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PHAGOCYTES
From the Imperial Cancer Research Fund Laboratories;
the Department of Cellular Science; and the Nuffield Department of
Medicine, University of Oxford, Institute of Molecular Medicine, John
Radcliffe Hospital, Oxford, United Kingdom; Imperial Cancer Research
Fund, London, United Kingdom; and Wellcome Trust Biocentre, Department
of Biochemistry, University of Dundee, Dundee, Scotland.
Sialoadhesin is a macrophage-restricted cellular interaction
molecule and a prototypic member of the Siglec family of sialic acid
binding immunoglobulin (Ig)-like lectins. So far, it has only
been characterized in rodents. Here, we report the molecular cloning,
binding properties, and expression pattern of human sialoadhesin. The
predicted protein sequences of human and mouse sialoadhesin are
about 72% identical, with the greatest similarity in the
extracellular region, which comprises 17 Ig domains in both species. A
recombinant protein consisting of the first 4 N-terminal domains of
human sialoadhesin fused to the Fc region of human IgG1
mediated sialic acid-dependent binding with a specificity similar to
its mouse counterpart, preferring sialic acid in the Macrophages constitute a heterogeneous population
of bone marrow-derived cells, present throughout the body, where they
perform myriad functions, both during steady-state conditions and in
pathology.1 This is reflected in the variable cell surface
phenotypes exhibited by macrophage subsets. Cell surface receptors
initiate and orchestrate many of the activities of macrophages, such as
growth, differentiation, adhesion, phagocytosis, activation,
chemotaxis, and apoptosis. Most such receptors are expressed on other
leukocytes, with only a restricted few being exclusive to macrophages
and hence implicated in macrophage-specific functions.
Sialoadhesin (Sn) is one such receptor. It was originally characterized
in the mouse on isolated resident bone marrow macrophages as a
nonphagocytic, sialic acid-dependent sheep erythrocyte
receptor.2 In situ, these macrophages bind avidly to
developing myeloid and erythroid cells and are therefore well
positioned to dispose of apoptotic cells and extruded erythroblast
nuclei that are generated during hemopoiesis3,4 and B
lymphopoiesis.5 Sialylated ligands for Sn are
displayed on the surface of the attached hemopoietic cells, and the
receptor is clustered at the contact points between macrophages and
developing myeloid cells.6 Sn is therefore likely to be
involved in macrophage-hemopoietic cell interactions in the bone
marrow. In addition to a scavenging function, resident macrophages may
contribute to the trophic microenvironment of the bone marrow, for
example, through recycling of heme-derived iron required for sustained
erythropoiesis.7
Immunocytochemical staining of tissue sections showed that Sn is also
expressed at high levels on discrete subsets of tissue macrophages,
especially those in secondary lymphoid organs.8 For
example, in the spleen of rodents, Sn is expressed mainly on
macrophages in the marginal zone, which have been implicated in
specialized functions in innate and acquired immunity.9 Lower levels of Sn were seen on many other tissue macrophage
populations, such as in the liver, gut, and lung, and certain
macrophage populations, notably the resident brain microglia, expressed
undetectable levels of the receptor.10 Studies on the ED3
antigen in the rat, which has a similar macrophage-restricted
distribution, showed that this molecule is the likely rat ortholog of
Sn.11,12
Mature, circulating neutrophils have been shown to express
high levels of Sn ligands, whereas other cells such as thymocytes and
resting T cells have relatively low levels.13 Because Sn is not a phagocytic receptor,2 it is unlikely to be
involved directly in scavenging functions, but it could cooperate with phagocytic receptors to increase the efficiency of recognition and
uptake as well as being involved in other types of cell-cell interactions. For example, recent findings in a murine model of allogeneic tumor rejection have shown that transferred, activated CD4
and CD8 T cells can cluster in vivo with Sn+ macrophages,
an association that may be important for T-cell effector
functions.14 Because potential sialylated ligands are also
present on molecules like laminin in the extracellular
matrix,15 Sn also may be involved in macrophage-matrix interactions.
Sn is the prototypic member of the Siglec (sialic acid
binding Ig-like lectin) family of cellular
interaction molecules and was designated Siglec-1.16
Siglecs possess a homologous N-terminal V-set domain that contains the
sialic acid binding site followed by varying numbers of C2-set domains.
