HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Brooke, G. Articles by Barclay, A. N. Search for Related Content PubMed PubMed Citation Articles by Brooke, G. Articles by Barclay, A. N.

Human Lymphocytes Interact Directly with CD47 through a Novel Member of the Signal Regulatory Protein (SIRP) Family¹

Gary Brooke^*, Joanna D. Holbrook, Marion H. Brown^* and A. Neil Barclay^2,*

^* Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom; and GlaxoSmithKline UK Ltd., Uxbridge, United Kingdom

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Two closely related proteins, signal regulatory protein (SIRP; SHPS-1/CD172) and SIRP, have been described in humans. The existence of a third SIRP protein has been suggested by cDNA sequence only. We show that this third SIRP is a separate gene that is expressed as a protein with unique characteristics from both and genes and suggest that this gene should be termed SIRP. We have expressed the extracellular region of SIRP as a soluble protein and have shown that, like SIRP, it binds CD47, but with a lower affinity (K_d, 23 µM) compared with SIRP (K_d, 2 µM). mAbs specific to SIRP show that it was not expressed on myeloid cells, in contrast to SIRP and -, being expressed instead on the majority of T cells and a proportion of B cells. The short cytoplasmic tail of SIRP does not contain any known signaling motifs, nor does it contain a characteristic lysine, as with SIRP, that is required for DAP12 interaction. DAP12 coexpression is a requirement for SIRP surface expression, whereas SIRP is expressed in its absence. The SIRP-CD47 interaction may therefore not be capable of bidirectional signaling as with the SIRP-CD47, but, instead, use unidirectional signaling via CD47 only.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The expression of signal regulatory protein (SIRP)³ (CD172) in humans and its homologues in other mammals has been observed on myeloid, neuronal, and endothelial cells. SIRP has been cloned several times in different species leading to a variety of names, including SIRP, SHPS-1, MyD-1, P84/BIT, and MFR (1, 2, 3, 4, 5, 6). The role of SIRP is generally assumed to be an inhibitory one, mainly because of its interaction via ITIM motifs in its cytoplasmic tail with the Src homology 2 domain-containing phosphatase 1 (Shp1) and Shp2 protein tyrosine phosphatases (1, 2, 7, 8), and there are functional data supporting this (9). SIRP can also have negative effects on the expression of inflammatory cytokines, especially TNF- (10, 11). The extracellular ligand for SIRP is CD47, an unusual five-pass transmembrane protein with a single Ig-like domain (12, 13). CD47 itself has been ascribed a wide variety of functions and is ubiquitously expressed. It interacts in a cis manner with cell surface integrins and was originally termed integrin-associated protein (14, 15). There is evidence that it affects cell behavior through an interaction with heterotrimeric G proteins (16, 17). CD47 has been shown to have effects on integrins, migration, phagocytosis, IL-12R expression, and T cell activation and conversely on anergy or cell death (18, 19, 20, 21, 22, 23, 24, 25, 26). Surprisingly, considering its ubiquitous expression and the multiple effects that CD47 ligation can involve, CD47^–/– mice are viable and healthy. An obvious phenotype is only apparent when mice are shown to succumb to bacterial infection more quickly than their wild-type relatives. This seems to be due to defects in neutrophil migration (27). CD47 has also been postulated to act as a marker of self, as CD47^–/– RBC are rapidly phagocytosed when injected into wild-type mice (28).

The role of CD47/SIRP is further complicated by the finding that thrombospondin-1 has been shown to interact with CD47 (29) via the C-terminal region. However, many of the effects seen may also be mediated by thrombospondin-1 adhesion with integrins or simultaneous ligation with integrins and CD47 (30). It is therefore important to discover other protein interactions in this system and their affinities and tissue distribution. In contrast to SIRP, SIRP appears to exert activatory stimuli by virtue of interacting with the DAP12 adapter protein via a charged lysine in the SIRP transmembrane region. DAP12 is thought to function via the binding and activation of Src family kinases such as Syk through ITAM motifs (31). However, despite the sequence similarity between SIRP and SIRP, SIRP · Fc fusion proteins do not bind to CD47 expressed on the cell surface (32). A third SIRP-related protein has been suggested at the cDNA level (33). In this study we show that SIRP arises from a unique gene that, at the amino acid level, is approximately equally conserved to both SIRP and SIRP. However, although the expressed protein has a truncated cytoplasmic tail, it is unlike SIRP in that it does not require DAP12 for surface expression and binds to CD47. Specific mAb show that it also has different cellular distributions to SIRP and -. Taken together, we suggest that the protein be named SIRP to differentiate it from SIRP and SIRP, but still indicate that it belongs to the same closely related protein family. Finally, we also show that the differences in affinity of interaction with CD47 may control the functional outcome of an interaction between different SIRP and SIRP.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Construction, expression, and purification of human SIRP and SIRP · CD4 soluble fusion proteins

The sequence representing the three Ig-like domains that comprise the extracellular region of the human SIRP was amplified by PCR using human peripheral blood leukocytes cDNA as a template. The oligonucleotides ATGATTCAGCCTGAGAAG (sense) and TCAGGTCTTCTGCTTCCAG (antisense) were designed using the known SIRP sequence (EMBL accession no. AB042624). For soluble SIRP the antisense primer used was GTAGCATCTGAGCTCTGG. The products were blunt end ligated into pCR2.1 (Invitrogen Life Technologies, Carlsbad, CA). For chimeric soluble proteins, the SIRP sequence was ligated into a pEFBOS-XB expression vector containing rat CD4 Ig-like domains 3 and 4 (henceforth referred to as CD4) and a biotinylation motif (34, 35). For construction of surface-expressed SIRP bearing the FLAG epitope, the N terminus of the three extracellular Ig-like domains of SIRP was ligated (SalI-BamHI) into a CD4Lflag-pEFBOS construct (using a rat CD4 leader (CD4^L) ending ... VVTTQG, followed by the FLAG epitope DYKDDDDKST). Protein expression was detected with anti-FLAG-M2 mAb (Sigma-Aldrich, Poole, U.K.). For chimeric constructs containing additional leader peptides, either human SIRP (SIRP^L) or rat CD4^L sequences were ligated onto the 5' end of SIRP. The resulting SIRP^L chimera had the following sequence at the join site: EEELQMIQP (SIRP^L sequence underlined). To produce the SIRP^L/SIRP construct, the SIRP signal peptide was joined to SIRP using an endogenous SIRP PstI site (the PstI site was inserted into the SIRP sequence using PCR). Soluble recombinant proteins with the rat CD4 domains were purified by OX68 mAb affinity chromatography (34).

