Blood-brain barrier tight junction disruption in human immunodeficiency virus-1 encephalitis

Abstract

The blood-brain barrier (BBB) plays a critical role in regulating cell trafficking through the central nervous system (CNS) due to several unique anatomical features, including the presence of interendothelial tight junctions that form impermeable seals between the cells. Previous studies have demonstrated BBB perturbations during human immunodeficiency virus encephalitis (HIVE); however, the basis of these permeability changes and its relationship to infiltration of human immunodeficiency virus type 1 (HIV-1)-infected monocytes, a critical event in the pathogenesis of the disease, remains unclear. In this study, we examined CNS tissue from HIV-1-seronegative patients and HIV-1-infected patients, both with and without encephalitis, for alterations in BBB integrity via immunohistochemical analysis of the tight junction membrane proteins, occludin and zonula occludens-1 (ZO-1). Significant tight junction disruption (P < 0.001), as demonstrated by fragmentation or absence of immunoreactivity for occludin and ZO-1, was observed within vessels from subcortical white matter, basal ganglia, and, to a lesser extent, cortical gray matter in patients who died with HIVE. These alterations were also associated with accumulation of activated, HIV-1-infected brain macrophages, fibrinogen leakage, and marked astrocytosis. In contrast, no significant changes (P > 0.05) were observed in cerebellar tissue from patients with HIVE compared to HIV-seronegative patients or HIV-1-infected patients without encephalitis. Our findings demonstrate that tight junction disruption is a key feature of HIVE that occurs in regions of histopathological alterations in association with perivascular accumulation of activated HIV-1-infected macrophages, serum protein extravasation, and marked astrocytosis. We propose that disruption of this key BBB structure serves as the main route of HIV-1-infected monocyte entry into the CNS.

Figure 1.

The distribution of tight junction proteins is disrupted in HIVE. A: In control sections, blood vessels show strong immunoreactivity for the tight junction-associated protein, occludin, in a continuous, interendothelial staining pattern. B: In HIV sections, isolated medium-sized vessels are weakly immunoreactive for occludin (arrowhead). C–F: In HIVE sections, numerous small and medium-sized vessels exhibit decreased to absent occludin immunoreactivity (C) in association with microglial nodules (D and F), mononuclear cell infiltrates (D and E), and multinucleated giant cells (E). Similar changes were observed in ZO-1-immunostained sections. D and E: Original magnification, ×200. A–C and F: Original magnification, ×400. CGM, cortical gray matter; CWM, cortical white matter; DGM, deep gray matter.

Figure 2.

Semiquantitative analysis of blood vessel immunoreactivity for the endothelial cell marker, CD34 (A), the tight junction proteins, occludin (B) and ZO-1 (C), and the intravascular serum protein, fibrinogen (D), in sections from control, HIV, and HIVE brains (average and standard error of five fields of blood vessels per 20× objective). Two-tailed P values of <0.01 (*) and <0.0001 (**) are noted.

Figure 3.

Tight junction protein disruption is associated with marked serum protein extravasation and astrocytosis in HIVE. A–C: In control sections, blood vessels display intact, ZO-1-positive tight junctions (red fluorochrome, A and B) and retain the intravascular serum protein, fibrinogen (green fluorochrome, A). Weakly staining, GFAP-reactive cell processes surround the intact BBB (green fluorochrome, B and C), however, do not overlap with either ZO-1-positive tight junctions (red fluorochrome, B) or intravascular fibrinogen (red fluorochrome, C). D–F: In HIV sections, scattered blood vessels with ZO-1-fragmented tight junctions (red fluorochrome, D) and, less often, intact ZO-1-positive tight junctions (red fluorochrome, E) overlap (yellow) with GFAP-reactive cell processes (green fluorochrome, D and E). In addition, GFAP-reactive astrocytes (green fluorochrome, F) colocalize focally (yellow) with extravasated fibrinogen (red fluorochrome, F) from the focally disrupted BBB. G–I: In HIVE sections, abundantly extravasated fibrinogen (green fluorochrome, G) and numerous hypertrophic, GFAP-reactive astrocytes (green fluorochrome, H), surround ZO-1-disrupted blood vessels (red fluorochrome, G and H). Colocalization of the two markers with ZO-1 is not observed due to extensive disruption of the tight junction protein (red fluorochrome, G and H). GFAP-reactive astrocytes (green fluorochrome, I), on the other hand, colocalize extensively (yellow) with extravasated fibrinogen (red fluorochrome, I) from the markedly disrupted BBB. Similar changes were observed in occludin immunostained sections. Double-label immunofluorescence laser confocal microscopy; original magnification, ×600. CGM, cortical gray matter; CWM, cortical white matter, DGM, deep gray matter.

