function of CNS capillary endothelial cells and capillary ... formed, nonproducer, sarcoma-positive, leukemia- negative cell line) ..... and follicular dendritic cells.
American Journal of Pathology Vol. 138, No. 3, March 1991 Copyright © American Association of Pathologists
Murine Retroviral Neurovirulence Correlates with an Enhanced Ability of Virus to Infect Selectively, Replicate in, and Activate Resident Microglial Cells T. V. Baszler and J. F. Zachary From the Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Illinois, Urbana, Illinois
To determine the biologic basis of tsl MoMuLV neurovirulence in vivo, newborn CFW/D mice were inoculated with neurovirulent tsl MoMuLV and nonneurovirulent wt MoMuLV and the temporal response to virus infection in the central nervous system (CNS), spleen, and thymus was studied comparatively. Experimental procedures included single and double labeling in situ immunohistochemistry with selective morphometric analyses, and steadystate immunoblotting of viral proteins. Cellular targets for virus infection were identical for both tsl and wt MoMuLV and consisted sequentially of 1) splenic megakaryocytes, 2) splenic and thymic lymphocytes, 3) CNS capillary endothelial cells, and 4) CNS pericytes and microglia Resident microglial cells served as the major reservoir and amplifier of virus infection in the CNS of tsl MoMuLV-infected mice; a similar but much less significant role was played by microglia in wt MoMuLV-infected mice. The genesis and progression of severe spongiform lesions in tsl MoMuLV-infected mice were both temporally and spatially correlated with amplified virus infection of microglia4 and hyperplasia and hypertrophy of both virus-infected and nonvirus-infected microglial cells. Direct virus infection of neurons was never observed The development of clinical neurologic disease and spongiform lesions in tsl MoMuLV-infected mice correlated with the accumulation of both viral gag and env gene products in the CNS; there was no selective accumulation of envprecursor polyprotein Pr8Oenv. When compared to wt MoMuLV-infected mice, the neurovirulence of tsl MoMuLV-infected mice occurred by an enhanced ability to replicate in the CNS and to infect and activate more microglig rather than by afundamental change in cellular tropism or topography of virus infection. (Am JPathol 1991, 138:655-671)
Neurovirulent tsl Moloney murine leukemia virus (MoMuLV) causes a progressive neurodegenerative disease when inoculated into susceptible strains of newborn mice.1 Infected mice develop generalized tremors and progressive hind-limb paralysis associated with noninflammatory spongiform lesions and loss of motor neurons in selected nuclei of the brainstem and ventral horns of the cervical and lumbar enlargements of the spinal cord.23 Interest in studying the murine neurovirulent retroviruses has increased with the emergence of neurologic diseases induced by human retroviruses such as human immunodeficiency virus (HIV), the causative agent of acquired immune deficiency syndrome (AIDS) dementia complex,4 and the human T-cell lymphotropic virus-1 (HTLV-1)-associated myeloneuropathies.56 In addition, the neurologic diseases induced by neurovirulent murine retroviruses share clinical and pathologic similarities with motor neuron diseases of unknown etiology such as amyotrophic lateral sclerosis.7 The pathogenesis of tsl MoMuLV-induced neurodegenerative disease is not well understood. Following either intraperitoneal or intracerebral inoculation of virus into newborn CFW/D or BALB/C mice, there is an initial replication in the spleen and thymus followed by plasma viremia, hematogenous spread to the central nervous system (CNS), and gradual elevation of CNS viral titers with the progression of neurologic disease.2 The spleen and thymus appear to play an essential role in the development of neurologic disease. The spleen serves as the major virus factory and the source of plasma viremia.2 The thymus appears to be involved in disease pathogenesis because athymic mice inoculated with tsl MoMuLV fail to develop neurologic disease.8 The cellular targets and regional distribution for virus infection in the spleen and thymus are not well characterized. Ultrastructural studies of tsl MoMuLV-infected mice revealed that Supported by the Muscular Dystrophy Association. Accepted for publication November 2, 1990. Address reprint requests to Dr. Timothy Baszler, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040.
655
656
Baszler and Zachary
AJP March 1991, Vol. 138, No. 3
splenic megakaryocytes were the dominant cell target in the spleen9; intravascular platelets and cell-free viremia were responsible for systemic spread of virus to the CNS.9 High viral titers in the CNS correlate with the progression of neurologic disease and degenerative spongiform changes in the CNS appear to be a direct consequence of virus replication and the accumulation of viral proteins.2 It is unclear whether neuronal degeneration is caused directly by infection of specific target neurons, or indirectly by infection of supportive neural cells such as endothelial cells, astroglia, oligodendroglia, or microglia. Some ultrastructrural studies of other neurovirulent murine retroviruses that cause a similar spongiform encephalopathy as tsl MoMuLV (Cas-Br-E MuLV and TsMoBA-1 MoMuLV) suggest that neuronal degeneration is secondary to metabolic alterations caused by virus-induced dysfunction of CNS capillary endothelial cells and capillary basal lamina.1011 Alternatively ultrastructural and in situ immunohistochemical studies of the Cas-Br-E MuLV model demonstrated virions and viral antigen within neurons and attributed neuronal degeneration to direct virus infection.12-14 Temporal ultrastructural studies of the tsl MoMuLV model indicated that spongiform lesions originated from vacuolar degeneration of both neuronal and oligodendroglial processes;9 spongiform neuronal degeneration was related directly to virus-infected microglial cells and was not associated with virus infection of neurons.9 These findings suggest that an indirect mechanism is responsible for neuronal dysfunction. The molecular basis of murine retroviral neurovirulence is understood partially. tsl MoMuLV has a unique env gene defect that impairs normal processing of envelope precursor polyprotein Pr8Oenv to gp7O and p15E and results in the intracellular accumulation of Pr8Oenv at the nonpermissive temperature in vitro.15 Wild type (wt) MoMuLV processes Pr8Oenv normally, is nonneurovirulent, and induces T-cell lymphoma when inoculated into similar strains of mice.2'15 These findings suggest that abnormal processing and accumulation of Pr8Oenv may play a central role in the development of neurologic disease in tsl MoMuLV-infected mice. The target cells for putative Pr8Oenv accumulation are not known but studies to date have focused on the neuron.2,16,17 A single in vitro study suggests that a single amino acid substitution in tsl MoMuLV Pr8Oenv is responsible for nonprocessing and neurovirulence17; however other studies demonstrated multiple disparate env gene mutations as well as changes in the viral long terminal repeat.18,19 The in vivo significance of selective intracellular accumulation of unprocessed Pr8Oenv in the CNS is unclear. Some temperature-sensitive mutants of Rauscher leukemia virus that cannot process Pr8Oenv in vitro do not cause neurologic disease when inoculated into mice.2021 In addition, some chimeric constructs be-
tween tsl and wt MoMuLV that contain the gene segment responsible for nonprocessing of Pr8Oenv (tsl wt-1 3 and tsl wt-32) do not cause paralysis.1718 These findings suggest that other env gene mutations, alone or in combination with the Pr8Oenv nonprocessing defect, are responsible for the neurovirulent phenotype.19 Like tsl MoMuLV, Cas-Br-E MuLV also has disparate env gene mutations that appear to be responsible for viral neurovirulence.22 Because env sequences determine receptor specificity, these findings suggest that receptor recognition may be involved in neurovirulence for both tsl MoMuLV and Cas-Br-E MuLV (altered cellular tropism or topographic distribution of virus in the CNS). In addition recent studies of the Cas-Br-E MuLV model have demonstrated that the long terminal repeat promoter and enhancer regions of the virus genome have a profound effect on the level of CNS infection and the rate of neurodegeneration.23 These findings suggest that quantitative factors also may be involved in neurovirulence of Cas-Br-E MuLV. The mechanisms of neurovirulent murine retroviralinduced neurologic disease could be defined more clearly by identifying cellular targets that are relevant to disease pathogenesis and by clarifying the role of virus load on the development of CNS lesions. Our laboratory designed a series of in vivo comparative studies that exploited the genetic differences between neurovirulent tsl MoMuLV and non-neurovirulent wt MoMuLV. The goals of these studies were to 1) define the lymphoid and neural cell targets for productive virus infection; 2) determine how virus gains access to the CNS and elucidate a sequence of virus entry, spread, and replication within the CNS; 3) differentiate between direct and indirect mechanisms of neuronal degeneration; and 4) determine what virus factors may be involved in the neurovirulence of tsl MoMuLV when compared to wt MoMuLV by contrasting changes in systemic and CNS cellular tropism, numbers of infected neural cells, topographic distribution of viral proteins in the CNS, and the differential accumulation of specific viral gene products in the CNS.
