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* Institute of Medical Microbiology and Immunology and Department of Medical Anatomy, University of Copenhagen, Copenhagen, Denmark; and Ina Sue Perlmutter Laboratory, Children’s Hospital and Harvard Medical School, Boston, MA 02115
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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producing) phenotype (Th1 cells) (11). However, recent observations suggest that CD8+ T cells with the same phenotype (Tc1 cells) also play a role (12, 13); this type of CD8+ T cells are known to be the primary effectors during viral infection (14). As Th1 and Tc1 cells appear to express a similar set of chemokine receptors (14), CCR5 and CXCR3 are also suspected to be involved in regulating the efficiency of CD8+ T cell-mediated immune surveillance of the CNS (15) and, consistent with this assumption coexpression of CXCR3 and CCR5, has been demonstrated recently for CD8+ T cells in cerebrospinal fluid (CSF) of HIV-infected patients (16).
For experimental studies on the role of chemokines in regulating CD8+ T cell-mediated antiviral immune surveillance of the CNS, intracerebral (i.c.) infection with lymphocytic choriomeningitis virus (LCMV) represent an ideal model system. LCMV is a noncytolytic virus. However, i.c. inoculation of LCMV leads to infection of the meninges and choroid plexus, and in immunocompetent mice, the antiviral CD8+ T cells cause severe tissue damage from which the animals succumb 
7–10 days postinfection (p.i.) (17, 18). Using this model, our group has demonstrated previously that failure to express CXCR3 delays and reduces T cell accumulation in critical areas of the brain parenchyma, and this is associated with markedly reduced mortality (19). Nevertheless, 30% of CXCR3-deficient mice still succumb to i.c. infection, indicating that other chemokine receptors also contribute significantly in the recruitment of CD8+ T cells. One important receptor may be CCR5 because recent results suggest that ligands of CCR5 and CXCR3 work synergistically in attracting T cells to the virus-infected CNS (20). We have shown previously that although CCR5 is expressed by the infiltrating cells, lack of this receptor does not in itself impair the LCMV-induced inflammatory process (21). However, because CCR5 is mostly expressed by a subpopulation of CXCR3+ T cells (10, 16, 22, 23), it is tempting to hypothesize that CCR5 would be redundant only when CXCR3 is normally expressed, i.e., concurrent inhibition of both chemokine receptors would lead to a severely impaired inflammatory response. Whether in fact a synergistic inhibitory effect on T cell-mediated inflammation will be obtained by combined blockade of CXCR3 and CCR5 is very important from a pharmacotherapeutic point of view considering that this receptor combination seems to characterize the majority of tissue-infiltrating lymphocytes (16, 24, 25, 26, 27). Therefore, to test our hypothesis, transgenic mice deficient in both CXCR3 and CCR5 were generated, and the outcome of LCMV infection in these mice was studied. Much to our surprise, we found that double-deficient mice are more susceptible to LCMV-induced T cell-mediated disease.
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Materials and Methods |
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The generation of CXCR3-deficient mice has been described before (28). The animals used in these experiments were the progeny of breeder pairs kept at the Panum Institute, University of Copenhagen. CCR5-deficient mice (B6,129P-CmKbr5<tm/Kn2>) were bred locally from breeder pairs obtained from The Jackson Laboratory. Mice deficient in CXCR3/CCR5 were produced in the following way: CCR5-deficient mice were mated with CXCR3-deficient mice to generate an F1 generation. These mice were then backcrossed to CCR5-deficient mice, and offspring heterozygous at the CXCR3 locus (+/–) and deficient for a functional CCR5 gene at both loci (–/–) were then selected and interbred. From the subsequent, offspring homozygous double-deficient mice were selected for further inbreeding. Assessment of genotypes was performed by PCR. Wild-type (WT) C57BL/6 mice were purchased from Taconic M&B. Mice from outside sources were always allowed to rest for at least a week before entering into experiments; by that time, the animals were 7–9 wk old. Animals were housed under controlled (specific pathogen free) conditions, and experiments were conducted according to national guidelines.
