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Reduced Macrophage Infiltration and Demyelination in Mice Lacking the Chemokine Receptor CCR5 Following Infection with a Neurotropic Coronavirus William G Glass Michael T Liu William A Kuziel and Thomas E Lane 1 Department of Molecular Biology and Biochemistry Reeve Irvine Research Center University of California Irvine California 92697 3900 and Section of Molecular Genetics and Microbiology and Institute for Cellular and Molecular Biology University of Texas Austin Texas 78712 Received April 6 2001 returned to author for revision May 21 2001 accepted June 5 2001 Studies were performed to investigate the contributions of the CC chemokine receptor CCR5 in host defense and disease development following intracranial infection with mouse hepatitis virus MHV T cell recruitment was impaired in MHV infected CCR5 2 2 mice at day 7 postinfection pi which correlated with increased P 0 03 titers within the brain However by day 12 pi T cell infiltration into the CNS of infected CCR5 2 2 and CCR5 1 1 mice was similar and both strains exhibited comparable viral titers indicating that CCR5 expression is not essential for host defense Following MHV infection of CCR5 1 1 mice greater than 50 of cells expressing CCR5 antigen were activated macrophage microglia determined by F4 80 antigen expression In addition infected CCR5 2 2 mice exhibited reduced P 0 02 macrophage CD45 high F4 80 1 infiltration which correlated with a significant reduction P 0 001 in the severity of demyelination compared to CCR5 1 1 mice These data indicate that CCR5 contributes to MHV induced demyelination by allowing macrophages to traffic into the CNS 2001 Academic Press Key Words chemokine chemokine receptor demyelination multiple sclerosis macrophage neuroimmunology INTRODUCTION CCR5 is a member of the CC chemokine receptor family that is expressed on various hematopoietic cells including lymphocytes and macrophages Raport et al 1996 Che mokines that are capable of binding to CCR5 include CCL3 MIP 1a CCL4 MIP 1b and CCL5 RANTES Boring et al 1996 Meyer et al 1996 Raport et al 1996 Zlotnik and Y oshie 2000 Recent studies have impli cated CCR5 as an important molecule in regulating the immune response as well as leukocyte trafficking follow ing microbial infection Mice deficient in CCR5 CCR5 2 2 exhibit altered T cell activity and impaired macrophage function Sato et al 1999 Zhou et al 1998 Furthermore macrophage trafficking in response to antigen is impaired in CCR5 2 2 mice indicating that CCR5 is required for mi gration of this population of cells Huffnagle et al 1999 Mouse hepatitis virus MHV is a positive strand RNA virus that is a member of the Coronaviridae family MHV infection of the CNS results in an acute encephalomyeli tis followed by a chronic demyelinating disease that is similar to the pathology observed in the human demyeli nating disease multiple sclerosis MS Houtman and Flemming 1996 Lane and Buchmeier 1997 A robust expression of chemokine genes occurs within the brains and spinal cords of MHV infected mice which precedes then accompanies leukocyte infiltration Lane et al 1998 Among the chemokines expressed within the CNS of MHV infected mice are the CCR5 ligands CCL3 CCL4 and CCL5 suggesting an important role for these che mokines in attracting leukocytes into the CNS In support of this are studies demonstrating that treatment of MHV infected mice with neutralizing antisera specific for CCL5 resulted in reduced lymphocyte infiltration into the brain and enhanced viral recovery Lane et al 2000 More over anti CCL5 treatment resulted in decreased macro phage infiltration which correlated with a significant decrease in the severity of demyelination Therefore these data suggested that CCL5 contributes to demyeli nation in MHV infected mice by attracting macrophages into the CNS following binding and signaling of chemo kine receptors expressed on the surface of these cells The present study was undertaken in order to