Sn contains the unusually large number of 16 C2-set domains, a feature
that may be important in its ability to mediate macrophage adhesive
functions.17 Apart from Sn, members of the Siglec family
include CD22 (Siglec-2) on B cells,18 CD33 (Siglec-3) on
immature myeloid cells and monocytes,19 myelin-associated
glycoprotein (MAG) (Siglec-4A) on Schwann cells and
oligodendrocytes,20 and Siglecs-5, -6, -7, -8, and -9 expressed on various hemopoietic subsets.21-26 The restricted expression patterns of different Siglecs implies that they
mediate distinct functions, some of which are likely to involve sialic
acid recognition. Each protein displays a binding preference for sialic
acid in either The molecular basis for carbohydrate binding by Sn has been
determined recently by X-ray crystallography29 in
conjunction with site-directed mutagenesis30 and nuclear
magnetic resonance analysis.31 In the crystal structure of
the Sn V-set domain complexed to 3'-sialyllactose, a highly conserved
arginine residue (Arg97 in mouse Sn) forms a bidentate salt bridge with
the carboxylate group of sialic acid, and 2 well-conserved aromatic
groups (both tryptophan for Sn) make hydrophobic interactions with the
N-acetyl and glycerol moieties of
N-acetylneuraminic acid. The crucial importance of the
arginine has been demonstrated by the finding that even a conservative
substitution with lysine leads to an approximate 10-fold loss in
binding affinity of Sn for sialic acid.31
Despite detailed knowledge of the expression pattern of Sn and the
molecular basis for its recognition of sialic acid, the biologic
functions of this receptor are relatively poorly understood. Insights
into the potential functions of a given protein can be obtained by
comparing the molecular properties and expression patterns of different
species' orthologs. In addition, for proteins expressed by cells of
the immune system, an understanding of their expression during disease
states can give valuable insight into potential roles in pathologic
situations. So far, Sn has only been characterized in rodents. The
purpose of the present study was to identify a human ortholog of Sn in
order to compare its structure and binding properties. The production
of specific antibodies then allowed us to investigate the
expression pattern in normal and diseased situations.
Materials
Cells
Preparation and screening of cDNA libraries A complementary DNA (cDNA) library in ZAPII (Stratagene,
Cambridge, United Kingdom) was prepared as described32
using human monocytic THP-1 cells cultured for 24 hours with 100 ng/mL
phorbol myristate acetate (PMA) as a source of messenger RNA (mRNA). A total of 6 × 105 plaque-forming units were screened with
a 1.35-kilobase (kb) BamHI/EcoRI fragment of
mouse Sn cDNA (nucleotides 4337-568732) labeled with
[ -32P]-dCTP by random priming as
described.32 One positive plaque was identified on
duplicate filters and the cDNA insert excised as a pBluescript
SK phagemid. The nucleotide sequence of the 2.9-kb cDNA
insert (pTH1) was determined by the dideoxynucleotide chain termination
method using double-stranded DNA templates with Sequenase Version 2.0 (United States Biochemical Corp, Cleveland, OH). A cDNA library was
also prepared from human spleen poly(A) RNA as described above but with
random priming.
Isolation of a full-length genomic clone A human genomic P1-derived artificial chromosome (PAC) library33 was used to isolate the Sn gene by polymerase chain reaction (PCR) using primers derived from the cDNA sequence of pTH1.34 A single clone was identified that, on Southern blotting, hybridized with a 5' EcoRI fragment of pTH1. When the Southern blot was hybridized with a 0.3-kb BamHI fragment of mouse Sn cDNA (encoding most of domain 1), 6.5-kb BamHI and 2.2-kb PstI fragments were identified. Hybridization with a 0.9-kb BamHI mouse cDNA probe (encoding domains 3-6) identified a fragment of 12 kb. These 3 restriction fragments were subcloned into pBluescript and sequenced using AmpliTaq FS and dye-labeled terminators (Perkin Elmer, Warrington, United Kingdom) followed by analysis on an ABI Prism 377 DNA Sequencer (Perkin Elmer). End-sequencing revealed that the 2.2-kb PstI fragment spanned from 5' of the gene to the N-terminal immunoglobulin (Ig)-like domain (domain 1); the 6.5-kb BamHI fragment spanned from 5' of the gene to domain 2; and the 12-kb BamHI fragment spanned from the intron between domains 3 and 4 to a BamHI site approximately 1 kb from the poly(A) tail. The nucleotide sequence of the leader peptide, domain 1, and most of domain 2 was obtained from the PstI and 6.5-kb BamHI genomic subclones. Repeated attempts to subclone a fragment encoding the 3' end of domain 2 and all of domain 3 were unsuccessful, and therefore this sequence was obtained from 3 independent PCR products spanning this region, using reverse-transcribed THP-1 mRNA as the template. The region encoding domains 4 to 13 was amplified by PCR from human spleen cDNA and sequenced. The sequence was confirmed using the 12-kb BamHI subclone as a genomic template.Computer analysis Nucleotide and amino acid comparisons were performed using Blast Version 2.0 (National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD). Protein motif searches were carried out using the Prosite database (Swiss Institute for Bioinformatics, Geneva, Switzerland). The amino acid sequences of human and mouse Sn were aligned using the ClustalW 1.7 software available on the Baylor College of Medicine server (http://dot.imgen.bcm.tmc.edu:9331/multi-align/multi-align.html).Northern blot analysis Poly(A) RNA was purified from various cell lines and from human spleen and electrophoresed in a 1% (wt/vol) agarose formaldehyde gel. The RNA was transferred to Hybond N+ nylon membrane (Amersham Phamacia Biotech) and hybridized with a 1.2-kb EcoRI fragment of pTH1 or human actin cDNA (Clontech, Palo Alto, CA) labeled with [-32P]-dCTP by random priming.