Materials and Methods Virus and Virus Assay tsl MoMuLV is a spontaneous mutant of wt MoMuLV-TB. The isolation and characterization of both viruses have been described previously,24 and both viruses have been single virus, single cell cloned by limiting dilution.25 Stock viruses were harvested from persistently infected thymus bone marrow (TB) cells26 and quantitated using a modified 15F direct focus forming assay (nontransformed, nonproducer, sarcoma-positive, leukemianegative cell line) to determine virus titers.27 All cell lines
Neurovirulent Retrovirus and Microglia
657
AJP March 1991, Vol. 138, No. 3
maintained in Dulbecco's modified eagle minimal essential medium supplemented with 10% fetal bovine serum, 50 lU/ml penicillin, and 50 mg/ml streptomycin.
were
Mice CFW/D mice were donated by Dr. J. K. Ball, University of Western Ontario, London, Ontario, Canada and were tested semiannually and determined to be free of exogenous virus antibody (Charles River Professional Services, Willmington, MA). Whole litters of randomly selected neonatal CFW/D mice (12 to 24 hours old) were divided into tsl and wt MoMuLV groups and inoculated intraperitoneally with 0.1 ml of virus suspension that contained 105 to 1 06 infectious units of respective stock virus grown in TB cell culture. Control mice received a similar volume of conditioned medium from noninfected TB cells. Mice were examined daily for clinical signs of neurologic disease. On days 5, 10, 15, 20, 25, 30, and 35 after inoculation (the entire disease period), at least three mice from each experimental and control group were killed and processed for light microscopy, immunohistochemistry, or immunoblotting. In total, at least 21 tsl MoMuLV-infected, 21 wt MoMuLV-infected, and 21 control mice were examined by each of these experimental methods.
CNS Immunoblotting Immunoblotting studies were performed only on CNS tissues. At 5-day intervals after inoculation, mice were anesthetized by ether inhalation and intracardially perfused with cold (40C) phosphate-buffered saline pH7.4 to flush residual blood from the CNS. The entire brain stem and spinal cord were dissected, immersed and minced in cold lysis buffer (10 mmol/l [millimolar] potassium phosphate, pH7.4, containing 0.5% Triton X-100 [Sigma Chemical Co., St. Louis, MO], 0.5% aprotinin [Sigma
Chemical Co], and 10 ,ug/ml leupeptin [Sigma Chemical Co.]), sonicated in an ultrasonic cleaner (Branson Cleaning Equipment Co., Shelton, CT), and centrifuged at 12,000g at 40C for 10 minutes. Central nervous system lysate supernatants subsequently were suspended in 4 volumes of 5X sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (315 mmol/l TRIS, pH6.8, 5% SDS [Bio-Rad Laboratories, Richmond, CA], 5% 2-mercaptoethanol, 25% glycerol, 0.01% bromophenol blue), heated for 5 minutes at 1 00°C, and frozen at - 800C. The concentration of protein from each brain homogenate was determined before the addition of SDS-PAGE sample buffer by the colorimetric method of Bradford with a commercial coomassie protein reagent assay (Pierce Chemical Co., Rockford, IL).28 A consistent quantity of CNS lysate (35 to 40 ,ug protein) from each sample was loaded onto an 8% to 16% SDSpolyacrylamide gel and electrophoresed under denaturing conditions using the one-dimensional, discontinuous method of Laemmli-9 and electrophoretically transferred onto nitrocellulose as described by Towbin.30 Immunodetection of viral antigens on the nitrocellulose blots was performed with both anti-MuLV gag and anti-gp7O (env) antibodies (Table 1) using the indirect avidin-biotin-complex method with conjugated alkaline phosphatase (Vector Laboratories, Burlingame, CA). The specificity of the antiviral antibodies was determined by immunoblotting against viral proteins obtained from lysates of tsl and wt MoMuLV-infected TB cell cultures. All incubations were carried out at room temperature. Nonspecific binding was blocked by incubation of the nitrocellulose sheet for 30 minutes in TRIS-buffered saline/ Tween 20 (TTBS) (0.1 mol/I [molar] TRIS, pH7.6, 0.15 mol/l NaCI, 0.1% Tween 20 [Sigma Chemical Co.]) that contained 3% bovine serum albumin (BSA) and 3% normal serum (from the species of animal in which the secondary antibody was obtained). TTBS with 3% BSA and 3% normal serum was also used as the diluent for the primary and secondary antibodies. The primary antiviral antibodies were absorbed against CNS lysates from con-
Table 1. Antibodies Usedfor Immunohistochemistry (IHC) and/or Immunoblotting (IB) Test Type Specificity
Antiviral Antibodies (dilution) Polyclonal anti-gp7O (NIH 81S000082) (1:200) Polyclonal anti-MuLV (5020) (1:400)
IHC; IB
Viral env proteins
IHC; IB
Viral gag proteins
CNS Cellular Markers (dilution) Biotinylated Ricinus Communis
IHC
Microglia
Polyclonal anti-glial fibrillary acidic protein (GFAP) (1:1000) Polyclonal anti-carboanhydrase-C (1:200)
IHC
Astroglia
IHC
Oligodendroglial soma
Monoclonal anti-nonphosphorylated neurofilament (SMI 32) (1:1000)
IHC
Neuronal soma
(RCA-I) (1:800)
Source
Microbiological Associates, Bethesda, MD Electro-Nucleonics, Silver Springs, MD Vector Laboratories, Burlingame, CA DAKO Corp., Carpinteria, CA CALBIOCHEM Corp., San Diego, CA
Sternberger-Meyer, Jarrettsville, MD
658
Baszler and Zachary
AJP March 1991, Vol. 138, No. 3
trol mice to reduce nonspecific binding to resident CNS proteins. The nitrocellulose sheets subsequently were incubated with the appropriate dilution of primary antibody for 1 hour, then a 1:200 dilution of biotinylated antispecies antibody (Vector Laboratories) for 30 minutes, and finally a 1:20 dilution of avidin-biotin-alkaline phosphatase complex (ABC-Alk-Phos; Vector Laboratories) for 30 minutes. Between each incubation step there was a thorough wash with TTBS (5 minutes; four times). Immunostaining was visualized by incubation of the nitrocellulose sheets for 3 to 5 minutes with an alkaline phosphatase substrate (alkaline phosphatase substrate kit 11; Vector Laboratories) diluted in 0.1 mol/l TRIS-HCI, pH 9.5. The sheets were washed in distilled water, air dried, and photographed. Negative controls consisted of replacement of the primary antibody with an equal concentration of normal serum from a homologous species, replacement of the primary antibody with TTBS alone, and incubation of the primary antibody on blots obtained from CNS tissue from control mice. Immunodetected bands were quantitated with a laser densitometer (LKB Ultroscan XL, Pharmacia LKB Biotechnology Inc., Piscataway, NJ) and evaluated by regression analysis for kinetic studies within each experimental group, and by the Student's T-test for comparison between experimental groups.
