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Originally published online as doi:10.1189/jlb.0107053 on June 27, 2007

Published online before print June 27, 2007
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(Journal of Leukocyte Biology. 2007;82:585-593.)
© 2007 by Society for Leukocyte Biology

Mannose receptor regulation of macrophage cell migration

Justin Sturge*,{dagger}, S. Katrina Todd*, Giolanta Kogianni{dagger}, Afshan McCarthy* and Clare M. Isacke*,1

* Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, London, United Kingdom; and
{dagger} Prostate Cancer Research Group, Department of Oncology, Division of Surgery, Oncology, Reproductive Biology and Anaesthesia, Faculty of Medicine, Imperial College London, Hammersmith Hospital Campus, London, United Kingdom

1 Correspondence: Institute of Cancer Research, Breakthrough Breast Cancer Research Centre, 237 Fulham Road, London, SW3 6JB UK. E-mail: clare.isacke{at}icr.ac.uk


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ABSTRACT
 
The migration of macrophages through peripheral tissues is an essential step in the host response to infection, inflammation, and ischemia as well as in tumor progression and tissue repair. The mannose receptor (MR; CD206, previously known as the macrophage MR) is a 175-kDa type I transmembrane glycoprotein and is a member of a family of four recycling endocytic receptors, which share a common extracellular domain structure but distinct ligand-binding properties and cell type expression patterns. MR has been shown to bind and internalize carbohydrate and collagen ligands and more recently, to have a role in myoblast motility and muscle growth. Given that the related Endo180 (CD280) receptor has also been shown to have a promigratory role, we hypothesized that MR may be involved in regulating macrophage migration and/or chemotaxis. Contrary to expectation, bone marrow-derived macrophages (BMM) from MR-deficient mice showed an increase in random cell migration and no impairment in chemotactic response to a gradient of CSF-1. To investigate whether the related promigratory Endo180 receptor might compensate for lack of MR, mice with homozygous deletions in MR and Endo180 were generated. These animals showed no obvious phenotypic abnormality, and their BMM, like those from MR-deficient mice, retained an enhanced migratory behavior. As MR is down-regulated during macrophage activation, these findings have implications for the regulation of macrophage migration during different stages of pathogenesis.

Key Words: collagen • Endo180 • mannose receptor • migration • macrophage


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INTRODUCTION
 
Macrophages are large, motile cells of the mononuclear phagocyte series. They arise from circulating monocytes, which extravasate across the blood vessel wall and migrate into the surrounding tissues, where they undergo differentiation into macrophages. This maturation process is driven by glycoprotein cytokines, which include CSF-1, GM-CSF, and IL-3 [1 ]. CSF-1 is the only macrophage-specific hematopoietin, and it can promote the development and subsequent proliferation of macrophages from bone marrow (BM)-derived cells in vitro in the absence of any other growth factor [2 ]. Macrophages have an essential role in the immune system, with involvement in the innate and adaptive responses, but have also been implicated in pathogenic processes, such as wound repair, angiogenesis, and the promotion of tumor progression, as well as tissue morphogenesis during development [3 , 4 ]. The migratory phenotype of macrophages is essential, not only in the active process of their differentiation but also for their recruitment to sites of infection, inflammation, and tissue remodeling [5 , 6 ].

The mannose receptor (MR; CD206, also known as the macrophage MR) is a 175-kDa transmembrane receptor comprised of an N-terminal, cysteine-rich domain, a fibronectin type II (FNII) domain, eight tandemly arranged C-type lectin-like domains (CTLDs), a transmembrane domain, and a cytoplasmic domain. This domain arrangement is also found in three other related receptors, Endo180 (CD280, urokinase plasminogen activator receptor-associated protein), the M-type phospholipase A2 receptor, and DEC-205 (CD205) [7 ]. This receptor family is characterized by the ability to recycle between the plasma membrane and the endosomal machinery, allowing them to deliver extracellular ligands to the cell. Despite this structural similarity, and in particular, the presence of multiple CTLDs, calcium-dependent binding of carbohydrate ligands is restricted to MR and Endo180 [8 9 10 11 ], making these the most functionally analogous members of this receptor family. In addition to their carbohydrate ligands, MR and Endo180 bind native collagens and denatured collagen (gelatin) via a calcium-independent interaction with the FNII domain [12 13 14 15 ]. When incubated with soluble collagens, this binding is followed by rapid endocytic internalization and degradation within the lysosomes [13 , 15 16 17 ], and in vivo studies have shown that in mice with a targeted deletion in Endo180, stromal cells within a collagen-rich tumor matrix have a reduced intracellular collagen accumulation [18 ].

