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* Sir William Dunn School of Pathology, Oxford University, Oxford, UK;
Department of Microbiology and Immunology, Chang Gung University, Gueishan, Taoyuan, Taiwan, Republic of China;
Department of Physiology and Pathology, University of Trieste, Trieste, Italy;
William Harvey Research Institute, University of London, London, UK;
|| St. Thomas’ Hospital, Kings College, London, UK; and
¶ Department of Experimental Immunology, University of Amsterdam, Amsterdam, Holland
1Correspondence: Sir William Dunn School of Pathology, Oxford University, Oxford, OX1 3RE, UK. E-mail: M.S., martin.stacey{at}path.ox.ac.uk; S.G., siamon.gordon{at}path.ox.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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150 different members of the adhesion-G protein-coupled receptor (GPCR) family have been identified in metazoans. Surprisingly, very little is known about their function, although they all possess large extracellular domains coupled to a seven-transmembrane domain, suggesting a potential role in cell adhesion and signaling. Here, we demonstrate how the human-restricted adhesion-GPCR, EMR2 (epidermal growth factor-like module-containing mucin-like hormone receptor), regulates neutrophil responses by potentiating the effects of a number of proinflammatory mediators and show that the transmembrane region is critical for adhesion-GPCR function. Using an anti-EMR2 antibody, ligation of EMR2 increases neutrophil adhesion and migration, and augments superoxide production and proteolytic enzyme degranulation. On neutrophil activation, EMR2 is rapidly translocated to membrane ruffles and the leading edge of the cell. Further supporting the role in neutrophil activation, EMR2 expression on circulating neutrophils is significantly increased in patients with systemic inflammation. These data illustrate a definitive function for a human adhesion-GPCR within the innate immune system and suggest an important role in potentiating the inflammatory response. Ligation of the adhesion-GPCR EMR2 regulates human neutrophil function.—Yona, S., Lin, H.-H., Dri, P., Davies, J. Q., Hayhoe, R. P. G., Lewis, S. M., Heinsbroek, S. E. M., Brown, K. A., Perretti, M., Hamann, J., Treacher, D. F., Gordon, S., Stacey, M. Ligation of the adhesion-GPCR EMR2 regulates human neutrophil function.
Key Words: inflammation • migration • sepsis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. It is now well established that leukocyte migration from the vasculature occurs as a multistep process, dictated by the sequential interactions and activation of cell adhesion receptors on both leukocytes and the endothelium (2
, 3)
. On arrival at the sites of infection, phagocytes recognize and migrate toward pathogens before potentially deploying their arsenal of antimicrobial weapons. These include reactive oxygen species (ROS) and a variety of cytotoxic proteolytic enzymes. It is now recognized that in order to prevent collateral host tissue damage, the majority of these events culminating in the release of cytotoxic mediators need to be tightly regulated (1)
. Phagocytes therefore often require high stimulus concentrations or multiple stimuli to potentiate cellular activation to ensure that the magnitude of their response is appropriate (4
, 5)
. The failure of such regulatory mechanisms is thought to underlie inflammatory diseases such as the organ failure associated with sepsis and systemic inflammatory response syndrome (SIRS) (6
, 7)
. Processes involved in the regulation of phagocyte activation and potentiation are mediated, in part, by an array of cell surface receptors. One group of neglected receptors that is likely to play an important role in phagocyte biology are the adhesion G protein-coupled receptors (GPCRs) (8)
.
At present 150 different adhesion-GPCRs have been identified in metazoans (8
, 9)
. All members of this family possess a large extracellular region often containing common protein modules, such as immunoglobulin, epidermal growth factor-like (EGF), lectin, or thrombospondin repeats, coupled to a seven-transmembrane (TM7) domain via a mucin-like stalk region (10
, 11)
. The mucin-like domain contains a conserved proteolytic motif known as the GPS (G protein-coupled receptor proteolytic cleavage site) (12
, 13)
. Mutations within this region of adhesion-GPCRs occur in a number of human diseases, including Usher’s syndrome, bilateral frontoparietal polymicrogyria, and polycystic kidney disease (14
15
16)
.
