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The Journal of Immunology, 2004, 172: 7610-7617.
Copyright © 2004 by The American Association of Immunologists

Neuroregulatory Events Follow Adaptive Immune-Mediated Elimination of HIV-1-Infected Macrophages: Studies in a Murine Model of Viral Encephalitis1

Larisa Poluektova2,*, Santhi Gorantla*, Jill Faraci, Kevin Birusingh*, Huanyu Dou* and Howard E. Gendelman*,{dagger}

* Laboratory of Neuroregeneration, Center for Neurovirology and Neurodegenerative Disorders, and Department of Pathology and Microbiology and {dagger} Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68198


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
HIV-1-specific cellular immunity serves to eliminate infected cells and disease. However, how this process specifically affects the CNS is poorly understood. To mirror the regulatory events that occur in human brain after HIV-1 infection, a murine model of viral encephalitis was used to study relationships, over time, among lymphocyte-mediated infected cell elimination, innate immune responses, and neuropathology. Nonobese diabetic SCID mice were reconstituted with human PBL and a focal encephalitis induced by intracranial injection of autologous HIV-1-infected, monocyte-derived macrophages (MDM). On days 7, 14, and 21 after MDM injection into the basal ganglia, the numbers of human lymphocytes and mouse monocytes, virus-infected MDM, glial (astrocyte and microglial) responses, cytokines, inducible NO (iNOS), neurotrophic factors, and neuronal Ags were determined in brain by immunohistochemistry, real-time PCR, and Western blot assays. Microglia activation, astrocytosis, proinflammatory cytokines, and iNOS expression accompanied the loss of neuronal Ags. This followed entry of human lymphocytes and mouse monocytes into the brain on days 7 and 14. Elimination of virus-infected human MDM, expression of IL-10, neurotropins, and a down-regulation of iNOS coincided with brain tissue restoration. Our results demonstrate that the degree of tissue damage and repair parallels the presence of infected macrophages and effectors of innate and adaptive immunity. This murine model of HIV-1 encephalitis can be useful in elucidating the role played by innate and adaptive immunity in disease progression and resolution.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Neurological manifestations produced as a consequence of HIV-1 infection can occur during the acute seroconversion reaction as a self-limited meningoencephalitis or after prolonged viral persistence and immunosuppression. The latter occurs as a subacute and progressive neurodegenerative process, commonly called HIV-1-associated dementia (HAD)3 (1, 2, 3, 4, 5, 6, 7). It is generally accepted that mononuclear phagocytes (MP; perivascular macrophages and microglia) are primary cellular targets for viral infection and sources of neurotoxins that affect neuronal function, and ultimately to cognitive, motor, and/or behavioral abnormalities (8, 9, 10, 11). Interestingly, not only meningoencephalitis, but also HAD are reversible. This occurs, for the former, as a consequence of effective innate and adaptive immunity (12, 13, 14, 15) and for the latter through control of ongoing viral replication and glial inflammatory responses by highly active, anti-retroviral therapy (16, 17, 18, 19, 20). These observations suggest that immunoregulatory events that are operative in the brain not only affect the disease process, but also lead to tissue repair after viral infection is controlled (21).

Little is known concerning the neuropathological, metabolic, and immunopathological mechanisms surrounding the elimination of HIV-1 in affected brain tissue. Eradication of infected cells and neuronal restorative processes are particularly relevant for the acute meningoencephalitis that occurs at a time when both innate and adaptive immune systems remain active. How this occurs could depend upon environmental cues in that MP activation can lead to neurotoxic or neurotrophic outcomes (22, 23, 24). Alternatively, but not mutually exclusive, CNS-specific autoreactive T cells could protect neural tissue from secondary injury (25, 26, 27, 28). Thus, tissue restitution after elimination of a viral insult could occur as a consequence of innate and adaptive immunity.

In an attempt to investigate this idea, we modified a SCID murine model of human HIV-1 encephalitis (HIVE) developed in our laboratories (29, 30) to reflect dynamic interactions between virus-infected MP and effectors of adaptive immunity (31). We repopulated the immune systems of nonobese diabetic (NOD) SCID mice with human PBL. One week after PBL transfer, HIVE was induced by injection of syngeneic HIV-1-infected, monocyte-derived macrophages (MDM) into the basal ganglia generating hu-PBL/HIVE mice. Cellular anti-retroviral immune responses were demonstrated by the presence of granzyme B-positive CD8+ cells in the brain and the appearance of HIV-1 gag- and pol-specific CTL in the spleen. This was shown by tetramer staining and IFN-{gamma} ELISPOT assays (31). The elimination of autologous infected MDM from the brains of hu-PBL/HIVE mice was accompanied by significantly diminished lymphocyte numbers (from 7–21 days), but led to sustained astrocyte and microglial responses, increased expression of proinflammatory cytokines (including IL-1{beta} and IL-6) and neurotropins (including brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor-2 (bFGF-2), nerve growth factor (NGF), epidermal growth factor (EGF), and neurotrophin-3 (NT-3)). These changes coincided with down-regulation of inducible NO synthase (iNOS), up-regulation of IL-10, and restoration of neuronal Ag expression (microtubule-associated protein 2 (MAP-2) and neuron-specific nuclear protein (NeuN)). Together, these observations provide unique insights into the pathological and immune consequences of HIV-1 infection of the brain during both progressive viral infection. In addition, potential mechanisms of neural injury and repair during brain infection are illustrated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and viruses