Although MR and Endo180 share ligand-binding properties, the distribution of these molecules in vivo is distinct. MR is expressed predominantly in macrophages, and additional sites of expression are in astrocytes, perivascular microglia, glomerular mesangial cells, retinal pigment epithelial, lymphatic endothelium, and endothelial cells within the spleen and liver [17 , 19 20 21 ]. In contrast, expression of Endo180 in vivo is restricted predominantly to stromal fibroblasts [12 , 18 , 22 23 24 ], and additional sites of expression are on chondrocytes at areas of active cartilage deposition and ossification [25 ], hepatic stellate cells [26 ], and basal gingival keratinocytes [27 ]. In addition, Endo180 expression has been reported on human dermal macrophages [10 , 27 ], as well as macrophages differentiated in vitro from human peripheral blood monocytes [10 , 28 ]. It is interesting that in these expressing cells, Endo180 has been demonstrated to regulate cell migration. Fibroblasts isolated from mice with a targeted deletion in Endo180 show a migration defect when placed in culture [12 , 29 ], and similarly, overexpression of Endo180 in Endo180-negative cell lines results in increased migration, whereas down-regulation of expression with small interfering RNA results in a migration impairment [30 , 31 ]. Recently, it has been demonstrated that myoblasts isolated from MR null (MR–/–) mice also show a migration defect and in addition, were unable to respond to a chemotactic gradient [17 ]. Here, we addressed the question as to whether the MR is involved in modulating the migration of BM-derived macrophages (BMM) isolated from wild-type (WT) and MR–/– mice. In addition, we have investigated whether the related Endo180 receptor has a redundant role in mediating migration and collagen binding in these cells by generating mice with homozygous-targeted deletions in MR and Endo180.


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MATERIALS AND METHODS
 
Reagents
Reagents include rabbit anti-mouse F4/80 and mouse anti-human MR (15-2; Serotec, Oxford, UK) and mouse anti-tubulin mAb (Sigma, Poole, UK). Rat anti-mouse MR mAb (MR5D3) was a kind gift from Luisa Martinez-Pomares (The University Of Nottingham, UK). Mouse anti-human Endo180 mAb, A5/158, is as described previously [10 ]. Rabbit polyclonal Endo180 mAb (CAT2) was produced by ImmunoKontact (AMS Biotechnology, Abingdon, UK) by immunization of rabbits with the keyhole limpet hemocyanin-conjugated peptide sequence CATEKNILVSDMEMNEQQE, located in the cytoplasmic tail of mouse Endo180, as an antigen. Antibody was purified from the immunized serum by Protein G purification. Alexa Fluor® 488-phalloidin and TO-PRO-3 were from Molecular Probes, Invitrogen (Paisley, UK). HRP-anti-mouse, anti-rat, and anti-rabbit Ig (Jackson ImmunoResearch, West Grove, PA, USA) and HRP-anti-rabbit Ig (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used. Oregon Green (OG)-gelatin and FITC-BSA (Molecular Probes, Invitrogen) were also used.

Mice
MR–/– mice [32 ] were a kind gift from Michel C. Nussenzweig (The Rockefeller University, New York, NY, USA). Endo180{Delta}Ex2–6/{Delta}Ex2–6 mice have been described previously [29 ]. MR–/– mice were crossed with Endo180{Delta}Ex2–6/{Delta}Ex2–6 mice to generate double heterozygotes (MR+/–;Endo180+/{Delta}Ex2–6). These were then crossed together to generate the double homozygotes (MR–/–;Endo180{Delta}Ex2–6/{Delta}Ex2–6), as well as the WT littermate controls. The mice were genotyped for the MR allele by PCR as described previously [32 ] and for the Endo180 allele by Southern blot analysis [29 ].