A subgroup of the adhesion-GPCRs is the predominantly leukocyte restricted EGF-TM7 family (10)
. The human EGF-TM7 family includes CD97 and EMR1, -2, -3 and -4, all of which contain EGF-like repeats in their extracellular domain and undergo alternative splicing (17
18
19
20
21
22
23
24)
. Because of their hybrid structure and restricted expression pattern, a dual adhesion and signaling role within the immune system has been postulated. This adhesion hypothesis has now been substantiated after a number of reports demonstrating cell surface and matrix ligands for several of these receptors (25
26
27
28
29
30
31)
. CD97 has been shown to bind a number of ligands, including the complement control protein CD55, chondroitin sulfate, and selected angiogenic integrins. More recently, CD97 has been shown to have multiple effects on tumor invasion and is up-regulated in chronic inflammatory diseases such as rheumatoid arthritis and multiple sclerosis and in human malignancies (28
, 32
, 33)
. CD97 has also been implicated in neutrophil migration in mouse models of colitis and the clearance of Streptococcus pneumonia (28
, 31
, 33)
.
In the present study, we report for the first time a functional role for the human EMR2 receptor. EMR2 is expressed on neutrophils, monocytes, macrophages, and dendritic cells (34)
and is known to bind chondroitin sulfate found on cells and matrix within tissues, suggesting a potential role during cell adhesion/migration (26)
. Here, we demonstrate that EMR2 ligation potentiates the activation and recruitment of human neutrophils. Isolated neutrophils ligated at the stalk region of EMR2 adhere and migrate to a greater degree under both static and shear stress conditions. In addition, we demonstrate that activation of this receptor acts in a synergistic manner with a number of proinflammatory mediators potentiating polymorphonuclear (PMN) leukocyte respiratory burst and degranulation. Using a cell transduction model, we also show that the transmembrane region of EMR2 is essential for receptor function, in the absence of antibody ligation. The current findings reveal an important new membrane protein regulator of PMN function and demonstrate the critical role of the transmembrane region in the family of adhesion-GPCRs.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Isolation of human PMN
PMNs were isolated from fresh venous blood donated by healthy volunteers. Blood was collected in 4 mM EDTA and isolated by Histopaque or Percoll density gradient centrifugation, as described previously (35)
. Subsequent hypotonic lysis of erythrocytes of the PMN-enriched fraction yielded a purity >98%. PMNs were resuspended in RPMI/0.2% BSA unless stated otherwise. This project was approved by the Oxford University Research Ethics Committee (MSD/IDREC/C1/2006/32).
Cell lines
Murine B-lymphoblast Baf/3b cells were grown in RPMI/4% WEHI3b cell-conditioned medium, whereas the human fibrosarcoma HT1080 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM). Both cell lines were supplemented with 10% fetal calf serum (FCS), 1 mM L-glutamine, 100 U/ml penicillin, and 0.01 mg/ml streptomycin, and grown at 37°C, 5% CO2, and 95% humidity.
Plasmid construction and stable cell line generation
Previously isolated full-length cDNA encoding EMR2 (125) and EMR2 (1–5) isoforms (where numbers denote the presence of particular EGF-repeats) (20)
were cloned into either pFBneo (Stratagene, Amsterdam, Holland) or IRES-GFP (Clontech, Cowley, UK) vectors. The EMR2-TM1 construct encoding the truncated EMR2 protein that contains the entire extracellular domain region plus the first TM domain and the first intracellular loop ending at TSTSL was made by PCR amplification using gene-specific primers, as well as subsequent cloning into the pFBneo vector. HT1080 cells were transfected with IRES-GFP constructs using lipofectamine. Stable cells were selected with 1 mg/ml G418- and GFP-positive cells subsequently sorted by FACS. Infectious pFB-neo EMR2 virions for retroviral transduction were generated using 293T 
NX ecotropic packaging cells, as described previously (36)
. Baf/3b cells to be transduced were resuspended (1x106 cells/ml) in viral supernatant, centrifuged at 500 g for 2 h at room temperature (RT), and selected with 1 mg/ml G418.
