Myelin down-regulates myelin phagocytosis by microglia and macrophages through interactions between CD47 on myelin and SIRPα (signal regulatory protein-α) on phagocytes

Abstract

Background: Traumatic injury to axons produces breakdown of axons and myelin at the site of the lesion and then further distal to this where Wallerian degeneration develops. The rapid removal of degenerated myelin by phagocytosis is advantageous for repair since molecules in myelin impede regeneration of severed axons. Thus, revealing mechanisms that regulate myelin phagocytosis by macrophages and microglia is important. We hypothesize that myelin regulates its own phagocytosis by simultaneous activation and down-regulation of microglial and macrophage responses. Activation follows myelin binding to receptors that mediate its phagocytosis (e.g. complement receptor-3), which has been previously studied. Down-regulation, which we test here, follows binding of myelin CD47 to the immune inhibitory receptor SIRPα (signal regulatory protein-α) on macrophages and microglia.

Methods: CD47 and SIRPα expression was studied by confocal immunofluorescence microscopy, and myelin phagocytosis by ELISA.

Results: We first document that myelin, oligodendrocytes and Schwann cells express CD47 without SIRPα and further confirm that microglia and macrophages express both CD47 and SIRPα. Thus, CD47 on myelin can bind to and subsequently activate SIRPα on phagocytes, a prerequisite for CD47/SIRPα-dependent down-regulation of CD47+/+ myelin phagocytosis by itself. We then demonstrate that phagocytosis of CD47+/+ myelin is augmented when binding between myelin CD47 and SIRPα on phagocytes is blocked by mAbs against CD47 and SIRPα, indicating that down-regulation of phagocytosis indeed depends on CD47-SIRPα binding. Further, phagocytosis in serum-free medium of CD47+/+ myelin is augmented after knocking down SIRPα levels (SIRPα-KD) in phagocytes by lentiviral infection with SIRPα-shRNA, whereas phagocytosis of myelin that lacks CD47 (CD47-/-) is not. Thus, myelin CD47 produces SIRPα-dependent down-regulation of CD47+/+ myelin phagocytosis in phagocytes. Unexpectedly, phagocytosis of CD47-/- myelin by SIRPα-KD phagocytes, which is not altered from normal when tested in serum-free medium, is augmented when serum is present. Therefore, both myelin CD47 and serum may each promote SIRPα-dependent down-regulation of myelin phagocytosis irrespective of the other.

Conclusions: Myelin down-regulates its own phagocytosis through CD47-SIRPα interactions. It may further be argued that CD47 functions normally as a marker of "self" that helps protect intact myelin and myelin-forming oligodendrocytes and Schwann cells from activated microglia and macrophages. However, the very same mechanism that impedes phagocytosis may turn disadvantageous when rapid clearance of degenerated myelin is helpful.

Figure 1

Myelin regulates its own phagocytosis by simultaneous activation and down-regulation - a schematic representation of the working hypothesis and experimental design. (A & B) Degenerated wild-type CD47^+/+ (CD47) myelin can simultaneously bind CR3 and SIRPα, which are expressed on microglia and macrophages. (A) Myelin binds CR3, which is the major receptor that mediates myelin phagocytosis in context of traumatic injury (see text), either directly or indirectly after being opsonized by complement protein C3bi (± C3bi). CR3 ligation initiates a signaling cascade that activates phagocytosis. (B) SIRPα ligation by myelin CD47 initiates a signaling cascade that inhibits phagocytosis by down-regulating signaling produced by CR3. (C & D) Augmentation of phagocytosis is anticipated if CD47 binding to SIRPα is blocked by either anti-SIRPα or anti-CD47 mAbs. (C) SIRPα on phagocytes is blocked by anti-SIRPα mAbs. (D) Myelin CD47 is blocked by anti-CD47 mAbs whose Fc-segments (black circle) are coated with anti-Fc-Fab₂ fragments that lack their own Fc-segments. (E) Myelin CD47 is blocked by anti-CD47 mAbs whose Fc-segments are exposed. Consequently, CD47 binding to SIRPα is blocked (as in D) but binding to and activation of phagocytosis through FcγR is possible. (F) However, if anti-CD47 Fc-segments are coated by anti-Fc-Fab₂ fragments (as in D), binding to and activation of phagocytosis through FcγR are blocked. Phagocytosis of (H) CD47^+/+ is expected to be augmented after reduction of SIRPα levels in phagocytes; i.e. compared to phagocytosis of CD47^+/+ by (G) phagocytes expressing normal SIRPα levels. Further, phagocytosis of CD47^-/- myelin by phagocytes expressing (I) normal or (J) reduced SIRPα levels are expected to be about the same.

