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The Journal of Immunology, 2004, 172: 1888-1895.
Copyright © 2004 by The American Association of Immunologists

Induction of C Chemokine XCL1 (Lymphotactin/Single C Motif-1{alpha}/Activation-Induced, T Cell-Derived and Chemokine-Related Cytokine) Expression by HIV-1 Tat Protein1

Byung Oh Kim*,{dagger}, Ying Liu*,{dagger}, Betty Y. Zhou*,{dagger} and Johnny J. He2,*,{dagger},{ddagger},§

* Department of Microbiology and Immunology, {dagger} Walther Oncology Center, and {ddagger} Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202; and § Walther Cancer Institute, Indianapolis, IN 46206


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
HIV-1 Tat has been proposed as a key agent in many AIDS-related disorders, including HIV-1-associated neurological diseases. We have recently shown that Tat expression induces a significant increase in T lymphocytes in the brains of Tat transgenic mice. The CNS infiltration of T lymphocytes has been noted in AIDS patients. In the present study using this unique genetic system we attempted to understand the underlying mechanisms of Tat expression-induced infiltration of T lymphocytes by examining chemokine expression. RNase protection assay revealed that in addition to CCL2 (monocyte chemoattractant protein-1), CCL3 (macrophage inflammatory protein-1{alpha} (MIP-1{alpha})), CCL4 (MIP-1{beta}), CCL5 (RANTES), CXCL2 (MIP-2), and CXCL10 (inducing protein-10), XCL1 (lymphotactin/single C motif-1{alpha}/activation-induced, T cell-derived and chemokine-related cytokine) was identified to be up-regulated by Tat expression. XCL1 is a C chemokine and plays a specific and important role in tissue-specific recruitment of T lymphocytes. Thus, we further determined the relationship between Tat and XCL1 expression. Tat-induced XCL1 expression was further confirmed by XCL1-specific RT-PCR and ELISA. Combined in situ hybridization and immunohistochemical staining identified astrocytes, monocytes, and macrophages/microglia as XCL1-producing cells in vivo. Using human astrocytes, U87.MG cells, as an in vitro model, activation of XCL1 expression was positively correlated with Tat expression. Moreover, the XCL1 promoter-driven reporter gene assay showed that Tat-induced XCL1 expression occurred at the transcriptional level. Taken together, these results demonstrate that Tat directly trans-activated XCL1 expression and suggest potential roles of Tat-induced XCL1 expression in the CNS infiltration of T lymphocytes during HIV-1 infection and subsequent HIV-1-induced neurological diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Human immunodeficiency virus type 1 infects the CNS of a majority of AIDS patients and causes neurologic symptoms, ranging from memory loss and impaired cognitive and motor functions to dementia (1, 2, 3, 4). HIV-1-associated neuropathologies are characterized by reactive astrocytosis, cerebellar atrophy, demyelination, formation of multinucleated giant cells, neuron death, and breakdown of the blood-brain barrier (1, 2, 4, 5, 6, 7, 8, 9). HIV-1 Tat protein has been proposed as a key neuropathogenic factor in HIV-1-associated neurological diseases. Possible molecular pathways involve modulation of host gene expression by Tat protein internalized or expressed, intracellular signaling elicited by Tat binding to cell surface receptors, or both (10). Supporting these hypotheses is the evidence that HIV-1 Tat is secreted from Tat-expressing cells (11, 12, 13, 14) and HIV-infected cells (15, 16) and is capable of interacting with molecules on the cell surface and entering cells in a biologically active form (17, 18, 19).

In vivo studies have directly implicated Tat protein in HIV-induced neuropathogenesis. When injected into the brain, Tat protein causes histological changes similar to those seen in patients with HIV-induced dementia (20, 21). The levels of Tat mRNA transcript have been linked to HIV- and SIV-induced encephalitis (22). Using an inducible and brain-targeted Tat transgenic mouse model, we have recently shown that targeted Tat expression in the absence of HIV-1 infection results in neuropathologies reminiscent of those noted in the brains of AIDS patients (14). In the same study we noted that Tat expression results in increased CNS infiltration of activated T lymphocytes as well as monocytes/macrophages, but to a lesser extent. Similar results have been obtained in a rat model by direct injection of recombinant Tat protein into the brain (20). The CNS infiltration of T lymphocytes has been one of the most common and consistent neuropathological features in a number of inflammatory neurological disorders, such as encephalitis of both bacterial and viral origins, multiple sclerosis, and ischemia. For example, in the brains of AIDS patients, T cell infiltrates have often been noted (23, 24, 25), and closely correlated with HIV-1-associated encephalitis (26). Thus, it is conceivable that Tat expression-induced infiltration of activated T lymphocytes also contributes to Tat neurotoxicity.