Cell adhesion molecule expression
Whole blood was incubated with or without platelet activating factor (PAF) (0.01–1µM) or formyl-Met-Leu-Phe (fMLP; 0.1–1µM) for 15 min at 37°C. Selected groups were preincubated with either of two EMR2 antibodies: 2A1 (5 µg/ml, which has previously been shown to bind to the stalk region of EMR2) (13
, 37)
, CLB-CD97/1 (5 µg/ml), which recognizes the N-terminal first EGF-like domain of both EMR2 and CD97, or vehicle for 10 min before addition of agonist. Samples were then labeled with anti-CD11b:FITC (5 µg/ml), or CD62L:FITC (5 µg/ml) for 1 h at 4°C. Following two washes in PBS, erythrocyte lysis was performed with Immuno-LyseTM (Coulter, Luton, UK). FACS was performed using a FACScan analyzer (Becton Dickinson, Cowley, UK); PMNs were gated according to their FSC/SSC properties, and data were analyzed using CellQuest software (BD Biosciences).
Chemotaxis assays
2.5 x 105 PMNs or transduced Baf/3b cells were placed in the upper chamber of a ChemoTx plate (Receptor Technologies, Oxford, UK), equipped with a 3-µm pore filter. Selected samples of cells were preincubated with either of two EMR2 antibodies: 2A1 (5 µg/ml), CLB-CD97/1 (5 µg/ml), or vehicle for 10 min. The chemoattractants fMLP (10 nM) or CXCL12 (10 ng/ml) in RPMI/0.2% BSA, were added to the bottom well and incubated for 2 h at 37°C. Migrated cells were incubated with Alamar blue for 4 h and quantified at 560 nm in a plate reader; cells were also stained with Turk’s solution and counted by microscopy. Data were expressed as a migration index (number of cells migrated to chemoattractant/number of cells migrated to vehicle). Cell invasion was examined using the ECMatrix transwell system (Chemicon). The ECMatrix inserts were cultured with transfected HT1080 cells in RPMI. Cellular invasion was initiated with the addition of RPMI/10% FCS in the lower chambers. Invasion was assayed after 24 h; migrated cells were stained with crystal violet and analyzed by microscopy and colorimetry at 590 nm.
Flow chamber assay
Confluent human umbilical vein endothelial cell (HUVEC) (Promocell, Heidelberg, Germany) monolayers were stimulated with TNF-
(10 ng/ml) for 4 h. Isolated PMNs (1x106 cells/ml) were resuspended in PBS supplemented with Ca2+ (0.95 mM) and Mg2+ (1.2 mM) and incubated with or without EMR2 mAbs 2A1 (5 µg/ml), or CLB-CD97/1 (5 µg/ml) before flow for 10 min at 37°C. PMNs were perfused over the endothelial monolayers at a constant shear of 1 dyn/cm2. Following 8 min of perfusion, 6 arbitrary fields of 10 s each were recorded using a Q-imaging Retiga EXi digital video camera (Q-imaging, Burnaby, BC, Canada) onto a computer running Streampix capture software (Norpix Inc., Montreal, QC, Canada). These video sequences were subsequently analyzed as described previously (38)
. Briefly, captured cells were defined as either adherent or rolling. Capture is the total tethers at time 0 (the beginning of recording; cells that appear stationary on the endothelial cells); rolling is characterized by those cells that move during the 10-s timeframe, and adherent cells are those that do not move.