Characterization of protein interactions by surface plasmon resonance

Surface plasmon resonance measurements were obtained using a BIAcore 2000 biosensor instrument (BIAcore, Stevenage, Herts, U.K.) using CM5 research grade chips. The streptavidin was immobilized directly via amine coupling in 10 mM sodium acetate, pH 4.5. Equilibrium affinity and kinetic measurements were conducted in 10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20 at 37°C using short injection times of 3 s (5 µl at 100 µl/min) to minimize the contribution of any aggregated material. For equilibrium binding, immediately before the experiment, purified CD47 · CD4 was size fractionated to exclude any aggregated protein. Increasing and decreasing concentrations of monomeric CD47 · CD4 were passed over SIRP, SIRP, or control CD4 (all coated on chip at 1000 response units (RU)). For off-rate determinations, CD47 · CD4 (40 µM) was passed over immobilized SIRP · CD4 (at 1600 and 800 RU), or control CD4 (1600 RU). K_d values were obtained by both nonlinear curve fitting of the Langmuir binding isotherm and Scatchard transformations of the binding data (Origin software, OriginLab, Northampton, MA). k_off values were determined by fitting a first-order exponential decay curve to normalized data after subtraction of the negative control values.

Generation of mAb

Six-week-old male BALB/c mice were immunized s.c. with 10–20 µg of purified SIRP · CD4 in CFA and then with IFA. A mouse generating good immune responses to the immunogen was boosted, and 4 days later the spleen was removed, and hybridomas were generated using standard procedures by fusing with the NS-1 cell line. Hybridoma supernatants were initially screened by ELISA, using SIRP · CD4, human SIRP.Fc (13), human SIRP · Fc, or CD4 to eliminate hybridomas with cross-reactivity to rat CD4, human SIRP, and SIRP. The remaining hybridoma supernatants were screened for the ability to stain SIRP-transfected 293T cells. Four hybridomas recognizing SIRP were named OX116–OX119 and were recloned. All mAb were of the mouse IgG1 isotype.

Cells

PBMC were purified from blood of healthy volunteers on Ficoll-Paque density gradient (Amersham Biosciences, Arlington Heights, IL) and cultured in tissue culture medium (RPMI 1640, 10% FCS, 2 mM glutamine, 50 µM 2-ME, 100 U/ml penicillin, and 100 µg/ml streptomycin). 293T cells were transiently transfected with human SIRP · pEFBOS, human SIRP · pcDNA6 (gift from M. Colonna, Washington University School of Medicine, St. Louis, MO) and human FLAG-tagged DAP12 · pREP10 (gift from J. Sedgwick, DNAX, Palo Alto, CA) using FuGene transfection reagent (Roche, Indianapolis, IN) and the manufacturer’s protocol. Surface expression of transfected cells was analyzed after 48 h in culture.

Flow cytometric analyses

Cells were labeled by indirect immunofluorescence at 4°C in the presence of 10 mM sodium azide according to standard procedures and were analyzed by flow cytometry on a FACScan (BD Biosciences, Mountain View, CA). All mAb, unless otherwise stated, were obtained from BD Pharmingen (San Diego, CA) and were directly conjugated to FITC or PE. Annexin-FITC (Roche)/propidium iodide (Sigma-Aldrich) staining was performed according to the manufacturer’s protocol. Two-color staining was performed using biotinylated SIRP mAb OX116 or OX119 with streptavidin-PE and a second, directly FITC-conjugated mAb (CD3, CD4, CD8, CD19, and CD25). Isotype-matched biotinylated or fluorochrome-labeled mAb were used as controls. Generation of multivalent SIRP reagents using avidin-coated fluorescent beads (Sphero beads; Biotechnologie, www.kisker-biotech.com) and subsequent binding assays were previously described (36, 37). For lymphocyte activation, cells were resuspended in 200 µl of RPMI 1640, 10% FCS, and 50 µM 2-ME with antibiotics in flat-bottom, 96-well tissue culture plates at 10⁶/ml. At the indicated time points, the cells were frozen in 10% DMSO/90% FCS at –80°C. All samples were thawed and resuspended in PBS with azide before analysis as described above.

Apoptosis assay

Jurkat or U937 cells at 4 x 10⁵/well in 96-well plates were cultured for 3 h with the indicated mAb or fusion proteins bound to avidin-coated fluorescent beads (Sphero) in tissue culture medium. Ten microliters of beads (streptavidin-coated, preincubated with 20 µl of biotinylated fusion protein) or mAb at 5 µg/ml (final concentration) were added per well. The mAb used were control mAb (OX2), CD3 (mAb OKT3), CD47 (mAb 1796; Cymbus Biotechnology (Southampton, Hants, U.K.) or mAb MCA911 (Serotec, Oxford, U.K.)), and CD51/61 (BD Pharmingen). For blocking assays soluble CD47 · CD4 or CD4 alone as a control was added at 100 µg/ml before addition of beads. Cells were pelleted and washed in binding buffer (10 mM HEPES/NaOH (pH 7.4), 150 mM NaCl, 5 mM KCl, 1 mM MgCl₂, and 1.8 mM CaCl₂). Cells were then incubated with annexin-PE/7-aminoactinomycin D (7-AAD) according to the manufacturer’s protocol (BD Pharmingen) and analyzed on a FACScan (BD Biosciences, Mountain View, CA).