Figure 2

Macrophages and microglia express CD47 and SIRPα whereas myelin, Schwann cells, oligodendrocytes and astrocytes express CD47 without SIRPα. Macrophages (MO) express (A) Galectin-3, cell surface (B) SIRPα, and (C) CD47. (D) Macrophages phagocytose myelin. F-actin (filamentous actin) is visualized by Alexa 488 labeled phalloidin (green) and myelin by anti-MBP mAb (red); overlap between the two is yellow. Myelin is present in the cytoplasm interior to cortical F-actin. Similar observations were made in microglia (not shown; see [29]). Microglia (MG) express (E) Galectin-3, and cell surface (F) SIRPα and (G) CD47. (H) MBP and (J) CD47 are expressed in myelin without (I) SIRPα. (K) Anti-Fc-Fab₂ fragments coat/block Fc-segments of anti-CD47 mAb that binds CD47 on myelin, thus preventing visualization of anti-CD47 mAb by a secondary Ab. (L) CD47 is expressed on spindle shaped bipolar Schwann cells (arrow) and flat fibroblasts (double arrow). (M) CD47 is expressed on oligodendrocytes (arrow) that extend elongated branched processes, and on flat astrocytes (double arrow). Galectin-3, CD47, SIRPα and MBP are visualized by immunofluorescence confocal microscopy using respective mAbs directed against each (red). Bars are 5 μm in A through K, and 50 μm in L and M.

Figure 3

Myelin phagocytosis is augmented in the presence of anti-CD47and anti-SIRPα function-blocking mAbs (see also Fig. 1). Myelin phagocytosis is augmented in (A) microglia and (B) macrophages in the presence of function-blocking anti-SIRPα and anti-CD47 mAbs. Phagocytosis was assayed using different paradigms for each mAb (see text). (i) Microglia and macrophages were pre-incubated in the presence of anti-SIRPα or matched control IgG (Cont-IgG), myelin was added and phagocytosis was assayed. Phagocytosis in the presence of anti-SIRPα was calculated as a percentage of the phagocytosis in control IgG, which was defined as 100%. (ii) Anti-CD47 or matched control IgGs that had their Fc-segments coated/blocked by anti-Fc Fab₂ fragments (Fab2-anti-CD47 and Cont-Fab2-IgG, respectively) were used to opsonize myelin that was added to phagocytes. Phagocytosis of myelin opsonized by Fab₂-anti-CD47 was calculated as a percentage of phagocytosis in the presence of Cont-Fab₂-IgG, which was defined as 100%. Values of individual experiments, each performed in triplicates, as well as averages ± SE are given; two tailed p-values of significance are *** p < 0.001.

Figure 4

CD47 on myelin and serum can each promote SIRPα-dependent down-regulation of myelin phagocytosis in microglia with reduced SIRPα levels. (A) Quantitation of SIRPα levels in Balb/C microglia infected with SIRPα-shRNA (SIRPα-KD) or non-target control Luciferase-shRNA (Con-Luc) based on (B) immunoblot analyses in which SIRPα and GAPDH levels were determined. An SIRPα to GAPDH ratio in SIRPα-KD microglia was calculated as a percentage of that ratio in Con-Luc microglia, which was defined as 100%. Values of individual experiments, averages ± SE and levels of significance are given; two tailed p-value of significance is *** p < 0.001. Phagocytosis of CD47^+/+ myelin is augmented in SIRPα-KD microglia compared to phagocytosis by Con-Luc microglia in both (C) the presence and (E) the absence of serum. Phagocytosis of CD47^-/- myelin is augmented in SIRPα-KD microglia compared to phagocytosis by Con-Luc microglia in (D) the presence but not (F) the absence of serum. Phagocytosis by SIRPα-KD microglia was calculated as a percentage of phagocytosis by Con-Luc microglia, which was defined as 100% (see Methods). Values of individual experiments, each performed in triplicates, averages ± SE and levels of significance are given; two tailed p-value of significance are ** p < 0.01 and *** p < 0.001.