Monocytes and PBL were obtained from HIV-1, HIV-2, and hepatitis B seronegative donor leukopaks and were purified by countercurrent centrifugal elutriation. Monocytes were cultivated in DMEM (Sigma-Aldrich, St. Louis, MO) supplemented with 10% heat-inactivated pooled human serum, 1% glutamine, 50 µg/ml gentamicin, 10 µg/ml ciprofloxacin (Sigma-Aldrich), and 1000 U/ml highly purified recombinant human macrophage CSF (gift from Genetics Institute, Cambridge, MA). After 7 days in culture, MDM were infected with HIV-1ADA at multiplicity of infection of 0.01 (32).

Hu-PBL/HIVE mice

Four-week-old male NOD/C.B-17 SCID mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Animals were maintained in sterile microisolator cages under pathogen-free conditions in accordance with ethical guidelines for the care of laboratory animals at University of Nebraska Medical Center and National Institutes of Health. All animals were injected i.p. with rat anti-CD122 (0.5 mg/mouse) 3 days before PBL transplantation and twice with rabbit asialo-GM1 Abs (0.2 mg/mouse; WAKO, Richmond, VA) 1 day before and 3 days after PBL injection (20–50 x 106 cells/mouse). HIV-1ADA-infected MDM (3 x 105 cells in 10 µl) were injected intracranially (i.c.) (30) 8 days after PBL reconstitution (n = 51; hu-PBL/HIVE) mice. NOD/SCID mice injected i.c. with HIV-1ADA-infected MDM (n = 50, HIVE), hu-PBL reconstituted mice injected i.c. with uninfected MDM (n = 20, hu-PBL/MDM), or media-injected mice (n = 9; sham-operated) served as controls. Animals were sacrificed 4, 7, 14, and 21 days after MDM injection. Mice with limited PBL engraftment (<10% of human cells in mouse spleen) or those with signs of graft-vs-host disease were excluded from analyses. Five experiments with cells from five different donors were included. Brain tissues from three experiments were used for immunohistochemistry. Brain samples from replicate animals were used for protein and RNA extractions. In each experiment four mice per group were sacrificed at 7, 14, and 21 days for histopathology and RNA and/or protein extractions. A single experiment was performed on day 4. Serum HIV-1 p24 levels were detected by ELISA (Beckman Coulter, Miami, FL).

FACS analysis of mononuclear cells derived from brain tissue

Cells were isolated from the injected hemispheres of four hu-PBL/HIVE mice and five HIVE mice on day 14 after MDM injection by previously published techniques (33). Briefly, brains were removed and washed in ice-cold PBS. The injected hemispheres were ground and strained through a 70-µm pore size mesh to generate single-cell suspensions. Cells were placed in 5 ml of RPMI 1640 medium supplemented with 10% FCS, then pelleted and resuspended in isotonic 30% Percoll (Amersham Pharmacia Biotech, Piscataway, NJ). The lysates were spun at 1300 x g for 30 min at 4°C. The Percoll and lipid layers were aspirated, and the cell pellets were washed twice and counted, then stained for flow cytometry. Abs for mouse leukocyte common Ag (LCA; CD45-FITC), NK cells (DX-5-FITC), and MHC class II (I-Ab-PE), and human Ags CD45RO-PE, CD4-PE, CD8-PE, CD56-PE, and CD3-allophycocyanin (BD PharMingen, San Diego, CA) were used. Data were analyzed with a FACSCalibur using CellQuest software (BD Immunocytometry Systems, Mountain View, CA).