In Situ Immunohistochemistry Single and Double Immunolabeling At 5-day intervals after inoculation, mice were killed by ether inhalation and the brain, spinal cord, spleen, and thymus were immersion fixed in B-5 fixative (6% mercuric chloride, 0.15 mol/l sodium acetate and 4% formaldehyde) for 3 or 4 hours and transferred to cold (40C) 70% ethanol until the tissues were processed. Earlier temporal histopathologic studies of the tsl MoMuLV system revealed that the brain stem, cerebellar peduncles, and the ventral gray horns and adjacent white matter funiculi of the cervical enlargement (CE) and lumbar enlargement (LE) of the spinal cord consistently developed spongiform lesions typical of the disease syndrome.3 These areas of the CNS and random areas from the spleen and thymus were targeted for immunohistochemical studies and were trimmed, dehydrated, embedded in paraffin, and sectioned at 5 ,um. Viral antigens and neural cells were localized by the indirect avidin-biotin-complex immunoperoxidase method (Vector Laboratories) with the primary antibodies and lectins listed in Table 1. Anti-viral antibodies were characterized by immunoblotting (as described above) against viral proteins obtained from MoMuLV-infected cell cultures. Paraffin-embedded tissue sections were mounted on
acid-rinsed slides, prewarmed to 60°C, deparaffinized with xylene, and rehydrated in graded ethanols. Mercury pigment was removed from the sections by incorporating 1% iodine in the xylene rinse. Endogenous peroxidase activity was quenched by adding 3% hydrogen peroxide to the 100% ethanol rinse. The wash buffer and antibody diluent for all subsequent incubations consisted of 0.125 mol/l TRIS pH7.6 that contained 0.35 mol/l NaCI and 0.025% Triton X-100 (TRIS-TX)(Sigma Chemical Co.); all incubations were done at 370C. Tissue sections were washed in TRIS-TX for 20 minutes and incubated for 20 minutes with 1.5% normal serum (from the species in which the secondary antibody was obtained) to block nonspecific binding sites. Sections subsequently were incubated with the appropriate dilution of primary antibody for 30 minutes, then with a 1:200 dilution of biotinylated antispecies antibody (Vector Laboratories) for 20 minutes, and finally with a 1:20 dilution of avidinbiotin-peroxidase complex (Vector Laboratories) for 20 minutes. For incubation with the biotinylated lectin RCA-I, the secondary antibody step was deleted. Between each of the incubations the tissue sections were washed in TRIS-TX for 10 minutes. The diluent for the primary and secondary antibodies consisted of TRIS-TX with 1.5% normal serum. Immunostaining was visualized by incubating sections for 1 to 5 minutes with 0.5% diaminobenzidine tetrahydrochloride substrate (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, PA) in 0.1 mol/l TRIS-HCI pH7.6 that contained 0.01% H202. Finally sections were washed in distilled water, counterstained with Mayer's hematoxylin (Sigma Diagnostics, St. Louis, MO), rapidly dehydrated in graded ethanols and xylene, and mounted with Permount (Fisher Scientific Co., Fair Lawn, NJ). Negative controls consisted of substitution of the primary antibody with an equal concentration of normal serum from a homologous species, substitution of the primary antibody with TRIS-TX only, and incubation of the primary antibody on tissue sections from control mice. In addition, the RCA-I lectin was incubated with 0.2 mol/l lactose before addition to the tissue section to ascertain sugar specificity. Anti-nonphosphorylated neuorfilament immunohistochemistry was done on tissues fixed with cold acetone (- 200C) and processed by the AMeX method to reduce the denaturing effect of formalin on neurofilament antigens.31 Double immunostaining for the dual visualization of viral antigens and CNS cell markers on the same tissue section was carried out using a modified indirect sequential peroxidase/alkaline phosphatase method.32 Briefly the first and second antigens were visualized sequentially by the method described above, except that in the second sequence avidin-biotin-peroxidase complex was replaced with avidin-biotin-alkaline phosphatase complex (Vector Laboratories) and immunostaining was visualized
Neurovirulent Retrovirus and Microglia
659
AJP March 1991, Vol. 138, No. 3
with an alkaline phosphatase substrate (Alkaline Phosphatase Substrate l; Vector Laboratories) diluted in 0.1 mol/l TRIS-HCI, pH 8.2. The two sequences were divided by a thorough 20-minute wash in TRIS-TX.
to the sections cut for immunohistochemical studies. Sections were dehydrated, embedded in paraffin, sectioned at 5 ,um, and stained with hematoxylin and eosin for routine histopathologic evaluation and comparison with previous studies.
Quantitative Immunohistochemistry Results The degree of immunostaining in the CNS of both tsl and wt MoMuLV-infected mice was quantitated by tissue morphometry. Morphometric analysis was done by counting the number of positive staining cells obtained from 10 randomly selected 625-,um2 fields within targeted areas of the brainstem (gigantocellular reticular nucleus) and spinal cord (CE, LE). The numerical counts were described graphically and evaluated by the Student's T-test for differences between experimental groups.
Light Microscopy Coronal slices of brain and spinal cord from the target areas were obtained for histopathologic examination from B-S-fixed tissue sections that were serially adjacent
Spleen Viral antigens, as indicated by positive immunostaining with monospecific antiviral gag and env MuLV antibodies, were initially detected in the spleen 5 days after inoculation. There was no evidence of virus infection in either the thymus or CNS at this time. Temporal cellular targets and quantity of virus infection were identical in both tsl and wt MoMuLV-infected mice throughout the period of virus infection (days 0 to 35 after inoculation) and consisted of megakaryocytes, followed by lymphocytes and reticulum (stromal) cells (Figure 1 a). Viral antigens were detected within both T-cell and B-cell areas, as indicated by immunostaining of periarteriolar lymphoid sheaths and germinal centers, respectively; viral immu-
Figu re 1. Immunoperoxidase staining of the spleen (A) and thymus (B) using anti-MoMuLVantibody. A: Spleen at day 10 after inoculation; viral antigen accumulation was diffusely abundant within megakaryocytes (arrowheads), moderate within germinal centers (g) (B lympbocytes), and scant within periarteriolar sheaths (p) (T lymphocytes). Hematoxylin counterstain (X90). B: Thymus at day 10 after inoculation; viral immunostaining was mild and limited to scattered cortical lymphocytes (small ar-roubeads) and stromal cells (large arrowheads). Thymic cortex (c) and thymic medulla (m) (hematoxylin counterstain, x300).
660
Baszler and Zachary
AJP March 1991, Vol. 138, No. 3
nostaining of B-cell areas was more abundant and intense than viral immunostaining in T-cell areas (Figure 1a). Precise determination of cell lineage of positively stained splenic cells was difficult with single-labeling immunohistochemistry. Viral antigen accumulation in B-cell (follicular) areas may have occurred in both lymphocytes and follicular dendritic cells. The degree of viral antigen accumulation in the spleen peaked at days 15 to 20 after inoculation and remained constant for the remaining period of virus infection. Unlike wt MoMuLV-infected mice, tsl MoMuLV-infected mice developed progressive splenic lymphoid atrophy during the terminal stages of neurodegenerative disease (days 25 to 35 after inoculation). Microscopically, when compared to wt MoMuLVinfected and control spleens, the spleens of tsl MoMuLVinfected mice had diffuse white pulp lymphoid depletion, lymphoid depletion from periarteriolar sheaths and mantle zones, decreased number and size of germinal centers, and stromal collapse. Splenic lymphoid atrophy did not correlate with virus infection in the spleen. Virusinduced cytopathology in virus-infected cells was never observed and wt MoMuLV-infected mice, which never developed splenic lymphoid atrophy, had similar cellular targets and the same degree of virus antigen accumulation as tsl MoMuLV-infected mice.
Thymus Cell targets and the degree of viral immunostaining in the thymus were identical in both tsl and wt MoMuLVinfected mice throughout the entire period of virus infection (days 0 to 35 after inoculation). Viral antigens first were detected in the thymus at day 10 after inoculation, subsequent to infection of the spleen; infection was restricted to the cortex and targeted to occasional thymic stromal cells and lymphocytes (Figure 1 b). The degree of viral antigen accumulation in the thymus was never abundant and peaked at days 15 to 20 after inoculation. tsl MoMuLV-infected mice developed severe, progressive lymphoid atrophy in the thymus during the terminal stages of neurodegenerative disease (days 25 to 35 after inoculation). Affected thymuses had marked diffuse thinning of the cortex associated with severe depletion of cortical lymphocytes and moderate, multifocal individual lymphocyte necrosis. Similar to splenic lymphoid atrophy, thymic lymphoid atrophy was not correlated with the degree of virus infection. Virus-infected lymphocytes had no cytopathologic changes and wt MoMuLV-infected mice, which did not develop thymic atrophy, had similar cellular targets and the same relative degree of viral antigen accumulation as tsl MoMuLV-infected mice.
