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Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Role of Macrophage Inflammatory Protein-1

in T-Cell-Mediated

Immunity to Viral Infection

Andreas N. Madsen, Anneline Nansen,† Jan P. Christensen, and Allan R. Thomsen*

Institute of Medical Microbiology and Immunology, University of Copenhagen, Copenhagen, Denmark

Received 4 June 2003/Accepted 28 July 2003

The immune response to lymphocytic choriomeningitis virus in mice lacking macrophage inflammatory protein-1(MIP-1) was evaluated. Generation of virus-specific effector T cells is unimpaired in MIP-1 -deficient mice. Furthermore, MIP-1is not required for T-cell-mediated virus control or virus-induced T-cell-dependent inflammation. Thus, MIP-1is not mandatory for T-cell-mediated antiviral immunity.

CD8⫹T cells play an important role in antiviral immunity

(11, 12). The main effector function of this cell subset is con-tact-dependent lysis (16, 35), but production of cytokines such as gamma interferon (IFN-␥) is also important (23, 26). Both of these effector mechanisms have a short action range, and cell-cell contact is therefore essential for virus-specific CD8⫹T

cells to exert their antiviral effector function (19). It has pre-viously been found that viral infections are often associated with local production of inflammatory chemokines known to function as chemoattractants of activated lymphocytes (4, 24). Therefore, chemokines are likely to play a pivotal role in di-recting antiviral effector T cells to sites of viral replication in vivo.

Macrophage inflammatory protein-1␣ (MIP-1␣) is one of the chemokines known to be produced in the context of many viral infections (8), and a number of studies have provided strong evidence indicating that MIP-1␣plays an important role in virus-induced inflammation and in some cases also in anti-viral immunity (8, 13, 15, 34). However, in the previous studies the cell population(s) responsible for reduction of viral titers has not been clearly defined, and the mechanisms underlying the observed inflammatory reactions are complex. Thus, to our knowledge no studies have previously addressed the role of MIP-1␣in the generation and recruitment of CD8⫹T

cyto-toxic type 1 (Tc1) effector cells to sites of viral infection. There is, however, evidence in the literature indicating that MIP-1␣

might be important for other types of CD8⫹T-cell-mediated

immunity (9, 30).

To study the role of MIP-1␣ in T-cell-mediated antiviral immunity, we used the murine lymphocytic choriomeningitis virus (LCMV) model (32). LCMV is a noncytopathic virus that causes little or no inflammation in the absence of a virus-specific T-cell response (2, 20). In immunocompetent mice, however, the appearance of effector T cells is associated with substantial inflammation in infected organs, and intracerebral (i.c.) infection leads to a fatal T-cell-mediated meningitis (10). Previous studies have revealed that the inflammatory exudate

consists mainly of activated (L-selectinlowLFA-1highvery late

antigen-4high) (3) CD8Tc1 cells and monocytes and

macro-phages, whereas it contains very few CD4⫹T lymphocytes (5,

10). By use of this model, our group has previously shown that MIP-1␣ is expressed in LCMV-infected organs on both the mRNA and protein levels (24). Furthermore, inflammatory exudate cells express chemokine receptors, which may bind MIP-1␣. Thus, virus-activated CD8⫹ T cells express CCR5,

and activated monocytes and macrophages express both CCR1 and CCR5 (24). It therefore appeared likely that MIP-1␣

might play a significant role in the recruitment of virus-specific Tc1 cells to sites of virus-induced inflammation. Consequently, the primary aim of this study was to define the importance of MIP-1␣ in directing Tc1 cells to sites of viral infection. In addition, we wanted to elucidate the contribution of Tc1 cells to MIP-1␣production in situ during viral inflammation. For those purposes, we compared various parameters of the LCMV-specific T-cell response in wild-type (C57BL/6) and MIP-1␣-deficient (MIP-1␣⫺/⫺) mice (B6.129P2-Scya3tm1Unc

backcrossed seven times to a C57BL/6 background and ob-tained as breeder pairs from the Jackson Laboratory [Bar Harbor, Maine]).