Immunocytochemistry

Brain tissue was collected on days 4, 7, 14, and 21 after i.c. injection, fixed in 4% phosphate-buffered paraformaldehyde, and embedded in paraffin or frozen for later use. Blocks were cut to identify the injection site. For each mouse, 30–100 serial (5-µm-thick) sections were cut from the human MDM injection site, and three to seven slides (10 sections apart) were analyzed. Brain sections were deparaffinized with xylene and hydrated in gradient alcohols. Immunohistochemical staining followed a basic indirect protocol, using Ag retrieval by heating to 95°C in 0.01mol/l citrate buffer for 30 min. Abs to vimentin intermediate filaments (1/100; clone VIM 3B4), CD45RO, CD79{alpha}, HLA-DR (1/100; clone CR3/43), and CD8 (clone 144B; 1/50; DAKO, Carpinteria, CA) were used to detect human cells. Murine microglia were detected by Griffonia simplicifolia isolectin B4(1/100; Vector Laboratories, Burlingame, CA) and CD45 Abs (1/50; BD PharMingen). Abs to glial fibrillary acidic protein (GFAP; 1/3000, DAKO), NeuN (1/100), and MAP-2 (1/1000; Chemicon International, Temecula, CA) were used to identify mouse astrocytes and neurons. The Vectastain Elite ABC kit (Vector Laboratories) and the DAKO EnVision polymer-based system developed the immunolabeling tests. All paraffin-embedded sections were counterstained with Mayer’s hematoxylin. Deletion of primary Ab or mouse IgG served as controls. The numbers of HIV-1 p24 (clone Kal-1; 1/10; DAKO) Ag-expressing lymphocytes and MDM were manually counted. Tissue examination was performed with an Eclipse 800 microscope (Nikon, Melville, NY). Glial and neuronal Ags were measured by computer-assisted image analysis (Image-ProPlus; Media Cybernetics, Silver Spring, MD) as previously described (30). At the injected and corresponding regions on contralateral sites, immunostained area was measured at x4 for astrocytes and x10 for microglial and neuronal Ags. Immunofluorescent staining was performed on paraformaldehyde-fixed, sucrose-processed, frozen and fresh-frozen brains fixed in acetone-methanol (1/1). Ten or 20-µm tissue sections were stained for 24 h with Abs to HAM56 (1/50; mouse IgM; DAKO), CD8 (1/50; clone 144B; DAKO), BDNF (1/500; Chemicon International), CD3 (1/100; rabbit polyclonal; DAKO), Ki-67 protein (pKi-67; 1/50; mouse IgG; BD PharMingen), and ionized calcium-binding adaptor molecule 1 for mouse microglial cells (1/500; rabbit polyclonal; provided by Dr. Y. Imai, Jutendo University School of Medicine, Tokyo, Japan). Secondary Abs conjugated with Alexa Fluoro-488 and -594 (1/100; Molecular Probes, Eugene, OR) were applied for 1 h. Fluorescent images were visualized with an LSM 410 confocal laser-scanning microscope (Zeiss, Goettinger, Germany) with argon/krypton at 488/568/647 nm. To identify human cells in mouse brains, Ab to vimentin intermediate filaments clone 3B4 (DAKO), which selectively stains the cytoplasm of human cells in paraffin-embedded tissue, was used. After citric buffer (pH 6.0) treatment, rare mouse astrocytes were detected with vimentin 3B4 Abs (30). To differentiate human MDM, lymphocytes, and HIV-1-infected cells, 5-µm serial sections of paraffin-embedded tissue were stained with Abs to CD68, CD45RO, CD8, and HIV-1 p24. Replicate frozen brain tissue was stained with Abs HAM56 and CD3 for human MDM and lymphocyte identification.

Western blot assays

Two-millimeter sections that included the site of injection were used for extraction of protein. Tissue sections corresponding to the site of injection in the contralateral hemisphere served as controls. Brain tissue was collected on days 7, 14, and 21 after i.c. MDM injection. The brain was homogenized in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, aprotinin, bestatin, leupeptin, pepstatin A, aminoethyl benzenesulfonylfluoride, and E-64. The total protein concentration was determined by the bicinchoninic acid assay (Pierce, Rockford, IL). Proteins were electrophoretically separated on SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes. Membranes were incubated with primary Abs to BDNF, NGF, NT-3, bFGF-2, EGF, GFAP, MAP-2, or actin. All Abs were obtained from Chemicon International, except for GFAP (DAKO) and EGF (Upstate Biotechnology, Charlottesville, VA). HRP-conjugated secondary Abs were used, and membranes were treated with chemiluminescent substrate, then exposed to x-ray film. Images were digitized with a densitometer (Molecular Dynamics, Sunnyvale, CA), and protein levels were expressed as a ratio to actin.

Real time RT-PCR

Total RNA from brain sections was extracted with TRIzol (Invitrogen, Carlsbad, CA). RNA was reverse transcribed with random hexamers, and real-time quantitative PCR was performed with cDNA using an ABI PRISM 7000 sequence detector (Applied Biosystems, Foster City, CA).