Confocal microscopy
Isolated PMNs in RPMI/0.2% BSA (5x105) were allowed to adhere to fibronectin-coated coverslips (100 µg/ml) for 20 min before addition of fMLP (10 nM). After 4 min, PMNs were fixed with 4% paraformaldehyde for 15 min. Cells were subsequently permeabilized for 1 h using PBS/0.2% saponin, 1% BSA, and 1% goat serum. Cells were first incubated at RT with primary antibodies for 1 h. Cells were then washed three times before incubation with AlexaFluor-488 donkey anti-mouse IgG (1:200; Molecular Probes, Paisley, UK) and TRITC phalloidin (1:1500). Slides were finally incubated with DAPI (1:10,000) for 10 min before mounting in fluorescent mounting medium. Analysis by confocal laser scanning microscopy was performed using a Zeiss LSM510 microscope (Carl Zeiss, Oberkochen, Germany). Representative pictures were collected and analyzed using MetaMorph software.
Reactive oxygen species generation
Freshly isolated PMNs (5x105 cells/ml) were resuspended in PBS supplemented with 0.2% BSA, 5 mM glucose, and 2 mM NaN3, and incubated with 2 µM dihydrorhodamine-123 (DHR123; Molecular Probes) for 30 min at RT. Cells were then incubated for 10 min with either of the two EMR2 mAb, 2A1 (5 µg/ml), CLB-CD97/1 (5 µg/ml), or with vehicle. The cells were then stimulated with either PMA (10 ng/ml) or fMLP (0.1 µM) for a further 15 min before being placed on ice. The accumulation of H2O2 was immediately analyzed by FACS.
Production of superoxide was measured by the superoxide dismutase (SOD) -inhibitable cytochrome c reduction assay. Isolated PMNs (10x106 cells/ml) were resuspended in HBSS/0.2% BSA, and 50 µl of cell suspension was added to 0.85 ml HBSS/0.2% BSA containing cytochrome c (1.2 mM) and either of the two EMR2 antibodies, 2A1 or CLB-CD97/1 (5 µg/ml). The cell suspension was prewarmed for 15 min at 37°C, and the reaction was started by addition of 100 µl fMLP (final concentration 0.1 µM). The kinetics of cytochrome c reduction was followed spectrophotometrically at 550 nm. SOD (10 µg/ml) was used to demonstrate that the reduction of cytochrome c was due to superoxide. An extinction coefficient of 21.1 mM/cm (reduced minus oxidized) was used to calculate the amount of reduced cytochrome c (39)
. Total cytochrome c reduction was determined as the area under the curve over the entire time course.
MPO release
Isolated PMNs (5x106 cells/ml) were incubated with or without fMLP (1 µM) for 10 min at 37°C. Selected groups of cells were preincubated with either 2A1 (5 µg/ml), CLB-CD97/1 (5 µg/ml), or vehicle. Cells were centrifuged and supernatant was added to a sodium-acetate buffer (0.1 M, pH 5.5) supplemented with 0.1% cetyltrimethyl-ammonium bromide, 2 mM TMB substrate and 0.7 mM H2O2. After 5 min at RT, the reaction was terminated by the addition of 50 µl sulfuric acid (0.1 M), and the absorbance was read at 450 nm.
Cell adhesion molecule expression of SIRS patients
Blood samples were collected in EDTA from healthy control subjects and patients at St. Thomas’ Hospital, London, who fulfilled the diagnosis for SIRS. Patients satisfied three of the following criteria: body temperature >38°C or <36°C; heart rate >90 beats/min; respiratory rate >20 breaths/min, and a white blood cell count >12 x 109 cells/L or <4 x 109 cells/L or the appearance of >10% immature cells in the circulation. All patients had evidence of at least one organ failing, and 50% of the patients had sepsis. All of the subjects provided consent under the guidelines approved by the Ethics Committee of St. Thomas’ Hospital.
