Journal of Leukocyte Biology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ahn, J. S.
Right arrow Articles by Davis, W. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ahn, J. S.
Right arrow Articles by Davis, W. C.
(Journal of Leukocyte Biology. 2002;72:382-390.)
© 2002 by Society for Leukocyte Biology

Scavenger receptor cysteine-rich domains 9 and 11 of WC1 are receptors for the WC1 counter receptor

J. S. Ahn*, A. Konno{dagger}, J. A. Gebe{ddagger}, A. Aruffo§, M. J. Hamilton*, Y. H. Park|| and W. C. Davis*

* Department of Veterinary Microbiology and Pathology, Washington State University, Pullman;
{dagger} Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan;
{ddagger} Benoaroya Research Institute, Virginia Mason Research Center, Seattle, Washington;
§ Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey; and
|| Department of Veterinary Microbiology, Seoul National University, Korea

Correspondence: W. C. Davis, Department of Veterinary Microbiology and Pathology College of Veterinary Medicine, Bustad Hall, Room 326, Washington State University, Pullman, WA 99164-7040. E-mail: davisw{at}mail.vetmed.wsu.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Workshop cluster 1 (WC1) is a member of the scavenger receptor cysteine-rich (SRCR) superfamily that includes CD5, CD6, CD163, and M160. Bovine WC1 consists of 11 SRCR domains, a unique domain 1, and two homologous 5 SRCR domain cassettes, WC1 domains 2–6 and 7–11. The porcine orthologue of WC1 contains five SRCR domains with a different domain arrangement. Although the function of WC1 is unknown, WC1 is proposed to be an accessory or homing molecule. Thus, identification of cells that express the counter receptor for WC1 (WC1-CR) is critical to understanding the function of WC1. For this reason, we constructed WC1-human immunoglobulin G1 fusion proteins to identify the binding domain of WC1 and cells that express the WC1-CR. Immunohistochemical analysis revealed WC1 domains 9 and 11 bind cells with macrophage and dendritic cell morphology and cells in ellipsoids in the spleen. These results and the finding of conserved signaling motifs in the cytoplasmic tail suggest WC1 may be an accessory molecule.

Key Words: bovine • {gamma}{delta} T lymphocytes • cell surface molecule • immunohistochemistry


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In most species examined thus far, {gamma}{delta} T lymphocytes comprise only a small proportion (<10%) of T lymphocytes in peripheral blood [1 ,2 ]. Their proportion in epithelial tissue at portals of entry of infectious agents is varied. Their early appearance and accumulation at sites of infection have suggested they play a role in the first line of defense as effector cells and, potentially, a role in the regulation of immune responses restricted by the major histocompatibility complex (MHC) mediated through {alpha}ß T cells [2 ,3 ]. The majority of {gamma}{delta} T cells express CD2, CD3, and CD5. Subsets express CD4 or CD8 [4 ]. Limited information has been obtained on expression of accessory molecules that provide second signals during activation of {gamma}{delta} T cells. During ontogeny, waves of {gamma}{delta} T cells develop with restricted use of V{gamma} segments and C{gamma} chains. In mice, such cells home to specific tissues [1 ,2 ]. Patterns of homing are less clear in humans, and it appears use of V{gamma} and C{gamma} chains differs from mice. In contrast to humans and mice, studies in Artiodactyla (mainly in cattle, sheep, goats, and pigs) have shown {gamma}{delta} T cells may comprise 50% or more of T cells in peripheral blood [5 6 7 ]. Proportions comparable to those in humans and mice are present in secondary lymphoid organs, except in the spleen, where they may comprise 35% or more of T lymphocytes present in the spleen [8 9 10 ]. They also vary in relative proportion of T lymphocytes in epithelial tissues. It is now clear that the noted differences are attributable to the presence of a unique subpopulation of {gamma}{delta} T lymphocytes, which is phenotypically distinct. It is comprised of subsets that express CD3, CD5, and lineage-restricted molecules, workshop cluster 1 (WC1), and GD3.5 [11 ,12 ]. A comparable population in pigs expresses the orthologue of WC1 (referred to as PoWC1 in this report) and lineage-restricted molecules, swine workshop cluster-defined molecules SWC4, SWC5, and SWC6 [13 ]. The relationship of these molecules to GD3.5 is unknown. Peripheral blood WC1+ {gamma}{delta} T cells in each species do not express CD2, CD6, CD4, or CD8. The subpopulation referred to throughout the text as WC1+ {gamma}{delta} T lymphocytes accounts for the large population of {gamma}{delta} T lymphocytes in peripheral blood [12 ]. A WC1- subpopulation, similar in phenotype and tissue distribution to {gamma}{delta} T lymphocytes in humans and mice, is low in frequency in peripheral blood and most secondary lymphoid organs. However, it accounts for the large proportion of {gamma}{delta} T lymphocytes in spleen. The WC1- {gamma}{delta} T lymphocytes express CD2, CD3, CD5, and CD6. A subset coexpresses CD8 [9 ]. They do not express GD3.5. In addition to the difference in phenotype and tissue distribution, the WC1+ {gamma}{delta} T lymphocytes also differ in V{gamma} and J{gamma} segment and C{gamma} chain usage [14 ].