Murine-specific primers pairs were: BDNF, 5'-CTGCAGCCTTCCTTGGTGTAA-3' and 5'-CCAAAGGCCAACTGAAGCA-3'; NT-3, 5'-CACAGGCTCTCACTGTCACACA-3' and 5'-CACCACGGAGGAAACGCTAT-3'; bFGF-2, 5'-ACCCCAAGCGGCTCTACTG-3' and 5'-GATGGATGCGCAGGAAGAAG-3'; EGF, 5'-AATGGCCTTTTTTGGTGATCG-3' and 5'-CCAAATCGC CTTGCTTTTCA-3'; IL-1{beta}, 5'-CACCCCGACTGAAGGTGACT-3' and 5'-CTGTTCCAGAAGCGCCATTAA-3'; IL-10, 5'-GTTGCCAAGCCTTATCGGAA-3' and 5'-CCAGGGAATTCAAATGCTCCT-3'; IL-6, 5'-TTCCATCCAGTTGCCTTCTTG-3' and 5'-CCCTGCCACAAGCAGGAAT-3'; and iNOS, 5'-GGCAGCCTGTGAGACCTTTG-3' and 5'-GAAGCGTTTCGGGATCTGAA-3'.

A SYBR Green I detection system was used, and the reactions generated a melting temperature dissociation curve enabling quantitation of the PCRs. All PCR reagents were obtained from Applied Biosystems. Gene expression was normalized to 18S ribosomal RNA as an endogenous control.

Statistical analysis

Data were analyzed using Excel (Macintosh, 1994) with Student’s t test for comparisons and ANOVA. A value of p < 0.05 was considered statistically significant. All results are presented as the mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Distribution of human lymphocytes and MDM in mouse brain tissue

Immunostaining of brain sections for vimentin (30) and CD8 demonstrated the distribution of human cells and cell-to-cell interactions over a 21-day period. In hu-PBL/MDM and hu-PBL HIVE mice, PBL were predominantly found in the areas surrounding human MDM (including the meninges, choroid plexus, and ventricles) after cell injection (Fig. 1, A and B). The most prominent lymphocyte brain infiltration in hu-PBL/HIVE mice was found on day 7 (Fig. 1, B–D), where 31 ± 6% of human leukocytes were HIV-1 p24+ (Fig. 1, F and H). Reduction of lymphocyte numbers occurred over 21 days and followed that of human MDM. On day 21, 4.1 ± 1.3% of lymphocytes were HIV-1 p24+. Human CD8+ T lymphocytes were seen in the affected hemisphere on day 7 (176 ± 31 cells/section). These lymphocytes were detected throughout the area of focal encephalitis and were associated with multinucleated giant cells (MNGC) undergoing apoptotic cell death (Fig. 1, C and E). Lymphocytes demonstrated a memory cell (CD45RO+) phenotype and readily expressed HLA-DR Ags (data not shown).



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  		FIGURE 1. Distribution of human lymphocytes and macrophages in brains of hu-PBL/HIVE mice. A, Low power (x1) view of coronal sections of mouse brain showing diffuse activation of astrocytes (GFAP immunostains, magenta) around the area of human cells (brown MDM and lymphocytes) 7 days after brain injection of HIV-1-infected MDM. B, Meningeal infiltration of lymphocytes is in the injected hemisphere. The inset shows lymphocytes within a perivascular cuff, and arrows indicate migrating cells (brown). Perivascular astrocytes were immunostained with GFAP. All paraffin-embedded tissue sections were counterstained with Mayer’s hematoxylin. Higher magnification images of the regions highlighted by black and white arrows in A are shown in B. C and E, CD8+ T lymphocytes and HIV-1-infected MNGC (condensed dark nuclei and shrunken cytoplasm, indicated by arrows) are shown in contact with CD8+ T lymphocytes. G, Human CD68+ macrophages were counted from five to seven sections from each mouse brain. D, Brain sections stained for CD45RO (brown) and CD68 (purple). F, Brain sections stained for CD45RO (brown) and HIV-1p24 (purple). Arrows show single lymphocytes. H, Total PBL and HIV-1 p24+ lymphocyte numbers were counted from five to seven sections per mouse (n = 6–11 mice/group). *, p < 0.01 for hu-PBL/HIVE vs HIVE. Magnification: B and C, x200; B (inset), D, E, and F, x1000.

 
Human MDM migrated from the site of injection and were often observed in perivascular spaces. A significant reduction of MDM was observed on day 14 in hu-PBL/HIVE compared with HIVE mice. In hu-PBL/HIVE mice, 54.7 ± 13.3 MDM/section were detected on day 7, and their number dropped to 6.9 ± 2.2 MDM/section on day 14 compared with HIVE mice (46.2 ± 7.5; p < 0.01; Fig. 1G). A majority of the MDM present after day 14 were MNGC (Fig. 1D). On day 21 after injection, HIV-1-infected MDM were detected in brains from three of 11 hu-PBL/HIVE mice. In these hu-PBL/HIVE animals, 20.1 ± 4.9 MDM/section and 90.3 ± 19.9 PBL/section were found. Few lymphocytes (27.0 ± 4.2/section) were observed in the eight animals in which human MDM were eliminated.