Migration of activated T cells toward MIP-1in vitro.To elucidate the potential role of MIP-1␣as a chemoattractant for virus-activated CD8⫹T cells, recruitment of splenocytes from

wild-type as well as MIP-1␣⫺/⫺mice was evaluated in an in

vitro chemotaxis assay (18). MIP-1␣was found to induce the migration of splenocytes from virus-infected mice (Fig. 1A), and flow cytometric analysis confirmed that the recruited cells were preferentially activated (very late antigen-4high) CD8T

cells (Fig. 1B through D). Similar results were obtained when the knockout mice were used (Fig. 1A and data not shown). The capacity of MIP-1␣to attract virus-activated CD8⫹T cells

together with the fact that MIP-1␣is known to be produced in LCMV-infected tissues (4, 24) prompted us to study the bio-logical importance of this chemokine in LCMV-induced inflammation.

CD8T-cell effector function ex vivo and virus clearance.

LCMV infection is primarily controlled through perforin-me-diated killing of virus-infected cells (16, 35). To investigate whether the absence of MIP-1␣ would influence the genera-tion of CTL effector cells, splenocytes from infected MIP-1␣⫺/⫺ and wild-type mice were assayed for ex vivo lysis of * Corresponding author. Mailing address: Institute of Medical

Mi-crobiology and Immunology, The Panum Institute, 3C Blegdamsvej, DK-2200 Copenhagen N, Denmark. Phone: 35327871. Fax: 45-35327891. E-mail: a.r.thomsen@immi.ku.dk.

† Present address: Statens Serum Institute, Laboratory of Molecular Virology, Copenhagen, Denmark.

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51Cr-labeled target cells coated with the immunodominant

class I restricted peptides GP33-41 and nucleoprotein (NP) 396-404; peptide-specific lysis was evaluated on days 7, 9, and 14 after intravenous (i.v.) infection. As can be seen in Fig. 2A, splenocytes from MIP-1␣⫺/⫺and wild-type mice were equally

cytotoxic against GP33-41-coated targets, and similar results were obtained for NP396-404-coated targets (data not shown). Enumerating virus-specific CD8⫹T cells through intracellular

staining for peptide-induced IFN-␥(22) confirmed that the absence of MIP-1␣ did not impair the afferent phase of the LCMV-induced immune response (data not shown). Together, these findings validate the subsequent use of these knockout mice to investigate the role of MIP-1␣in directing the migra-tion of effector T cells to sites of viral infecmigra-tion.

A critical measure of the ability of CD8⫹effector T cells to

function in vivo is virus clearance from infected organs. Elim-ination of virus occurs most rapidly between day 7 and day 10 postinfection (p.i.), except in the lungs, where clearance is slightly delayed compared to the kinetics in organs like the spleen and liver (23, 31). To study the role of MIP-1␣in CD8⫹

T-cell-mediated clearance of virus, virus levels in MIP-1␣⫺/⫺

mice and wild-type controls were compared. Mice were in-fected i.v. with 200 PFU of LCMV Traub, and on day 9 p.i., groups of three mice were sacrificed, and the spleen, lungs, and liver were removed for determination of virus content. Since virus clearance is well under way but not complete at this point, virus levels measured at this time point would appropriately reveal any delay in virus clearance (7). As seen in Fig. 2B, similar levels of virus were found in MIP-1␣⫺/⫺and wild-type

mice regardless of the organ site investigated. To further eval-uate the influence of MIP-1␣on the ability to clear the infec-tion in nonlymphoid organs, lung and liver virus titers were also determined on day 14 p.i., and again similar (and com-pared to day 9 p.i., lower) levels of virus were observed, re-gardless of the genotype of the host.