better understand how CCR5 contributes to disease following MHV infection of the CNS To this end we have evalu ated the severity of neurologic disease following MHV infection of CCR5 2 2 mice Andres et al 2000 The results presented indicate that MHV infection of the CNS of CCR5 2 2 mice results in the development of an effec tive immune response as characterized by cytokine and chemokine gene expression and clearance of virus from the CNS However both macrophage infiltration and de myelination was significantly reduced in MHV infected CCR5 2 2 mice compared to CCR5 1 1 mice indicating that CCR5 contributes to the pathogenesis of MHV induced demyelination by attracting macrophages into the CNS 1 To whom correspondence and reprint requests should be ad dressed at Department of Molecular Biology and Biochemistry Biolog ical Sciences II University of California Irvine CA 92697 3900 Fax 949 824 8551 E mail tlane uci edu Virology 288 8 17 2001 doi 10 1006 viro 2001 1050 available online at on 0042 6822 01 35 00 Copyright 2001 by Academic Press All rights of reproduction in any form reserved 8RESULTS MHV induced CNS disease and CCR5 expression MHV infection of C57BL 6 mice results in an acute encephalomyelitis characterized by viral replication in neurons and glial cells Approximately 40 of infected mice die during the acute stage of disease and surviving mice develop a severe demyelinating disease character ized by persistence of viral RNA and antigen within white matter tracts accompanied by mononuclear cell infiltra tion and white matter destruction Chemokines are ex pressed within the CNS of MHV infected mice and these molecules contribute to both host defense and disease development through attraction of T cells and macro phages Lane et al 1998 2000 Liu et al 2000 2001 In order to characterize the chemokine receptor profile in the brains of MHV infected mice total RNA was isolated at day 7 postinfection pi and subjected to RPA using a multitemplate probe set containing mouse chemokine receptor riboprobes The results shown in Fig 1 clearly demonstrate increased expression of mRNA transcripts for CCR1 CCR2 and CCR5 in CCR5 1 1 mice following MHV infection of the CNS As expected CCR5 transcripts were not expressed in CCR5 2 2 mice In addition there was an approximate twofold reduction in mRNA tran script levels for both CCR1 and CCR2 in CCR5 2 2 mice Neither sham infected CCR5 1 1 nor CCR5 2 2 mice exhib ited increased expression for any of the chemokine re ceptors tested Confocal microscopy revealed that greater than 55 of cells positive for CCR5 also ex pressed F4 80 an antigen expressed by activated micro glia macrophages Fig 2 CCR5 antigen was not de tected on F4 80 positive cells within the brains of CCR5 2 2 mice Although not determined it is likely that T cells as well as activated astrocytes constitute the re maining population of CCR5 positive cells as these cells have been shown to express this receptor Mack et al 2001 Dorf et al 2000 MHV infection of CCR5 2 2 mice To determine the contribution of CCR5 to host defense and neurologic disease following MHV infection of the CNS CCR5 2 2 and CCR5 1 1 mice were infected with MHV and the severity of disease was evaluated There were no marked differences in either clinical disease severity or mortality with approximately 60 of CCR5 2 2 FIG 1 A Chemokine receptor mRNA expression in the brain following MHV infection CCR5 1 1 and CCR5 2 2 mice were infected with MHV or sterile saline sham brains isolated at 7 days pi and total RNA was subjected to RPA The free probe set is indicated in the lefthand margin These fragments run higher than protected fragments due to additional cloning sequences Each lane indicates a single mouse and infected V5A13 1 or sham mice are indicated at the top of the autoradiograph Note the decreased levels of mRNA transcripts for CCR1 and CCR2 within infected CCR5 2 2 mice compared to CCR5 1 1 mice CCR5 transcripts are absent in CCR5 2 2 mice The RPA shown is