- Email: [email protected]
The antigen recognized by MOMA-I is sialoadhesin
Immunology Letters 106 (2006) 96–98
Short communication
The antigen recognized by MOMA-I is sialoadhesin Cornelia Oetke a,∗ , Georg Kraal b , Paul R. Crocker a a b
The Wellcome Trust Biocentre, Division of Cell Biology and Immunology, School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK Department of Molecular Cell Biology and Immunology, Vrije University Medical Center, PO Box 7057, 1007 MB Amsterdam, The Netherlands Received 2 April 2006; accepted 9 April 2006 Available online 8 May 2006
Abstract The monoclonal antibody MOMA-1 is a widely-used marker for marginal metallophilic macrophages in spleen and some other subsets of macrophages. The antigen recognized by MOMA-1 has yet to be characterized, but its expression pattern is similar to that of sialoadhesin (Sn, CD169, Siglec-1), a member of the sialic acid binding Ig-like lectin (Siglec) family. Using flow cytometry of Sn-transfected cells and staining of lymphoid tissue sections from Sn-deficient mice, we demonstrate here that the antigen recognized by MOMA-1 is Sn. © 2006 Elsevier B.V. All rights reserved. Keywords: Sialoadhesin; Monoclonal antibody; Macrophages
1. Introduction Resident tissue macrophages show a high degree of heterogeneity due to their functional and regional specialization. This is well-illustrated in the spleen which contains red pulp macrophages, tingible-body macrophages, marginal zone macrophages (MZM) and marginal metallophilic macrophages (MMM), each population varying in cell surface receptors, localization, morphology and function [1]. MMM are localized at the inner lining of the marginal sinus and form a clearly distinct population from marginal zone macrophages inside the marginal zone. The first antibody recognizing these cells, MOMA-1, was described in 1986 [2]. Beside MMM, MOMA-1 also stains red pulp macrophages in spleen, albeit less strongly, subcapsular sinus macrophages and medullary macrophages in lymph nodes, lamina propria macrophages, macrophages in Peyer’s patches [2] and macrophage subsets in the developing and adult central nervous system [3]. Although this marker has been frequently used in immunohistochemistry and immunofluroescence [4,5], its molecular identity has remained unknown. Sialoadhesin (CD169, Siglec-1, Sn) shows a strikingly similar expression pattern to the MOMA-1 antigen [6]. Sn is a ∗ Corresponding author at: The Wellcome Trust Biocentre, University of Dundee, Dow Street, Dundee DD1 5EH, UK. Tel.: +44 1382 385 778; fax: +44 1382 385 783. E-mail address: [email protected] (C. Oetke).
0165-2478/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2006.04.004
member of the sialic acid-binding Ig-like lectin (Siglec) family that was originally described as a sheep erythrocyte receptor (SER) [7,8]. Although it has been suggested that the antigen recognized by the monoclonal antibody MOMA-1 might be Sn [9], this has never been confirmed experimentally. In fact MOMA-1 and anti-Sn antibodies have been used in some studies in parallel as independent markers [10,11]. In this study, we provide definitive evidence that MOMA-1 recognizes Sn. We used stably transfected CHO cells expressing either full-length or a truncated form of Sn and analyzed MOMA-1 binding by flow cytometry. Furthermore, we demonstrate that MOMA-1 staining was lost in tissue sections of Sn-deficient mice. 2. Material and methods 2.1. Reagents, antibodies and cell lines Unless otherwise stated, all reagents were purchased from Sigma–Aldrich (Dorset, UK). Throughout the study the following antibodies and detecting reagents were used: Goat antimouse IgM (biotin), rat IgG2a mAb isotype control (eBR2a, pure, eBiosciences, San Diego, CA), mAb anti-rat IgG2a (RG7/1.30, biotin, BD Pharmingen, Oxford, UK), streptavidin (APC, BD Pharmingen), rabbit anti-rat (HRP, Dakocytomation, Cambridgeshire, UK), MOMA-1 (IgG2a, Serotec, Oxford, UK), affinity-purified monoclonal anti-Sn antibody 1C2 and tissue culture supernatant containing SER-4 were generated
C. Oetke et al. / Immunology Letters 106 (2006) 96–98