Virus-induced delayed-type hypersensitivity in absence of MIP-1.The above results indicate that CD8⫹effector T cells

are capable of targeting sites of viral infection independently of whether or not MIP-1␣is produced locally. However, to more directly study the kinetics of CD8⫹effector T-cell migration

into a solid tissue, the primary LCMV-induced footpad swell-ing reaction was used. This is a classical model of T-cell-mediated inflammation that represents the CD8⫹

[image:2.603.54.274.65.355.2]

T-cell-me-diated response to subdermal virus challenge (6). To study the

FIG. 1. Activated T cells migrate toward a MIP-1␣gradient. Mice were infected with 200 PFU of LCMV Traub. (A) Spleen cells (1.5⫻

106) from mice infected (Inf.) 7 days earlier or from naive animals were

allowed to migrate for 3 h using Transwell chambers (Corning Costar Corp.) with culture inserts of 6.5 mm and a 5-␮m pore size; lower chambers contained MIP-1␣(50␮g/ml; R&D Systems) or medium for control. The total number of migrated cells was determined, and re-sults are presented as percentage of migrated cells; columns represent average⫾standard deviation (SD) (n⫽3). Representative fluores-cence-activated cell sorter plots of input cells (B), transmigrated cells (MIP-1␣) (C), and transmigrated cells (medium) from an infected mouse (D). Results of one of two similar experiments are presented.

FIG. 2. LCMV-specific Tc activity and organ virus titers in MIP-1␣⫺/⫺and wild-type mice. (A) Mice were infected i.v. with 200 PFU of LCMV, and cytotoxic activity was assayed in a51Cr release assay by use

of GP33-41 peptide-pulsed EL-4 cells as target cells. Unpulsed EL-4 cells served as control target cells. Results from individual mice are depicted; results of one of two similar experiments are presented. E/T, effector/target ratio. (B) Organs (spleen, lungs, and liver) were har-vested on the days indicated, and organ virus titers were determined. Points represent individual mice. ID50, 50% infective dose; D.L.,

de-tection limit.

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role of MIP-1␣ in this delayed-type hypersensitivity (DTH)-like reaction, MIP-1␣⫺/⫺ mice and wild-type mice were

in-fected in the right hind footpad, and the difference in thickness of the infected right and the uninfected left foot was deter-mined between days 6 and 13 p.i. We observed no significant difference in the response pattern of MIP-1␣⫺/⫺and wild-type

mice (Fig. 3A), indicating that a subdermal inflammatory re-sponse can also proceed normally in the absence of MIP-1␣

expression.

Role of MIP-1in LCMV-induced meningitis.When mice are infected i.c. with LCMV, a fatal T-cell-mediated meningitis is induced (2, 6, 10, 20). To investigate whether MIP-1␣ is necessary to recruit activated T cells to the brain, we infected MIP-1␣⫺/⫺mice and wild-type mice i.c., and for a portion of

the mice we recorded the resulting mortality. In parallel ex-periments, we determined the number and composition of cells recovered from the cerebrospinal fluid (CSF) of infected mice. Regardless of the genotype, infected animals died about 8 to 10 days after i.c. challenge, and there was no difference in cell numbers or composition of inflammatory cells in the CSF (Fig. 3B). Thus, the recruitment of mononuclear effector cells to the LCMV-infected meninges is unimpaired in the absence of MIP-1␣.

Taken together, the above results provide strong evidence that effector T-cell homing to virus-infected organs does not require MIP-1␣. However, the redundancy of MIP-1␣could be explained by compensatory mechanisms, e.g., the upregulation of other chemokines or cytokines at the inflammatory sites in LCMV-infected knockout mice. To investigate this possibility, expression of chemokine and cytokine genes was evaluated by RNase protection assays on total RNA (22, 24). RNA was extracted by use of the RNeasy kit (Qiagen, Hilden, Germany) from brains of MIP-1␣⫺/⫺and wild-type mice that were

in-fected i.c. with LCMV 3 and 7 days earlier; these time points reflect the innate and T-cell-dependent phases of the inflam-matory response, respectively (4, 24). Control animals of either strain were included to show basal transcript levels (Fig. 4). MIP-1␣⫺/⫺mice constitutively expressed mRNA for MIP-1

and TCA-3 in the brain, while there was no detectable expres-sion of chemokines in the brains of uninfected wild-type mice. This difference in the basal expression of two chemokines, and of course the lack of MIP-1␣, were the only differences that we observed regarding the composition and kinetics of mRNA for chemokines (Fig. 4) and cytokines (data not shown). To ex-plore whether TCA-3 could be involved in some form of com-pensatory circuit, a separate RNase protection assay was run in which we looked for expression of CCR8, the receptor for TCA-3. No expression was observed in any group (data not shown). This finding, together with the fact that the onset of inflammation was associated with little or no increase in TCA-3 expression, led us to conclude that TCA-3 expression does not explain the redundancy of MIP-1␣.