representative of three separate experiments Sample sizes are as follows CCR5 1 1 n 5 2 CCR5 2 2 n 5 2 sham n 5 2 for both groups of mice B Quantitative analysis of chemokine receptor mRNA transcripts Densitometric analysis of each lane representing a brain sample from an individual mouse was performed on the scanned autoradiograph A Data are presented as the average 6SEM 9 CCR5 AND VIRAL INDUCED DEMYELINATION10 GLASS ET AL and CCR5 1 1 mice surviving until day 12 pi Analysis of viral titers within the brains revealed a fivefold increase in virus present P 0 03 in CCR5 2 2 mice 5 9 6 0 1 PFU g Log 10 n 5 12 compared to CCR5 1 1 mice 5 1 6 0 1 PFU g n 5 9 at day 7 pi Table 1 However by day 12 pi there was not a significant difference in viral burden between CCR5 2 2 mice 2 5 6 0 1 PFU g n 5 9 and CCR5 1 1 mice 2 2 6 0 2 PFU g n 5 12 Table 1 In addition no differences were detected in the distribution of viral antigen within the brains of infected CCR5 1 1 or CCR5 2 2 mice at either day 7 or day 12 pi indicating that lack of CCR5 did not alter the cellular tropism of the virus data not shown Collectively these data indicate that CCR5 is not essential in host defense or clearance of virus from the brains of MHV infected mice Mononuclear cell infiltration The increased viral titers within the brains of CCR5 2 2 mice at day 7 pi suggested that there may be reduced levels of infiltrating T cells present during acute disease as both CD4 1 and CD8 1 T cells are required for optimal clearance of virus from the CNS Williamson and Stohl man 1990 Lane et al 2000 FACS analysis of infiltrating mononuclear cells at day 7 pi revealed decreased infil tration P 0 03 of CD4 1 T cells 12 2 6 1 9 n 5 8 and CD8 1 T cells 14 1 6 1 6 n 5 8 into the brains of CCR5 2 2 mice compared to CCR5 1 1 mice CD4 1 19 5 6 1 3 n 5 13 CD8 1 22 1 6 1 6 n 5 13 Table 1 By day 12 pi however there were no significant differences in the infiltration of CD4 1 or CD8 1 T cells into the CNS of infected CCR5 2 2 or CCR5 1 1 mice Table 1 Macro phage microglial staining as assessed by F4 80 antigen expression was decreased by 30 and 60 in CCR5 2 2 mice at day 7 CCR5 1 1 2 7 26 2 4 n 5 13 CCR5 2 2 18 8 6 2 3 n 5 8 P 0 03 and day 12 pi CCR5 1 1 29 1 6 1 5 n 5 3 CCR5 2 2 1 1 86 0 7 n 5 3 P 0 001 respectively Table 1 Collectively these data indicate that T cell infiltration into the CNS is affected during acute disease in CCR5 2 2 mice yet these cells are ultimately able to enter the CNS and participate in elimination of infectious virus Further these data indi cate that macrophage microglial activation and or infil tration is compromised in the absence of CCR5 Reduced macrophage infiltration and myelin destruction in CCR5 2 2 mice Previous studies have demonstrated that macro phages are important contributors to demyelination in MHV infected mice Lane et al 2000 Wu and Perlman 1999 Wu et al 2000 In an effort to distinguish between infiltrating macrophages and resident microglia flow analysis was performed using FITC conjugated F4 80 antibodies and PE conjugated CD45 antibodies Infiltrat ing macrophages have been found to exhibit a CD45 high F4 80 1 phenotype whereas microglia are CD45 low F4 80 1 Ford et al 1995 Fife et al 2000 Such FIG 2 F4 80 positive cells express CCR5 CCR5 expression on F4 80 positive cells was demonstrated using confocal microscopy Representative staining from brains of MHV infected CCR5 1 1 and CCR5 2 2 mice at day 7 pi for F4 80 alone red cells CCR5 alone green cells or dual labeled F480 CCR5 positive cells yellow cells is shown Cells shown were present within the brain parenchyma of infected mice Original magnification 3400 FIG 3 Demyelination is reduced in CCR5 2 2 mice Representative LFB staining of spinal cords of MHV infected CCR5 1 1 and CCR5 2 2 mice at 12 days postinfection CCR5 1 1 mice A display mononuclear infiltration and myelin destruction in contrast to CCR5 2 2 mice B which have limited cellular infiltration and compact myelin Original magnification 3200 FIG 