Isolation and culture of cells
The method for generating BMM was modified from that described previously [33 , 34 ]. Briefly, BM was removed from the femurs and tibias of sibling 6- to 8-week-old, WT, MR–/–, Endo180{Delta}Ex2–6/{Delta}Ex2–6 and MR–/–;Endo180{Delta}Ex2–6/{Delta}Ex2–6 mice and suspended in RPMI-1640, supplemented with 10,000 units/ml penicillin and streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 1x nonessential amino acids, 50 µM 2-ME, 10% heat-inactivated FBS, and 5 ng/ml recombinant human CSF-1 (R&D Systems, Abingdon, UK). Cells were seeded at 2 x 105 cells/cm2 in 75 cm2 tissue-culture flasks (Nunc, Fisher Scientific, Loughborough, UK). After 48 h, the nonadherent progenitor cells were collected and resuspended in identical medium as described above with inclusion of 18 ng/ml CSF-1 (BMM growth medium). Progenitor cells were seeded at 105 cells/ml in bacteriological plates (Falcon, BD Biosciences, Oxford, UK) and grown for 5–7 days in BMM growth medium to allow differentiation into a migratory macrophage lineage [34 ]. Prior to experimental use, BMM, no greater than 9 days in culture, were harvested using AccutaseTM (TCS Cellworks, Buckingham, UK). To control for variation between animals, BMM isolates from three different mice were used in all experiments, unless otherwise stated. Murine embryonic fibroblasts were isolated and cultured as described previously [29 ]. Human BM was obtained with full patient consent and ethical approval. Human BMM were generated as described above for mouse BMM. Human BM-derived stromal fibroblasts were isolated as described previously [35 ].

Immunoblotting and collagen internalization
Cells were lysed directly in 2x nonreducing sample buffer and immunoblotted as described previously [31 ]. The ability of cells to internalize collagen was assessed as described previously [13 , 15 ]. In brief, cells were starved for 1 h in serum-free RPMI-1640 medium at 37°C, followed by addition of 20 µg/ml OG-gelatin for 2 h at 37°C. Cells were washed twice in PBS, harvested by exposure to AccutaseTM for 1 min at 37°C, and fixed with 1% paraformaldehyde in PBS. Internalization was analyzed using a Becton Dickinson FACSCalibur flow cytometer and CellQuest software.

Real-time quantitative PCR (qPCR)
RNA was extracted from BMM and WT mouse embryonic fibroblasts using Trizol (Molecular Probes, Invitrogen). RNA clean-up and DNase digestion were performed using the RNeasy Micro kit (Qiagen, Crawley, UK), and 500 ng total RNA was used in the RT reactions. qPCR was performed on the ABI Prism 7900HT sequence detection system (Applied Biosystems, Foster City, CA, USA) using Taqman® gene expression assays (Applied Biosystems). The reference numbers for the assays used were: 4352339E (GAPDH), Mm00485148_m1 (MR), and Mm00485184_m1 (Endo180). GAPDH was set as the endogenous control. Assays were conducted on two separate occasions. On each occasion, qPCR reactions were run in four separate plates with triplicate reactions in each plate. Applied Biosystems SDS v2.2 software was used to analyze results, generating relative quantification expression levels (RQ values) for each plate, relative to mouse embryonic fibroblasts (comparator). Mean RQ values and SD were determined in Microsoft Excel software for the paired cDNA samples across all the experiments.

Time-lapse microscopy
To study cell migration or chemotaxis, cells were seeded at a density of 2 x 104 cells/ml in BMM growth medium on glass coverslips. Cells were left for 24 h if measuring random cell migration or 16 h if being used to assess chemotaxis. For random cell migration, coverslips were placed onto Dunn chambers (Hawksley Technology, Lancing, UK) with the inner and outer wells containing BMM growth medium. To assess the ability of cells to detect a chemotactic gradient, BMM were first starved of CSF-1 for 8 h before being placed onto Dunn chambers with CSF-1-free medium in the inner well and BMM growth medium containing 18 ng/ml CSF-1 in the outer well [34 , 36 ]. Images of cells were digitally recorded at a time-lapse interval of 10 min for 3–5 h using an Olympus IX70 microscope fitted with a humidified 37°C incubation chamber, an Olympus x20 lens (0.4 numerical aperture, dry), and Simple PCI acquisition software (Digital Pixel, UK). Mean ± SEM cell migratory speed and directionality were calculated using Motion Analysis software (Kinetic Imaging Ltd., Nottingham, UK) to track cells and Mathematica software (Wolfram Research Ltd., Champaign, IL, USA) using a notebook written by Daniel Zicha (Cancer Research UK) for statistical analysis, as described previously [37 ]. A significant chemotactic response is shown on the Rayleigh plots as an arrow and wedge, which indicate the mean direction of migration and corresponding 95% confidence interval, respectively.