Virus infection
Mice were infected i.c. with a virus dose of 3 x 103 LD50 of LCMV Traub in a volume of 0.03 ml. Intracerebral infection induces a fatal CD8+ T cell-mediated meningitis from which immunocompetent mice succumb on days 7–10 p.i. (29). In a few cases the same dose of virus was injected i.v.; in this case no disease is induced.
Survival study
Mortality was used to evaluate the clinical severity of acute LCMV-induced meningitis. Mice were checked twice daily for a minimum of 2 wk after i.c. inoculation; deaths occurring <5 days after infection were excluded from analysis.
Organ virus titers
To determine virus titers in organs, these were first homogenized in PBS to yield 10% (v/w) organ suspensions, and serial 10-fold dilutions were prepared. Each dilution was then plated in duplicates on MC57G cells. Forty-eight hours after infection, infected cell clusters were detected using monoclonal rat anti-LCMV (VL-4) Ab, peroxidase-labeled goat anti-rat Ab, and o-phenylenediamine (substrate) (30). The numbers of PFU were counted, and results were expressed as PFU/g tissue.
Cell preparations
Single-cell suspensions of spleen cells were obtained by pressing the organs through a fine steel mesh and, when required, erythrocytes were lysed by 0.83% NH4Cl treatment.
CSF cell count
CSF was obtained from the fourth ventricle of mice deeply anesthetized and exsanguinated. The total number of inflammatory cells (background level in uninfected mice is <100 cells/µl) was determining by counting in hemocytometer, and phenotypic analysis was conducted by flow cytometry (see Flow cytometric analysis).
Abs for flow cytometry
The following mAbs were purchased from BD Pharmingen as rat anti-mouse Ab: PE- and CyChrome-conjugated anti-CD8, allophycocyanin-conjugated anti-CD4, FITC-conjugated anti-VLA-4 (CD49d), FITC-conjugated anti-CD44, FITC-conjugated anti-Mac-1 (CD11b), FITC-conjugated anti-L-selectin (CD62L), PE-conjugated anti-B220 (CD45R), PE-conjugated anti-CCR5, PE-conjugated anti-IFN-, and PE-conjugated IgG1 isotype standard. Rabbit anti-CXCR3 Ab and biotin-SP-conjugated goat anti-rabbit Ab were purchased from Zymed Laboratories. Streptavidin-Tricolor was purchased from Caltag Laboratories.
Flow cytometric analysis
Staining of cells for flow cytometry was performed according to standard laboratory procedure (31, 32). For enumeration of LCMV-specific CD8+ T cells, splenocytes were incubated in vitro for 5 h at 37°C in 5% CO2 with gp33-41 peptide (0.1 µg/ml) in the presence of monensin (3 µM; Sigma-Aldrich) and murine rIL-2 (10 U/well; R&D Systems Europe). After incubation cells were surface stained, washed, and permeabilized using 0.5% saponin. Cells were then stained with anti-IFN- or IgG1 isotype control for 20 min at 4°C. Samples were analyzed using a BD Biosciences FACSCalibur, and at least 104 mononuclear cells were gated using a combination of low angle and side scatter to exclude dead cells and debris. Data analysis was conducted using CellQuest Pro (BD Biosciences).
