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Journal of Virology, July 2003, p. 7393-7400, Vol. 77, No. 13
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.13.7393-7400.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

The Virus-Encoded Chemokine vMIP-II Inhibits Virus-Induced Tc1-Driven Inflammation

Morten Lindow,1,{dagger} Anneline Nansen,2,{ddagger} Christina Bartholdy,2 Annette Stryhn,2,{ddagger} Nils J. V. Hansen,2,§ Thomas P. Boesen,1,|| Timothy N. C. Wells,3 Thue W. Schwartz,1 and Allan R. Thomsen2*

Laboratory for Molecular Pharmacology,1 Institute of Medical Microbiology and Immunology, The Panum Institute, University of Copenhagen, Copenhagen, Denmark,2 Serono International SA, Geneva, Switzerland3

Received 24 January 2003/ Accepted 8 April 2003


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ABSTRACT
 
The human herpesvirus 8-encoded protein vMIP-II is a potent in vitro antagonist of many chemokine receptors believed to be associated with attraction of T cells with a type 1 cytokine profile. For the present report we have studied the in vivo potential of this viral chemokine antagonist to inhibit virus-induced T-cell-mediated inflammation. This was done by use of the well-established model system murine lymphocytic choriomeningitis virus infection. Mice were infected in the footpad, and the induced CD8+ T-cell-dependent inflammation was evaluated in mice subjected to treatment with vMIP-II. We found that inflammation was markedly inhibited in mice treated during the efferent phase of the antiviral immune response. In vitro studies revealed that vMIP-II inhibited chemokine-induced migration of activated CD8+ T cells, but not T-cell-target cell contact, granule exocytosis, or cytokine release. Consistent with these in vitro findings treatment with vMIP-II inhibited the adoptive transfer of a virus-specific delayed-type hypersensitivity response in vivo, but only when antigen-primed donor cells were transferred via the intravenous route and required to migrate actively, not when the cells were injected directly into the test site. In contrast to the marked inhibition of the effector phase, the presence of vMIP-II during the afferent phase of the immune response did not result in significant suppression of virus-induced inflammation. Taken together, these results indicate that chemokine-induced signals are pivotal in directing antiviral effector cells toward virus-infected organ sites and that vMIP-II is a potent inhibitor of type 1 T-cell-mediated inflammation.


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INTRODUCTION
 
Because the immune system is composed of single cells, its functional integrity is entirely dependent on the capacity of these cells to traffic, localize, and interact with each other in a timely and precisely controlled manner. Chemokines, capable of activating leukocytes rolling along the endothelium and generating chemotactic gradients in the extracellular space, are key coordinators of these processes. In addition to these physiological roles, it is becoming increasingly evident that chemokines play a crucial role in the pathophysiology of inflammatory and autoimmune diseases (16), and they are regarded as rational targets for the development of anti-inflammatory agents (25).

However, antichemokine therapy is hampered by the general redundancy and pleiotropy within the chemokine network (i.e., most chemokines bind more than one receptor, most receptors bind more than one chemokine, and most leukocytes express and respond to more than one chemokine receptor), rendering the chemokine network very robust against intervention (26). Molecules that have the capacity to bind and antagonize multiple types of chemokine receptors could provide a tool to overcome this potential redundancy. Such a protein has evolved in human herpesvirus 8 (HHV-8) and been termed viral macrophage inflammatory protein II (vMIP-II) (27). vMIP-II has been characterized as a functional in vitro antagonist on the receptors CCR1, CCR2, CCR5, CCR8, CCR10, CXCR3, and CXCR4 in addition to the lymphotactin (XCR1) and fractalkine (CX3CR1) receptors (13, 19, 23, 24), while it is an agonist on CCR3 (5). Thus, vMIP-II acts on receptors for all the chemokine subfamilies. So far most of the existing information on the function of vMIP-II has come from in vitro studies. Fortunately, however, chemokines and their receptors are very well conserved and usually cross-reactive between species (reference 22 and references therein), and this permits the use of animal models to conduct in vivo experiments. Thus, in a recent in vivo trial of vMIP-II in a rat model of glomerulonephritis, it has been found that treatment with vMIP-II intravenously (i.v.) was able to inhibit leukocyte infiltration and attenuate proteinuria (7). However, vMIP-II has not yet been evaluated in a model system imitating the setting that it probably was evolved to work in, namely, virus-induced inflammation.

