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. 2006 Mar 15;107(6):2548-56.
doi: 10.1182/blood-2005-04-1463. Epub 2005 Nov 15.

Species- and cell type-specific interactions between CD47 and human SIRPalpha

Shyamsundar Subramanian  1 , Ranganath Parthasarathy, Shamik Sen, Eric T Boder, Dennis E Discher
Affiliations

Species- and cell type-specific interactions between CD47 and human SIRPalpha

Shyamsundar Subramanian et al. Blood. .

Abstract

CD47 on red blood cells (RBCs) reportedly signals "self" by binding SIRPalpha on phagocytes, at least in mice. Such interactions across and within species, from mouse to human, are not yet clear and neither is the relation to cell adhesion. Using human SIRPalpha1 as a probe, antibody-inhibitable binding to CD47 was found only with human and pig RBCs (not mouse, rat, or cow). In addition, CD47-mediated adhesion of human and pig RBCs to SIRPalpha1 surfaces resists sustained forces in centrifugation (as confirmed by atomic force microscopy) but only at SIRPalpha-coating densities far above those measurable on human neutrophils, monocytes, and THP-1 macrophages. While interactions strengthen with deglycosylation of SIRPalpha1, low copy numbers explain the absence of RBC adhesion to phagocytes under physiologic conditions and imply that the interaction being studied is not responsible for red cell clearance in humans. Evidence of clustering nonetheless suggests mechanisms of avidity enhancement. Finally, using the same CD47 antibodies and soluble SIRPalpha1, bone marrow-derived mesenchymal stem cells were assayed and found to display CD47 but not bind SIRPalpha1 significantly. The results thus demonstrate that SIRPalpha-CD47 interactions, which reportedly define self, exhibit cell type specificity and limited cross-species reactivity.