Detection of mRNA in the brain
Brains from mice deeply anesthetized and exsanguinated were immediately removed, snap frozen in liquid nitrogen, and stored in a liquid nitrogen freezer. Total RNA was extracted from homogenized brains by use of a RNeasy Midi kit (Qiagen). Transcription levels were studied using the RiboQuant multiprobe RNase protection assay (RPA) system (BD Pharmingen) (21, 33). The following templates sets (from BD Pharmingen) were used: T cell marker mRNA (CD3
, CD4, CD11b, CD8
, and F4/80), cytokine marker mRNA (TNF-, LT, TNF-
, IL-6, IFN-, IFN-, TGF-1–3, and MIF) chemokine receptor marker mRNA (CCR1, CCR3, CCR4, CCR5, CCR2, CCR7, CCR8, CXCR3, CXCR2, CXCR4, and CXCR5), and chemokine marker mRNA (XCL1 (lymphotactin), CCL5 (RANTES), CCL11 (eotaxin), CCL4 (MIP-1), CCL3 (MIP-1), CXCL1–2 (MIP-2), CXCL10 (inflammatory protein-10), CCL2 (MCP-1), and CCL1 (TCA-3)). All sets of probes included templates for the housekeeping genes L-32 and GADPH to serve as loading controls. The RPA was performed according to the manufacturer’s instructions. Briefly, [-32P]UTP-labeled antisense RNA transcript was generated from the template sets using T7 RNA polymerase. RNA from each sample was allowed to hybridize to the labeled probe for 16–20 h at 56°C. Single-stranded RNA was digested with an RNase/T1 mixture, and the hybrids were analyzed on a denaturing urea-polyacrylamide gel. For qualitative and quantitative results, gels were subjected to PhosphorImager analysis (Amersham Biosciences), and the data were subsequently analyzed using ImageMaster TotalLab software (Amersham Biosciences).
Immunohistochemistry
Mice were deeply anesthetized with tribromoethanol (Sigma-Aldrich) i.p. and decapitated. Brains were then processed for immunohistochemistry (19). Ten-micrometer cryostat sections fixed in acetone were washed three times for 5 min in 0.1 M PBS (pH 7.4), followed by incubation with 5% normal swine serum (X0901; DakoCytomation) diluted in 1% BSA in PBS/0.3% Triton X-100 (BSA/TX100) to block nonspecific binding by the Abs. Next, sections were incubated overnight at 4°C with monoclonal rat anti-mouse CD8a (550281; BD Biosciences) diluted 1/50 in BSA/TX100, or monoclonal rat anti-mouse LCMV Ab (VL-4, kindly provided by R. M. Zinkernagel, Zurich, Switzerland) (30, 34) diluted 1/100 was used to detect viral protein in the brain. Specific binding of the primary Abs was verified using HRP-streptavidin-biotin complex followed by diaminobenzidine.
Quantitative morphological analysis
Counts of CD8+ T cells were performed in sections using a standardized protocol for estimating cell density, which involved counting the number of CD8+ T cells in a 10 x 10-mm frame overlaying the part of the corpus callosum situated above the body of the lateral ventricle at x250 magnification equivalent to an area of 10,000 µm2. Three sections with an individual distance of 300 µm were examined in brains of WT- and CCR5-deficient mice on day 7 p.i. and CXCR3-deficient and CXCR3/CCR5 double-deficient mice on days 7 and 9 p.i. Data were examined by ANOVA, and means were tested with the Student-Newman-Keuls test for differences between individual means. Evidence of statistically significant difference between mean values was considered to be a p < 0.05.
Statistical analysis
For survival curves comparisons were performed using the Mantel-Cox test; quantitative results were compared using the Mann-Whitney U test. A value of p < 0.05 was considered as evidence of statistical significance.
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Results |
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We initially asked whether CXCR3 and CCR5 are coexpressed on CD8+ T cells during acute viral infection. To address this question, mice were infected i.c. with LCMV, and on day 7, p.i. splenocytes were isolated from WT as well as single and double knockout mice, and chemokine receptor expression on CD8+ T cells was evaluated using flow cytometry. From an earlier study, we know that a subset of the CD8+ T cells from naive mice express CXCR3. The majority of these cells are CD44high, indicating that they represent primed (effector/memory) cells (19). Extending this observation, we now find that 15–20% of the CD8+ T cells in young naive mice express CXCR3, whereas none are CCR5+ (Fig. 1 (numbers seen in brackets)). In mice infected 7 days earlier, 50–80% of splenic CD8+ T cells have an activated (CD44high) phenotype (19), and more than half of the CD8+ T cells express CXCR3 (Ref.19 ; Fig. 1). Furthermore, about one-fifth of splenic CD8+ T cells from recently infected mice express CCR5, and these cells all coexpress CXCR3. These finding are consistent with the hypothesis that CXCR3 might compensate for the lack of CCR5 and that CXCR3 should be more important in effector T cell homing. Notably, the results in Fig. 1 also confirm the phenotypes of the different knockout strains used in this study.