Most viral infections are controlled through cell-mediated immunity, and since T cells require close contact in order to exert their function, rapid and efficient targeting of effector T cells to areas of viral replication is pivotal to an optimal antiviral immune response. The recruitment of effector cells to sites of viral infection is generally thought to follow the normal sequence of leukocyte extravasation, i.e., rolling along the endothelium, followed by chemokine-triggered attachment and transmigration, and further directed migration along chemotactic gradients. However, despite the likelihood that chemokines are centrally placed in the regulation of virus-induced effector cell recruitment, our understanding of the biological relevance of chemokines in antiviral immunity is still quite limited. Again analysis of the role of individual chemokines is hampered by the redundancy of the system, often leading to negative findings.

To obtain a better understanding of how effector T cells are targeted to sites of inflammation, we have for a number of years been using the murine lymphocytic choriomeningitis virus (LCMV) model. LCMV is a noncytolytic virus that causes no inflammation unless virus-specific T cells are present (14). However, the presence of virus-specific effector T cells leads to substantial inflammation of the infected area. The inflammatory exudate is well characterized and consists predominantly of CD8+ Tc1 cells, whereas few CD4+ Th cells are found in infected organs during acute infection (9, 11, 14). The formation of this Tc1-dependent inflammatory exudate is intimately associated with both virus clearance and immunopathology (21, 28), and previous studies have revealed that inhibition of T-cell migration by using antibodies to block critical adhesion molecules (VLA-4, LFA-1, and Mac-1) leads to a reduced inflammatory response evaluated in terms of virus-specific delayed-type hypersensitivity (DTH) (35). Notably, we have also previously found that the inflammatory exudate is dominated by cells expressing CCR1, CCR2, and CCR5 (31), but functional analysis has not been carried out, except that we have found CCR5 to be redundant based on studies in knockout mice (29). As negative findings, however, are likely to reflect the functional redundancy within the chemokine system, we have taken the opportunity to test whether simultaneous inhibition of multiple chemokine-receptor interactions will result in suppression of LCMV-induced inflammation. In the present report we have therefore investigated the ability of vMIP-II to inhibit Tc1-cell-mediated inflammation by using this well-established viral model system, in order to probe a truly broad-spectrum blocker of chemokine receptors.


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MATERIALS AND METHODS
 
Mice. Female C57BL/6 (B6) mice were purchased from Taconic M & B (Ry, Denmark). CCR5-deficient mice (CCR5-/-) (B6;129P-CmKbr5<tm/Kn2>) were bred locally at The Panum Institute from breeding pairs obtained from The Jackson Laboratory (Bar Harbor, Maine). The animals were always allowed to acclimatize to the local environment for at least 1 week before starting experiments; by that time they were 7 to 10 weeks old. Mice were housed under specific-pathogen-free conditions as validated by screening of sentinels according to Federation of European Laboratory Animal Science Associations criteria. All animal experiments were conducted in accordance with national guidelines.

Virus. LCMV of the Traub or Armstrong (Arm) strain was used; viral stocks were produced, stored, and quantitated as previously described (30).

DNA vaccination. The eukaryotic expression vector pcDNA3.1/zeo+ (Invitrogen, Carlsbad, Calif.) containing the cytomegalovirus immediate-early gene enhancer and promoter was used. A vector containing a major histocompatibility complex (MHC) class I-restricted immunogenic peptide epitope (GP33-41) connected to human ß2-microglobulin through a linker of 10 amino acids was used (8). The construct also included the leader sequence of murine ß2-microglobulin inserted ahead of the immunogenic epitope to ensure translocation of the protein into the endoplasmic reticulum. Here, the encoded peptide-human ß2-microglobulin fusion product can assemble with MHC class I heavy chain to produce the intact trimeric MHC class I complex which stimulates CD8+ T cells. The vMIP-II vector was constructed from DNA cloned from Kaposi's sarcoma skin lesions as described previously (19) and matched the sequence from GenBank (accession no. U75698). The DNA, complete with signal peptide, was cloned into pcDNA3.1/Zeo+ as described above. For preparation of vaccines used in this study, purified GP33-41 DNA was mixed with equal amounts of DNA encoding vMIP-II or empty vector for control. DNA was then coated onto 1.6-µm-diameter gold particles at a concentration of 2 µg of DNA/mg of gold, and the DNA-gold complex was applied as a coating to plastic tubes so that 0.5 mg of gold was delivered to the mouse per shot (1 µg of DNA per shot). These procedures were performed according to the manufacturer's instructions (Bio-Rad Laboratories, Hercules, Calif.). Mice were immunized on shaved abdominal skin with the handheld GeneGun device employing compressed helium (400 lb/in2) as the particle motive force. Animals were immunized four times at intervals of 1 week and then allowed to rest for 1 week before direct ex vivo analysis.

Cell preparations. Spleens were aseptically removed and transferred to Hanks' balanced salt solution. Single-cell suspensions were obtained by pressing the organs through a fine sterile steel mesh, and when necessary, erythrocytes were lysed by 0.83% NH4Cl treatment.