Antigen-specific CD8T cells are the main source of MIP-1in the LCMV-infected central nervous system.

MIP-1␣may be produced by a variety of cell types (21), in-cluding CD8⫹ T cells (9). To investigate whether

[image:3.603.304.534.74.570.2]

LCMV-specific T cells produce MIP-1␣, splenocytes from naive mice and mice infected 9 days earlier were stimulated in vitro with a mixture of the immunodominant LCMV class I restricted peptides GP33-41 and NP396-404. Large amounts of MIP-1␣

FIG. 3. Kinetics of LCMV-induced inflammation in MIP-1␣⫺/⫺ and wild-type mice. (A) A primary footpad swelling was elicited by infecting mice with 200 PFU of LCMV Armstrong in the right hind footpad. Points represent individual mice, and group medians are connected; results of one of two similar experiments are presented. (B) LCMV-induced meningitis was studied in MIP-1␣⫺/⫺and wild-type mice infected i.c. with 200 PFU of LCMV Traub. For some of the mice, mortality was registered (curves). For the remainder, CSF was harvested from the fourth ventricle of mice that had been ether anes-thetized and exsanguinated, and the total number of cells present was determined (columns); background level in uninfected mice is⬍100 cells/␮l. The number of mice per group is indicated in the figure, and results are presented as averages⫾SD. Flow cytometric analysis (not depicted) revealed an identical composition of CSF cells (CD8⫹T cells and Mac-1⫹mononuclear phagocytes) regardless of genotype.

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(as determined by a sandwich enzyme-linked immunosorbent assay [R&D Systems, Minneapolis, Minn.]) were produced by LCMV-primed cells when stimulated with these class I re-stricted peptides for 6 h (data not shown). Virus-primed spleen cells left alone or stimulated with an irrelevant peptide did not produce detectable amounts of MIP-1␣, nor did naive spleen cells stimulated with the same peptides. Thus, LCMV-primed cells produce MIP-1␣upon recognition of cognate antigen.

To investigate the role of CD8⫹ T cells as producers of

MIP-1␣in vivo, we used an adoptive transfer setup, in which MIP-1␣⫺/⫺recipients received a small number of donor cells

from TCR318 mice, which express a T-cell receptor (TCR) directed against GP33-41 on 50 to 60% of their CD8⫹T cells

(25). One day prior to i.c. infection with LCMV, MIP-1␣⫺/⫺

mice received either 3 ⫻ 106 whole spleen cells, the same

number of splenocytes from which CD8⫹T cells were depleted

(by using anti-CD8 and rabbit complement [Cederlane, Hornby, Ontario, Canada]) or an equivalent number of puri-fied (⬎90% pure through depletion of major histocompatibil-ity class II⫹, Ig, and CD4cells using monoclonal rat

anti-bodies and magnetic beads coupled with sheep anti-rat and anti-mouse immunoglobulin G [Dynal, Oslo, Norway]) CD8⫹

T cells from naive TCR318 mice. Five days later (these mice developed meningitis with accelerated kinetics and died around day 6 p.i.), total RNA from the brains of the infected animals was extracted and analyzed with regard to chemokine transcripts (Fig. 5). Control mice and untransplanted mice infected i.c. 7 days earlier were included as references with regard to constitutive and maximal expression of chemokine transcripts in the brains of the two strains. As shown in Fig. 5, it is evident that both groups of recipients which received donor cells that included TCR⫹CD8T cells have completely

regained the capacity to express MIP-1␣mRNA. This is doc-umented by visual inspection as well as by quantification of band intensities (Fig. 5B); in the latter case, quantifications of

mRNA for IP-10 and RANTES have been included as positive controls for an inflammatory response. Recipients that re-ceived splenocytes from which CD8⫹T cells were depleted

before cell transfer had no MIP-1␣mRNA expression in the brain. Thus, our results indicate that CD8⫹T cells are potent

producers of MIP-1␣and are the main source of this chemo-kine in the brains of LCMV-infected mice, although a minor contribution of parenchymal MIP-1␣ production cannot be ruled out.