Central Nervous System Qualitative Immunohistochemistry Virus infection of the CNS occurred subsequent to infection of both the spleen and thymus. Both viral gag and env antisera labeled virus-infected neural cells equally. The degree of viral antigen accumulation and the sequence of virus infection were identical in both tsl and wt MoMuLV-infected mice until day 20 after inoculation. During this early period of virus infection, cellular targets for virus infection (first detected on day 15 after inoculation) were capillary endothelial cells and occasional microglia within targeted areas of the brainstem and spinal cord (Figure 2). Early virus infection of CNS capillary endothelial cells was temporally and spatially correlated with mild, local, vasocentric spongiform lesions and neuronal swelling in both wt and tsl MoMuLV-infected mice (Figure 2), but infection of endothelial cells was not associated with neurologic disease. Direct virus infection of neurons was not observed during the inception of early neuronal degeneration. There was occasional viral antigen immunostaining of capillary endothelial cells and Bergman's glial cells in the cerebellum; these areas of the CNS never developed spongiform lesions. The development of severe CNS spongiform changes and clinical signs of progressive neurologic disease occurred only in tsl MoMuLV-infected mice from days 20 to 35 after inoculation. During this late period of virus infection, the development of severe spongiform lesions correlated with virus spread to the neuropil, abundant localization of virus in resident CNS microglia, and hypertrophy and hyperplasia of resident microglial cells (Figure 3). Viral antigens localized to neural cells with a robust, branching morphology typical of activated (ramified) microglia (Figure 3). Perivascular or intravascular accumulation of blood-derived inflammatory cells (mononuclear or polymorphonuclear leukocytes) was never observed. During this same late period of virus infection, wt MoMuLV-infected mice had similar CNS cell targets but there was a significantly reduced quantity of viral antigen immunostaining and reduced number of virus-infected cells present within the targeted CNS areas; these mice never developed severe spongiform lesions or clinical neurologic disease. Microglia were identified definitively as the primary CNS cellular target for virus infection by double-labeling immunohistochemistry using monospecific antiviral antibody with a battery of neural cell markers. These studies showed near-total colocalization of viral antigens with neural cells that labeled with Ricinus communis-l (RCA-I) (Figure 4a). RCA-1 recognizes alpha-D galactose glycoconjugates that are unique to microglial cells and not present on other types of neuroglia
Neurovirulent Retrovirus and Microglia 661 AJP March 199J1, Vol. 138, No. 3
Figure 2. Immunoperoxidase staining of the brainstem at day 15 after inoculation using anti-MoMuLVantibody: Viral immunostaining of
capillary endothelia (large arrowheads) and mild vasocentric neuronal swelling and neuropil spongiosis. Occasional viral immunostaining of microglia (small arrowheads). No viral immunostaining of neurons (hematoxylin counterstain, X300).
or neurons. Although RCA-1 recognizes CNS endothelia and choroid plexus epithelial cells, staining of these cells can be distinguished readily morphologically.34 There also was minimal colocalization of viral antigens with an oligodendroglial somal marker (carbonic anhydrase C); this occurred late in the disease process (days 30 to 35 after inoculation) subsequent to the development of severe spongiform changes (Figure 4b). No localization of viral antigen was detected in neurons or astrocytes with single-labeling immunohistochemistry, nor was there colocalization of viral antigens with markers for neurons (nonphosphorylated neurofilament) or astrocytes (glial fibrillary acidic protein, GFAP) with double-labeling immunohistochemistry (Figure 4c and d). Both tsl and wt MoMuLV-infected mice had astrogliosis within targeted areas of the CNS that was proportional to the degree of local tissue damage (mild in wt MoMuLV-infected mice and marked in tsl MoMuLV-infected mice). There was a distinct temporal and spatial correlation between the degree of viral antigen accumulation (virus infection) in the brain stem and spinal cord, the severity of
CNS spongiform lesions, and the development of neurologic disease. wt MoMuLV-infected mice, which had only mild CNS spongiform lesions and never developed neurologic signs, had mild accumulation of viral antigen and few virus-infected neural cells (microglia) (Figure 5a). tsl MoMuLV-infected mice, which developed severe CNS spongiform lesions and severe neurologic signs, had abundant accumulation of viral antigen and many virusinfected neural cells (microglia) (Figure 5b). From days 20 to 35 after inoculation, tsl MoMuLV-infected mice had a greater relative abundance of viral antigen accumulation and much more severe CNS spongiform lesions when compared to wt MoMuLV-infected mice; viral antigen accumulation paralleled the degree of CNS spongiform lesions (Figure 5c). Spongiform changes in wt MoMuLV-infected mice had a similar topographic distribution as spongiform lesions in tsl MoMuLV-infected mice but were much less severe, did not progress past day 25 after inoculation, and regressed from days 25 to 35 after inoculation. A finding unique to wt MoMuLV-infected mice was the for-
662
Baszler and Zachary
AJP March 1991, Vol. 138, No. 3
Figure 3. Immunoperoxidase staining of the brainstem of a tsl mouse at day 30 after inoculation using antiMoMuLV antibody: Abundant viral immunostaining of resident microglial cells (arrowheads) that often had a robust, branching morphology. Virus-infected microgliaffrequentlv were directly adjacent to swollen degenerate neurons and areas ofneuropil spongiosis (hematoxylin counterstain, X 400).
MoMuLV-infected
mation of multinucleate syncytial cells during this period of spongiform lesion regression. The multinucleate syncytial cells stained with microglial-specific lectin (RCA-I), indicating their microglial origin. In addition, there was positive viral antigen immunostaining of syncytial cells due either to productive viral infection or to the phagocytosis of viral proteins. Multinucleate syncytia were not observed in tsl MoMuLV-infected mice. Quantitative Immunohistochemistry Tissue morphometry of anti-MuLV gag and envexpressing cells in both tsl and wt MoMuLV-infected mice indicated an approximately equal number of virusinfected neural cells through day 20 after inoculation (Figure 6). From days 20 to 35 after inoculation, the number of virus-infected cells in tsl MoMuLV-infected mice had a marked and continuous increase until the terminal stages of neurologic disease, whereas the number of virusinfected cells in wt MoMuLV-infected mice increased only slightly and then decreased during the same time period. By days 30 to 35 after infection there was a significant (unpaired t-test; P = 0.001) three- to four-fold increase in the number of virus-infected cells in the targeted areas of the CNS of tsl MoMuLV-infected mice when compared to wt MoMuLV-infected mice. Both viral
gag- and env-encoded antigens followed parallel curves and suggested general virus replication rather than selective accumulation of one viral gene product over another. Quantitative counts of microglial cells in the targeted areas of the CNS in both tsl and wt MoMuLV-infected mice, as indicated by positive staining with RCA-I lectin, revealed a similar temporal distribution pattern of cellular proliferation as had morphometry of virus-infected neural cells. tsl MoMuLV-infected mice had an increased number of microglia and more severe spongiform lesions when compared to wt MoMuLV-infected mice (Figures 7a and b). Double-staining immunohistochemistry revealed that RCA-1labeled microglia were by far the predominate cell target for virus infection in the CNS. The quantitative difference between the number of RCAL-labeled cells and the number of viral antigen-labeled cells represented the number of nonvirus-infected microglia within the targeted areas of the CNS (microglial cells that expressed only RCA-I versus microglial cells that coexpressed viral antigen and RCA-1). The numbers of nonvirus-infected microglial cells in the CNS of wt MoMuLV-infected mice was never high and paralleled the increase in virus-infected microglial cells throughout the period of virus infection (Figure 7c). In contrast, the number of nonvirus-infected microglial cells in tsl MoMuLVinfected mice was high and increased at a greater rate than the number of virus-infected microglial cells from days 25 to 35 after inoculation (Figure 7c). Hyperplastic microglia had a robust, branching morphology characteristic of activated cells (Figure 7b). These findings indicated that many virus-infected microglia and hypertrophied, nonvirus-infected microglia were associated with the development of severe CNS spongiform lesions in tsl MoMuLV-infected mice.