Assay of IP-10 (CXCL10)-induced migration. Cell suspensions from mice infected with 200 PFU of LCMV Traub 7 days earlier were obtained as described above. After the final wash the cells were resuspended in culture medium (RPMI medium containing 10% fetal calf serum, 2-mercaptoethanol, and L-glutamine) and subsequently incubated for 1 h in 5% CO2 at 37°C with or without vMIP-II in concentrations varying from 10-8 to 10-6 M. Migration assays were performed with Transwell chambers with culture inserts of 6.5-mm and 5-µm pore size (Corning Costar Corp., Acton, Mass.). Recombinant murine IP-10-CXCL10 (R & D Systems, Minneapolis, Minn.) was diluted in assay medium to a pretitrated optimal concentration of 100 ng/ml, and chemokine solution or assay medium alone was added to the lower chamber of Transwells in a final volume of 600 µl. Then filter inserts were placed in the wells, 1.5 x 106 splenocytes were added to the top chamber in a volume of 100 µl, and the chamber was incubated for 3 h in 5% CO2 at 37°C. The total number of migrated cells was counted. To identify activated CD8+ T cells, a fraction of the cells were stained for surface expression of CD8 and VLA-4 and analyzed by flow cytometry. Results are presented as % transmigrated CD8+ VLA-4high cells = (number of transmigrated CD8+ VLA-4high cells/total number of input CD8+ VLA-4high cells) x 100. All determinations were performed in triplicate.

Cytotoxic assay. Virus-specific cytotoxic T-lymphocyte activity was assayed in a standard 51Cr release assay. Effector cells were splenocytes from mice infected 8 days earlier with 200 PFU of LCMV Traub. Target cells were histocompatible EL-4 cells pulsed for 1 h with either LCMV GP33-41 or LCMV NP396-404. Unpulsed EL-4 cells served as control targets. To study the inhibitory effect of vMIP-II, recombinant protein was added to the wells to final concentrations of 1 and 0.1 µg/ml. The assay time was 6 h, and percent specific 51Cr release was calculated as described previously (29). All determinations were performed in triplicate.

In vitro stimulation and enzyme-linked immunosorbent assay for MIP-1{alpha} (CCL3). T-cell production of MIP-1{alpha} was analyzed by in vitro restimulation of LCMV-specific T cells with cognate peptide. Briefly, splenocytes (2 x 105 cells per well in round-bottomed 96-well plates) from mice infected 8 days earlier with 200 PFU of LCMV Traub were stimulated in vitro with immunodominant class I-restricted peptide (LCMV GP33-41 or NP396-404, 0.1 µg/ml) or left unstimulated. To study the inhibitory effect of vMIP-II, recombinant protein was added to triplicate wells at concentrations of 1 and 0.1 µg/ml. After 6 h of stimulation cell-free supernatants were harvested and stored at -70°C until analysis. Cytokine levels were determined by a sandwich enzyme-linked immunosorbent assay (R & D Systems).

Monoclonal antibodies for flow cytometry. The following monoclonal antibodies were all purchased from PharMingen (San Diego, Calif.) as rat anti-mouse antibodies: fluorescein isothiocyanate-conjugated anti-CD44, fluorescein isothiocyanate-conjugated anti-VLA-4, Cy-Chrome-conjugated anti-CD8a, and phycoerythrin-conjugated anti-gamma interferon (IFN-{gamma}).

Flow cytometric analysis. For enumeration of LCMV-specific (IFN-{gamma}-producing) CD8+ cells, 1 x 106 to 2 x 106 splenocytes were incubated for 5 h at 37°C in 0.2 ml of complete RPMI medium supplemented with 50 U of murine recombinant interleukin-2 (R & D Systems)/ml, 3 µM monensin (Sigma Chemical Co., St. Louis, Mo.), and 0.1 µg of relevant peptide per ml. MHC class I (H-2Db)-restricted GP33-41 or NP396-404 was used as the peptide; wells without peptide served as background controls. After incubation cells were surface stained, washed, permeabilized, and stained with IFN-{gamma}-specific monoclonal antibody (3).

Cells were analyzed with a FACSCalibur (Becton Dickinson, San Jose, Calif.), and at least 104 cells were gated by using a combination of low angle and side scatter to exclude dead cells and debris. Data analysis was conducted with Cell-Quest software.