Immunostaining and confocal imaging
Staining of the actin cytoskeleton was carried out as described previously [31 , 38 ]. For confocal imaging, cells were fixed, stained, and mounted in Vectashield H-1000 (Vector Laboratories, Peterborough, UK) at room temperature. Images were captured at room temperature with a Leica TCS SP2 confocal microscope and Leica Confocal software using a Leica 63x (1.40 numerical aperture; oil) lens and Immersol 518F oil (Carl Zeiss Ltd., Hertfordshire, UK). Images were imported into Adobe Photoshop Version 8.0 for processing.

Quantification of membrane ruffles
BMM were starved of CSF-1 for 8 h and then restimulated with CSF-1 for 2 min, followed by fixation and staining with Alexa Fluor® 488-phalloidin and TO-PRO-3. A cell was counted as having membrane ruffles when these structures covered more than 25% of its dorsal surface.


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RESULTS
 
BMM from MR–/– mice display an increase in random cell migration
To determine whether the MR plays a role in the migration of macrophages, cultures of BMM from MR–/– mice and WT littermate-matched control mice were established. All of the cultured cells used were determined by flow cytometry to be 85–95% F4/80-positive, which not only confirms their correct lineage but also that a lack of MR does not affect differentiation of BM-derived precursors to macrophages. WT BMM expressed MR, and as expected, BMM harvested and cultured from MR–/– mice were MR-deficient (Fig. 1 ).


Figure 1
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  		Figure 1. Generation and characterization of BMM from MR-deficient mice. Lysates of BMM from WT and MR–/– mice were immunoblotted with anti-MR mAb followed by HRP-anti-rat Ig (upper panel) or antitubulin antibody followed by HRP-anti-mouse Ig (lower panel). The blot shows expression levels in BMM from two WT (Lanes 1 and 2) and two MR–/– (Lanes 3 and 4) mice.

A recent study defined a previously unappreciated role for MR in the promotion of cell migration, through which it promotes the fusion of myoblasts during muscle development [17 ]. To investigate whether MR has a functional role during macrophage migration, WT and MR–/– BMM were assayed for their migratory capacity using time-lapse microscopy. Experiments were performed using BMM derived from three WT and three MR–/– mice. In contrast to the decreased migration of MR–/– myoblasts [17 ], BMM cultured from the three MR–/– mice consistently migrated greater distances over the 3-h duration of the assay than those from WT mice. An example of the single cell tracks of ~200 BMM assayed in one representative experiment demonstrating the increase in migratory distance traveled by MR–/– BMM compared with WT BMM is shown in Figure 2A . This increase in distance traveled by migrating MR–/– cells was consistent with an overall increase in mean migratory speed for the pooled data of >750 cells from the three mice assayed (Fig. 2B) .


Figure 2
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  		Figure 2. MR–/– BMM display increased random cell migration. The migratory capacity of BMM from WT and MR–/– mice cultured on uncoated glass coverslips was assessed by time-lapse microscopy. (A) Migratory tracks of cells assayed from one WT (n=206 cells) and one MR–/– (n=194 cells) mice are shown. (B) Data shown are mean cell speed in µm/h ± SD from three WT (n=777 cells) and three MR–/– (n=1050 cells) mice. *, Statistical difference from control levels, P < 0.0001.

MR is not required for macrophage chemotaxis or membrane ruffling in response to CSF-1
The sensing of chemotactic signals and directional migration is one of the most important biological responses of macrophages during pathologies, which involve an immunological response [5 ]. Jansen and Pavlath [17 ] have demonstrated recently that the MR was not only involved in the promotion of increased speed but also in directional chemosensing during myoblast migration in a gradient of conditioned medium from nascent myotubes. Consequently, although the MR appears to be a negative regulator of macrophage migratory speed, it was important to investigate whether MR has a similar regulatory function in macrophage chemosensing. To assess this, we used a well-established model of macrophage chemotaxis in which BMM are visualized directly in a gradient of CSF-1 in Dunn chemotaxis chambers [34 , 36 , 39 ]. In this model, Bac1.2f5 macrophages [39 ] and BMM [34 , 36 ], which have been starved of CSF-1, respond and migrate directionally in a gradient of this growth factor, mimicking their physiological responses, which occur in vivo [5 , 40 ]. As expected, WT BMM were consistently able to sense and migrate up a chemotactic gradient of CSF-1 (Fig. 3A ). The speed at which WT cells migrated up a gradient of CSF-1 (Fig. 3B) was similar to that measured for WT cells migrating randomly in growth medium containing the same concentration of CSF-1 (Fig. 2B) . BMM lacking MR were also able to chemotax efficiently toward the source of CSF-1 (Fig. 3A) . As measured in randomly migrating MR–/– BMM (Fig. 2B) , these MR–/– cells also displayed an increase in speed compared with their WT counterparts when migrating up a gradient of CSF-1 (Fig. 3B) .


