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Janus-like opposing roles of CD47 in autoimmune brain inflammation in humans and mice.

Han MH, Lundgren DH, Jaiswal S, Chao M, Graham KL, Garris CS, Axtell RC, Ho PP, Lock CB, Woodard JI, Brownell SE, Zoudilova M, Hunt JF, Baranzini SE, Butcher EC, Raine CS, Sobel RA, Han DK, Weissman I, Steinman L - J. Exp. Med. (2012)

Bottom Line: Immunohistochemical studies demonstrate that CD47 is expressed in normal myelin and in foamy macrophages and reactive astrocytes within active MS lesions.In vitro assays demonstrate that blocking CD47 also promotes phagocytosis of myelin and that this effect is dependent on signal regulatory protein α (SIRP-α).Depending on the cell type, location, and disease stage, CD47 has Janus-like roles, with opposing effects on EAE pathogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA 94305, USA. mayhan@stanford.edu

ABSTRACT
Comparison of transcriptomic and proteomic data from pathologically similar multiple sclerosis (MS) lesions reveals down-regulation of CD47 at the messenger RNA level and low abundance at the protein level. Immunohistochemical studies demonstrate that CD47 is expressed in normal myelin and in foamy macrophages and reactive astrocytes within active MS lesions. We demonstrate that CD47(-/-) mice are refractory to experimental autoimmune encephalomyelitis (EAE), primarily as the result of failure of immune cell activation after immunization with myelin antigen. In contrast, blocking with a monoclonal antibody against CD47 in mice at the peak of paralysis worsens EAE severity and enhances immune activation in the peripheral immune system. In vitro assays demonstrate that blocking CD47 also promotes phagocytosis of myelin and that this effect is dependent on signal regulatory protein α (SIRP-α). Immune regulation and phagocytosis are mechanisms for CD47 signaling in autoimmune neuroinflammation. Depending on the cell type, location, and disease stage, CD47 has Janus-like roles, with opposing effects on EAE pathogenesis.

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Down-regulation of CD47 enhances myelin and EAE immune cell phagocytosis by a SIRP-α–dependent mechanism. (A and B) Graph depicting CD47 expression in different myelin fractions measured by spectral count. (C) A representative spectra of CD47 peptide ions identified by mass spectrometry. (D) Purified human myelin fraction was stained with CSFE and incubated with mature mouse macrophages in the presence of control IgG, anti-CD47 blocking mAb, or anti–SIRP-α, and then phagocytosis of myelin was visualized by fluorescent microscopy. (E and F) CD47 blockade analysis of EAE immune cell phagocytosis. (E) Graph depicts EAE splenocytes phagocytosis in the presence of the indicated antibodies. Error bars were calculated from standard error of triplicate samples. *, P < 0.05. (F) Splenocytes from either naive or WT C57BL/6 mice immunized with myelin peptide were stained with CSFE and then pretreated with either isotype control IgG or blocking antibodies against either CD47 or SIRP-α before incubation with mature differentiated mouse macrophages. The phagocytosis index was measured by a fluorescent microscope.
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fig6: Down-regulation of CD47 enhances myelin and EAE immune cell phagocytosis by a SIRP-α–dependent mechanism. (A and B) Graph depicting CD47 expression in different myelin fractions measured by spectral count. (C) A representative spectra of CD47 peptide ions identified by mass spectrometry. (D) Purified human myelin fraction was stained with CSFE and incubated with mature mouse macrophages in the presence of control IgG, anti-CD47 blocking mAb, or anti–SIRP-α, and then phagocytosis of myelin was visualized by fluorescent microscopy. (E and F) CD47 blockade analysis of EAE immune cell phagocytosis. (E) Graph depicts EAE splenocytes phagocytosis in the presence of the indicated antibodies. Error bars were calculated from standard error of triplicate samples. *, P < 0.05. (F) Splenocytes from either naive or WT C57BL/6 mice immunized with myelin peptide were stained with CSFE and then pretreated with either isotype control IgG or blocking antibodies against either CD47 or SIRP-α before incubation with mature differentiated mouse macrophages. The phagocytosis index was measured by a fluorescent microscope.

Mentions: Engagement of CD47 to its receptor SIRP-α relays the “don’t eat me” signal and prevents cells from being phagocytosed by macrophages (Jaiswal et al., 2009). Immune regulation is one of the major mechanisms by which CD47 participates in the pathogenesis of EAE. However, other potential mechanisms such as phagocytosis may also play an important role in neuroinflammation. To test this hypothesis, we identified CD47 protein in the human brain myelin fractions by mass spectrometry. Human brain myelin was fractionated by gradient centrifugation (Menon et al., 2003). CD47 protein was colocalized in several fractions containing myelin (main band, dispersion, and pellet fractions; Fig. 6, A–C). We then tested the effects of down-regulation of CD47 on myelin phagocytosis in an in vitro assay. Purified human myelin fraction was incubated with activated mouse macrophages, in the presence of either media alone or with blocking antibodies against CD47, SIRP-α, or isotype control IgG. There was increased phagocytosis of the myelin fraction when blocked with mAb CD47 compared with media or isotype control (Fig. 6 D). Similarly, myelin phagocytosis was also increased when blocked with anti–SIRP-α. This suggested that down-regulation of CD47 promotes demyelination in a SIRP-α–dependent mechanism in the CNS, explaining a potential mechanism (among many) for worsening EAE with down-regulation of CD47.