Analysis of EMR2, CD97, and CD64 expression on PMNs in whole blood was undertaken at 4°C. Whole blood was incubated for 20 min with LDS-751 nuclear stain (10 µg/ml; Molecular Probes) and 1% mouse serum. Samples were then subdivided and labeled with either anti-CD64:FITC (0.5 µg/ml), CD97:FITC (0.6 µg/ml), EMR2:FITC (0.4 µg/ml), or CD66b:FITC (1 µg/ml) for 20 min in the dark. Results were recorded as a percentage of the cells expressing surface molecules. PMNs were gated on their FSC/SSC properties and by nuclear staining with LDS-751; population gating was confirmed by CD66b staining.
Statistical analysis
All values are expressed as means ± SE, with number of experiments (n) per group conducted in triplicate (flow chamber means ± SE were calculated from means of 6 readings from each experiment). Differences between groups were determined by Student’s t test. In all cases, a probability value of P < 0.05 was accepted to reject the null hypothesis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A and Tables 1
and 2
). The addition of fMLP to peripheral blood aliquots for 15 min led to a concentration-dependent increase in PMN CD11b surface expression and shedding of CD62L. Interestingly, the up-regulation of CD11b and CD62L shedding was enhanced by preincubation with an anti-EMR2 antibody, 2A1 (5 µg/ml), which binds to the stalk region of EMR2 (13
, 37)
(Fig. 1A
and Table 1
). These experiments were also performed in whole blood, using another EMR2 antibody CLB-CD97/1, which recognizes the N-terminal EGF-like domain, and CLB-CD97/3 (5 µg/ml, an isotype control, which binds specifically to a CD97 epitope; data not shown). As no potentiation was detected with the other two antibodies, this ruled out any Fc-mediated effects. This phenomenon was not observed when blood was incubated with the antibodies alone. The pattern of PMN activation was also reproduced with PAF (Table 2)
. The greatest synergistic effect of the 2A1 antibody was observed with 0.3 µM PAF (Table 2)
for CD11b up-regulation.
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Taken together these experiments indicate a selective alteration of PMN cell adhesion molecules during activation, by ligation of the EMR2 stalk region (2A1), but not the most N-terminal EGF-like domain (CLB-CD97/1). Because the changes were not linked to the use of a single stimulus, it is likely that 2A1 binding to EMR2 affects pleiotropic downstream molecules common to more than one signaling pathway.
EMR2 regulates PMN migration under both static and shear conditions
To determine whether EMR2 could modify PMN migration, chemotaxis assays were conducted in a modified Boyden chamber. As expected, purified PMNs responded with directed locomotion toward the chemokine CXCL12/SDF1- (10 ng/ml) or fMLP (20 nM; Fig. 1B
). As chemotaxis is known to follow a bell-shaped dose response, these concentrations were selected for their submaximal response (data not shown). Preincubation of PMNs for 5 min before chemotaxis with the EMR2 antibody 2A1 produced a significant increase in the extent of migration toward either CXCL12 or fMLP (Fig. 1B
). In contrast, incubation with the control EMR2 antibody CLB-CD97/1 did not modify cell migration (Fig. 1B
). Cells incubated with antibodies in the absence of either chemoattractant, CXCL12 or fMLP, did not migrate above basal levels (data not shown). Again, enhanced activation was associated with binding to the stalk region of EMR2. To determine whether the observed effects were due to the EMR2 antibody possessing chemotactic, fugetactic, or chemokinetic properties, a checkerboard analysis was performed in which antibodies were incubated in both the top and bottom chambers separately or simultaneously. As equal migration was observed in all cases in which 2A1 was present (data not shown), it was presumed that 2A1 had a chemokinetic rather than a chemotactic or fugetactic effect.
To confirm and expand these data under more physiological conditions, cellular kinetics were examined in a shear stress environment. Isolated PMNs were perfused over confluent TNF--treated (10 ng/ml) HUVECs at a shear of 1 dyn/cm2 over an 8-min period; cells were defined as rolling or firmly attached. Interestingly, PMN incubation with the stalk region antibody 2A1 increased PMN adhesion, but not rolling (Fig. 1C
). By contrast, the EMR2 EGF-like binding antibody CLB-CD97/1 did not alter the extent of PMN rolling or adhesion.