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Figures

Figure 1.
Figure 1.
Adhesive interactions of SIRPα-CD47 on cell surfaces. Based on mouse studies, binding between CD47's Ig domain on RBCs and the N-terminal Ig domain of SIRPα (or SHPS-1, P84) on phagocytes is thought to trigger clustering of SIRPα. Phosphorylation events at SIRPα's cytoplasmic tail are then believed to ultimately signal “self” and inhibit phagocytosis of RBCs. This occurs through tyrosine phosphorylation of SIRPα and subsequent recruitment of phosphatases (SHP-1 predominantly).
Figure 2.
Figure 2.
Human SIRPα 1ex binding to RBCs is CD47 specific and species specific. Human RBC (A) or Ig-CD47-coated beads (B) were incubated with soluble SIRPα1ex or GST, and bound protein was detected in flow cytometry using fluorescent anti-GST. SIRPα1ex specifically binds to human RBCs whereas GST does not. Blocking antibodies (B6H12 and BRIC126) inhibit the CD47-SIRPα interaction. 2D3, a nonblocking antibody, enhances SIRPα1ex binding to RBCs but not Ig-CD47 beads, probably by ability to cluster CD47 (C). Using the same methodology, RBCs from 5 mammalian species were labeled with SIRPα1ex or GST (D) under standardized conditions of cell number and reagent concentration. Human SIRPα1ex binds to human RBCs as expected and does not bind to RBCs from cow, mouse, or rat. Human SIRPα1ex also binds to pig RBCs and results in higher intensity in comparison to human RBCs. Note that * indicates a slight signal above background.
Figure 3.
Figure 3.
CD47-mediated adhesion of human RBCs to human SIRPα1ex-coated surfaces. (A) Human RBCs were allowed to adhere at modest density to SIRPα1excoated wells in a 96-well plate for 10 minutes and centrifuged inverted for 10 minutes at 100g (cells remained wet). (B) Microscopy showed significant, intact adhesion of human RBCs to SIRPα1ex-coated wells but minimal adhesion to GST. (C) The fraction of well area covered by adherent RBCs after centrifugation was very small for bovine serum albumin (BSA)- and glutathione S-transferase (GST)-compared with SIRPα1ex-coated surfaces. (D) Prelabeling of human RBCs with blocking antibodies (B6H12 and BRIC126) prevented cell adhesion, whereas 2D3 did not inhibit or significantly enhance adhesion. The G-forces used here exert a sustained peeling force of approximately 100 pN on a human red cell (volume = 95 fL; density = 1.09 g/mL). Error bars indicate plus or minus 1 SD from multiple experiments.
Figure 4.
Figure 4.
Species-restricted adhesion of RBCs to human SIRPα1ex-coated surfaces. (A) RBCs from 5 mammalian species were allowed to adhere to either human SIRPα1ex- or GST-coated wells (∼10 000 molecules/μm2) and the fraction of well area covered by adherent RBCs after centrifugation was determined. Adhesion results closely matched the results from the flow cytometry-based SIRPα1ex binding assay. Only RBCs from human and pig showed significant adhesion to SIRPα1excoated wells among the species tested. The ratio of RBC area coverage on SIRPα1ex to that on GST is indicated. (B) Human and rat RBCs are probed using atomic force microscopy using human SIRPα1ex-coated tips, leading to almost 100% adhesion with human cells. Almost no adhesion is seen with rat cells. The force required to disrupt single bonds of CD47-SIRPα is approximately 70 pN. See Document S1 and Figures S1-S3 for details on AFM experiments. Error bars represent 1 SD from multiple experiments.
Figure 5.
Figure 5.
Characterization of glycosylation in recombinant and native SIRPα and effects on binding to RBCs. (A) Purified soluble human SIRPα1ex produced in COS-1 cells (in the presence or absence of tunicamycin) or from Lec-1 cells detected using anti-GST is shown. The Lec-1 product is Endo H sensitive, whereas PNGase is required to remove all glycans from the COS-1 product (without inhibitors). Human RBCs (B) or pig RBCs (C) were labeled with complex glycosylated, core glycosylated, and aglycosylated human SIRPα1ex complexed with fluorescent anti-GST. Alteration in the type of N-linked glycans (complex/hybrid to mannose) or removal of N-linked glycans enhances binding to both human and pig RBCs. (D) Recombinant human SIRPα1 from COS-1 cells and native SIRPα from human monocytes and THP-1 cells is detected using a C-terminus peptide-specific anti-SIRPα. Recombinant and native SIRPα show limited Endo H sensitivity, requiring PNGase for complete deglycosylation, indicating complex/hybrid glycans. Extent of glycosylation is also similar between these forms of SIRPα. See Document S1 and Figures S1-S3 for Western blotting details.
Figure 6.
Figure 6.
RBC adhesion is dependent on immobilized ligand density. Immobilized ligand density was varied to measure the effect on RBC adhesion in centrifugation. (A) Human RBCs adhere to human SIRPα1ex-coated wells with half-max adhesion at C50 = 1670 sites/μm2. Inset shows log scale with the average physiologic range of SIRPα1ex on various phagocytes, per Table 3. (B) Pig RBCs adhere to human SIRPα1ex-coated wells more tightly than human RBCs. (C) Human RBCs adhere more strongly to B6H12 antibody-coated wells than to human SIRPα1ex. Averages (± SEM) of % area covered by RBCs after centrifugation from multiple experiments are shown and the ligand density at half-maximal adhesion (C50) was determined by data fit. Note that SIRPα1 immobilized here prevents clustering and limits avidity effects, consistent with the low Hill coefficients. Error bars represent plus or minus 1 SEM from multiple experiments.
Figure 7.
Figure 7.
Ligand-induced clustering of SIRPα on phagocytes. Freshly isolated human neutrophils and monocytes as well as undifferentiated THP-1 cells and PMA-differentiated THP-1 cells were labeled (at RT, without fixation) with antibody P3C4 against human SIRPα (A-D). Bound antibody was detected with R-phycoerythrin-tagged secondary antibody. Inset images magnify the membrane association. (E-F) Neutrophils and monocytes were also stained with biotinylated Ig-CD47 and fluorescent extravidin. Ligand-induced SIRPα clusters are clearly visible for both ligands and for all phagocytes. Average pixel intensity in unclustered regions was calculated to determine the fraction of cell area with at least 2- or 5-fold the mean unclustered pixel intensity. Antibody-mediated preclustering of SIRPα with P3C4 enhances Ig-CD47 binding (G).
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