Infiltration of monocytes/macrophages and lymphocytes is mediated mainly by the expression of chemokines and chemokine receptors. Chemokines are grouped into four subfamilies, i.e., CXC, CC, C, and CX3C, primarily based on the organization of the N-terminal conserved cysteine (C) residues (27). CXC chemokines, including the IFN-inducible 10-kDa protein CXCL10 (inducing protein-10), mainly attract neutrophils and T cells, whereas CC chemokines such as CCL2 (monocyte chemoattractant protein-1), CCL3 (macrophage inflammatory protein-1{alpha} (MIP-1{alpha})3), CCL4 (MIP-1{beta}), CCL5 (RANTES), CCL11 (eotaxin), and CXC chemokine CXCL2 (MIP-2) are chemoattractants for monocytes, eosinophils, basophils, and T cells. There are multiple members in both CXC and CC chemokine subfamilies, but only one in C chemokine subfamily, i.e., XCL1 (lymphotactin/single C motif-1{alpha}/activation-induced T cell-derived and chemokine-related cytokine) and one in CX3C subfamily, i.e., fractalkine. Unlike CXC and CC chemokines that are chemoattractant to different types of cells, both C and CX3C chemokines are highly specific for lymphocytes, in particular, T lymphocytes. In the brains of HIV-1-infected individuals, several chemokines, including CCL2, CCL3, CCL4, CXCL10, and CX3CL1 (fractalkine), have been demonstrated to be up-regulated (28, 29, 30, 31, 32). One of the chemokines, i.e., CCL2, has been shown to be directly trans-activated by HIV-1 Tat protein and positively correlated with HIV-1-associated encephalitis (33). In addition, Tat protein itself has been demonstrated to be a strong chemoattractant for monocytes (34, 35, 36, 37). Indeed, the chemoattractant activity of Tat protein has been shown to modulate cytokines and growth factor production, which subsequently leads to recruitment of more monocytes/macrophages (38, 39, 40, 41).

In view of the increased CNS infiltration of activated T lymphocytes, Tat chemoattractant activity, and chemokine expression induced by Tat, we examined chemokine expression in the brain of the unique inducible and brain-targeted Tat transgenic mouse model. Besides those chemokines previously identified to be up-regulated by Tat, our results have shown for the first time that Tat expression up-regulated XCL1 expression and suggest potential involvement of XCL1 in the CNS infiltration of T lymphocytes and eventual neuronal injury in the brains of AIDS patients.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Inducible and brain-targeted HIV-1 Tat transgenic mice and treatment of doxycycline (Dox)

The creation and genotyping of the inducible and brain-targeted HIV-1 Tat transgenic mice were described previously (14). All mice were housed under pathogen-free conditions at Indiana University School of Medicine Animal Care and Use Center, in accordance with the National Institutes of Health and Animal Care and Use Center institutional guidelines for animal care. To express Tat86, inducible Tat transgenic and wild-type mice (21 days old) were given Dox (Sigma-Aldrich, St. Louis, MO)-containing drinking water at a concentration of 6 mg/ml in light-protected bottles for 7 days. Empty vehicle treatment was included as a control. Mice were sacrificed on the last day, i.e., postnatal day 28, of Dox treatment, and the brain were removed and divided sagittally. One hemibrain was processed for RNA isolation analysis. The other hemibrain was fixed overnight in PBS-buffered 4% paraformaldehyde and processed for paraffin embedding.

RNase protection assay (RPA)

The multiprobe RPA was performed using the mCK-5 probe set (BD PharMingen, San Diego, CA) according to the manufacturer’s instructions. Total RNAs from the brain were isolated using the TRIzol reagents (Life Technologies, Gaithersburg, MD) and were used in the RPA assay. Briefly, 20 µg of RNA was hybridized with a 32P-labeled RNA probe (3.6 x 105 cpm/sample) prepared using the RiboQuant in vitro transcription kit (BD PharMingen). After hybridization, the samples were treated with RNases A and T1, and the RNases were inactivated using the proteinase K mixture (390 µl of proteinase K buffer, 30 µl of proteinase K, and 15 µg of yeast tRNA/20 samples). Then the samples were precipitated at -70°C for 30 min by 4 M ammonium acetate and ethanol and centrifuged at 14,000 x g at 4°C for 15 min. The pellets were suspended in 5 µl of sample buffer and subjected to 5% PAGE as recommended by the manufacturer. Autoradiographs were obtained using high performance autoradiographic film (Amersham Pharmacia Biotech, Arlington Heights, IL) at -70°C for 4 days.