On day 7 the frequency of HIV-1 p24 Ag-positive MDM per section was higher in hu-PBL/HIVE animals (81.9 ± 6.6%) compared with HIVE mice (68.7 ± 7.7%), but differences did not reach statistical significance. At later time points (days 14 and 21), ≥98% of MDM in hu-PBL/HIVE and HIVE animals expressed HIV-1 p24 Ags. The spread of HIV-1 from brain to blood was first detected in hu-PBL/HIVE mice on day 14 and reached 1428 ± 424 pg/ml HIV-1 p24 Ag in plasma on day 21. In HIVE animals, the level of HIV-1 p24 was <78 pg/ml.

Lymphocytes were infrequently observed around cerebral vessels in sham-operated mice. Increased numbers of human lymphocytes were observed in hu-PBL/HIVE compared with hu-PBL/MDM mice starting on day 4 (Fig. 2, A and B). This difference reached a maximum of 8- to 10-fold on day 14 after MDM injection. Mice injected with uninfected MDM on day 21 demonstrated minimal PBL infiltration and human cells formed lymphoid-like clusters (Fig. 2C).



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  		FIGURE 2. Human lymphocytes in brains of hu-PBL/HIVE mice. Immunofluorescent staining of frozen brain tissue 4 days after injection of HIV-1-infected (A) and uninfected (B) MDM. HAM56-positive human MDM are in red, and CD3+ T lymphocytes are in green. C, Vimentin (brown) immunostaining of paraffin-embedded brain tissue demonstrates macrophage-lymphocyte clusters in association with astrocytes (GFAP; red) at 21 days after injection of uninfected MDM. Magnification, x400.

 
Quantitative characterization of mouse microglia and human leukocytes by FACS analysis

Staining of brain tissue with G. simplicifolia isolectin B4 showed the presence of murine macrophages in perivascular areas and parenchyma where human cells had accumulated (Fig. 3, A–C). To compare the levels of activation of murine macrophages/microglia, we performed flow cytometric analysis of mononuclear cells recovered from the injected hemispheres of hu-PBL/HIVE and HIVE mice (Fig. 3, E and F). Mouse CD45+ cells isolated from hu-PBL/HIVE brain contained two cell populations, CD45low (microglia, 24.3%) and CD45high (peripheral macrophages, 2.9%), compared with HIVE animals, in which CD45high cells were rare (0.5%). These data confirmed an influx of macrophages in brains of hu-PBL/HIVE mice. The proportion of MHC class II-positive microglial cells was also higher in hu-PBL/HIVE than in HIVE animals. Colocalization of mouse class II and CD45 showed that hu-PBL/HIVE had larger numbers of class II-positive cells than HIVE mice. Flow cytometric analysis of brain-infiltrating lymphocytes showed principally human CD8+ cells (Fig. 3, G and H). The frequency of human NK (NK, CD3–/CD56+) cells did not exceed 3%. Mouse NK (DX-5+) cells were <0.3% of the total.



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  		FIGURE 3. Distribution of human leukocytes and murine macrophages in perivascular and parenchymal compartments of encephalitic brain. A and B, High power view (x600) of murine macrophages (G. simplicifolia; brown round cells; A), human leukocytes (vimentin+; brown cells) and activated astrocytes (GFAP+; purple; B) on adjusted 5-µm sections. C, Artificially masked mouse (green) and human cells (yellow) with no overlap (red). D, Serial section to C demonstrates HIV-1 p24 human lymphocytes in the same region. Arrows show vascular lumens. E–H, FACS analyses of leukocytes isolated from the injected hemispheres of hu-PBL/HIVE and HIVE mouse brain tissue. The flow cytometric histograms of murine microglia and macrophages in hu-PBL/HIVE (E) and HIVE (F) mice are shown. Human CD4+ (H) and CD8+ (G) lymphocyte profiles are illustrated.

 
Microglial reactions and astrogliosis

Enlarged lectin-stained areas were observed around human MDM. Microglial activation in hu-PBL/HIVE peaked on day 14 (Figs. 4 and 5D). The high level of microglial activation (50.1 ± 9.0% of G. simplicifolia isolectin B4-stained field of view) persisted in the brain where HIV-1-infected MDM was sustained (three animals total). Microglia activation persisted in hu-PBL/HIVE mice throughout the study. Lymphocytes that entered the cell cycle (pKi-67+) and proliferating microglia in hu-PBL/HIVE animals were also detected on day 7 (data not shown).



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  		FIGURE 4. Neuropathology and immunological analysis of hu-PBL/HIVE and HIVE mice. Relationships between human leukocytes and mouse microglia, astrocytes, and neurons are shown for days 7 and 21 after injection of HIV-1-infected MDM. Brain tissue sections were immunostained with Abs to vimentin (brown; human cells) and GFAP (purple), demonstrating significant mouse astrocyte response in hu-PBL/HIVE mice. HIV-1-infected cells were detected by staining with Abs to p24 Ag (brown). Brain tissue sections stained with G. simplicifolia (brown) demonstrate persistence murine microglial activation. Brain tissue sections immunostained with Abs to NeuN (brown) illustrate neuronal cells and show a reduction in the levels of neural damage over time (red line). All sections were counterstained with Mayer’s hematoxylin. Original magnification, x200. Arrows indicate the presence of infected MDM.