The generation of EMR2 expressing cell lines to investigate cell migration
Because of the absence of a murine EMR2 ortholog and the difficulty in manipulating PMNs, cell lines were generated expressing EMR2 isoforms, to investigate further EMR2’s involvement in leukocyte migration. Murine B-lymphoblast Baf/3b and human fibrosarcoma HT1080 cells were transduced/transfected with vectors containing EMR2 (125), EMR2 (1–5), where numbers denote the presence of particular EGF-repeats (20)
or EMR2 possessing only one transmembrane region (Fig. 2
A). To allow for objective comparisons between cells throughout the study, the levels of expression for each construct were checked by FACS analysis in comparison with cells containing empty vectors (Fig. 2B, C
). Surface expression for EMR2 (125), EMR2 (1
2
3
4
5)
, and EMR2 (TM1 mutant) isoforms was comparable in both cell lines, for example, 64 ± 6, 62 ± 8, and 61 ± 8 mean fluorescence intensity (MFI), respectively, in Baf/3b cells (Fig. 2C
).
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To dissect EMR2’s involvement in cell migration, transduced Baf/3b cells expressing the naturally occurring EMR2 isoforms were incubated in a Boyden chamber using CXCL12 (10 ng/ml) as a chemotactic agent. Following a 2-h incubation, cells transduced with either EMR2 (125) or EMR2 (1–5) demonstrated an enhanced migration toward CXCL12, even in the absence of the EMR2 antibody, compared with cells transduced with empty plasmid (Fig. 2D
). However, EMR2-mediated migration was completely abrogated in the truncated EMR2 TM1 mutant, demonstrating that possessing just the adhesion moiety was not sufficient for migration, but the transmembrane region was also essential for receptor function. Finally, cellular invasion through an extracellular matrix was explored using transfected HT1080 cells in the ECMatrix transwell system. Again, migration of cells transfected with EMR2 (1–5) was significantly augmented compared with WT cell migration (1.03±0.11 compared with 0.4±0.1, P<0.05, respectively, Fig. 2E
).
These data together imply that the naturally occurring full-length EMR2 isoform plays an important role in regulating leukocyte migration, under static and shear stress conditions and that the seven-span transmembrane region is crucial for this phenomenon.
EMR2 redistribution during PMN migration
During cell migration in response to chemoattractants, the interaction of cell surface receptors and cytoskeletal components is essential for cellular polarization and the formation of leading edges and uropods. The involvement and localization of EMR2 in this process were examined using confocal microscopy. Immunofluorescent labeling of EMR2 and a related EGF-TM7 receptor CD97 on resting human PMNs showed punctate staining distributed uniformly over the entire surface of the cell (Fig. 3
). In addition, CD162 and Rac were evenly distributed on the plasma membrane (Fig. 3)
. When PMNs were treated with as little as 10 nM fMLP for 4 min, EMR2 and Rac were rapidly translocated to the leading edge and other lamellipodia and colocalized with actin, yet CD97 expression showed no sign of a similar redistribution. In contrast to EMR2 and Rac, CD162 was redistributed to the uropod on stimulation (Fig. 3)
. This observation is consistent with a selective role for EMR2 at the leading edge during chemotaxis.