Figure 3
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  		Figure 3. MR–/– BMM display increased migration up a gradient of CSF-1. BMM from WT and MR–/– mice cultured on uncoated glass coverslips were starved of CSF-1 for 8 h and then placed on a Dunn chemotaxis chamber containing 18 ng/ml CSF-1 in the outer chamber. Directional migration was assessed by time-lapse microscopy. (A) Statistically significant directionality up a gradient of CSF-1 is indicated on the Rayleigh plots by an arrow and wedge (P<0.00001). (B) Data shown are mean cell speed in µm/h ± SD from four WT (n=1336 cells) and four MR–/– (n=1096 cells) mice. *, Statistical difference from control levels, P < 0.0001.

The migratory response of macrophages in a gradient of CSF-1 involves a distinct series of actin cytoskeletal responses including filopodial protrusions, membrane ruffles, spreading, F-actin accumulation at the cell periphery, and polarization [36 , 41 ]. Filopodial protrusions are formed through activation of the Cdc42-Wiskott Aldrich syndrome protein pathway [41 42 43 ], which is necessary for the directionality of macrophages in chemotactic gradients [39 , 44 , 45 ]. Membrane ruffles in macrophages are formed through the activation of Rac [34 , 41 ], which is a signaling event that has been associated with the promotion of cell speed during macrophage migration [39 ]. To ascertain whether the increased migratory speed of MR–/– BMM correlated with activation of Rac signaling, membrane ruffling in WT and MR–/– BMM in response to CSF-1 was quantified. As reported previously [36 ], membrane ruffling could be visualized on the dorsal surface of cells, and this was increased significantly in response to CSF-1 (Fig. 4A and 4B ). As WT and MR–/– BMM displayed a similar level of membrane ruffling in response to CSF-1, we deemed it unlikely that Rac activation was altered in MR–/– BMM or involved in their increased migration.


Figure 4
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  		Figure 4. Membrane ruffles stimulated by CSF-1 are not altered in MR–/– BMM. (A) BMM from WT and MR–/– mice cultured on uncoated glass coverslips were starved of CSF-1 for 8 h prior to stimulation with CSF=1 for 2 min. BMM were fixed and stained with phalloidin-Alexa488, and confocal microscopy was used to visualize actin-rich membrane ruffles on the dorsal surface of cells. (A) Representative confocal images showing extensive ruffling in WT and MR–/– BMM following CSF-1 treatment; original scale bar = 25 µm. (B) Data shown are membrane ruffles quantified from three WT and three MR–/– mice (>500 cells assayed). A nonsignificant (n.s.) statistical difference is indicated.

Increased migration of MR–/– BMM does not involve up-regulation of Endo180
The related MR family member Endo180 has been established recently as an important component in the migratory machinery of fibroblasts and a number of tumor cell lines [12 , 29 30 31 ]. As Endo180 expression has been detected on human monocyte-derived macrophages in vitro as well as on macrophages localized in human dermis [10 , 27 , 28 ], we considered the possibility that up-regulation of Endo180 might be a mechanism to compensate for the lack of MR. To investigate this possibility, four approaches were taken.