RT-PCR analysis of XCL1 expression

Total RNA was isolated as described above. RNA (0.5 µg) was used for XCL1 expression analysis by the Titan one-tube RT-PCR system kit (Roche, Indianapolis, IN) with XCL1-specific primers 5'-TTCTCCTCCTGACTTTCCTG-3' and 5'-AACTGAGATGAGCTGCTTAAC-3'. The reactions were performed at 50°C for 30 min and at 94°C for 3 min, followed by 25 cycles at 94°C for 30 s, 52°C for 30 s, and 72°C for 30 s, then the reaction was completed at 72°C for 7 min. Parallel reactions were conducted in the absence of reverse transcriptase as a control for genomic DNA contamination. RT-PCR using the GAPDH-specific primers 5'-CTCAGTGTAGCCCAGGATGC-3' and 5'-ACCACCATGGAGAAGGCTGG-3' was also included.

ELISA for XCL1

To determine the XCL1 expression level in the brains of Tat-expressing mice, each brain was homogenized in 25 mM HEPES, pH 7.4, containing a mixture of protease inhibitor Complete (Roche) on ice for 10 min. The homogenates were centrifuged at 2,000 x g at 4°C for 5 min. The supernatants were collected and centrifuged at 13,000 x g for additional 30 min to remove tissue debris and were used for XCL1 determination. For XCL1 production in cell culture medium, culture medium of Tat-expressing cells was collected 48 h after transfection. The XCL1 level in the supernatants was determined using an Ab sandwich ELISA with a rabbit anti-mouse XCL1 polyclonal Ab as the capture Ab (R&D Systems, Minneapolis, MN) and biotinylated anti-mouse XCL1 Ab as the detector Ab (R&D Systems). Serial dilutions of the supernatants were performed to achieve measurements within the linear range. The total amounts of proteins in the supernatants of brain homogenates or in the lysates of Tat-expressing cells were determined using the Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA) and were used to calculate the XCL1 concentration.

Combined in situ hybridization and immunohistochemistry

Paraffin-embedded brain sections (5 µm) were cut on a microtome and mounted directly on glass slides for immunohistochemical staining. After the sections were deparaffinized, in situ hybridization was performed. A 281-bp DNA fragment of the mouse XCL1 cDNA (GenBank accession no. NM_008510) was first cloned into a pCR4-TOPO (Invitrogen, Carlsbad, CA) using primers 5'-TGGGGACTGAGTCCTAGAA-3' and 5'-TTACCCAGTCAGGGTTACTGCTGTG-3', and then PCR-amplified to contain a T3 promoter at the 5' end and a T7 promoter at the 3' end using primers 5'-ATTAACCCTCACTAAAGGGA-3' and 5'-TAATACGACTCACTATAGGG-3'. The PCR DNA product was used as a template to synthesize by in vitro transcription the 32P-labeled sense riboprobes using T3 RNA polymerase or the 32P-labeled antisense riboprobes using T7 RNA polymerase. Both labeled riboprobes were further passed through a Centrospin column (Princeton Separations, Adelphia, NJ) to remove unincorporated [32P]UTP. Sections were allowed to hybridize overnight at 60°C in the hybridization buffer containing 40% formamide, 10% dextran sulfate, 2x SSC (20x SSC = 3 M NaCl and 0.3 M sodium citrate, pH 7.0), 1x Denhardt’s solution (50x = 1% each of BSA, Ficoll, and polyvinylpyrrolidone), 200 µg/ml salmon sperm DNA, and ~1–3 x 106 cpm/slide). After hybridization, slides were rinsed in 2x SSC and then in PBS at room temperature, and were treated with RNase A (100 µg/ml) at 37°C for 1 h. Sections were washed in 2, 1, 0.5, and 0.1x SSC at 60°C, each for 30 min.

In some cases in situ hybridization was followed by immunohistochemical staining, which was performed using the Immunocruz staining system (Santa Cruz Biotechnology, Santa Cruz, CA) under the humidified condition. Briefly, the sections were placed in methanol containing 0.3% hydrogen peroxide for 5 min to quench endogenous peroxidase, preabsorbed in normal serum, and then incubated with primary Abs. The Abs included rabbit anti-mouse Abs against glial fibrillary acid protein (GFAP; a marker for astrocytes; 1/50; Sigma-Aldrich) and CD14 (a marker for monocytes; 1/50; Santa Cruz Biotechnology), and goat anti-mouse Abs against microtubule-associated protein (MAP-2; a marker for neurons; 1/20, Santa Cruz Biotechnology) and CD68 (a marker for macrophages/microglia; 1/50; Santa Cruz Biotechnology). The sections were extensively rinsed in PBS between each step described above. The sections were then incubated with the appropriate biotinylated secondary Abs and HRP-streptavidin complex, each for 30 min. Peroxidase activity was visualized with 3,3'-diaminobenzidine as the substrates. Omission of the primary Ab was included as a negative control. Sections were dried and exposed on high performance autoradiographic film (Amersham Pharmacia Biotech) at -70°C for 15 days. After the slides were developed, the sections were counterstained with Mayer’s hematoxylin. The brightfield micrographic images were captured with a Zeiss color camera mounted on a Zeiss Axiovert M200 immunofluorescence microscope using a x20 plan apochrosomat objective. Image analysis was performed using an Axiovision 3.0 software package (Zeiss, Thornwood, NY).