Limited information is available on the ontogeny and functional relation of WC1+ and WC1- cells in host defense. No information is available that accounts for the evolution of this unique population of {gamma}{delta} T cells. Although DNA sequences related to WC1 have been identified in humans and other species, {gamma}{delta} T cells that express orthologues of WC1 have only been identified in suborders of Artiodactyla (Ruminantia, Suiformes, and Tylopoda) [12 ,15 16 17 18 19 ]. The function of WC1 is unknown. However, it has been suggested that it might function as a coreceptor similar to CD4 and CD8 or as a homing receptor [20 ]. WC1 is a member of the scavenger receptor cysteine-rich (SRCR) superfamily of molecules that includes CD5, CD6, CD163, and M160 [21 ,22 ]. The extracellular portions of CD5 and CD6 consist of three SRCR domains. CD163 and M160 consist of 9 and 12 domains, respectively [22 ,23 ]. Bovine WC1 (BoWC1) and the ovine orthologues have 11 SRCR domains, and the porcine orthologue of WC1 has 5 domains [15 ,24 ,25 ]. Comparative analysis has suggested that the difference in size between the ruminant and porcine molecules is attributable to an internal duplication of a five-member cassette of SRCR domains in the ruminant form of WC1 [23 ]. Multiple copies of the gene are present in the genomes of cattle and sheep [24 ] and may be present in pig [25 ]. At least two monoclonal antibody-defined isoforms of WC1 are expressed on mutually exclusive subsets of {gamma}{delta} T lymphocytes, WC1-N3 and WC1-N4 [9 ,16 ]. Two genes, WC1.1 and WC1.2, have been expressed in fibroblasts that encode for isoforms that express WC1-N3 (WC1.2) or WC1-N4 (WC1.1) [9 ,26 ]. To gain further insight into the potential function of WC1, we constructed WC1-human immunoglobulin G1 (IgG1; hIg) fusion proteins for use in immunohistochemistry to determine which domains of WC1 bind the WC1-counter receptor (CR) and which cells express the WC1-CR. We compared the SRCR domain sequences in BoWC1 with the sequences in the porcine orthologue to determine if we could predict which porcine domain contains receptor activity. In addition, we identified and compared motifs in the cytoplasmic tails of the respective molecules that might be involved in signaling.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals, tissues, and thymocyte preparation
The tissues used in this study were obtained from four healthy Holstein Frisian calves at necropsy. Tissue samples were collected from thymus, popliteal lymph node, spleen, lung, ileum, liver, and skin. The tissues were embedded in OCT compound (Miles Laboratories, Elkhart, IN) and then snap frozen in liquid nitrogen.

cDNA synthesis and polymerase chain reaction (PCR) amplification of WC1
The cell line NBC13 was used as a source of mRNA to construct WC1-hIgG1 (WC1.hIg) fusion plasmids [27 ]. mRNA was isolated with Trizol (Gibco-BRL, Grand Island, NY) and oligo-dT cellulose columns. cDNA was prepared with 4 µl oligo-dT15 (0.12 mg/ml), 5 µl diethylpyrocarbonate water, 2 µl poly(A) RNA (0.4 mg/ml), 4 µl 5 x room temperature (RT) buffer, 2 µl dNTPs (10 mM), 1 µl RNasin (40 units/µl; Promega, Madison, WI), and 2 µl M-multilamellar vesicales RT (200 units/µl; Gibco-BRL). This mixture was incubated at 42°C for 60 min. PCR was performed in 100 µl solution containing 50 mM KCl, 10 mM Tris-HCl (pH 8.4), 1.5 mM MgCl2, 0.01% gelatin, 100 µM dNTPs, 15 pmole PCR primers, and 2.5 units Taq polymerase (Gibco-BRL). As WC1.D1-3hIg was available [28 ], only WC1 D4–D11 were amplified by PCR and used to make fusion proteins. WC1 D4–D8 were amplified with forward primer WC1.D4F2 and reverse primer WC1.D8R2 (Table 1 ). This PCR product was reamplified with WC1.D4hIgF and WC1.D8hIgR, WC1.D4hIgF and WC1.D4hIgR, WC1.D5hIgF and WC1.D5hIgR, WC1.D6hIgF and WC1.D6hIgR, and WC1.D7hIgF and WC1.D8hIgR to construct WC1.D4-8hIg, WC1.D4hIg, WC1.D5hIg, WC1.D6hIg, and WC1.D7-8hIg fusion proteins, respectively. WC1 D9–D11 were amplified with WC1.D9hIgF and WC1.D11hIgR to construct WC1.D9–11hIg. The PCR product was reamplified with WC1.D10hIgF and WC1.D11hIgR2, WC1.D9hIgF and WC1.D9hIgR, WC1.D10hIgF and WC1D.10hIgR, and WC1.D11hIgF and WC1.D11hIgR2 to construct WC1.D10-11hIg, WC1.D9hIg, WC1.D10hIg, and WC1.D11hIg fusion proteins, respectively. Every reverse primer except WC1.D8hIgR had the same TC ACC TGC CTC TTC CTT TTT AGC CTC TTC CTT TTT TCC anchor sequence to remove possible steric hindrance effects of WC1 domains on the hIg fusion cassette. As the WC1.D8hIgR primer site is located within the 35 amino acid (aa) spacer between D8 and D9, no additional anchor sequence was needed. The design of the reverse primer WC1.D11hIgR2 was based on the anchor sequences of WC1.D11hIgR (Table 1) . Each PCR cycle was repeated 25 times with appropriate PCR conditions. Every PCR product was cloned into PCR2.1 (Invitrogen, Carlsbad, CA) and sequenced. Sequenced WC1 domains were subcloned into plasmid pCDM7b- containing the hIg fusion cassette [29 ].


View this table:
[in this window]
[in a new window]
  		Table 1. PCR Primers Used to Construct WC1 and Human IgG1 Fusion Proteins

 
Transfection of COS1 cells with WC1.hIg fusion plasmids
WC1.hIg fusion plasmids were transformed into Escherichia coli DH5{alpha} by electroporation with a Cell-Porator (Gibco-BRL) and purified with a plasmid purification kit (Qiagen, Valencia, CA). At the time of use, 20 µg plasmid was added to 24 ml Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% heat-inactivated Nu serum (Becton Dickinson Labware, Franklin Lakes, NJ), penicillin (100 units/ml), streptomycin (100 µg/ml), and 4 mM L-glutamine. Then, 1 ml prewarmed 25x transfection solution [diethylaminoethyl/dextran 1 g, chloroquine diphosphate 0.129 g, in 100 ml phosphate-buffered saline (PBS)] was added and mixed well.

COS1 cells were cultured in 135 ml tissue culture flasks. When the cultures became 60–70% confluent, old medium was removed and, following washing with PBS, replaced with 25 ml transfection cocktail. The flasks were incubated at 37°C for 3–4 h. For a final shock, the transfection cocktail was aspirated off and replaced with 10% dimethyl sulfoxide in PBS for 2 min; then, shock solution was replaced with 25 ml DMEM supplemented with 5% fetal bovine serum and 4 mM L-glutamine. The cultures were incubated overnight at 37°C. The next day, medium was removed, and the cells were rinsed with PBS three to four times. Serum free DMEM (25 ml) supplemented with 4 mM L-glutamine was added. On day 10, supernatant was harvested and centrifuged at 4000 rpm for 10 min. The supernatant was filtered through a sterile 0.2 µm filter and refrigerated until processed.