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EMR2 potentiates antimicrobial mediator production
As 2A1 potentiates PMN locomotion, experiments were performed to determine whether ligation of EMR2 could have similar effects on the PMN respiratory burst and degranulation. The respiratory burst and its derivatives were measured by a FACS assay (Fig. 4
A) and confirmed by measuring the kinetics of superoxide production by means of superoxide dismutase (SOD) -inhibitable cytochrome c reduction (Fig. 4B
). The addition of inflammatory mediators to isolated neutrophils or whole blood led to an increase in fluorescence (Fig. 4A
) indicating the generation of H2O2. Furthermore, when PMNs were preincubated with 2A1, the respiratory burst was significantly augmented (Fig. 4A, B
). Preincubation with control antibodies binding to the first EGF-like domain of EMR2 (CLB-CD97/1) had no effect on ROS production. PMN degranulation was assessed by monitoring myeloperoxidase (MPO) release from primary granules following PMN activation. Addition of as little as 0.1 µM fMLP for 10 min led to a significant release of MPO from isolated PMNs (Fig. 4C
). When PMNs were preincubated with the EMR2 antibody 2A1, the release of MPO was significantly enhanced. This effect was absent in cells preincubated with the control antibody, binding to EGF-like domain (Fig. 4C
).
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SIRS patient PMNs express augmented EMR2 levels
To investigate whether EGF-TM7 membrane expression was up-regulated on PMNs during systemic inflammation, FACS analysis was performed on whole blood samples from patients with SIRS. As expected, PMNs from SIRS patients exhibited an up-regulation of CD64 (40)
(Fig. 5
). Interestingly, in comparison with control subjects there was an increased expression of EMR2 on PMNs in patients with SIRS, 15 ± 2 vs. 51 ± 5% PMNs expressing EMR2, respectively, P < 0.05 (Fig. 5)
, yet no significant difference was observed with CD97 expression.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. However, very little is known about the adhesion-GPCR subgroup; in particular, the EGF-TM7 family. Here we have shown for the first time that ligation of EMR2 results in the potentiation of PMN migration, degranulation, and ROS production toward a number of inflammatory stimuli. By truncating EMR2, we also demonstrate that the transmembrane region is critical for migration. This observation, in addition to previous studies of CD97 (32)
, indicates that the transmembrane region of the other 150 members of the adhesion-GPCR family may also be crucial for receptor function.
GPCR activation is known to regulate many cellular functions in PMNs, including chemotaxis and degranulation (42
, 43)
. The synergistic effect observed here by EMR2 ligation on CD11b/CD62L expression levels provides the first evidence that receptor ligation of EGF-TM7 molecules results in intracellular signaling. Whether signaling is via classical heterotrimeric G proteins or is G protein independent, as is the case for a growing number of 7TM proteins (44)
, remains to be seen. The human-restricted EMR2-mediated potentiation observed here, may also suggest another level of PMN control in humans and further extend the list of differences observed in rodent and human leukocyte migration (45
, 46)
.
In addition to CD11b/CD62L modulation, modified Boyden chamber analysis demonstrated that ligation of EMR2 at the stalk region, but not at the N-terminus, mediates an increase in cell migration toward a variety of chemoattractants, implicating EMR2 signaling via pleiotropic downstream effector molecules. Interestingly, we also observed that the transduction of whole-length EMR2 into cell lines promoted enhanced cell migration, even without ligation of the receptor, ruling out that this phenomenon was due to Fc-mediated effects, indicating a potential constitutive basal activity of the receptor.
Ligation of EMR2 enhanced the adhesion but not the rolling of PMNs on TNF- stimulated HUVECs under shear conditions. This augmented attachment may be explained, in part, by 2A1’s promotion of CD62L shedding and CD11b up-regulation. The proteolytic shedding of CD62L observed, can have a large impact on the subsequent stages of leukocyte migration, by promoting leukocyte arrest through the increased valency and activation of CD11b, especially if other costimulatory signals such as chemokines are present (47)
. Recent advances in cell adhesion molecule function have illustrated that intraluminal crawling of PMNs on endothelial cells is largely regulated by CD11b, in contrast to adhesion of unstimulated PMNs, which is generally CD11a dependent (48
, 49)
. The increased migration observed in this study might be regulated by both the extracellular and intracellular component of EMR2. Cellular anchoring to matrix or endothelial cells could occur via the extracellular region, whereas the intracellular signaling by EMR2 may up-regulate CD11b expression and CD62L shedding. It is tempting to speculate that the binding of EMR2 by its ligand chondroitin sulfate (26)
mediates PMN migration through the ECM to sites of inflammation. Indeed, high levels of both EMR2 and chondroitin sulfate have been found together in the intimal layer and synovial sublining of tissues from rheumatoid arthritis patients (50)
, and aberrant EMR2 expression is observed in in situ and invasive breast carcinomas (unpublished data). These data suggest that this receptor can regulate cell activation and migration in myeloid and abnormal nonmyeloid cells.
