffer and incubated with 2 µl BirA enzyme and supplied substrates (Avidity, Denver, CO) overnight at room temperature. Excess biotin was subsequently removed by dialysis against 10 mM Tris-HCl (pH 7.3) buffer containing 10 mM CaCl2 and 150 mM NaCl. The biotinylated proteins were then aliquoted and stored at –80°C after quantification by Bradford assay.

Flow cytometry using biotinylated protein-coupled fluorescent beads
Cell-binding assays using biotinylated Fc proteins coupled to fluorescent beads were performed as described previously [23 ]. Briefly, 10 µl avidin-coated fluorescent beads (Spherotech Inc., Libertyville, IL) were washed with phosphate-buffered saline (PBS)/0.5% bovine serum albumin (BSA) and incubated with saturating amounts (>1 µg) of biotinylated recombinant protein in a volume of 10 µl. After 1 h, nonbinding protein was removed by washing with PBS/0.5% BSA. The bead-protein complexes were sonicated immediately before addition to the cells (0.5x106 cells/50 µl PBS/0.5% BSA). For blocking studies, 1 µg mAb was added to the bead-protein complexes and incubated for 10 min at 4°C before adding the complexes to the cells. The cell-bead mixture, in a 96-well flat-bottom plate, was spun at 1000 g at 4°C for 10 min, incubated for another 50 min at 4°C, and finally resuspended in 300 µl PBS/0.5% BSA for flow cytometric analysis. All flow cytometry was performed on a FACSCalibur (Becton Dickinson, San Jose, CA).

GAG assays
Cell-binding assays were done as described above. Cells were treated with chondroitinase ABC (Sigma Chemical Co.; 0.8 U/4x106 cells/ml) or heparinase III (Sigma Chemical Co.; 4 U/4x106 cells/0.3 ml) or incubated in buffer without enzyme as control. After enzymatic treatment, some samples were incubated for 10 min with 1 µg CD55 mAb (CLB-CD97L/1; ref. [16 ]) to block CD55-mediated binding. For GAG competition assays, 0.5 x 106 cells were incubated in increasing concentrations of exogenous CS-A (C-8529, Sigma Chemical Co.), CS-B (C-3788, Sigma Chemical Co.), or CS-C (C-4384, Sigma Chemical Co.) before addition of protein-bead complexes.

Functional assays
PBMC were enriched for B cells by depletion of T cells, natural killer (NK) cells, and monocytes using CD3 (CLB-1x1, CLB, Amsterdam, The Netherlands), CD14 (CLB-8C3, CLB), and CD16 (CLB-5D2, CLB) mAb and goat-anti-mouse IgG M-450 Dynabeads (Dynal Technology, Oslo, Norway) according to the manufacturer’s instructions. Resulting cell suspension contained 80–95% B cells. Cells were cultured with or without anti-IgM (CLB-MH15, ascites 1:1000, CLB), anti-CD40 (CLB-14G7, 5 µg/ml, CLB), interleukin (IL)-4 (10 ng/ml, Strathmann Biotech GmbH, Hamburg, Germany), and different CD97 or EMR2 probes (10 µl-coated beads/ml) for 1–7 days. Cellular activation was determined after overnight culture by measuring CD69 expression. Proliferation of B cells was determined by labeling the cells with carboxyfluoroscein succinimidyl ester (CFSE) according to the manufacturer’s instructions and assessing CFSE levels after 5–7 days of culture. Ig secretion and class-switching were measured after 7 days of culture using IgM and IgG enzyme-linked immunosorbent assay [24 ].

Flow cytometry using Ab
mAb against CD97 and EMR2 were described previously. CLB-CD97/3 [25 ] and 2A1 [26 ] recognize the stalk region of CD97 and EMR2, respectively. CLB-CD97/1 [16 ] recognizes EGF domain 1 of CD97 and EMR2, and 1B5 [19 ] recognizes EGF domain 4 of CD97 and EMR2.

Ab were obtained from Becton Dickinson unless otherwise specified. CD3-phycoerythrin (PE), CD3-allophycocyanin (APC), CD4-peridinin chlorophyll protein (PerCP), CD8-PerCP, CD14-PE, CD14-APC (PharMingen, San Diego, CA), CD16-fluorescein isothioycanate (FITC; CLB), CD19-PerCP-Cy5.5, CD56-APC, CD69-APC (Caltag Laboratories, Burlingame, CA), and biotinylated mouse IgG1 (DAKO A/S, Glostrup, Denmark) were used. To characterize monocyte-derived cell subsets, three-color flow cytometry was performed on human PBMC, which were incubated in a first step with biotinylated 1B5, 2A1, or CLB-CD97/3, followed by a second step with mAb to CD14 (PE-labeled) and CD16 (FITC-labeled) and streptavidin-APC (PharMingen). To analyze 1B5 expression, human whole blood samples were incubated in a first step with biotinylated CLB-CD97/3, 2A1, 1B5, or control IgG1, followed by a second step with streptavidin-APC. Prior to flow cytometry, erythrocytes were lysed using FACS lysing solution (Becton Dickinson). Leukocyte populations were defined by forward- and side-scatter.