Virus-induced DTH. LCMV-specific DTH was assessed either in intact mice infected locally in a hind footpad or following adoptive transfer of virus-primed donor cells to naive recipients infected shortly before the cell transfer (32). For analysis of intact animals, mice were infected in the right hind footpad with 200 PFU of LCMV Arm in 0.03 ml, and footpad thickness was measured from day 5 postinfection (p.i.). For adoptive transfer of primed cells, donor mice were infected i.v. with approximately 200 PFU of LCMV Traub 8 days prior to transplantation. Before adoptive transfer the donor cells were depleted of adherent cells (mainly macrophages and stromal cells) by incubation in tissue culture flasks for 60 min at 37°C, followed by gentle resuspension and recovery of nonadherent cells. In addition, the cells were treated with mitomycin C (25 µg/ml for 30 min at 37°C) to prevent further clonal expansion in the recipients. Recipients were naive mice infected in the footpad with approximately 2 x 106 PFU of LCMV Traub in 0.03 ml 4 h before cell transfer. Two types of adoptive transfer systems were applied. (i) For systemic transfer, recipients were infected in the right hind footpad and subsequently received 50 x 106 virus-primed donor cells i.v.; footpad thickness was measured 16, 24, 48, and 72 h after cell transfer. (ii) For local transfer, both hind footpads were infected as described above. Four hours later, 3 x 106 LCMV-primed donor cells in 0.03 ml were injected into the right hind footpad, and 3 x 106 similarly treated naive spleen cells were injected into the left hind footpad as a control. Footpad thickness was again measured 16, 24, 48, and 72 h after cell transfer. In all experiments footpad thickness was measured with a dial caliper (Mitutoyo Co., Tokyo, Japan), and virus-specific swelling was calculated as the difference in thickness between the hind feet.

RNase protection assay. Total RNA was extracted from the organs of LCMV-infected and control mice as previously described (31). Chemokine transcripts were analyzed with the mCK-5 multitemplate probe set (PharMingen). Chemokine receptor transcripts were analyzed with a custom-made template set (also from PharMingen) enabling detection of CCR1, -2, -3, -4, -5, -7, and -8 and CXCR3. Both template sets included probes for the housekeeping genes L32 and glyceraldehyde-3-phosphate dehydrogenase; these served as loading controls. The assays were performed according to the manufacturer's instructions, and protected fragments were visualized by autoradiography as previously described (31).

vMIP-II and Met-RANTES. vMIP-II was cloned into the pET-24d expression vector and expressed in the Escherichia coli strain BL21(DE3)pLysS growing in brain heart infusion medium. The protein was refolded from inclusion body material and subsequently purified by cation-exchange and reverse-phase chromatography as previously described (15). The mature and refolded protein had the sequence LGASWHRPDKCCLGYQKRPLPQVLLSSWYPTSQLCSKPGVIFLTKRGRQVCADKSKDWVKKLMQQLPVTAR. Met-RANTES was produced as previously described (33).

Statistics. Groups of treated and untreated animals were compared, where applicable at each time point, with the Mann-Whitney rank sum test.


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RESULTS
 
Characterization of potential chemokine-chemokine receptor interactions in LCMV-infected organs. From previous studies we know that the majority of LCMV-specific CD8+ T cells and monocytes/macrophages invade infected organs around day 7 after infection (29, 31). Therefore, to determine the spectrum of potential chemokine-receptor interactions involved in directing antiviral effector cells to sites of LCMV replication, total RNA was extracted from brains, lungs, and livers of uninfected control mice and mice infected with LCMV 7 days earlier. Using RNase protection assays, we compared expression of chemokines and chemokine receptors in these organs. This analysis (Fig. 1) revealed that, while some chemokines might be produced to a higher degree in certain organs, the overall pattern was that the range of chemokines transcribed in LCMV-infected organs is essentially independent of the tissue of origin and primarily involves RANTES (CCL5), IP-10 (CXCL10), MCP-1 (CCL2), and to a lesser extent MIP-1{alpha} (CCL3) and -ß (CCL4). Corresponding to this chemokine profile, the chemokine receptors found to dominate in inflamed organs are CCR1, -2, and -5 together with CXCR3. In addition we found CCR7 (a chemokine receptor required for entry into lymph nodes and mostly expressed by naive cells) to be expressed in the lungs. However, the expression of this receptor was not upregulated as a result of inflammation, and its presence might reflect the fact that the bronchial lymph node (mostly naive cells) is included in the lung tissue used to prepare the lung RNA samples. Taken together, these results indicate that most chemokine-receptor interactions likely to play a role in effector cell recruitment to LCMV-infected organ sites would be inhibited by vMIP-II.