Hybridization was performed using 50% formamide in the hybridization
buffer and washing at 55°C with 0.1 × SSC.
Fc protein production A soluble, truncated form of human Sn was produced consisting of the first 4 N-terminal domains (d1-4) of the receptor fused to the Fc portion of human IgG. Human Sn d1-4 was generated by the PCR from spleen cDNA using the following primers: forward, 5'-AGGAATTCGCTATGGGCTTCTTGCCCAAGCTTCTC-3'; reverse, 5'-AGGAATTCACTTACCTGTGTTGACTACCACGCTGACAGG-3'; and cloned into the pIG1 vector provided by Dr D. L. Simmons.35 Recombinant Fc protein was produced in transiently transfected COS cells and purified on protein A-Sepharose (Amersham Pharmacia Biotech) as previously described.35 The protein was dialysed against 20 mM Tris (pH 8.0), concentrated, and filter-sterilized. Control Fc proteins, CD22-Fc, MAG-Fc, and neural cell adhesion molecule (NCAM)-Fc were prepared as described.20Binding assays to RBCs and polyacrylamide glycoconjugates RBC binding assays were performed as detailed elsewhere.20 For polyacrylamide (PAA) assays, various concentrations of biotinylated PAA glycoconjugates carrying either NeuAc2,3Gal 1,4Glc (2,3-PAA) or NeuAc2,6Gal1,4Glc (2,6-PAA)
(Syntesome, Munich, Germany) were added to wells that had been coated
with Fc proteins as described above and incubated on ice for 1 hour.
After washing, horse radish peroxidase (HRP)-conjugated streptavidin
was incubated in the wells for a further hour, and then
o-phenylenediamine dihydrochloride substrate was added and
the optical density measured at 450 nm.
Fc protein binding to leukocytes Mononuclear cells and granulocytes were separated from peripheral blood by sedimentation of RBCs on Dextran T-500 (Amersham Pharmacia Biotech) followed by centrifugation on Lymphoprep (Nycomed Pharma, Oslo, Norway). The leukocytes were labeled immediately or following incubation at 37°C for 2 hours in RPMI 1640 with or without 0.1 U/mL Vibrio cholerae sialidase. All subsequent steps were performed at 4°C. Cells were incubated with 10 µg/mL Fc protein and mAbs for 30 minutes, followed by fluorescein isothiocynate (FITC)-conjugated goat antihuman IgG and PE-conjugated F(ab')2 goat antimouse Ig (Dako), to detect CD3 and CD4. In the case of CD8, CD19, CD16, and CD69, labeling was carried out directly using PE-conjugated mAbs. The cells were analyzed immediately on a FACScan (Becton Dickinson, Oxford, United Kingdom). The monocytes and lymphocyte/natural killer (NK) cell fractions were distinguished by their characteristic forward and side scatter properties. Data were routinely collected for 20 000 to 50 000 mononuclear cells and 20 000 granulocytes.Monoclonal antibody production Mice were immunized with 5 injections of 10 µg purified hSn(d1-4)Fc protein and fusions carried out 4 days after the last immunization. Hybridoma supernatants were screened for reactivity with hSn(d1-4)Fc but not with an irrelevant Fc protein, MAG(d1-3)Fc. Two positive hybridomas, clones 7D2 and 8H2, were identified from 2 separate fusions and the mAbs, both IgG1, designated HSN1 and HSN2, respectively.Immunoprecipitation THP-1 cells (4 × 107) were surface-biotinylated for 10 minutes at 37°C with 0.5 mM sulfo-NHS-biotin (Pierce and Warriner, Chester, United Kingdom). Immunoprecipitations of Nonidet P-40 lysates were carried out as described8 and samples run out under reducing and nonreducing conditions on 6.5% PAA gels. Following transfer to nitrocellulose, immunoprecipitated proteins were detected with HRP-conjugated streptavidin and enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech).Monocyte-derived macrophages Monocytes were purified from granulocyte-depleted blood leukocytes by adhesion to plastic Petri dishes as described.36 Cells were cultured in RPMI plus 10% autologous serum alone for 6 days or with interferon- (500 U/mL) and
TNF- (10 ng/mL) for 2 days. Cells were lifted with
phosphate-buffered saline containing 5 mM ethylenediaminetetraacetic
acid immediately prior to immunostaining and fluorescence-activated
cell sorter (FACS) analysis.