Purification of WC1.hIg fusion proteins
Immobilized protein A-sepharose (Repligen, Needham, MA) was used to purify WC1.hIg fusion proteins. The presence of fusion protein was confirmed by dot blot using goat anti-hIg. Final protein concentration was determined by UV spectrophotometry.

Immunohistochemistry
Thick cryostat sections (8 µm) were prepared from thymus, popliteal lymph node, spleen, ileal Peyer’s patch, skin, lung, and liver. The sections were mounted on organosilane (Sigma Chemical Co., St. Louis, MO) -coated glass slides and fixed in ice-cold absolute ethanol for 10 min. To block nonspecific binding, the tissue sections were incubated with 5–10% normal goat serum in PBS for 30 min at room temperature. The tissue sections were incubated with WC1.hIg fusion proteins (5 µg/ml) for 1 h at 37°C and then rinsed with PBS. Tissue sections were incubated with biotin-labeled goat anti-hIg (Caltag Laboratories, Burlingame, CA) for 30 min and rinsed with PBS. The ABC kit (Vector Laboratories, Burlingame, CA) and diaminobenzidine were used for labeling.

Monoclonal antibodies (mAb)
The following mAb were used in immunohistochemistry: BAQ4A (IgG1, specific for a determinant, WC1-N2, expressed on the majority of WC1 isoforms), DH59B (IgG1, specific for SWC3, a molecule expressed on monocytes, macrophages, and dendritic cells), CAM36A (IgG1, specific for CD14), and TH14B (IgG2a, specific for the bovine orthologue of MHC class II DR{alpha} chain) [8 ,9 ,16 ,26 ,30 31 32 33 ].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
aa Sequence comparison of WC1 SRCR domains
Analysis of CD6 binding to cells using SRCR domain-Ig fusion proteins has shown that the membrane-proximal SRCR domain binds the CR CD166 [34 ]. Initial analysis of the WC1 DNA sequences indicated that an internal duplication of SRCR domains had occurred, because the homology of D1–D5 (394–1776 bp) and D6–D10 (2059–3441 bp) was 85% [15 ]. However, based on the aa sequence homology of SRCR domains and the location of conserved spacers, it was later proposed that WC1 D2–D6 and D7–D11 were the duplicated domains [23 ]. In addition, WC1 D4, D6, D9, and D11 were grouped together based on homology. To determine the exact homology between the SRCR domains of WC1 and to predict which domain(s) binds the WC1-CR pairwise, aa comparisons of the domains were performed. There was homology of greater than 86% between D2 and D7, D3 and D8, D4 and D9, and D5 and D10 and greater than 80% between D4, D6, and D9; however, less than 60% between D4, D6, D9, and D11 (Table 2 ). This comparison revealed that WC1 D11 is different from D4, D6, and D9.


View this table:
[in this window]
[in a new window]
  		Table 2. Pairwise Sequence Alignment of WC1 Domains at the Amino Acid Level

 
Further evidence of the uniqueness of WC1 D11 was obtained by comparison with PoWC1, which contains only five SRCR domains. Comparing pairs of aa sequences demonstrated greater than 58% sequence identity between WC1 D1 and PoWC1 D1; WC1 D2, D7, and PoWC1 D2; WC1 D3, D8, and PoWC1 D3; WC1 D5, D10, and PoWC1 D4; and WC1 D4, D6, D9, and PoWC1 D5 (Table 3 ). Interestingly, the comparison revealed PoWC1 D5 has 92% sequence identity with WC1 D11 but less than 60% with WC1 D4, D5, and D6.


View this table:
[in this window]
[in a new window]
  		Table 3. Pairwise Sequence Alignment of WC1 and PoWC1 Domains at the Amino Acid Level

 
Comparison of cytoplasmic tails of WC1, ovine WC1 (OvWC1), and PoWC1
Although studies to determine the function of WC1 have yielded contradictory results, probably due to different experimental conditions and the use of mAb specific for different epitopes on WC1, cumulative studies have suggested that WC1 may be associated with cell signaling. In the presence of secondary stimulation induced by anti-CD3 or anti-CD5 mAb, anti-WC1 mAb, IL-A29 enhanced the proliferation of WC1+ {gamma}{delta} T lymphocytes [35 ]. However, without secondary stimulation, anti-WC1 mAb, SC-29, induced reversible growth arrest and activated, multiple protein tyrosine phosphatases in IL-2-dependent cell lines of WC1+ {gamma}{delta} T lymphocytes [36 ,37 ]. To gain further insight that might explain these contrasting results, we examined the cytoplasmic tail of WC1 for conserved signaling motifs that might indicate the potential function of WC1. The aa sequence of the cytoplasmic tail of WC1 was compared with the cytoplasmic tail of other known molecules using the Baylor College of Medicine (BCM) search launcher, Multiple Sequence Alignment program "ClustalW 1.8," provided by BCM Human Genome Sequencing Center (http://searchlauncher.bcm.tmc.edu/multi-align/multi-align.html). Although SRCR domains of WC1 are homologous to those of other SRCR superfamily members, comparative examination revealed only the cytoplasmic tails of OvWC1 and PoWC1 are closely related to WC1. Amino acid sequence alignment of the cytoplasmic tails of WC1, OvWC1, and PoWC1 revealed the presence of several highly conserved motifs for signal transduction such as protein kinase C phosphorylation site S/T-X-K/R [38 ], casein kinase II phosphorylation site S/T-X-X-D/E [39 ], immunoreceptor tyrosine-based activation mofit (ITAM)-like Y-X-X-I/L [40 ], and immunoreceptor tyrosine-based inhibitory motif (ITIM)-like Y-X-X-L/V [41 ]. WC1, OvWC1, and PoWC1 also have a Y-X-X-A motif that is a potential site for interaction with SH2 domain-containing proteins [42 ] (Fig. 1 ).



View larger version (45K):
[in this window]
[in a new window]
  		Figure 1. ClustalW multiple sequence alignment of cytoplasmic tails of BoWC1 (GenBank accession no. X63723), OvWC1 (GenBank accession no. S76313), and PoWC1 (GenBank accession no. X99336). WC1 and the orthologues have highly conserved signal motifs S/T-X-K/R, S/T-X-X-D/E, Y-X-X-I/L, and Y-X-X-L/V.