Lymphocyte activation
For lymphocyte activation studies, PBMC were cultured at 1 x 106 cells/ml in IMDM with or without 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.


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RESULTS
 
CD97-5EGF has a ligand specifically expressed on B cells
A multivalent assay, developed by Brown et al. [27 ] for the investigation of low-affinity interactions between leukocyte cell-surface molecules, has recently proved successful in studying EGF-TM7 receptors [12 , 18 , 19 , 23 ]. This approach was therefore used to study the interactions of CD97 and its ligands. Soluble recombinant Fc proteins of the ligand-binding extracellular part of all naturally occurring splice variants of CD97 and EMR2 were generated to compare their ligand-binding capacity. These proteins were biotinylated and coupled to avidin-coated fluorescent beads for use in flow cytometry.

As depicted in Figure 1A , the three CD97 isoform probes displayed different binding to PBMC. In general, the CD97-3EGF probe showed the highest binding to PBMC, the CD97-4EGF probe, intermediate levels, and the CD97-5EGF probe caused only a modest shift in fluorescence. This is in complete concurrence with the reported affinities of the different isoforms of CD97 for CD55 [17 , 18 ]. Binding of the CD97-3EGF and CD97-4EGF probes could be prevented entirely by preincubating the cells with a mAb against CD55, demonstrating the CD55 specificity of these interactions (data not shown).



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  		Figure 1. CD97 isoforms differentially bind to PBMC. Binding of multivalent fluorescent probes as indicated to (A) PBMC and to (B) monocytes, B cells, T cells, and NK cells (defined by forward- and side-scatter and the expression of CD14, CD19, CD3, and CD56, respectively) was analyzed by flow cytometry. The smallest isoform of EMR2 was used as a negative control for binding [19 ]. Probe binding to CD4+ and CD8+ T cells was similar (data not shown). NK cells do not express CD55 [20 ]. (C) Binding of multivalent fluorescent probes to spleen cells, treated with CD55 mAb or in the presence of EGTA (EGF domains in EGF-TM7 receptors are stabilized by Ca2+ ions) as indicated.

It is surprising that costaining PBMC with cell type-specific markers for monocytes (CD14), B cells (CD19), T cells (CD3, CD4, and CD8), and NK cells (CD56) revealed that CD97-5EGF probe binding to B cells exceeded the binding of the CD97-3EGF probe to these cells (Fig. 1B) . In a similar test, specific binding to B cells was also observed for EMR2-5EGF. Preincubation of B cells with CD55 mAb only slightly reduced CD97-5EGF probe binding and did not affect EMR2-5EGF binding, indicating the interaction with a different structure on B cells (Fig. 1C) .

CD97-5EGF binds CS-B on B cells
We showed previously that CS expressed on fibroblastic cell lines is a ligand for the largest isoform of EMR2 [19 ]. To evaluate the contribution of B cell GAG to CD97-5EGF binding, B cells were treated with GAG-degrading enzymes (Fig. 2A ). Whereas heparinase III treatment had no effect, chondroitinase ABC digestion of B cells strongly diminished CD97-5EGF binding and completely blocked EMR2-5EGF binding. Treatment with either enzyme did not affect CD97-3EGF probe binding, indicating that binding to CD55 is not influenced by the GAG expression of the cell.



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  		Figure 2. CD97-5EGF binds to CS present on primary B cells. (A) Binding of multivalent fluorescent probes to spleen cells, treated with heparinase III, chondroitinase ABC, and CD55 mAb as indicated, was measured by flow cytometry. (B) Effect of exogenous CS on CD97-3EGF, CD97-5EGF, and EMR2-5EGF ligand binding. Bars represent binding of multivalent fluorescent probes to spleen cells. In the upper panel, cells were incubated with 10 µg/ml CS. Data shown are mean fluorescence intensity ± SD in percent from three separate experiments. The lower panel shows CD97-5EGF ligand binding in response to increasing doses of CS. Data are mean fluorescence intensity in percent.