FACS analysis A total of 100 µL whole blood or cell suspensions at 5 × 106/mL were incubated with 10 µL mAb culture supernatant or purified mAb at 5 µg/mL for 30 minutes on ice, washed, and incubated for a further 30 minutes with FITC-conjugated F(ab')2 goat antimouse Ig (Dako). Samples were then treated with Becton Dickinson FACS lysing solution prior to analysis on a FACScan. Data were collected for 1000 THP-1 cells and cultured macrophages or 2500 whole leukocytes and cell populations gated according to their characteristic forward and side scatter profiles.Immunohistochemistry Acetone-fixed, 6 µm frozen sections of various human tissues were incubated with HSN1 or HSN2 culture supernatant or control antibodies diluted in phosphate-buffered saline containing 10% normal human serum. Binding of mAbs was detected by incubating sections with biotinylated goat antimouse IgG (Vector Labs, Burlingame, CA), followed by biotin-avidin complexes (ABC, Vector Labs) conjugated to either peroxidase or alkaline phosphatase and then diaminobenzidine or Fast Red substrates, respectively. Samples of synovial tissue from patients with seropositive rheumatoid arthritis were obtained during routine surgical operations and biopsies performed at the Nuffield Orthopaedic Centre, Oxford Radcliffe Trust Hospital, United Kingdom. Cryostat sections measuring 8 µm were stained with the following primary mAbs at 10 µg/mL: HSN1 or Y182A anti-CD68,37 followed by peroxidase-conjugated secondary antibodies. Omission of the primary antibody was used as a negative control. All sections were counterstained with hematoxylin prior to mounting.
Molecular cloning of human Sn Clone pTH1 was isolated from a cDNA library prepared from PMA-treated THP-1 cells by low-stringency hybridization with a murine Sn probe. The 2.9-kb insert from pTH1 had significant homology to mouse Sn and corresponded to a region between domain 14 and the 3' untranslated sequence. However, further clones isolated from either the THP-1 library or a human spleen cDNA library did not extend beyond the 5' end of pTH1. As an alternative strategy, a clone containing the entire human Sn gene was isolated from a PAC library.33 Sequencing revealed an open reading frame of 5127 base pairs (bp) encoding a protein of 1709 amino acids (Figure 1) and a 3' untranslated region of 1593 bp. Northern blot analysis showed a single transcript of 7.5 kb in the spleen and certain cell lines (Figure 2), suggesting that the 5' untranslated region is about 780 bp. Northern analysis was consistent with Sn gene expression being restricted to macrophages. Signals were detected with THP-1 cells (monocytic), both unstimulated and after culture with PMA to promote macrophage differentiation. In comparision, U937 cells (promonocytic) failed to express detectable Sn mRNA unless they were induced to differentiate with PMA (Figure 2). The Daudi B-cell line and the K562 myeloerythroid cell line did not express detectable Sn mRNA (Figure 2).
Sequence comparisons with mouse Sn The long open reading frame encodes a type 1 transmembrane glycoprotein that is 72% identical to mouse Sn. It consists of a leader peptide of 19 amino acids, a large extracellular domain of 1622 amino acids, and a short cytoplasmic tail of 47 amino acids (Figure 1, Table 1). Like the mouse protein,32 the extracellular region of human Sn consists of 17 Ig-like domains, comprising an N-terminal V-set domain and 16 C2-set domains that show an alternating pattern of long and short domains (Table 1). The size of each Ig-like domain is well conserved between the human and murine receptors, with only 4 of the 17 domains differing in amino acid length. The Ig-like domains are 60% to 80% identical between species, the N-terminal domain being the most highly conserved (Table 1). There are 14 potential N-linked glycosylation sites, 12 of which are present in the mouse (Figure 1). The cytoplasmic tail is poorly conserved, being 12 amino acids longer than its mouse counterpart and only 30% identical over the full length (Table 1). There are 2 potential phosphorylation sites, one for protein kinase C at position 1664 and one for casein kinase-2 at position 1674, only the former being conserved between species.