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To evaluate the redundancy of CXCR3 and CCR5 in regulating the migration of leukocytes to the infected CNS, WT and single and double knockout mice were inoculated i.c. with 3 x 103 LD50 of LCMV, and the number of leukocytes present in the CSF of mice infected 7 days earlier was quantified (Fig. 2A). Confirming earlier reports (19, 21), lack of CXCR3 or CCR5 individually did not significantly delay the accumulation of leukocytes in this CNS compartment. The average cell infiltrate in double knockout mice matched that in CXCR3-deficient mice. However, a lesser interindividual variation was observed, which could indicate that meningeal cell accumulation might be more consistently delayed in these mice. Therefore, to study in greater detail the kinetics of cell accumulation in the CSF of double knockout mice, these mice and WT mice were analyzed in parallel at several different time points after i.c. infection. As seen in Fig. 2B, we observed no difference in the number of cells in the CSF between WT and double knockout mice on day 6 p.i. In contrast, on day 7 p.i., we could detect a small but statistically significant reduction in the number of infiltrating mononuclear cells in CXCR3/CCR5-deficient mice compared with WT mice (p < 0.05). At this time point, the composition of the inflammatory exudate was also investigated (Fig. 2C), and in both mouse strains, CD8+ T cells and macrophages made up the majority of cells in the CSF, whereas few CD4+ T cells and B cells were present. Most importantly, no significant differences between the genotypes with regard to composition of the infiltrate were observed. On day 9 p.i., all WT mice had succumbed to LCMV disease, and in the double knockout mice, the number of inflammatory cells reached its maximum, which together with the results from day 7 could point to a slight delay in the accumulation of cells in the CSF of these mice. Nonetheless, it is evident that CXCR3 and CCR5 neither individually nor together are pivotal for the accumulation of mononuclear cells in the CSF.
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To further study the importance of CXCR3 and CCR5 in the recruitment of effector T cells to the virus-infected CNS, WT and single and double knockout mice were again infected i.c. with 3 x 103 LD50 of LCMV, and for the next 14 days, their mortality was registered (Fig. 3). WT mice succumb to LCMV-induced CD8+ T cell-mediated meningitis around days 7–10 p.i., and a similar pattern is observed in CCR5-deficient mice confirming earlier results by our group (21). As recently reported (19), CXCR3-deficient mice are less susceptible to i.c. infection with LCMV, and 60% of these mice survive i.c. infection. Surprisingly, mice lacking both chemokine receptors present a disease phenotype intermediate to that of CCR5 and CXCR3 single-deficient mice, and only 30% of i.c. infected double knockout mice survived the infection. This reduction in the survival rate, relative to CXCR3-deficient mice, is small but statistically significant (p < 0.05; Mantel-Cox). Analysis of the viral load in the brain of i.c. infected mice on days 4 and 7 p.i. did not reveal any consistent genotype-related differences, indicating that the differences in survival rate do not reflect differences in the extent of viral replication in the brain (see also Fig. 8 for immunohistochemical analysis). Thus, our results indicate that CCR5 is not redundant, but instead of the expected positive influence on survival in absence of CXCR3 expression, the clinical evaluation reveals that the two receptors exert opposite effects on the severity of LCMV-induced disease.