To determine which type of CS binds CD97-5EGF on B cells, the different types of soluble CS were used as blocking agents. CS-A (chondroitin-4-sulfate), CS-B (dermatan sulfate), or CS-C (chondroitin-6-sulfate) were added to the probes before addition to the cells. As can be seen in Figure 2B , CS-B reduced the binding of the CD97-5EGF probe to B cells in a dose-dependant way to ~60%. A similar effect on binding of the EMR2-5EGF probe was observed. The discrepancy in blocking efficiency, seen between chondroitinase ABC and purified CS, is likely a result of heterogeneity of animal-derived GAG preparations (see Discussion). In conclusion, the EGF domains of the largest isoforms of CD97 and EMR2 bind to CS-B expressed specifically on B cells.

CD97-5EGF has dual ligand specificity for CS and CD55
It was noted that not all binding of the CD97-5EGF probe was prevented by chondroitinase ABC treatment of B cells (Fig. 2A) . Furthermore, a small shift in fluorescence was seen when B cells were preincubated with CD55 mAb (Fig. 1C) . This indicated that CD55 also contributed to CD97-5EGF binding to B cells. To test whether EGF domain 1 (binding CD55) and EGF domain 4 (binding CS) cooperate in cell binding, we used the human fibroblast cell line HEK293 (Fig. 3 ), which expresses high amounts of CD55 and CS, facilitating detection of both interactions. CD97-3EGF, CD97-5EGF, and EMR2-5EGF probes all bound to HEK293 cells. We made use of mAb against the first and the fourth EGF domain of CD97 and EMR2 to analyze the role of CD55 and CS in probe binding, respectively. Binding of CD97-3EGF to HEK293 cells was completely prevented when the probe was preincubated with CLB-CD97/1 (anti-EGF domain 1), whereas 1B5 (anti-EGF domain 4) had no effect. EMR2-5EGF showed the opposite. Probe binding was completely blocked by 1B5 (anti-EGF domain 4), and CLB-CD97/1 (anti-EGF domain 1) had no effect. For CD97-5EGF, a strikingly different pattern was observed. Thirty-seven percent of binding was blocked when the probe was incubated with CLB-CD97/1, showing the effect of loss of CD55 binding. A reduction of 16% was seen when the CD97-5EGF probes were incubated with 1B5, which blocks the CS-binding site. When both mAb were added, binding was completely abolished. This shows that CD97-5EGF can simultaneously bind CS and CD55 on the same cell.



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  		Figure 3. CD97-5EGF can bind to CD55 and CS present on one cell. Bars represent binding of multivalent fluorescent probes to HEK293 cells. Probes were preincubated with mAb to EGF domain 1 (CD55-binding site) and EGF domain 4 (CS-binding site) as indicated. Data shown are mean fluorescence intensity ± SD in percent from three separate experiments.

CD97-5EGF and EMR2-5EGF do not signal to B cells
Several in vitro experiments were done to investigate the consequence of the CD97/EMR2-5EGF ligand interaction on cellular B cell responses. PBMC-derived B cells were stimulated (suboptimal) through the B cell receptor and/or CD40 and in addition, triggered with CD97- or EMR2-coated beads. First, induction of cellular activation was monitored by analyzing CD69 expression levels after overnight stimulation. Second, B cell proliferation was measured after 5–7 days of culture. Third, Ig secretion and class switching were analyzed by measuring IgG and IgM levels after 7 days of stimulation. No effect of CD97 or EMR2 in any of these assays could be detected (data not shown).

CD97-5EGF and EMR2-5EGF are expressed on myeloid cells and on activated lymphocytes
To analyze the cellular distribution of the largest, CS-binding isoform of CD97 and EMR2, we used the mAb 1B5, which is directed against the fourth EGF domain of both molecules, in combination with CLB-CD97/3 (anti-CD97 stalk) and 2A1 (anti-EMR2 stalk). Flow cytometric analysis of PBMC is shown in Figure 4 . CD97 is expressed on all cell types, and EMR2 expression is restricted to myeloid cells. The CS-binding isoform of both receptors was detected only on monocytes and granulocytes.



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  		Figure 4. Monocytes and granulocytes express the largest isoform of CD97 and EMR2. PBMC were analyzed by flow cytometry with mAb as indicated. Streptavidin-APC was used as a second-step reagent. Leukocyte lineages were defined by forward- and side-scatter.