Immunoblotting of Viral Proteins in the CNS Immunoblots from the CNS of tsl MoMuLV-infected mice during the entire period of virus infection (days 0 to 35 after inoculation) revealed a gradual and simultaneous increase of both the major viral gag (p30)- and envencoded (gp70) proteins from days 10 to 35 after inoculation (Figure 8 a and b). The increase in viral proteins was slow from days 10 to 20 after inoculation but rose rapidly from days 20 to 35 after inoculation. Accumulation of viral proteins in the CNS was correlated temporally with the development of severe CNS spongiform lesions and neurologic signs. Linear regression analysis indicated an exponential increase in both viral gag and env-encoded proteins from days 20 to 35 after inoculation, a finding consistent with in situ viral replication in CNS tissue. Immunoblots from the CNS of wt MoMuLV-infected mice revealed a similar simultaneous increase of both viral gag and env-encoded proteins from days 0 to 20 after inoc-
Neurovirulent Retrovirus and Microglia 663 AJP March 1991, Vol. 138, No. 3
N
I39 I
2
4,
g
Figure 4. Double-labeling immunoperaxidase/immunoalkalinephosphatase staining of the brainstem of tsl MoMuLV-infected mice at day 30 after inoculation (bematoxylin counterstain, x 750). A: Abundant diffuse colocalization (arrowbeads) of viral antigen (brown chromogen) with the microglial cell-specific marker, RCA-I fred chromogen). Virus-infected and non-virs-infected microglia surround a degenerate neuron (n). B: Occasional colocalization (arrowbead) of viral antigen fred chromogen) witb the oligodendroglial somal marker, anti-carboanbydrase C (brown chromogen). C: No colocalization of viral antigen (brown chromogen) with the astroglial cell marker, anti-GFAP (red chromogen). D: No colocalization of viral antigen (brown chromogen) with the neuronal somal marker, antinonphobsporylated neurofilament fred chromogen).
i..
A.
Figure 5. Immunoperoxidase staining of the brainstem of wt (A) and tsl MoMuLV-(B) infected mice at day 30 after inoculation using anti-MoMuLVantibody. A: Brainstem of a wt MoMuL V-infected mouse at the level of the gigantocellular reticular nucleus (GCRN = 1) and medial vestibular nucleus (MVN = 2) with scant virus-infected cells (arrowheads) that were spatially correlated with mild spongiform lesions. B: Brainstem of a tsl MoMuLV-infected mouse at the level of the GCRN (1) and MVN (2) with a distinct parallelism between abundant viral antigen accumulation and virus-infected cells (arrowheads) and severe spongiform lesions. C: Temporal comparison of the relative degree of viral immunostaining and the relative severity ofspongiform lesions within the targeted areas of the CNS in wt and tsl MoMuLV-infected and control mice. The relative degree of viral immunostaining parallels the severity of spongiform lesions. 1 = minimal, 2 = mild, 3 = moderate, 4 = marked, 5 = severe.
6-
-----C
0) o C.)
----I
3.
U) U1)
2-
cc
1* 0 -1
_S,K~
665 AJP March 1991, Vol. 138, No. 3
.
0
10
:
I
30
20
40
Figure 5. (Continued).
ulation, with a subsequent decrease in to both viral gag and env-encoded proteins frompdays 25to 35 after inoculation that was correlated temporally with spongiform lesion regression (Figure 8b). The quantity of viral protein in the CNS of wt MoMuLV-inf( significantly lower than the quantity of vir al protein in the days 25 25 to 35 CNS of tsl MoMuLV-infected mice from daYs after inoculation (unpaired T-test; P = C synthesis occurred in conjunction with o\verall viral replication in both tsl and wt MoMuLV-infecte,d mice. tsl MoMuLV-infected mice did not have an exc(essive accumuvirus I foealvirus lation of Pr80env (compared to the level of overall replication in the CNS) and processing of Pr8Oenv to gp7O appeared to be normal in vivo. a) :
C.)
0
20
----a---
18
----U---
1614
0 U2)
12-
Ii) a) .0
10-
-U---
8U) CU
z
themquantiy
oe35 verallvi)alPr8epli
ts1 gag wt gag tsl env wt env
-l--- Virus control
0
EL
J,,-
6-
II'' ;
4.
_ __ _
20-2
..
0
..I
10
.
.
.
.
Discussion The comparative aspects of the immunohistochemical and immunoblotting findings between tsl and wt MoMuLV-infected mice are summarized in Table 2. The cellular targets for virus infection were identical in the spleen, thymus, and CNS of both tsl and wt MoMuLV-infected mice. The difference in CNS virus infection between tsl and wt MoMuLV-infected mice was quantitative, that is, tsl MoMuLV-infected mice had an enhanced ability to
Days postinoculation
C
_
I
Virus-tsl
54-
Neurovirulent Retrovirus and Microglia
_~~~~~~~~~~
ofx
.
20
30
40D
Days postinoculation
Figure 6. Temporal morphometrc analysis of tviral gag and envantigen expressing microglial cells in the brainmstem of wt and tsi MoMuLV-infected mice and control mice. DurinIg the development of severe spongiform lesions and progressive wurologic disease (day 25-35 after inoculation), tsl MoMuLV-injfected mice had a increase significant (unpaired t-test; P = 0.001) three- to in the number ofvirus-infected microglia within ithe targeted areas of the CNS when compared to wtMoMuLV-infect ed mice. The numbers of both viral gag- and envespressing cells followed parallel (simultaneous) curves. (tsl and wt gag = gagjprotein expressing microglia; tsl and wt env = env-protein-eVprensing microglia.)
tfoufold
IC! +r..t 11- -k-hr rrsl;^~~+kf;rs %1^12 ;rtrrr In ine UN..j wnen ana to inTeCi more neurai celis repiicaLe compared to wt MoMuLV-infected mice. Immunohistochemical findings demonstrated that virus spread tem%
.
porally from splenic megakaryocytes, to splenic and thymic lymphocytes, to CNS capillary endothelial cells, and finally to resident CNS microglia. tsl and wt MoMuLVinfected mice had the same topography of viral protein accumulation in the CNS, and topography of early, nonclinical, CNS spongiform lesions. These findings indicated that the neurovirulence of tsl MoMuLV when compared to non-neurovirulent wt MoMuLV did not depend on altered cellular tropism or topography of virus infection in the CNS but depended on quantitative factors. The possibility exists that the antisera used for the present in situ immunohistochemistry studies may crossreact with antigens of endogenous murine retrovirus activated by local tissue damage. We believe that the virus detected in these studies was input virus and not actideetdithsstdewainuviuannoacvated endogenous virus because the virus recovered from the CNS of paralyzed, tsl MoMuLV-infected mice;
1) has the same phenotype as the input virus (in vitro temperature sensitivity for Pr80env processing,2 15) and 2) does not form foci in mink cell lines (mink focus formation is typical of recombinants of exogenous ecotropic and endogenous xenotropic murine retroviruses3). In addition, ultrastructural studies of tsl MoMuLV-infected mice did not reveal virions with the aberrant morphology characteristic of endogenous retrovirus.9 There were direct parallels between the degree of virus infection in the CNS, the severity of CNS spongiform lesions, and the progression of clinical neurologic disease in tsl MoMuLV-infected mice. The local accumulation of viral proteins within specific regions of the CNS was necessary for the development of pathologic changes. Immunoblotting of specific viral proteins in the CNS revealed that tsl MoMuLV neurovirulence correlated with the simultaneous accumulation of both major gag-encoded (p30) and env-encoded (gp7O) proteins in the CNS, and suggested that tsl MoMuLV had an overall enhanced CNS replicative ability when compared to wt MoMuLV. These findings were supported by previous virologic studies that demonstrated higher viral titers in the CNS of tsl MoMuLV-infected mice compared to wt Mo2 MuLV-infected mice, as well as ultrastructural studies
666
Baszler and Zachary
AJP March 1991, Vol. 138, No. 3
.. ",;;
--
----Ce--
-I.........'... ..." -_--.,-
...
----
...
-1
ts.lga
t..
--"t--
*S 2 -
..
i.
wt gag
."
..
-1
-.-
-1;. '0
35.-
.0
0Q-
O.. 0l
'C. *:C
*T:7.71-"M. *7 i.-. 071".'.o.O.O..
0
;tpI
": ', o-. n
* 10.
.-
-.
"
-!. -.i!,., 77, ., .1; "'i'O
I
T
..'; "
01 O", i!" ", .,7
30.. D'ypos-'-':-oula':'f'on' Os
that showed abundant virions budding from the microglia of tsl MoMuLV-infected mice and only scant budding virions in wt MoMuLV-infected mice.9 The molecular basis for the enhanced replicative abil-
.