Alignment of the N-terminal region of human and mouse Sn showed that all of the characteristic structural features of Siglecs are present (Figure 1). In particular, amino acids important for sialic acid binding29 are identical in human and mouse Sn. These include an invariant arginine on the F strand at position 116 and 2 aromatic residues (both tryptophan in Sn) at positions 21 and 125 (Figure 1). In addition, there is conservation of the unusual pattern of cysteine residues that is characteristic of the Siglec family and thought to give rise to an intrasheet disulfide bond within domain 1 and a disulfide bond between domains 1 and 2.29 Sialic acid-dependent binding properties of recombinant human Sn To study the binding properties of human Sn, a soluble, truncated form of the receptor consisting of the first 4 N-terminal domains (d1-4) fused to the Fc portion of human IgG (hSn[d1-4]Fc) was produced in COS cells. Human RBCs, which have abundant cell surface sialic acid, were used as indicator cells to investigate whether human Sn could mediate sialic acid-dependent binding to cells similar to that seen with murine Sn.13 Untreated, but not sialidase-treated, RBCs bound avidly to hSn(d1-4)Fc immobilized on microtiter wells (data not shown).To determine the sialic acid linkage preference of human Sn, binding
assays were carried out with biotinylated PAA carrying either 3'- or
6'-sialyllactose (2,3-PAA or 2,6-PAA). The hSn(d1-4)Fc exhibited
dose-dependent binding of 2,3-PAA (Figure
3A) but, under the same conditions, only
low levels of binding were seen with 2,6-PAA (Figure 3B). CD22-Fc was
also included as a control and, as expected, bound strongly to 2,6-PAA
but not to 2,3-PAA (Figure 3). No binding was seen with NCAM-Fc used as
a nonsialic acid binding control protein. Therefore, human Sn has a
specificity similar to that of murine Sn, preferring
Binding of recombinant Sn to human peripheral blood leukocytes In flow cytometric assays with human peripheral blood leukocytes, hSn(d1-4)Fc showed strong binding to granulocytes and intermediate levels of binding to monocytes compared with the control protein, NCAM-Fc. Within the lymphocyte/NK cell fraction, a subset of cells showed background binding to NCAM-Fc, but binding was clearly stronger with hSn(d1-4)Fc (Figure 4A). Sn-specific binding to all 3 populations was sialic acid-dependent because sialidase pretreatment of the leukocytes reduced hSn(d1-4)Fc binding to a level similar to that seen with the control protein, NCAM-Fc (Figure 4). To characterize the lymphocyte/NK cell-reactive subset in more detail, double labeling was carried out by combining hSn(d1-4)Fc labeling with staining for CD3 (pan T cell), CD4 (helper T cells), CD8 (cytotoxic T cells plus NK cell subset), CD19 (B cells), and CD16 (NK cells) (Figure 4B). This showed that hSn(d1-4)Fc bound specifically to most CD19+ B cells and CD16+ NK cells and about 30% of the CD3+ T cells, most of which belonged to the CD8+ subset (Figure 4B). Most CD4+ T cells were unlabeled. To investigate the possibility that hSn(d1-4)Fc interacts with activated cells, double labeling was carried out for CD69, an early activation marker of lymphocytes. This showed that although only about 4% of lymphocytes expressed CD69, most of these activated cells were specifically recognized by hSn(d1-4)Fc (Figure 4B).
Antibody production and immunoprecipitation Two mouse mAbs, HSN1 and HSN2, were raised to hSn(d1-4)Fc. Using lysates from surface-biotinylated THP-1 cells, HSN1 immunoprecipitated a single protein corresponding to human Sn, which migrated at about 200 kd on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and about 180 kd under nonreducing conditions (Figure 5). This is similar to the mouse protein8 and is consistent with the presence of multiple intramolecular disulfide bonds.
Immunostaining of blood leukocytes and cultured monocytes Northern blot analysis (Figure 2) suggested that expression of human Sn was likely to be restricted to macrophages, as is the case in mouse8 and rat.38 To investigate this further, FACS analysis was initially performed with peripheral blood leukocytes and THP-1 cells. Using either HSN1 or HSN2, Sn was found to be expressed weakly by THP-1 cells but not by blood monocytes, although CD14 and CD18 expression was comparable on both cell types (Figure 6). Neutrophils and lymphocytes were also found to be negative for Sn expression (data not shown).
