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Human adenovirus E3 genes as immunomodulators in the CNS
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Poster Abstracts / Journal of Neuroimmunology 90 (1998) 13-105
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I n c r e a s e d Expression o f CDIIb ( M a c - l ) on C D 8 + T cells in Patients with HAM/TSP G.J. Buckle~ V. Batra, D.A. Hailer, P. Hollsberg, BrighamandWomen'sHospital, USA
C h e m o k i n e Expression D u r i n g M o u s e A d e n o v i r u s Type 1 I n d u c e d Hemorrhagic Enecphalopathy P.C. Charles, X. Chen, C.F. Brosnan, AlbertEinsteinCollege ofMedicine, USA
HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic viral disease of the central nervous system characterized by profound immune dysregulation and by a high frequency of virus-reactive CD8+ T cells in the blood and spinal fluid, expression of the surface molecules CDI lb (Mac-I) and CD28 may define reciprocal subsets of CD8+ T cells. CD28 appears to identify a "naive" population ofCD8+ T cells and CDI lb appears to represent an oligoclonally expanded "memory" phenotype, which may contain virusspecific CD8+T cells. We used magnetic bead separation and flow cytometry to assess a variety of surface markers on CD8+ T cells from HAM/TSP patients, patients with multiple sclerosis (MS), and healthy controls. We found drumaticallyincreased expression of C D I I b on CD8+ T cells of patients with HAM/TSP as compared to age-matched patients with MS or normal controls. As in murine models of LCMV infection, CDII b may identify a human CDS+ "memory" pool in chronic viral infection and may play an important role in the pathogenesis of HAM/TSP.
Mouse Adeeovirus Type-1 (MAV-1) causes a fatal, hemorrhagic encephalomyelitisin some strains of mice. Analysis ofthe disease course in C57BIJ6j (susceptible) and BALB/c (resistant) mice revealed that hemorrhage and infarction within the CNS of susceptible mice is the result of viral infection of the CNS microvasculature. Immunohistochemistry and ultrestructurel pathologic examination demonstrated early and extensive replication of MAV-1 in endothelial ceils of the cerebral vasculature in C57BI/B mice, whereas viral replication within the CNS of BALB/c mice was delayed and at much reduced Jevels. Replication of virus in C57BL/6 mice was accompanied by disorganization of microvascular structures and sloughing of infected endothelial cells into vessel lumens. Damage to cerebral vessels appears to be the direct result viral replication, with little detectable immunologic contribution. Compahson of cytokine mRNA profiles from the brains of MAV-1 infected susceptible and resistant mice revealed little difference between the two. In both cases, laurels of IL-lb, TNFoa, LT, and IL-8 mRNA were elevated by 96 hours post inoculation, consistent with mild inflammation in both strains of mice. in contrast, C57BI/6 mice showed prominent and early expression of IP-10 in both the spleen and CNS whereas infected BALB/c mice expressed predominantly MCP-t. Both strains showed an early upregulation of MIP-2 in the CNS but not the spleen. These data demonstrate striking strain-specific differences in chemokine expression in response to a CNS endothelial infection by a viral pathogen.
354
357
Astroeyte T a r g e t e d Expression o f I n t e r f e r o n - a l p h a l Protects Mice f r o m A c u t e O c u l a r H e r p e s Simplex V i r u s Type 1 Infection D.J.J. Carl LouisianaState UniversiiyMedical Center,USA, EL, Campbell, Scripps Researchlnstitute, USA
H u m a n A d e n o v i r u s E3 G e n e s as l m m u n o m o d u l a t o r s in the C N S EC. Charles~ X. Chen, C.F. Brosnan, AIbertEinsteinCollege ofMedicine, USA
Type I IFNs (i.e., IFN-o~ and IFN-[~) play a key role in the host's innate defense against viral pathogens. To examine the biological relevance of IFN-c~ to a viral pathogen within the confines of the nen, eus system, IFN..c~I transgenic mice whose transgene is under the control of the glial fibrillary acidic protein promoter (GFAP-IFN~, astrucyte specific) were examined for resistance to an ocular herpes simplex virus type 1 (HSV-I) infection. GFAPIFNct mice expressed significantly higher levels of IFN-c,./[3 (533 units) in the trigeminal ganglion compared to non-transgenic mice (70 units) 72 hr post infection that corresponded with a significant reduction in the mRNA expression of the HSV-I immediate early gene 1CP27 and the chemokioes MCP-1 and CRG-2 in the eye and trigeminal ganglion of GFAP-IFNo~ transgenics. Six days post infection, the viral load and the expression of ICP27, CD8, RANTES, 1FN-),, and IFN-c¢ mRNA levels were reduced in the trigeminal ganglion of GFAP-IFN~ mice compared to the non-transgenic mice. Collectively, the results show that the IFN-~I transgenic mice are less susceptible to acute HSV-I infection and the establishment of viral latency.