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as described previously (IgG2a [6,12]), Vectastain® ABC-AP Reagent (Vector laboratories, Peterborough, UK). CHO cells stably transfected with either full-length Sn containing all 17 domains, full-length Sn carrying a mutation in the sialic acid binding pocket (R97A) or truncated Sn containing domain 1–2 were generated as described previously [13]. 2.2. Mice Sn-deficient mice were generated as described [14] and bred on a C57Bl/6 genetic background for 17 generations. Animals used were intercross offspring of heterozygotes from the same litter, age- and sex-matched at 8–12 weeks of age. Mice were bred and maintained under specific pathogen-free conditions and under the UK Home Office Project Licence PPL 60/3187. 2.3. Flow cytometry CHO cells were cultured in F12 Ham’s Medium (Invitrogen, Paisley, UK), 5% FBS (PAA, Somerset, UK), 1 mM l-glutamine (Invitrogen), at 37 ◦ C, 5% CO2 . Cells were detached using trypsin/EDTA (Invitrogen) and 1 × 106 cells per sample were incubated with 5 g/ml 1C2, MOMA-1, IgG2a isotype control or SER-4 (five-fold diluted tissue culture supernatant) in PBA (PBS, 1% BSA, 0.05% azide) for 30 min at 4 ◦ C. After washing, cells were labelled with biotinylated anti-rat IgG2a (0.65 g/ml) followed by allophycocyanin-conjugated streptavidin (0.65 g/ml) for 30 min at 4 ◦ C each. Cells were finally resuspended in PBA containing 7-amino actinomycin D (Invitrogen) to exclude dead cells and analysed using a FACSCalibur (Becton Dickinson, Oxford, UK) and FlowJo (Tree Star, Inc., Ashland, OR) software. 2.4. Immunohistochemistry Spleens and mesenteric lymph nodes were embedded in OCT (Agar Scientific, Essex, UK) and frozen in 2-methylbutane, solidified in liquid N2 . 7 m cryostat sections were fixed for 10 min in acetone and stored at −20 ◦ C. Slides were blocked with 2% rabbit serum in PBS and incubated with either 1C2 or MOMA-1 in TBS (150 mM NaCl, 100 mM Tris pH 7.4), followed by HRP-conjugated rabbit anti-rat (25 g/ml, 5% mouse serum). Diaminobenzidine tablets (Sigma) were used as peroxidate substrate following the manufacturer protocol. Slides were rinsed in H2 O and mounted in glycerol gelatine (BDH, Pool, UK) before they were analyzed using an Axioskope microscope (Zeiss, Munich, Germany) and Axio vision 3.0 software. 3. Results and discussion The substantial similarity in staining patterns of MOMA-1 and anti-Sn antibodies on tissue sections suggests that they might recognize the same antigen. We therefore used CHO cell stably transfected with Sn cDNA to analyze MOMA-1 binding to Sn by flow cytometry. Binding to CHO cells expressing full-length
Fig. 1. MOMA-1 binds to CHO cells stably transfected with Sn. CHO cells stably transfected with either full-length Sn cDNA (CHO Snd1-17), full-length Sn cDNA containing a point mutation in the sialic acid binding pocket (CHO Snd1-17 R97A) or truncated Sn cDNA comprising the first two domains (CHO Snd1-2) and WT CHO cells were stained with either the MOMA-1 antibody, or with the anti-Sn antibodies 1C2 or SER-4, or with an IgG2a isotype control and analyzed by flow cytometry. Shown are representative profiles from 3 independent experiments.
Sn containing all 17 domains (CHO Snd1-17) was compared to cells expressing full-length Sn carrying a mutation in the sialic acid binding pocket (CHO Snd1-17R97A), truncated Sn containing domain 1–2 (CHO Snd1–2) or wt CHO cells. FACS analysis showed that MOMA-I recognized both forms of fulllength Sn (Fig. 1), but failed to recognize the truncated form of Sn, containing domain 1 and 2 (Fig. 1, thin line). This is strong evidence that MOMA-1 is an anti-Sn antibody that recognizes an epitope lying outside the first two domains. The staining profile of MOMA-1 was very similar to SER-4 (Fig. 1), an anti-Sn mAb whose epitope has previously been mapped to domain 3 [15]. In contrast the anti-Sn blocking antibody, 1C2, recognizes the first, sialic acid binding domain of Sn and consequently also bound to the truncated Sn (Fig. 1) [15]. We next asked if MOMA-1 staining is lost in Sn-deficient mice. We therefore stained spleen (Fig. 2A) and lymph node (Fig. 2B) sections from wt and Sn-deficient mice with either MOMA-1 or the monoclonal anti-Sn antibody 1C2. Highest expression of Sn is on MMM in spleen and subcapsular sinus and medullary macrophages in lymph nodes. For both tissues, no detectable MOMA-1 staining was seen with Sn-deficient mice, whereas typical staining profiles were observed in the wild-type tissues (Fig. 2). Taken together, these results provide compelling evidence that the antigen recognized by MOMA-1 is Sn. This knowledge will be helpful in future studies using these antibodies as macrophage markers. It may also allow reinterpretation of previous results using MOMA-1 antibodies and may contribute to our overall understanding of the biological functions of Sn, especially in immune regulation in tissues like spleen and lymph node.