Next, we analyzed whether CD97-5EGF is up-regulated during lymphocyte activation, as has been shown previously for CD97 with mAb that do not differentiate between isoforms [14 , 26 ]. Cells were stimulated with PHA, PMA, or CD3 mAb and analyzed after 4 and 24 h (Fig. 5 ). Increased staining with 1B5 was detected after 24 h. As no up-regulation of EMR2 expression on lymphocytes was detected, consistent with previous findings [26 ], 1B5 staining solely reflects the up-regulation of CD97-5EGF expression.



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  		Figure 5. Expression of CD97-5EGF is up-regulated on activated lymphocytes. PBMC were stimulated with PHA, PMA, or CD3 mAb. Expression of CD97 and EMR2 was analyzed at different time-points by flow cytometry with mAb as indicated. Streptavidin-APC was used as a second-step reagent. Staining for CD69 was used as a positive control for cellular activation. Lymphocytes were defined by forward- and side-scatter.

Previously, EMR2 expression levels were found to increase during differentiation of myeloid cells [26 ]. We determined whether expression of the CS-binding isoforms is specifically regulated within the myeloid lineage. Based on expression of CD14 and CD16, peripheral blood monocytes can be subdivided into common monocytes (CD14++CD16–), monocytes with properties of tissue macrophages (CD14++CD16+), and monocytes that exhibit characteristics of dendritic cells (DC; CD14+CD16+) [28 , 29 ]. CD16+ monocytes transmigrate through layers of resting endothelial cells and have recently been suggested to give rise to resident myeloid cells [30 , 31 ]. Analysis of 1B5 staining in these subpopulations revealed an increase of 1B5 staining on CD16+ populations compared with the CD16– population (Fig. 6 ). As CD97 expression was relatively stable, and EMR2 expression increased, increased 1B5 staining likely reflected up-regulation of EMR2-5EGF.



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  		Figure 6. Expression of the largest isoform of EMR2 increases during differentiation of myeloid cells. Blood monocyte subsets, defined by the expression of CD14 and CD16 (A), were analyzed by three-color flow cytometry for expression of CD97 and EMR2 (B). CD14 and CD16 were stained with conjugated mAb, CD97 and EMR2, with biotinylated mAb, followed by streptavidin-APC as a second-step reagent. Monocytes were defined by forward- and side-scatter. Results are mean fluorescence intensity ± SEM in percent of 10 experiments. For CD14++CD16– monocytes, absolute numbers of mean fluorescent intensity are given in parentheses.


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DISCUSSION
 
Previous studies have demonstrated that EGF-TM7 receptors bind cellular ligands. CD97 can interact through its first and second EGF domain with CD55. More recently, we reported that the fourth EGF domain of EMR2 and CD97 specifically binds the GAG CS [19 ]. We here demonstrate that both interactions can be engaged cooperatively by the largest isoform of CD97. Furthermore, we show that the interaction with CS may play a role in the adhesion of activated lymphocytes and differentiated myeloid cells to B cells

We previously found that the EMR2-5EGF probe binds CS on fibroblast cell lines and within connective tissue and the extracellular matrix (ECM) of various tissues [19 ]. In addition, binding was observed to areas within the white pulp of the spleen, suggesting that leukocytes also express the ligand. This study shows that B cells, but no other leukocytes, express CS, bound by the fourth EGF domain of CD97 and EMR2. This B cell specificity could be a result of at least two potential mechanisms. First, the recognized CS is attached to a B cell-specific core protein. Second, the CS is modified in a B cell-specific manner. Variability in total length of the polysaccharide chain, in iduronic acid placement and in sulfation, dictates a high level of complexity of CS [32 ]. This fine structure is tightly regulated in a tissue- and cell type-specific manner, generating high regional heterogeneity of CS. We previously reported binding of EMR2-5EGF probes to transfectants of the lymphoblastoid B cell line ARH-77, stably expressing syndecan-1, -2, and -4 and glypican-1 [19 ]. In contrast to this, the CD97/EMR2-5EGF probes did not bind to syndecan-1 when transfected into the Burkitt’s lymphoma cell line Namalwa (data not shown). In addition, major CS-bearing molecules such as syndecans are differentially expressed during B cell development [33 , 34 ]. The CS bound by CD97/EMR2-5EGF, however, is rather evenly distributed on B cells as shown by similar probe binding to CD27-negative and CD27-positive (memory) B cells (data not shown). These observations suggest that CD97 and EMR2 bind B cell-specific CS rather than core protein-specific CS. The importance of glycosylation in shaping the specificity of molecular interactions is increasingly recognized. For example, cell-specific glycosylation determines whether intercellular adhesion molecule-2 (ICAM-2) and ICAM-3 are recognized by the C-type lectin DC-specific ICAM-3-grabbing nonintegrin [35 ].