Plasmids

A pBabe-based retrovirus vector, pMX, was described previously (42). The pTat-Myc was constructed to express HXB2 Tat protein with a Myc epitope for detection (18). The green fluorescence protein (GFP) gene (gfp), obtained by digestion of pEGFP.N3 (BD PharMingen) with EcoRI and NotI enzymes, was cloned into the backbone of pMX linearized by the same enzymes. The resulting plasmid was designated as pMX.GFP. HXB2 Tat86 cDNA was PCR-amplified with primers 5'-GTCGGGATCCTAATGGAGCCAGTAGATTCCT-3' and 5'-GTCGGGATCCTTCCTTCGGGCCTGTCGG-3' (BamHI site underlined), and then digested and cloned in-frame into the pMX.GFP linearized by the same enzyme. For human XCL1 promoter-driven luciferase (Luc) reporter plasmid pGL3.XCL1p-Luc, the human XCL1 promoter fragment was obtained by PCR of human genomic DNA using primers 5'-CGAGCTCAAGCTTCTATAAATGTGTATGTTA-3' (SacI site underlined) and 5'-CCCTCGAGCTGTGCAAGGAGAGTGAGGAT-3' (XhoI site underlined) according to the published sequence (43) and digested with SacI and XhoI enzymes, then cloned into pGL3 (Promega, Madison, WI) linearized by the same enzymes. All recombinant plasmids were confirmed by sequencing, except for the XCL1 promoter, which was found to be different from the published sequence by only a few nucleotides (see the GenBank entry under accession no. AY170619).

Cell culture, transfection, retrovirus-mediated transduction, and Luc reporter gene assay

Astroglial cells (U87.MG) and ampho-pheonix cells were maintained in DMEM supplemented with 10% FBS at 37°C with 5% CO2. Cell transfections were performed by the calcium phosphate precipitation method, which usually gave rise to 80% or higher transfection efficiency in ampho-pheonix cells and ~5–8% in U87.MG cells. To prepare MX-based retroviruses, ampho-pheonix cells were transfected with pMX DNA or its derivatives and replaced with fresh culture medium 8–10 h after transfection. Culture medium was collected 48 h after the medium change, centrifuged to remove cell debris, and saved as the virus stock. For transduction, U87.MG cells were plated at a density of 2 x 105/well in a six-well plate and allowed to grow for 18–24 h. The cells were then incubated with viruses of 100,000 cpm of reverse transcriptase activity in the presence of 8 µg/ml polybrene at 37°C for 2–4 h, and incubation was continued in fresh medium for additional 48 h for determination of XCL1 production in the culture supernatant.

For the Luc reporter gene assay, U87.MG cells were plated at a density of 2 x 105 cells/well in a six-well plate and allowed to grow to 18–24 h. The cells were then transfected with 1.2 µg of pXCL1promo-Luc and pTat-myc at various amounts as indicated. pCMV{beta}Gal was also included to normalize variations among transfections, and pcDNA3 was used to equal the total amount of DNAs in each transfection. The cells were replaced with fresh DMEM 24 h after transfection, and culture was continued for an additional 48 h. Then cells were washed twice with PBS and suspended in the lysis buffer included in the Luc assay system (Promega), and Luc activity was measured and expressed as light units using an Opticomp luminometer (MGM Instruments, Hamden, CT).

Statistical analysis

Values were expressed as the mean ± SEM. Comparisons among groups were made using Student’s t test. A value of p < 0.05 was considered statistically significant, and p < 0.001 was considered highly significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Induction of chemokines in the brains of inducible and brain-targeted HIV-1 Tat transgenic mice

To examine chemokine expression in the brain, we took advantage of the inducible and brain-targeted Tat transgenic mouse model (14). We isolated total RNAs from the brains of Tat-expressing mice and age-matched controls and performed RPA using a multiprobe mouse chemokine set (mCK-5; BD PharMingen). This multiprobe chemokine kit allows simultaneous analysis of transcripts of several important mouse chemokine genes, such as CCL1, CCL2, CCL3, CCL4, CCL5, CCL11, CXCL2, CXCL10, and XCL1. The representative results showed that Tat expression in the brains of Tat transgenic mice (GT-Tg) by treatment with Dox resulted in up-regulation of XCL1, CCL2, CCL3, CCL4, CCL5, CXCL2, and CXCL10 (Fig. 1A). Induction of CXCL2, CCL4, CCL5, and XCL1 was higher than that of CCL2, CCL3, and CXCL10 (Fig. 1B), but little change in CCL1 and CCL11 expression was noted in the brains of age-matched wild-type controls, Tat transgenic mice without Dox treatment, or Tat transgenic mice receiving Dox treatment (Fig. 1B). Induction of CCL2, CCL3, CCL4, CCL5, CXCL2, and CXCL10 has been demonstrated in HIV-1 infection, whereas some of these chemokines, such as CCL2 and CXCL10, have been directly linked to Tat expression. In disagreement with the previous studies of Tat-expressing astrocytes, Tat-induced CCL2 expression was very modest in this system. This may be due to use of the whole brain for the RNA isolation.