 


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  		FIGURE 5. Quantitative microglia, astroglial, and neuronal staining in hu-PBL/HIVE and HIVE mice. Morphometric analysis was performed with G. simplicifolia, GFAP, and NeuN in brain tissue of hu-PBL/HIVE and HIVE mice from 7–21 days after brain injection of HIV-1 infected MDM. A, Schematic presentation of analyzed brain regions on the injected ipsilateral and contralateral hemispheres. B, Areas stained for activated GFAP astrocytes were analyzed under x4 magnification. C and D, G. simplicifolia+ and NeuN+ areas were analyzed under x10 magnification. n = 6–11 mice/group. *, p < 0.01 for hu-PBL/HIVE vs HIVE.

 
Levels of astrocytosis were distinct in hu-PBL/HIVE and HIVE animals (Figs. 4 and 5B). GFAP immunostaining showed hypertrophy and branching astrocytes, which persisted in hu-PBL/HIVE mice, but not in HIVE mice, during the entire study. GFAP immunostaining of astrocyte foot processes was increased around microvessels, demonstrating lymphocyte adhesion to endothelial cells. A diffuse increase in GFAP immunostaining was found in hu-PBL/HIVE mice compared with the more localized reaction seen in HIVE mice.

Neuronal Ag expression

The extent of neuronal damage in areas of human cells was examined by immunostaining with NeuN and MAP-2 antibodies. Neuronal staining intensity was expressed as a percentage of the ipsilateral compared with the contralateral hemisphere. On day 7 after MDM injection, the neuronal Ag staining intensity of the ipsilateral hemisphere was reduced to 61.5 ± 11.7% in hu-PBL/HIVE mice and 89.7 ± 3.5% in HIVE mice (p < 0.04). The reduction in neuronal protein expression corresponded to the area occupied by activated microglia and murine macrophages (Figs. 4 and 5D). On day 14, the area of NeuN staining in the ipsilateral hemisphere of hu-PBL/HIVE mice had increased to 84.7 ± 3.8% and reached 92.6 ± 1.7% on day 21. Elimination of MDM coincided with the restoration of tissue morphology, with the appearance of NeuN-positive cells in close proximity to the scar. Restitution of MAP-2 staining was also demonstrated by Western blot tests (data not shown).

Cytokines and iNOS

On day 7 after MDM injection, significant elevations of IL-1{beta} and IL-6 mRNA expression were observed in brains of hu-PBL/HIVE mice compared with HIVE mice (Fig. 6). On day 21 IL-1{beta} and IL-6 expression in hu-PBL/HIVE mice decreased and did not reach the level of expression observed in the contralateral hemisphere (2.2 ± 0.8; Fig. 6A). Notably, iNOS expression increased on day 7 in both hu-PBL/HIVE and HIVE mice, but diminished on day 21. Also, a statistically significant difference in iNOS expression between hu-PBL/HIVE (7.5 ± 1.3) and HIVE mice (3.3 ± 1.5) was observed on day 14 (p < 0.05; Fig. 6A). IL-10 expression was significantly elevated in hu-PBL/HIVE mice compared with HIVE mice only on day 21. Concurrent with up-regulation of IL-10 expression and tissue restitution in hu-PBL/HIVE mice, we observed down-regulation of proinflammatory cytokines (IL-1{beta} and IL-6) and iNOS expression.



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  		FIGURE 6. Real-time PCR analysis for pro- and anti-inflammatory cytokines and iNOS in hu-PBL/HIVE and HIVE mice. Levels of IL-1{beta}, IL-6, and IL-10 and iNOS mRNAs are shown 7–21 days after injection of infected MDM. RNA was isolated from 2-mm-thick brain slices. *, p < 0.05; **, p < 0.01 (hu-PBL/HIVE vs HIVE). n = 4–6 mice/group.

 
Neurotrophic factors

In hu-PBL/HIVE mice, immunofluorescent staining of fresh-frozen tissues showed wide distribution of BDNF signal and colocalization with human cells (Fig. 7). BDNF, NT-3, and NGF proteins, as determined by Western blot analysis, persisted from days 7–21 in hu-PBL/HIVE compared with HIVE mice (Fig. 8, B and C). NT-3 (p < 0.05) and NGF (p < 0.01) levels in hu-PBL/HIVE animals attained statistical significance to HIVE mice only on day 21. We speculated that human cells considerably contributed to the total amount of neurotropins on days 7 and 14 when the presence of human lymphocytes was significantly higher. However, mouse-specific, real-time PCR analysis confirmed that the prolonged overexpression of BDNF and NT-3, as well as nonneuronal growth factors bFGF-2 and EGF was predominately of mouse origin (Fig. 8A). Surprisingly, the expression of all four tested trophic factors was not statistically different between reconstituted and nonreconstituted animals during the acute period of damage (first 2 wk after MDM injection). Moreover, in hu-PBL/HIVE mice, the expression of NT-3, bFGF-2, and EGF peaked on day 21, at which time in nonreconstituted mice these parameters were indistinguishable from the unaffected control hemisphere. These changes paralleled the up-regulation of IL-10 and tissue restoration in hu-PBL/HIVE mice.



