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Regulation of T cell migration during viral infection: role of adhesion molecules and chemokines
Immunology Letters 85 (2003) 119 /127 www.elsevier.com/locate/
Review
Regulation of T cell migration during viral infection: role of adhesion molecules and chemokines Allan Randrup Thomsen , Anneline Nansen, Andreas Nygaard Madsen, Christina Bartholdy, Jan Pravsgaard Christensen Institute of Medical Microbiology and Immunology, University of Copenhagen, Copenhagen, Denmark
Abstract T cell mediated immunity and in particular CD8/ T cells are pivotal for the control of most viral infections. T cells exclusively exert their antiviral effect through close cellular interaction with relevant virus-infected target cells in vivo. It is therefore imperative that efficient mechanisms exist, which will rapidly direct newly generated effector T cells to sites of viral replication. In the present report we have reviewed our present knowledge concerning the molecular interactions, which are important in targeting of effector CD8/ T cells to sites of viral infection. # 2002 Elsevier Science B.V. All rights reserved. Keywords: T cells; Antiviral immunity; Virus-induced inflammation; Adhesion molecules; Chemokines
1. Introduction When a parasite enters a vertebrate host it is normally met by two different defence systems. The first line of defence (innate immunity) comprises a number of nonspecific effector systems that work with little delay to limit the replication of the infecting agent. A characteristic feature of the early effector systems of innate immunity is that recognition of non-self is based on general motifs (e.g. LPS and dsRNA) associated with large groups of parasites [1] and thus does not involve the highly specific receptor-mediated recognition characteristic of the adaptive immune response. Basically, this is what allows the very short reaction time of innate defences. In contrast, highly refined and specific recognition as found for T and B cells, necessitates a clonal distribution of the involved receptors, and therefore a delay in action time due to the need for clonal expansion (and differentiation). Since this delay clearly puts a major constraint on the defensive value of adaptive immunity in relation to very rapidly replicating para Corresponding author. Address: Institute of Medical Microbiology and Immunology, Panum Institute, University of Copenhagen, 3C Blegdamsvej, DK-2200 Copenhagen N, Denmark. Tel.: /45-35-32-78-71; fax: /45-35-32-78-91. E-mail address: [email protected] (A.R. Thomsen).
sites, it is imperative that the potential of the adaptive response is exploited to its fullest. This requires at least two things: (a) effective mechanisms for presentation of antigen to as many lymphocytes as possible; this is needed to ensure that most of the (limited number of) cells with appropriate receptors are actually brought into action, and (b) once initial antigen-driven triggering and expansion has taken place, there also has to be efficient mechanisms for rapidly focussing the generated effector cells at and around sites of microbial replication. Bearing this in mind, much about the design of lymphocyte circulation becomes easier to understand. First, it is obvious that lymphocytes have to be able to migrate in a highly regulated fashion, and secondly, that the migration patterns required must be different before and after initial antigen-specific encounter [2,3]. Initially the naive lymphocyte has to recognise the antigen under conditions, which allow for an efficient response to invading non-self. This occurs optimally in the secondary lymphoid organs [4] e.g. the spleen and regional lymph nodes. Antigen is also transported to these organs, typically by way of dendritic cells, which mature in the process [5]. Thus, the secondary lymphoid organs represent critical intersections between the migrational pathways of antigen-presenting cells and naive lymphocytes. Because antigen-presentation does not involve cells with clonally distributed receptors, these are not
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limiting, and the above ‘design’ therefore allows for rapid and efficient presentation of parasitic intruders, while at the same time ensuring that any part of the body becomes frequently, albeit indirectly, monitored by the entire pool of naive lymphocytes. However, while stringent regulation of antigen-induced triggering of naive lymphocytes is essential, it is just as important that any host cells can be targeted by at least one type of effector cell since otherwise there would be potential hiding places for smart pathogens. Consequently, once clonal expansion has taken place, the generated effector and memory cells must be able to monitor for antigen in an anatomically unrestricted fashion. Furthermore, to guarantee rapid focussing of the adaptive response, one result of antigen encounter should be the release of mediators leading to the recruitment of more effector cells to the site. The interface between circulating lymphocytes and vascular endothelium together with extravascular chemokine gradients probably constitutes the most important gatekeeper in regulating lymphocyte migration, and extravasation in itself involves a complex set of interactions between adhesion molecules and their ligands and between chemokines and their receptors [3,6 /8]. In this brief review we will try to present an overview of the mechanisms involved in regulating CD8/ T cell migration as this is required for an optimal adaptive immune response to a viral infection. The focus is on our own studies using functional parameters: virus control and immunopathology, and represents our present views, based in particular on analysis of gene-targeted mice.
2. The extravasation paradigm All leukocytes are believed to extravasate through a series of steps (for a graphic representation see Fig. 1) that are considered to be essentially similar, independent of the type of leukocyte involved [6]. First, the leukocyte must adhere to the endothelium of blood vessels in the relevant organ site. The initial, reversible interaction is normally mediated by the interaction of selectin receptors with glycoconjugate ligands. The selectin family comprises three members which falls into two functionally distinct groups [9]: (1) L-selectin, which is expressed by many leukocytes including naive T cells, but not recently primed T cells, and (2) E- and P-selectins both of which are expressed by activated endothelium. The selectin-glycoprotein interaction causes the leukocyte in the post-capillary venules to roll along the inner endothelial surface and thus creates the basis for further adhesive interactions: firm adhesion and transmigration. These latter events require leukocyte integrins and endothelial expression of relevant Ig superfamily ligands. However, for the leukocyte to establish firm binding, chemokine-mediated triggering is assumed to
Fig. 1. The extravasation paradigm. Following free flow in the larger vessels, extravasation of leukocytes occurs through a stepwise interaction with molecules expressed on the inner vascular surface of the postcapillary venules. Initially, the leukocyte tethers to the endothelial surface through selectin/glycoconjugate interactions. This binding is not sufficiently strong to stop cell movement, but the leukocyte rolls slowly along the inner surface of the venule, continually making and breaking contact. However, the slowing down of leukocyte movement allows for interactions between leukocyte integrins (e.g. LFA-1 and VLA-4) and vascular ligands belonging to the superimmunoglobulin family (CAMs, e.g. ICAM-1 and VCAM-1). This results in tight binding provided that the leukocyte is activated by chemokines. These can be presented to the leucocyte bound to heparin-like glycosaminoglycans on the surface of endothelial cells close to the area of microbial challenge. The tight binding totally stops cell movement, and leads the leukocyte to squeeze out between adjacent endothelial cells (diapedesis). Finally, the cells migrate along gradients of chemoattractants, e.g. chemokines, towards the site of maximal microbial activity.
be critical [10]. The chemokine-induced activation leads to a conformational change in the integrins that increases their affinity for their appropriate ligand very substantially, and this seems to be a precondition for firm adhesion and subsequent transmigration. The final positioning of the extravasated leukocyte in the extracellular matrix is probably regulated by gradients of chemoattractants, e.g. chemokines, in conjunction with integrin-based adhesive interactions with common matrix proteins, e.g. collagen. While this is the general picture for leukocyte migration in any tissue, surprisingly few studies addressing the molecular requirements for effector T lymphocyte migration in vivo have been carried out */at least in the context of relevant biological challenges such as viral infections, against which this lymphocyte subset is known to play a major role in controlling infection. Our group has been studying antiviral immunity for many years, in particular the role of cell-mediated immunity and cytotoxic (CD8/) T cells. Since these cells require close contact with their infected targets in order to exert any antiviral function be it killing or cytokine release, T cell migration is clearly a central issue in viral immunology. It was therefore obvious for us to ask which mechanisms
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regulate effector T cell migration into sites of viral infection.