Immunoprecipitation

PBMC were isolated by Ficoll density gradient, surface-biotinylated, and lysed at 2 x 10⁷/ml in lysis buffer (150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1% Nonidet P-40, 1 mM PMSF, 50 mM benzamidine, 1 mg/ml leupeptin, 1 mg/ml aprotinin, and 1 mg/ml pepstatin) for 30 min, and insoluble material was spun down (13 krpm, 10 min). For all preclearing and immunoprecipitate steps, 10⁷ goat anti-mouse conjugated Dynal beads (Dynal Biotech, Great Neck, NJ) preincubated with 3 µg of mAb/2 x 10⁷ cells were used. After immunoprecipitation, Dynal beads were resuspended in reducing sample buffer by boiling for 5 min and loaded onto a 4–12% gradient polyacrylamide gel (NOVEX, San Diego, CA). After electrophoresis, the gel was blotted onto polyvinylidene difluoride, which was then blocked in 1% BSA before incubation with extra-avidin conjugated to peroxidase and developed with ECL reagents (Amersham Biosciences).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
SIRP is closely associated with other SIRP genes in the human genome

Analysis of the genomic sequence using the University of California Santa Cruz human genome browser (http://genome.ucsc.edu) showed that SIRP is present on chromosome 20p13 and has six possible exons (Table I). This also shows a predicted signal peptide that shows a high degree of homology with SIRP peptide (Fig. 1a). There are at least two transcript variants of SIRP shown by established sequence tags (ESTs). One form has exons 1–6 (variant 1), whereas the other has exons 1, 2, 5, and 6 (variant 2). Both variants 1 and 2 are predicted to have the same transmembrane domain. Variant 1 consists of an N-terminal, Ig-like V domain and two Ig-like C domains, whereas variant 2 only contains the N-terminal, Ig-like V domain (Fig. 1b). The existence of both forms (at least at the mRNA level) was confirmed using PCR on cDNA from PBLs (data not shown). However, transient expression of constructs containing variant 2 failed to produce surface protein, and immunoprecipitation data with available mAb (see below) did not indicate the presence of a protein corresponding to the predicted m.w. of variant 2. Therefore, surface expression of this protein by leukocytes seems unlikely. SIRP variant 1 is highly related to SIRP and SIRP, as shown by the comparison of amino acid sequences in Fig. 1a. Over the Ig-like regions, there is an equal level of conservation (79%). Very low levels of conservation were seen in the transmembrane regions and cytoplasmic tail with either SIRP or SIRP.

View larger version (57K): [in this window] [in a new window]   FIGURE 1. A, Alignment of amino acid sequences of human SIRP (SWISSPROT accession no. P78324), SIRP (SWISSPROT accession no. Q9P1W8), and SIRP (SWISSPROT accession no. O00241) using GCG (49 ) and GeneDoc software (http://psc.edu/biomed/genedoc/). B, Alignment of human SIRP long form with human SIRP short form (-v2, middle) and bovine SIRP (EMBL accession no. AJ563808). Conserved residues are indicated by dots, lack of sequence is shown by a dash, and predicted leader and transmembrane regions are underlined.

View larger version (26K): [in this window] [in a new window]   FIGURE 2. SIRP gene prediction and genomic localization. A, Diagram showing a map of the genes and approximate locations of the SIRP cluster on human chromosome 20p13. B, Diagramatic representation of the five members of the SIRP gene family in humans, showing predicted structural features (50 51 52 ). Also shown are the two possible splice variants of SIRP predicted from cDNA.

SIRP conservation in mammals

Human SIRP showed a high degree of conservation (78% amino acid identity) with a partial cDNA from another large mammal, cattle (Fig. 1b). Although encoding a stop codon, the cDNA does not encode a transmembrane region and may encode a soluble protein. Database searches for genomic and EST data have shown no evidence yet of SIRP in mice or rats.

Cloning and expression of human SIRP

The published SIRP sequence appeared truncated at the N terminus (amino acid sequence commenced MIQP..) (33). SIRP was amplified by PCR from leukocyte cDNA using primers based on this sequence, but protein could not be expressed (data not shown). A leader sequence from SIRP or CD4 was inserted, and SIRP was expressed both at the cell surface, as detected by the FLAG label, and with SIRP mAb B1D5 (gift from H. G. Buhring, University of Tubingen, Tubingen, Germany), and as a soluble chimeric construct with rat CD4 domains 3 and 4 (data not shown). Recently, a full-length protein sequence for SIRP has been described (SWISSPROT accession no. Q9P1W8) that confirmed that the original cDNA sequence lacked a leader peptide. This sequence also confirmed that the constructs we produced with the SIRP signal peptide did not confer any SIRP-specific amino acids to the predicted mature SIRP protein that could have affected the data.

Human SIRP is a CD47 ligand

Given the sequence similarity between SIRP and SIRP, we wanted to establish whether there is also a direct interaction between SIRP and CD47. The binding of purified soluble recombinant CD47 · CD4 to immobilized SIRP was analyzed using surface plasmon resonance with a BIAcore. Similar amounts of biotinylated SIRP · CD4, SIRP · CD4, SIRP · CD4, and control CD4 were immobilized on separate flow cells via binding to covalently attached streptavidin. Fig. 3 shows that SIRP, like SIRP, does bind to CD47, but the lower equilibrium binding indicates that it interacts with a lower affinity than SIRP. SIRP gave no signal above the control CD4 background level, indicating that it does not react with CD47, in agreement with cell binding data (32) (E. Vernon Wilson, D. Voulgaraki, M. H. Brown, A. N. Barclay, and G. Brooke, unpublished observations).

View larger version (15K): [in this window] [in a new window]   FIGURE 3. Affinity and dissociation rate of soluble human CD47 binding to human SIRP. A,- BIAcore trace showing CD47 · CD4 binding to SIRP · CD4 and SIRP · CD4, but not SIRP · CD4 or CD4 control, which were immobilized onto streptavidin-coated flow cells at 1000 RU. B, For affinity measurements at 37°C, biotinylated SIRP · CD4 (1600 RU) or CD4 control (1400 RU) were immobilized onto streptavidin-coated flow cells. The indicated concentrations of CD47 · CD4 were injected over SIRP · CD4 (solid line) or CD4 (dotted line). C, The difference between the response at equilibrium in the SIRP · CD4 and the control flow cell was plotted against the CD47 · CD4 concentration. A Kd of 23 µM was calculated by nonlinear curve fitting of the Langmuir isotherm (line) to data (•) from B with the negative control subtracted. The inset shows Scatchard plots of binding data for both SIRP (circles) and SIRP (squares), where a linear fit indicates monomeric binding. D, The dissociation of soluble CD47 · CD4 from SIRP · CD4 (squares; 1600 RU), SIRP · CD4 (diamonds; 1000 RU) or control CD4 (triangles; 1600 RU). The response in the control flow cell was subtracted from that in the SIRP cells for calculation of Koff values. The data were then normalized; thus, 100% represents the start of the dissociation phase, and first-order exponential decay curves were fitted to the data.