First, BMM lysates were subject to immunoblotting with a polyclonal antibody, CAT2, raised against a conserved peptide in the Endo180 cytoplasmic domain (see Materials and Methods). As a positive control, this antibody was shown to recognize a characteristic 180-kDa band in mouse embryo fibroblasts isolated from WT mice. In contrast, no Endo180 expression was detected in BMM derived from WT or MR–/– mice (Fig. 5A ), even after longer exposure of the immunoblot (Fig. 5B) . MR–/– mice were also crossed with mice with targeted deletion Endo180 (Endo180{Delta}Ex2–6/{Delta}Ex2–6 mice) [29 ] to generate double-homozygotes MR–/–;Endo180{Delta}Ex2–6/{Delta}Ex2–6 animals. The MR–/–;Endo180{Delta}Ex2–6/{Delta}Ex2–6 mice appear to be phenotypically normal, healthy, and fertile, and no Endo180 expression was detected in the BMM derived from these double-homozygote mice or from the Endo180{Delta}Ex2–6/{Delta}Ex2–6 mice (Fig. 5A and 5B) . Second, transcript levels of Endo180 and MR were analyzed in BMM by real-time qPCR. Endo180 transcripts were readily detectable in mouse embryonic fibroblasts, but for all BMM samples, the amplification plots did not cross the CT within the 40-cycle reaction, thus confirming that Endo180 is not up-regulated to compensate for the targeted deletion in MR. In addition, equivalent levels of MR transcripts were detected in WT and Endo180{Delta}Ex2–6/{Delta}Ex2–6 BMM, demonstrating that targeted deletion of Endo180 does not result in altered expression of MR. The RQ values for MR amplification in MR–/– and MR–/–;Endo180{Delta}Ex2–6/{Delta}Ex2–6 cells were 1.51 x 10–3 and 1.48 x 10–3, respectively, which is equivalent to background levels of fluorescence (Fig. 5C) . This lack of Endo180 expression on BMM is not confined to mouse cells. No Endo180 expression was detected in human BMM, whereas Endo180 protein was detected readily in human BM-derived fibroblasts (Fig. 5D) . Third, the collagen-binding capacity of BMM from WT, MR–/–, Endo180{Delta}Ex2–6/{Delta}Ex2–6, and MR–/–;Endo180{Delta}Ex2–6/{Delta}Ex2–6 mice was tested by incubating cells with OG-conjugated, denatured collagen (OG-gelatin). Only those BMM derived from MR–/– and MR–/–;Endo180{Delta}Ex2–6/{Delta}Ex2–6 mice were defective in the binding and internalization of OG-gelatin (Fig. 6 ), in agreement with a previous report that MR has a nonredundant role in collagen internalization by macrophages [15 ]. As all BMM macrophages showed a similar profile of FITC-BSA binding and uptake, it can also be concluded that loss of MR expression does not alter nonspecific internalization pathways. Finally, the migratory capacity of BMM from Endo180{Delta}Ex2–6/{Delta}Ex2–6 and MR–/–;Endo180{Delta}Ex2–6/{Delta}Ex2–6 mice was tested. It was found that Endo180{Delta}Ex2–6/{Delta}Ex2–6 BMM translocated at the same speed as WT cells but that a significant increase in speed was observed in BMM derived from MR–/–;Endo180{Delta}Ex2–6/{Delta}Ex2–6 mice (Fig. 7 ). Together, these data provide conclusive validation that BMM do not express Endo180 and that Endo180 is not up-regulated in MR–/– BMM. Consequently, it can be concluded that Endo180 is not involved in promoting the observed increase in random and chemotactic migration of BMM with a targeted deletion in the MR.


Figure 5
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  		Figure 5. MR and Endo180 expression in BMM. WT (Lane 1), Endo180{Delta}Ex2–6/{Delta}Ex2–6 (Lane 2), MR–/– (Lane 3), MR–/–;Endo180{Delta}Ex2–6/{Delta}Ex2–6 (Lane 4), and WT mouse embryonic fibroblasts (Lane 5) were analyzed as follows. (A) Cell lysates were immunoblotted using the anti-mouse Endo180 antibody CAT2 followed by HRP-anti-rabbit Ig (upper panel) or antitubulin antibody followed by HRP-anti-mouse Ig (lower panel). (B) Longer exposure of immunoblot shown in A. (C) qPCR analysis to determine fold changes in expression of MR in BMM compared with WT mouse embryonic fibroblasts (comparator). Figure shows relative MR expression of WT (sample 1), Endo180{Delta}Ex2–6/{Delta}Ex2–6 (sample 2), MR–/– (sample 3), and Endo180{Delta}Ex2–6/{Delta}Ex2–6 (sample 4) BMM. Data shown are the mean ± SD of fold–relative expression in two independent experiments. Note: In MR–/– and MR–/–;Endo180{Delta}Ex2–6/{Delta}Ex2–6 BMM samples, RQ values were 1.51 x 10–3 and 1.48 x 10–3, respectively, which is equivalent to background levels of fluorescence. For Endo180, the amplification plots did not cross the cycle threshold (CT) within the 40-cycle reaction for any of the BMM samples (RQ values <2x10–2). Endo180 was amplified robustly from mouse embryonic fibroblasts. (D) Lysates of human BMM (Lane 1) and human BM fibroblasts (Lane 2) were immunoblotted using the anti-human Endo180 antibody A5158 (top panel), anti-human MR antibody 15-2 (middle panel), or antitubulin antibody (bottom panel), followed by HRP-anti-mouse Ig.