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  		FIG. 1. RNase protection assays showing chemokine (A) and chemokine receptor (B) transcription in inflamed LCMV-infected organs. Mice were infected with 200 PFU of LCMV Traub either intracerebrally (for brain samples) or i.v. (for liver and lung samples), and 7 days later (7 days p.i.) organs were harvested from these mice and uninfected controls (uninf.). Total RNA was isolated, and 20 µg was subjected to RNase protection analysis. Each lane represents a single mouse. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

vMIP-II attenuates LCMV-induced footpad swelling. To study the ability of vMIP-II to inhibit virus-induced inflammation, LCMV-induced footpad swelling was used as a model system. Injection of LCMV into the footpad of adult, immunocompetent mice induces a marked inflammatory response that becomes measurable around day 6 p.i. This DTH reaction closely models virus-induced inflammation in infected organs that is dependent on virus-specific CD8+ T effector cells recognizing viral antigen in the local environment (10, 12). To investigate the impact of vMIP-II on this T-cell-mediated response, mice were infected with LCMV, and approximately 24 h before the expected onset of inflammation, i.v. treatment with vMIP-II was initiated. Treatment was repeated every 12 h, and a total of four injections were given. Five micrograms of vMIP-II per i.v. injection was unable to significantly reduce the swelling (Fig. 2A). However, when the dose was increased to 20 µg per injection, the treatment markedly delayed and attenuated the DTH reaction (Fig. 2B), thus demonstrating the anti-inflammatory potential of vMIP-II.



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  		FIG. 2. Influence of vMIP-II dose on the ability to inhibit LCMV-induced footpad swelling. B6 mice were infected with 200 PFU of LCMV Arm in the right hind footpad, and at the indicated time points half the mice were injected i.v. with vMIP-II or vehicle. Four consecutive injections of 5 µg did not affect inflammation (A), while 20 µg markedly delayed and attenuated the swelling (B). Values represent medians and ranges of five animals. *, P < 0.05.

vMIP-II does not inhibit the afferent phase of Tc1-mediated immunity. In the above experiment treatment was administered primarily during the efferent phase of the antiviral immune response; however, we also wanted to examine whether vMIP-II might modulate the afferent phase, i.e., the induction of the antiviral Tc1 response. To study that, two different approaches were used.

First, mice were systemically treated with vMIP-II protein as described above, but this time a group of animals was included in which the mice were subjected to four injections of vMIP-II during the first 2 days of the infection. This vMIP-II treatment during the induction phase of the immune response did not lead to significant reduction in the inflammatory response, while similar treatment (similar doses and intervals) during the efferent phase again proved effective (Fig. 3A).



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  		FIG. 3. Effect of vMIP-II treatment on footpad swelling and antigen-specific T-cell frequencies following treatment during different phases of the immune response. Mice were infected with 200 PFU of LCMV Arm in the footpads and treated with i.v. injections of 20 µg of vMIP-II either early (days 0.5, 1, 1.5, and 2 p.i.), late (days 5.5, 6, 6.5, and 7 p.i.), or not at all (control). (A) LCMV-induced footpad swelling; values represent medians and ranges of five animals from one representative experiment. *, P < 0.05. (B) Frequency of LCMV-specific T cells. On day 9 p.i., mice were sacrificed, and splenocytes were stimulated with either of two immunodominant LCMV epitopes (GP33-41 and NP396-404). After 5 h of stimulation cells were stained for CD8, permeabilized, and stained for IFN-{gamma} intracellularly. Points represent frequencies for individual animals; gates have been set for CD8+ cells. Without peptide stimulation <0.2% of the cells stained positive for IFN-{gamma}.

To directly evaluate the degree of CD8+ T-cell priming in vMIP-II treated mice, the mice were euthanized on day 9 p.i., and the total number of splenic CD8+ T cells as well as the frequency of CD8+ cells specific for two immunodominant viral epitopes (GP33-41 and NP396-404) was determined by intracellular cytokine staining and flow cytometry. As the frequency of virus-specific T cells in the spleen of mice infected via the footpad increases from about background to a readily detectable level between day 7 and 9 p.i., analysis on day 9 p.i. readily reveals any delay in T-cell priming (e.g., in L-selectin-deficient mice in which priming in the peripheral lymph nodes is abolished [J. P. Christensen and A. R. Thomsen, unpublished results]). Similar numbers of splenic CD8+ T cells were found in all groups (~4 x 107/spleen; P > 0.1). As regards the frequencies of antigen-specific CD8+ T cells (Fig. 3B), the only statistically significant difference was observed between controls and mice treated late, and only with regard to one epitope (GP33-41). However, as no significant differences were noted between the two treated groups yet inhibition of inflammation was observed only in the latter, we conclude that inhibition of effector T-cell generation is not the major mechanism through which vMIP-II inhibits LCMV-induced inflammation.

