CD163, a marker of perivascular macrophages, is up-regulated by microglia in simian immunodeficiency virus encephalitis after haptoglobin-hemoglobin complex stimulation and is suggestive of breakdown of the blood-brain barrier

Abstract

Macrophages and microglia are the major cell types infected by human immunodeficiency virus and simian immunodeficiency virus (SIV) in the central nervous system. Microglia are likely infected in vivo, but evidence of widespread productive infection (ie, presence of viral RNA and protein) is lacking. This conclusion is controversial because, unlike lymphocytes, macrophages and microglia cannot be discreetly immunophenotyped. Of particular interest in the search for additional monocyte/macrophage-lineage cell markers is CD163; this receptor for haptoglobin-hemoglobin (Hp-Hb) complex, which forms in plasma following erythrolysis, is expressed exclusively on cells of monocyte/macrophage lineage. We examined CD163 expression in vitro and in vivo by multiple techniques and at varying times after SIV infection in macaques with or without encephalitis. In normal and acutely SIV-infected animals, and in SIV-infected animals without encephalitis, CD163 expression was detected in cells of monocyte/macrophage lineage, including perivascular macrophages, but not in parenchymal microglia. However, in chronically infected animals with encephalitis, CD163 expression was detected in activated microglia surrounding SIV encephalitis lesions in the presence of Hp-Hb complex, suggesting leakage of the blood-brain barrier. CD163 expression was also induced on microglia in vitro after stimulation with Hp-Hb complex. We conclude that CD163 is a selective marker of perivascular macrophages in normal macaques and during the early phases of SIV infection; however, later in infection in animals with encephalitis, CD163 is also expressed by microglia, which are probably activated as a result of vascular compromise.

Figure 1

Immunohistochemistry of brain macrophage populations. In normal brain immunohistochemistry reveals CD163 expression by meningeal macrophages (A), choroid plexus macrophages (B), and perivascular macrophages (C), but not cells morphologically compatible with microglia. D: Immunohistochemistry for Iba1 labels resting microglia throughout the parenchyma characterized by elongated nuclei with scanty perikaryon and multiple cytoplasmic extensions. E: Double-label immunohistochemistry in normal brain clearly shows that CD163⁺ perivascular macrophages (blue, vector blue chromogen) and Iba1⁺ microglia (red, AEC chromogen) are mutually exclusive. F: In contrast to normal brain, immunohistochemistry for CD163 in animals with SIVE reveals focally extensive aggregates of cells morphologically consistent with microglia in the areas surrounding SIVE lesions. Original magnifications: ×200 (A, B, F); ×1000 (C); ×400 (D).

Figure 2

Multilabel confocal microscopy of CD163 in SIVE brain. Individual channels are on the left with a larger merged image on the right. A: Triple-label confocal microscopy showing co-localization of CD163 (green) and Iba1 (red), indicating that CD163 is expressed on microglia during SIVE (CD163 with Alexa 488, green; Iba1 with Alexa 568, red; and cell nuclei with Topro3, co-localization of both markers results in a yellow-orange). B: Double-label confocal microscopy showing that CD163 is expressed by individual macrophages and multinucleated giant cells, and these cells are often infected with SIV (CD163 with Alexa 488, green; SIV in situ hybridization with Fast Red, red; and differential interference contrast, DIC). C: Double-label confocal microscopy for CD163 and HLA-DR to assess immune activation in SIVE. Extensive co-localization of CD163 and HLA-DR can be observed. In addition to CD163⁺HLA-DR⁺ cells with a round to oval morphology within the lesion, cells with a more ramified morphology are present at the margins of the lesion. These cells are consistent with parenchymal microglia (CD163 with Alexa 488, green; and HLA-DR with Alexa 568, red).

Figure 3

Multilabel confocal microscopy demonstrating co-localization of CD163, Iba1, and HLA-DR indicative of CD163 expression on activated microglia. Images for individual channels from the same field (Iba1 with Alexa 488, green; HLA-DR with Alexa 568, red; and CD163 with Alexa 633, blue) are shown on the left and a larger merged image containing all three channels showing co-localization of the three markers is on the right. Microglia (Iba1⁺ cells) expressing HLA-DR appear yellow, CD163⁺ cells expressing HLA-DR appear purple, and the co-localization of CD163 and Iba1 appear light blue. Note that the co-localization of all three colors indicates that CD163 is expressed by activated microglia.

Figure 4

Multilabel confocal microscopy for Hp in animals without (A) or with (B–D) encephalitis. A: In normal animals, acutely SIV-infected animals and SIV-infected animals without SIVE, labeling for Hp was limited to the lumen of vessels. Images for individual channels (Hp with Alexa 488, green; CD163 with Alexa 568, red; and cell nuclei with Topro3, blue and DIC) are shown on the left and a larger merged image is on the right. B: Hp is present in the brain parenchyma and co-localizes with microglia (Iba1⁺) resulting in an orange-yellow color. Images for individual channels [Hp with Alexa 488, green; Iba1 (chicken polyclonal) with Alexa 568, red; and DIC] are shown on the left and a larger merged image on the right. C: Similar to B, this image shows the presence of Hp that co-localizes with CD163 in cells morphologically compatible with microglia giving an orange-yellow color. Images for individual channels (Hp with Alexa 488, green; CD163 with Alexa 568, red) are shown on the left and a larger merged image is on the right. D: Co-localization of Hp (green), CD163 (red), and Iba1 (blue) can be seen in this image. Images for individual channels from the same field [Hp with Alexa 488, green; CD163 with Alexa 568, red; and Iba1 (chicken polyclonal) with Alexa 633, blue] are shown on the left with a larger merged image on the right.

Figure 5

CD163 protein induction in vitro in microglia. A: Untreated microglia in an eight-well chamber labeled with Iba1 (Iba1 with Alexa 568 red). B: Microglial cells were either treated for 2 hours with Hp-Hb complexes and cultured in vitro for 6, 12, or 18 hours or constantly treated for 18 hours. After incubation, cell homogenates were immunoprecipitated and immunoblotted with a rabbit anti-human CD163 polyclonal antibody. The figure inset on the top of the histogram shows protein bands of CD163 and β-actin used as a loading control for Western blotting. The graph depicts the density value for each treatment as a ratio of CD163 to β-actin levels. 0 = no addition of Hp-Hb; 2 + 6 hours = treated 2 hours with Hp-Hb, wash, and 6 hours of incubation; 2 + 12 = treated 2 hours with Hp-Hb, wash, and 12 hours of incubation; 2 + 18 = treated 2 hours with Hp-Hb, wash, and 18 hours of incubation; 18 hours constant = 18 hours of incubation with Hp-Hb. C: Microglia in an eight-well chamber labeled for Iba1 (red) and CD163 (green) after treatment with the Hp-Hb complex showing co-labeling for both Iba1 and CD163. CD163 expression is predominantly on the cell surface in green. This demonstrates up-regulation of CD163 in cultured microglia in response to stimulation with Hp-Hb complex. (Iba1 with Alexa 568, red; CD163 with Alexa 488, green; and cell nuclei with Topro3, blue).