Janus-like opposing roles of CD47 in autoimmune brain inflammation in humans and mice.

Han MH, Lundgren DH, Jaiswal S, Chao M, Graham KL, Garris CS, Axtell RC, Ho PP, Lock CB, Woodard JI, Brownell SE, Zoudilova M, Hunt JF, Baranzini SE, Butcher EC, Raine CS, Sobel RA, Han DK, Weissman I, Steinman L - J. Exp. Med. (2012)

Down-regulation of CD47 enhances myelin and EAE immune cell phagocytosis by a SIRP-α–dependent mechanism. (A and B) Graph depicting CD47 expression in different myelin fractions measured by spectral count. (C) A representative spectra of CD47 peptide ions identified by mass spectrometry. (D) Purified human myelin fraction was stained with CSFE and incubated with mature mouse macrophages in the presence of control IgG, anti-CD47 blocking mAb, or anti–SIRP-α, and then phagocytosis of myelin was visualized by fluorescent microscopy. (E and F) CD47 blockade analysis of EAE immune cell phagocytosis. (E) Graph depicts EAE splenocytes phagocytosis in the presence of the indicated antibodies. Error bars were calculated from standard error of triplicate samples. *, P < 0.05. (F) Splenocytes from either naive or WT C57BL/6 mice immunized with myelin peptide were stained with CSFE and then pretreated with either isotype control IgG or blocking antibodies against either CD47 or SIRP-α before incubation with mature differentiated mouse macrophages. The phagocytosis index was measured by a fluorescent microscope.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC3405500&req=5

fig6: Down-regulation of CD47 enhances myelin and EAE immune cell phagocytosis by a SIRP-α–dependent mechanism. (A and B) Graph depicting CD47 expression in different myelin fractions measured by spectral count. (C) A representative spectra of CD47 peptide ions identified by mass spectrometry. (D) Purified human myelin fraction was stained with CSFE and incubated with mature mouse macrophages in the presence of control IgG, anti-CD47 blocking mAb, or anti–SIRP-α, and then phagocytosis of myelin was visualized by fluorescent microscopy. (E and F) CD47 blockade analysis of EAE immune cell phagocytosis. (E) Graph depicts EAE splenocytes phagocytosis in the presence of the indicated antibodies. Error bars were calculated from standard error of triplicate samples. *, P < 0.05. (F) Splenocytes from either naive or WT C57BL/6 mice immunized with myelin peptide were stained with CSFE and then pretreated with either isotype control IgG or blocking antibodies against either CD47 or SIRP-α before incubation with mature differentiated mouse macrophages. The phagocytosis index was measured by a fluorescent microscope.
Mentions: Engagement of CD47 to its receptor SIRP-α relays the “don’t eat me” signal and prevents cells from being phagocytosed by macrophages (Jaiswal et al., 2009). Immune regulation is one of the major mechanisms by which CD47 participates in the pathogenesis of EAE. However, other potential mechanisms such as phagocytosis may also play an important role in neuroinflammation. To test this hypothesis, we identified CD47 protein in the human brain myelin fractions by mass spectrometry. Human brain myelin was fractionated by gradient centrifugation (Menon et al., 2003). CD47 protein was colocalized in several fractions containing myelin (main band, dispersion, and pellet fractions; Fig. 6, A–C). We then tested the effects of down-regulation of CD47 on myelin phagocytosis in an in vitro assay. Purified human myelin fraction was incubated with activated mouse macrophages, in the presence of either media alone or with blocking antibodies against CD47, SIRP-α, or isotype control IgG. There was increased phagocytosis of the myelin fraction when blocked with mAb CD47 compared with media or isotype control (Fig. 6 D). Similarly, myelin phagocytosis was also increased when blocked with anti–SIRP-α. This suggested that down-regulation of CD47 promotes demyelination in a SIRP-α–dependent mechanism in the CNS, explaining a potential mechanism (among many) for worsening EAE with down-regulation of CD47.

Bottom Line: Immunohistochemical studies demonstrate that CD47 is expressed in normal myelin and in foamy macrophages and reactive astrocytes within active MS lesions.In vitro assays demonstrate that blocking CD47 also promotes phagocytosis of myelin and that this effect is dependent on signal regulatory protein α (SIRP-α).Depending on the cell type, location, and disease stage, CD47 has Janus-like roles, with opposing effects on EAE pathogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA 94305, USA. mayhan@stanford.edu

ABSTRACT
Comparison of transcriptomic and proteomic data from pathologically similar multiple sclerosis (MS) lesions reveals down-regulation of CD47 at the messenger RNA level and low abundance at the protein level. Immunohistochemical studies demonstrate that CD47 is expressed in normal myelin and in foamy macrophages and reactive astrocytes within active MS lesions. We demonstrate that CD47(-/-) mice are refractory to experimental autoimmune encephalomyelitis (EAE), primarily as the result of failure of immune cell activation after immunization with myelin antigen. In contrast, blocking with a monoclonal antibody against CD47 in mice at the peak of paralysis worsens EAE severity and enhances immune activation in the peripheral immune system. In vitro assays demonstrate that blocking CD47 also promotes phagocytosis of myelin and that this effect is dependent on signal regulatory protein α (SIRP-α). Immune regulation and phagocytosis are mechanisms for CD47 signaling in autoimmune neuroinflammation. Depending on the cell type, location, and disease stage, CD47 has Janus-like roles, with opposing effects on EAE pathogenesis.

Show MeSH
Related in: MedlinePlus