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  		FIGURE 7. BDNF in hu-PBL/HIVE and HIVE mice 7 days after injection of infected MDM. Immunofluorescent staining of fresh-frozen brain tissue derived from the injected hemisphere of hu-PBL/HIVE and HIVE mice is shown using Abs to BDNF, CD8, and HAM56. All magnifications are x200.

 


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  		FIGURE 8. Levels of neurotrophic and growth factors in brain tissue of hu-PBL/HIVE and HIVE mice by real-time PCR and Western blot tests. A, Real-time PCR quantitated expression of bFGF-2, NT-3, BDNF, and EGF. B, Quantitated protein levels are shown from injected hemispheres of hu-PBL/HIVE mice ({square}) and HIVE mice ({blacksquare}). , Protein levels from the contralateral hemispheres. *, p < 0.05 for hu-PBL/HIVE vs HIVE mice. n = 4–6 mice/group. C, A representative immunoblot was prepared from brain tissue stained for BDNF, NT-3, NGF, and actin.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
HAD is a metabolic encephalopathy fueled by productive viral replication in brain MP and sustained through the amplification of glial inflammatory neurotoxic products (21, 34, 35). Both viral and cellular toxins affect the neurodegenerative process, leading ultimately to devastating cognitive, motor, and behavioral dysfunction (36). Innate and adaptive immunities in most infected people serve to curtail productive viral replication and ameliorate subsequent clinical neurological sequelae (37). During the acute seroconversion reaction, effective immune responses clear infection, and neurological impairment is transient. However, during progressive viral disease, immune surveillance breaks down, and a portion of HIV-infected individuals ultimately progress to develop significant nervous system impairments (38). For the most part, clinical recovery of disease is linked closely to elimination of infected cells in the brain, whether it occurs as a consequence of effective host immunity or after anti-retroviral therapy (39). Using a murine model of human HIVE, we investigated neuropathological and immunopathological events that follow elimination of infected macrophages. We show that restoration of brain injury is accompanied by continued microglia and astrocyte activation, diminished iNOS, increased IL-10, and significant production of a wide range of neurotropins. The data demonstrate that mere elimination of infected cells is one step in a compensatory immune response that affects brain restitution. This is the first time, to our knowledge, that such complex pathogenic events have been studied in an animal model of human HIVE.

Mechanisms that affect viral replication in susceptible MP lineage cells and support neuronal survival are more difficult to study in SIV-infected rhesus macaques (40). The dynamics of infected cell clearance, the diffuse nature of disease, subtle changes in innate and adaptive immunity, and complex neuropathological events necessitate the use of large numbers of animals to obtain meaningful results. Our murine model allows the investigation of cell-to-cell interactions as they occur during HIV-induced CNS disease. In particular, interactions of infected MP with effectors of adaptive immunity over a range of disease stages can be investigated.

For example, placement of HIV-1-infected human MDM in brains of NOD/SCID mice reconstituted with autologous lymphocytes allowed observations of cell-to-cell interactions among lymphocytes, HIV-1-infected MDM, microglia, and astrocytes during ongoing tissue damage and restitution. Moreover, lymphocytes are recruited to and proliferate within the brain in association with HIV-1-infected human MDM through the generation of a chemokine gradient and recognition of viral Ags. This is probably a consequence of glial inflammation induced by infected MDM and not of a xenogeneic reaction against mouse Ags. This is supported by the observation that only limited numbers of lymphocytes are found in brains of animals injected with uninfected MDM. PBL transendothelial migration into and proliferation within the brain were virtually abrogated after the elimination of infected human MDM from the brain. Together, the de novo innate and cellular immune responses generated as a consequence of HIV-1-infected macrophages can be studied in this animal model.