Redundancy of MIP-1does not require infectious antigen.

To test whether the redundancy of MIP-1␣reflected the use of live antigen as a local trigger of inflammation, mice were in-fected i.v. and challenged in the footpad with LCMV-specific peptide (GP33-41); this method has previously been estab-lished as a valid way to assess CD8⫹T-cell-mediated

inflam-mation (17, 22). As can be seen in Fig. 6A, a substantial inflammatory response was induced in all MIP-1␣⫺/⫺ mice,

and no major difference was observed between MIP-1␣⫺/⫺and

wild-type mice, although marginal reductions were noted in some experiments. Finally, to see if live, replicating antigen was required at any point during the immune response, mice were immunized subcutaneously with inert antigen (sheep red blood cells [SRBC]) and challenged 5 days later with this antigen in the footpad (Fig. 6B). In the context of this CD4⫹

T-cell-mediated DTH reaction, we also found MIP-1␣to be redundant, suggesting that the redundancy we observed for LCMV-specific CD8⫹T cells is not a unique situation.

At first glance, this conclusion appears to be at odds with findings from other viral model systems. For example, it has previously been found that after influenza (8) or paramyxovi-rus infection (13), MIP-1␣⫺/⫺mice have increased lung virus

titers compared to wild-type mice. Furthermore, it has been documented that MIP-1␣⫺/⫺mice are resistant to

coxsackievi-rus-induced myocarditis (8) and that such mice have a reduc-tion in lung inflammareduc-tion when infected with respiratory syn-cytial virus (15). However, the cellular and molecular events involved in the pathogenesis of those infections tend to be more complex, and the mechanisms underlying virus clearance are not as strongly related to the activity of a single effector cell population, namely effector Tc1 cells, as is true in the case of LCMV. This is clearly exemplified by analysis of murine cyto-megalovirus-induced hepatitis. In this model, a complex series of cellular interactions is triggered by the viral infection, which at one particular step critically involves MIP-1␣(27, 28): NK cells are crucial for early IFN-␥ production, which again is required for local T-cell accumulation via a regulatory effect on Mig production and is critical for the initiation of this cascade early MIP-1␣production at the site of viral replication. How-ever, in the LCMV model, NK cells are not essential for in-flammation (1), and the elicitation of an inflammatory re-sponse is entirely dependent on the generation and local accumulation of virus-specific CD8⫹T cells (10, 20), which in

[image:4.603.49.280.69.229.2]

this case can apparently function without involving MIP-1␣at any level.⌻his comparison immediately raises questions as to the generality of our findings: is the chemokine profile unique to this infection explaining the redundancy, or can our results be extended to other cases of T-cell-mediated inflammation? At least two observations support the assumption that effector T-cell migration may often bypass a need for MIP-1␣. First, the chemokine profile that is found in the context of the LCMV

FIG. 4. Cerebral chemokine transcripts in MIP-1␣⫺/⫺ and wild-type mice following i.c. infection with LCMV. Mice were infected i.c. with 200 PFU of LCMV Traub. On the indicated days (0, 3, or 7 days p.i.), total RNA was isolated from the brain, and 20␮g of total RNA was subjected to RNase protection assay using the following kits ob-tained from PharMingen (San Diego, Calif.): RiboQuant in vitro tran-scription kit, mouse multiprobe template set mCK-5 (including the template for IP-10), and the RiboQuant RNase protection assay kit. Results from individual mice are presented.