3. Activation-induced changes in T-cell phenotype Naive T cells gain access to the lymph nodes through specialized high endothelial venules (HEV). Expression of L-selectin on the lymphocyte is pivotal for binding to peripheral-node addressin and initial contact to HEV [11,12]. In addition, successful extravasation at this stage involves T cell activation with a chemokine named secondary lymphoid-tissue chemokine [13]; this chemokine binds to a receptor named CCR7 [14,15]. Thus naive T cells are characterized by expression of Lselectin and CCR7. These molecules are superfluous for migration to inflammatory sites and are down-regulated upon activation of the T cell [16,17]. In contrast integrins like VLA-4 and LFA-1 are upregulated when the T cell is properly activated [17,18]. Thus, in the case of acute systemic viral infection, close to 90% of all CD8/ T cells present at the time of the primary immune response may be L-selectinlow, VLA-4high and LFA-1high, and all virus-specific CD8/ T cells are contained within this subset [19 /21]. The expression of chemokine receptors also changes markedly, and receptors responsive to inflammatory chemokines are now expressed (see below). Together these changes (Fig. 2) in the expression of surface molecules constitute the molecular foundation for the profound change in lymphocyte circulation that is essential for effective targeting of activated T cells to sites of microbial challenge.
4. Role of selectins To study the relevance of selectins for CD8/ T cell migration in the context of viral infection, gene-targeted mice lacking one or more selectins [22 /24] were infected with a non-cytolytic virus: lymphocytic choriomeningitis virus (LCMV). Since this virus causes little or no cellular damage on its own, both virus-induced pathology and virus control reflect the efficiency of the immune response in situ [21]. Furthermore, a great number of
Fig. 2. Phenotypic changes affecting cell migration induced as result of T-cell activation and differentiation towards type 1 (IL-2 and IFN-g producing) effector cells.
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studies have clearly established that CD8/ effector T cells completely dominate the initial immune response both as mediators of virus control and immunopathology. Therefore this model system is ideally suited to study effector CD8/ T cell migration to infected organs, since both the inflammatory response and virus clearance constitute very relevant functional read-outs for appropriate effector cell migration [25]. Using E- or P-selectin knock-out mice as well as E/P-selectin double knock-outs, we found little role for endothelial expression of selectins [26]. Effector CD8/ T cell responses were effectively generated in these mice, and at best a slight delay in effector cell migration was observed when virus-primed wild-type donor T cells were adoptively transferred into these knock-outs. Since the effector CD8/ T cells express little L-selectin [19,20], these results suggested that selectin-based rolling was not pivotal for migration of CD8/ T cells into a virusinfected area. However, a comparison of the level of Lselectin expression on effector cells from wildtype and Lselectin knock-out mice (Christensen et al., in preparation) suggested that residual L-selectin expression could play a role. To test this experimentally, adoptive transfer analysis was conducted giving E/P-selectin knock-out mice virus-primed effector T cells from L-selectin deficient as well as wildtype donors. However, even under these conditions virus-induced T-cell mediated inflammation was induced with little delay compared to wild-type recipients given wild-type effector cells, and this delay mostly reflected the slightly reduced responsiveness of the E/P-selectin recipients in general. Therefore, we conclude that selectin-based rolling is not essential for effector CD8/ T cell migration to foci of virus-infection, and that other molecules apparently suffice for the initial tethering of CD8/ effector T lymphocytes in the post-capillary venules of inflamed areas. This conclusion is supported by a recent study analysing the role of adhesion molecules in CD8/ T cell rolling in cytokine activated venules [27]. Here it was clearly demonstrated that VLA-4, but not selectins were responsible for rolling of CD8/ T cells in M. cremaster venules activated with TNF-a and IFN-g. It is not clear why VLA-4 should suffice for rolling of activated CD8/ T cells. One reason might be the high expression of this integrin on activated CD8/ T cells as compared to the level of expression on neutrophils. This does not, however, explain why CD4/ T cells appear to be more dependent on endothelial expression of selectins than CD8/ T cells [28], since at least for virus-specific CD4/ and CD8/ T cells, levels of VLA-4 expression are similar [19,29]. The above findings should not, however, be taken to indicate that selectins are completely superfluous in the context of antiviral T cell mediated immunity. L-selectin knock-out mice are more susceptible to influenza virus infection than matched wildtypes (unpublished observa-
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tion), although this does not prove that L-selectin expression on the T cells plays a role. More importantly, if L-selectin knock-out mice are infected with LCMV by a peripheral route (via the footpad), T-cell priming is significantly delayed (Christensen et al., in preparation). This does not reflect a general decrease in the ability of these mice to respond to this virus as systemic infection (i.v. inoculation) primes L-selectin knock-out mice and wild-type controls with equal efficiency. Therefore, even with a rapidly disseminating virus like LCMV, initial priming in the regional lymph nodes plays a significant role, although efficient priming will eventually take place in the spleen. The latter priming may in part reflect dissemination of live virus. However, migration of antigen-presenting cells to the spleen is also likely to contribute since dermal application (using a gene-gun) of a non-replicating DNA vector also leads to T-cell priming in L-selectin knock-out mice */albeit with somewhat reduced efficiency (Christensen et al., in preparation). Interestingly, it has recently been claimed that Lselectin is important for lymphocyte migration into the skin. Thus, in contrast to E- and P-selectin double knock-out mice, fucosyltransferease VII-deficient mice with defective E-, P-, and L-selectin ligands were found to have impaired primary LCMV-induced footpad swelling, while no deficit of T cell migration to virusinfected visceral organs (of systemically-infected mice!) was observed [30]. This led the authors of the latter report to suggest a role for L-selectin in skin homing of CD8/ T cells. However, in adoptive transfer experiments we find that L-selectin negative effector cells readily migrate to virus-infected skin (Christensen et al., in preparation). The most likely explanation for the impaired footpad swelling in fucosyltransferease VIIdeficient mice is therefore that peripheral infection, which is needed for induction of a primary LCMVinduced footpad swelling reaction, leads to impaired priming in mice lacking relevant ligands for L-selectin similar to that observed in L-selectin knock-out mice. While effector T cells express little L-selectin, memory cells are heterogenous in this respect [31 /34], and analysis of splenic (unselected) virus-specific CD8/ T cells have shown that re-expression of this molecule increases with time after virus inoculation [32] (Christensen et al., in preparation). Furthermore, parallel analysis reveals that in the early phase after control of a systemic viral infection, antigen-specific CD8/ T cells are to be found almost exclusively in the spleen, whereas few can be found in peripheral lymph nodes. However, with time, redistribution to the peripheral lymph nodes gradually takes place, and this coincides with an increase in the fraction of L-selectin expressing cells (Christensen et al., in preparation). Thus L-selectin regulates not only the migration of naive T cells, but also seems to be involved in controlling the distribution of primed
CD8/ T cells between different compartments of the secondary lymphoid organs.