Expression of SIRP on human peripheral blood leukocytes

Recombinant soluble SIRP · CD4 protein was used to raise mAb in mice. The resulting mAb were screened against the three known SIRP proteins by ELISA. Four mAb (mAb OX116–OX119) were picked for further study by flow cytometry using transfected cells. None of the mAb stained untransfected 293T cells (data not shown). From the mAb obtained, only one, mAb OX119, appeared to be largely specific for SIRP (Fig. 4), although some marginal binding to SIRP transfectants was observed. The specificity of OX119 was confirmed by immunoprecipitation, because this showed a single immunoprecipitated band of 55 kDa (Fig. 4). The positive control mAb SE5A5 bound with a band of the expected size for SIRP and also a particularly prominent band corresponding to SIRP. Studies with transfectants confirmed that this mAb binds SIRP and SIRP, but not SIRP (Table II), although the prominence of the SIRP band was unexpected. Data produced from transfected cells with some of the mAb may not reflect the specificity of staining on ex vivo cells, because although mAb OX116 apparently stained SIRP-transfected 293T cells, it did not cross-react with SIRP by ELISA and did not stain the U937 cell line (that expresses SIRP and SIRP) or PBLs. It may be that alternative glycosylation by some cell lines (e.g., 293T) can alter the specificity of some mAb. Of the other mAb, OX117 cross-reacted with SIRP (SIRP/DAP12-transfected RBL cells were gifts from E. Tomasello and E. Vivier, Centre d’Immunologie de Marseille-Luminy, Marseille, France), and OX118 cross-reacted with SIRP (Fig. 4). A summary and comparison of SIRP mAb specificity is shown in Table II (other mAb were SE5A5 and B1D5 (32) and ILA24 (3)). The expression of SIRP was equally expressed in the presence or the absence of DAP12, in marked contrast to SIRP, which requires coexpression of DAP12 for surface expression (Fig. 4).

View larger version (45K): [in this window] [in a new window]   FIGURE 4. Specificity of SIRP mAb and staining on human peripheral blood leukocytes. A, Flow cytometric analysis of mAb OX116, OX117, OX118, and OX119 staining () or an isotype-matched control mAb () on SIRP- and SIRP-transfected 293T cells, SIRP/DAP12-transfected RBL cells and the U937 cell line. B, Immunoprecipitation of SIRP from human PBMC with mAb as indicated. C, Staining with the SIRP/SIRP-reacting mAb OX117 on 293T cells transfected with either SIRP or SIRP alone or transfected together with DAP12. D, For flow cytometry of human PBMC, granulocytes, monocytes, and lymphocytes were gated on the basis of forward and side scatter. Human peripheral blood granulocytes and monocytes from two individuals (i and ii) were stained with mAb OX119 or SE5A5 () or an isotype-matched control mAb (). Two-color analysis of lymphocyte gated cells are represented by dot plots with the indicated mAb. Quadrants were set with isotype-matched control mAb, and the percentage of events within the quadrant is indicated, apart from CD8, where only CD8high T cells were gated to avoid CD8low NK cells. E, Expression of SIRP on Con A-activated PBMC. Histograms show SIRP staining on CD25+-gated cells.

View this table: [in this window] [in a new window]   Table II. A comparison of SIRP mAb specificity between different human SIRP proteins based on staining of transfected 293T cells (SIRP and SIRP) or SIRP/DAP12-transfected RBL

Both SIRP and SIRP can induce a functional interaction through CD47

To show that SIRP can influence the behavior of CD47 on T cells, an apoptosis assay was used in a similar manner to that described by Manna and Frazier (40). To overcome the low affinity of the SIRP-CD47 interaction and achieve binding of recombinant proteins to cells, a high avidity system of biotinylated SIRP fusion protein bound to avidin-Sphero beads was used. These, or mAb (also bound to beads as a control), were incubated with Jurkat or U937 cells in tissue culture medium for 2–3 h at 37°C before levels of apoptosis were measured with annexin. As expected, the CD47 mAb (clone 1796; Cymbus Biotechnology) induced high levels of apoptosis. The results show that SIRP and SIRP induced apoptosis at much higher levels than with control beads in both Jurkat and U937 cells (Fig. 5a). The slight reduction in apoptosis induction by SIRP vs SIRP is consistent with lower affinity binding to CD47. Levels of apoptosis induction were comparable to the CD47 mAb itself (Fig. 5, b and c). The CD47 mAb MCA911-coated beads show that not all CD47 mAb are capable of inducing apoptosis as has been previously described (40). Abs that cross-linked the CD47-associated integrin CD51/61 had no effect on apoptosis, indicating a lack of direct involvement by CD47-associating integrins. The effect could also be specifically blocked by either excess soluble CD47 protein (CD4 chimera) or the OX119 mAb (Fig. 5c).

View larger version (35K): [in this window] [in a new window]   FIGURE 5. SIRP ligation of CD47 can induce apoptosis in Jurkat and U937 cells. a, Two-color flow cytometric analysis of cells stained with 7-AAD and annexin-PE after incubation with the indicated coated Sphero beads. b, Histograms show annexin-PE binding on Jurkat cells after incubation with Sphero beads coated with the indicated fusion protein (CD4, SIRP · CD4, or SIRP · CD4) or mAb (CD3, CD47 (1796 and MCA911), or CD51/61). The cells were gated to remove 7-AAD-stained dead cells. c, Assay to show specificity of effects of bead binding on Jurkat cells, using either soluble CD47 protein (CD4 as a control) or OX119 mAb (isotype-matched control mAb as control).