4 F4 80 staining is decreased in white matter tracts of MHV infected CCR5 2 2 mice Representative staining for F4 80 antigen within white matter tracts of MHV infected CCR5 1 1 mice B or CCR5 2 2 mice D F4 80 positive cells are stained brown Note heavy infiltration within white matter tracts of CCR5 1 1 mice compared to CCR5 2 2 mice F4 80 staining of sham infected CCR5 1 1 mice A and CCR5 2 2 mice C are included for controls Original magnification 3200 TABLE 1 Reduced Leukocyte Infiltration in CCR5 2 2 Mice Mouse type Days postinfection Brain titer PFU gram tissue Log 10 mean 6 SEM Average cell recovery CD4 1 mean 6 SEM CD8 1 mean 6 SEM F4 80 1 mean 6 SEM CCR5 1 1 7 5 1 6 0 1 9 a 9 9 3 10 5 19 5 6 1 3 13 c 22 1 6 1 6 13 27 2 6 2 4 13 12 2 2 6 0 2 12 7 8 3 10 5 21 6 6 1 7 3 16 5 6 0 9 3 29 1 6 1 5 3 CCR5 2 2 7 5 9 6 0 1 12 b 7 7 3 10 5 12 2 6 1 9 8 b 14 1 6 1 6 8 b 18 8 6 2 3 8 b 12 2 5 6 0 1 9 1 1 3 10 6 21 0 6 2 3 3 21 5 6 3 7 3 11 8 6 0 7 3 d a Parentheses indicate numbers of mice used Data presented represent five independent experiments b P 0 03 when compared to CCR5 1 1 mice c Data are presented as the percentage of positive cells within the gated population d P 0 001 when compared to CCR5 1 1 mice 11 CCR5 AND VIRAL INDUCED DEMYELINATIONanalysis revealed an approximate threefold reduction P 0 02 in numbers of infiltrating macrophages present within the CNS of infected CCR5 2 2 mice at day 12 4 5 3 10 4 6 8 2 3 10 3 n 5 5 compared CCR5 1 1 mice 1 3 3 10 5 6 1 2 3 10 4 n 5 4 Table 2 In order to determine if limited macrophage infiltration into the CNS of infected CCR5 2 2 mice correlated with a reduction in demyelination spinal cords from infected CCR5 1 1 and CCR5 2 2 mice at day 12 pi were stained with luxol fast blue LFB and the severity of demyelination was evalu ated As shown in Fig 3B infected CCR5 2 2 mice dis played reduced mononuclear cell infiltration into white matter tracts which was accompanied by a marked re duction in the severity of demyelination compared to CCR5 1 1 mice Fig 3A Quantitative analysis revealed a significant reduction P 0 001 in demyelination in CCR5 2 2 mice 0 9 6 0 4 n 5 11 when compared to CCR5 1 1 mice 2 2 6 0 6 n 5 9 at day 12 pi Table 2 Immunohistochemical staining was performed on spinal cord sections from both strains of mice to determine if macrophage microglial infiltration into white matter tracts was reduced The data presented in Fig 4 reveal intense F4 80 staining within white matter tracts of CCR5 1 1 mice while staining in CCR5 2 2 mice is notably reduced Cytokine and chemokine gene expression in CCR5 1 1 and CCR5 2 2 mice We next evaluated the cytokine and chemokine gene expression patterns in the CNS of MHV infected CCR5 1 1 and CCR5 2 2 mice in an attempt to correlate the reduc tion in demyelination in CCR5 2 2 mice with the presence and or the absence of specific mRNA signals There was no obvious difference in the cytokine transcript profile between the two strains of mice at either 7 or 12 days pi Fig 5A Moreover quantification of signal intensities revealed similar levels of transcripts for the cytokines LT b TNF a IFN g and IFN b at days 7 and 12 pi be tween the strains of mice Fig 5B MHV infected CCR5 1 1 and CCR5 2 2 mice expressed a similar chemo kine profile with CCL2 MCP 1 CCL3 CCL5 and CXCL10 IP 10 being expressed at days 7 and 12 pi Fig 6A Analysis of transcript levels revealed comparable mRNA abundance for each chemokine at day 7 in both strains of mice Fig 6B However by day 12 pi CCR5 2 2 mice exhibited increased levels approximately twofold of mRNA transcripts for CCL5 compared to CCR5 1 1 mice DISCUSSION Animal models of demyelination including experimen tal autoimmune encephalomyelitis EAE Theiler s virus induced demyelination and MHV induced demyelina tion have provided valuable information with regard to understanding the immunopathological mechanisms contributing to human demyelinating diseases such as MS We have used the MHV model of viral induced demyelination to