Many pathogenic viruses contain genes that serve to subvert immunological responses of the host. For example, the early region 3 (E3) of human adenovirus 2 (AD2) contains genes that inhibit the action of tumor necrosis factor and block cell surface expression of MHC class I. In this study we tested whether the AD2 E3 region can protect, and perhaps facilitate repair, of the central nervous system against immune-mediated damage. To test for this, we made a construct with the AD2-E3 region under the control of the mouse MBP promoter, transfected it into a mouse eligodendrocyte cell line and established tranegenic mice. In the mouse line, the MBP/AD2E3 construct blocked the induction of MHC class I by IFNg, indicating that the gene construct was functional. In mice, expression of the AD2E3 mRNA transcripts could be detected at high levels in the CNS and immunohistochemicel staining revealed the presence of the AD2E3-gpf9K protein within oligodendrocytes and white matter tracts. Animals were then sensitized to develop experimental allergic encephalomyelitis with myelin bligodendrocyte glycoprotein in CFA. Animals expressing the MBP/AD2E3 tranegene showed a delayed onset, and a reduction in both clinical and histological evidenceof disease, particularly in the acute phase of the disease. These data indicate that the AD2E3 gene region may represent a novel therapeutic approach to the regulation of central nervous system autoimmune disease.
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Restricted HIV-1 Expression in Astrocytes: I n v o l v e m e n t of Cellular Inhibition of R e v Function E Ceccherini- Silberstein. E. Ludwig, M. Neumann, A. Ludvigsen, E Willnauer, V. Erflc, IL Brack-Werner, GSF-NationaIResearchCenterforEnvironmentandHealth, Germany
A n t i - b a c t e r i a l a n d A n t i - p a r a s i t i c Effecter M e c h a n i s m s in H u m a n B r a i n Cells W. Daubener. C.R. Mackenzie, U, Haddhlg, In~'tituteforMedicatMicrobiologyand Virology,H¢inrich-H¢ine-Universitat,Dusseldorf Germany
In the majority of individuals with AIDS, HIV invades the CNS and provides neurologic and psychiatric dysfunction in both adults and children. Gila cells can be important mediators of HlV-associated damage to the CNS. In vitro and in vivo studies demonstrate that HIV actively replicates in microgtial cells, while astrecytes are target cells for nonproductive HIV persistence. We established an astrocytoma cell line harboring a biologically functional HIV-provlms as a cellular model system for persistently HIV-I infected astrocytes. In these cells, HIV replication and virion production are severely reduced and only a small proportion of Rev-dependent transcripts are expressed, suggesting a failure of the intracellular environment of astrocytes to support Rev functions. Rev is a viral regulatory protein that plays an essential role in temporal regulation of geec expression by enhancing nudeocytoplasmic transport and translation of HIV-I mRNAs encoding the viral structural proteins. Functional comparison of Rev in astrocytes and eonglial cells demonstrated reduced Rev function in various human astrocytoma/glioblastoma cell lines and in primary human fetal astrocytes. Intracellular localization with Rev-GFP revealed distinct cytoplasmic localization of Rev in astrocytes, while in nonglial control cells Rev is contained predominantly in nuclear/nucleol~ compartment. These results suggest that attenuation of Rev function contributes to suppression of HIV production by infected astrocytes. Moreover, cultured human astrecytes are a suitable model control system to investigate molecular mechanisms underlying cell-directed restriction of HtV replication.
Several infectious organisms, such as viruses, bacteria and parasites, are able to induce inflammation within the central nervous system. Interfemn-y is one of the most prominent cytokines inducing protection against these pathogens. Within the brain two types of antimicrobial effector cells are known: astrocytes and microglia cells. There are also two different cellular effector mechanisms described which are active against a variety of microorganisms: the induction of indoleamine 2,3-dioxygenase activity and the activation of nitric oxyde synthase activity. W e found that in human cells the induction of [ndolamine 2,3 dioxygenase activity is responsible for a strong antiparasitic effect against Toxop/aama gondii. In contrast in murine ceils IDO activation is not involved in the antimiorobial defense, which is mediated in these cells by the production of nitric oxide.IDO activation is responsible for the cellular defense against toxoplasma and chlamydia defense in several human cell lines including astrocytes, glioblastoma cells, flbroblasts and macropbages. In addition we found that besides these two obJigate intracel]ular pathogens the growth of extracellular bacteria (i.e. group B streptococci), is also inhibited by IFN-y activated astrocytoma ceils. These streptococci were clearly inhibited in their growth by IFN-y induced IDO activation with subsequent tryptophan degradation, and the addition of excess amounts of tryptophan completely blocked the antibacterial effect.