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C. Oetke et al. / Immunology Letters 106 (2006) 96–98
Fig. 2. MOMA-1 staining is lost in tissue sections from Sn-deficient mice. Cryosections from spleen (A) and lymph nodes (B) of wt (upper row) and Sn-deficient (lower row) mice were stained with anti-Sn (1C2) antibody or MOMA-1. Representative pictures of two mice are shown. The size bar corresponds to 100 m.
Acknowledgments This work was supported by the Wellcome Trust Senior Research Fellowship. We thank Kirsten McLeod for her contribution to this project.
[8]
[9]
References [1] Taylor PR, Martinez-Pomares L, Stacey M, Lin HH, Brown GD, Gordon S. Macrophage receptors and immune recognition. Annu Rev Immunol 2005;23:901–44. [2] Kraal G, Janse M. Marginal metallophilic cells of the mouse spleen identified by a monoclonal antibody. Immunology 1986;58:665–9. [3] de Groot CJ, Huppes W, Sminia T, Kraal G, Dijkstra CD. Determination of the origin and nature of brain macrophages and microglial cells in mouse central nervous system, using non-radioactive in situ hybridization and immunoperoxidase techniques. Glia 1992;6:301–9. [4] Guinamard R, Okigaki M, Schlessinger J, Ravetch JV. Absence of marginal zone B cells in Pyk-2-deficient mice defines their role in the humoral response. Nat Immunol 2000;1:31–6. [5] Martin F, Oliver AM, Kearney JF. Marginal zone and B1 B cells unite in the early response against T-independent blood-borne particulate antigens. Immunity 2001;14:617–29. [6] Crocker PR, Gordon S. Mouse macrophage hemagglutinin (sheep erythrocyte receptor) with specificity for sialylated glycoconjugates characterized by a monoclonal antibody. J Exp Med 1989;169:1333–46. [7] Crocker PR, Kelm S, Dubois C, Martin B, McWilliam AS, Shotton DM, et al. Purification and properties of sialoadhesin, a sialic
[10]
[11] [12]
[13]
[14]
[15]
acid-binding receptor of murine tissue macrophages. Embo J 1991;10: 1661–9. Crocker PR, Gordon S. Properties and distribution of a lectinlike hemagglutinin differentially expressed by murine stromal tissue macrophages. J Exp Med 1986;164:1862–75. Gijbels MJ, van der Cammen M, van der Laan LJ, Emeis JJ, Havekes LM, Hofker MH, et al. Progression and regression of atherosclerosis in APOE3-Leiden transgenic mice: an immunohistochemical study. Atherosclerosis 1999;143:15–25. Ciavarra RP, Taylor L, Greene AR, Yousefieh N, Horeth D, van Rooijen N, et al. Impact of macrophage and dendritic cell subset elimination on antiviral immunity, viral clearance and production of type 1 interferon. Virology 2005;342:177–89. Bilyk N, Holt PG. The surface phenotypic characterization of lung macrophages in C3H/HeJ mice. Immunology 1991;74:645–51. Crocker PR, Freeman S, Gordon S, Kelm S. Sialoadhesin binds preferentially to cells of the granulocytic lineage. J Clin Invest 1995;95:635– 43. Crocker PR, Mucklow S, Bouckson V, McWilliam A, Willis AC, Gordon S, et al. Sialoadhesin a macrophage sialic acid binding receptor for haemopoietic cells with 17 immunoglobulin-like domains. Embo J 1994;13:4490–503. Oetke C, Vinson MC, Jones C, Crocker PR. Sialoadhesin-deficient mice exhibit subtle changes in B- and T-cell populations and reduced immunoglobulin M levels. Mol Cell Biol 2006;26:1549–57. Nath D, van der Merwe PA, Kelm S, Bradfield P, Crocker PR. The amino-terminal immunoglobulin-like domain of sialoadhesin contains the sialic acid binding site. Comparison with CD22. J Biol Chem 1995;270:26184–91.
