Kuebler DJ, Martin LN, Baskin GB: 1991. Serial patho- genesis study of SIV ... Zack PM, Elkins WR, Desrosiers RC, Eddy GA: 1994. Titration and characterization ...
American Journal of Pathology, Vol. 146, No. 4, April 1995 Copyright © American Society for Investigative Pathology
Transfer of Neuropathogenic Simian Immunodeficiency Virus with Naturally Infected Microglia
Debbie Watry, Thomas E. Lane, Marla Streb, and Howard S. Fox From the Department of Neuropharmacology, The Scripps Research Institute, La Jolla, California
The central nervous system (CNS) is a targetfor human immunodeficiency virus infection, and, in individuals with acquired immune deficiency syndrome, this can lead to a devastating dementia. Only certain viral variants appear capable of invading the CNS and infecting microglia and brain macrophages. To determine whether the virus entering the brain may beparticularlypathogenic to the CNS, we isolated microgliafrom the brains of simian immunodefciency virus-infected rhesus monkeys. Serial transfer of these ceUs into naive animals indicated that productive simian immunodeficiency virus infection could indeed be transferred. Furthermore, CNS infection occurred within a relatively short time span and was associated with viral gene expression in the brain and pathology characteristic of human immunodeficiency virus encephalitis. While demonstrating that neuropathogenic variants partition into the CNS, our approach will allow the dissection of functional neuropathogenic elements present in these viruses. (Am J Pathol 1995,
146:914-923) The simian immunodeficiency virus (SIV) is very similar to human immunodeficiency virus (H IV), the cause of acquired immune deficiency syndrome (AIDS).1' 2 Experimental inoculation of SIV into rhesus monkeys resulted in an acquired immunodeficiency syndrome that closely resembled human AIDS. The targets for infection by these viruses are CD4+ T cells as well as tissue macrophages. In the organs, except lymph nodes, cells of the macrophage lineage are the predominant cell types infected. Both HIV and SIV have been shown to infect the central nervous system (CNS) with HIV causing the HIV-1-associated
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cognitive/motor complex, also known as the AIDS dementia complex. Hopes that the arrival of antiretroviral therapy would diminish the incidence of dementia have not been realized, and approximately one-third of infected individuals suffer this debilitating consequence.35 At postmortem examination, the brains of such patients frequently show parenchymal changes that include microglial nodules, infiltrating macrophages, and multinucleated giant cells.6 10 In many studies, the neuropathology of SIV infection closely resembled that of HIV infection.11-14 In the CNS, the infected cells are predominantly infiltrating macrophages and microglia, bone marrow-derived cells that share many characteristics with tissue macrophages.3'6'11 In rhesus monkeys injected with SIV, within 1 week, virus is visible in the brain. 12,13 Early during this infection, virus localizes mainly in perivascular mononuclear cells but later is evident in microglia, infiltrating macrophages, and multinucleated giant cells. Although a recent report described HIV sequences in neurons, identified during a late stage of infection by using the in situ polymerase chain reaction technique,15 the vast majority of such infected cells in the CNS are macrophages and microglia. In terms of cellular tropism, studies of both HIV and SIV have revealed a strong correlation between neuroinvasive viruses and macrophage tropism; these properties also correlate with sequences in the viral envelope gene.16-19 Although numerous mechanisms enable viruses to enter the brain, the Trojan horse model for lentivirus infection has much support.3'20 This holds that infected immune cells travel to the brain, carrying virus with them and leading to CNS infection. In rodents, both T cells and monocytes normally can enter the CNS from the blood.21'22 T cells can migrate through the CNS parenchyma, and monocytes may pass Supported by National Institute of Mental Health Grant MH 47680. Accepted for publication January 12, 1995. Address reprint requests to Dr. Howard Fox, Department of Neuropharmacology, CVN-8, The Scripps Research Institute, 10666 N. Torrey Pines Road, La Jolla, CA 92037.