 
Identification of WC1 domains that bind the WC1-CR
As the SRCR domains of WC1 were duplicated in the course of evolution, at least one domain in the five-domain cassette and its homologous domains in the duplicated cassette were expected to potentially bind the WC1-CR. As WC1.D1-3hIg was available [28 ], a fusion protein containing the membrane proximal domains D9–D11 was constructed. These two fusion proteins were examined first to see if they contained the WC1-CR-binding domains, as WC1 domains D2–D3 are homologous with D7–D8, and D4–D6 are homologous with D9–D11. The fusion protein cassette containing no insert was used as a control in the initial studies. Preliminary studies revealed that WC1.D1-3hIg caused weak background labeling similar to labeling caused by the control fusion protein that only contained the empty hIg cassette (Fig. 2a ), whereas WC1.D9–11hIg yielded strong, specific labeling of a restricted number of cells in the lymph node in the subcapsular and medullary sinuses (Fig. 2b) . A few widely distributed, positive cells were also found in the cortex. Additional fusion proteins were constructed to identify which domains of WC1 bound the WC1-CR. Immunohistochemical analysis showed specific labeling with WC1.D10-11hIg (not shown), WC1.D9hIg (Fig. 2c) , and WC1.D11hIg (Fig. 2d) , but not with WC1.D10hIg (not shown). As both WC1 D9 and D11 bound the WC1-CR, other fusion proteins were constructed to determine whether internal, homologous domains also bind the WC1-CR. None of the fusion proteins, including WC1.D4hIg, WC1.D5hIg, WC1.D6hIg, WC1D7-8hIg, and WC1.D4-8hIg, showed specific labeling (not shown). Based on these data, the WC1.D9–11hIg fusion protein was used in subsequent immunohistochemistry experiments. Because of the expense of color reproductions, only selected examples of labeling of tissue are presented to show the patterns or reactivity of WC1 fusion protein and mAb with cells in different tissues.



View larger version (124K):
[in this window]
[in a new window]
  		Figure 2. Sections of calf lymph node labeled with (a) WC1.D1–3hIg x 6.3, (b) WC1.D9–11hIg x 6.3, (c) WC1.D9hIg x 80, and (d) WC1.D11hIg x 80. (a) Little or nominal background labeling was observed with WC1.D1–3hIg and the empty hIg fusion cassette. (b) Strong labeling of cells in the subcapsular and medullary sinuses was observed following reaction with WC1.D9–11hIg. WC1.D9–11hIg also labeled cells widely distributed in the cortex. Equally strong labeling was obtained in the same areas with WC1.D9hIg (c) and WC1.D11hIg (d).

 
Tissue distribution of WC1-CR-expressing cells
The distribution of cells binding WC1.D9–11hIg in lymphoid organs was evaluated. In the thymus, WC1.D9–11hIg+ cells were sparsely distributed in the cortex and medulla, but densely distributed at the junction of the cortex and medulla (Fig. 3a ). In lymph node, WC1.D9–11hIg bound cells lining the subcapsular sinus (Fig. 3b) and cells lining the intermediate and medullary sinuses and adjacent areas (Fig. 3c) . In spleen, WC1.D9–11hIg bound strongly to reticular cells in the ellipsoids (sheathed capillaries) (Fig. 3d) . WC1.D9–11hIg binding cells were also seen in the marginal zone and at the junction of the marginal zone and red pulp (not shown). In the distal ileum, WC1.D9–11hIg bound to cells in the interfollicular spaces of Peyer’s patches in the submucosa (Fig. 3e) and to rare cells in the intestinal mucosa (not shown).



View larger version (91K):
[in this window]
[in a new window]
  		Figure 3. Sections of (a) calf thymus, (b) lymph node x 80, (c) spleen x 40, (d) ileum x 80, (e) skin x 80, and (f) lung x 40 labeled with WC1.D9–11hIg. High power magnification showing labeling of cells in the thymus present at the junction of the cortex and medulla (a, arrows) of subcapsular sinus of lymph node (b, arrow) and medullary sinuses of lymph node (c, arrow). High power magnification showing labeling of cells in the capillary sheath of spleen (d, arrow), cells in the space between lymphoid follicles in the submucosa of Peyer’s patches in the ileum (e, arrow), the papillary and reticular dermis of skin (f, arrows), and the interstitium of alveoli in the lung (g, arrow).

 
Cells in nonlymphoid tissues were also examined for reactivity with WC1.D9–11hIg. In the skin, WC1.D9–11hIg+ cells were sparsely distributed in the papillary dermis and more frequently distributed in the reticular dermis, but not present in the epidermis (Fig. 3f) . In the liver, WC1.D9–11hIg bound cells lining sinusoids, presumably Kupffer cells (not shown). In the lung, WC1.D9–11hIg+, cells were present in the lamina propria of the bronchus, bronchioles, and interstitium of alveoli (Fig. 3g) .

Identification of cells that express WC1-CR
WC1.D9–11hIg fusion protein and mAb to cell surface markers were used in immunohistochemistry to more clearly establish which cells express the WC1-CR: DH59B (monocytes, macrophages, and dendritic cells), CAM36A (monocytes and macrophages), and TH14B (B lymphocytes, monocytes, macrophages, and dendritic cells). TH14B exhibited a broad pattern of reactivity that overlapped the pattern of reactivity observed with the fusion protein (not shown), and DH59B and CAM36A exhibited more restricted patterns of reactivity similar to the pattern of labeling obtained with the fusion protein. The pattern of reactivity of DH59B closely approximated the pattern of labeling with the fusion protein, and CAM36A approximated the pattern of reactivity only in areas of tissue containing macrophages. In thymus, DH59B and CAM36A labeled cells in the same areas labeled with WC1.D9–11hIg (Fig. 4a ). In lymph node, DH59B labeled cells lining the sinuses in a pattern similar to that obtained with WC1.D9–11hIg (compare Figs. 4b with 3b and Figs. 4c with 3c ). CAM36A did not react with cells lining the sinuses. Both mAb and the fusion protein yielded similar patterns of labeling in areas containing macrophages (Fig. 4d ; data shown only for CAM36A). In the spleen, the WC1.D9–11hIg+ reticular cells in the ellipsoid (Fig. 3d) were TH14B-, DH59B-, and CAM36A- (not shown). In the distal ileum, DH59B and CAM36A yielded a pattern of labeling similar to that obtained with WC1.D9–11hIg (not shown).