Short communication
The antigen recognized by MOMA-I is sialoadhesin Cornelia Oetke a,∗ , Georg Kraal b , Paul R. Crocker a a b
The Wellcome Trust Biocentre, Division of Cell Biology and Immunology, School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK Department of Molecular Cell Biology and Immunology, Vrije University Medical Center, PO Box 7057, 1007 MB Amsterdam, The Netherlands Received 2 April 2006; accepted 9 April 2006 Available online 8 May 2006
Abstract The monoclonal antibody MOMA-1 is a widely-used marker for marginal metallophilic macrophages in spleen and some other subsets of macrophages. The antigen recognized by MOMA-1 has yet to be characterized, but its expression pattern is similar to that of sialoadhesin (Sn, CD169, Siglec-1), a member of the sialic acid binding Ig-like lectin (Siglec) family. Using flow cytometry of Sn-transfected cells and staining of lymphoid tissue sections from Sn-deficient mice, we demonstrate here that the antigen recognized by MOMA-1 is Sn. © 2006 Elsevier B.V. All rights reserved. Keywords: Sialoadhesin; Monoclonal antibody; Macrophages
1. Introduction Resident tissue macrophages show a high degree of heterogeneity due to their functional and regional specialization. This is well-illustrated in the spleen which contains red pulp macrophages, tingible-body macrophages, marginal zone macrophages (MZM) and marginal metallophilic macrophages (MMM), each population varying in cell surface receptors, localization, morphology and function [1]. MMM are localized at the inner lining of the marginal sinus and form a clearly distinct population from marginal zone macrophages inside the marginal zone. The first antibody recognizing these cells, MOMA-1, was described in 1986 [2]. Beside MMM, MOMA-1 also stains red pulp macrophages in spleen, albeit less strongly, subcapsular sinus macrophages and medullary macrophages in lymph nodes, lamina propria macrophages, macrophages in Peyer’s patches [2] and macrophage subsets in the developing and adult central nervous system [3]. Although this marker has been frequently used in immunohistochemistry and immunofluroescence [4,5], its molecular identity has remained unknown. Sialoadhesin (CD169, Siglec-1, Sn) shows a strikingly similar expression pattern to the MOMA-1 antigen [6]. Sn is a ∗ Corresponding author at: The Wellcome Trust Biocentre, University of Dundee, Dow Street, Dundee DD1 5EH, UK. Tel.: +44 1382 385 778; fax: +44 1382 385 783. E-mail address: [email protected] (C. Oetke).
0165-2478/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2006.04.004
member of the sialic acid-binding Ig-like lectin (Siglec) family that was originally described as a sheep erythrocyte receptor (SER) [7,8]. Although it has been suggested that the antigen recognized by the monoclonal antibody MOMA-1 might be Sn [9], this has never been confirmed experimentally. In fact MOMA-1 and anti-Sn antibodies have been used in some studies in parallel as independent markers [10,11]. In this study, we provide definitive evidence that MOMA-1 recognizes Sn. We used stably transfected CHO cells expressing either full-length or a truncated form of Sn and analyzed MOMA-1 binding by flow cytometry. Furthermore, we demonstrate that MOMA-1 staining was lost in tissue sections of Sn-deficient mice. 2. Material and methods 2.1. Reagents, antibodies and cell lines Unless otherwise stated, all reagents were purchased from Sigma–Aldrich (Dorset, UK). Throughout the study the following antibodies and detecting reagents were used: Goat antimouse IgM (biotin), rat IgG2a mAb isotype control (eBR2a, pure, eBiosciences, San Diego, CA), mAb anti-rat IgG2a (RG7/1.30, biotin, BD Pharmingen, Oxford, UK), streptavidin (APC, BD Pharmingen), rabbit anti-rat (HRP, Dakocytomation, Cambridgeshire, UK), MOMA-1 (IgG2a, Serotec, Oxford, UK), affinity-purified monoclonal anti-Sn antibody 1C2 and tissue culture supernatant containing SER-4 were generated
C. Oetke et al. / Immunology Letters 106 (2006) 96–98
97