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  		FIGURE 1. Up-regulation of chemokines in the brain of Tat-expressing mice. A, RPA. Levels of chemokine mRNAs were determined in the brains of inducible and brain-targeted Tat transgenic mice treated with and without Dox and in age-matched wild-type control mice treated with Dox using RPA. The free RNA probes used in the RPA were shown as size markers (M). Samples included control mouse RNA (Ctrl), RNA from wild-type mice treated with Dox (Wt), and RNA from inducible and brain-targeted Tat transgenic mice treated with Dox (GT-Tg + Dox) and without Dox (GT-Tg - Dox). <, protected RNAs, which are XCL1, CCL5, CCL4, CCL3, CXCL2, CXCL10, and CCL2 from top to bottom. The RPA was representative of at least three independent experiments. B, Quantitative analysis of the RPA. The RPA image was scanned by a laser densitometer and calculated as the fold induction of chemokine expression in inducible and brain-targeted Tat transgenic mice treated without Dox () and treated with Dox ({blacksquare}) relative to that in wild-type mice treated with Dox ({square}).

 
Up-regulation of XCL1 mRNA and protein by HIV-1 Tat expression

XCL1 is a T lymphocyte-specific chemokine and plays an important role in the recruitment of T lymphocytes as part of early host response to inflammation. Thus, we decided to further characterize the relationship between Tat expression and XCL1 induction. To ascertain up-regulated expression of XCL1 in the brains of Tat-expressing mice, we performed RT-PCR using XCL1-specific primers and determined XCL1 mRNA levels. RT-PCR using primers specific for the mouse GAPDH gene was also included as a control. The results confirmed that levels of XCL1 mRNA were indeed increased in the brains of Tat-expressing mice, whereas little XCL1 mRNA was detected in the brains of age-matched wild type controls or in Tat transgenic mice treated without Dox (Fig. 2A). There was no apparent difference in GAPDH mRNA levels among those mice. The difference in XCL1 mRNA levels was not due to any nonspecific effects, such as genomic DNA contamination in particular RNA preparations, as RT-PCR containing no reverse transcriptase was also performed, and no amplifications were detected (data not shown). To determine whether the increased XCL1 mRNA expression was translated into XCL1 protein synthesis, we performed the direct Ab ELISA using XCL1-specific capture and detection Abs and measured the levels of XCL1 protein in the brain tissues. In agreement with the results obtained from the RPA and RT-PCR analysis, XCL1 production was increased significantly (p < 0.001) from 7.1 ± 1.2 ng/mg protein in the brains of Tat transgenic mice treated without Dox to 28.1 ± 7.9 ng/mg protein in the brains of Tat transgenic mice treated with Dox (Fig. 2B). Meanwhile, there was no significant change in the brains of age-matched wild-type mice treated with Dox (2.3 ± 2.1 ng/mg protein) or in those not treated with Dox (5.9 ± 1.8 ng/mg protein).



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  		FIGURE 2. XCL1 expression induced by HIV-1 Tat. A, Induction of XCL1 mRNA. RT-PCR using XCL1-specific primers was performed, and the DNA products were electrophoretically separated in a 1.5% agarose gel. RT-PCR using GAPDH-specific primers was also performed as a control (bottom panel). The data are representative of four independent experiments. M, 1-kb DNA marker. B, Induction of XCL1 protein. Brain homogenates were prepared, and XCL1 protein in the supernatants was determined using direct Ab ELISA. The data are the mean ± SEM of three animals per group and are representative of three independent experiments. Mock, homogenate buffer only; Wt, age-matched wild-type control mice; GT-Tg, inducible and brain-targeted Tat transgenic mice; +, treated with Dox; -, treated with empty vehicle only; **, p < 0.001.