View larger version (117K):
[in this window]
[in a new window]
  		Figure 4. Sections of (a) thymus, (b and c) lymph node, (e) skin, and (f) lung labeled with DH59B x 80 (arrows). (d) Lymph node labeled with CAM36A x 80 (arrow). The pattern of labeling with DH59B (antimacrophage and dendritic cells) is similar to labeling with WC1.D9–11hIg in the thymus and the subcapsular and medullary sinuses, suggesting macrophages and dendritic are the cells that express the WC1-CR. The pattern of labeling with CAM36A (anti-CD14) is similar to the areas labeled with WC1.D9–11hIg (d). CD14 is only expressed on macrophages in this area. The pattern of labeling with DH59B overlaps areas labeled with WC1.D9–11hIg in the papillary and reticular dermis (e). DH59B also labeled cells in the epidermis that did not react with WC1.D9–11hIg. DH59B labeled cells in the interstitium of alveoli in same area as WC1.D9–11hIg (f). Cells labeled with CAM36A had a similar distribution (not shown). (a) c, Cortex; m, medulla.

 
In skin, DH59B yielded a pattern of labeling similar to the pattern obtained with WC1.D9–11hIg (compare Figs. 4e with 3f ) in the papillary and reticular dermis but not the epidermis. As mentioned previously, no fusion protein positive cells were seen in the epidermis. CAM36A bound cells only in the reticular dermis (not shown). In the liver, TH14B labeled cells in the sinusoids (Kupffer cells) similar to the pattern of labeling obtained with WC1.D9–11hIg (not shown). Cells in this area of the liver showed variable labeling with DH59B and CAM36A (not shown). In the lung, DH59B and CAM36A labeled cells in the bronchus, bronchioles, and interstitium of alveoli in a pattern similar to that obtained with WC1.D9–11hIg (compare Figs. 4f with 3g ; not shown for CAM36A).

Tissue distribution of WC1+ {gamma}{delta} T lymphocytes
To compare the distribution of cells that expressed the WC1-CR and WC1, Pan-WC1 mAb, BAQ4A, was used for labeling. Immunohistochemical analysis showed WC1+ {gamma}{delta} T lymphocytes were distributed in tissues as previously described [5 ,8 ,43 44 45 ]. These distributions were in the same general areas of tissue as WC1.D9–11hIg binding cells described above except for a major difference in the liver. WC1.D9—11hIg-labeled cells (presumably Kupffer cells) present in sinusoids; however, no WC1+ {gamma}{delta} T lymphocytes were detected in the liver. In the thymus, WC1+ {gamma}{delta} T lymphocytes were densely distributed in the medulla and at the junction of the medulla and cortex, but sparsely distributed in the cortex (Fig. 5a ). In the lymph node, WC1+ {gamma}{delta} T lymphocytes were present in the superficial cortex adjacent to the subcapsular sinus and sparsely distributed in the medullary sinuses (Fig. 5b and 5c) . In the spleen, WC1+ lymphocytes were present in the marginal zone (not shown). In the skin, numerous WC1+ lymphocytes were seen in the reticular dermis, but rarely seen in the epidermis and papillary dermis (Fig. 5d) . In the ileum, a few WC1+ lymphocytes were seen in the lamina propria (not shown). In the lung, WC1+ {gamma}{delta} T lymphocytes were sparsely scattered in the interstitium of alveoli and the lamina propria of bronchioles (Fig. 5e) .



View larger version (113K):
[in this window]
[in a new window]
  		Figure 5. (a) Thymus x 80, (b and c) lymph node x 40, (d) skin x 80, and (e) lung x 40 labeled with BAQ4A (anti-WC1). WC1+ cells are localized predominantly in the medulla of the thymus (a, arrow) and the superficial cortex adjacent to the subcapsular sinus in the lymph node (b, arrow) and are diffusely distributed in the medullary sinuses of the lymph node (c, arrow). WC1+ cells are present in the reticular dermis of skin (d, arrow). WC1+ cells are diffusely distributed in the interstitium of alveoli in the lung (e, arrow).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
WC1 is a highly glycosylated protein uniquely expressed on a subpopulation of {gamma}{delta} T lymphocytes in ruminants, pigs, and camelids. WC1 is a member of the SRCR superfamily that contains one or more copies of a highly conserved ~110 aa domain [15 ,16 ]. The SRCR superfamily can be divided into two subfamilies, A and B [21 ]. The domains of group A SRCR contain six cysteine residues, and group B, with some exceptions, contains eight cysteine residues. WC1 is a member of the group B SRCR superfamily, which includes CD5, CD6, CD163, and M160 [21 22 23 ]. WC1 contains 11 SRCR domains including two homologous 5 SRCR domain cassettes. Based on the aa sequence homologies of the 11 SRCR domains, WC1 was previously subgrouped as a(D1)-[b(D2)-c(D3)-d(D4)-e(D5)-d(D6)]-[b(D7)-c(D8)-d(D9)-e(D10)-d(D11)] [23 ]. It was suggested that the WC1 SRCR domains D4, D6, D9, and D11 are homologous and consequently can be grouped together. However, in this study, a comparison of the aa sequence identity of WC1 domains demonstrated that WC1 D11 is different from D4, D6, and D9. Thus, we propose that the SRCR domains of WC1 should be grouped as a-[b-c-d-e-d]-[b-c-d-e-D]. Comparing pairs of WC1 with PoWC1 provided further evidence of the uniqueness of WC1 D11. As shown in Table 3 and the dendogram (Fig. 6 ), WC1 D11 has the highest homology with PoWC1 D5 (92%), while WC1 D4, D6, and D9 have less than 60% homology with PoWC1 D5.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 6. Dendogram showing the relation of WC1 domains with the orthologous porcine WC1 domains. The PHYLIP program was used to generate the dendogram [49 ].