In vivo studies with blocking mAb recently revealed a pivotal role for CD97 in the migration of neutrophils [4 ]. Based on this study and the ability of CD97 to bind CS, we proposed a model according to which CD97 binds to the ECM and thereby facilitates binding of ECM-attached molecules to their cellular receptors. CS and other GAG bind a diverse range of molecules including growth factors, protease inhibitors, cytokines, chemokines, and pathogen virulence factors [21 , 22 , 36 ]. It has been shown that GAG on the endothelial cell surface immobilize and enhance local concentrations of chemokines, promoting the presentation of these chemokines to their receptors [37 ]. In addition, soluble GAG-chemokine complexes can form chemokine gradients in tissues [38 ]. It seems possible that CD97 and EMR2 may play a crucial role in the capture of such complexes.

The cellular distribution of CD55 partially overlaps with that of CS. CD55 is found on endothelial cells and on virtually all types of leukocytes [20 ]. Expression is up-regulated during physiological and pathological conditions, as indicated by the presence on fibroblast-like synoviocytes and various malignant cells [39 40 41 ]. In addition, CD55 is abundantly present in the extracellular matrix [41 , 42 ]. Recent evidence that CD97 has a role in cell migration [4 ] is not conclusive about the role of the different ligands. Blocking of the CD55- and the presumed CS-binding site of mouse CD97 with mAb similarly impaired neutrophil migration.

The broad distribution of CD97 raises the question how ligand interactions are regulated. Expression of CD97 has been analyzed extensively [8 , 14 , 15 , 26 , 43 ]. Whereas expression on myeloid cells is constitutive and hardly affected by activation or differentiation, CD97 on lymphocytes is rapidly up-regulated during cellular stimulation. We found no evidence that expression of the binding sites for CD55 and CS is independently regulated. Consequently, activated lymphocytes and most myeloid cells can engage with CD55 and CS through CD97. Thereby, the interaction with CD55 is mediated mainly by the smaller isoforms, whereas CS binding is restricted to the largest isoform. The ability of the largest isoform to interact weakly with CD55 is likely to be of little physiological relevance, as CD97+ cells usually express the smaller isoforms, which have much higher affinities for CD55 [17 , 18 ], more abundantly [43 , 44 ]. In addition, CD55 expression levels on PBMC and on synovial fibroblasts (Else Kop, Mark Kwakkenbos, Gwendoline Teske, Maarten Kraan, Tom Smeets, Martin Stacey, His-Hsien Lin, Jörg Hamann, and Paul Peter Tak, submitted for publication) are too low to allow for efficient binding of multivalent CD97-5EGF probes.

We demonstrated that CD97 binding does not stimulate B cells proliferation, Ig synthesis, or class switching, independently of whether CD97 isoforms preferentially bind to CD55 (CD97-3EGF) or CS (CD97-5EGF). These findings raise the possibility that a main function of CD97 could be the generation of adhesion contacts with B cells as well as the capturing of B cell-released ECM fragments. Such interactions might be engaged in the migration of activated T cells, DC, and macrophages toward B cell follicles in lymphoid organs [45 46 47 ]. These cell types are indispensable for activation, proliferation, maturation, and homeostasis of B cells. DC and macrophages could rely, in their contacts with B cells, on EMR2 as a second receptor. It is tempting to speculate that CD97 and EMR2 facilitate the recognition of chemokines secreted by B cells such as IL-8, regulated on activation, normal T expressed and secreted macrophage-inflammatory protein-1{alpha} (MIP-1{alpha}), and MIP-1ß [48 , 49 ]. In the future, the investigation of genetically targeted mice should provide the means to investigate the consequences of CD97 interactions with its ligands in detail. For EMR2, data will need to be extrapolated, as this molecular twin of CD97 has no murine ortholog [3 ].


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ACKNOWLEDGEMENTS
 
This work was supported by grants from the Netherlands Organization for Scientific Research (M. J. K.), the Landsteiner Foundation for Bloodtransfusion Research (W. P. and M. M.), the Wellcome Trust (M. S.), and the British Heart Foundation (H-H. L.). S. G. is supported by grants from the Medical Research Council, UK. J. H. is a fellow of the Royal Netherlands Academy of Arts and Sciences. The Renal Transplant Unit of the Academic Medical Centre of the University of Amsterdam kindly provided us with surplus human splenocytes. We thank Dr. Guido David, Dr. Reinhard Schwartz-Albiez, and Richard Bende for their comments and suggestions and Dr. Martijn Nolte for critical reading of the manuscript.

Received July 15, 2004; revised September 14, 2004; accepted September 23, 2004.


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