Previously, it was found that cultivation of murine monocytes and
macrophages in autologous serum can lead to induction of Sn
expression.39 To determine whether human monocyte-derived macrophages behaved similarly, human monocytes were cultured for 6 days
in autologous serum and FACS analysis performed. Despite repeated
attempts, Sn could only be detected at the cell surface at very low
levels under these conditions (Figure 6). Interestingly, however,
addition of interferon- Expression of Sn in normal human tissues In the mouse, Sn is expressed at high levels on distinctive populations of macrophages in spleen and lymph node as well as stromal macrophages in the bone marrow.8 Lower levels are found on many other tissue macrophages although certain macrophages, notably resident brain microglia, express undetectable levels of Sn.10 When immunohistochemistry was performed on frozen sections of human bone marrow using HSN1, a reticular staining pattern of resident macrophages was observed (Figure 7A) that was strikingly similar to the previous description in the mouse.8 In the spleen, a subset of macrophages was intensely stained (Figure 7B), in a region defined as the perifollicular zone,40 whereas red pulp macrophages were stained only weakly. Lymph node perifollicular sinusoidal macrophages stained strongly with mAb HSN1 (Figure 7C). In addition, other resident macrophages in various human tissues were found to be Sn+, including liver Kupffer cells (Figure 7D), macrophages in the lamina propria of the colon (Figure 7E), and alveolar and interstitial macrophages in the lung (Figure 7F). In human brain sections, perivascular macrophages were intensely positive, but the resident microglia did not express detectable levels of Sn (not shown). In conclusion, expression of human Sn is restricted to stromal tissue macrophage subsets in a remarkably similar way to the mouse protein.
Expression of Sn in rheumatoid arthritis To investigate the expression of Sn under inflammatory conditions, immunohistochemistry was carried out using tissue sections from patients with rheumatoid arthritis, a disease in which inflammatory macrophages are believed to play key etiologic roles.41 Parallel staining with HSN1 and the panmacrophage marker CD68 revealed both resident macrophages within the synovial membrane and infiltrating macrophages present in the subintima as strongly positive for Sn. However, macrophages within the T- and B-cell-rich extralymphoid follicles that were positive for CD68 were largely negative for Sn (Figure 8). Interestingly, the follicular macrophages also expressed several other monocyte/macrophage markers, including CD14, the high-affinity IgG Fc receptor CD64, and CD163 (data not shown). This differential staining pattern within an area of active lymphoid proliferation indicates that Sn is expressed by a functionally distinct macrophage subpopulation in inflammatory tissue.
The most striking feature of the data presented here is the remarkable degree to which Sn is conserved between mouse and man. This is apparent at all levels examined, namely sequence similarity, domain organization, binding properties, and expression patterns. Interestingly, the highest degree of sequence similarity was seen in the extracellular region, particularly within the V-set domain that contains the sialic acid binding site. In comparison, the cytoplasmic tail was poorly conserved. Taken together, these observations indicate that Sn has evolved primarily to mediate extracellular functions, namely cell-cell or cell-matrix interactions. The interspecies conservation of all 17 Ig domains is relevant in this regard, because it has been proposed that this large number of domains could be important in extending the binding site of the molecule away from the plasma membrane, which then favors cell-cell interactions.17 Similar conclusions have been made regarding the importance of length for the adhesion molecule, P-selectin,42 which has 9 complement control protein domains in addition to an EGF-like domain and the ligand binding C-type lectin domain.42 Although the ligand binding studies of human Sn are not as extensive as
those carried out previously for the mouse protein, the results
presented here show that human Sn has very similar properties. It binds
strongly to human RBCs in a sialic acid-dependent manner, prefers
Neu5Ac in Despite the marked differences in binding to autologous RBCs, human and
mouse Sn exhibit similar strong, sialic acid-dependent binding to
autologous neutrophils. Although the physiologic significance of this
is unknown, one possibility is that Sn contributes to the clearance of
these cells following apoptosis or senescence in tissues like liver,
spleen, and lymph nodes, for example, in conjunction with defined
phagocytic receptors like CD36 and the vitronectin receptor,
The generation of specific mAbs to Sn has allowed us to investigate the
expression pattern of the human protein. The immunocytochemical and
FACS studies show that Sn is not detectable on monocytes but is
expressed on a wide variety of tissue macrophages in both healthy and
diseased tissues. As in the mouse and rat, human Sn appears to be
exquisitely macrophage-restricted but, importantly, the expression is
heterogeneous and not all macrophages express the receptor. As in the
mouse, subsets of stromal macrophages in lymphoid and hemopoietic
tissues express high levels, whereas Sn is undetectable on the resident
microglia in the brain. It has been shown previously in the mouse that
a factor(s) in plasma is important in regulating Sn expression in
vitro.39 This could provide a partial explanation why
macrophages not exposed to plasma proteins, such as those behind the
blood-brain barrier, do not express Sn. Other factors such as
glucocorticosteroids and interferon- Here, we examined expression of Sn in rheumatoid arthritis, a commonly
occurring inflammatory disorder in which macrophages are thought to be
important.52 We found that most macrophages expressed high
levels of Sn along with other markers like CD68 and CD163. These
findings raise the possibility that Sn contributes to the cell-cell and
cell-matrix interactions of macrophages during inflammatory reactions.