Activation of T cells modulates CD47 expression and, hence, SIRP/SIRP binding

Human PBMC were stimulated with Con A for up to 4 days. To ensure consistency in the results, cells were frozen at each time point, and all staining was conducted at the same time and with the same preparations of reagents. To measure binding of SIRP proteins with CD47, the high avidity system of avidin-coated, fluorescently labeled beads saturated with biotinylated SIRP · CD4, SIRP · CD4, or CD4 control was used. SIRP binding to PBMC was mediated solely by CD47, as a CD47 mAb blocked binding of the beads to the cells (Fig. 6a). The results presented in Fig. 6 show that PBMC at time zero had weak binding to SIRP or SIRP fluorescent beads despite expression of CD47. Upon Con A activation, PBMC expression of CD47 increased 2-fold until day 2 before falling to lower levels by day 4. Despite this modest increase in CD47 expression, binding with both SIRP and SIRP beads increased dramatically. However, SIRP binding decreased more quickly once CD47 levels started falling. Thus, the reduction in affinity between SIRP and SIRP does not alter the overall amount bound at optimum levels of CD47, but, instead, changes the threshold of binding at intermediate concentrations of CD47. Thus, a threshold of CD47 expression is required before SIRP beads can remain bound. Around the threshold concentration, small changes in CD47 lead to dramatic differences in SIRP binding, as shown in Fig. 6. The higher affinity SIRP interaction allows a lower threshold, leading to smaller changes in binding relative to the initial binding seen on day 0. These findings are in agreement with other low affinity cell surface molecular interactions, such as that between CD2 and CD48 (36).

View larger version (20K): [in this window] [in a new window]   FIGURE 6. SIRP and SIRP binding is sensitive to CD47 expression levels on T cells. A, Binding of SIRP is blocked by CD67 mAb. B, Human PBMC were stimulated with Con A for up to 4 days, and cells were frozen at each time point indicated to allow simultaneous bead and mAb binding assays. Histograms in the left column represent flow cytometric analysis of binding assays with avidin Sphero beads FITC-conjugated to biotinylated SIRP · CD4, SIRP · CD4, or CD4 control protein (as indicated) and PBMC at the time points indicated. Histograms in the right column show PBMC stained with mAb for CD47 or an isotype-matched control mAb at the time points indicated.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Of the known expressed SIRP genes, all have very different intracellular signaling potential. SIRP seems to signal via interaction of phosphotyrosines in cytoplasmic ITIM motifs with the Shp-1 and Shp-2 protein tyrosine phosphatases (2), whereas SIRP can impart signals via interaction with the DAP12 adapter (41), possibly through subsequent activation of Src family type tyrosine kinases. SIRP is unlikely to transmit a signal to the cytoplasm, because it has a negligible cytoplasmic tail with no tyrosines capable of being phosphorylated and no charged transmembrane amino acids capable of interacting with other transmembrane signaling proteins, such as DAP12.

SIRP was originally described as SIRP-b2 (33), which suggests that it is a subtype of the SIRP gene. We have termed it SIRP, because it is a separate gene product distinct from SIRP or SIRP. Analysis of the sequence from the mature expressed protein shows the same level of sequence identity between the genes, although the signal sequence of SIRP is more similar to that of SIRP. As SIRP is well conserved between mice and humans, it seems likely that duplication of an ancestral SIRP gene gave rise to a common CD47 binding precursor of SIRP and SIRP, followed by further duplication and divergence of SIRP into a non-CD47-binding protein, possibly driven by a viral pathogen. However, the low identity (57%) of the putative SIRP orthologue in mice (EMBL accession no. XM_283831) may indicate that it duplicated independently in this species, and the apparent lack of an SIRP gene indicates that mouse SIRP evolved directly from SIRP. The identity of a SIRP ligand remains unknown, but it is possible that it binds a pathogen, from the analogy with Ly49 in mice. Most mouse strains have an inhibitory Ly49 form, but some, in addition, have a closely related activatory form that can bind a CMV protein and confer resistance to this virus (42, 43).

The finding of two related proteins that bind the same ligand with differing affinities has also been described for other cell surface receptors. CD80 binds to both CD28 and CTLA-4 with K_d of 4 and 0.4 µM, respectively, and CD80 favors CTLA-4 engagement over CD28 (44, 45). It is also notable that the higher affinity interaction (CD80-CTLA4) is inhibitory, and this parallels the higher affinity SIRP-CD47 inhibitory interaction. The threshold effect observed with multivalent binding of SIRP to CD47 on activation of T cells implies that in vivo, resting T cells would not signal to each other via CD47 due to the low affinity/avidity of SIRP, even if cell-cell contact were taking place. However, activation, leading to increased CD47 expression, would allow for the SIRP-CD47 interaction to take place. This would be transient, and the interaction would be lost on chronically activated T cells when their CD47 levels decrease. This means that freshly activated CD47^high T cells may be on a "knife edge," where any extra signals through CD47 could lead to their destruction and uptake by neighboring myeloid cells (46, 47). Indeed, ligation of CD47 has been associated with apoptosis and uptake of cells in some experimental systems (48). This functional consequence of CD47 ligation was reproduced in this study using SIRP fusion protein on Jurkat cells. The existence of SIRP on T cells means that they have another mechanism for inducing signaling through CD47 with possible multiple effects on integrin function and T cell behavior. Thus, the ability of T cells to directly send signals via CD47 has important implications for T cell biology.

	Acknowledgments

 
We are grateful to Michael J. Puklavec and Steve Simmonds for help with mAb production, to H. J. Buhring for providing mAb SE5A5 and B1D5, to E. Tomasello and E. Vivier for SIRP/DAP12 transfectants and M. Colonna for SIRP cDNA, and to J. Sedgwick for DAP12 vector.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by the Medical Research Council, the Arthritis Rheumatism Council, and GlaxoSmithKline Pharmaceuticals.

² Address correspondence and reprint requests to Dr. A. Neil Barclay, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, U.K. OX1 3RE. E-mail address: neil.barclay{at}path.ox.ac.uk

³ Abbreviations used in this paper: SIRP, signal regulatory protein; 7-AAD, 7-aminoactinomycin D; EST, established sequence tag; RU, response unit; Shp1, Src homology 2 domain-containing phosphatase 1.