It is noteworthy to review the limitations of these mice in studies of immune functions. NOD/SCID mice have reduced NK activity, diminished production of IL-1{beta} by macrophages after LPS stimulation, and lowered C5 components (41). The mice lack a functional DNA repair mechanism for VDJ gene segment recombination (42). Neurons in adult mice are often more susceptible to apoptotic death (43, 44), and tissue recovery after traumatic and inflammatory insults can be delayed (45, 46). Despite these limitations, the model permitted the study of key features of HIVE and HAD pathogenesis. First, HIV-1-infected human MDM elicited a glial inflammatory response that produced a chemokine gradient, permitting the attraction of human lymphocytes in murine brain tissue (47). Second, both an innate and an adaptive immune response followed the injection of HIV-1-infected MDM, eliciting clearance of infected cells through generation of CTL (31) and the persistance of microglial and astrocyte immune activation responses. Third, mouse monocytes were recruited into the brains of hu-PBL/HIVE animals. Such penetration of a murine blood-brain barrier by human lymphocytes and retention of cells by brain tissue could not be recapitulated by simple injection of chemokines and cytokines into recipient animals (48). Fourth, expansion of human lymphocytes occurred as a result of Ag presentation by infected human macrophages. Fifth, significant neural damage ensued as a consequence of infected macrophages and an induced glial immune response, which was restored after elimination of infected cells. Sixth, a profound neurotrophic and anti-inflammatory cytokine response was induced, with a marked and significant reduction in iNOS that paralleled tissue restitution. Thus, despite inherent limitations, the model was successful in recapitulating a number of important aspects of immune and neural responses that are operative during human disease. The duration of HIV-1 infection and lymphocyte attraction as well as human cell and viral persistence in a murine environment await further investigation.

The neuropathological findings in this study were not the result of trauma. Stereotactic injection induced minimal injury, as previously shown (30, 49). Moreover, the most significant neural damage, 7 days after injection of infected MDM, reflected the high level of expression of viral and cellular products from human macrophages, lymphocytes, and mouse glia rather than from trauma. The diminished expression of neuronal proteins around the injection site in hu-PBL/HIVE mice compared with HIVE or sham mice supported this idea. This was shown in hu-PBL/HIVE mice by the relationship between increased numbers of infected human cells and glial activation. Despite significant reductions in infected MDM on day 14, the presence of human lymphocytes facilitated the attraction of mouse peripheral monocytes and led to sustained expression of iNOS and proinflammatory cytokines. Interestingly, by day 21 tissue restitution was observed, probably due to several, but not mutually exclusive, mechanisms. Significant activation of microglia and influx of peripheral murine monocytes provided a clearing of tissue debris from the area of injection. The ensuing neurotrophic factors served to facilitate neural repair (50, 51). We found an up-regulation of neurotrophic factors, including the nonneuronal growth factors, bFGF-2 and EGF. These two factors are involved in in vivo proliferation and migration of neuronal progenitor cells and can provide additional pathways for brain repair (52). Overexpression of IL-10 served in part to affect microglia inflammatory responses. IL-10 is induced in microglial cells during phagocytosis of apoptotic lymphocytes (53) and might have an even broader role in neuronal repair through its abilities to induce neurotrophic factor production (54). Lymphocytes entering the brain may accelerate the process of neuronal injury. Nonetheless, mounting data from several laboratories demonstrate a neuroprotective role for lymphocytes in a number of experimental models for neurodegenerative diseases (27, 28).

In conclusion, our hu-PBL/HIVE mouse model reflects novel aspects of HIV-1 neurological disease not easily studied in other systems or animals. The observations recorded in this study support a multifaceted role for HIV-1-specific and activated lymphocytes in regulating disease during viral infection and immune clearance from the brain. This includes induction of neural damage and tissue repair. Certainly the use of NOD/SCID mice in studies of viral neuropathogenesis will provide a unique tool for the evaluation of disease mechanisms operative in the infected human host.


   Acknowledgments
 
We thank Drs. Jenae Limoges, Yuri Persidsky, Anuja Ghorpade, and Ximena Paez for critical review of the manuscript, and Robin Taylor for excellent administrative assistance. Technical assistance provided by Janice Taylor for confocal laser scanning microscopy and by Linda Wilkei and Dr. Charles Kusynski for flow cytometry is thankfully appreciated.


   Footnotes
 
1 This work was supported by National Institutes of Health Research Grants P01MH57556, P01NS31492, R01NS34239, and R37NS36136 (to H.E.G.) and National Center for Research Resources P20RR15635 (to L.P. and H.E.G). Back

2 Address correspondence and reprint requests to Dr. Larisa Poluektova, 985215 Nebraska Medical Center, Omaha, NE 68198-5215. E-mail address: lpoluekt{at}unmc.edu Back

3 Abbreviations used in this paper: HAD, HIV-1-associated dementia; BDNF, brain-derived neurotrophic factor; bFGF-2, basic fibroblast growth factor-2; EGF, epidermal growth factor; HIVE, HIV encephalitis; hu-PBL/HIVE, human PBL, HIV encephalitis; i.c., intracranially; iNOS, inducible NO; LCA, leukocyte common Ag; MAP, microtubule-associated protein; MDM, monocyte-derived macrophage; MNGC, multinucleated giant cell; MP, mononuclear phagocyte; NeuN, neuron-specific nuclear protein; NGF, nerve growth factor; NOD, nonobese; NT-3, neurotropin-3. Back

Received for publication January 23, 2004. Accepted for publication April 13, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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