In rheumatoid arthritis, macrophages, together with T cells, are the
predominant cell types within the hyperplastic synovial intima. It is
thought that macrophages are recruited from the circulation rather than
produced locally. The synovial lining, which normally consists of a
bilayer, is increased in thickness by 3- to 4-fold, mostly by activated
macrophages. Synovial membrane and rheumatoid pannus macrophages are
implicated in a number of key events, including production of
interleukin-1 and TNF- From a pragmatic perspective, the antihuman Sn mAbs described here are useful reagents for detecting human tissue macrophages under a variety of different conditions. In mice, several macrophage-restricted markers have been described in addition to Sn, including F4/80 antigen,55 macrosialin,56 MARCO,57 MOMA-1,58 MOMA-2,59 and ERTR9.60 However, in humans only a few macrophage-specific mAbs have been defined, such as those against CD6861 and CD163.62 Although CD68 is apparently expressed on all human macrophage populations, the antigen can also be detected on other myeloid cells and also on certain nonmyeloid cells.37,61 In addition, CD68 is an intracellular antigen that, unlike Sn, does not allow the visualization of plasma membrane processes in tissue sections. Thus, the use of anti-Sn mAbs in conjunction with others like anti-CD163 and anti-CD68 mAbs may be a useful way to maximally visualize macrophages in normal and diseased human tissues.
Human sialoadhesin has been designated CD169 at the recent CD antigen workshop (HLDA7) held at Harrogate, United Kingdom, June 20-24, 2000.
We are grateful to Roger Cox for PAC library screening, Jonathon Fawcett for providing human spleen, Kevin Gatter for access to human tissue samples, and Jiquan Zhang for help with recombinant protein production.
Submitted February 24, 2000; accepted September 5, 2000.
Supported by the Imperial Cancer Research Fund and the Wellcome Trust.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: P.R. Crocker, MSI/WTB Complex, Department of Biochemistry University of Dundee, Dow Street, Dundee DD1 5EH, Scotland; e-mail: p.r.crocker{at}dundee.ac.uk.
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© 2001 by The American Society of Hematology.
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  C. Wu, U. Rauch, E. Korpos, J. Song, K. Loser, P. R. Crocker, and L. M. Sorokin Sialoadhesin-Positive Macrophages Bind Regulatory T Cells, Negatively Controlling Their Expansion and Autoimmune Disease Progression J. Immunol., May 15, 2009; 182(10): 6508 - 6516. [Abstract] [Full Text] [PDF]
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 P. L. Delputte, W. Van Breedam, I. Delrue, C. Oetke, P. R. Crocker, and H. J. Nauwynck Porcine Arterivirus Attachment to the Macrophage-Specific Receptor Sialoadhesin Is Dependent on the Sialic Acid-Binding Activity of the N-Terminal Immunoglobulin Domain of Sialoadhesin J. Virol., September 1, 2007; 81(17): 9546 - 9550. [Abstract] [Full Text] [PDF]
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H.-R. Jiang, L. Hwenda, K. Makinen, C. Oetke, P. R. Crocker, and J. V. Forrester Sialoadhesin Promotes the Inflammatory Response in Experimental Autoimmune Uveoretinitis J. Immunol., August 15, 2006; 177(4): 2258 - 2264. [Abstract] [Full Text] [PDF]
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 M. Skoberne, S. Somersan, W. Almodovar, T. Truong, K. Petrova, P. M. Henson, and N. Bhardwaj The apoptotic-cell receptor CR3, but not {alpha}vbeta5, is a regulator of human dendritic-cell immunostimulatory function Blood, August 1, 2006; 108(3): 947 - 955. [Abstract] [Full Text] [PDF]
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 T. Avril, E. R. Wagner, H. J. Willison, and P. R. Crocker Sialic Acid-Binding Immunoglobulin-Like Lectin 7 Mediates Selective Recognition of Sialylated Glycans Expressed on Campylobacter jejuni Lipooligosaccharides Infect. Immun., July 1, 2006; 74(7): 4133 - 4141. [Abstract] [Full Text] [PDF]
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J. Zhang, A. Raper, N. Sugita, R. Hingorani, M. Salio, M. J. Palmowski, V. Cerundolo, and P. R. Crocker Characterization of Siglec-H as a novel endocytic receptor expressed on murine plasmacytoid dendritic cell precursors Blood, May 1, 2006; 107(9): 3600 - 3608. [Abstract] [Full Text] [PDF]
