Interferon-independent, human immunodeficiency virus type 1 gp120-mediated induction of CXCL10/IP-10 gene expression by astrocytes in vivo and in vitro

Abstract

The CXC chemokine gamma interferon (IFN-gamma)-inducible protein CXCL10/IP-10 is markedly elevated in cerebrospinal fluid and brain of individuals infected with human immunodeficiency virus type 1 (HIV-1) and is implicated in the pathogenesis of HIV-associated dementia (HAD). To explore the possible role of CXCL10/IP-10 in HAD, we examined the expression of this and other chemokines in the central nervous system (CNS) of transgenic mice with astrocyte-targeted expression of HIV gp120 under the control of the glial fibrillary acidic protein (GFAP) promoter, a murine model for HIV-1 encephalopathy. Compared with wild-type controls, CNS expression of the CC chemokine gene CCL2/MCP-1 and the CXC chemokine genes CXCL10/IP-10 and CXCL9/Mig was induced in the GFAP-HIV gp120 mice. CXCL10/IP-10 RNA expression was increased most and overlapped the expression of the transgene-encoded HIV gp120 gene. Astrocytes and to a lesser extent microglia were identified as the major cellular sites for CXCL10/IP-10 gene expression. There was no detectable expression of any class of IFN or their responsive genes. In astrocyte cultures, soluble recombinant HIV gp120 protein was capable of directly inducing CXCL10/IP-10 gene expression a process that was independent of STAT1. These findings highlight a novel IFN- and STAT1-independent mechanism for the regulation of CXCL10/IP-10 expression and directly link expression of HIV gp120 to the induction of CXCL10/IP-10 that is found in HIV infection of the CNS. Finally, one function of IP-10 expression may be the recruitment of leukocytes to the CNS, since the brain of GFAP-HIV gp120 mice had increased numbers of CD3(+) T cells that were found in close proximity to sites of CXCL10/IP-10 RNA expression.

FIG. 1

(A) Chemokine mRNA expression in the brain of GFAP-HIV gp120 transgenic mice at different ages. In this representative experiment, poly(A)⁺ RNA was isolated from the whole brain of wild-type or transgenic mice, and 1 μg was analyzed by RPA as described in Materials and Methods. (B) Quantitative analysis of CXCL10/IP-10 and CCL2/MCP-1 gene expression. Densitometric analysis of each lane was performed on scanned autoradiographs using NIH Image 1.57 software. Expression of different chemokine mRNAs was normalized to the respective expression of rp132 mRNA, and the mean plus standard deviation was calculated using Microsoft Excel 98.

FIG. 2

(A) Expression of mRNAs for CXCL9/Mig, CXCL10/IP-10, CXCL11/I-TAC, and their common receptor CXCR3 in the brain of GFAP-HIV gp120 transgenic mice. Poly(A) RNA was isolated from the whole brain of wild-type (wt) or transgenic mice at 4 and 15 month of age, and 1 μg was analyzed by RPA as described in Materials and Methods. (B) Quantitative analysis of RPA autoradiographs was performed as described for Fig. 1.

FIG. 3

Anatomical localization of CXCL10/IP-10 and HIV gp120 RNA in the brain of GFAP-HIV gp120 transgenic mice. Mice were anesthetized, and the brains removed, processed, and analyzed by in situ hybridization as described previously using ³³P-labeled-gp120 IIIB and CXCL10/IP-10 riboprobes (4). Examples of overlap in the brain for the hybridization pattern of these two different probes are represented by arrows (expression) and asterisks (no expression).

FIG. 4

Cellular localization of CXCL10/IP-10 RNA in the brain of GFAP-HIV gp120 transgenic mice. After in situ hybridization, some sections were further subjected to dual-label analysis to identify the cellular localization for CXCL10/IP-10 RNA expression. Immunostaining for GFAP (to label astrocytes) and neurofilament (NF; to label neurons) or binding of tomato lectin (to label microglia) was performed after in situ hybridization as described in Materials and Methods. Top panels, immunohistochemistry for wild-type animals. In the GFAP-HIV gp120 transgenic mice, numerous cells positive for CXCL10/IP-10 colabeled with the GFAP-positive cells (left panel, arrows). Few cells positive for the tomato lectin and representing the microglia were colabeled with CXCL10/IP-10 RNA expression (right panel, arrowhead). In contrast, no neurofilament-positive cells that were also positive for CXCL10/IP-10 were detected (middle panel).

FIG. 5

Analysis of IFN mRNA expression in brain from GFAP-HIV gp120 mice of different ages or GFAP-IFN-α transgenic mice (lane A) and the spleen from a mouse infected with LCMV and killed at day 3 after infection (lane B). Poly(A)⁺ RNA was isolated from the whole brain of wild-type or GFAP-HIV gp120 mice at 4 and 15 month of age and from symptomatic GFAP-IFN-α mice, and 1 μg was analyzed by RPA as described in Materials and Methods.

FIG. 6

Expression of various IFN-regulated genes in the brain of GFAP-IFN-α (A) and GFAP-HIV gp120 (B) transgenic mice. Poly(A)⁺ RNA was isolated from the whole brain of wild-type (wt) or transgenic mice at 4 and 15 months of age for the GFAP-HIV gp120 mice and from symptomatic GFAP-IFN-α mice, and 1 μg was analyzed by RPA as described in Materials and Methods. Quantitative analysis of RPA autoradiographs (C) was performed as described above for Fig. 1.

FIG. 7

(A) Induction of CXCL10/IP-10 RNA expression in astrocyte cell culture by soluble recombinant HIV gp120 protein. The astrocyte cultures were incubated with medium alone or with different doses (0.02 to 1 nM) of HIV gp120 protein (gp120 IIIB) for 6 h. Specificity for gp120 IIIB was shown by incubation of gp120 IIIB with an anti-gp120 (α-gp120) neutralizing antibody (B) for 1 h at room temperature prior to addition to the astrocyte culture. A control isotype antibody (human IgG1) was used in parallel (α-IgG). LPS, lipopolysaccharide.

FIG. 8

Examination of role of STAT1 in induction of CXCL10/IP-10 RNA expression by soluble recombinant HIV gp120 protein. Astrocyte cultures derived from wild-type or STAT1-null mice were incubated in medium alone or with gp120 IIIB or murine recombinant IFN-γ proteins for 6 h. Each treatment was done in triplicate. Following treatment, the astrocyte cultures were washed twice in PBS, and RNA was extracted with Trizol reagent according to the manufacturer's instructions. For RPA, 5 μg of total RNA was analyzed as described in Materials and Methods. Quantitative analysis of RPA autoradiographs was performed as described for Fig. 1.

FIG. 9

(A) Analysis of numbers of CD3⁺ cells in the brain. Six different brain sections containing specimens from nontransgenic littermates or GFAP-HIV gp120 mice were immunostained for CD3 and analyzed by two different individuals. The average cell count was calculated, and statistical significance was determined using the Student t-test. The presence of CD3⁺ T cells in the olfactory bulb (∗, P < 0.07), neocortex (∗, P < 0.00007), and subcortex (∗, P < 0.002) was significantly increased compared with the wild-type mice. (B) Colocalization of infiltrating CD3⁺ lymphocytes and CXCL10/IP-10 RNA in the brain of GFAP-HIV gp120 transgenic mice. Sections from a wild-type control and a GFAP-HIV gp120 mouse were hybridized with the CXCL10/IP-10 antisense probe and immunostained for CD3 as described in Materials and Methods. Original magnification, ×450.