 
Based on previous studies of CD6 binding to its ligand [34 ] and an aa comparison of WC1 SRCR domains with those in CD6, we predicted WC1 domain 11 would bind to the putative WC1-CR. Immunohistochemical analysis revealed that WC1.D9hIg as well as WC1.D11hIg exhibited equally strong, specific binding to the WC1-CR, while fusion proteins containing the other WC1 domains did not. Based on the close homology with WC1 D9, D4 and D6 were expected to bind the WC1-CR also. However, neither D4 nor D6 fusion proteins bound to any of the tissues tested. As the N terminal half of SRCR domains are more conserved than the C-terminal half, it was proposed that the C terminal half determines the specificity of binding of these domains to their CRs. Site-directed, mutational studies with CD6 showed that a single aa substitution in the C terminus of the membrane proximal SRCR domain abolished binding of CD6 to CD166 [46 ]. Thus, differences in the aa sequence in the C terminal end of the domains may be the reason why WC1 D4 and D6 did not bind the WC1-CR.

Preliminary immunohistochemical studies showed WC1.D9–11hIg bound cells in pig lymph node tissue similar to binding in bovine tissue (not shown). Based on the homology between WC1 and PoWC1, PoWC1 D5 is predicted to bind the orthologue of the WC1-CR in pigs. Further studies on PoWC1 are needed to verify and extend these observations. Interestingly, studies in sheep indicate that CD4+ {alpha}ß T cells may also express a WC1 isoform that lacks D10 and D11 [47 ]. This suggests WC1 may be a member of a subfamily of the SRCR superfamily with different patterns of expression on lymphocyte subsets and may play a common role in the activation of T lymphocytes.

Immunohistochemical analysis of lymphoid and nonlymphoid tissues revealed that macrophages appear to be the predominant cell type expressing the WC1-CR. Macrophages were CAM36A+, TH14B+, and DH59B+ in the lymph node. WC1.D9–11hIg also bound to cells lining the sinus in the lymph node, suggesting that some dendritic cells may also express a receptor for WC1 [48 ]. These cells were DH59B+, TH14B+, and CAM36A-. A third nonlymphoid cell type in the ellipsoids of the spleen that expressed the WC1-CR was DH59B-, TH14B-, and CAM36A-. These observations indicate that macrophage lineage cells and some nonlymphoid cells express a receptor for WC1. Further studies are in progress to clone the gene encoding the WC1-CR and verify that these are the primary cell types that constitutively express the WC1-CR.

In conclusion, we have shown that membrane proximal SRCR domains D9 and D11 of WC1 contain receptor activity. The data show that WC1 D9 and D11 bind to a putative CR on cells with macrophage and dendritic cell morphology as well as nonlymphoid cells in spleen ellipsoids. Comparison of the distribution of WC1.hIg+ cells with the distribution of WC1+ {gamma}{delta} T cells revealed WC1.hIg positive cells were concentrated in the same general areas of tissue as WC1+ {gamma}{delta} T cells, except in the liver. These findings suggest that WC1 might serve as an accessory molecule involved in cell signaling and cell proliferation. The identification of conserved signaling motifs in the cytoplasmic tail of WC1 also supports this premise. The finding of ITAM- and ITIM-like motifs suggests WC1 could be involved in positive and negative signaling.


   ACKNOWLEDGEMENTS
 
This study was supported in part by grants from the United States Department of Agriculture-National Research Initiative Competitive Grants Program (no. 98-02-02480), the College of Veterinary Medicine Animal Health Research Center intramural grant (no. WNV-00138), Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture, Japan (no. 11760199), and the Washington State Monoclonal Antibody Center.