Poster Abstracts / Journal of Neuroimmunology 90 (1998) 13-105
353
356
I n c r e a s e d Expression o f CDIIb ( M a c - l ) on C D 8 + T cells in Patients with HAM/TSP G.J. Buckle~ V. Batra, D.A. Hailer, P. Hollsberg, BrighamandWomen'sHospital, USA
C h e m o k i n e Expression D u r i n g M o u s e A d e n o v i r u s Type 1 I n d u c e d Hemorrhagic Enecphalopathy P.C. Charles, X. Chen, C.F. Brosnan, AlbertEinsteinCollege ofMedicine, USA
HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic viral disease of the central nervous system characterized by profound immune dysregulation and by a high frequency of virus-reactive CD8+ T cells in the blood and spinal fluid, expression of the surface molecules CDI lb (Mac-I) and CD28 may define reciprocal subsets of CD8+ T cells. CD28 appears to identify a "naive" population ofCD8+ T cells and CDI lb appears to represent an oligoclonally expanded "memory" phenotype, which may contain virusspecific CD8+T cells. We used magnetic bead separation and flow cytometry to assess a variety of surface markers on CD8+ T cells from HAM/TSP patients, patients with multiple sclerosis (MS), and healthy controls. We found drumaticallyincreased expression of C D I I b on CD8+ T cells of patients with HAM/TSP as compared to age-matched patients with MS or normal controls. As in murine models of LCMV infection, CDII b may identify a human CDS+ "memory" pool in chronic viral infection and may play an important role in the pathogenesis of HAM/TSP.
Mouse Adeeovirus Type-1 (MAV-1) causes a fatal, hemorrhagic encephalomyelitisin some strains of mice. Analysis ofthe disease course in C57BIJ6j (susceptible) and BALB/c (resistant) mice revealed that hemorrhage and infarction within the CNS of susceptible mice is the result of viral infection of the CNS microvasculature. Immunohistochemistry and ultrestructurel pathologic examination demonstrated early and extensive replication of MAV-1 in endothelial ceils of the cerebral vasculature in C57BI/B mice, whereas viral replication within the CNS of BALB/c mice was delayed and at much reduced Jevels. Replication of virus in C57BL/6 mice was accompanied by disorganization of microvascular structures and sloughing of infected endothelial cells into vessel lumens. Damage to cerebral vessels appears to be the direct result viral replication, with little detectable immunologic contribution. Compahson of cytokine mRNA profiles from the brains of MAV-1 infected susceptible and resistant mice revealed little difference between the two. In both cases, laurels of IL-lb, TNFoa, LT, and IL-8 mRNA were elevated by 96 hours post inoculation, consistent with mild inflammation in both strains of mice. in contrast, C57BI/6 mice showed prominent and early expression of IP-10 in both the spleen and CNS whereas infected BALB/c mice expressed predominantly MCP-t. Both strains showed an early upregulation of MIP-2 in the CNS but not the spleen. These data demonstrate striking strain-specific differences in chemokine expression in response to a CNS endothelial infection by a viral pathogen.
354
357
Astroeyte T a r g e t e d Expression o f I n t e r f e r o n - a l p h a l Protects Mice f r o m A c u t e O c u l a r H e r p e s Simplex V i r u s Type 1 Infection D.J.J. Carl LouisianaState UniversiiyMedical Center,USA, EL, Campbell, Scripps Researchlnstitute, USA
H u m a n A d e n o v i r u s E3 G e n e s as l m m u n o m o d u l a t o r s in the C N S EC. Charles~ X. Chen, C.F. Brosnan, AIbertEinsteinCollege ofMedicine, USA
Type I IFNs (i.e., IFN-o~ and IFN-[~) play a key role in the host's innate defense against viral pathogens. To examine the biological relevance of IFN-c~ to a viral pathogen within the confines of the nen, eus system, IFN..c~I transgenic mice whose transgene is under the control of the glial fibrillary acidic protein promoter (GFAP-IFN~, astrucyte specific) were examined for resistance to an ocular herpes simplex virus type 1 (HSV-I) infection. GFAPIFNct mice expressed significantly higher levels of IFN-c,./[3 (533 units) in the trigeminal ganglion compared to non-transgenic mice (70 units) 72 hr post infection that corresponded with a significant reduction in the mRNA expression of the HSV-I immediate early gene 1CP27 and the chemokioes MCP-1 and CRG-2 in the eye and trigeminal ganglion of GFAP-IFNo~ transgenics. Six days post infection, the viral load and the expression of ICP27, CD8, RANTES, 1FN-),, and IFN-c¢ mRNA levels were reduced in the trigeminal ganglion of GFAP-IFN~ mice compared to the non-transgenic mice. Collectively, the results show that the IFN-~I transgenic mice are less susceptible to acute HSV-I infection and the establishment of viral latency.