'I
*4X,
ity of tsl MoMuLV is unclear. It may simply reflect a failure to prevent superinfection at the receptor level within permissive CNS cells. Lack of viral envelope proteins on the surface of infected cells secondary to nonprocessing of
Neurovirulent Retrovirus and Microglia 667 AJP March 1991, Vol. 138, No. 3
Figure 8. Immunoblots from the CNS of tsl MoMuLV-infected mice using MuLV gag and env-protein-specific antibody probes (ABCalkaline phosphatase). A: Gradual accumulation of both viral gag (p30) and env (gp70)encoded proteins with marked accumulation of p30 and gp70 from days 25 to 35 after inoculation. Normal processing of Pr80env to gp70. Lane 1, 2, 3, 4, 5, 6, 7, = days 5, 10, 15, 20, 25, 30, 35, respectively. B: Comparative temporal quantitative densitometric analysis of viral gag and env-encoded proteins in the CNS of wt and tsl MoMuLVinfected and control mice. From days 25 to 35 after inoculation, the CNS of tsl MoMuLVinfected mice had a significant (unpaired ttest; P = 0.001) three- tofivefoldfold increase in accumulation of viral gag and envencoded proteins when compared to the CNS ofwtMoMuLV-infected mice. Abundant accumulation of viral protein in the CNVS of tsl MoMuLV-infected mice was correlated temporally with the development of severe spongiform lesions and progressive clinical neurologic disease. The increase of both viral gag and env-encoded proteins followed roughly simultaneous curves. Increased Pr80env in tsl MoMuLV-infected mice was proportionate with the overall level of virus replication in the CNS. (tsl and wt/gag-avg = p30; tsl and wt/ gp70-avg = gp70; tsl and wt/Pr80env avg = Pr80env; control = CNS of control mice.)
0.40
----a--- tsl/gag-avg
0.35
.6- -
0
0.30 *,,_ M
.25
:R . t
.15
*.
-
go.
..
-
U)
..0
0
i
*.E
0.10 J.0
-
W._ *
'0.05
o.O .
A
.IF; 1_
0 ED
flo
envelope precursor molecules was demonstrated to 100-fold increase in superinfection in MuLV infected cells in vitro.61Because tsl MoMuLV has disparate amino acid substitutions of env-encoded gp7O, structural changes could account for a similar effect in vivo within infected CNS cells. The normal viral clearance mechanisms in the CNS may be overwhelmed by excessive production of virus and viral proteins. Alternatively, because this study does not reveal enhanced productive replication of tsl MoMuLV over wt MoMuLV within cells outside the CNS (splenic and thymic cells), tsl MoMuLV cause a
O
11
1o
*vs n20 30 .Da+. postinocujlation
40
may have other genetic alterations that allow for more efficient infection of CNS microglia.'9 The development of severe CNS spongiform lesions and clinical neurologic disease occurred only in tsl MoMuLV-infected mice and correlated with a markedly increased accumulation of all the major viral-encoded proteins in the CNS, as well as the infection and activation of many resident CNS microglia. Direct virus infection of neurons was not observed. Resident microglia served as the major reservoir and amplifier of virus infection in the CNS. Central nervous system lesions and neuronal de-
Baszler and Zachary
668
AJP March 1991, Vol 138, No. 3
Table 2. Summary of Conparative tsl and wt MoMuLVFindings
tsl MoMuLV-infected mice Cellular target and degree of virus infection Splenic megakaryocytes Splenic lymphocytes Thymic lymphocytes CNS capillary endothelia CNS microglia Neurons Quantity of viral protein in the CNS Microglial activation Spongiform lesions Viral protein accumulation parallels lesion progression Selective accumulation of Pr80env Clinical neurologic disease -
wt MoMuLV-infected mice
+++ + + +++ ++++
+++ + + +++
++++ yes
+ no
+
severe/progressive
mild/regressive
yes
yes no none
no
severe/fatal
= none, + = minimal, + + = mild, + + + = moderate, + + + + = marked.
generation, from the inception of neuronal degeneration to cell death, were correlated spatially and temporally with high levels of viral protein and large numbers of virus-infected and nonvirus-infected microglia. Microglial activation was indicated by a robust, branching morphology and a local virus-associated hyperplasia of resident microglia that was disproportionate for the degree of local tissue damage. Concurrent ultrastructural morphologic studies demonstrated virus-infected microglia with long, branching cell processes and ruffled plasma membranes, characteristics consistent with activated cells.9 Similar virus-associated activation of macrophages recently was shown to correlate with lentivirus-induced diseases. In humans, the spinal cords of HIV-infected patients with the vacuolar myelopathy of AIDS-dementia complex had an accentuated expression of class II major histocompatibility complex molecules in both virusinfected and noninfected microglia that correlated with local CNS tissue lesions.37 In goats, the severity of arthritis induced by Caprine-arthritis-encephalitis virus is associated with the local activation of macrophages within the affected synovia.' These findings in diverse species suggest that virus-induced activation of tissue macrophages may be a common, widely applicable pathogenetic mechanism of retroviral-induced diseases. The increased number of microglia in the CNS of tsl MoMuLV-infected mice probably originated by in situ multiplication of resident cells. There was no intracapillary or perivascular accumulation of monocytic cells to suggest recruitment of blood-derived monocytes as the source of proliferating microglial cells; concurrent ultrastructural studies supported these findings.9 The in situ proliferative capacity of resident microglia has been demonstrated with electron microscopic autoradiographic studies of the axonal reaction.39 In addition, other mesodermal-derived counterparts of brain macrophages (tissue macrophages) have inherent proliferative capacity.40 In addition to microglial proliferation, the targeted areas of the CNS in tsl MoMuLV-infected mice also had a
striking astrogliosis. These findings are consistent with previous morphologic studies of tsl MoMuLV-infected mice.3 Although astrocytes did not serve as a site of viral replication or viral antigen accumulation (productive infection), they could be involved indirectly in disease pathogenesis by producing growth factors for microglial cells. Astrocytes are the main source of interleukin-3, a known growth factor for microglial cells in vitro.41 In situ activation of astrocytes, either as a physiologic response to local tissue damage or as a virus-induced activation secondary to insertion of strong viral promoters into the astrocytic genome through nonproductive infection, may provide a local mitogenic stimulus for resident microglia. Thus, although resident microglia appear to play a central role in tsl MoMuLV-induced neurologic disease, there may be more complex cellular interactions between neuroglia that ultimately result in neuronal dysfunction. tsl MoMuLV-infected mice had viral antigen accumulation in low numbers of oligodendroglia during the terminal stages of paralytic disease. The significance of oligodendroglia as a cell target relevant to the pathogenesis of neurologic disease is unclear because there was not a temporal correlation between virus infection and lesion development. Companion ultrastructural studies of tsl MoMuLV-infected mice revealed that oligodendroglial degeneration (myelin vacuolization of distal axons) occurred early during the inception of spongiform lesions (day 15 after inoculation),9 while the present immunohistochemistry studies showed that viral antigen accumulation occurred very late in the disease process (day 30 after inoculation). In vitro, tsl MoMuLV is defective in processing Pr8Oenv to gp7O and p1 5E at the nonpermissive temperature (39°C), resulting in the selective intracellular accumulation of unprocessed Pr80env within infected cells.15 The present immunoblotting studies of tsl MoMuLVinfected mice suggest that there is not a selective accumulation of nonprocessed Pr80env in vivo (Pr80env processing appeared normal in the CNS). Although the
Neurovirulent Retrovirus and Microglia
669
AJP March 1991, Vol. 138, No. 3