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infection seems to be shared with several other viral infections (14, 24, 29, 33), indicating that there is nothing unique about this infection. Furthermore, even when inert antigen, namely SRBC, was used for both immunization and local challenge, we did not see any major impairment of T-cell-mediated inflam-mation, and the same was true when i.v. infected MIP-1␣⫺/⫺

[image:5.603.132.450.67.573.2]

mice were challenged locally with GP33-41 peptide. Taken together, these findings strongly indicate that the redundancy of MIP-1␣is not a reflection of a special chemokine profile related to this particular infection or even to the use of live virus. In conclusion, our results strongly indicate that although Tc1 effector cells are a relevant local source of MIP-1␣ and

FIG. 5. Antigen-specific T cells are the main source of MIP-1␣production in the brains of LCMV-infected mice. (A) Mice were infected i.c. with 200 PFU of LCMV Traub, total RNA was isolated from the brain, and 20␮g of total RNA was subjected to RNase protection assay. Groups 1 to 4, brains assayed on the indicated days; groups 5 to 7, MIP-1␣⫺/⫺mice infected i.c. with LCMV Traub 24 h after they had been injected i.v. with splenocytes from TCR 318 mice (group 5, 3⫻105splenocytes enriched for CD8T cells; group 6, 3106total splenocytes; and group 7,

3⫻106splenocytes from which CD8T cells were depleted using anti-CD8 and complement). (B) Quantitative analysis of chemokine mRNA expression. Columns represent averages⫾SD of the results from two to four mice.

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may migrate in response to this chemokine, the presence of MIP-1␣ is not mandatory for T-cell-mediated inflammation. For this reason, we propose that prior studies indicating a role for this chemokine in virus-induced inflammation be reinter-preted. Thus, it is possible that MIP-1␣ may serve a more critical role in attracting effector cells other than T cells, and the critical involvement of this chemokine in the context of certain viral infections may therefore reflect a pivotal role played by non-T-effector cells, e.g., NK cells, in the murine cytomegalovirus infection model (27, 28).

This study was supported in part by the Danish Medical Research Foundation and the Novo-Nordisk Foundation. A.N.M. is the recipi-ent of a scholarship from the Novo Nordisk Foundation. J.P.C. is the recipient of a research fellowship from the Alfred Benzon Foundation, Copenhagen, Denmark.

We thank Grethe Thørner Andersen and Lone Malte for expert technical assistance.

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[image:6.603.48.275.67.241.2]

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FIG. 6. Outcome of peptide-induced and SRBC-induced DTH. (A) Peptide-induced footpad swelling was elicited in mice infected with 200 PFU of LCMV Traub 8 days earlier. Mice were challenged in their right hind footpads with 30␮l of LCMV GP33-41 peptide (10

␮g/ml), and the induced footpad swelling was measured at 16, 24, 48, and 72 h after peptide injection. (B) Mice were immunized subcuta-neously with 0.1 ml of a freshly washed suspension of SRBC (2% in saline). Five days later, mice were challenged in their right hind foot-pads with 30␮l of 1.25% SRBC, and footpad swelling was measured at 16, 24, and 48 h after challenge. Averages⫾SD of four to five mice are presented. Results of one of two or three similar experiments are presented.

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lymphocyte and delayed-type hypersensitivity mediating T lymphocyte activ-ity in parallel. J. Immunol.142:1333–1341.

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35. Walsh, C. M., M. Matloubian, C. C. Liu, R. Ueda, C. G. Kurahara, J. L. Christensen, M. T. Huang, J. D. Young, R. Ahmed, and W. R. Clark.1994. Immune function in mice lacking the perforin gene. Proc. Natl. Acad. Sci. USA91:10854–10858.

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Figure

FIG. 1. Activated T cells migrate toward a MIP-1�10were infected with 200 PFU of LCMV Traub
FIG. 3. Kinetics of LCMV-induced inflammation in MIP-1�depicted) revealed an identical composition of CSF cells (CD8and wild-type mice
FIG. 4. Cerebral chemokine transcripts in MIP-1�type mice following i.c. infection with LCMV
FIG. 5. Antigen-specific T cells are the main source of MIP-1�with 200 PFU of LCMV Traub, total RNA was isolated from the brain, and 203expression
+2

References

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