Lymphocytic Choriomeningitis Virus-Induced Central Nervous System ...

JOURNAL OF VIROLOGY, Jan. 2009, p. 20–28 0022-538X/09/$08.00⫹0 doi:10.1128/JVI.00682-08 Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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MINIREVIEW Lymphocytic Choriomeningitis Virus-Induced Central Nervous System Disease: a Model for Studying the Role of Chemokines in Regulating the Acute Antiviral CD8⫹ T-Cell Response in an Immune-Privileged Organ䌤 Allan Randrup Thomsen* Institute of International Health, Immunology, and Microbiology, University of Copenhagen, The Panum Institute, 3C Blegdamsvej, DK-2200 Copenhagen N, Denmark protective status, the CNS is not exempt from immunological surveillance. Thus, unless CNS infection is entirely confined to the neuroparenchyma (70), an immune response will be raised and effector cells will invade the CNS. However, just as in any other organ site, leukocyte recruitment is the result of a tightly controlled multistep process.

CNS AS A SITE OF IMMUNE PRIVILEGE The ability of leukocytes to extravasate and enter infected tissue is a crucial element in the host immune response to invading pathogens. Locally, a complex inflammatory response is played out, the purpose of which being the elimination of the intruding pathogen and the initiation of the process of tissue repair. However, in the context of relevant antimicrobial activities, collateral tissue damage may be induced, which in certain situations may pose a serious threat to the survival of the host. Probably for this reason, certain crucial organs/organ sites seem to be particularly well protected against an overzealous immune attack; these sites have been named immuneprivileged sites. The central nervous system (CNS) represents such a site, with its high content of postmitotic, nonrenewable cells. Nevertheless, the CNS may become the site of life-threatening viral infections, which makes it imperative for the immune system to be able to monitor this organ site. Therefore, insight into how the local inflammatory response within the CNS is regulated is a key to understanding the pathogenesis of viral infections in the CNS. The concept of the brain as an immunologically privileged organ stems from the seminal observation of Sir Peter Medawar that allogeneic cells were not rejected from the brain (57). This observation was followed by several complementary findings, namely, that there are no classical antigen-presenting cells, such as dendritic cells, within the CNS, lymphatic vessels are absent, and constitutive expression of major histocompatibility complex classes I and II on parenchymal cells is low or missing. Moreover, the environment of the CNS is structurally protected by the blood-brain and the blood-cerebrospinal fluid (CSF) barriers. As a consequence, immunoglobulin levels in the CSF are very low and complement components virtually absent in the uninfected host. However, the concept of immune privilege has greatly evolved since the original observations, and it now widely accepted that while the special anatomy clearly provides the cells of the CNS with a certain

LEUKOCYTE EXTRAVASATION According to the multistep paradigm (10), leukocyte extravasation is the result of a sequence of leukocyte/endothelial cell contacts, each regulated by its specific set of receptor/ ligand interactions. At sites of inflammation, the initial contact between circulating leukocytes and the vascular endothelium is established primarily through interactions between carbohydrate ligands on the leukocytes and selectins highly expressed at sites of endothelial activation (P and E selectin). This results in a tethering of the circulating leukocyte and causes it to roll along the activated endothelium. Uniquely, lymphocytes can also roll via interactions between the integrin VLA-4 on the lymphocyte and VCAM-1 on the endothelium (3, 9). While slowly rolling along the endothelium, the leukocytes then have the opportunity to sense the presence of chemokines exhibited on the luminal surface of the endothelium. If a chemokine is detected for which matching receptors are expressed on the cell surface of the rolling leukocyte, a signal cascade is initiated, leading to a conformational change of the leukocyte integrins LFA-1 and VLA-4, which are highly expressed on recently activated lymphocytes. The affinity of these integrins for their ligands of the immunoglobulin superfamily is markedly increased, and a tight binding of the leukocyte to the activated endothelium is established. Ultimately, leukocyte diapedesis is observed. Notably, it is the activation stage, rather than specificity, which determines lymphocyte extravasation, according to this model. However, in order to retain the emigrated cells locally in the tissue, recognition of specific antigen appears to be pivotal, and this would favor the preferential accumulation of primed T cells with specificity for the invading pathogen.

* Mailing address: Institute of International Health, Immunology, and Microbiology, University of Copenhagen, The Panum Institute, Building 22.5.18, 3C Blegdamsvej, DK-2200 Copenhagen N, Denmark. Phone: 45-35-32-78-71. Fax: 45-35-32-78-91. E-mail: a.r.thomsen @immi.ku.dk. 䌤 Published ahead of print on 10 September 2008.

EXPERIMENTAL MODEL Based on the above description, it is evident that chemokines are in a key position to function as gatekeepers with 20

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regard to virus-induced inflammation in the CNS; for this reason, this family of cytokines has recently been the focus of research on T-cell-mediated immune surveillance of the virusinfected CNS. Several well-established models for viral infection of the CNS are currently in use and have generated interesting results. However, the most prevalent model is perhaps intracerebral (i.c.) infection with murine lymphocytic choriomenigitis virus (LCMV). The basic pathogenesis was already resolved about 30 years ago. The virus itself is essentially noncytolytic in vitro and in vivo (34), but in the immunocompetent host, i.c. inoculation results in a potent T-cell-mediated inflammatory response inside the virus-infected CNS (20), which causes the death of the host animal in about 7 to 9 days. Subsequent studies revealed that the central T-cell population is the CD8⫹ T-cell subset (24, 46), and kinetics analyses indicated that a fatal outcome in essence was the unfortunate result of a normal antiviral CD8⫹ T-cell response losing out to the rapid replication of virus in the brain of the i.c.-infected host (75). Regarding the cellular events underlying the disease, it was for many years widely assumed that CD8⫹ T-cell-mediated destruction of the blood-brain and blood-CSF barriers represented a key element of this disease (4, 26, 54, 67). Precisely how the virus-specific CD8⫹ T cells induce lethal disease in i.c.-infected mice has been the subject of controversy (4, 67), but based on the relative resistance of perforin-deficient mice to the induction of fatal CNS disease, a widely accepted view has been that perforin-dependent killing of virus-infected cells primarily in the meninges is essential to a fatal outcome of this infection (36). Recently, however, various observations point to the necessity of reevaluating this hypothesis. As will be discussed in greater detail below, a fatal outcome is better correlated with lymphocyte infiltration of the neuroparenchyma than with meningeal inflammation, at least in some cases (16). Despite these few outstanding issues, this model is extremely well suited for the study of the regulation of virusinduced T-cell recruitment to the CNS, because the clinical outcome is directly related to the infiltration of critical CNS regions with virus-specific CD8⫹ T cells; immune-deficient or, more specifically, CD8⫹ T-cell-depleted mice invariably survive i.c. infection (19, 46). LCMV is the prototypical member of the Arenaviridae family, and extensive viral replication is observed for many organs of its natural host, the common house mouse, Mus musculus or Mus domesticus. The outcome of infection varies with the inoculation route and the mouse-virus strain combination, but clinical symptoms are almost invariably the result of the host reacting to viral invasion. In i.c.-infected mice, fatal CNS disease is observed, but virus replication is found in many other organ sites, including the secondary lymphoid organs, and this is probably one of the reasons that such a very potent T-cell response is induced (see further discussion below). In fact, it is generally assumed that ⬎90% of the inoculum, due to the volume and pressure applied during i.c. inoculation, escapes into the blood (58), making this model unsuited for studies regarding details of the afferent phase of viral infection in the CNS. On the other hand, the advantage of this overflow is that the spleen is the major site of effector T-cell development; therefore, the central phase of the immune response can readily be monitored through phenotypic and functional anal-

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ysis of the splenic T-cell population. From this organ and the peripheral lymph nodes, the differentiated effector T cells are released into the circulation and subsequently recruited to sites of infection in nonlymphoid organs, such as the brain. Indeed, it has been shown that surgical removal of the spleen in the early phase of infection significantly delays the appearance of clinical symptoms and death in mice infected i.c. with LCMV (26). Histological examination of the CNS 6 to 7 days after i.c. inoculation reveals severe inflammation of the leptomeniges, the choriod plexus, and the ependyma lining the ventricles. The brain parenchyma is not extensively involved, but in regions surrounding the ventricles, some cell infiltration is seen. The cellular infiltrate is dominated by macrophages and CD8⫹ T cells (2, 12), whereas CD4⫹ T cells are present in very limited numbers unless the infection is allowed to become chronic (e.g., in T-cell low responders) (17). Similar infiltrations are found in other virus-infected organs at this time, but it is the CNS inflammation which sets the i.c. infection apart. The simplest way to monitor disease progression is to study the cellular infiltration of the CSF (25), and a detailed analysis has revealed that a significant infiltrate starts forming at around 4 to 5 days after virus inoculation; from then on, the number of infiltrating cells increases exponentially until the host dies at around 7 to 9 days postinfection (p.i.) (2). For many years, CSF cell numbers have therefore been used as a surrogate marker for disease severity. However, as mentioned above, parenchymal infiltration may actually be more important, as discussed later. When normal, immunocompetent mice are infected with LCMV, a very substantial T-cell response is initiated, and at the peak of the response, as much as 80 to 90% of the expanded splenic CD8⫹ T-cell population represents virus-specific CD8⫹ T cells (40, 55). These cells are functionally highly activated cells (41, 55, 73) with a surface phenotype that matches their propensity to migrate into sites of inflammation. Thus, primary LCMV-specific CD8⫹ T cells are predominantly low L-selectin , CD44high, VLA-4high, and LFA-1high (73); a subset of these cells also expresses the integrins CD11b and CD11c (14, 47). Additionally, tissue-infiltrating T cells, including those in the CSF, are enriched in cells with this phenotype (73), and expression of the corresponding ligands, VCAM-1 and ICAM-1, on the local endothelium of inflamed areas has been demonstrated (17, 53). These findings led to the assumption that integrins play a critical role in the recruitment of activated CD8⫹ T cells to areas of LCMV infection, and subsequent analyses using blocking antibodies and gene-targeted mice clearly support this model (5, 17, 18, 74). Therefore, since the affinity of integrins is closely regulated by chemokines, a number of recent studies have focused on the role of chemokines in LCMV-induced inflammation. CHEMOKINE SYSTEM Chemokines comprise a large group of small, structurally related, chemotactic cytokines that are involved in regulating the normal trafficking of leukocytes to both lymphoid and nonlymphoid organs and in recruiting leukocytes to sites of injury and infection (64, 80). In addition, chemokines play an important role in immune regulation; thus, chemokines have

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been reported to influence activation, costimulation, and differentiation of T lymphocytes and monocytes during innate and adaptive immune responses (11, 50). The ⬃50 human and murine chemokines are classified according to the motif of amino acids around the N-terminal cysteine(s), and they are currently subdivided into four families (CC, CXC, CX3C, and C) (80). From a functional point of view, chemokines have been divided into three groups: (i) constitutive chemokines, also called homeostatic or lymphoid chemokines, which fulfill housekeeping functions and are involved in normal leukocyte trafficking, (ii) inflammatory or inducible chemokines found at sites of inflammation, and (iii) dual-function chemokines that may be expressed constitutively but also become upregulated during inflammation. For the remainder of this review, the focus will be on groups ii and iii, i.e., chemokines with the potential to regulate the effector phase of the immune response. The biologic effects of chemokines are mediated via their interaction with a large group of seven-transmembrane-spanning, G-protein-coupled cell surface receptors. The two major families of chemokine receptors are the CXC chemokine receptors (CXCR) and the CC chemokine receptors (CCR), which bind CXC and CC chemokines, respectively (80). Many leukocytes express a distinct pattern of chemokine receptors, and it is important to note that the expression of many receptors varies with the differentiation state of the cell type in question (65). Thus, as T lymphocytes mature and undergo activation and differentiation, they develop new patterns of chemokine receptor expression, which are associated with changes in their route of migration. As a result, naive T cells are directed through the secondary lymphoid organs, guided by the constitutive chemokines CCL19 and CCL21 and their corresponding receptor CCR7. In contrast, effector T cells patrol the nonlymphoid organs and accumulate at sites of inflammation, guided by inflammatory chemokines and matching receptors. Moreover, type 1 (Th1, Tc1) and type 2 (Th2, Tc2) polarized T cells tend to express distinct sets of receptors favoring the preferential accumulation of these functionally different cell types at different sites of inflammation (65), e.g., predominantly Th1 and Tc1 cells at sites of virus-induced inflammation (13, 74), guided by receptors such as CXCR3 and CCR5. Notably, the chemokine network is generally characterized by a high degree of promiscuity and functional redundancy (52). Thus, one receptor typically binds more than one ligand, and one ligand typically binds to more than one receptor. Additionally, chemokines or receptors with parallel functions in vivo are typically expressed in concert. While obviously representing a nuisance to the investigator trying to work out how individual chemokines function in vivo, this pattern serves to underscore the biological importance of the chemokine network (52); the redundancy is likely to represent an evolutionary adaption making it much more difficult for any pathogen to bypass or block the system. This view is reinforced by the fact that several viruses encode gene products resembling chemokines or chemokine receptors, probably with the purpose of interfering with the proper functioning of this defense system (32, 59).

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BIOLOGICAL ROLE OF CHEMOKINES DURING VIRAL INFECTION When a mammalian host is infected, there are essentially two waves of chemokines being produced in the infected tissues (74). The first wave comprises part of the innate immune response, whose purpose is to attract dendritic cells and effector cells of the innate defense system, e.g., NK cells, to sites of initial virus replication. In this manner, the infection is contained while the adaptive immune system acquires the time required to make use of its more-refined antiviral machinery, e.g., antigen-specific cytolytic CD8⫹ T cells. The second wave starts when the first antigen-specific T cells enter the infection site, recognize viral antigen, and release their effector cytokines. Some of these may themselves have chemotactic activity (27, 63, 66): some chemokines are actually found in the cytolytic granules together with granzyme A (76); others, such as gamma interferon (IFN-␥), may act in a paracrine fashion to induce neighboring cells, be they resident or recently recruited cells, to produce additional chemokines. Together, this leads to an amplification of the local inflammatory response and the recruitment of more antiviral effector cells. During LCMV infection, essentially the same chemokines are produced during the first and second waves, albeit in different amounts (6, 61). A few days after virus inoculation, mRNA for CCL2 to -5 and CXCL9 to -11 is already detected in the virus-infected tissues, including the CNS (6, 48, 61) (initially, only CXCL10 was found, but subsequent analyses revealed an essentially similar kinetics of expression regarding CXCL9 and CXCL11); this range of chemokines more or less characterizes viral infection in any organ (48, 74). The initial level is maintained until about day 5 p.i., at which time T cells start to appear in the infected tissue, and clinical symptoms become noticeable. Around day 6 to 7 p.i., a very marked increase in expression of the chemokines is observed (6, 61), and some additional chemokines become clearly detectable, e.g., XCL1 and CXCL16. This burst in chemokine activity is associated with a further increase in the T-cell infiltrate, and typically, the i.c.-infected mice succumb to the infection on day 7 or 8 p.i. A similar analysis of chemokine receptor expression reveals local expression of CCR1, -2, and -5 as well as CXCR3 around the time when infiltrating T cells can be found in the tissues (48, 61). Analyses of sorted cells from the inflammatory exudate indicate that T cells express CCR2, CCR5, and CXCR3, whereas macrophages are the predominant cell type expressing CCR1 (16, 48, 61). OUTCOME OF i.c. INFECTION WITH LCMV IN CHEMOKINE OR CHEMOKINE RECEPTORDEFICIENT MICE To directly evaluate the role of these potential chemokine/ chemokine receptor interactions, a number of gene-targeted mouse strains have been evaluated with respect to critical parameters of LCMV disease. Neither CCL3- nor CCR5-deficient mice had any distinctive phenotype when the clinical outcome of i.c. infection was used as an end point (51, 60). CCR1- and CCR2-deficient mice also readily succumbed to LCMV-induced CNS disease, but a detailed analysis of the CSF infiltrate revealed a reduced frequency of CD11b⫹ mac-

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rophage phenotype cells (J. E. Christensen and A. R. Thomsen, unpublished observations), thus supporting older reports that macrophages are not essential in the pathogenesis of LCMV-induced CNS disease (2). In contrast, 50 to 60% of CXCR3-deficient mice survived i.c. infection despite similar numbers and localization of virus-infected target cells in the brain (16). Since the outcome of i.c. infection with LCMV is decided by the severity of the local CD8⫹ T-cell-mediated inflammatory reaction, the reduced mortality of these knockouts strongly suggested that interaction of CXCR3 with its ligands significantly influences the degree and/or localization of the CD8⫹ T-cell infiltration in the LCMV-infected CNS. ROLE OF CXCR3 IN LCMV-INDUCED CNS DISEASE CXCR3 is the receptor for CXCL9 to -11 (49, 77), all of which are IFN-regulated gene products and therefore are typically expressed in the context of viral infection, including i.c. infection with LCMV (6, 61). In humans, the expression of this receptor is consistently found on the majority of tissue-infiltrating T cells, including those in multiple sclerosis lesions (7, 29, 43, 49, 69), thus suggesting a key role of this receptor in regulating the accumulation of effector T cells in inflamed organs. In the context of LCMV infection, CXCR3 is expressed by the majority of CD8⫹ T cells with an activated (CD44high) phenotype (16), including T cells with known specificity for the virus as determined by staining with fluorescent major histocompatibility complex/peptide multimers (15). Furthermore, cells with this phenotype are enriched in the CSF from i.c.-infected mice. In vitro, in Transwell experiments evaluating cell migration, CD8⫹ T cells with an activated phenotype are preferentially recruited in response to recombinant CXCL10 in the lower chamber (48). Similar cells from CXCR3-deficient mice do not respond to CXCL10 but retain the capacity to respond to another chemokine, CCL3 (16). The above findings convincingly point to a critical role for CXCR3 in regulating the accumulation of effector T cells at sites of inflammation, but definitive evidence was lacking until the advent of CXCR3-deficient mice. However, in 2000, Hancock et al. (31) showed that allograft rejection was severely impaired in mice lacking CXCR3 expression, demonstrating for the first time the importance of this receptor in the development of T-cell-mediated inflammation. Using the same strain of mice, Christensen et al. subsequently demonstrated a reduced susceptibility to lethal infection with LCMV (16). Importantly, the finding of a reduced susceptibility to i.c. infection with LCMV does not in itself show that CXCR3 is involved in regulating the accumulation of virus-specific effector CD8⫹ T cells in the virus-infected CNS. However, adoptive transfer of wild-type (i.e., CXCR3⫹) CD8⫹ T cells could restore the susceptibility to i.c. disease in CXCR3-deficient mice (16), supporting the contention that receptor expression on the CD8⫹ T cells was the key element. Nonetheless, based on the fact that LCMV disease is dependent on an intact T-cell response, other functions of chemokines have to be considered. While they are defined as chemotactic, chemokines now very clearly have in vivo functions other than the regulation of chemotaxis. Thus, chemokines may be important during the afferent phase of the immune response, controlling successful T-cell/antigen-presenting cell interaction (11, 79), and some

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chemokines may also be directly involved in T-cell activation and polarization (28, 50, 78). It is pertinent, therefore, that a detailed analysis of effector T-cell development in LCMVinfected mice did not reveal any requirement for the expression of CXCR3 during the expansion and differentiation phases of the LCMV-specific CD8⫹ T-cell response (16). Moreover, normal development of a CSF infiltrate is observed for i.c.-infected CXCR3-deficient mice (16), demonstrating that effector T cells not only are being generated in CXCR3deficient mice but also seem capable of crossing the blood-CSF barrier. Similarly, primed effector T cells from wild-type and CXCR3-deficient mice were found to be equally efficient in reducing early viral replication in the CNS upon adoptive transfer to naive recipients at the time of i.c. challenge (Christensen and Thomsen, unpublished). At first sight, both of these observations were very puzzling, given that meningeal infiltration has classically been taken to reflect the severity of LCMVinduced CNS disease (2). However, detailed histological examinations revealed a new layer of complexity with regard to the pathogenesis of this disease. Thus, in wild-type mice, CD8⫹ T cells were found preferentially in the leptomeninges and choroid plexus, but also in some parenchymal regions, such the corpus callosum, internal capsule, and the white matter of the cerebellum (16). However, while the meningeal CD8⫹ T-cell infiltration was reproduced in CXCR3-deficient mice, the accumulation of these cells in parenchymal regions near the ventricular surfaces was significantly delayed (16). Therefore, it seemed that CXCR3 was redundant in regard to CD8⫹ T-cell extravasation and meningeal infiltration, but the accumulation of these cells in critical parenchymal regions was delayed in the absence of this receptor. The finding that adoptive transfer of effector cells from CXCR3-deficient mice was as efficient as that of wild-type cells in reducing early viral replication in the brain also makes perfect sense if one takes into account a difference in the requirements for localization of the effector T cells to the leptomeninges versus the neuroparenchyma. Early in the infection, most virus replication takes place in the meninges and choroid plexus. Therefore, it is the ability of the primed T cells to reach these sites that matters in terms of a reduced viral load, and not the ability to rapidly penetrate deeper into the neuroparenchyma. To summarize, the expression of CXCR3 is redundant during the afferent and central phases of the LCMV-specific T-cell response, and even CXCR3-deficient effector cells are capable of leaving the lymphoid organs and causing meningeal inflammation. However, CXCR3 is expressed on most virus-specific CD8⫹ T cells in LCMV-infected mice, and expression of this receptor plays a pivotal role in regulating the accumulation of these cells in critical regions of the neuroparenchyma, and this correlates with a reduced susceptibility of CXCR3-deficient mice to fatal LCMV-induced T-cell-mediated disease. ROLE OF CXCL10 IN LCMV-INDUCED CNS DISEASE There are three known ligands of CXCR3, CXCL9 to -11, all of which can be found in virus-infected tissues, including the LCMV-infected CNS. All follow the same kinetics of expression in i.c.-infected mice, and peak activity is detected at the same times and immediately before the mice succumb to the disease (15). Important questions, therefore, are if these che-

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mokines overlap in their biological functions in vivo or if one is more important than the others during LCMV-induced CNS disease. The answer to these questions comes from a recent study of LCMV infection in CXCL10-deficient mice, and it quite clear from this analysis that CXC10 is the key mediator (15). Thus, all the essential findings from CXCR3-deficient mice are exactly reproduced in mice deficient in CXCL10 (15): about 50 to 60% of i.c.-infected mice survive a virus dose that is invariably lethal to wild-type mice, there is no impairment of effector T-cell generation in CXCL10-deficient mice, and CSF infiltration is unimpaired, but the accumulation of CD8⫹ T cells in parenchymal regions such as the corpus callosum is reduced. Finally, the production of the alternative ligands is only slightly reduced (CXCL9) or increased (CXCL11). Therefore, CXCL10 appears to be a key mediator of severe LCMVinduced CNS disease attracting CXCR3⫹ CD8⫹ T cells to critical parenchymal regions. In contrast, at least CXCL11 seems to be redundant, as the increased level of CXCL11 did not seem to have any compensatory effect in vivo. This is quite interesting given that CXCL11 is the most potent ligand of CXCR3 in vitro (21). Evidently, chemokines with similar functions in vitro may differ markedly in their roles in vivo. How the production of CXCL10 is induced and regulated in the LCMV-infected CNS is not yet clearly established. However, ongoing studies in my laboratory support the concept of a biphasic response (J. E. Christensen, S. Simonsen, C. Fenger, M. R. Sorensen, T. Moos, J. P. Christensen, B. Finsen, and A. R. Thomsen, submitted for publication). Thus, despite very limited expression of type I IFNs in the brain early after LCMV infection, IFN-␣/␤ nevertheless appears to control initial CXCL10 production through a STAT1- and STAT2-dependent signaling pathway (Fig. 1). However, limited expression of CXCL10 is observed in the late phase of infection in T-cell- or IFN-␥-deficient mice. This is not because the T cells themselves are producing CXCL10. However, expression can be restored by transfer of wild-type CD8⫹ T cells into IFN-␥deficient but not IFN-␥ receptor (IFN-␥R)-deficient mice. Thus, these results indicate that once virus-specific CD8⫹ T cells have been generated and enter the infection site, local production of type II IFN by the antigen-specific T cells provides strong positive feedback, which brings about further, marked upregulation of CXCL10 expression in the CNS. In other words, the full-blown inflammatory reaction associated with fatal immunopathology is the expression of a bidirectional interaction between local cytokines/chemokines and the recruited CD8⫹ T cells. Hence, these findings serve to underscore the intimate interplay between innate and adaptive immunity in the antiviral inflammatory response. Interestingly, results from bone marrow chimeras reveal that the main CXCL10-producing cell types throughout this response are long-lived resident cells of the CNS. Consistent with this, an immunohistochemical analysis reveals that the predominant producers in both the early and late phases of infection are cells of the meninges and choroid plexus together with periventricular astrocytes. In contrast, bone marrow-derived cells (i.e., the inflammatory cells) do not directly contribute (Christensen et al., submitted). This is different from the case of CCL3, another chemokine produced in the LCMV-infected CNS. In this case, the T cells themselves release the chemokine (upon recognition of cognate antigen) and appear to be the

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FIG. 1. Schematic overview of the current working hypothesis regarding the events underlying LCMV-induced CNS inflammation and death following intracerebral infection. (A) Innate response. Initially, viral invasion is sensed by pattern-recognizing receptors (PRR) most likely expressed by resident cells of the CNS at their cell surface, in endosomal compartments or in the cytosol. This triggers the production and secretion of type I IFN, which seems to act in an autocrine and paracrine fashion to amplify its own production as well as the production and release of CXCL10. (B) Adaptive response. Once virus-specific CXCR3⫹ CD8⫹ T cells become available in the circulation, they are recruited to the sites of infection, and following recognition of virus-infected cells by the incoming antigen-specific T cells (mostly CD8⫹), type II IFN is produced and released. Rapidly, type II IFN takes over as the dominant regulator of CXCL10 production. As a result, the production of CXCL10 is markedly increased, which further augments the recruitment of CXCR3⫹ T cells. Together, this reciprocal interaction spirals into the full-blown virus-induced inflammatory response and, ultimately, the death of the host animal. STAT1/2, STAT1 and STAT2.

main producers in the CNS during the T-cell-dependent phase of the host immune response to LCMV (51). Therefore, perhaps not surprisingly, it can be concluded that chemokines present at the same time in a site of viral inflammation may reflect a mixture of cellular sources working in concert to provide the optimal inflammatory environment.

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It is still not clear at the molecular level how LCMV infection of the CNS is translated into a local type I IFN response. Cells sense and respond to RNA virus infection through innate nucleic acid sensors, including Toll-like receptors (TLRs) (8). Only five TLRs, TLR3, TLR4, TLR 7, TLR8, and TLR9, are known to induce production of type I IFN as a consequence of their ligation (1), and preliminary results indicate that neither MyD88 (an adaptor molecule for most TLRs) nor TLR3 (which does not use MyD88 as an adaptor) are of critical importance (J. E. Christensen, S. Simonsen, C. Fenger, M. R. Sorensen, T. Moos, J. P. Christensen, B. Finsen, and A. R. Thomsen, unpublished data). This could suggest that TLRs are not pivotal for the detection of LCMV infection in cells of the CNS and that the critical sensor system may instead have to be found at the cytoplasmic level. A putative cytosolic sensor might be the IFN-inducible RNA helicase RIG-I, which is activated by the presence of triphosphates at the 5⬘ end of single- and double-stranded RNA (33, 62). Alternatively, the cytosolic sensor could be another RNA helicase, MDA-5, the agonist of which is still not firmly identified; these two sensors signal through a common and recently identified downstream adaptor, IPS-1 (37, 42). Finally, the possibility exists that functional redundancy within the involved sensor systems may blur the picture and impede unequivocal identification of the participating receptors. Matching the uncertainty at the molecular level, the cells involved in sensing LCMV infection of the CNS and producing type I IFN also have not yet been identified. Indeed, it is not clear whether these are predominantly long-lived resident cells or cells of peripheral origin. Importantly, because initial LCMV replication takes place predominantly in the meninges, it should be considered that cell types not normally found in the CNS proper, e.g., plasmacytoid dendritic cells (56), might also participate in the early IFN response; however, the apparent dominance of IFN-␤ (Christensen et al., unpublished) suggests that resident cells are the predominant producers. POTENTIAL ROLE OF OTHER CHEMOKINES While the above findings point to a critical role for CXCL10/ CXCR3 in regulating the accumulation of CD8⫹ T cells at sites with LCMV-induced CNS inflammation, it is also quite evident that other chemotactic mediators must play a role. Thus, meningeal inflammation is unimpaired in CXCL10- and CXCR3deficient mice, and parenchymal infiltration is delayed, but not absent, in these mice. Similarly, 30 to 50% of i.c.-infected mice die despite a deficiency in either CXCL10 or CXCR3. The obvious question is therefore whether this reflects biological redundancy of the chemokines already mentioned or whether there are additional critical mediators. To answer the first part of the above question, I and members of my laboratory have started to generate mice with multiple chemokine system defects. At the time of writing, only one strain of double-deficient mice, CCR5/CXCR3-deficient mice, has been thoroughly evaluated by using the LCMV model. As mentioned above, we have previously shown that lack of CCR5 does not in itself impair the LCMV-induced inflammatory process (60). However, since CCR5 is expressed mostly by a subpopulation of CXCR3⫹ T cells (22, 29, 68), and recent results suggest that ligands of CCR5 and CXCR3 work

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synergistically in attracting T cells to the virus-infected CNS (30), we considered it possible that CCR5 would be redundant only when CXCR3 is normally expressed. If this assumption were correct, concurrent inhibition of both chemokine receptors would lead to a severely impaired inflammatory response. However, contrary to expectations, we found that the susceptibility of CCR5/CXCR3-deficient mice with regard to a lethal outcome of i.c. infection with LCMV was in between that of single CXCR3-deficient (low-mortality) and CCR5-deficient (high-mortality) mice (22). Furthermore, while the accumulation of CD8⫹ T cells in the neural parenchyma was significantly delayed in both CXCR3- and CCR5/CXCR3-deficient mice, more CD8⫹ T cells were found in the parenchymas of double-deficient mice when they were analyzed around the time when the difference in clinical outcome manifests itself (22). Based on these results, it was evident that the working hypothesis was wrong, but how could this new pattern be explained? Analyzing the generation of cytokine producing, virus-specific effector CD8⫹ T cells, I and members of my laboratory found that the immune response was augmented in CCR5-deficient mice. Moreover, when the capacity of the individual effector CD8⫹ T cell to produce IFN-␥ was evaluated, it was noted that, at least in vitro, CD8⫹ T cells from CCR5deficient mice continued to produce cytokine independently of known stimulation for a much longer time than did matched cells from wild-type mice (22). Taken together, these findings indicate that the redundancy of CCR5 does not reflect a functional overlap with CXCR3. Moreover, the increased susceptibility of double-deficient mice serves to underscore that CXCR3 is only one of the factors involved in regulating local T-cell accumulation and that an augmented immune response may reduce the rate-limiting effect of CXCR3 deficiency. Notably, we also found that meningeal inflammation was only marginally reduced in double-deficient mice, and so from these results, it is evident that further studies are needed to identify additional critical receptor/ligand pairs important during T-cell extravasation and localization within the LCMV-infected CNS. IMPLICATIONS FOR OUR UNDERSTANDING OF THE PATHOGENESIS OF LCMV-INDUCED CNS DISEASE Although the main focus of this review has been to provide an overview regarding the role of chemokines in CNS targeting and localization of effector T cells, the results presented here also have clear implications with respect to our insight into the molecular pathogenesis of LCMV-induced meningoencephalitis in the i.c.-infected host animal. Thus, as mentioned earlier, the now-classical view has been that CD8⫹ T-cell-mediated killing of virus-infected cells in the meninges represents the key element in causing a lethal outcome. However, the finding that mice with defects in the CXCL10/CXCR3 pathway are significantly more resistant to LCMV-induced CNS disease than are wild types despite virtually identical meningeal infiltration casts doubt on this simple model and points to the neural parenchyma as playing a much more important role than previously thought. Also, with regard to the final molecular effector molecule(s) involved in disease induction, new studies question the classical dogma. Originally, the absence of mortality in perforin-deficient mice infected i.c. with a viscero-

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tropic virus was taken to indicate that perforin was directly involved in the CNS pathogenesis (36). However, great care should be exerted in interpreting this experimental setup, as the virus spread is extremely poorly controlled by perforindeficient effector CD8⫹ T cells. This creates a situation where there is much more virus in the viscera of perforin-deficient mice than in those of wild-type mice, and a nonlethal outcome of i.c. infection in the former mice could therefore simply reflect the sequestering of the majority of virus-specific effector T cells in the heavily infected visceral organs. To circumvent this argument, I and coworkers recently repeated the experiment using neurotropic LCMV to minimize the possibility of visceral sequestering in the perforin-deficient mice. In this situation, we did see a delay in the mortality of perforindeficient mice, but eventually all i.c.-infected mice succumbed with symptoms of LCMV-related CNS disease (72). Thus, while the presence of perforin clearly accelerates the progression of disease, it is not absolutely pivotal, as was previously thought. A remaining question is therefore which effector molecule(s) besides perforin may be involved in mediating this disease. Unfortunately, experiments with perforin/type II IFN double-deficient mice proved to be inconclusive as even i.v.inoculated mice succumbed from the LCMV infection (72), probably reflecting overwhelming immunopathology in mice that were severely impaired in their ability to control the systemic infection (since depletion of CD8⫹ T cells saves the mice). Therefore, all that can be concluded with certainty at the time of writing is that several T-cell-dependent molecular pathways may initiate the relevant cellular damage in i.c.-infected mice and that perforin is an important, but not the only, mediator in this context. CHEMOKINE REGULATION IN A WIDER PERSPECTIVE From the above study and similar studies involving other neurotropic viruses, it is evident that viral infection of the CNS is associated with marked and coordinated expression of a range of inflammatory chemokines with the capacity to interact with cellular receptors expressed on relevant antiviral effector cells, such as monocytes, NK cells, and Th1 and Tc1 cells. Thus, far from being exempt from immune surveillance, the virus-infected CNS is monitored actively and efficiently, a fact not entirely surprising given the fatal consequences that destructive infection in this organ might have. Although it is not always clear precisely how the presence of virus in the CNS is detected, it is pertinent that resident cells of the CNS seem to have the capacity to sense viral infection and respond with production of the central mediators of innate inflammation, such as type I IFN (23) and inflammatory chemokines (38; Christensen et al., unpublished). This is crucial because current data support the contention that the appropriate induction of innate inflammation is pivotal to the optimal recruitment of effector T cells and to subsequent virus clearance in addition to its importance as an essential early antiviral defense (45). One key chemokine/receptor pair identified in several studies of viral meningoencephalitis is CXCL10/CXCR3. The ligand is prominently expressed during the early phase of most viral infections, and its production is commonly further upregulated during the adaptive immune response. Moreover,

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CXCR3 is found to be expressed on virus-specific CD8⫹ T cells in several model systems (15, 71). This interaction plays a pivotal role in regulating the local CD8⫹ T-cell response inside the CNS not only in LCMV-infected mice but also following infection with West Nile virus (38) and Dengue virus (35). Also, in humans, CXCL10 represents a major chemokine found in the CSF of patients with viral meningoencephalitis (39, 44). Therefore, although CXCL10/CXCR3 interaction may not always be essential for CD8⫹ T-cell recruitment following viral infection of the CNS (71), I believe that there is now sufficient evidence to recommend great caution in targeting this receptor/ligand system as part of a therapeutic strategy to prevent or suppress unwanted inflammation, e.g., in the context of autoimmune disease or allograft rejection. Blocking this interaction may interfere with essential components of the normal antiviral host response, and in the worst case, fatal disease may be the result. ACKNOWLEDGMENTS I acknowledge the expert contributions made by many past and present members of the laboratory, in particular A. Nansen, Carina de Lemos, Stine Simonsen, J. Erbo Christensen, and Jan Pravsgaard Christensen. I am also grateful to the collaborators (T. Moos, C. Fenger, B. Finsen, T. Owens, A. Luster, B. Lu, C. Gerard, and R. Zinkernagel) who have provided technological expertise and genedeficient mice essential to the project. The financial support of the Danish Medical Research council, the Novo-Nordisk Foundation, the Lundbeck Foundation, and the Leo Pharma Research Foundation is acknowledged. REFERENCES 1. Akira, S., and K. Takeda. 2004. Toll-like receptor signalling. Nat. Rev. Immunol. 4:499–511. 2. Allan, J. E., J. E. Dixon, and P. C. Doherty. 1987. Nature of the inflammatory process in the central nervous system of mice infected with lymphocytic choriomeningitis virus. Curr. Top. Microbiol. Immunol. 134:131–143. 3. Alon, R., P. D. Kassner, M. W. Carr, E. B. Finger, M. E. Hemler, and T. A. Springer. 1995. The integrin VLA-4 supports tethering and rolling in flow on VCAM-1. J. Cell Biol. 128:1243–1253. 4. Andersen, I. H., O. Marker, and A. R. Thomsen. 1991. Breakdown of bloodbrain barrier function in the murine lymphocytic choriomeningitis virus infection mediated by virus-specific CD8⫹ T cells. J. Neuroimmunol. 31:155– 163. 5. Andersson, E. C., J. P. Christensen, A. Scheynius, O. Marker, and A. R. Thomsen. 1995. Lymphocytic choriomeningitis virus infection is associated with long-standing perturbation of LFA-1 expression on CD8⫹ T cells. Scand. J. Immunol. 42:110–118. 6. Asensio, V. C., and I. L. Campbell. 1997. Chemokine gene expression in the brains of mice with lymphocytic choriomeningitis. J. Virol. 71:7832–7840. 7. Balashov, K. E., J. B. Rottman, H. L. Weiner, and W. W. Hancock. 1999. CCR5⫹ and CXCR3⫹ T cells are increased in multiple sclerosis and their ligands MIP-1alpha and IP-10 are expressed in demyelinating brain lesions. Proc. Natl. Acad. Sci. USA 96:6873–6878. 8. Barton, G. M., and R. Medzhitov. 2003. Linking Toll-like receptors to IFNalpha/beta expression. Nat. Immunol. 4:432–433. 9. Berlin, C., R. F. Bargatze, J. J. Campbell, U. H. von Andrian, M. C. Szabo, S. R. Hasslen, R. D. Nelson, E. L. Berg, S. L. Erlandsen, and E. C. Butcher. 1995. ␣4 integrins mediate lymphocyte attachment and rolling under physiologic flow. Cell 80:413–422. 10. Butcher, E. C. 1991. Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity. Cell 67:1033–1036. 11. Castellino, F., A. Y. Huang, G. Altan-Bonnet, S. Stoll, C. Scheinecker, and R. N. Germain. 2006. Chemokines enhance immunity by guiding naive CD8⫹ T cells to sites of CD4⫹ T cell-dendritic cell interaction. Nature 440:890–895. 12. Ceredig, R., J. E. Allan, Z. Tabi, F. Lynch, and P. C. Doherty. 1987. Phenotypic analysis of the inflammatory exudate in murine lymphocytic choriomeningitis. J. Exp. Med. 165:1539–1551. 13. Cerwenka, A., T. M. Morgan, A. G. Harmsen, and R. W. Dutton. 1999. Migration kinetics and final destination of type 1 and type 2 CD8 effector cells predict protection against pulmonary virus infection. J. Exp. Med. 189:423–434. 14. Christensen, J. E., S. O. Andreasen, J. P. Christensen, and A. R. Thomsen. 2001. CD11b expression as a marker to distinguish between recently activated effector CD8⫹ T cells and memory cells. Int. Immunol. 13:593–600.

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