Received for publication June 17, 2003. Accepted for publication June 10, 2004.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Kharitonenkov, A., Z. Chen, I. Sures, H. Wang, J. Schilling, A. Ullrich. 1997. A family of proteins that inhibit signalling through tyrosine kinase receptors. Nature 386:181.[Medline]
2. Fujioka, Y., T. Matozaki, T. Noguchi, A. Iwamatsu, T. Yamao, N. Takahashi, M. Tsuda, T. Takada, M. Kasuga. 1996. A novel membrane glycoprotein, SHPS-1, that binds the SH2-domain-containing protein tyrosine phosphatase SHP-2 in response to mitogens and cell adhesion. Mol. Cell. Biol. 16:6887.[Abstract]
3. Brooke, G. P., K. R. Parsons, C. J. Howard. 1998. Cloning of two members of the SIRP family of protein tyrosine phosphatase binding proteins in cattle that are expressed on monocytes and a subpopulation of dendritic cells and which mediate binding to CD4 T cells. Eur. J. Immunol. 28:1.[Medline]
4. Comu, S., W. Weng, S. Olinsky, P. Ishwad, Z. Mi, J. Hempel, S. Watkins, C. F. Lagenaur, V. Narayanan. 1997. The murine P84 neural adhesion molecule is SHPS-1, a member of the phosphatase-binding protein family. J. Neurosci. 17:8702.[Abstract/Free Full Text]
5. Sano, S., H. Ohnishi, A. Omori, J. Hasegawa, M. Kubota. 1997. BIT, an immune antigen receptor-like molecule in the brain. FEBS Lett. 411:327.[Medline]
6. Saginario, C., H. Sterling, C. Beckers, R. Kobayashi, M. Solimena, E. Ullu, A. Vignery. 1998. MFR, a putative receptor mediating the fusion of macrophages. Mol. Cell. Biol. 18:6213.[Abstract/Free Full Text]
7. Takada, T., T. Matozaki, H. Takeda, K. Fukunaga, T. Noguchi, Y. Fujioka, I. Okazaki, M. Tsuda, T. Yamao, F. Ochi, et al 1998. Roles of the complex formation of SHPS-1 with SHP-2 in insulin-stimulated mitogen-activated protein kinase activation. J. Biol. Chem. 273:9234.[Abstract/Free Full Text]
8. Veillette, A., E. Thibaudeau, S. Latour. 1998. High expression of inhibitory receptor SHPS-1 and its association with protein-tyrosine phosphatase SHP-1 in macrophages. J. Biol. Chem. 273:22719.[Abstract/Free Full Text]
9. Lienard, H., P. Bruhns, O. Malbec, W. H. Fridman, M. Daeron. 1999. Signal regulatory proteins negatively regulate immunoreceptor-dependent cell activation. J. Biol. Chem. 274:32493.[Abstract/Free Full Text]
10. Patel, V., R. E. Smith, A. Serra, G. Brooke, C. J. Howard, K. P. Rigley. 2002. MyD-1 (SIRP) regulates T cell function in the absence of exogenous danger signals, via a TNF-dependent pathway. Eur. J. Immunol. 32:1865.[Medline]
11. Smith, R. E., V. Patel, S. D. Seatter, M. R. Deehan, M. H. Brown, G. P. Brooke, H. S. Goodridge, C. J. Howard, K. P. Rigley, W. Harnett, et al 2003. A novel MyD-1 (SIRP-1) signaling pathway that inhibits LPS-induced TNF production by monocytes. Blood 102:2532.[Abstract/Free Full Text]
12. Seiffert, M., C. Cant, Z. Chen, I. Rappold, W. Brugger, L. Kanz, E. J. Brown, A. Ullrich, H. J. Buhring. 1999. Human signal-regulatory protein is expressed on normal, but not on subsets of leukemic myeloid cells and mediates cellular adhesion involving its counterreceptor CD47. Blood 94:3633.[Abstract/Free Full Text]
13. Vernon-Wilson, E. F., W. J. Kee, A. C. Willis, A. N. Barclay, D. L. Simmons, M. H. Brown. 2000. CD47 is a ligand for rat macrophage membrane signal regulatory protein SIRP (OX41) and human SIRP1. Eur. J. Immunol. 30:2130.[Medline]
14. Lindberg, F. P., D. M. Lublin, M. J. Telen, R. A. Veile, Y. E. Miller, H. Donis-Keller, E. J. Brown. 1994. Rh-related antigen CD47 is the signal-transducer integrin-associated protein. J. Biol. Chem. 269:1567.[Abstract/Free Full Text]
15. Mawby, W. J., C. H. Holmes, D. J. Anstee, F. A. Spring, M. J. Tanner. 1994. Isolation and characterization of CD47 glycoprotein: a multispanning membrane protein which is the same as integrin-associated protein (IAP) and the ovarian tumour marker OA3. Biochem. J. 304:525.
16. Frazier, W. A., A. G. Gao, J. Dimitry, J. Chung, E. J. Brown, F. P. Lindberg, M. E. Linder. 1999. The thrombospondin receptor integrin-associated protein (CD47) functionally couples to heterotrimeric G_i. J. Biol. Chem. 274:8554.[Abstract/Free Full Text]
17. Green, J. M., A. Zhelesnyak, J. Chung, F. P. Lindberg, M. Sarfati, W. A. Frazier, E. J. Brown. 1999. Role of cholesterol in formation and function of a signaling complex involving _v3, integrin-associated protein (CD47), and heterotrimeric G proteins. J. Cell Biol. 146:673.[Abstract/Free Full Text]
18. Cooper, D., F. P. Lindberg, J. R. Gamble, E. J. Brown, M. A. Vadas. 1995. Transendothelial migration of neutrophils involves integrin-associated protein (CD47). Proc. Natl. Acad. Sci. USA 92:3978.[Abstract/Free Full Text]
19. Pettersen, R. D., K. Hestdal, M. K. Olafsen, S. O. Lie, F. P. Lindberg. 1999. CD47 signals T cell death. J. Immunol. 162:7031.[Abstract/Free Full Text]
20. Latour, S., H. Tanaka, C. Demeure, V. Mateo, M. Rubio, E. J. Brown, C. Maliszewski, F. P. Lindberg, A. Oldenborg, A. Ullrich, et al 2001. Bidirectional negative regulation of human T and dendritic cells by CD47 and its cognate receptor signal-regulator protein-: down-regulation of IL-12 responsiveness and inhibition of dendritic cell activation. J. Immunol. 167:2547.[Abstract/Free Full Text]
21. Avice, M. N., M. Rubio, M. Sergerie, G. Delespesse, M. Sarfati. 2001. Role of CD47 in the induction of human naive T cell anergy. J. Immunol. 167:2459.[Abstract/Free Full Text]
22. Oldenborg, P. A., H. D. Gresham, F. P. Lindberg. 2001. CD47-signal regulatory protein (SIRP) regulates Fc and complement receptor-mediated phagocytosis. J. Exp. Med. 193:855.[Abstract/Free Full Text]
23. Ticchioni, M., M. Deckert, F. Mary, G. Bernard, E. J. Brown, A. Bernard. 1997. Integrin-associated protein (CD47) is a comitogenic molecule on CD3-activated human T cells. J. Immunol. 158:677.[Abstract]
24. Waclavicek, M., O. Majdic, T. Stulnig, M. Berger, T. Baumruker, W. Knapp, W. F. Pickl. 1997. T cell stimulation via CD47: agonistic and antagonistic effects of CD47 monoclonal antibody 1/1A4. J. Immunol. 159:5345.[Abstract]