5. Critical role of integrins Whereas selectins play little role in targeting effector CD8/ T cells to sites of viral infection, integrins are very important. This has been shown primarily through blocking of various integrins in their ability to interact with their appropriate ligands. In intact virus-infected animals, treatment with non-depleting anti-VLA-4 antibody significantly delays virus-induced T cell mediated inflammation, indicating that one or more of the involved cell types utilizes this integrin during elicitation of inflammation (unpublished observation). Directly demonstrating a role for integrins in the migration of CD8/ effector T cell to sites of viral replication, we have found that pre-incubation of effector CD8/ T cells with anti-Mac-1, anti-LFA-1 or anti-VLA-4 all significantly inhibits the ability of these cells to adoptively induce virus-specific T-cell mediated inflammation [20,35,36] Notably, this is the case only when the effector T cells are required to actively migrate to the virus-infected site. Thus, when effector cells pre-incubated with antibody are injected directly into the test site, no inhibition is observed. Complementing the antibody based analysis, a reduced inflammatory response is also found in ICAM-1 deficient recipients [37], and the residual inflammatory response in these mice is completely eliminated [26] by inhibition of VCAM-1 binding using soluble molecules (sVCAM-1) [38]. In order to highlight the functional impact of integrins versus selectins on the migration of CD8/ effector T cells to sites of viral replication, we conducted a set of parallel adoptive transfer experiments [26], each involving the inhibition of two sets of molecular interactions (see Fig. 3a /c): either (a) binding to Eand P-selectin was prevented; alternatively, (b) one selectin (P-selectin) and one integrin (ICAM-1) was eliminated, and as the third possibility (c) binding to both major ligands of integrins, ICAM-1 and VCAM-1, was prevented. It is evident from the results obtained that while failure to interact with endothelial selectins is of little functional relevance, integrins are pivotal for a virus-induced T cell mediated inflammatory response.
6. The role of chemokines and chemokine receptors Whereas selectins are constitutively active, integrins need to be activated to mediate efficient binding. The important role played by integrins therefore drew our attention to another important set of molecular players involved in regulating leukocyte migration, namely the chemokines. Chemokines are low m.w. chemotactic
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Fig. 3. Comparison of the role of selectins and integrins in virus-induced T-cell mediated inflammation. Groups of gene-targeted and matched wildtype mice were inoculated with 105 ID50 in their right hind footpad. Four hours later these recipients were injected intravenously (i.v.) with 50/ 106 adherent cell depleted, mitomycin C treated (to prevent donor cell proliferation subsequent to cell transfer) splenocytes from wildtype donors infected 8 days earlier with 103 ID50 i.v. Groups consisted of 4 /6 mice; medians and ranges are depicted. () denotes P B/0.05 relative to wildtype recipients (Mann /Whitney rank sum test) (taken from Ref. [26]).
cytokines that bind to specific 7-transmembrane G protein-coupled cell surface receptors [39,40]. While some chemokines appear to play a major role in regulating adhesive interactions with the vascular endothelium [10], others may control subsequent migration and positioning within the extravascular space [3,8]. Spatiotemporal patterns of chemokine and chemokine receptor expression are therefore supposed to critically influence immune responses [3,7,8]. Chemokines are classified into four families based on the configuration of cysteine residues near the Nterminus [39 /41]. In CXC chemokines two cysteines are separated by another amino acid (X) while CC chemokines have two adjacent cysteines. Both of these families comprise several members, while the two last families, named CX3C and XC receptors, have only one member each. The nomenclature of the corresponding receptors follows that of their ligand with the addition of R and a number, e.g. CXCR3 [40,41]. Generally, the chemokine network is characterized by a high degree of functional redundancy, which hampers the analysis of functional relevance in vivo. Thus, one chemokine may often bind to several members of the same receptor family and, similarly, one receptor may bind several related chemokines (for relevant examples see Fig. 6) [40]. While constituting a nuisance to the researcher, this phenomenon is biologically very understandable because it makes the chemokine system much more robust to interference by pathogens. From a functional point of view, chemokines are divided into (a) constitutive (alternatively called homeostatic or lymphoid) and (b) inflammatory (or inducible) [8,39]. Constitutive chemokines are involved in regulating normal lymphocyte migration and positioning within the lymphoid tissues, while the inflammatory
chemokines are critical for attracting a diverse set of effector cells to inflammatory sites. Matching this functional division of labour between different sets of chemokines, chemokine receptor expression changes markedly with the state of lymphocyte differentiation [42,3,39] (Fig. 4). For peripheral T cells this is very striking. While naive T cells express CCR7, which is essential for lymph node homing, effector T cells express receptors for inflammatory chemokines. Even more important, different effector subsets appear to express different receptors. Thus, type 2 CD4/ effector T cells (Th2 cells) primarily express CCR3 and CCR4, whereas the phenotype of Th1 cells involves high expression of CCR5 and CXCR3 [3,39,42]. CD8/ effector T cells have not been as extensively characterized with regard to chemokine receptor expression, but they seem to follow the same pattern as CD4/ effector T cells i.e., receptor expression correlates with the cytokine profile
Fig. 4. Chemokine receptor expression as a function of T-cell differentiation. Based on the existing literature and our own studies we have tried to make a consensus picture of chemokine receptors expressed by type 1 and type 2 effector T cells regardless of their coreceptor phenotype (CD4 or CD8). Given that there are some inconsistencies in the literature, and most studies have involved in vitro generated lines/clones, the picture is not yet complete and may be subject to change with more information from direct ex vivo analysis.