 
XCL1 expression in astrocytes, monocytes, and macrophages/microglia

The expression of XCL1 mRNA has been detected in certain subsets of T cells, including activated CD8+ and CD4+ T cells, intraepithelial {gamma}{delta} T cells, and {alpha}{beta}-type thymocytes, mast cells, and NK cells (44). In the mouse, it is also detected at low levels in the spleen and thymus, but little is found in heart, brain, lung, liver, kidney, testis, or skeletal muscle (45). Thus, we next determined the types of cells producing XCL1 in the brains of Tat-expressing mice. We first examined XCL1 expression in astrocytes, as Tat has been targeted to the brain under the astrocyte-specific GFAP promoter (14). To this end, we performed in situ hybridization using an antisense XCL1 RNA probe in combination with immunohistochemical staining for GFAP expression. In situ hybridization analysis showed increased XCL1 expression in the brains of Tat transgenic mice treated with Dox, specifically in the cortex and cerebellar regions (Fig. 3C), but little in the brains of age-matched wild-type controls (Fig. 3A), or Tat transgenic mice treated without Dox (Fig. 3B). Combined immunohistochemical staining revealed that a majority of GFAP-positive cells, i.e., astrocytes was XCL1-positive in the brains of Tat-expressing mice (Fig. 3F). To determine whether XCL1 was also expressed in other types of brain cells, we performed combined immunohistochemical staining using Abs against CD14 (a marker for monocytes), MAP-2 (a marker for neurons), or CD68 (a marker for macrophages/microglia). The results showed that XCL1 was expressed in a fraction of monocytes (Fig. 3H) and macrophages/microglia (Fig. 3I), but not in neurons (Fig. 3G). To ensure the specificity of XCL1 detection, in situ hybridization of brain sections with a sense XCL1 RNA probe was also performed. There were no hybridization signals noted (data not shown). The apparent difference in cell size (Fig. 3, B and C) may be due to cell morphological changes associated with Tat expression. Taken together, these data demonstrated that Tat expression up-regulated XCL1 production not only in Tat-expressing astrocytes, but also in monocytes and macrophages/microglia. The latter was probably due to Tat secretion from Tat-expressing astrocytes and subsequent internalization into monocytes and macrophages/microglia.



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  		FIGURE 3. Induction of XCL1 expression in astrocytes, monocytes, and macrophages/microglia. In situ hybridization (ISH) was performed on brain sections using 32P-labeled antisense XCL1 RNA probes for XCL1 expression, and immunohistochemical staining (IHC) was performed using cell marker-specific Abs for different cell types. ISH was performed either alone (A–C) or in combination with IHC (D–I). Brain sections were from age-matched, wild-type mice treated without Dox (Wt + Dox; A and D), inducible and brain-targeted Tat transgenic mice treated without Dox (GT-Tg – Dox; B and E), and inducible and brain-targeted Tat transgenic mice treated with Dox (GT-Tg + Dox; C and F–I). The cell marker used in IHC was GFAP for astrocytes (D–F), MAP-2 for neurons (G), CD14 for monocytes (H), and CD68 for microglia/macrophages (I). CXL1-positive cells by ISH are shown in brown (open arrows), and cell marker-positively stained cells are shown in red. Closed arrows represented cells that were both XCL1-positive and specific cell marker-positive.

 
Induction of XCL1 expression in Tat-expressing astrocyte U87.MG cells

Although HIV-1 infects astrocytes in an inefficient and restricted manner, the expression of completely spliced HIV-1 viral transcripts such as Tat has been found to be abundant. Thus, we next determined whether Tat expression was sufficient to up-regulate constitutive XCL1 expression in astrocytes. To this end, we expressed Tat in a well-characterized human astroglial cell line, U87.MG, and determined XCL1 production. We expressed Tat as a Tat-GFP fusion protein (TatGFP). The use of TatGFP allows an accurate and sensitive determination of Tat gene expression based on GFP expression. TatGFP protein has been shown to trans-activate HIV-1 long terminal repeat promoter-driven reporter gene expression to the same extent as native Tat protein (Y. Zhou and J. He, unpublished observations). In addition, to overcome the low efficiency in U87.MG cells by the conventional calcium phosphate transfection method, we adapted a retrovirus-mediated gene transfer strategy (42). This approach gives rise to 40–60% efficiency in U87.MG cells (data not shown). Thus, we infected U87.MG cells with retroviruses carrying the TatGFP fusion gene and determined XCL1 production in the culture supernatant. We also included retroviruses carrying the GFP gene alone or empty vector as controls in the experiments. Infection efficiency and expression of TatGFP and GFP were found to be comparable (Fig. 4A). As expected, TatGFP was mainly expressed in the nucleus, whereas GFP was distributed throughout the cell. The direct Ab ELISA of the culture supernatants using anti-XCL1 Abs showed that TatGFP expression resulted in XCL1 production of 86.9 ± 8.1 ng/mg protein, significantly higher than that by GFP-expressing cells and cells expressing the empty vector control (30.2 ± 2.6 and 21.8 ± 4.7 ng/mg protein, respectively; Fig. 4B).