Figure 6
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  		Figure 6. Collagen-binding capacity of BMM. Cells from WT, MR–/–, Endo180{Delta}Ex2–6/{Delta}Ex2–6, and MR–/–;Endo180{Delta}Ex2–6/{Delta}Ex2–6 mice, were cultured for 1 h in serum-free DME and then incubated with OG-gelatin (open plots) or FITC-BSA (filled plots) at 20 µg/ml in serum-free DME for 2 h at 37°C prior to washing and analysis by flow cytometry. The percent of cells binding OG-gelatin is shown.


Figure 7
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  		Figure 7. MR–/–;Endo180{Delta}Ex2–6/{Delta}Ex2–6 but not Endo180{Delta}Ex2–6/{Delta}Ex2–6 BMM display increased random cell migration. BMM from WT, Endo180{Delta}Ex2–6/{Delta}Ex2–6, and MR–/–;Endo180{Delta}Ex2–6/{Delta}Ex2–6 mice were cultured on uncoated glass coverslips, and random cell migration was assessed using time-lapse microscopy. Data shown are mean cell speed in µm/h ± SD. All data are from two mice (n>500 cells assayed). *, Statistical difference from control levels, P < 0.0001.


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DISCUSSION
 
The results of this study demonstrate that MR has an inhibitory influence during the important biological process of macrophage migration. Although the mechanism underlying the negative regulation by MR on migratory speed is not yet clear, it is notable that it occurs in macrophages responding to nonlinear and linear chemotactic gradients. In particular, it was confirmed that BMM derived from MR–/– mice display significant increases in random motility when responding to the nonlinear, migratory cues present in growth medium containing CSF-1. A similar increase in migratory speed was also evoked when MR–/– BMM, starved of CSF-1, were exposed to a linear chemotactic gradient of this chemoattractant growth factor in Dunn chambers. As similar levels of membrane ruffling were induced by CSF-1 on the dorsal surface of WT and MR–/– BMM, it is unlikely that pathways leading to Rac activation drive this migratory response, which compliments reports that Rac-1 and -2 isoforms are redundant in BMM migration [46 , 47 ]. It was also determined that mouse BMM migration does not involve the related MR family receptor Endo180, and a first report that MR–/–;Endo180{Delta}Ex2–6/{Delta}Ex2–6 mice are phenotypically normal, healthy, and fertile is also provided. Nevertheless, the BMM derived from MR–/–;Endo180{Delta}Ex2–6/{Delta}Ex2–6 mice displayed an increase in migratory speed, equivalent to that observed in BMM from MR–/– mice. Together, these data demonstrate that Endo180 up-regulation does not compensate for a loss of MR expression.

Cell migration is a highly complex process, in which balanced adhesion and de-adhesion of cell surface receptor and matrix interactions are responsible for the dynamics of protrusion at the leading edge and concomitant release at the cell rear [48 ]. It should be appreciated that too little or too much cell adhesion, through a multiple array of receptor-matrix interactions, can equate to an overall reduction in migratory capacity [49 ]. Here, it may be that the altered adhesive capacity of MR–/– BMM underlies their increased cell migration, particularly as MR has been confirmed recently to bind the major extracellular matrix component collagen directly [15 ]. However, this notion remains speculative and also contrasts with the positive regulatory role reported for MR during myoblast migration and chemotaxis [17 ] and the positive regulatory role established for the related MR family member Endo180 in the regulation of cell-matrix adhesions during fibroblast and tumor cell migration [12 , 29 30 31 ]. In the experiments described here, uncoated glass coverslips were used to study BMM migration, whereas a collagen-containing matrix was used for similar experiments in the myoblast study [17 ], which is the only other to investigate a functional role for MR in cell migration. It is possible that the different migratory outcomes of MR absence in these two cell types are the result of the different substrata used and/or that there are different adhesive structures used by macrophages and mesenchymal cells to facilitate migration. Unlike mesenchymal cells, macrophages do not migrate via the assembly and disassembly of focal adhesions and associated actin stress fibers but instead, use the much smaller and relatively less stable focal contacts together with highly dynamic, actin-rich units, called podosomes, of which the latter are restricted mainly to myeloid cells or their precursors [5 , 38 ] (for review, see ref. [50 ]). How the direct interaction between MR and collagen might impact cell adhesion and migration poses a particularly interesting question for future consideration. The recent finding that Endo180 functions to disassemble focal adhesions through localization of Rho/Rho-kinase/regulatory myosin light chain-based contractile signals at the cell rear [31 ] indicates the possible existence of a similar role for MR in myoblasts or macrophages. Indeed, dynamin-rich endosomes, which are likely to contain MR, are seen to accumulate adjacent to podosomes in osteoclasts and macrophages [38 , 51 ]. Although we find MR did not promote the generation of membrane ruffles in BMM, a response that requires Rac activity [41 , 46 , 47 ], we cannot rule out the possibility that MR may have some regulatory effects on Rho GTPases, such as Cdc42 and RhoB, which have been identified as downstream targets of MR during phagocytosis [52 ]. Whether MR functions in regulating podosome function through binding, adhesion, and internalization of collagen and/or the generation of localized signals will be an interesting avenue to explore. In this respect, it is of interest that there is a growing body of evidence that podosomes can degrade collagen and other matrix proteins [50 ] and that their assembly and/or disassembly is under the regulation of Rho signaling [53 54 55 ].