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[43]. Thus, it would be predicted that it is the expression of chemokine receptors induced during T cell activation together with locally produced chemokines, which in the end determines the composition of the inflammatory exudate. When viruses infect a mammalian host essentially two waves of chemokines are produced at the site of viral invasion [44,45]. The first wave comprises part of the innate response to a viral infection. The precise signals which elicits this response are not clear, but virusinduced perturbation of normal cell metabolism may be one way in which this response is elicited. However, a significant chemokine response may be seen in the absence of outright cell damage e.g. after LCMV infection [44,45]. In addition, at least some chemokines are partially regulated by other signal molecules generated in the context of the innate response to viral invasion, e.g. IFN-a/b [45]. The second wave comprises chemokines produced as a result of a local cell-mediated immune response [44,45]. Some of these chemokines are synthesized by the incoming T cells themselves (e.g. MIP-1a [46]), while others are being produced by a variety of cell types (in particular macrophages, e.g. MCP-1) under the influence of T-cell dependent cytokines such as IFN-g [10,44,47]. Together these two waves of chemokine production allow for (1) rapid focussing of early effector cells to the site of viral invasion and (2) effective enhancement of the response once viral antigen is detected by specific cell types. To define the chemokines likely to play a role in viral infections, we have studied the chemokines produced in a number of organs (liver, lungs and CNS) infected by different viruses (LCMV, influenza virus and vesicular stomatitis virus) differing in their biological properties and cell tropism. A representative result of this survey is seen in Fig. 5, and another example can be found in Ref. [45]. Although differences may be observed, particularly in the relative magnitude of production of various chemokines, the overall impression is how uniform the virus-induced chemokine profile actually is: RANTES, MIP-1a and b, IP-10 and MCP-1 appear to dominate independent of virus, organ site or phase of infection (at present we have only concentrated on acute infections which of course means that the picture may be different in the context of chronic viral infections). In a set of parallel experiments we found that the chemokine receptors expressed at sites of virus-induced T-cell mediated inflammation match the observed chemokine profile very well, thus CCR1, 2 and 5 together with CXCR3 receptor expression was observed (for relevant chemokine/receptor interactions, see Fig. 6). Separation of inflammatory exudate cells into adherent and nonadherent subsets revealed that adherent cells (mostly macrophages) expressed all of the above mentioned receptors, while sorted, activated (VLA-4high) CD8/ T cells expressed primarily CCR2, 5 and CXCR3 [45]. Our
interpretation of these findings, which is also supported by a survey of the relevant literature, is that essentially the same effector cell subsets are required to control the majority of viral infections, and these are exactly the effector cell types expressing the chemokine receptors, which match the virus-induced chemokine profile. Consistent with this view, the chemokine receptor profile of effector cell types already known to be relevant in virus control (macrophages, NK cells, Tc1 and Th1 cells) is also found to be very uniform and to include the above mentioned chemokine receptors (CCR1, 2 and 5 and CXCR3). More importantly, this descriptive analysis is supported by migration studies both in vitro and in vivo. First, we have found that chemokines normally produced in the context of viral infections (e.g. MIP-1a and IP-10) also exert chemotactic activity upon virus-specific effector Tc1 cells in vitro, and this pattern is independent of the virus used for priming (unpublished observation). Second, when in vivo migration of in vitro generated Tc1 (expressing CCR2 and 5) and Tc2 (expressing CCR3) cells were compared, Tc2 cells were found to be less efficient in virus control, and this correlated with an impaired ability to reach foci of infection [43]. Thus while there is every reason to assume that chemokines critically influence the efficiency of the antiviral immune response, relatively few studies have directly addressed the functional importance of chemokines in directing effector T cells to sites of viral infection. Indeed, when compared to neutrophils and monocytes, lymphocytes were long considered poor targets for inflammatory chemokines. This view has changed, though, based on the realization that activation of lymphocytes is essential for induction of responsiveness to this category of chemokines. One problem facing researchers trying to study the role of chemokines in vivo is that successful antibodymediated neutralization may be difficult to obtain. This could reflect the fact that chemokines often function in compartments not easily accessible to circulating antibodies, but it might also reflect functional redundancy, which */as already pointed out*/is a frequently-occurring event within the chemokine network. For this reason, gene targeted mice constitute the most informative tool when trying to study the functional impact of chemokines in vivo. Using LCMV infection as our primary model system, we have initiated experiments aimed at evaluating the functional importance of those chemokines and chemokine receptors, which we have previously found to be upregulated before and during T cell infiltration into virus infected organs. CCR5 deficient mice were selected as the first mouse strain to be studied. This choice was in part based on fact that CCR5 is an important coreceptor for HIV, and thus a focus for pharmacological modulation as part of the treatment against this viral infection. If CCR5 were to play a significant role in the normal antiviral immune
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Fig. 5. Chemokine and chemokine receptor mRNA expression in virus-infected lungs as a function of time. Mice were infected with either 103 ID50 of LCMV intravenously or 106.8 EID50 of influenza virus intranasally; both these viruses actively replicate in the lungs, and T cells are required for virus control. On the indicated days, total RNA was isolated from the lungs and subjected to RNAse protection assays using probes for chemokines (A) or chemokine receptors (B). For LCMV-infected mice day 3 represent the innate response, and day 7 marks the point of maximal increase in Tcell influx. For influenza-virus infected mice, day 5 is also analysed because in this case the separation into distinct phases is less apparent (Madsen et al., unpublished observation).
Fig. 6. Chemokines and chemokine receptors found to be most prominent in relation to virus-induced inflammation. In addition, the potential interactions between virus-induced chemokines and their relevant receptors are depicted. A full arrow denotes a strong interaction whereas a dotted arrow denotes weaker interactions.
response, blocking of CCR5 might hamper anti-HIV immune reactivity and thus nullify beneficial effects obtained through a direct inhibition of viral spreading. Fortunately extensive analysis did not reveal any evidence indicating that absence of CCR5 negatively
influenced either virus clearance or effector cell migration to LCMV infected organs [48]. In fact, we observed a tendency towards an augmentation of the primary T cell response in CCR5 deficient mice. This was the case particularly for the generation of Th1 effector cells, but at the time of peak primary immune reactivity a similar trend was noted also for Tc1 cells. Thus targeting of CCR5 may be a safe way to reduce HIV spreading in the infected host. This conclusion is further supported by similar studies in MIP-1a knockout mice (Madsen et al., in preparation); MIP-1a is a primary ligand of CCR5 and is a prominent chemokine found at sites of viral infection. Using the MIP-1a knockout mice, we found that although virus-primed T cells normally produce high amounts of this chemokine and, at least in the case of LCMV infection, represent the major local source, no functional deficiency regarding virus-induced T-cell
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mediated inflammation was observed in MIP-1a deficient mice (Madsen et al., in preparation). Complicating matters, however, studies involving other viruses have yielded conflicting results. For example, in mice infected intracerebrally with neurotropic mouse hepatitis virus, absence of CCR5 seems to reduce CNS cell infiltration particularly of monocytes/macrophages [49]. Furthermore, a slight increase in peak viral titers was observed, although this had no clinical consequences in terms of increased mortality. In contrast, CCR5 deficient mice were found to be more susceptible to influenza virusinduced pneumonitis than wildtype mice, and this correlated with an augmented early influx of mononuclear phagocytes, although viral titers were similar in receptor knockouts and wildtype controls [50]. The problem with both of these studies, however, is that the effects of CCR5-deficiency on specific components of the immune response were poorly characterized, making it difficult to separate effects on the afferent and efferent phases of the specific immune response. Furthermore, both of these models are less transparent than the LCMV system, since both the viruses used and the host responses they induce contribute to pathology. This is the advantage of the LCMV model, in that although an innate immune response is seen, it is weak and not associated with any pathology [25]. Therefore T-cell dependent effector responses can be clearly quantified together with their clinical correlates. Nevertheless, the fact that findings from different viral models may support apparently conflicting conclusions, serve to underscore how much work remains to be carried out, before we can hope to understand the role of chemokines in regulating antiviral immune responses.
7. Concluding remarks Migration of effector T cells to sites of viral infection is a tightly regulated process controlled through a number of receptor/ligand interactions. Although we are getting better at defining the essential molecules in this respect, much still remains to be learnt. While we appear to have a pretty good picture of the adhesion molecules involved in the extravasation process, little is known about the cell/cell and cell/matrix interactions required for appropriate positioning within the extracellular space; integrins such as VLA-1 and -2 are suspected to play a role at this stage. Furthermore, our understanding of the role of chemokines in physiologically relevant inflammatory processes is still very far from being complete. One problem in this context is the complexicity and high degree of functional redundancy within the chemokine system, which often leads to negative experimental findings in vivo. However, the fact that chemokines are very likely to play a critical role in deciding the virus/host balance is perhaps best
indicated by the fact that several large, complex viruses (pox and herpes viruses) appear to have pirated genes encoding chemokine receptors and antagonistic ligands [51]. Assuming that interference with the chemokine system is indeed the major function of these molecules in vivo, this pattern strongly suggests that successful inhibition/misdirection of the host’s chemokine response carries a survival value for the virus. Supporting this conclusion we have recently obtained results indicating that a virally encoded chemokine antagonist, vMIP-II, has the ability to inhibit the in vivo effector phase of an antiviral CD8/ Tc1 response (Lindow et al., in preparation).
Acknowledgements The authors would like to acknowledge the expert contributions made by several past and present members of our research group involved with these and related studies (C. Andersson, J.E. Christensen, S.Ø. Kauffmann, and H.V. Nielsen). We would also like to thank G.T. Andersen and L. Malte for their excellent technical assistance.