as described previously (IgG2a [6,12]), Vectastain® ABC-AP Reagent (Vector laboratories, Peterborough, UK). CHO cells stably transfected with either full-length Sn containing all 17 domains, full-length Sn carrying a mutation in the sialic acid binding pocket (R97A) or truncated Sn containing domain 1–2 were generated as described previously [13]. 2.2. Mice Sn-deficient mice were generated as described [14] and bred on a C57Bl/6 genetic background for 17 generations. Animals used were intercross offspring of heterozygotes from the same litter, age- and sex-matched at 8–12 weeks of age. Mice were bred and maintained under specific pathogen-free conditions and under the UK Home Office Project Licence PPL 60/3187. 2.3. Flow cytometry CHO cells were cultured in F12 Ham’s Medium (Invitrogen, Paisley, UK), 5% FBS (PAA, Somerset, UK), 1 mM l-glutamine (Invitrogen), at 37 ◦ C, 5% CO2 . Cells were detached using trypsin/EDTA (Invitrogen) and 1 × 106 cells per sample were incubated with 5 g/ml 1C2, MOMA-1, IgG2a isotype control or SER-4 (five-fold diluted tissue culture supernatant) in PBA (PBS, 1% BSA, 0.05% azide) for 30 min at 4 ◦ C. After washing, cells were labelled with biotinylated anti-rat IgG2a (0.65 g/ml) followed by allophycocyanin-conjugated streptavidin (0.65 g/ml) for 30 min at 4 ◦ C each. Cells were finally resuspended in PBA containing 7-amino actinomycin D (Invitrogen) to exclude dead cells and analysed using a FACSCalibur (Becton Dickinson, Oxford, UK) and FlowJo (Tree Star, Inc., Ashland, OR) software. 2.4. Immunohistochemistry Spleens and mesenteric lymph nodes were embedded in OCT (Agar Scientific, Essex, UK) and frozen in 2-methylbutane, solidified in liquid N2 . 7 m cryostat sections were fixed for 10 min in acetone and stored at −20 ◦ C. Slides were blocked with 2% rabbit serum in PBS and incubated with either 1C2 or MOMA-1 in TBS (150 mM NaCl, 100 mM Tris pH 7.4), followed by HRP-conjugated rabbit anti-rat (25 g/ml, 5% mouse serum). Diaminobenzidine tablets (Sigma) were used as peroxidate substrate following the manufacturer protocol. Slides were rinsed in H2 O and mounted in glycerol gelatine (BDH, Pool, UK) before they were analyzed using an Axioskope microscope (Zeiss, Munich, Germany) and Axio vision 3.0 software. 3. Results and discussion The substantial similarity in staining patterns of MOMA-1 and anti-Sn antibodies on tissue sections suggests that they might recognize the same antigen. We therefore used CHO cell stably transfected with Sn cDNA to analyze MOMA-1 binding to Sn by flow cytometry. Binding to CHO cells expressing full-length
Fig. 1. MOMA-1 binds to CHO cells stably transfected with Sn. CHO cells stably transfected with either full-length Sn cDNA (CHO Snd1-17), full-length Sn cDNA containing a point mutation in the sialic acid binding pocket (CHO Snd1-17 R97A) or truncated Sn cDNA comprising the first two domains (CHO Snd1-2) and WT CHO cells were stained with either the MOMA-1 antibody, or with the anti-Sn antibodies 1C2 or SER-4, or with an IgG2a isotype control and analyzed by flow cytometry. Shown are representative profiles from 3 independent experiments.
Sn containing all 17 domains (CHO Snd1-17) was compared to cells expressing full-length Sn carrying a mutation in the sialic acid binding pocket (CHO Snd1-17R97A), truncated Sn containing domain 1–2 (CHO Snd1–2) or wt CHO cells. FACS analysis showed that MOMA-I recognized both forms of fulllength Sn (Fig. 1), but failed to recognize the truncated form of Sn, containing domain 1 and 2 (Fig. 1, thin line). This is strong evidence that MOMA-1 is an anti-Sn antibody that recognizes an epitope lying outside the first two domains. The staining profile of MOMA-1 was very similar to SER-4 (Fig. 1), an anti-Sn mAb whose epitope has previously been mapped to domain 3 [15]. In contrast the anti-Sn blocking antibody, 1C2, recognizes the first, sialic acid binding domain of Sn and consequently also bound to the truncated Sn (Fig. 1) [15]. We next asked if MOMA-1 staining is lost in Sn-deficient mice. We therefore stained spleen (Fig. 2A) and lymph node (Fig. 2B) sections from wt and Sn-deficient mice with either MOMA-1 or the monoclonal anti-Sn antibody 1C2. Highest expression of Sn is on MMM in spleen and subcapsular sinus and medullary macrophages in lymph nodes. For both tissues, no detectable MOMA-1 staining was seen with Sn-deficient mice, whereas typical staining profiles were observed in the wild-type tissues (Fig. 2). Taken together, these results provide compelling evidence that the antigen recognized by MOMA-1 is Sn. This knowledge will be helpful in future studies using these antibodies as macrophage markers. It may also allow reinterpretation of previous results using MOMA-1 antibodies and may contribute to our overall understanding of the biological functions of Sn, especially in immune regulation in tissues like spleen and lymph node.