In addition, to more closely simulate a viral infection and have vMIP-II produced at the same site as the antigen, we performed direct GeneGun-mediated transfections of the abdominal skin with naked DNA. All mice were transfected with DNA encoding the major LCMV epitope GP33-41 under the control of the cytomegalovirus promoter. Half the animals were cotransfected with a plasmid encoding vMIP-II, while the other half were cotransfected with empty vector for a control. As the plasmids were applied together as a coating to the gold particles, all cells taking up a gold particle should express both constructs. Mice were vaccinated four times with 1-week intervals, and 1 week after the last immunization, frequencies of GP33-41-specific CD8+ T cells were determined by flow cytometry. While GP33-41-specific cells were undetectable in unvaccinated mice (<0.2%), a distinct population of primed (CD44high) GP33-41-specific cells was found in both groups of vaccinated mice, and no significant difference was observed between mice covaccinated with vMIP-II (2.7% ± 0.7%) and control mice (3.8% ± 1.9%) (mean ± standard deviation [SD] of four mice/group; P > 0.1). No response above background was seen when mice were tested with NP396-404 as control peptide (all ≤0.2%). Thus, we could find no evidence indicating that vMIP-II significantly inhibited the induction of an antigen-specific CD8+ T-cell response.

vMIP-II inhibits migration, but not effector mechanisms, of virus-specific CD8+ T cells. The above results suggest that vMIP-II inhibits the effector phase of the virus-specific CD8+ T-cell response. However, it is not clear whether this is due to inhibition of cell migration toward the infected site or whether vMIP-II directly interferes with CD8+ T-cell-mediated effector functions such as cell-contact-dependent target cell killing or release of cytokines. To evaluate these possibilities, we tested the ability of vMIP-II to inhibit (i) migration of activated CD8+ T cells in vitro, (ii) cell-cell interaction and target cell killing, and (iii) cytokine release. Previous studies have shown that activated CD8+ T cells are characterized by high expression of VLA-4, and CD8+ T cells with this phenotype dominate in LCMV-infected organs (1, 9); most of these cells are LCMV specific as revealed by tetramer staining or intracellular staining for IFN-{gamma} (2, 11). Using a double-chamber assay to evaluate cell migration, we found that relatively low doses of vMIP-II inhibited IP-10 (CXCL10)-induced recruitment of VLA-4high CD8+ T cells (Fig. 4) (in a repeat experiment 10-7 M also inhibited migration). In contrast, vMIP-II did not inhibit target cell lysis evaluated in a conventional 51Cr release assay (Fig. 5, upper panel). Nor did these levels of vMIP-II inhibit peptide-induced release of MIP-1{alpha} (Fig. 5, lower panel), one of the involved proinflammatory cytokines.



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  		FIG. 4. vMIP-II inhibits chemokine-induced in vitro migration of virus-activated CD8+ T cells. Splenocytes from mice infected with 200 PFU of LCMV Traub 7 days earlier were incubated with or without vMIP-II for 1 h and then allowed to migrate toward IP-10 for 3 h in a double-chamber assay. The percentages of CD8+ VLA-4high cells that have migrated into the lower, chemokine-containing chamber are presented. Averages ± SDs are presented.



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  		FIG. 5. vMIP-II does not inhibit target cell lysis or chemokine production-release in vitro. Splenocytes from mice infected with 200 PFU of LCMV Traub 8 days earlier were incubated with peptide-coated (GP33-41 or NP396-404) or uncoated target cells for 6 h in the presence or absence of vMIP-II (upper panel). Alternatively, splenocytes were incubated with these peptides (0.1 µg/ml) for 6 h and the level of chemokine released into the supernatant was determined (lower panel); if no peptide was added, <100 pg/ml was produced (indicated by stippled lines). Averages (± SDs) are presented; for the cytotoxicity assay SDs were too small to be readily depicted.

vMIP-II effectively inhibits DTH by blocking effector cell migration. To confirm the ability of vMIP-II to inhibit the effector phase of Tc1-mediated inflammation, we isolated the efferent phase of the cellular immune response, by employing an adoptive transfer system (32). Virus-specific primary effector T cells were generated in donor mice infected with the virus i.v. Before cell transfer, donor splenocytes were depleted of macrophages by incubation in plastic flasks, and only nonadherent cells were transferred to the recipients. In addition donor cells were treated with mitomycin C—a mitosis blocker—to prevent clonal expansion in the recipients. Four hours before cell transfer, recipients were injected with LCMV in their footpads. Previous analysis has shown that no inflammation is detectable for at least 5 days after infection unless virus-primed splenocytes (including antigen-specific CD8+ T cells) are used for adoptive transfer (32) To evaluate the effect of vMIP-II, recipients were injected with 5 µg of vMIP-II i.v. 4 h before transplantation, at the time of transplantation (mixed with the donor cells), and after a further 16 h. Under this regimen the marked attenuation of virus-specific DTH observed in intact, virus-infected mice was fully reproduced in the adoptive model (Fig. 6A).