quantity of Pr80env in the CNS of tsl MoMuLV-infected mice is higher than wt MoMuLV-infected mice, the level is in proportion with the higher levels of other viral proteins (p30, gp7O) due to higher overall virus replication. In addition, virus-infected microglia have no ultrastructural cytopathologic changes to suggest aberrant intracellular accumulation of unprocessed viral proteins.9 The apparent paradox of Pr80env processing in vitro and in vivo is not resolved easily. It is likely that other env gene mutations, alone or in combination with Pr80env nonprocessing, also are involved in tsl MoMuLV neurovirulence. Alternatively the CNS lysate procedure or anti-viral antisera used in the present in vivo immunoblotting studies may not have been sensitive enough for the detection of subtle viral processing defects. A recent study with Cas-Br-E MuLV reported that neurons are the major target cell for viral infection and impaired post-translational synthesis of the env protein, despite normal synthesis of env mRNA, results in abortive retroviral infection of neurons and spongiform CNS degeneration.14 Although ultrastructural and immunohistochemistry studies of the tsl MoMuLV model reveal no productive virus infection of neurons, neuronal degeneration secondary to nonproductive virus infection could not be ruled out. Nonproductive virus infection could cause neuronal degeneration through excessive accumulation of unintegrated, extrachromasomal proviral DNA42 or insertional mutagenesis. In situ hybridization studies using tsl MoMuLV-specific probes are underway in our laboratory to define the involvement of nonproductive neuronal infection in the pathogenesis of neuronal dysfunction. The differences in cell targets for virus infection, viral-encoded protein synthesis, and probable pathogeneses of spongiform CNS degeneration between the Cas-Br-E MuLV model and the tsl MoMULV model, in light of similar neurologic, histologic, and select ultrastructural findings, suggest that differences in host mouse strain or subtle env gene differences also may play important roles in determining degenerative mechanisms. The finding of primary microglial cell infection in tsl MoMuLV-infected mice is similar to findings of many in situ studies of AIDS-dementia-complex that indicate that HI V-infected brain macrophages, microglia, and multinucleate syncytial cells are the major reservoir for CNS virus infection.445 Studies of the vacuolar myelopathy of AIDS-dementia complex, which shares some morphologic similarities with the white matter spongiform lesions of tsl MoMuLV spongiform myelopathy (myelin vacuolization and degeneration), also demonstrated a direct correlation between spongiform lesions and local infection of microglia.37 Because direct virus infection of neurons is not observed, these studies also suggest an indirect mechanism of neuronal degeneration. The cause-
effect relationship of neuronal degeneration, CNS spongiform changes, and enhanced localization of virus to CNS microglia is unclear. Increased secretion of viruscoded or cell-coded molecules from virus-infected mircoglia, either as a physiologic cellular reaction to infection or as a virus-induced alteration in cell metabolism, could be responsible for CNS tissue damage through direct neuronal toxicity, interference with neuronal metabolic processes, excessive stimulation of cell-surface receptors, or interference with neuronal trophic factors. In vitro, natural and recombinant HIV envelope protein gp120 can induce neuronal cell injury in the absence of infectivity.4647 Recent studies demonstrated that the injurious effect on neurons is mediated by a gpl20induced increase in intracellular calcium.47 If similar mechanisms apply in vivo, microglia could serve as a continuous, local source of virus-encoded neurotoxic molecules. Alternatively excessive microglial cellencoded molecules from virus-infected microglial cells also could mediate cytotoxicity to neighboring neural cells. Emerging studies conclude that microglia are major immune effector cells in the CNS that are capable of secreting a wide array of cytokines and immune mediator molecules such as tumor necrosis factor-alpha, prostaglandins, leukotrienes, oxidative radicals, and various proteases.451 In vitro, recombinant TNF-alpha induces oligodendrocytes necrosis and demyelination and provides an example for TNF-mediated damage to neural
cells.52 Previous studies revealed that mice inoculated with tsl MoMuLV developed severe thymic atrophy in the terminal stages of viral-induced neurologic disease53 and that athymic nu/nu mice failed to develop neurologic disease.8 These findings suggested that the thymus may play a crucial role in tsl MoMuLV-induced neurologic disease. The present studies demonstrated that the spleen served as the major virus factory and the systemic source of viremia before infection of the CNS, findings that correlate with previous virologic studies of a number of neurovirulent murine retroviruses.2 5 Thymic infection occurred subsequent to virus infection of the spleen, was never abundant, and did not correlate temporally with pathologic spongiform changes in the CNS or with neurologic signs. The severe splenic and thymic lymphoid atrophy that occurred in tsl MoMuLV-infected mice did not correlate with the degree of virus infection in the respective organs. Both tsl and wt MoMuLV-infected mice had an equal degree of splenic and thymic virus infection, yet only tsl MoMuLV-infected mice developed lymphoid atrophy. It is concluded that the chronic debilitating neurologic disease induced by tsl MoMuLV was the cause and not the effect of lymphoid atrophy in the spleen and thymus. The stress-induced release of endogenous glucocorticoids in the response to a variety of
670
Baszler and Zachary
AJP March 1991, Vol. 138, No. 3
chronic diseases is a known cause of lymphocyte necrosis and atrophy of lymphoid organs.55 Pathogenetic studies of the tsl MoMuLV model provide rare insight into the sequence of events that occur in retroviral-induced neurologic disease from initial infection to the development of severe CNS lesions during progressive neurologic- disease. It is hoped that findings from present and future investigations of murine retroviralinduced neurodegenerative disease will elucidate pathogenetic mechanisms that are common with emerging retroviral-induced neurologic diseases in humans such as AIDS-dementia complex and the HTLV-1 associated myeloneuropathies, as well as motor neuron diseases of undetermined etiology, such as amyotrophic lateral sclerosis.
10.
11.
12.
13.
Acknowledgments
14.
The authors thank Drs. Gail Scherba, Stuart Shapiro, and Mark Kuhlenschmidt for collaboration, and Donna Epps and Valerie Todd for their diligent and skilled technical assistance.
15.
References 1. McCarter JA, Ball JK, Frie JV: Lower limb paralysis induced in mice by a temperature sensitive mutant of Moloney murine leukemia virus. J Natl Canc Inst 1977, 59:179-183 2. Wong PKY, Knupp C, Yuen PH, Zachary JF, Tompkins WAF: Tsl, a paralytogenic mutant of Moloney murine leukemia virus TB, has an enhanced ability to replicate in the central nervous system and primary nerve cell culture. J Virol 1985, 55:760-767 3. Zachary JF, Knupp CH, Wong PKY: Noninflammatory spongiform polioencephalomyelopathy caused by a neurotropic temperature-sensitive mutant of Moloney murine leukemia virus TB. Am J Pathol 1986, 124:457-468 4. Price RW, Brew B, Sidtis J, Rosenblum M, Scheck A, Cleary P: The brain and AIDS: Central nervous system HIV-1 infection and AIDS dementia complex. Science 1988, 239:586592 5. Kramer A, Blattner W A: The HTLV-1 model and chronic demyelinating neurological diseases, Concepts in Viral Pathogenesis Ill. Edited by AC Notkins, MB Oldstone. New York, Springer-Verlag, 1989, pp 204-214 6. Rodgers-Johnson PE, Garruto RM, Gajdusek DC: Tropical myeloneuropathies-A new aetiology. Trend Neurosci 1988, 11:526-532 7. Tandan R, Bradley WG: Amyotrophic lateral sclerosis: Part 1. Clinical features, pathology, and ethical issues in management. Ann Neurol 1985,18:271-280 8. Prasad G, Stoica G, Wong PKY: The role of the thymus in the pathogenesis of hind-limb paralysis induced by tsl, a mutant of Moloney murine leukemia virus-TB. J Virol 1989, 169:332-340 9. Baszler T, Zachary JF: Murine retroviral-induced spongiform
16.
17.
18.
19.
20.
21.
22.
