Self inhibition of phagocytosis: the affinity of 'marker of self' CD47 for SIRPalpha dictates potency of inhibition but only at low expression levels

Abstract

Phagocytes engulf foreign cells but not 'self' in part because self cells express CD47 as a ligand for signal regulatory protein SIRPalpha, which inhibits phagocytosis. Motivated by reports of upregulation of CD47 on both normal and cancerous stem cells [1: Jaiswal et al., 2009] and also by polymorphisms in SIRPalpha [2: Takenaka et al., 2007], we show here that inhibition of engulfment correlates with affinity of CD47 for SIRPalpha - but only at low levels of CD47. One common human polymorph of SIRPalpha is studied and binds more strongly to human-CD47 than to mouse-CD47 (K(d) approximately 0.12 microM and 6.9 microM, respectively) and does not bind sheep red blood cells (RBCs) - which are well-established targets of human macrophages; in comparison, a common mouse polymorph of SIRPalpha binds with similar affinity to human and mouse CD47 (K(d) approximately 0.22 microM). Using immunoglobulin (IgG)-opsonized particles with varying levels of either human- or mouse-CD47, the effective inhibition constants K(i) for blocking phagocytosis are then determined with both human- and mouse-derived macrophages. Only human phagocytes show significant differences in man versus mouse K(i)'s and only at CD47 levels below normal densities for RBCs. While phospho-signaling through human-SIRPalpha shows similar trends, consistent again with the affinity differences, saturating levels of CD47 (>K(i)) can signal and inhibit phagocytosis regardless of man versus mouse. Quantitative analyses here prompt more complete characterizations of both CD47 levels and SIRPalpha polymorphisms when attempting to study in vivo effects of these key proteins in innate immunity.

Figure 1. Species-specific binding of soluble human-SIRPα to various species RBCs and CD47-coated beads

(A) Fresh human, sheep, and mouse RBC binding to soluble hSIRPα (4μM of GST conjugate), as detected by FITC-anti-GST. ‘Bkgd’ is obtained with RBC plus anti-GST. Species-specific affinities of hCD47- and mCD47-coated beads binding to soluble (B) hSIRPα or (C) mSIRPα based on mean fluorescence in flow cytometry. Saturation binding fits gave the indicated dissociation constants, K_d.

Figure 2. Phagocytosis of Ig-opsonized sheep-RBC and human-RBC by human-THP-1 macrophages

(A) Fresh human and sheep RBC were incubated with antiserum at different dilution ratios and detected by fluorescent secondary antibodies, exhibiting a linear opsonization based on mean fluorescence in flow cytometry. (B) Phagocytosis of shRBC by human-derived THP-1 macrophages at different opsonization level observed by DIC microscopy, which showed a saturable type of response (C). Addition of soluble human-CD47 at μM concentrations showed at most a slight inhibition of phagocytosis of sh-RBC by THP-1 macrophages but not enough to reach hRBC phagocytosis levels at 1/900 antiserum dilution ratio.

Figure 3. CD47 is sufficient to inhibit phagocytosis

(A) Microbeads with streptavidin were coated with anti-streptavidin IgG as the opsonin ± biotinylated human-CD47 or mouse-CD47. Phagocytosis of beads (red) by THP-1 or J774A.1 cells was assessed in DIC and fluorescence with non-ingested beads (green) visible with rabbit anti-streptavidin plus a second, fluorescent goat anti-rabbit antibody. White arrow indicates non-phagocytosed beads. Scale bar, 10μm. Streptavidin beads coated with both anti-streptavidin IgG as the opsonin and ± biotinylated (B) human CD47 particle targets with THP-1 macrophages or (C) mouse CD47 particle targets with J774A.1 in a phagocytosis assay.

Figure 4. Inhibition of phagocytosis by CD47 is density dependent

IgG-opsonized particles with human (solid line) and mouse CD47 (dashed line) were used as targets for (A) human THP-1 macrophages or (B) mouse J744A.1 macrophages. Grey shaded vertical bar indicates normal RBC density (~250 CD47/μm²). Species-specific differences are quantitated by fits to y = a – b x^m / (K_i^m + x^m) with effective K_i [molecules/mm²] that differ by 5-fold at high opsonin, although the maximum inhibition is similar (see Table 1). A Hill coefficient of m = 2 suggests cooperativity.

Figure 5. Species-specific signaling through SIRPα

hCD47 or mCD47 were bound at varying densities to opsonized beads and phagocytosed by THP-1 macrophages. From macrophage lysates, SIRPα was immunoprecipitated and immunoblotted (inset) for quantitation of phospho-Tyr and total SIRPα for normalization. Fits of the data gave effective signaling constant K_s for each species that depends on the CD47 density; all densities are scaled by hCD47's inhibitory constant (K_i-h) as determined in Figure 4.3A at the same opsonization.

Figure 6. Potency and binding strength depend on species-specific CD47-SIRPα interactions

IgG-opsonization modulates the density-dependent activity of hCD47 or mCD47 with either human THP-1 macrophages (hSIRPα) or mouse J774A.1 macrophages (mSIRPα). Each point on the plot represents a K_d obtained from binding studies per Figure 4.1B and a Ki obtained per Figure 4.3A as fitted by a Hill-type model. Larger symbols indicate results with higher opsonin, which promotes synapse formation and thereby more potent inhibition than at lower opsonin.