   FOOTNOTES
 
J. S. A. and A. K. contributed equally to this study and should be considered as equal first authors.

Received December 17, 2001; revised March 14, 2002; accepted March 26, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Haas, W., Pereira, P., Tonegawa, S. (1993) Gamma/delta cells Annu. Rev. Immunol. 11,637-685[Medline]
  2. Born, W., Cady, C., Jones-Carson, J., Mukasa, A., Lahn, M., O’Brien, R. (1999) Immunoregulatory functions of gamma delta T cells Adv. Immunol. 71,77-144[Medline]
  3. Hayday, A. C. (2000) {gamma}{delta} Cells: a right time and a right place for a conserved third way of protection Annu. Rev. Immunol. 18,975-1026[Medline]
  4. Kaufmann, S. H. (1996) Gamma/delta and other unconventional T lymphocytes: what do they see and what do they do? Proc. Natl. Acad. Sci. USA 93,2272-2279[Abstract/Free Full Text]
  5. Mackay, C. R., Beya, M. F., Matzinger, P. (1989) Gamma/delta T cells express a unique surface molecule appearing late during thymic development Eur. J. Immunol. 19,1477-1483[Medline]
  6. Mackay, C. R., Hein, W. R. (1989) A large proportion of bovine T cells express the gamma delta T cell receptor and show a distinct tissue distribution and surface phenotype Int. Immunol. 1,540-545[Abstract/Free Full Text]
  7. Binns, R. M., Duncan, I. A., Powis, S. J., Hutchings, A., Butcher, G. W. (1992) Subsets of null and gamma delta T-cell receptor+ T lymphocytes in the blood of young pigs identified by specific monoclonal antibodies Immunology 77,219-227[Medline]
  8. MacHugh, N. D., Mburu, J. K., Carol, M. J., Wyatt, C. R., Orden, J. A., Davis, W. C. (1997) Identification of two distinct subsets of bovine gamma delta T cells with unique cell surface phenotype and tissue distribution Immunology 92,340-345[Medline]
  9. Davis, W. C., Brown, W. C., Hamilton, M. J., Wyatt, C. R., Orden, J. A., Khalid, A. M., Naessens, J. (1996) Analysis of monoclonal antibodies specific for the gamma delta TcR Vet. Immunol. Immunopathol. 52,275-283[Medline]
  10. Wilson, E., Walcheck, B., Davis, W. C., Jutila, M. A. (1998) Preferential tissue localization of bovine {gamma}{delta} T cell subsets defined by anti-T cell receptor for antigen antibodies Immunol. Lett. 64,39-44[Medline]
  11. Jones, W. M., Walcheck, B., Jutila, M. A. (1996) Generation of a new gamma delta T cell-specific monoclonal antibody (GD3.5). Biochemical comparisons of GD3.5 antigen with the previously described workshop cluster 1 (WC1) family J. Immunol. 156,3772-3779[Abstract]
  12. Morrison, W. I., Davis, W. C. (1991) Individual antigens of cattle. Differentiation antigens expressed predominantly on CD4- CD8- T lymphocytes (WC1, WC2) Vet. Immunol. Immunopathol. 27,71-76[Medline]
  13. Davis, W. C., Zuckermann, F. A., Hamilton, M. J., Barbosa, J. I., Saalmuller, A., Binns, R. M., Licence, S. T. (1998) Analysis of monoclonal antibodies that recognize gamma delta T/null cells Vet. Immunol. Immunopathol. 60,305-316[Medline]
  14. Hein, W. R., Dudler, L. (1997) TCR gamma delta+ cells are prominent in normal bovine skin and express a diverse repertoire of antigen receptors Immunology 91,58-64[Medline]
  15. Wijngaard, P. L., Metzelaar, M. J., MacHugh, N. D., Morrison, W. I., Clevers, H. C. (1992) Molecular characterization of the WC1 antigen expressed specifically on bovine CD4-CD8- gamma delta T lymphocytes J. Immunol. 149,3273-3277[Abstract]
  16. Wijngaard, P. L., MacHugh, N. D., Metzelaar, M. J., Romberg, S., Bensaid, A., Pepin, L., Davis, W. C., Clevers, H. C. (1994) Members of thenovel WC1 gene family are differentially expressed on subsets of bovine CD4-CD8- gamma delta T lymphocytes J. Immunol. 152,3476-3482[Abstract]
  17. Carr, M. M., Howard, C. J., Sopp, P., Manser, J. M., Parsons, K. R. (1994) Expression on porcine gamma delta lymphocytes of a phylogenetically conserved surface antigen previously restricted in expression to ruminant gamma delta T lymphocytes Immunology 81,36-40[Medline]
  18. Davis, W. C., Heirman, L. R., Hamilton, M. J., Parish, S. M., Barrington, G. M., Loftis, A., Rogers, M. (2000) Flow cytometric analysis of an immunodeficiency disorder affecting juvenile llamas Vet. Immunol. Immunopathol. 74,103-120[Medline]
  19. Zidan, M., Schuberth, H-J., Pabst, R. (2000) Immunohistology of the splenic compartments of the one humped camel (Camelus dromedarius) Vet. Immunol. Immunopathol. 74,17-29[Medline]
  20. Hein, W. R., Mackay, C. R. (1991) Prominence of gamma delta T cells in the ruminant immune system Immunol. Today 12,30-34[Medline]
  21. Aruffo, A., Bowen, M. A., Patel, D. D., Haynes, B. F., Starling, G. C., Gebe, J. A., Bajorath, J. (1997) CD6-ligand interactions: a paradigm for SRCR domain function? Immunol. Today 18,498-504[Medline]
  22. Grønlund, J., Vitved, L., Lausen, M., Skjødt, K., Holmskov, U. (2000) Cloning of a novel scavenger receptor cysteine-rich type I transmembrane molecule (M160) expressed by human macrophages J. Immunol. 165,6406-6415[Abstract/Free Full Text]
  23. Resnick, D., Pearson, A., Krieger, M. (1994) The SRCR superfamily: a family reminiscent of the Ig superfamily Trends Biochem. Sci. 19,5-8[Medline]
  24. Walker, I. D., Glew, M. D., O’Keeffe, M. A., Metcalfe, S. A., Clevers, H. C., Wijngaard, P. L., Adams, T. E., Hein, W. R. (1994) A novel multi-gene family of sheep gamma delta T cells Immunology 83,517-523[Medline]
  25. Kanan, J. H., Nayeem, N., Binns, R. M., Chain, B. M. (1997) Mechanisms for variability in a member of the scavenger-receptor cysteine-rich superfamily Immunogenetics 46,276-282[Medline]
  26. MacHugh, N. D., Wijngaard, P. L., Clevers, H. C., Davis, W. C. (1993) Clustering of monoclonal antibodies recognizing different members of the WC1 gene family Vet. Immunol. Immunopathol. 39,155-160[Medline]
  27. Ferrer, J. F., Stock, N. D., Lin, P. (1971) Detection of replicating C-type viruses in continuous cell cultures established from cows with leukemia: effect of the culture medium J. Natl. Cancer Inst. 47,613-621
  28. Gebe, J. A., Kiener, P. A., Ring, H. Z., Li, X., Francke, U., Aruffo, A. (1997) Molecular cloning, mapping to human chromosome 1 q21–q23, and cell binding characteristics of Spalpha, a new member of the scavenger receptor cysteine-rich (SRCR) family of proteins J. Biol. Chem. 272,6151-6158[Abstract/Free Full Text]
  29. Edwards, C. P., Aruffo, A. (1993) Current applications of COS cell based transient expression systems Curr. Opin. Biotechnol. 4,558-563[Medline]
  30. Ababou, A., Davis, W. C., Levy, D. (1993) The DA6-147 monoclonal antibody raised against the HLA-DR{alpha} chain identifies a cryptic epitope on the BoLA-DR{alpha} chain Ann. Rech. Vet. 24,402-407
  31. Sager, H., Davis, W., Dobbelaere, D., Jungi, T. (1997) Macrophage-parasite relationship in theileriosis. Reversible phenotypic and functional dedifferentiation of macrophages infected with Theileria annulata J. Leukoc. Biol. 61,459-468[Abstract]
  32. Howard, C. J., Morrison, W. I., Bensaid, A., Davis, W. C., Eskra, L., Gerdes, J., Hadam, M., Hurley, D., Leibold, W., Letesson, J-J., MacHugh, N., Naessens, J., O’Reilly, K. O., Parsons, K. R., Schlote, D., Sopp, P., Splitter, G., Wilson, R. (1991) Summary of workshop findings for leukocyte antigens of cattle Vet. Immunol. Immunopathol. 27,21-27[Medline]
  33. Alvarez, B., Sánchez, C., Bullido, R., Marina, A., Lunney, J., Alonso, F., Ezquerra, A., Domínguez, J. (2000) A porcine cell surface receptor identified by monoclonal antibodies to SWC3 is a member of the signal regulatory protein family and associates with protein-tyrosine phosphatase SHP-1 Tissue Antigens 55,342-351[Medline]
  34. Bowen, M. A., Bajorath, J., Siadak, A. W., Modrell, B., Malacko, A. R., Marquardt, H., Nadler, S. G., Aruffo, A. (1996) The amino-terminal immunoglobulin-like domain of activated leukocyte cell adhesion molecule binds specifically to the membrane-proximal scavenger receptor cysteine-rich domain of CD6 with a 1:1 stoichiometry J. Biol. Chem. 271,17390-17396[Abstract/Free Full Text]
  35. Hanby-Flarida, M. D., Trask, O. J., Yang, T. J., Baldwin, C. L. (1996) Modulation of WC1, a lineage-specific cell surface molecule of gamma/delta T cells augments cellular proliferation Immunology 88,116-123[Medline]
  36. Kirkham, P. A., Takamatsu, H. H., Parkhouse, R. M. (1997) Growth arrest of gamma delta T cells induced by monoclonal antibody against WC1 correlates with activation of multiple tyrosine phosphatases and dephosphorylation of MAP kinase erk2 Eur. J. Immunol. 27,717-725[Medline]
  37. Takamatsu, H. H., Kirkham, P. A., Parkhouse, R. M. (1997) A gamma delta T cell specific surface receptor (WC1) signaling G0/G1 cell cycle arrest Eur. J. Immunol. 27,105-110[Medline]
  38. Woodgett, J. R., Gould, K. L., Hunter, T. (1986) Substrate specificity of protein kinase C. Use of synthetic peptides corresponding to physiological sites as probes for substrate recognition requirements Eur. J. Biochem. 161,177-184[Medline]
  39. Pinna, L. A. (1990) Casein kinase 2: an "eminence grise" in cellular regulation? Biochim. Biophys. Acta 1054,267-284[Medline]
  40. Isakov, N. (1997) Immunoreceptor tyrosine-based activation motif (ITAM), a unique module linking antigen and Fc receptors to their signaling cascades J. Leukoc. Biol. 61,6-16[Abstract]
  41. Vivier, E., Daeron, M. (1997) Immunoreceptor tyrosine-based inhibition motifs Immunol. Today 18,286-291[Medline]
  42. Burkhardt, A. L., Costa, T., Misulovin, Z., Stealy, B., Bolen, J. B., Nussenzweig, M. C. (1994) Ig alpha and Ig beta are functionally homologous to the signaling proteins of the T-cell receptor Mol. Cell. Biol. 14,1095-1103[Abstract/Free Full Text]
  43. Clevers, H., MacHugh, N. D., Bensaid, A., Dunlap, S., Baldwin, C. L., Kaushal, A., Iams, K., Howard, C. J., Morrison, W. I. (1990) Identification of a bovine surface antigen uniquely expressed on CD4- CD8- T cell receptor gamma/delta+ T lymphocytes Eur. J. Immunol. 20,809-817[Medline]
  44. Doherty, M. L., Bassett, H. F., Quinn, P. J., Davis, W. C., Kelly, A. P., Monaghan, M. L. (1996) A sequential study of the bovine tuberculin reaction Immunology 87,9-14[Medline]
  45. Thomas, L. H., Cook, R. S., Howard, C. J., Gaddum, R. M., Taylor, G. (1996) Influence of selective T-lymphocyte depletion on the lung pathology of gnotobiotic calves and the distribution of different T-lymphocyte subsets following challenge with bovine respiratory syncytial virus Res. Vet. Sci. 61,38-44[Medline]
  46. Rose, T. M., Schultz, E. R., Henikoff, J. G., Pietrokovski, S., McCallum, C. M., Henikoff, S. (1998) Consensus-degenerate hybrid oligonucleotide primers for amplification of distantly related sequences Nucleic Acids Res. 26,1628-1635[Abstract/Free Full Text]
  47. Bodian, D. L., Skonier, J. E., Bowen, M. A., Neubauer, M., Siadak, A. W., Aruffo, A., Bajorath, J. (1997) Identification of residues in CD6 which are critical for ligand binding Biochemistry 36,2637-2641[Medline]
  48. O’Keeffe, M. A., Metcalfe, S. A., Cunningham, C. P., Walker, I. D. (1999) Sheep CD4(+) alphabeta T cells express novel members of the T19 multigene family Immunogenetics 49,45-55[Medline]
  49. Wacker, H-H., Frahm, S. O., Heidebrecht, H. J., Parwaresch, R. (1997) Sinus-lining cells of the lymph nodes recognized as dendritic cell type by the new monoclonal antibody Ki-M9 Am. J. Pathol. 151,423-434[Abstract]



