CD163 identifies perivascular macrophages in normal and viral encephalitic brains and potential precursors to perivascular macrophages in blood

Abstract

Perivascular macrophages are uniquely situated at the intersection between the nervous and immune systems. Although combined myeloid marker detection differentiates perivascular from resident brain macrophages (parenchymal microglia), no single marker distinguishes perivascular macrophages in humans and mice. Here, we present the macrophage scavenger receptor CD163 as a marker for perivascular macrophages in humans, monkeys, and mice. CD163 was primarily confined to perivascular macrophages and populations of meningeal and choroid plexus macrophages in normal brains and in brains of humans and monkeys with human immunodeficiency virus or simian immunodeficiency virus (SIV) encephalitis. Scattered microglia in SIV encephalitis lesions and multinucleated giant cells were also CD163 positive. Consistent with prior findings that perivascular macrophages are primary targets of human immunodeficiency virus and SIV, all SIV-infected cells in the brain were CD163 positive. Using fluorescent dyes that definitively and selectively label perivascular macrophages in vivo, we confirmed that dye-labeled simian perivascular macrophages were CD163 positive and able to repopulate the central nervous system within 24 hours. Flow cytometric studies demonstrated a subset of monocytes (CD163(+)CD14(+)CD16(+)) that were immunophenotypically similar to brain perivascular macrophages. These findings recognize CD163(+) blood monocytes/macrophages as a source of brain perivascular macrophages and underscore the utility of this molecule in studying the biology of perivascular macrophages and their precursors in humans, monkeys, and mice.

Figure 1

CD163 expression in HIV encephalitis brains. A: Perivascular macrophages in brains of control uninfected humans selectively labeled with anti-human CD163 antibody. B: Association of CD163⁺ cells (DAB; brown) with Glut-1⁺ CNS vessels (Fast Red; red) in HIVE brains. C: Accumulation of CD163⁺ cells in HIVE lesions. CD163 immunoreactivity (DAB; brown) was found on CNS perivascular macrophages and intimately associated with Glut-1⁺ CNS vessels (Fast Red; red). MNGCs and populations of macrophages in the meninges and choroid plexus were also stained with anti-CD163 antibody (data not shown). Original magnifications, ×400 (A and C) and ×200 (B).

Figure 2

SIV-infected cells in the CNS are CD163 positive. A: Brains of control uninfected macaques stained with anti-human CD163 antibody. B: Association of CD163⁺ cells with CNS vessels in SIVE brains. C: Accumulation of CD163⁺ cells in SIVE lesions. Virtually all CD163⁺ cells, although morphologically diverse, were found intimately associated with Glut-1⁺ vessels (Fast Red; red). Note the presence of CD163⁺ MNGCs in the SIVE lesions in C (arrow). CD163 expression levels and the number of CD163⁺ cells in SIVE brains were higher than those found in the CNS of noninfected controls. D: In situ hybridization for SIV RNA combined with immunohistochemistry for CD163. CD163⁺ (brown) and SIV RNA⁺ (blue) perivascular macrophages (arrowheads) and MNGCs (inset). Virtually all SIV RNA⁺ productively infected cells detected were CD163 positive. E: In situ hybridization for SIV RNA combined with immunofluorescence for CD163. A representative confocal image of SIV RNA⁺ (red), CD163⁺ (green) perivascular macrophages near a CNS vessel (Glut-1; blue).

Figure 3

Localization and turnover of CD163⁺ perivascular and meningeal macrophages labeled by dextran amine dye injected into the CSF of live monkeys. Confocal microscopy studies of brain sections from normal noninfected rhesus macaques injected intracisternally with fluorescent dextran amine dye (Fluoro-Emerald; green), showing localization of dye-labeled CD14⁺CD163⁺ perivascular macrophages in the perivascular space (A, B, and C). A: Dextran amine dye specifically labels CNS perivascular macrophages in normal noninfected rhesus macaques. Perivascular macrophages (green) adjacent to a CNS microvessel (Glut-1; red). Astrocytes (GFAP; yellow) and cell nuclei (ToPro3; blue). B: Dye-labeled perivascular macrophages (green) are CD163 positive (red) in CNS of a normal noninfected rhesus macaque. C: Dye-labeled perivascular macrophages (green) are CD14 positive (red). Confocal images from a noninfected animal subjected to consecutive injections of Fluoro-Emerald (green) followed by biotinylated dextran amine (BDA-10,000; visualized with streptavidin-Alexa Fluor 633; blue) 7 days later (D, E, and F). The animal was sacrificed at 24 hours after BDA-10,000 injection. D: Sequential labeling of perivascular macrophages by consecutive injections of Fluoro-Emerald (green; top leftpanel) followed 7 days later by biotinylated dextran (blue; bottom leftpanel) detects double-positive (green and blue) and single-positive (blue) perivascular macrophages, both of which are CD163 positive (red). The sequential labeling identifies two populations of CD163⁺ perivascular macrophages in perivascular spaces: perivascular macrophages that do not turnover in a 7-day period (green-blue) and perivascular macrophages that have recently arrived in the CNS (blue only). E: Sequential labeling by consecutive injections of Fluoro-Emerald (green) followed 7 days later by biotinylated dextran (blue) demonstrates double-positive (green-blue) and single-positive (blue) meningeal macrophages, all of which express CD163. The majority of cells are double positive, with few single-positive cells that have recently arrived. F: Magnification of E. High-power magnification view demonstrates double-positive (green-blue) and single-positive (blue) meningeal macrophages in the same field of meninges (horizontally flipped rear view image of E). Arrows point to the identical CD163⁺ meningeal macrophage that does not contain Fluoro-Emerald but BDA-10,000 (blue only).

Figure 4

Co-expression of CD14 and CD16 on CD163⁺ perivascular macrophages in human and monkey brains. Triple-label confocal microscopic studies of HIVE brains (A, B, C, and D) and SIVE brains (E and F) demonstrate perivascular macrophages (CD163, green) that are CD14 positive (red; A, C, and E) and CD16 positive (red; B, D, and F) next to a CNS vessel (Glut-1, blue). A large number of CD163⁺ perivascular macrophages were positive for CD14 and CD16. These CD163⁺CD14⁺CD16⁺ perivascular macrophages accumulated in the perivascular space, which was markedly enlarged (A, B, and C). B, inset: A typical HIVE lesion with a collapsed vessel at its center. CD163⁺CD14⁺CD16⁺ cells were often found within the lumen of a vessel (D and F). It is possible that such peripheral blood monocytes/macrophages were in the process of trafficking to the perivascular space.