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Because the clinical outcome of i.c. LCMV infection directly reflects cerebral CD8+ T cell-mediated immunopathology and we have previously obtained results suggesting an augmented T cell response in LCMV-infected CCR5-deficient mice (21), a simple explanation for the increased mortality of i.c. infected double knockout mice compared with CXCR3-deficient mice could be the generation of a stronger antiviral CD8+ T cell response in the former mice. To test this possibility, we compared the virus-specific CD8+ T cell response in CXCR3- and CXCR3/CCR5-deficient mice, including WT- and CCR5-deficient mice for further comparison. On days 7 and 9 p.i., cells were isolated from the spleen, which is the predominant site of effector T cell generation (35), and CD8+ T cells specific for one of the immunodominant epitopes were measured as the fraction of CD8+ T cells, which respond to stimulation with the MHC class I-restricted LCMV peptide (gp31-44) by production of IFN-. As WT- and CCR5-deficient mice would all have succumbed by day 9 after i.c. infection, i.v. infection was used for these mice. This approach is valid because in i.c. infected mice 90% of the inoculum in fact goes i.v. due to a combination of the pressure and volume applied.
Seven days after infection, similar virus-specific CD8+ T cell responses were measured in the spleens of CXCR3-deficient and WT mice (Fig. 4). In contrast, more virus-specific CD8+ T cells were found in the spleens of CCR5- and CXCR3/CCR5-deficient mice. Additional experiments (data not shown) confirmed that these differences were reproducible and statistically significant.
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To see whether early differences in the viral load in the spleen was the cause of the genotype-related differences in the kinetics of the antiviral T cell response, spleen virus titers were determined in mice infected 4 days earlier, and this roughly marks the last day before onset of the T cell response; in all cases, the virus titer was 0.5–1.0 x 106 PFU/g organ, indicating that innate defenses were not significantly influenced by the absence of CCR5.
In addition to cell numbers, the quality of the virus-specific CD8+ T cells was also compared. Using intracellular cytokine staining, we did not find any difference in the capacity of the cells to produce IFN- following standard peptide stimulation (Fig. 5). However, interestingly, unstimulated CD8+ T cells from virus-infected double-deficient mice "spontaneously" produced low amounts of IFN-. Thus, cytokine production in absence of overt stimulation was consistently higher in mice lacking CCR5 expression. However, because the virus infection in the spleen is not completely eliminated at this time, these results may simply indicate that CD8+ T cells from CCR5-deficient mice are more easily triggered by the remaining infected cells (notably, we have no evidence indicating delayed virus clearance in the spleen as a result of CCR5 deficiency (21)). Consistent with this interpretation, neither naive cells nor virus-primed cells from CCR5-deficient mice taken 
14 days p.i. produced IFN- unless stimulated (data not shown). If CD8+ T cells from CCR5-deficient mice were more responsive in vivo, this would further augment the inflammatory reaction in these mice.
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To evaluate the local immune response in the brain, we quantified transcripts for cell subset markers and proinflammatory cytokines by RPAs. mRNA was isolated from the brains of WT and single and double knockout mice 7 days after i.c. infection and from matched control mice inoculated i.c. with PBS. The level of CD8 and CD3 mRNA is reduced in the CXCR3- and CXCR3/CCR5-deficient mice compared with WT- and the CCR5-deficient mice (Fig. 6A). The lower expression of transcripts for T cell markers probably reflects delayed accumulation of leukocytes in the CNS of CXCR3- and CXCR3/CCR-deficient mice. Furthermore, we find that the level of TNF--specific transcripts is similarly increased in all infected mice, whereas the level of transcripts for IFN- is lower in double knockout and CXCR3-deficient mice (Fig. 6B), which may be explained by the reduced level of CD8+ T cells. Other proinflammatory cytokines were either not detected (TNF- and LT) or only expressed at a very low level (IL-6) (data not shown).