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  		FIG. 6. Influence of vMIP-II treatment on adoptively transferred DTH. Recipient mice were infected with 2 x 106 PFU of LCMV Traub in the footpad 4 h before adoptive transfer of day 8 virus-primed, adherent cell-depleted splenocytes. Either cells were transplanted i.v. and recipients were treated i.v. (A), or cells and treatment were administered at the site of viral challenge (B). Values represent medians and ranges of five animals. *, P < 0.05.

If our in vitro analysis correctly reflected the mechanism underlying vMIP-II-mediated inhibition of the virus-induced T-cell-mediated inflammatory response, vMIP-II should inhibit the effector response only if the antigen-primed effector cells were required to actively home to areas of infection, but not if they were coinjected with the cells directly into the test site. To test the latter part of this prediction, an additional adoptive transfer assay was carried out, but instead of the donor cells being transplanted i.v., they along with vMIP-II were injected locally in the footpad at the site of viral challenge. In this case no inhibitory effect of vMIP-II was detected (Fig. 6B).

Finally, to compare the in vivo potency of vMIP-II with that of another chemokine antagonist sharing the ability to block CCR1 and CCR5, transfer experiments were conducted in which Met-RANTES was substituted for vMIP-II. However, with doses of Met-RANTES of up to 100 µg per treatment we did not see any inhibition (Fig. 7A), which should be compared to the fact that, in parallel experiments, doses of 5 µg of vMIP-II almost completely inhibited adoptive transfer of LCMV-specific DTH. Similarly CCR5-/- mice treated with Met-RANTES did not show any attenuation of DTH (Fig. 7B).



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  		FIG. 7. Influence of Met-RANTES treatment on adoptively transferred DTH in wild-type B6 mice (A) or CCR5-/- mice (B). Values represent medians and ranges of five animals; open symbols represent untreated mice, while closed symbols represent Met-RANTES-treated animals.


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DISCUSSION
 
In this report we have shown that the virus-encoded chemokine vMIP-II is capable of dramatically reducing antigen-specific T-cell-mediated inflammation in a classical model for virus-induced inflammation.

In an adoptive transfer system, we find that virus-induced DTH can be almost completely blocked by vMIP-II, when primed donor cells are transplanted i.v. In contrast, no blocking effect is observed when the primed cells are injected directly into the site of viral challenge. This indicates that cells already on location are fully functional in the presence of vMIP-II and points to the conclusion that the major effect of vMIP-II lies in an ability to inhibit migration of effector cells to the infected footpad. This interpretation is fully consistent with our in vitro experiments showing that vMIP-II inhibits chemokine-induced migration of activated CD8+ T cells but not the interaction of effector T cells with target cells nor granule exocytosis and cytokine release. The precise cellular target of the inhibitory effect in vivo is not absolutely clear, as both CD8+ effector cells and monocytes/macrophages express receptors (31) that may be blocked by vMIP-II. However, the in vitro inhibition of CD8+ T-cell migration suggests that this population is a relevant target also in vivo.

Analysis of the number and proportion of antigen-specific T cells in animals treated with vMIP-II during both the induction and the effector phases of the antiviral immune response showed that vMIP-II had little or no effect on the activation, expansion, and differentiation of these cells, indicating that the viral protein when administered systemically primarily interacts with the effector cells of the immune system.

By administering DNA encoding vMIP-II via direct transfection of the skin, we tried to create a pseudoinfection, where vMIP-II is expressed at the same time and place as a proven antigen. However, both vMIP-II- and empty-vector-treated animals were able to mount a CD8+ T-cell response against the encoded antigenic epitope, which indicates that vMIP-II does not inhibit induction of antiviral cell-mediated immunity. This is in agreement with previous reports showing that vMIP-II is capable of reducing inflammation in models not associated with the generation of antigen-specific T cells.

Thus, it has previously been demonstrated that vMIP-II infusion is capable of reducing disease parameters in a rat model of glomerulonephritis (7) and damage after spinal cord contusion (17). However, the nature of the inflammatory response in our model is clearly different, because it involves recognition of a specific (viral) antigen, i.e., antigen-specific CD8+ Tc1 cells play a key role in LCMV-induced DTH (10, 32). Nevertheless, both LCMV-induced DTH and the glomerulonephritis model (18) are characterized by an accumulation of CD8+ cells and monocytes/macrophages at the site of inflammation.