This article has been cited by other articles:


Home page
 Vet PatholHome page
M. Y. Gulbahar, W. C. Davis, T. Guvenc, M. Yarim, U. Parlak, and Y. B. Kabak
Myocarditis Associated with Foot-and-Mouth Disease Virus Type O in Lambs
Vet. Pathol., September 1, 2007; 44(5): 589 - 599.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
B. O. Fabriek, M. M. J. Polfliet, R. P. M. Vloet, R. C. van der Schors, A. J. M. Ligtenberg, L. K. Weaver, C. Geest, K. Matsuno, S. K. Moestrup, C. D. Dijkstra, et al.
The macrophage CD163 surface glycoprotein is an erythroblast adhesion receptor
Blood, June 15, 2007; 109(12): 5223 - 5229.
[Abstract] [Full Text] [PDF]

Home page
 Eur Heart JHome page
G. De Visscher, I. Vranken, A. Lebacq, C. Van Kerrebroeck, J. Ganame, E. Verbeken, and W. Flameng
In vivo cellularization of a cross-linked matrix by intraperitoneal implantation: a new tool in heart valve tissue engineering
Eur. Heart J., June 1, 2007; 28(11): 1389 - 1396.
[Abstract] [Full Text] [PDF]

Home page
 Vet PatholHome page
M. Y. Gulbahar, W. C. Davis, H. Yuksel, and M. Cabalar
Immunohistochemical Evaluation of Inflammatory Infiltrate in the Skin and Lung of Lambs Naturally Infected with Sheeppox Virus
Vet. Pathol., January 1, 2006; 43(1): 67 - 75.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Madsen, H. J. Moller, M. J. Nielsen, C. Jacobsen, J. H. Graversen, T. van den Berg, and S. K. Moestrup
Molecular Characterization of the Haptoglobin{middle dot}Hemoglobin Receptor CD163: LIGAND BINDING PROPERTIES OF THE SCAVENGER RECEPTOR CYSTEINE-RICH DOMAIN REGION
J. Biol. Chem., December 3, 2004; 279(49): 51561 - 51567.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ahn, J. S.
Right arrow Articles by Davis, W. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ahn, J. S.
Right arrow Articles by Davis, W. C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS