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. 1999 Nov;155(5):1599-611.
doi: 10.1016/S0002-9440(10)65476-4.

Microglial and astrocyte chemokines regulate monocyte migration through the blood-brain barrier in human immunodeficiency virus-1 encephalitis

Y Persidsky  1 , A Ghorpade, J Rasmussen, J Limoges, X J Liu, M Stins, M Fiala, D Way, K S Kim, M H Witte, M Weinand, L Carhart, H E Gendelman
Affiliations

Microglial and astrocyte chemokines regulate monocyte migration through the blood-brain barrier in human immunodeficiency virus-1 encephalitis

Y Persidsky et al. Am J Pathol. 1999 Nov.

Abstract

The numbers of immune-activated brain mononuclear phagocytes (MPs) affect the progression of human immunodeficiency virus (HIV)-1-associated dementia (HAD). Such MPs originate, in measure, from a pool of circulating monocytes. To address the mechanism(s) for monocyte penetration across the blood-brain barrier (BBB), we performed cross-validating laboratory, animal model, and human brain tissue investigations into HAD pathogenesis. First, an artificial BBB was constructed in which human brain microvascular endothelial and glial cells-astrocytes, microglia, and/or monocyte-derived macrophages (MDM)-were placed on opposite sides of a matrix-coated porous membrane. Second, a SCID mouse model of HIV-1 encephalitis (HIVE) was used to determine in vivo monocyte blood-to-brain migration. Third, immunohistochemical analyses of human HIVE tissue defined the relationships between astrogliosis, activation of microglia, virus infection, monocyte brain infiltration, and beta-chemokine expression. The results, taken together, showed that HIV-1-infected microglia increased monocyte migration through an artificial BBB 2 to 3.5 times more than replicate numbers of MDM. In the HIVE SCID mice, a marked accumulation of murine MDM was found in areas surrounding virus-infected human microglia but not MDM. For human HIVE, microglial activation and virus infection correlated with astrogliosis, monocyte transendothelial migration, and beta-chemokine expression. Pure cultures of virus-infected and activated microglia or astrocytes exposed to microglial conditioned media produced significant quantities of beta-chemokines. We conclude that microglial activation alone and/or through its interactions with astrocytes induces beta-chemokine-mediated monocyte migration in HAD.

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Figures

Figure 1.
Figure 1.
Effect of microglia/MDM on monocyte migration across a model of the BBB. HIV-1ADA-infected/control uninfected MDM (7 × 105) or virus-infected/control microglia cells (105) were placed in the lower chamber. Fresh blood monocytes were applied to the upper chamber of the BBB model (105). Numbers reflect migrated monocytes attached to coverslips in the lower chamber at 48 hours. Twenty random fields (objective ×20) of each coverslip were assayed. Experiments were performed in duplicate. Data presented (mean ± SE) are from one of two independent experiments.
Figure 2.
Figure 2.
Production of chemokines by microglia and MDM. Adherent MDM and microglia were cultured on 96-well plates at a density of 10 and 5 × 10 cells/well, respectively, for 7 days before infection with HIV-1ADA at multiplicity of infection of 0.1. Replicate microglia and monocytes were stimulated with LPS (1 g/ml), and culture fluids from infected or uninfected cells were collected 24 hours later. The MCP-1 (A), MIP-1α (B), MIP-1β (C), and RANTES (D) were assayed using the Quantikine ELISA kits (R&D Systems). The levels of chemokines were normalized to cell numbers by measuring cell viability at the end of the sample collection by the MTT assay. The normalized values of chemokines per 10 cells were used to perform statistical analysis by the two-tailed Student’s t-test. Data presented in a log scale (mean ± SE) are from one of two independent experiments. Comparison of microglia- versus MDM-treated similarly yielded statistically significant differences (P < 0.0001).
Figure 3.
Figure 3.
Microglia-mediated changes in SCID mice with HIV-1 encephalitis. Equal numbers of HIV-1-infected MDM (A) and microglia (B) were stereotactically inoculated into basal ganglia. Both MDM (C) and microglia (D) express high levels of HIV-1 p24 antigen in their cytoplasm. Pronounced accumulation of mouse macrophages and microglia is found in and around the location of virus-infected human microglia (F). This is infrequently observed in mouse brains with HIV-1-infected MDM (E). More pronounced astrogliosis (GFAP immunostaining) is detected in areas contained microglia (H) as compared with MDM (G). A–D, G and H and G and B, D, and H present serial coronal sections immunostained with anti-CD68 (A and B), HIV-1 p24 (C and D), and GFAP antibodies (G and H). E and F are stained with biotinylated Griffonia simplicifolica lectin-isolectin B4. Primary Abs or lectin-stained cells are detected by Vectastain Elite Kit with DAB as a substrate. Tissue sections were counterstained with Mayer’s hematoxylin. Original magnifications, ×200 (A-F) and ×100 (G and H).
Figure 4.
Figure 4.
HIV-1 infection and immune activation of microglia in HIVE. Significant accumulation of monocytes (CD68-positive) around microvessels in the subcortical white matter (A) coincides with HIV-1 infection of microglia (HIV-1 p24 positive) (B) and its immune activation (as detected by HLA-DR expression) (C). Same areas of the brain tissue are characterized by prominent astrogliosis as identified by GFAP immunoreactivity (D). HIV-1-infected multinucleated giant cells (E) express markers of immune activation (HLA-DR-positive, F) and are especially prominent in zones of significant monocyte infiltration. A-D and E-F present serial sections of brain tissue from cases 1 and 2, respectively. Sections are immunostained with anti-CD68 (A), HIV-1 p24 (B and E), HLA-DR (C and F), and GFAP antibodies (D). Primary Abs are detected by Vectastain Elite Kit with DAB as a substrate. Tissue sections were counterstained with Mayer’s hematoxylin. Original magnification, ×200.
Figure 5.
Figure 5.
Microglia and reactive astrocytes express MCP-1 in HIVE. In severe HIVE (case 3), microglial cells (red) expressing MCP-1 (green) are stained yellow in microglial nodules (A) whereas control brain tissue (case 16) without HIV-1 infection shows no MCP-1 in microglia (red; B). Part of reactive astrocytes (red) in severe HIVE (case 3) express MCP-1 (green) resulting in yellow cytoplasmic staining, whereas astrocytes (red) are MCP-1-negative in controls (case 16). MCP-1 and microglia are identified by double immunolabeling on frozen sections with antibodies to MCP-1 (fluoroscein isothiocyanate) and HAM-56 (TRITC) (A and B), respectively. MCP-1 expression in astrocytes was detected with antibodies to MCP-1 (fluoroscein isothiocyanate) and GFAP (rhodamine) (C and D). Antigen expression was detected by indirect immunofluorescence. Original magnification, ×200.
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