Compared to antagonism and targeted deletion of cell adhesion molecules—which are also important in leukocyte migration—vMIP-II is at least as effective at inhibiting LCMV-induced inflammation as is blocking of VLA-4, which previously held the record of maximal DTH reduction in this LCMV model (9, 35). However, it is noteworthy that neither CCR5 knockouts (Fig. 7B) (29) nor mice treated with Met-RANTES (a CCR1 and CCR5 antagonist) show any significant reduction in LCMV-induced DTH. This could indicate that, although CCR1 and CCR5 are among the receptors interacting with vMIP-II, blocking these receptors is not sufficient to inhibit DTH. Whether a combined CCR1/CCR5 block is necessary could be investigated by using combinations of more selective antagonists and/or knockout animals.

As already mentioned, many studies indicate that more than one chemokine (and chemokine receptor) is responsible for the recruitment of any individual cell type in inflammatory reactions. Given that chemokines are rarely produced individually, this redundancy makes it essential to block multiple receptor-ligand interactions in order to obtain a significant effect, and this is probably the reason why vMIP-II is so uniquely effective. In this respect it should be noted that most of the chemokines known to be produced in the context of acute viral infection, including LCMV (e.g., RANTES (CCL5), IP-10 (CXCR3), MCP-1 (CCL2), and MIP-1{alpha} (CCL3) [4, 31]) interact with receptors blocked by vMIP-II. Further underscoring the inhibitory potential of vMIP-II, we find that the dominant chemokine receptors transcribed in association with T-cell-mediated inflammation in virus-infected organs are CCR1, -2, and -5 (17) together with CXCR3 (Fig. 1).

The Th1 and Tc1 subsets of CD4+ or CD8+ T cells are known to play an important role in many types of T-cell-mediated inflammation, and type 1 effector cells are in most cases crucial for an optimal antiviral immune response (6). These cells express several different chemokine receptors. However, a differential-preferential expression of chemokine receptors on both in vivo and in vitro polarized type 1 and type 2 effector T lymphocytes has been reported (reviewed by Syrbe et al. [34]). If one matches the receptor-binding profile of vMIP-II with this picture of differential chemokine receptor expression on activated T cells, an appealing picture emerges.

vMIP-II antagonizes CCR1, CCR2, and CXCR4, which all have been found to be expressed on both type 1 and type 2 effector T cells. But it also antagonizes CCR5 and CXCR3—two receptors that seem to be preferentially expressed on type 1 cells. Thus, vMIP-II would presumably preferentially block T cells with a type 1 cytokine profile. Furthermore, when vMIP-II is produced locally by HHV-8-infected cells its agonistic activity on CCR3 could further shift the local type 1/type 2 balance by recruiting CCR3-bearing type 2 T cells (37), provided that cells with the latter phenotype were generated during the antiviral T-cell response. Since antiviral immunity generally depends heavily on Tc1 cells (recently shown also for HHV-8 [36]), it is tempting to speculate that HHV-8 has evolved a highly efficient countermeasure preventing influx of this important effector cell type. The molecular piracy of a mammalian chemokine gene combined with a rapid genetic cycle has made it possible for the virus to develop efficient anti-inflammatory strategies by screening and selection on the host immune system. Evasion of the immune system is well known and has already been described for many microorganisms.

Even though we cannot as yet be sure that the above interpretation regarding the biological role of vMIP-II is correct—clearly vMIP-II would not normally be found in the general circulation—our study shows that potentially very valuable immunopharmacological information may be extracted from the genomes of human pathogens in general, and HHV-8 in particular. Therapeutically, it could be useful to exploit vMIP-II—the "viral drug"—to shift the local immune response away from a type 1 response. Th1-Tc1 cells are thought to be important mediators of autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and diabetes type I (20). It will be interesting to see if vMIP-II will be able to affect the pathogenesis of these important diseases.


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

This work was supported in part by the Danish Medical Research Council, the Biotechnology Center for Cellular Communication, and the Novo Nordisk Foundation.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Medical Microbiology and Immunology, University of Copenhagen, The Panum Institute 22.5.16, 3C Blegdamsvej, DK-2200 Copenhagen N, Denmark. Phone: 45 35 32 78 71. Fax: 45 35 32 78 91. E-mail: A.R.Thomsen{at}immi.ku.dk. Back

{dagger} Present address: Bioinformatics Centre, University of Copenhagen, Copenhagen, Denmark. Back

{ddagger} Present address: Statens Serum Institut, Copenhagen, Denmark. Back

§ Present address: Praecis Pharmaceuticals Inc., Waltham, Mass. Back

|| Present address: Maxygen A/S, Hørsholm, Denmark. Back


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Journal of Virology, July 2003, p. 7393-7400, Vol. 77, No. 13
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.13.7393-7400.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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