Microglia-Associated SIV 915 AJP Apil 1995, Vol. 146, No. 4
through or take up residence as meningeal or perivascular macrophages. Rarely, or perhaps slowly, microglia cells are also replaced by new bone marrow-derived cells. Similarly, in humans receiving bone marrow transplants, donor cells are found frequently in perivascular locations and may occasionally replace microglia.23 Only a minority of individuals develop HIV-induced neuropathology in AIDS. Similarly, only a minority of animals show distinct neuropathology after infection with SIV, hampering its study. The variation in disease patterns induced by SIV in monkeys undoubtedly results from differences in the stocks and clones used for infection, in normal biological responses in vivo, and in the genetic background of animals obtained from different sources.24 After infection by HIV or SIV, related but distinct quasispecies of virus exist in the blood and tissues. However, both HIV and SIV isolated from the brain are relatively unique, with restricted heterogeneity.1725-28 It is clear from other systems, particularly mouse retroviruses, that certain viral strains have unique potentials to induce CNS disease.2>33 We therefore questioned whether we could select for virus capable of inducing CNS disease by enriching for naturally neuroinvasive SIV. Additionally, as microglia and brain macrophages are the targets of CNS infection, we speculated that the virus associated with these cells might yield a neuropathogenic variant. By using an in vivo selection scheme, with microglia as the source of virus, we obtained such a strain, yielding neuroinvasion and neurovirulence. These studies demonstrate that functionally neuropathogenic SIV virions partition into the CNS compartment during infection. Our approach will allow dissection of the factors causing the devastating CNS disease observed with these viruses.
Materials and Methods Animals and Tissues Rhesus monkeys, free of type D simian retroviruses and herpes B virus, were obtained from an isolated colony on Key Lois (Charles River Laboratories, Wilmington, MA), housed in a containment facility, and handled after anesthesia with ketamine. They were infected by injection into the saphenous vein and bled from the femoral vein. These animals were sacrificed by lethal anesthesia, followed by intracardiac perfusion with phosphate-buffered saline (PBS). Microglia were isolated from one-half of their PBS-perfused brains by a modification of a recently described technique.34 After removal of the meninges, the brains were sterilely minced with scalpels in the presence of
Hanks' balanced salt solution (HBSS) containing 2% human AB' serum (GIBCO BRL, Gaithersburg, MD), then pushed through a 100-mesh screen; the resulting suspension was washed by centrifugation in HBSS. This suspension was then digested in HBSS containing 40 U of collagenase 11 (GIBCO BRL) and 50 U of DNase (Sigma Chemical Co., St. Louis, MO) per ml for 1 hour at 37 C. After being washed twice in HBSS, the suspension was brought to 1.03 g/ml with Percoll (Pharmacia, Uppsala, Sweden) and layered over Percoll at 1.088 g/ml. The single step gradients were centrifuged at 800 x g for 20 minutes; cells harvested from the interface were then diluted with RPMI 1640 medium and washed twice with RPMI 1640. For cryopreservation, cells were frozen slowly in 80% RPMI 1640, 10% human AB' serum, and 10% dimethylsulfoxide, then stored under liquid nitrogen. Peripheral blood mononuclear cells (PBMC) were isolated from the monkeys' heparinized blood by dilution with an equal volume of HBSS and centrifugation over Ficoll-Hypaque (Sigma) at 400 x gfor 20 minutes. For FACS analysis, cells were incubated with optimized concentrations of fluorescein isothiocyanate or phycoerythrin-labeled anti-CD4 (Ortho Diagnostic Systems, Raritan, NJ), anti-CD8, anti-CD11 b, or an irrelevant antibody (all from Becton Dickinson, San Jose, CA) washed, fixed in paraformaldehyde, and analyzed on a FACScan (Becton Dickinson, Mountain View, CA).
SIV Infection of Rhesus Macaques The first group of six animals was infected intravenously (i.v.) with 800 rhesus monkey infectious doses of SlVmac251 (kindly provided by Dr. R. Desrosiers, N.E. Primate Research Center, Harvard University, Boston, MA). Animals were sacrificed at 6 to 16 months after infection (three animals were sacrificed because of their AIDS-like disease, and the other three were scheduled sacrifices). In the initial animal infected with the microglial preparation, 10 x 106 cells freshly isolated from animal 183 (15 months after infection) were injected i.v. into animal 230. For subsequent infections in the second round of transfer, cryopreserved microglia that had been freshly isolated from animal 230 were thawed, washed twice in RPMI 1640, suspended in a 1-ml volume, and injected i.v. into animals 181 (0.5 x 106 cells), 182 (1 .25 x 106 cells), and 185 (2.0 x 106 cells). Regardless of the source of virus, all animals were productively infected, manifested by an immune response, presence of viral antigen in the plasma, and recovery of virus from PBMC.