Figure 4.

Tight junction protein disruption and BBB permeability are associated with mononuclear cell infiltrates and microglial nodules in HIVE. A: In control sections, infrequent blood vessels are infiltrated (yellow) by CD68-positive mononuclear cells (green fluorochrome) with no apparent disruption of ZO-1-positive tight junctions (red fluorochrome). B, C, and G: In HIV sections, increased CD68-positive mononuclear cells and microglial processes (green fluorochrome, B) are present among and colocalize focally (arrowheads, yellow) with ZO-1-positive blood vessels (red fluorochrome, B). In addition, scattered ZO-1-fragmented (red fluorochrome, C) and fibrinogen-permeable blood vessels (red fluorochrome, G) are infiltrated (arrowheads, yellow) by CD68-positive mononuclear cells (green fluorochrome, C and G). D–F, H, and I: In HIVE sections, numerous ZO-1-disrupted (red fluorochrome, D) and fibrinogen-permeable blood vessels (red fluorochrome, H) are associated with CD68-positive mononuclear cell aggregates and microglial nodules (green fluorochrome, D and H) and microglial nodules. CD68-reactive cells (green fluorochrome, E, F, and I) both surround (E) and infiltrate (yellow, F and I) the disrupted blood vessels (red fluorochrome: ZO-1, E and F; fibrinogen, I). Similar changes were observed in occludin-immunostained sections. B, D, G, and H: Double-label immunofluorescence laser confocal microscopy; original magnification, ×200. A and C: Double-label immunofluorescence laser confocal microscopy; original magnification, ×400. E, F, and I: Double-label immunofluorescence laser confocal microscopy, original magnification, ×600. CGM, cortical gray matter; DGM, deep gray matter.

Figure 5.

BBB disruption in HIVE is associated with mononuclear cell expression of Class II MHC activation markers but not HIV envelope protein. In control (A) and HIV (B) sections labeled for endothelial cells (green fluorochrome), infrequent infiltrating (yellow) mononuclear cells express the MHC Class II activation marker, HLA-DR (red fluorochrome). C: In HIVE sections, perivascular and parenchymal expression of the MHC Class II activation marker (red fluorochrome) is pronounced. D: In HIV sections, gp41-positive cells (green fluorochrome) infiltrate (yellow) and surround blood vessels with intact ZO-1-positive tight junctions (red fluorochrome). E: In HIVE sections, infiltrating CD68-positive mononuclear cells (green fluorochrome) colocalize focally (yellow) with the HIV envelope protein, gp41 (red fluorochrome). F and G: In HIVE sections, gp41-positive cells (green fluorochrome) associate with both intact and fragmented (large arrowhead) ZO-1-positive tight junctions (red fluorochrome). Likewise, some fragmented tight junctions show no association with gp41-positive cells (small arrowhead, F). H–I: In HIVE sections, fibrinogen-permeable blood vessels (red fluorochrome) are also variably associated with gp41-positive cells (green fluorochrome). A–D, F, and G: Double-label immunofluorescence microscopy; original magnification, ×200. E: Double-label immunofluorescence laser confocal microscopy, original magnification, ×600. H and I: Double-label immunofluorescence laser confocal microscopy, original magnification, ×200. CGM, cortical gray matter; DGM, deep gray matter.