Many pathogenic viruses contain genes that serve to subvert immunological responses of the host. For example, the early region 3 (E3) of human adenovirus 2 (AD2) contains genes that inhibit the action of tumor necrosis factor and block cell surface expression of MHC class I. In this study we tested whether the AD2 E3 region can protect, and perhaps facilitate repair, of the central nervous system against immune-mediated damage. To test for this, we made a construct with the AD2-E3 region under the control of the mouse MBP promoter, transfected it into a mouse eligodendrocyte cell line and established tranegenic mice. In the mouse line, the MBP/AD2E3 construct blocked the induction of MHC class I by IFNg, indicating that the gene construct was functional. In mice, expression of the AD2E3 mRNA transcripts could be detected at high levels in the CNS and immunohistochemicel staining revealed the presence of the AD2E3-gpf9K protein within oligodendrocytes and white matter tracts. Animals were then sensitized to develop experimental allergic encephalomyelitis with myelin bligodendrocyte glycoprotein in CFA. Animals expressing the MBP/AD2E3 tranegene showed a delayed onset, and a reduction in both clinical and histological evidenceof disease, particularly in the acute phase of the disease. These data indicate that the AD2E3 gene region may represent a novel therapeutic approach to the regulation of central nervous system autoimmune disease.
355
358
Restricted HIV-1 Expression in Astrocytes: I n v o l v e m e n t of Cellular Inhibition of R e v Function E Ceccherini- Silberstein. E. Ludwig, M. Neumann, A. Ludvigsen, E Willnauer, V. Erflc, IL Brack-Werner, GSF-NationaIResearchCenterforEnvironmentandHealth, Germany
A n t i - b a c t e r i a l a n d A n t i - p a r a s i t i c Effecter M e c h a n i s m s in H u m a n B r a i n Cells W. Daubener. C.R. Mackenzie, U, Haddhlg, In~'tituteforMedicatMicrobiologyand Virology,H¢inrich-H¢ine-Universitat,Dusseldorf Germany
In the majority of individuals with AIDS, HIV invades the CNS and provides neurologic and psychiatric dysfunction in both adults and children. Gila cells can be important mediators of HlV-associated damage to the CNS. In vitro and in vivo studies demonstrate that HIV actively replicates in microgtial cells, while astrecytes are target cells for nonproductive HIV persistence. We established an astrocytoma cell line harboring a biologically functional HIV-provlms as a cellular model system for persistently HIV-I infected astrocytes. In these cells, HIV replication and virion production are severely reduced and only a small proportion of Rev-dependent transcripts are expressed, suggesting a failure of the intracellular environment of astrocytes to support Rev functions. Rev is a viral regulatory protein that plays an essential role in temporal regulation of geec expression by enhancing nudeocytoplasmic transport and translation of HIV-I mRNAs encoding the viral structural proteins. Functional comparison of Rev in astrocytes and eonglial cells demonstrated reduced Rev function in various human astrocytoma/glioblastoma cell lines and in primary human fetal astrocytes. Intracellular localization with Rev-GFP revealed distinct cytoplasmic localization of Rev in astrocytes, while in nonglial control cells Rev is contained predominantly in nuclear/nucleol~ compartment. These results suggest that attenuation of Rev function contributes to suppression of HIV production by infected astrocytes. Moreover, cultured human astrecytes are a suitable model control system to investigate molecular mechanisms underlying cell-directed restriction of HtV replication.
Several infectious organisms, such as viruses, bacteria and parasites, are able to induce inflammation within the central nervous system. Interfemn-y is one of the most prominent cytokines inducing protection against these pathogens. Within the brain two types of antimicrobial effector cells are known: astrocytes and microglia cells. There are also two different cellular effector mechanisms described which are active against a variety of microorganisms: the induction of indoleamine 2,3-dioxygenase activity and the activation of nitric oxyde synthase activity. W e found that in human cells the induction of [ndolamine 2,3 dioxygenase activity is responsible for a strong antiparasitic effect against Toxop/aama gondii. In contrast in murine ceils IDO activation is not involved in the antimiorobial defense, which is mediated in these cells by the production of nitric oxide.IDO activation is responsible for the cellular defense against toxoplasma and chlamydia defense in several human cell lines including astrocytes, glioblastoma cells, flbroblasts and macropbages. In addition we found that besides these two obJigate intracel]ular pathogens the growth of extracellular bacteria (i.e. group B streptococci), is also inhibited by IFN-y activated astrocytoma ceils. These streptococci were clearly inhibited in their growth by IFN-y induced IDO activation with subsequent tryptophan degradation, and the addition of excess amounts of tryptophan completely blocked the antibacterial effect.