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AJP April 1995, Vol. 146, No. 4
Cell and Virus Cultures Microglia were cultured in Mac-SFM (GIBCO BRL) containing either 1 ng/ml granulocyte-macrophage colony-stimulating factor (100 U/ml, Collaborative Biomedical Products, Bedford, MA) or 10% Mac-Stim (Collaborative Biomedical Products) containing macrophage and granulocyte-macrophage colonystimulating factor in a humidified 37 C, 6% CO2 incubator. Primary cultures were washed with warm PBS after 5 days in culture, and the medium was replenished on the remaining adherent cells every 3 days. Uptake of fluorescently labeled acetylated low density lipoprotein (Dil-Ac-LDL; Biomedical Technologies, Stoughton, MA) was determined by incubation of cultures with 10 pg/ml Dil-Ac-LDL for 4 hours, followed by washing in PBS, fixation in 3% paraformaldehyde, and examination by fluorescence microscopy. Immunocytochemistry for glial fibrillary acidic protein (GFAP) was performed on cultures fixed in 70% ethanol, reacted with polyclonal rabbit anti-GFAP (Dako Corp., Carpinteria, CA) followed by fluorescein-labeled goat anti-rabbit immunoglobulin G, and examined by fluorescence microscopy. For coculture experiments with rhesus macrophages, macrophages were grown as adherent cells from rhesus PBMC in 90% Mac-SFM and 10% Mac-Stim. Such culture yields >95% Dil-Ac-LDL-positive cells. For viral recovery, PBMC from infected animals were stimulated for 4 days with 5 pg/mI PHA-M (CalBiochem, La Jolla, CA) and 50 U/ml interleukin-2 (Boehringer Mannheim, Indianapolis, IN). Stimulated PBMC were then added to an equal quantity of 174 x CEM cells and examined for syncytia formation and/or the supernatant was analyzed for SIV p27 production. The 50% tissue culture infectious dose viral titers were calculated by the Reed-Muench method after 7 and 14 days of culture. Plasma and tissue culture SIV p27 was measured by enzyme-linked immunosorbent assay with a commercially available kit (Coulter Corp., Hialnah, FL).
Histopathology Tissues were fixed in 10% neutral-buffered formalin and stained with hematoxylin and eosin (H&E) for histopathological analysis. Sections were obtained from paraffin blocks for all organs and multiple blocks from the CNS; luxol fast blue stains were used for selected CNS tissue blocks. Immunocytochemical stains were applied to paraffin-embedded material. Tissues were deparaffinized, digested in trypsin for 30 minutes (for Mac387 and CD3 staining only), blocked, and incubated with optimized dilutions of primary antibody (1/
300 for Mac387, anti-myelomonocytic Li protein (Dako); 1/300 for anti-CD3 (Dako); 1/750 for antiGFAP (Dako) and 1/3000 for FA2 anti-SIV p2735). After washing, species-specific biotinylated secondary antibody was added; then incubation and washing were followed by incubation with peroxidase-labeled avidin-biotin complex (Vector Laboratories, Burlingame, CA) and washing. Color was then developed with the VIP stain (Vector); slides were counterstained with Gill #1 hematoxylin and coverslipped. Transmission electron microscopy was performed on specimens fixed in modified Karnovsky's fixative (2% paraformaldehyde, 1.5% glutaraldehyde, 0.1 mol/L cacodylate buffer, pH 7.4) for 2 hours, post-fixed in 1% OS04 for 1 hour, dehydrated in graded ethanol, and embedded in epoxy resin Taab 812 (Taab Laboratory Equipment, Reading, UK). Specimens were stained with uranyl acetate and lead citrate and viewed with a Hitachi HU 12 A electron microscope.
Results Infection with the SlVmac251 Stock Initially, we examined rhesus macaques infected with the SlVmac251 virus stock. The result of infection was typical SIV-induced pathology, including giant cell pneumonitis, protozoan enterocolitis, noma, lymphoid hyperplasia and depletion, and lymphoproliferative disorders. Yet none of the animals had the classic neuropathological lesions of multinucleated giant cells and microglial nodules; the brain of only one animal contained a single, poorly formed multinucleated cell. To obtain a more neurovirulent strain, we then attempted to enrich (select) for a limited set of neuroinvasive quasispecies of virus, derived from the brain of infected animals, that could then infect the CNS of other monkeys through the i.v. route of inoculation.
Isolation of In Vivo Infected CNS Cells As most studies find most HIV- or SIV-infected cells in the CNS are microglia and macrophages, a recently described procedure was adapted to isolate microglia from the brains of infected animals (see Materials and Methods). FACS analysis for the CD1 1 b cell surface antigen showed that 50% of the isolated cells were microglia or macrophages (Figure 1). Of the remaining cells, 10% stained for the CD8 marker, which we have found to include SIV-specific cytotoxic T cells (M von Herrath, MBA Oldstone, and HS Fox, J Immunol, in press), whereas no significant staining for CD4 was seen. Electron microscopy revealed cells
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