better understand the complex mecha nisms by which chemokines and chemokine receptors contribute to neuroinflammation host defense and my elin destruction Through use of specific neutralizing antibodies we have demonstrated important roles for CXCL9 Mig and CXCL10 in promoting a protective Th1 response following MHV infection characterized by CD4 1 and CD8 1 T cell infiltration and IFN g production Liu et al 2000 2001 Further we have shown that CCL5 serves to attract macrophages into the CNS which then contribute to myelin destruction Lane et al 2000 Therefore chemokines clearly influence leukocyte mi gration and infiltration into the CNS following MHV infec tion and have a profound effect on the outcome of dis ease with regard to host defense and demyelination The present study was undertaken to provide informa tion regarding the role of chemokine receptors to MHV induced neurologic disease Instillation of MHV into the brains of C57BL 6 mice resulted in increased mRNA expression of the chemokine receptors CCR1 CCR2 and CCR5 Fig 1 Ligands for these receptors that are ex pressed within the CNS of MHV infected mice include CCL2 CCL3 CCL4 CCL5 and CCL7 MCP 3 Lane et al 1998 Therefore these data suggest that expression of chemokine receptors by either resident cells of the CNS and or inflammatory cells in response to chemo kine expression may influence the immune response We TABLE 2 Diminished Macrophage Infiltration Correlates with Decreased Demyelination in CCR5 2 2 Mice Mouse type Day postinfection Average cell recovery CD45 high F4801 b mean 6 SEM DM c mean 6 SEM CCR5 1 1 12 9 33 3 10 5 4 a 1 3 3 10 5 6 1 2 3 10 4 2 2 6 0 6 9 CCR5 2 2 12 8 21 3 10 5 5 4 5 3 10 4 6 8 2 3 10 3 d 0 9 6 0 4 11 e a Parentheses indicate number of mice used b Data are presented as the percentage of positive cells within the gated population c Data presented are from three independent experiments d P 0 02 when compared to CCR5 1 1 mice e P 0 001 when compared to CCR5 1 1 mice 12 GLASS ET AL elected to examine the role of CCR5 in MHV induced disease in more detail as we have recently determined that CCL5 is important in both host defense and disease development Lane et al 2000 In addition the CCR5 ligand CCL3 is expressed albeit at lower levels during acute disease suggesting that this chemokine may also FIG 5 A Cytokine gene expression in CCR5 1 1 and CCR5 2 2 mice Mice were infected with either MHV or sterile saline sham by ic injection brains isolated at days 7 or 12 pi and total RNA was subjected to RPA analysis Shown is a representative RPA from two separate experiments Sample sizes are as follows day 7 pi CCR5 1 1 n 5 2 CCR5 2 2 n 5 4 day 12 pi CCR5 1 1 n 5 2 CCR5 2 2 n 5 2 Results from sham mice are shown from both groups at days 7 and 12 pi n 5 2 B Quantitative analysis of cytokine mRNA transcripts expressed Densitometric analysis of each lane representing a brain sample from an individual mouse was performed on the scanned autoradiograph A Data are presented as the average 6SEM FIG 6 A Chemokine gene expression in CCR5 1 1 and CCR5 2 2 mice Representative RPA showing chemokine gene expression in either CCR5 1 1 or CCR5 2 2 mice at days 7 and 12 pi with MHV RNA samples from Fig 5 were used to examine chemokine mRNA transcripts in the autoradiograph presented B Quantitative analysis of chemokine mRNA transcripts expressed is shown Densitometric analysis of each lane representing a brain sample from an individual mouse was performed on the scanned autoradiograph A Note the approximate twofold increase in the level of CCL5 mRNA transcripts in CCR5 2 2 mice compared to CCR5 1 1 at day 12 pi Data are presented as the average 6SEM 13 CCR5 AND VIRAL INDUCED DEMYELINATIONcontribute to neuroinflammation following viral infection of the CNS In support of a role for CCR5 in host defense following viral infection are studies by Dawson et al 2000 that have shown increased mortality in CCR5 2 2 mice following influenza A viral infection Further CCR5 expression is cons