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  		FIGURE 4. Direct induction of XCL1 expression in U87.MG astrocytes expressing Tat protein. U87.MG cells were transduced with MX viruses, MX.GFP viruses, or MX.TatGFP viruses and allowed to grow for an additional 48 h. GFP or TatGFP expression was determined using a Zeiss digital immunofluorescence microscope (A), whereas the cell culture supernatants were collected to determine XCL1 production using direct Ab ELISA (B). The transduction efficiency was estimated to be ~18% based on the percentage of GFP-positive cells. The data are the mean ± SEM of triplicate determinations and are representative of three independent experiments. Trans, images under transmission light; FITC, images under an FITC filter; **, p < 0.001.

 
Direct activation of the XCL1 promoter by HIV-1 Tat protein

HIV-1 Tat protein has been shown to modulate the expression of a number of cellular genes, both negatively and positively, through its indirect interaction with the promoter elements of target genes. Tat has been shown to repress the transcription of the MHC class I gene (46, 47), human mannose receptor (48), and IL-12 (41). Tat has also been shown to activate the expression of CCL2, IL-8, and CXCL10. Thus, we next investigated the effects of Tat expression on transcription of the XCL1 promoter. To this end, we cloned the human XCL1 promoter and constructed a human XCL1 promoter-driven Luc reporter gene. We transfected U87.MG cells with the reporter plasmid alone or in the presence of increased amounts of HIV-1 Tat expression plasmid and determined Luc activity of the reporter gene. The results showed that Tat expression increased XCL1 promoter-driven Luc activity by a maximum of 3-fold (Fig. 5). The reduction of XCL1 promoter activity by higher amounts of Tat expression plasmid was probably due to Tat expression-induced adverse effects on astrocytes, such as growth inhibition and differentiation (19). There was no Luc activity obtained when the empty vector pGL3-basic was used (data not shown).



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  		FIGURE 5. Trans-activational activity of HIV-1 Tat protein on XCL1 promoter. U87.MG cells were transfected with human XCL1 promoter-driven Luc reporter pGL3.XCL1p-Luc (1.2 µg) and various amounts of Tat-expressing plasmid pTat.myc and were harvested 48 h thereafter for the Luc activity assay. The data are the mean ± SEM of triplicate determinations, are representative of multiple independent experiments, and are expressed as the percentage of the highest Luc activity obtained. **, p < 0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
HIV-1 infection of the CNS often causes dementia and other CNS injury and damage. Although a myriad of mechanisms have been proposed, the general consensus is that neuronal injury and damage are results of the production of soluble factors by and dysfunctions of HIV-infected brain cells, such as macrophages/microglia and astrocytes. It is becoming increasingly evident that HIV-1 infection-associated neuropathogenesis is a complex process, possibly mediated by multiple pathogenic factors. Among these factors is the CNS infiltration of monocytes/macrophages and T lymphocytes. In fact, the CNS infiltration of monocytes/macrophages and activated T lymphocytes has been one of the most common and consistent neuropathological features in a number of inflammatory neurological disorders. In AIDS, trafficking of these inflammatory cells into the CNS is often noted as an inflammatory response at the early stage of HIV-1 infection and at later stages of the viral infection, in particular when the integrity of the blood-brain barrier is compromised. Chemokines are known to play a crucial role in this process. These small inducible chemoattractants are early response genes to inflammatory mediators and, in concert with other cytokines and growth factors, may contribute significantly to the inflammatory responses. HIV-1 Tat, a soluble protein that is released from HIV-1-infected cells, can increase the expression of chemokine genes, such as CCL3 and CCL4 in macrophages, and CCL2 and CXCL10 in astrocytes. Using the unique Tat-transgenic mouse model, the expression of several major chemokines was examined. The results showed increased expression of most chemokines examined, including CXC chemokines CXCL2 and CXCL10; CC chemokines, CCL2, CCL3, CCL4, CCL5, and CCL11; and the C chemokine, XCL1. These results provide direct evidence that the HIV-1 Tat protein is central to the recruitment and extravasation of inflammatory cells, such as monocytes/macrophages and T lymphocytes, to the brains of HIV-1 infected individuals and suggest that multiple chemokines are involved in Tat expression-induced CNS infiltration of T lymphocytes and monocytes/macrophages.