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To study T cell accumulation in CNS more directly, the localization of CD8+ T cells was evaluated through immunohistological analysis. WT-, CXCR3-, CCR5-, and CXCR3/CCR5-deficient mice were infected i.c. with LCMV, and brain sections were analyzed 7 and 9 days after infection. As previously reported, the meninges of i.c. infected mice exhibited prominent signs of inflammation, including multiple CD8+ immunoreactive cells. WT- and CCR5-deficient mice also harbored CD8+ cells in many gray and white matter regions in close vicinity of the surfaces of the ventricular system (Fig. 8A and Table I). By contrast, this finding was not recapitulated in brains of CXCR3- and CXCR3/CCR5-deficient mice (Fig. 8B and Table I). Hence, a careful examination of the corpus callosum revealed very few CD8+ cells in this area in CXCR3- and CXCR3/CCR5-deficient mice infected 7 days earlier (Table I). When examined 2 days later, around the time when part of these mice succumbs to LCMV-induced disease, more cells were found in the corpus callosum of these strains. Furthermore, consistent with the higher mortality of double-deficient mice, the number of CD8+ T cells was also significantly higher in these mice (Table I).
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Discussion |
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Chemokines are thought to be important gatekeepers in leukocyte extravasation and migration into the different compartments of the brain, and based on studies of MS patients and related animal models, CXCR3 and CCR5 are believed to be key receptors in the accumulation of Th1 cells at sites of inflammation inside the CNS (e.g., Refs.7, 10, 22). The same chemokine receptors are expressed by Tc1 cells, and we have demonstrated recently that CXCR3 expression is essential for optimal Tc1 surveillance of the neural parenchyma (19). In contrast, expression of CCR5 does not seem to be essential in the recruitment of Tc1 cells to the virus-infected CNS (21, 36). One reason why CCR5 might be redundant is that other chemokine/receptor pairs exert a similar function in effector cell recruitment. Because CCR5 is often expressed on a subset of activated T cells, which also express CXCR3, we hypothesized that lack of both these receptors might result in a nearly complete block in CD8+ T cell migration to the infected CNS. Therefore, to unravel the biological functions and possible interplay of CXCR3 and CCR5, double knockout mice were generated.
For studying the mechanism of CD8+ T cell migration into the virus-infected CNS, LCMV infection is an ideal model system because the virus itself is noncytolytic, and a fatal outcome is therefore directly related to the invasion of the CNS by virus-specific cytotoxic CD8+ T cells (17, 18). Using this model, it has been demonstrated previously that ligands of both CXCR3 and CCR5 act as chemoattractants of relevant effector cells (CD8+ T cells and monocytes/macrophages) in vitro (37, 38, 39).
To evaluate the in vivo consequences of lacking expression of both CXCR3 and CCR5, the ability of the inflammatory cells to accumulate in the virus-infected CNS and cause lethal disease was analyzed.
Following invasion by two of the routes through which leukocytes may access the CNS, the infiltrating cells end up in the CSF (3). Therefore, accumulation of mononuclear cells in the CSF was evaluated both quantitatively and qualitatively. Except for a slight delay, no difference between WT and double knockout mice was observed. Thus, contrary to expectations (16), CD8+ T cells deficient in both CXCR3 and CCR5 are capable of crossing the blood-CSF barrier, albeit with a minor delay. A delay in the influx of CD8+ T cells is also reflected in a reduced level of transcripts for CD8+ T cell markers and the proinflammatory cytokine IFN-, which is produced by the infiltrating CD8+ T cells (40, 41).
Surprisingly, mice lacking both CXCR3 and CCR5 were less resistant to the CD8+ T cell-mediated meningitis than CXCR3-deficient mice (Fig. 3). This increase in the susceptibility toward the LCMV infection was not readily explained by up-regulation of new chemokine receptors or chemokines (Fig. 6C and 7). Immunohistological analysis of virus-infected brains on day 7 p.i. provided results, which conform with our recent finding that the accumulation of CD8+ T cells in the neural parenchyma is delayed in the absence of CXCR3. However, approximately one-third of CXCR3-deficient mice and two-thirds of double knockout mice eventually succumb to i.c. infection, underscoring that lack of CXCR3 expression only provides partial protection. This is consistent with the result of histological analysis performed around the time of maximal disease intensity in CXCR3-deficient strains. Thus, 9 days after infection, CD8+ T cells were found also in the brain parenchyma of mice with CXCR3–CD8+ T cells, i.e., CXCR3- and CXCR3/CCR5-deficient mice. Furthermore, in accordance with the higher mortality of double-deficient mice, significantly more CD8+ T cells were detected in the neural parenchyma from the latter mice.