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 C. Oetke, M. C. Vinson, C. Jones, and P. R. Crocker Sialoadhesin-Deficient Mice Exhibit Subtle Changes in B- and T-Cell Populations and Reduced Immunoglobulin M Levels Mol. Cell. Biol., February 15, 2006; 26(4): 1549 - 1557. [Abstract] [Full Text] [PDF]
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P. Tyrer, A. R. Foxwell, A. W. Cripps, M. A. Apicella, and J. M. Kyd Microbial Pattern Recognition Receptors Mediate M-Cell Uptake of a Gram-Negative Bacterium Infect. Immun., January 1, 2006; 74(1): 625 - 631. [Abstract] [Full Text] [PDF]
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 A. Varki and T. Angata Siglecs--the major subfamily of I-type lectins Glycobiology, January 1, 2006; 16(1): 1R - 27R. [Abstract] [Full Text] [PDF]
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S. Kirchberger, O. Majdic, P. Steinberger, S. Bluml, K. Pfistershammer, G. Zlabinger, L. Deszcz, E. Kuechler, W. Knapp, and J. Stockl Human Rhinoviruses Inhibit the Accessory Function of Dendritic Cells by Inducing Sialoadhesin and B7-H1 Expression J. Immunol., July 15, 2005; 175(2): 1145 - 1152. [Abstract] [Full Text] [PDF]
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W.-H. Kwan, A.-M. Helt, C. Maranon, J.-B. Barbaroux, A. Hosmalin, E. Harris, W. H. Fridman, and C. G. F. Mueller Dendritic Cell Precursors Are Permissive to Dengue Virus and Human Immunodeficiency Virus Infection J. Virol., June 15, 2005; 79(12): 7291 - 7299. [Abstract] [Full Text] [PDF]
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P. L. Delputte and H. J. Nauwynck Porcine Arterivirus Infection of Alveolar Macrophages Is Mediated by Sialic Acid on the Virus J. Virol., August 1, 2004; 78(15): 8094 - 8101. [Abstract] [Full Text] [PDF]
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B. Slobedman, J. L. Stern, A. L. Cunningham, A. Abendroth, D. A. Abate, and E. S. Mocarski Impact of Human Cytomegalovirus Latent Infection on Myeloid Progenitor Cell Gene Expression J. Virol., April 15, 2004; 78(8): 4054 - 4062. [Abstract] [Full Text] [PDF]
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N. Vanderheijden, P. L. Delputte, H. W. Favoreel, J. Vandekerckhove, J. Van Damme, P. A. van Woensel, and H. J. Nauwynck Involvement of Sialoadhesin in Entry of Porcine Reproductive and Respiratory Syndrome Virus into Porcine Alveolar Macrophages J. Virol., August 1, 2003; 77(15): 8207 - 8215. [Abstract] [Full Text] [PDF]
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 E. H. Hughes, F. C. Schlichtenbrede, C. C. Murphy, G.-M. Sarra, P. J. Luthert, R. R. Ali, and A. D. Dick Generation of Activated Sialoadhesin-Positive Microglia during Retinal Degeneration Invest. Ophthalmol. Vis. Sci., May 1, 2003; 44(5): 2229 - 2234. [Abstract] [Full Text] [PDF]
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K. Nakamura, T. Yamaji, P. R. Crocker, A. Suzuki, and Y. Hashimoto Lymph node macrophages, but not spleen macrophages, express high levels of unmasked sialoadhesin: implication for the adhesive properties of macrophages in vivo Glycobiology, March 1, 2002; 12(3): 209 - 216. [Abstract] [Full Text] [PDF]
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C. Stahl-Hennig, R. M. Steinman, P. Ten Haaft, K. Uberla, N. Stolte, S. Saeland, K. Tenner-Racz, and P. Racz The Simian Immunodeficiency Virus {Delta}nef Vaccine, after Application to the Tonsils of Rhesus Macaques, Replicates Primarily within CD4+ T Cells and Elicits a Local Perforin-Positive CD8+ T-Cell Response J. Virol., January 15, 2002; 76(2): 688 - 696. [Abstract] [Full Text] [PDF]
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T. K. van den Berg, D. Nath, H. J. Ziltener, D. Vestweber, M. Fukuda, I. van Die, and P. R. Crocker Cutting Edge: CD43 Functions as a T Cell Counterreceptor for the Macrophage Adhesion Receptor Sialoadhesin (Siglec-1) J. Immunol., March 15, 2001; 166(6): 3637 - 3640. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
indicates hSn(d1-4)Fc, and
comparisons were made with CD22-Fc (
) that binds specifically to
2,6-PAA and NCAM-Fc (
), a control protein that does not mediate
sialic acid binding. Data show mean absorbance values ± range of
duplicates and are representative of 2 experiments performed.
A Practical Approach. Oxford, England: IRL Press; 1993:93-128.