Several chemokines have been shown to be up-regulated in the brains of AIDS patients and are mostly linked to CNS infiltration of monocytes/macrophages and subsequent HIV-1 infection-induced encephalitis and other neurological diseases. Tat expression, along with these {alpha} and {beta} chemokines induced by Tat expression, primarily contributes to recruitment of monocytes/macrophages in the brains of HIV-1-infected individuals (3, 34, 35, 36, 38, 39, 40, 41). Although most biological functions of the C chemokine XCL1 are still unknown, XCL1 expression has been detected at inflammatory sites of several chronic inflammatory diseases. It has been demonstrated that XCL1 expression is involved in the recruitment of T cells in the pathogenesis of Crohn’s disease (49). The CNS infiltration of T lymphocytes has also often been noted in AIDS patients with evident neurological symptoms (23, 24, 25, 26). Our studies as well as those by others have shown a close association between Tat expression and the CNS infiltration of T lymphocytes (14, 20). To investigate the potential role of XCL1, a potent chemokine that predominantly and specifically attracts T lymphocytes, the expression of XCL1 mRNA and protein in relation to Tat expression was examined. Cell types expressing XCL1 in the brain were further characterized. The results showed that Tat expression activated XCL1 expression at both mRNA and protein levels (Fig. 2), and that that XCL1 was mainly expressed in astrocytes as well as monocytes and macrophages/microglia, but to a lesser extent (Figs. 3 and 4). Although we have not determined XCL1 expression in the CNS or cerebral spinal fluid of HIV-infected individuals, our results suggest the strong possibility of increased XCL1 levels in these tissues where Tat expression has been well documented.

Under normal conditions, very few activated T lymphocytes enter the CNS. This is due to the integrity of a tight blood-brain barrier as well as the low production of chemokines in the brain (50). Our results raise the possibility that XCL1 up-regulation in the brain resulting from HIV-1 infection and Tat expression initiates the presence of a minimal number of activated T lymphocytes in the brain during the early viral infection, which, in turn, secret more chemokines, including XCL1, and cause more CNS infiltration of T lymphocytes as well as monocytes/macrophages in the later stages of viral infection. The infiltration of activated T lymphocytes could cause CNS injury and damage in various ways. Like monocytes/macrophages, activated T lymphocytes secrete bioactive substances with complex interactions that alter a wide range of neurotoxic cytokines, such as TNF-{alpha}. In addition, direct contact of T lymphocytes with neurons has been shown to affect neuronal survival. Coculture of activated CD8+ cells with neurons induces neuronal apoptosis (51). Furthermore, CNS infiltration of T lymphocytes may account for the presence of T-tropic HIV-1 viruses in the brains of AIDS patients. Nevertheless, to what extent each chemokine contributes to the CNS infiltration of T lymphocytes and/or monocytes/macrophages and how CNS infiltration of these cells contributes to HIV-associated neuropathogenesis remain to be defined.

The control of XCL1 gene expression is not well understood. The human XCL1 promoter has recently been characterized to contain the NF-AT core sequence, TTTCC, within an E1 box, the major regulatory element responsive to mitogen-induced XCL1 expression in human T lymphocytes (43). Our XCL1 promoter-driven reporter gene assay showed that Tat-induced XCL1 expression occurred at the transcriptional level (Fig. 5). HIV-1 Tat protein has been shown to modulate a number of viral and cellular gene expression through direct trans-activation or interaction with upstream enhancer elements within the target gene promoters or through intracellular signaling elicited by Tat interaction with cellular receptors present on the cell surfaces. Interestingly, several studies have suggested possible involvement of interaction of HIV-1 Tat protein with NF-AT in modulation of gene expression (52, 53, 54). Thus, it is likely that the trans-activational activity of Tat on XCL1 promoter is mediated by Tat interaction with NF-AT within the XCL1 promoter. Furthermore, in our Tat transgenic mouse model, Tat is exclusively and inducibly expressed in astrocytes (14). Up-regulation of XCL1 in monocytes and macrophages/microglia in the presence of Tat expression indirectly supports the idea that HIV-1 Tat protein is secreted from astrocytes as a soluble factor and is taken up by neighboring cells such as monocytes, macrophages/microglia, and neurons (18).


   Footnotes
 
1 This work was supported by National Institutes of Health Grants R01NS39804 (to J.J.H.) and R01MH65158 (to J.J.H.) and a research award from the Ralph W. and Grace M. Showalter Trust Foundation (to J.J.H.). Back

2 Address correspondence and reprint requests to Dr. Johnny J. He, Department of Microbiology and Immunology, Indiana University School of Medicine, R2 302, 950 West Walnut Street, Indianapolis, IN 46202. E-mail address: jjhe{at}iupui.edu Back

3 Abbreviations used in this paper: MIP, macrophage inflammatory protein; Dox, doxycycline; GFAP, glial fibrillary acid protein; GFP, green fluorescence protein; GT-Tg, inducible and brain-targeted Tat transgenic mice; IHC, immunohistochemical staining; Luc, luciferase; MAP, microtubule-associated protein; RPA, RNase protection assay; XCL1, lymphotactin. Back

Received for publication August 29, 2003. Accepted for publication November 18, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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