Corresponding to the augmented local inflammatory response in double knockout mice, a stronger virus-specific CD8+ T cell response was detected in the spleen of these mice compared with matched WT1 and CXCR3-deficient mice.
The increased availability of effector CD8+ T cells early during the infection could explain the increased mortality in double-deficient mice. Evidently other chemokine receptors suffice to allow the accumulation of CD8+ T cells in critical areas within the virus-infected CNS, albeit with reduced efficiency. Under such conditions the mice are likely to be exquisitely sensitive to even a small increase in the T cell response.
As to the mechanism(s) underlying the accelerated response in double knockout mice, several explanations have to be considered. First, remaining genetic heterogeneity in the double knock-out mice could in theory play a role because the parental CCR5-deficient mice were on a mixed B6,129 background. However, comparison of B6 and (B6 x 129)F2 mice have not indicated that that admixture of 129-derived genes should influence the immune response to LCMV (unpublished observation). Altered tissue distribution could also play a role; delayed accumulation in infected, nonlymphoid tissues could result in a build-up of effector cells in the spleen. However, we have preliminary data showing that CD8+ T cell homing to the virus-infected liver is not impaired in double-deficient mice (P. Holst et al., manuscript in preparation), and changes in the localization to other organs are very unlikely to have a substantial impact on T cell numbers in the spleen. Finally, we did not observe critical differences in the early viral load in the spleen, indicating that the augmented response in CCR5-deficient mice does not relate to increased Ag presentation. Therefore, the most likely explanation is that absence of CCR5 somehow augments the generation of effector CD8+ T cells. This is in agreement with a number of recent reports suggesting a negative regulatory function for CCR5 (21, 36, 42, 43) and indicates that this chemokine receptor serves a negative regulator of effector CD8+ T cell generation. Using the mouse hepatitis virus model, Glass and Lane (36) have previously found that CCR5 signaling imparts a downstream effect on the effector capacity of the individual effector CD8+ T cell. Our studies have not confirmed this effect (Ref.21 and data not shown). However, we did find that primary effector T cells generated in absence of CCR5 produce more IFN- in the absence of overt stimulation. Because this phenomenon was only observed during the period when virus-infected cells were still present in the spleen, this could indicate that CD8+ T cells from CCR5-deficient mice are more easily triggered by the low number of virus-infected cells present at this time. If this is the case also in vivo, it could add further to the observed numerical difference, creating the basis for more intense inflammation in double-deficient mice.
In conclusion, the principle finding of this study is that inhibition of CCR5/ligand interaction fails to further suppress T cell-mediated inflammation even in a situation where CXCR3/ligand interaction is already inhibited. Hence, the redundancy of CCR5 (21) does not reflect a functional overlap with CXCR3. This information is highly pertinent from a pharmacotherapeutic perspective because these receptors tend to characterize most tissue-infiltrating T cells, e.g., during allograft rejection (27) and autoimmune disease (7, 25).
Additionally, based on the above results in combination with our prior analysis of mice lacking the individual chemokine receptors (19, 21), we are inclined to suggest that CXCR3 and CCR5 predominantly influences different stages in the immune response to viral invasion of CNS. Thus, while CXCR3 critically influences local T cell accumulation, particularly within the neural parenchyma, CCR5 seems primarily to influence the generation of effector T cells. Notably, the present data also reveal that while CXCR3 is clearly involved in promoting tissue-accumulation, the protective impact of blocking this receptor may be reduced, if the availability of effector T lymphocytes is increased. Together with the histological analysis, this underscores that T cell surface receptors other than CXCR3 and CCR5 play a role in controlling CNS invasion. Molecules that could play a role are the chemokine receptors CCR2 (33) and CXCR6 (44), as well as receptors for leukotrienes; leukotriene B4 has been identified recently as a potent nonchemokine chemoattractant of cytotoxic effector T cells (45, 46).