25. Parkos, C. A., S. P. Colgan, T. W. Liang, A. Nusrat, A. E. Bacarra, D. K. Carnes, J. L. Madara. 1996. CD47 mediates post-adhesive events required for neutrophil migration across polarized intestinal epithelia. J. Cell Biol. 132:437.[Abstract/Free Full Text]
26. de Vries, H. E., J. J. Hendriks, H. Honing, C. R. De Lavalette, S. M. van der Pol, E. Hooijberg, C. D. Dijkstra, T. K. van den Berg. 2002. Signal-regulatory protein -CD47 interactions are required for the transmigration of monocytes across cerebral endothelium. J. Immunol. 168:5832.[Abstract/Free Full Text]
27. Lindberg, F. P., D. C. Bullard, T. E. Caver, H. D. Gresham, A. L. Beaudet, E. J. Brown. 1996. Decreased resistance to bacterial infection and granulocyte defects in IAP-deficient mice. Science 274:795.[Abstract/Free Full Text]
28. Oldenborg, P. A., A. Zheleznyak, Y. F. Fang, C. F. Lagenaur, H. D. Gresham, F. P. Lindberg. 2000. Role of CD47 as a marker of self on red blood cells. Science 288:2051.[Abstract/Free Full Text]
29. Gao, A. G., F. P. Lindberg, M. B. Finn, S. D. Blystone, E. J. Brown, W. A. Frazier. 1996. Integrin-associated protein is a receptor for the C-terminal domain of thrombospondin. J. Biol. Chem. 271:21.[Abstract/Free Full Text]
30. Li, Z., M. J. Calzada, J. M. Sipes, J. A. Cashel, H. C. Krutzsch, D. S. Annis, D. F. Mosher, D. D. Roberts. 2002. Interactions of thrombospondins with ₄₁ integrin and CD47 differentially modulate T cell behavior. J. Cell Biol. 157:509.[Abstract/Free Full Text]
31. Lanier, L. L., B. C. Corliss, J. Wu, C. Leong, J. H. Phillips. 1998. Immunoreceptor DAP12 bearing a tyrosine-based activation motif is involved in activating NK cells. Nature 391:703.[Medline]
32. Seiffert, M., P. Brossart, C. Cant, M. Cella, M. Colonna, W. Brugger, L. Kanz, A. Ullrich, H. J. Buhring. 2001. Signal-regulatory protein (SIRP) but not SIRP is involved in T-cell activation, binds to CD47 with high affinity, and is expressed on immature CD34⁺CD38^– hematopoietic cells. Blood 97:2741.[Abstract/Free Full Text]
33. Ichigotani, Y., S. Matsuda, K. Machida, K. Oshima, T. Iwamoto, K. Yamaki, T. Hayakawa, M. Hamaguchi. 2000. Molecular cloning of a novel human gene (SIRP-B2) which encodes a new member of the SIRP/SHPS-1 protein family. J. Hum. Gen. 45:378.
34. Brown, M. H., A. N. Barclay. 1994. Expression of immunoglobulin and scavenger receptor superfamily domains as chimeric proteins with domains 3 and 4 of CD4 for ligand analysis. Protein Eng. 7:515.[Abstract/Free Full Text]
35. Brown, M. H., K. Boles, P. A. van der Merwe, V. Kumar, P. A. Mathew, A. N. Barclay. 1998. 2B4, the natural killer and T cell immunoglobulin superfamily surface protein, is a ligand for CD48. J. Exp. Med. 188:2083.[Abstract/Free Full Text]
36. Brown, M. H., S. Preston, A. N. Barclay. 1995. A sensitive assay for detecting low-affinity interactions at the cell surface reveals no additional ligands for the adhesion pair rat CD2 and CD48. Eur. J. Immunol. 25:3222.[Medline]
37. Preston, S., G. J. Wright, K. Starr, A. N. Barclay, M. H. Brown. 1997. The leukocyte/neuron cell surface antigen OX2 binds to a ligand on macrophages. Eur. J. Immunol. 27:1911.[Medline]
38. van der Merwe, P. A., S. J. Davis. 2003. Molecular interactions mediating T cell antigen recognition. Annu. Rev. Immunol. 21:659.[Medline]
39. van der Merwe, P. A., A. N. Barclay, D. W. Mason, E. A. Davies, B. P. Morgan, M. Tone, A. K. Krishnam, C. Ianelli, S. J. Davis. 1994. Human cell-adhesion molecule CD2 binds CD58 (LFA-3) with a very low affinity and an extremely fast dissociation rate but does not bind CD48 or CD59. Biochemistry 33:10149.[Medline]
40. Manna, P. P., W. A. Frazier. 2003. The mechanism of CD47-dependent killing of T cells: heterotrimeric G_i-dependent inhibition of protein kinase A. J. Immunol. 170:3544.[Abstract/Free Full Text]
41. Dietrich, J., M. Cella, M. Seiffert, H. J. Buhring, M. Colonna. 2000. Cutting edge: signal-regulatory protein 1 is a DAP12-associated activating receptor expressed in myeloid cells. J. Immunol. 164:9.[Abstract/Free Full Text]
42. Arase, H., E. S. Mocarski, A. E. Campbell, A. B. Hill, L. L. Lanier. 2002. Direct recognition of cytomegalovirus by activating and inhibitory NK cell receptors. Science 296:1323.[Abstract/Free Full Text]
43. Smith, H. R., J. W. Heusel, I. K. Mehta, S. Kim, B. G. Dorner, O. V. Naidenko, K. Iizuka, H. Furukawa, D. L. Beckman, J. T. Pingel, et al 2002. Recognition of a virus-encoded ligand by a natural killer cell activation receptor. Proc. Natl. Acad. Sci. USA 99:8826.[Abstract/Free Full Text]
44. Collins, A. V., D. W. Brodie, R. J. Gilbert, A. Iaboni, R. Manso-Sancho, B. Walse, D. I. Stuart, P. A. van der Merwe, S. J. Davis. 2002. The interaction properties of costimulatory molecules revisited. Immunity 17:201.[Medline]
45. van der Merwe, P. A., D. L. Bodian, S. Daenke, P. Linsley, S. J. Davis. 1997. CD80 (B7-1) binds both CD28 and CTLA-4 with a low affinity and very fast kinetics. J. Exp. Med. 185:393.[Abstract/Free Full Text]
46. Mateo, V., L. Lagneaux, D. Bron, G. Biron, M. Armant, G. Delespesse, M. Sarfati. 1999. CD47 ligation induces caspase-independent cell death in chronic lymphocytic leukemia. Nat. Med. 5:1277.[Medline]
47. Yamao, T., T. Noguchi, O. Takeuchi, U. Nishiyama, H. Morita, T. Hagiwara, H. Akahori, T. Kato, K. Inagaki, H. Okazawa, et al 2002. Negative regulation of platelet clearance and of the macrophage phagocytic response by the transmembrane glycoprotein SHPS-1. J. Biol. Chem. 277:39833.[Abstract/Free Full Text]
48. Pettersen, R. D.. 2000. CD47 and death signaling in the immune system. Apoptosis 5:299.[Medline]
49. Butler, B. A.. 1998. Sequence analysis using GCG. Methods Biochem. Anal. 39:74.[Medline]
50. Bateman, A., E. Birney, L. Cerruti, R. Durbin, L. Etwiller, S. R. Eddy, S. Griffiths-Jones, K. L. Howe, M. Marshall, E. L. L. Sonnhammer. 2002. The PFAM protein family database. Nucleic Acids Res. 30:276.[Abstract/Free Full Text]
51. Nielsen, H., J. Engelbrecht, S. Brunak, G. von Heijne. 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10:1.[Abstract/Free Full Text]
52. Krogh, A., G. Larsson, G. von Heijne, E. L. L. Sonnhammer. 2001. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J. Mol. Biol. 305:567.[Medline]