98
C. Oetke et al. / Immunology Letters 106 (2006) 96–98
Fig. 2. MOMA-1 staining is lost in tissue sections from Sn-deficient mice. Cryosections from spleen (A) and lymph nodes (B) of wt (upper row) and Sn-deficient (lower row) mice were stained with anti-Sn (1C2) antibody or MOMA-1. Representative pictures of two mice are shown. The size bar corresponds to 100 m.
Acknowledgments This work was supported by the Wellcome Trust Senior Research Fellowship. We thank Kirsten McLeod for her contribution to this project.
[8]
[9]
References [1] Taylor PR, Martinez-Pomares L, Stacey M, Lin HH, Brown GD, Gordon S. Macrophage receptors and immune recognition. Annu Rev Immunol 2005;23:901–44. [2] Kraal G, Janse M. Marginal metallophilic cells of the mouse spleen identified by a monoclonal antibody. Immunology 1986;58:665–9. [3] de Groot CJ, Huppes W, Sminia T, Kraal G, Dijkstra CD. Determination of the origin and nature of brain macrophages and microglial cells in mouse central nervous system, using non-radioactive in situ hybridization and immunoperoxidase techniques. Glia 1992;6:301–9. [4] Guinamard R, Okigaki M, Schlessinger J, Ravetch JV. Absence of marginal zone B cells in Pyk-2-deficient mice defines their role in the humoral response. Nat Immunol 2000;1:31–6. [5] Martin F, Oliver AM, Kearney JF. Marginal zone and B1 B cells unite in the early response against T-independent blood-borne particulate antigens. Immunity 2001;14:617–29. [6] Crocker PR, Gordon S. Mouse macrophage hemagglutinin (sheep erythrocyte receptor) with specificity for sialylated glycoconjugates characterized by a monoclonal antibody. J Exp Med 1989;169:1333–46. [7] Crocker PR, Kelm S, Dubois C, Martin B, McWilliam AS, Shotton DM, et al. Purification and properties of sialoadhesin, a sialic
[10]
[11] [12]
[13]
[14]
[15]
acid-binding receptor of murine tissue macrophages. Embo J 1991;10: 1661–9. Crocker PR, Gordon S. Properties and distribution of a lectinlike hemagglutinin differentially expressed by murine stromal tissue macrophages. J Exp Med 1986;164:1862–75. Gijbels MJ, van der Cammen M, van der Laan LJ, Emeis JJ, Havekes LM, Hofker MH, et al. Progression and regression of atherosclerosis in APOE3-Leiden transgenic mice: an immunohistochemical study. Atherosclerosis 1999;143:15–25. Ciavarra RP, Taylor L, Greene AR, Yousefieh N, Horeth D, van Rooijen N, et al. Impact of macrophage and dendritic cell subset elimination on antiviral immunity, viral clearance and production of type 1 interferon. Virology 2005;342:177–89. Bilyk N, Holt PG. The surface phenotypic characterization of lung macrophages in C3H/HeJ mice. Immunology 1991;74:645–51. Crocker PR, Freeman S, Gordon S, Kelm S. Sialoadhesin binds preferentially to cells of the granulocytic lineage. J Clin Invest 1995;95:635– 43. Crocker PR, Mucklow S, Bouckson V, McWilliam A, Willis AC, Gordon S, et al. Sialoadhesin a macrophage sialic acid binding receptor for haemopoietic cells with 17 immunoglobulin-like domains. Embo J 1994;13:4490–503. Oetke C, Vinson MC, Jones C, Crocker PR. Sialoadhesin-deficient mice exhibit subtle changes in B- and T-cell populations and reduced immunoglobulin M levels. Mol Cell Biol 2006;26:1549–57. Nath D, van der Merwe PA, Kelm S, Bradfield P, Crocker PR. The amino-terminal immunoglobulin-like domain of sialoadhesin contains the sialic acid binding site. Comparison with CD22. J Biol Chem 1995;270:26184–91.