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Acknowledgments |
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Disclosures |
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Footnotes |
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1 This work was supported in part by the Danish Medical Research Council, the Lundbeck Foundation, the Leo Pharma Research Foundation, and the Novo Nordisk Foundation. J.E.C. is the recipient of a Ph.D. scholarship from the Faculty of Health Science, University of Copenhagen. 
2 Address correspondence and reprint requests to Dr. Allan Randrup Thomsen, Institute of Medical Microbiology and Immunology, University of Copenhagen, The Panum Institute, 3C Blegdamsvej, DK-2200 Copenhagen N, Denmark. E-mail address: A.R.Thomsen{at}immi.ku.dk
3 Abbreviations used in this paper: MS, multiple sclerosis; i.c., intracerebral or intracerebrally; LCMV, lymphocytic choriomeningitis virus; CSF, cerebrospinal fluid; p.i., postinfection; WT, wild type; RPA, RNase protection assay.
Received for publication February 2, 2005. Accepted for publication May 25, 2005.
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References |
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and IP-10 are expressed in demyelinating brain lesions. Proc. Natl. Acad. Sci. USA 96: 6873-6878. Cells are responsible for CXCR3-mediated T cell infiltration in insulitis. Nat. Med. 8: 1414-1420.[Medline]
in T cell-mediated immunity to viral infection. J. Virol. 77: 12378-12384.-producing effector cells. J. Neuroimmunol. 127: 96-105.[Medline]
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K. B. Walsh, R. A. Edwards, K. M. Romero, M. V. Kotlajich, S. A. Stohlman, and T. E. Lane Expression of CXC Chemokine Ligand 10 from the Mouse Hepatitis Virus Genome Results in Protection from Viral-Induced Neurological and Liver Disease J. Immunol., July 15, 2007; 179(2): 1155 - 1165. [Abstract] [Full Text] [PDF]
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M. Thapa, W. A. Kuziel, and D. J. J. Carr Susceptibility of CCR5-Deficient Mice to Genital Herpes Simplex Virus Type 2 Is Linked to NK Cell Mobilization J. Virol., April 15, 2007; 81(8): 3704 - 3713. [Abstract] [Full Text] [PDF]
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 J. Zhang, E. Rao, M. Dioszegi, R. Kondru, A. DeRosier, E. Chan, S. Schwoerer, N. Cammack, M. Brandt, S. Sankuratri, et al. The Second Extracellular Loop of CCR5 Contains the Dominant Epitopes for Highly Potent Anti-Human Immunodeficiency Virus Monoclonal Antibodies Antimicrob. Agents Chemother., April 1, 2007; 51(4): 1386 - 1397. [Abstract] [Full Text] [PDF]
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L. N. Stiles, J. L. Hardison, C. S. Schaumburg, L. M. Whitman, and T. E. Lane T Cell Antiviral Effector Function Is Not Dependent on CXCL10 Following Murine Coronavirus Infection J. Immunol., December 15, 2006; 177(12): 8372 - 8380. [Abstract] [Full Text] [PDF]
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J. E. Christensen, C. de Lemos, T. Moos, J. P. Christensen, and A. R. Thomsen CXCL10 Is the Key Ligand for CXCR3 on CD8+ Effector T Cells Involved in Immune Surveillance of the Lymphocytic Choriomeningitis Virus-Infected Central Nervous System J. Immunol., April 1, 2006; 176(7): 4235 - 4243. [Abstract] [Full Text] [PDF]
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 D. J. J. Carr, J. Ash, T. E. Lane, and W. A. Kuziel Abnormal immune response of CCR5-deficient mice to ocular infection with herpes simplex virus type 1. J. Gen. Virol., March 1, 2006; 87(Pt 3): 489 - 499. [Abstract] [Full Text] [PDF]
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