As discussed, chemoattractants induce PMN activity through a full range of events including adhesion molecule expression, chemotaxis, ROS production, and degranulation. One of the earliest events to occur is the change in cellular morphology. For directed cell locomotion to take place, the differential formation of lamellipodia and membrane protrusions is required at both the leading edge and uropod of PMNs. Crawling neutrophils are known to redistribute CD162 to the uropod and Rac to the leading edge (51
, 52)
, we show that EMR2 is rapidly translocated to the leading edge unlike a related receptor CD97. The contribution of EMR2 in regulating PMN function and cytoskeletal reorganization may therefore be of physiological significance during the initial phase of leukocyte polarization and migration.
One of the final stages during an inflammatory response is the elimination of foreign microbes. This process is primarily controlled by the degranulation of cytotoxic mediators and the generation of reactive oxygen species by PMNs (53)
. Individuals suffering from neutropenia or patients who have genetic defects in NADPH oxidase species have profound immuno-deficiencies, which lead to recurrent and often fatal fungal and bacterial infections (54)
. Here, we observed that the enhanced respiratory burst and degranulation mediated by 2A1 ligation correlates with other observations of antibody priming of PMNs. Waddell et al. (55)
have shown that binding of CD62L antibodies leads to an increase in the respiratory burst and intracellular calcium levels, when stimulated by fMLP. Other studies with antibodies against the myeloid receptor TREM-1 have also demonstrated an increase in PMN degranulation and respiratory burst (56
, 57)
. These effects were similar to those observed here. In both studies, the potentiation of the respiratory burst was due to an increase in the rate of H2O2 and O2–production, rather than in the duration of the oxidative burst.
Finally, patients with SIRS expressed a three-fold increase in EMR2 expression on PMNs. This increased expression may be explained as either a cause of systemic inflammation inducing EMR2 expression; the increased EMR2 expression could also contribute to systemic inflammation. It is important to note that all of the patients studied were receiving ventricular support and that in the severe forms of lung dysfunction associated with SIRS, pulmonary damage is initiated by the extracellular release of proteolytic enzymes and ROS from the infiltrating PMNs (7
, 58)
. From the current study, we show that the ligation of EMR2 potentiates PMN endothelial adhesion, migration, and tissue-damaging mediators, it appears that EMR2 may be a molecule of potential pathological significance.
In conclusion, this study illustrates that the ligation of EMR2 at a distinct binding site on the stalk region or overexpression of this receptor, drives the early stages of the inflammatory response. Further work on monocytes, macrophages, and dendritic cells may elucidate the functional importance of this receptor during both the innate and acquired immune response.
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ACKNOWLEDGMENTS |
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Received for publication July 31, 2007. Accepted for publication September 6, 2007.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-chain phosphorylation of the human leukocyte CD11b/CD18 (Mac-1) integrin is pivotal for integrin activation to bind ICAMs and leukocyte extravasation in vivo. Blood 108,3379-3386This article has been cited by other articles:
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  H. Veninga, S. Becker, R. M. Hoek, M. Wobus, E. Wandel, J. van der Kaa, M. van der Valk, A. F. de Vos, H. Haase, B. Owens, et al. Analysis of CD97 Expression and Manipulation: Antibody Treatment but Not Gene Targeting Curtails Granulocyte Migration J. Immunol., November 1, 2008; 181(9): 6574 - 6583. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
P < 0.05 vs. EMR2 mediated migration.