23.
neuronal degeneration parallels resident microglial cell infection: Ultrastructural findings. Lab Invest 1990, 63:612623 Bilello JA, Pitts OM, Hoffman PM: Characterization of a progressive neurodegenerative disease induced by a temperature-sensitive Moloney murine leukemia virus infection. Virol 1986, 59:234-241 Pitts OM, Powers JM, Bilello JA, Hoffman PM: Ultrastructural changes associated with retroviral replication in the central nervous system capillary endothelial cells. Lab Invest 1987, 56:401-409 Andrews JM, Gardner MB: Lower motor neuron degeneration associated with type-C RNA virus infection: neuropathological features. J Neuropathol Exp Neurol 1974, 33:285307 Oldstone MBA, Lampert PW, Lee S, Dixon FJ: Pathogenesis of slow disease of the central nervous system associated with WM 1504 E virus. Am J Pathol 1977, 88:193-206 Sharpe AH, Hunter JJ, Chassler P, Jaenisch R: Role of abortive retroviral infection of neurons in spongiform CNS degeneration. Nature 1990, 346:181-183 Wong PKY, Soong MM, MacLeod R, Gallick GE, Yuen PH: A group of temperature sensitive mutants of Moloney murine leukemia virus which is defective in cleavage of env precursor polypeptide in infected cells, also induces hindlimb paralysis in newborn CFW/D mice. Virol 1983,125:513-518 Soong MM, Tompkins WAF: Role of cell cytoskeleton in MoMuLV env transport and processing: Implications in tsl neuropathology. Exp Molec Pathol 1987, 46:294-311 Szurek PF, Yuen PH, Ball JK, Wong PKY: A Val-25-to-lle substitution in the envelope precursor polyprotein, gPr80env, is responsible for the temperature sensitivity, inefficient processing of gPr8oenv, and neurovirulence of tsl, a mutant of Moloney murine leukemia virus TB. J Virol 1990, 64:467-475 Szurek PF, Yuen PH, Jerzy R, Wong PKY: Identification of point mutations in the envelope gene of Moloney murine leukemia virus TB temperature-sensitive paralytogenic mutant tsl: Molecular determinants for neurovirulence. J Virol 1988, 62:357-360 Yuen PH, Tzeng E, Knupp C, Wong PKY: The neurovirulent determinants of tsl, a paralytogenic mutant of Moloney murine leukemia virus TB, are localized in at least two functionally distinct regions of the genome. J Virol 1986, 59:59-65 Greenberger JS, Stephenson JR, Aaronson SA: Temperature-sensitive mutants of murine leukemia virus. V. Impaired leukemogenic activity in vivo. Int J Cancer 1975, 15:10091015 Ruta M, Murray MJ, Well MC, Kabat D: A murine leukemia virus mutant with a temperature-sensitive defect in membrane glycoprotein synthesis. Cell 1979, 16:77-88 Paquette Y, Hanna Z, Savard P, Brousseau R, Robitaille Y, Jolicoeur P: Retrovirus-induced murine motor neuron disease: Mapping the determinant of spongiform degeneration within the envelope gene. Proc Natl Acad Sci 86:3896,1989 Portis JL, Czub S, Garon CF, McAtee FJ: Neurodegenerative disease induced by the wild mouse ecotropic retrovirus is markedly accelerated by long terminal repeat and gag-
Neurovirulent Retrovirus and Microglia
671
AJP March 1991, Vol. 138, No. 3
24.
25.
26.
27.
28.
29. 30.
31.
32.
33. 34.
35. 36. 37.
38.
pol sequences from nondefective Friend murine leukemia virus. J Virol 1990, 1648-1656 Wong PKY, Russ U, McCarter JA: Rapid, selective procedure for isolation of spontaneous temperature-sensitive mutants of Moloney murine leukemia virus. Virol 1973, 51:424431 Yuen PH, Malehorn D, Nau C, Soong MM, Wong PKY: Molecular cloning of two paralytogenic temperature-sensitive mutants, tsl and ts7, and the parental wild-type Moloney murine leukemia virus. J Virol 1985, 54:178-185 Ball JK, Huh TY, McCarter JA: On the statistical distribution of epidermal papillomata in mice. Brit J Cancer 1964, 18:120-123 Wong PKY, Soong MM, Yuen PH: Replication of murine leukemia virus in heterologous cells; interaction between ecotropic and xenotropic viruses. Virology 1981, 109:366378 Bradford M: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976, 72:248-254 Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227:680-685 Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci 1979, 76:1855-1859 Yuichi S, Mukai K, Watanabe S, Goto M, Shimosato Y: The AmeX Method: A simplified technique of tissue processing and paraffin embedding with improved preservation of antigens for immunostaining. Am J Pathol 1986,125:431-435 Sternberger LA, Joseph SA: The unlabelled antibody method: Contrasting color staining of pituitary hormones without antibody removal. J Histochem Cytochem 1979, 27:1424-1429 Streit WJ, Kreutzberg GW: Lectin binding by resting and reactive micriglia. J Neurocytol 1987. 16:249-260 Mannoji H, Yeger H, Becker LE: A specific histochemical marker (lectin Ricinus communis agglutinin-1) for normal human microglia, and application to routine histopathology. Acta Neuropathol (Berl) 1986, 71:341-343 Joklik WK: Tumor viruses. Edited by WK Joklik. Norwalk, CT, Appleton-Century-Crofts, 1985, ppl 39-155 Freed EO, Risser R: The role of envelope glycoprotein processing in murine leukemia virus infection. J Virol 1987, 61:2852-2856 Eilbott DJ, Peress N, Burger H, LaNeve D, Orenstein J, Gendelman HE, Seidman R, Weiser B: Human immunodeficiency virus type 1 in spinal cords of acquired immnodeficiency syndrome patients with myelopathy: Expression and replication in macrophages. Proc Natl Acad Sci 1989, 86:33373341 Banks KL, Jutila MA, Jacobs CA, Michaels FH: Augmentation of lymphocyte and macrophage proliferation by caprine arthritis-encephalitis virus contributes to the development of progressive arthritis. Rheumatol Int 1989, 9:123-128
39. Graeber MB, Streit WJ, Kreutzberg GW: Microglial cells but not astrocytes undergo mitosis following rat facial nerve axotomy. Neurosci Lett 1988, 85:317-321 40. Papadimitriou JM, Ashman RB: Macrophages: Current views on their differentiation, structure, and function. Ultrastruc Pathol 1989, 13:343-372 41. Frei K, Bodmer S, Schwerdel C, Fontana A: Astrocytederived interleukin 3 as a growth factor for microglia cells and peritoneal macrophages. J Immunol 1986, 137:35213527 42. Temin HM: Mechanism of cell killing/cytopathologic effects by nonhuman retroviruses. 1988 10:399-405 43. Michaels J, Price RW, Rosenblum MK: Microglia in the giant cell encephalitis of acquired immune deficiency syndrome: Proliferation, infection and fusion. Acta Neuropathol 1988, 76:373-370 44. Koenig S, Gendelman HE, Orenstein JM, Dal Canto MC, Pezeshkpour GH, Yungbluth M, Janotta F, Aksamit A, Martin MA, Pauci AS: Detection of AIDS virus in macrophages in brain tissue from AIDS patients with encephalopathy. Science 1986, 233:1089-1094 45. Wiley CA, Schrier RD, Nelson JA, Lampert PW, Oldstone MBA: Cellular localization of human immunodeficiency virus infection within brains of acquired immunodeficiency syndrome patients. Proc NatI Acad Sci 1986, 83:7089-7093 46. Brenneman DE, Westbrook GL, Fitzgerald SP, Ennist DL, Elkins KL, Ruft MR, Pert CB: Neuronal cell killing by the envelope protein of HIV and its prevention by vasoactive intestinal peptide. Nature 1988, 335:639-542 47. Dreyer EB, Kaiser PK, Offermann JT, Lipton SA: HIV-1 coat protein neurotoxicity prevented by calcium channel antagonists. Science 1990, 248:364-367 48. Perry VH, Gordon S: Macrophages and microglia in the nervous system. Trend Neurosci 1988, 11:273-277 49. Streit WJ, Graeber MB, Kreutzberg GW: Functional plasticity of microglia: A review. Glia 1988,1:301-307 50. Frei K, Siepl C, Groscurth P, Bodmer S, Fontana A: Immunobiology of microglial cells. An NY Acad Sci 1988, 540:218-227 51. Wolfe LS: Eicosinoids, Basic Neurochemistry: Molecular, Cellular, and Medical Aspects. Edited by GJ Seigel. New York, Raven Press, 1989, pp 399-414 52. Selmaj KW, Raine CS: Tumor necrosis factor mediates myelin and oligodendrocyte damage in vitro. Ann Neurol 1988,
23:339-346 53. Wong PKY, Prasad G, Hansen J, Yuen PH: tsl, A mutant of Moloney murine leukemia virus-TB, causes both immunodeficiency and neurologic disorders in BALB/c mice. Virol 1989,170:450-459 54. Brooks BR, Swarz JR, Johnson RT: Spongiform polioencephalo-myelopathy caused by a murine retrovirus 1. Pathogenesis of infection in newborn mice. Lab Invest 1980, 43:480-486 55. Robbins SL: Thymus, Pathologic Basis of Disease. Edited by RS Cotran, V Kumar, SL Robbins. Philadelphia, W.B. Saunders Co., 1989, pp 1268-1271