The finding that functional Endo180 is not expressed on mouse BMM is in agreement with the findings of Martinez-Pomares et al. [15 ], in which MR was found to be the only receptor required for collagen binding by this cell type. Moreover, as this finding indicated that a species difference may exist for the distribution of Endo180 on cells of a myeloid lineage, we investigated this possibility further. In earlier studies, Endo180 expression was detected on the human U937 monocytic cell line, human peripheral monocyte-derived macrophages, and in macrophages localized within human dermal tissue [10 , 27 , 28 , 56 ]. It was for this reason that Endo180 expression was considered as a potential, compensatory mechanism, which facilitates migration in the absence of MR. The lack of expression of Endo180 on BMM derived from WT or MR–/– mice or in human BMM confirmed that this was not the case. However, we cannot rule out the possibility that MR/Endo180 expression levels are sequentially altered in monocytes during migration from their site of production in the BM, intravasation, adhesion to the vascular endothelium, extravasation, conversion to macrophages and migration to sites of tissue infection, and phagocytosis at specific inflammatory tissue sites. A more extensive cross-species evaluation will be required to clarify the expression of Endo180 in this context.

Monocytes, which cross the vascular endothelium and differentiate into macrophages, then migrate to sites of inflammation, where the primary activity driven by actin cytoskeletal dynamics switches from the promotion of cell migration to the process of phagocytosis [57 ]. It is interesting that the reverse of this process occurs during Myobacterium tuberculosis infection of human macrophages, whereby the phagocytic uptake of this bacterium is associated with an increase in adhesive and migratory properties and a marked decrease in phagocytic behavior [58 ]. These functional changes not only coincide with an increase in cell surface expression of the proadhesion molecules ICAM-1 and LFA-1 but also, a decrease in the cell surface expression of complement receptors and MR. This latter observation lends support to a correlation between loss of MR and the increased motility of macrophages under physiological conditions.

In vivo studies using MR–/– mice confirmed that normal recruitment of macrophages occurs during Candida albicans infection of the kidney, liver, and lung; however, the method used to determine macrophage recruitment was only semiquantitative [59 ]. In a similar study, a fully quantitative analysis revealed that a greater influx of macrophages in alveoli of MR–/– mice infected with Pneumocystis carnii, which was accompanied by an increase in pulmonary dysfunction [60 ]. Although it was hypothesized that this increase in phagocyte recruitment could represent a compensatory response for the concomitant decrease in pathogen clearance, the mechanism involved was not established. Hence, the increased motility of MR–/– BMM reported here may well contribute to the enhanced recruitment of macrophages observed in these infected animals. It is also feasible to predict that the inability of MR–/– BMM to bind collagen [15 ] may be important in the ability of macrophages to speed up their migration, not only in the in vitro model used in this study but also during microorganism infection or the wounding of tissues in vivo. Indeed, the down-regulation of MR observed in macrophages exposed to infectious pathogens [58 , 61 62 63 64 ] may function to facilitate their increased migration through tissues as part of the normal host defense response. Finally, the negative regulation of migration through maintained expression of MR could help to retain macrophages once they have been recruited successfully to sites of infection or tissue damage.


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ACKNOWLEDGEMENTS
 
Breakthrough Breast Cancer and the Association of International Cancer Research funded this work. We thank Michel Nussenzweig (The Rockefeller University, New York, NY, USA) for the generous provision of the MR–/– mice, Justin Cobb (Imperial College London, UK), who isolated the human bone marrow, Daniel Zicha (Cancer Research UK, London Research Institute, London, UK) for providing us with the Mathematica analysis notebook, Luisa Martinez-Pomares for the mannose receptor antibody, and Elizabeth Garner for help with the cell tracking.

Received January 4, 2007; revised May 30, 2007; accepted May 30, 2007.


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