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Review
Regulation of T cell migration during viral infection: role of adhesion molecules and chemokines Allan Randrup Thomsen , Anneline Nansen, Andreas Nygaard Madsen, Christina Bartholdy, Jan Pravsgaard Christensen Institute of Medical Microbiology and Immunology, University of Copenhagen, Copenhagen, Denmark
Abstract T cell mediated immunity and in particular CD8/ T cells are pivotal for the control of most viral infections. T cells exclusively exert their antiviral effect through close cellular interaction with relevant virus-infected target cells in vivo. It is therefore imperative that efficient mechanisms exist, which will rapidly direct newly generated effector T cells to sites of viral replication. In the present report we have reviewed our present knowledge concerning the molecular interactions, which are important in targeting of effector CD8/ T cells to sites of viral infection. # 2002 Elsevier Science B.V. All rights reserved. Keywords: T cells; Antiviral immunity; Virus-induced inflammation; Adhesion molecules; Chemokines
1. Introduction When a parasite enters a vertebrate host it is normally met by two different defence systems. The first line of defence (innate immunity) comprises a number of nonspecific effector systems that work with little delay to limit the replication of the infecting agent. A characteristic feature of the early effector systems of innate immunity is that recognition of non-self is based on general motifs (e.g. LPS and dsRNA) associated with large groups of parasites [1] and thus does not involve the highly specific receptor-mediated recognition characteristic of the adaptive immune response. Basically, this is what allows the very short reaction time of innate defences. In contrast, highly refined and specific recognition as found for T and B cells, necessitates a clonal distribution of the involved receptors, and therefore a delay in action time due to the need for clonal expansion (and differentiation). Since this delay clearly puts a major constraint on the defensive value of adaptive immunity in relation to very rapidly replicating para Corresponding author. Address: Institute of Medical Microbiology and Immunology, Panum Institute, University of Copenhagen, 3C Blegdamsvej, DK-2200 Copenhagen N, Denmark. Tel.: /45-35-32-78-71; fax: /45-35-32-78-91. E-mail address: [email protected] (A.R. Thomsen).
sites, it is imperative that the potential of the adaptive response is exploited to its fullest. This requires at least two things: (a) effective mechanisms for presentation of antigen to as many lymphocytes as possible; this is needed to ensure that most of the (limited number of) cells with appropriate receptors are actually brought into action, and (b) once initial antigen-driven triggering and expansion has taken place, there also has to be efficient mechanisms for rapidly focussing the generated effector cells at and around sites of microbial replication. Bearing this in mind, much about the design of lymphocyte circulation becomes easier to understand. First, it is obvious that lymphocytes have to be able to migrate in a highly regulated fashion, and secondly, that the migration patterns required must be different before and after initial antigen-specific encounter [2,3]. Initially the naive lymphocyte has to recognise the antigen under conditions, which allow for an efficient response to invading non-self. This occurs optimally in the secondary lymphoid organs [4] e.g. the spleen and regional lymph nodes. Antigen is also transported to these organs, typically by way of dendritic cells, which mature in the process [5]. Thus, the secondary lymphoid organs represent critical intersections between the migrational pathways of antigen-presenting cells and naive lymphocytes. Because antigen-presentation does not involve cells with clonally distributed receptors, these are not
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limiting, and the above ‘design’ therefore allows for rapid and efficient presentation of parasitic intruders, while at the same time ensuring that any part of the body becomes frequently, albeit indirectly, monitored by the entire pool of naive lymphocytes. However, while stringent regulation of antigen-induced triggering of naive lymphocytes is essential, it is just as important that any host cells can be targeted by at least one type of effector cell since otherwise there would be potential hiding places for smart pathogens. Consequently, once clonal expansion has taken place, the generated effector and memory cells must be able to monitor for antigen in an anatomically unrestricted fashion. Furthermore, to guarantee rapid focussing of the adaptive response, one result of antigen encounter should be the release of mediators leading to the recruitment of more effector cells to the site. The interface between circulating lymphocytes and vascular endothelium together with extravascular chemokine gradients probably constitutes the most important gatekeeper in regulating lymphocyte migration, and extravasation in itself involves a complex set of interactions between adhesion molecules and their ligands and between chemokines and their receptors [3,6 /8]. In this brief review we will try to present an overview of the mechanisms involved in regulating CD8/ T cell migration as this is required for an optimal adaptive immune response to a viral infection. The focus is on our own studies using functional parameters: virus control and immunopathology, and represents our present views, based in particular on analysis of gene-targeted mice.
2. The extravasation paradigm All leukocytes are believed to extravasate through a series of steps (for a graphic representation see Fig. 1) that are considered to be essentially similar, independent of the type of leukocyte involved [6]. First, the leukocyte must adhere to the endothelium of blood vessels in the relevant organ site. The initial, reversible interaction is normally mediated by the interaction of selectin receptors with glycoconjugate ligands. The selectin family comprises three members which falls into two functionally distinct groups [9]: (1) L-selectin, which is expressed by many leukocytes including naive T cells, but not recently primed T cells, and (2) E- and P-selectins both of which are expressed by activated endothelium. The selectin-glycoprotein interaction causes the leukocyte in the post-capillary venules to roll along the inner endothelial surface and thus creates the basis for further adhesive interactions: firm adhesion and transmigration. These latter events require leukocyte integrins and endothelial expression of relevant Ig superfamily ligands. However, for the leukocyte to establish firm binding, chemokine-mediated triggering is assumed to
Fig. 1. The extravasation paradigm. Following free flow in the larger vessels, extravasation of leukocytes occurs through a stepwise interaction with molecules expressed on the inner vascular surface of the postcapillary venules. Initially, the leukocyte tethers to the endothelial surface through selectin/glycoconjugate interactions. This binding is not sufficiently strong to stop cell movement, but the leukocyte rolls slowly along the inner surface of the venule, continually making and breaking contact. However, the slowing down of leukocyte movement allows for interactions between leukocyte integrins (e.g. LFA-1 and VLA-4) and vascular ligands belonging to the superimmunoglobulin family (CAMs, e.g. ICAM-1 and VCAM-1). This results in tight binding provided that the leukocyte is activated by chemokines. These can be presented to the leucocyte bound to heparin-like glycosaminoglycans on the surface of endothelial cells close to the area of microbial challenge. The tight binding totally stops cell movement, and leads the leukocyte to squeeze out between adjacent endothelial cells (diapedesis). Finally, the cells migrate along gradients of chemoattractants, e.g. chemokines, towards the site of maximal microbial activity.
be critical [10]. The chemokine-induced activation leads to a conformational change in the integrins that increases their affinity for their appropriate ligand very substantially, and this seems to be a precondition for firm adhesion and subsequent transmigration. The final positioning of the extravasated leukocyte in the extracellular matrix is probably regulated by gradients of chemoattractants, e.g. chemokines, in conjunction with integrin-based adhesive interactions with common matrix proteins, e.g. collagen. While this is the general picture for leukocyte migration in any tissue, surprisingly few studies addressing the molecular requirements for effector T lymphocyte migration in vivo have been carried out */at least in the context of relevant biological challenges such as viral infections, against which this lymphocyte subset is known to play a major role in controlling infection. Our group has been studying antiviral immunity for many years, in particular the role of cell-mediated immunity and cytotoxic (CD8/) T cells. Since these cells require close contact with their infected targets in order to exert any antiviral function be it killing or cytokine release, T cell migration is clearly a central issue in viral immunology. It was therefore obvious for us to ask which mechanisms
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regulate effector T cell migration into sites of viral infection.
3. Activation-induced changes in T-cell phenotype Naive T cells gain access to the lymph nodes through specialized high endothelial venules (HEV). Expression of L-selectin on the lymphocyte is pivotal for binding to peripheral-node addressin and initial contact to HEV [11,12]. In addition, successful extravasation at this stage involves T cell activation with a chemokine named secondary lymphoid-tissue chemokine [13]; this chemokine binds to a receptor named CCR7 [14,15]. Thus naive T cells are characterized by expression of Lselectin and CCR7. These molecules are superfluous for migration to inflammatory sites and are down-regulated upon activation of the T cell [16,17]. In contrast integrins like VLA-4 and LFA-1 are upregulated when the T cell is properly activated [17,18]. Thus, in the case of acute systemic viral infection, close to 90% of all CD8/ T cells present at the time of the primary immune response may be L-selectinlow, VLA-4high and LFA-1high, and all virus-specific CD8/ T cells are contained within this subset [19 /21]. The expression of chemokine receptors also changes markedly, and receptors responsive to inflammatory chemokines are now expressed (see below). Together these changes (Fig. 2) in the expression of surface molecules constitute the molecular foundation for the profound change in lymphocyte circulation that is essential for effective targeting of activated T cells to sites of microbial challenge.
4. Role of selectins To study the relevance of selectins for CD8/ T cell migration in the context of viral infection, gene-targeted mice lacking one or more selectins [22 /24] were infected with a non-cytolytic virus: lymphocytic choriomeningitis virus (LCMV). Since this virus causes little or no cellular damage on its own, both virus-induced pathology and virus control reflect the efficiency of the immune response in situ [21]. Furthermore, a great number of
Fig. 2. Phenotypic changes affecting cell migration induced as result of T-cell activation and differentiation towards type 1 (IL-2 and IFN-g producing) effector cells.
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studies have clearly established that CD8/ effector T cells completely dominate the initial immune response both as mediators of virus control and immunopathology. Therefore this model system is ideally suited to study effector CD8/ T cell migration to infected organs, since both the inflammatory response and virus clearance constitute very relevant functional read-outs for appropriate effector cell migration [25]. Using E- or P-selectin knock-out mice as well as E/P-selectin double knock-outs, we found little role for endothelial expression of selectins [26]. Effector CD8/ T cell responses were effectively generated in these mice, and at best a slight delay in effector cell migration was observed when virus-primed wild-type donor T cells were adoptively transferred into these knock-outs. Since the effector CD8/ T cells express little L-selectin [19,20], these results suggested that selectin-based rolling was not pivotal for migration of CD8/ T cells into a virusinfected area. However, a comparison of the level of Lselectin expression on effector cells from wildtype and Lselectin knock-out mice (Christensen et al., in preparation) suggested that residual L-selectin expression could play a role. To test this experimentally, adoptive transfer analysis was conducted giving E/P-selectin knock-out mice virus-primed effector T cells from L-selectin deficient as well as wildtype donors. However, even under these conditions virus-induced T-cell mediated inflammation was induced with little delay compared to wild-type recipients given wild-type effector cells, and this delay mostly reflected the slightly reduced responsiveness of the E/P-selectin recipients in general. Therefore, we conclude that selectin-based rolling is not essential for effector CD8/ T cell migration to foci of virus-infection, and that other molecules apparently suffice for the initial tethering of CD8/ effector T lymphocytes in the post-capillary venules of inflamed areas. This conclusion is supported by a recent study analysing the role of adhesion molecules in CD8/ T cell rolling in cytokine activated venules [27]. Here it was clearly demonstrated that VLA-4, but not selectins were responsible for rolling of CD8/ T cells in M. cremaster venules activated with TNF-a and IFN-g. It is not clear why VLA-4 should suffice for rolling of activated CD8/ T cells. One reason might be the high expression of this integrin on activated CD8/ T cells as compared to the level of expression on neutrophils. This does not, however, explain why CD4/ T cells appear to be more dependent on endothelial expression of selectins than CD8/ T cells [28], since at least for virus-specific CD4/ and CD8/ T cells, levels of VLA-4 expression are similar [19,29]. The above findings should not, however, be taken to indicate that selectins are completely superfluous in the context of antiviral T cell mediated immunity. L-selectin knock-out mice are more susceptible to influenza virus infection than matched wildtypes (unpublished observa-
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tion), although this does not prove that L-selectin expression on the T cells plays a role. More importantly, if L-selectin knock-out mice are infected with LCMV by a peripheral route (via the footpad), T-cell priming is significantly delayed (Christensen et al., in preparation). This does not reflect a general decrease in the ability of these mice to respond to this virus as systemic infection (i.v. inoculation) primes L-selectin knock-out mice and wild-type controls with equal efficiency. Therefore, even with a rapidly disseminating virus like LCMV, initial priming in the regional lymph nodes plays a significant role, although efficient priming will eventually take place in the spleen. The latter priming may in part reflect dissemination of live virus. However, migration of antigen-presenting cells to the spleen is also likely to contribute since dermal application (using a gene-gun) of a non-replicating DNA vector also leads to T-cell priming in L-selectin knock-out mice */albeit with somewhat reduced efficiency (Christensen et al., in preparation). Interestingly, it has recently been claimed that Lselectin is important for lymphocyte migration into the skin. Thus, in contrast to E- and P-selectin double knock-out mice, fucosyltransferease VII-deficient mice with defective E-, P-, and L-selectin ligands were found to have impaired primary LCMV-induced footpad swelling, while no deficit of T cell migration to virusinfected visceral organs (of systemically-infected mice!) was observed [30]. This led the authors of the latter report to suggest a role for L-selectin in skin homing of CD8/ T cells. However, in adoptive transfer experiments we find that L-selectin negative effector cells readily migrate to virus-infected skin (Christensen et al., in preparation). The most likely explanation for the impaired footpad swelling in fucosyltransferease VIIdeficient mice is therefore that peripheral infection, which is needed for induction of a primary LCMVinduced footpad swelling reaction, leads to impaired priming in mice lacking relevant ligands for L-selectin similar to that observed in L-selectin knock-out mice. While effector T cells express little L-selectin, memory cells are heterogenous in this respect [31 /34], and analysis of splenic (unselected) virus-specific CD8/ T cells have shown that re-expression of this molecule increases with time after virus inoculation [32] (Christensen et al., in preparation). Furthermore, parallel analysis reveals that in the early phase after control of a systemic viral infection, antigen-specific CD8/ T cells are to be found almost exclusively in the spleen, whereas few can be found in peripheral lymph nodes. However, with time, redistribution to the peripheral lymph nodes gradually takes place, and this coincides with an increase in the fraction of L-selectin expressing cells (Christensen et al., in preparation). Thus L-selectin regulates not only the migration of naive T cells, but also seems to be involved in controlling the distribution of primed
CD8/ T cells between different compartments of the secondary lymphoid organs.
5. Critical role of integrins Whereas selectins play little role in targeting effector CD8/ T cells to sites of viral infection, integrins are very important. This has been shown primarily through blocking of various integrins in their ability to interact with their appropriate ligands. In intact virus-infected animals, treatment with non-depleting anti-VLA-4 antibody significantly delays virus-induced T cell mediated inflammation, indicating that one or more of the involved cell types utilizes this integrin during elicitation of inflammation (unpublished observation). Directly demonstrating a role for integrins in the migration of CD8/ effector T cell to sites of viral replication, we have found that pre-incubation of effector CD8/ T cells with anti-Mac-1, anti-LFA-1 or anti-VLA-4 all significantly inhibits the ability of these cells to adoptively induce virus-specific T-cell mediated inflammation [20,35,36] Notably, this is the case only when the effector T cells are required to actively migrate to the virus-infected site. Thus, when effector cells pre-incubated with antibody are injected directly into the test site, no inhibition is observed. Complementing the antibody based analysis, a reduced inflammatory response is also found in ICAM-1 deficient recipients [37], and the residual inflammatory response in these mice is completely eliminated [26] by inhibition of VCAM-1 binding using soluble molecules (sVCAM-1) [38]. In order to highlight the functional impact of integrins versus selectins on the migration of CD8/ effector T cells to sites of viral replication, we conducted a set of parallel adoptive transfer experiments [26], each involving the inhibition of two sets of molecular interactions (see Fig. 3a /c): either (a) binding to Eand P-selectin was prevented; alternatively, (b) one selectin (P-selectin) and one integrin (ICAM-1) was eliminated, and as the third possibility (c) binding to both major ligands of integrins, ICAM-1 and VCAM-1, was prevented. It is evident from the results obtained that while failure to interact with endothelial selectins is of little functional relevance, integrins are pivotal for a virus-induced T cell mediated inflammatory response.
6. The role of chemokines and chemokine receptors Whereas selectins are constitutively active, integrins need to be activated to mediate efficient binding. The important role played by integrins therefore drew our attention to another important set of molecular players involved in regulating leukocyte migration, namely the chemokines. Chemokines are low m.w. chemotactic
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Fig. 3. Comparison of the role of selectins and integrins in virus-induced T-cell mediated inflammation. Groups of gene-targeted and matched wildtype mice were inoculated with 105 ID50 in their right hind footpad. Four hours later these recipients were injected intravenously (i.v.) with 50/ 106 adherent cell depleted, mitomycin C treated (to prevent donor cell proliferation subsequent to cell transfer) splenocytes from wildtype donors infected 8 days earlier with 103 ID50 i.v. Groups consisted of 4 /6 mice; medians and ranges are depicted. () denotes P B/0.05 relative to wildtype recipients (Mann /Whitney rank sum test) (taken from Ref. [26]).
cytokines that bind to specific 7-transmembrane G protein-coupled cell surface receptors [39,40]. While some chemokines appear to play a major role in regulating adhesive interactions with the vascular endothelium [10], others may control subsequent migration and positioning within the extravascular space [3,8]. Spatiotemporal patterns of chemokine and chemokine receptor expression are therefore supposed to critically influence immune responses [3,7,8]. Chemokines are classified into four families based on the configuration of cysteine residues near the Nterminus [39 /41]. In CXC chemokines two cysteines are separated by another amino acid (X) while CC chemokines have two adjacent cysteines. Both of these families comprise several members, while the two last families, named CX3C and XC receptors, have only one member each. The nomenclature of the corresponding receptors follows that of their ligand with the addition of R and a number, e.g. CXCR3 [40,41]. Generally, the chemokine network is characterized by a high degree of functional redundancy, which hampers the analysis of functional relevance in vivo. Thus, one chemokine may often bind to several members of the same receptor family and, similarly, one receptor may bind several related chemokines (for relevant examples see Fig. 6) [40]. While constituting a nuisance to the researcher, this phenomenon is biologically very understandable because it makes the chemokine system much more robust to interference by pathogens. From a functional point of view, chemokines are divided into (a) constitutive (alternatively called homeostatic or lymphoid) and (b) inflammatory (or inducible) [8,39]. Constitutive chemokines are involved in regulating normal lymphocyte migration and positioning within the lymphoid tissues, while the inflammatory
chemokines are critical for attracting a diverse set of effector cells to inflammatory sites. Matching this functional division of labour between different sets of chemokines, chemokine receptor expression changes markedly with the state of lymphocyte differentiation [42,3,39] (Fig. 4). For peripheral T cells this is very striking. While naive T cells express CCR7, which is essential for lymph node homing, effector T cells express receptors for inflammatory chemokines. Even more important, different effector subsets appear to express different receptors. Thus, type 2 CD4/ effector T cells (Th2 cells) primarily express CCR3 and CCR4, whereas the phenotype of Th1 cells involves high expression of CCR5 and CXCR3 [3,39,42]. CD8/ effector T cells have not been as extensively characterized with regard to chemokine receptor expression, but they seem to follow the same pattern as CD4/ effector T cells i.e., receptor expression correlates with the cytokine profile
Fig. 4. Chemokine receptor expression as a function of T-cell differentiation. Based on the existing literature and our own studies we have tried to make a consensus picture of chemokine receptors expressed by type 1 and type 2 effector T cells regardless of their coreceptor phenotype (CD4 or CD8). Given that there are some inconsistencies in the literature, and most studies have involved in vitro generated lines/clones, the picture is not yet complete and may be subject to change with more information from direct ex vivo analysis.
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[43]. Thus, it would be predicted that it is the expression of chemokine receptors induced during T cell activation together with locally produced chemokines, which in the end determines the composition of the inflammatory exudate. When viruses infect a mammalian host essentially two waves of chemokines are produced at the site of viral invasion [44,45]. The first wave comprises part of the innate response to a viral infection. The precise signals which elicits this response are not clear, but virusinduced perturbation of normal cell metabolism may be one way in which this response is elicited. However, a significant chemokine response may be seen in the absence of outright cell damage e.g. after LCMV infection [44,45]. In addition, at least some chemokines are partially regulated by other signal molecules generated in the context of the innate response to viral invasion, e.g. IFN-a/b [45]. The second wave comprises chemokines produced as a result of a local cell-mediated immune response [44,45]. Some of these chemokines are synthesized by the incoming T cells themselves (e.g. MIP-1a [46]), while others are being produced by a variety of cell types (in particular macrophages, e.g. MCP-1) under the influence of T-cell dependent cytokines such as IFN-g [10,44,47]. Together these two waves of chemokine production allow for (1) rapid focussing of early effector cells to the site of viral invasion and (2) effective enhancement of the response once viral antigen is detected by specific cell types. To define the chemokines likely to play a role in viral infections, we have studied the chemokines produced in a number of organs (liver, lungs and CNS) infected by different viruses (LCMV, influenza virus and vesicular stomatitis virus) differing in their biological properties and cell tropism. A representative result of this survey is seen in Fig. 5, and another example can be found in Ref. [45]. Although differences may be observed, particularly in the relative magnitude of production of various chemokines, the overall impression is how uniform the virus-induced chemokine profile actually is: RANTES, MIP-1a and b, IP-10 and MCP-1 appear to dominate independent of virus, organ site or phase of infection (at present we have only concentrated on acute infections which of course means that the picture may be different in the context of chronic viral infections). In a set of parallel experiments we found that the chemokine receptors expressed at sites of virus-induced T-cell mediated inflammation match the observed chemokine profile very well, thus CCR1, 2 and 5 together with CXCR3 receptor expression was observed (for relevant chemokine/receptor interactions, see Fig. 6). Separation of inflammatory exudate cells into adherent and nonadherent subsets revealed that adherent cells (mostly macrophages) expressed all of the above mentioned receptors, while sorted, activated (VLA-4high) CD8/ T cells expressed primarily CCR2, 5 and CXCR3 [45]. Our
interpretation of these findings, which is also supported by a survey of the relevant literature, is that essentially the same effector cell subsets are required to control the majority of viral infections, and these are exactly the effector cell types expressing the chemokine receptors, which match the virus-induced chemokine profile. Consistent with this view, the chemokine receptor profile of effector cell types already known to be relevant in virus control (macrophages, NK cells, Tc1 and Th1 cells) is also found to be very uniform and to include the above mentioned chemokine receptors (CCR1, 2 and 5 and CXCR3). More importantly, this descriptive analysis is supported by migration studies both in vitro and in vivo. First, we have found that chemokines normally produced in the context of viral infections (e.g. MIP-1a and IP-10) also exert chemotactic activity upon virus-specific effector Tc1 cells in vitro, and this pattern is independent of the virus used for priming (unpublished observation). Second, when in vivo migration of in vitro generated Tc1 (expressing CCR2 and 5) and Tc2 (expressing CCR3) cells were compared, Tc2 cells were found to be less efficient in virus control, and this correlated with an impaired ability to reach foci of infection [43]. Thus while there is every reason to assume that chemokines critically influence the efficiency of the antiviral immune response, relatively few studies have directly addressed the functional importance of chemokines in directing effector T cells to sites of viral infection. Indeed, when compared to neutrophils and monocytes, lymphocytes were long considered poor targets for inflammatory chemokines. This view has changed, though, based on the realization that activation of lymphocytes is essential for induction of responsiveness to this category of chemokines. One problem facing researchers trying to study the role of chemokines in vivo is that successful antibodymediated neutralization may be difficult to obtain. This could reflect the fact that chemokines often function in compartments not easily accessible to circulating antibodies, but it might also reflect functional redundancy, which */as already pointed out*/is a frequently-occurring event within the chemokine network. For this reason, gene targeted mice constitute the most informative tool when trying to study the functional impact of chemokines in vivo. Using LCMV infection as our primary model system, we have initiated experiments aimed at evaluating the functional importance of those chemokines and chemokine receptors, which we have previously found to be upregulated before and during T cell infiltration into virus infected organs. CCR5 deficient mice were selected as the first mouse strain to be studied. This choice was in part based on fact that CCR5 is an important coreceptor for HIV, and thus a focus for pharmacological modulation as part of the treatment against this viral infection. If CCR5 were to play a significant role in the normal antiviral immune
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Fig. 5. Chemokine and chemokine receptor mRNA expression in virus-infected lungs as a function of time. Mice were infected with either 103 ID50 of LCMV intravenously or 106.8 EID50 of influenza virus intranasally; both these viruses actively replicate in the lungs, and T cells are required for virus control. On the indicated days, total RNA was isolated from the lungs and subjected to RNAse protection assays using probes for chemokines (A) or chemokine receptors (B). For LCMV-infected mice day 3 represent the innate response, and day 7 marks the point of maximal increase in Tcell influx. For influenza-virus infected mice, day 5 is also analysed because in this case the separation into distinct phases is less apparent (Madsen et al., unpublished observation).
Fig. 6. Chemokines and chemokine receptors found to be most prominent in relation to virus-induced inflammation. In addition, the potential interactions between virus-induced chemokines and their relevant receptors are depicted. A full arrow denotes a strong interaction whereas a dotted arrow denotes weaker interactions.
response, blocking of CCR5 might hamper anti-HIV immune reactivity and thus nullify beneficial effects obtained through a direct inhibition of viral spreading. Fortunately extensive analysis did not reveal any evidence indicating that absence of CCR5 negatively
influenced either virus clearance or effector cell migration to LCMV infected organs [48]. In fact, we observed a tendency towards an augmentation of the primary T cell response in CCR5 deficient mice. This was the case particularly for the generation of Th1 effector cells, but at the time of peak primary immune reactivity a similar trend was noted also for Tc1 cells. Thus targeting of CCR5 may be a safe way to reduce HIV spreading in the infected host. This conclusion is further supported by similar studies in MIP-1a knockout mice (Madsen et al., in preparation); MIP-1a is a primary ligand of CCR5 and is a prominent chemokine found at sites of viral infection. Using the MIP-1a knockout mice, we found that although virus-primed T cells normally produce high amounts of this chemokine and, at least in the case of LCMV infection, represent the major local source, no functional deficiency regarding virus-induced T-cell
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mediated inflammation was observed in MIP-1a deficient mice (Madsen et al., in preparation). Complicating matters, however, studies involving other viruses have yielded conflicting results. For example, in mice infected intracerebrally with neurotropic mouse hepatitis virus, absence of CCR5 seems to reduce CNS cell infiltration particularly of monocytes/macrophages [49]. Furthermore, a slight increase in peak viral titers was observed, although this had no clinical consequences in terms of increased mortality. In contrast, CCR5 deficient mice were found to be more susceptible to influenza virusinduced pneumonitis than wildtype mice, and this correlated with an augmented early influx of mononuclear phagocytes, although viral titers were similar in receptor knockouts and wildtype controls [50]. The problem with both of these studies, however, is that the effects of CCR5-deficiency on specific components of the immune response were poorly characterized, making it difficult to separate effects on the afferent and efferent phases of the specific immune response. Furthermore, both of these models are less transparent than the LCMV system, since both the viruses used and the host responses they induce contribute to pathology. This is the advantage of the LCMV model, in that although an innate immune response is seen, it is weak and not associated with any pathology [25]. Therefore T-cell dependent effector responses can be clearly quantified together with their clinical correlates. Nevertheless, the fact that findings from different viral models may support apparently conflicting conclusions, serve to underscore how much work remains to be carried out, before we can hope to understand the role of chemokines in regulating antiviral immune responses.
7. Concluding remarks Migration of effector T cells to sites of viral infection is a tightly regulated process controlled through a number of receptor/ligand interactions. Although we are getting better at defining the essential molecules in this respect, much still remains to be learnt. While we appear to have a pretty good picture of the adhesion molecules involved in the extravasation process, little is known about the cell/cell and cell/matrix interactions required for appropriate positioning within the extracellular space; integrins such as VLA-1 and -2 are suspected to play a role at this stage. Furthermore, our understanding of the role of chemokines in physiologically relevant inflammatory processes is still very far from being complete. One problem in this context is the complexicity and high degree of functional redundancy within the chemokine system, which often leads to negative experimental findings in vivo. However, the fact that chemokines are very likely to play a critical role in deciding the virus/host balance is perhaps best
indicated by the fact that several large, complex viruses (pox and herpes viruses) appear to have pirated genes encoding chemokine receptors and antagonistic ligands [51]. Assuming that interference with the chemokine system is indeed the major function of these molecules in vivo, this pattern strongly suggests that successful inhibition/misdirection of the host’s chemokine response carries a survival value for the virus. Supporting this conclusion we have recently obtained results indicating that a virally encoded chemokine antagonist, vMIP-II, has the ability to inhibit the in vivo effector phase of an antiviral CD8/ Tc1 response (Lindow et al., in preparation).
Acknowledgements The authors would like to acknowledge the expert contributions made by several past and present members of our research group involved with these and related studies (C. Andersson, J.E. Christensen, S.Ø. Kauffmann, and H.V. Nielsen). We would also like to thank G.T. Andersen and L. Malte for their excellent technical assistance.
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