This article has been cited by other articles:

				H. Umemori and J. R. Sanes Signal Regulatory Proteins (SIRPS) Are Secreted Presynaptic Organizing Molecules J. Biol. Chem., December 5, 2008; 283(49): 34053 - 34061. [Abstract] [Full Text] [PDF]

				M. Stefanidakis, G. Newton, W. Y. Lee, C. A. Parkos, and F. W. Luscinskas Endothelial CD47 interaction with SIRP{gamma} is required for human T-cell transendothelial migration under shear flow conditions in vitro Blood, August 15, 2008; 112(4): 1280 - 1289. [Abstract] [Full Text] [PDF]

				D. Hatherley, K. Harlos, D. C. Dunlop, D. I. Stuart, and A. N. Barclay The Structure of the Macrophage Signal Regulatory Protein {alpha} (SIRP{alpha}) Inhibitory Receptor Reveals a Binding Face Reminiscent of That Used by T Cell Receptors J. Biol. Chem., May 11, 2007; 282(19): 14567 - 14575. [Abstract] [Full Text] [PDF]

				S. Subramanian, E. T. Boder, and D. E. Discher Phylogenetic Divergence of CD47 Interactions with Human Signal Regulatory Protein {alpha} Reveals Locus of Species Specificity: IMPLICATIONS FOR THE BINDING SITE J. Biol. Chem., January 19, 2007; 282(3): 1805 - 1818. [Abstract] [Full Text] [PDF]

				D. Braun, L. Galibert, T. Nakajima, H. Saito, V. V. Quang, M. Rubio, and M. Sarfati Semimature Stage: A Checkpoint in a Dendritic Cell Maturation Program That Allows for Functional Reversion after Signal-Regulatory Protein-{alpha} Ligation and Maturation Signals J. Immunol., December 15, 2006; 177(12): 8550 - 8559. [Abstract] [Full Text] [PDF]

				M. H. Lahoud, A. I. Proietto, K. H. Gartlan, S. Kitsoulis, J. Curtis, J. Wettenhall, M. Sofi, C. Daunt, M. O'Keeffe, I. Caminschi, et al. Signal Regulatory Protein Molecules Are Differentially Expressed by CD8- Dendritic Cells J. Immunol., July 1, 2006; 177(1): 372 - 382. [Abstract] [Full Text] [PDF]

				E. M. van Beek, F. Cochrane, A. N. Barclay, and T. K. van den Berg Signal Regulatory Proteins in the Immune System J. Immunol., December 15, 2005; 175(12): 7781 - 7787. [Abstract] [Full Text] [PDF]

				Y. Liu, I. Soto, Q. Tong, A. Chin, H.-J. Buhring, T. Wu, K. Zen, and C. A. Parkos SIRP{beta}1 Is Expressed as a Disulfide-linked Homodimer in Leukocytes and Positively Regulates Neutrophil Transepithelial Migration J. Biol. Chem., October 28, 2005; 280(43): 36132 - 36140. [Abstract] [Full Text] [PDF]

				J. Alblas, H. Honing, C. Renardel de Lavalette, M. H. Brown, C. D. Dijkstra, and T. K. van den Berg Signal Regulatory Protein {alpha} Ligation Induces Macrophage Nitric Oxide Production through JAK/STAT- and Phosphatidylinositol 3-Kinase/Rac1/NAPDH Oxidase/H2O2-Dependent Pathways Mol. Cell. Biol., August 15, 2005; 25(16): 7181 - 7192. [Abstract] [Full Text] [PDF]

				D. Hatherley, H. M. Cherwinski, M. Moshref, and A. N. Barclay Recombinant CD200 Protein Does Not Bind Activating Proteins Closely Related to CD200 Receptor J. Immunol., August 15, 2005; 175(4): 2469 - 2474. [Abstract] [Full Text] [PDF]

				P.-A. Oldenborg CD47 and SIRPs: new openings Blood, March 15, 2005; 105(6): 2245 - 2246. [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Brooke, G. Articles by Barclay, A. N. Search for Related Content PubMed PubMed Citation Articles by Brooke, G. Articles by Barclay, A. N.