Vaccinia Virus Interleukin-18-Binding Protein Promotes Virulence by Reducing Gamma Interferon Production and Natural Killer and T-Cell Activity

Cells and viruses.Human TK⁻143B and D98OR cells and monkey BS-C-1 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (DMEM₁₀). The origins of the orthopoxviruses used here and their growth, titration, and purification were described previously (5).

Assay for virus virulence.Groups of female BALB/c mice between 6 and 8 weeks of age were anesthetized and infected intranasally with 10⁴ PFU of VV in 20 μl of phosphate-buffered saline (PBS). Each day, mice were weighed individually and monitored for signs of illness (2), and those suffering a severe infection or having lost >25% of their original body weight were sacrificed. To determine virus titers in organs, mice were sacrificed and their lungs, brains, and spleens were removed, Dounce homogenized, frozen and thawed three times, and sonicated. The titer of infectious virus was determined by plaquing on BS-C-1 cells.

Recovery of immune cells for flow cytometry and cytotoxic assays.BAL fluid and lung, spleen, and mediastinal lymph nodes (MLNs) were obtained from mock-infected and VV-infected mice 3 and 7 days postinfection (p.i.). To obtain BAL, mice were sacrificed, and the lungs of each mouse were inflated five times with a 1-ml volume of PBS containing 10 U of heparin per ml through a blunted 23-gauge needle inserted into the trachea. BAL was centrifuged at 3,000 rpm for 10 min in a Beckman bench-top centrifuge, and the supernatant was removed and frozen at −20°C for analysis of cytokines by enzyme-linked immunosorbent assay (ELISA). BAL cells were resuspended in Tris-NH₄Cl (0.14 M NH₄Cl in 17 mM Tris, adjusted to pH 7.2) to lyse erythrocytes, washed and resuspended in cold RPMI 1640 medium containing 10% fetal bovine serum. Single-cell suspensions of lung tissue, spleen, and MLN were prepared by sieving through a 100-μm-pore-diameter nylon mesh followed by hypotonic shock to lyse erythrocytes. Cell viability in all samples was assessed by trypan blue exclusion.

Cytotoxic assays.NK cell cytotoxicity and VV-specific CTL activity in single-cell suspensions from BAL, lung, spleen, and MLN was assayed with a standard ⁵¹Cr-release assay. NK-mediated lysis was tested on YAC-1 cells, while P815 cells (H-2^d, mastocytoma) were used as targets for VV-specific CTL lysis. Prior to labeling with Na₂⁵¹CrO₄ (150 μCi per 3 × 10⁶ cells), P815 cells were mock infected or infected with VV WR at 10 PFU per cell for 2 h at 37°C. Uninfected YAC-1 cells were labeled as described above. Serial dilutions of effector cells were incubated in triplicate cultures with either noninfected or VV-infected target cells in 100 μl of RF₁₀ in 96-well V-bottomed plates at 37°C in 5% CO₂. After 4 h (YAC-1) or 6 h (P815) cells were collected by centrifugation, and 50 μl of supernatant was transferred to a Lumaplate-96 (Packard Instrument Company, Inc.) and counted. The percentage of specific ⁵¹Cr release was calculated as specific lysis = [(experimental release − spontaneous release)]/(total detergent release − spontaneous release)] × 100. The spontaneous release values were always <10% of total lysis.

In some experiments, asialo-GM₁⁺ or CD8⁺ cells were depleted from lung cell suspensions by incubation at 37°C with an anti-CD8 monoclonal antibody (MAb) (clone 3.115 [47]) or with anti-asialo-GM₁ (Wako Pure Chemicals) in the presence of human complement. Analysis by flow cytometry revealed selective depletion of the desired cell populations. Depleted cells were added to cytotoxicity assays without adjustment for the depletion in cell number.

In vitro infection of lung macrophages.To obtain lung macrophages, a BAL procedure was carried out on naive BALB/c mice as described above. Approximately 0.5 × 10⁵ to 1 × 10⁵ cells were consistently obtained, and more than 95% of these cells were macrophages. These cells were resuspended in DMEM₁₀ and cultured in 96-well plates at 0.1 × 10⁶ cells per well. After 4 h, nonadherent cells were removed by three vigorous washes and the adherent macrophages were cultured with VV at 10 PFU per cell for 2 h. Cells were washed three times and cultured for 24 h before medium was removed, centrifuged at 3,000 rpm for 10 min, and the clarified supernatants frozen at −20°C.

ELISA for cytokines.Cytokines were measured in BAL and macrophage culture supernatants by ELISA. ELISA kits were from Pharmingen (IFN-γ, IL-10, and IL-4) or R&D Systems (IL-18).

Flow cytometric analysis of cell surface and intracellular antigens.Single-cell supensions of BAL, lung, spleen, or MLN cells were blocked with 10% normal rat serum and 0.5 μg of Fc block (Pharmingen) in fluorescence-activated cell sorter (FACS) buffer (PBS containing 0.1% bovine serum albumin and 0.1% sodium azide) on ice for 20 min. Cells were stained with appropriate combinations of fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, or quantum red (QR)-labeled anti-CD3, anti-CD8, anti-CD4, or anti-pan NK (DX5) and the relevant isotype antibody controls (all from Pharmingen, San Diego, Calif.). The distribution of cell surface markers was determined on a FACScan flow cytometer with CellQUEST software (Becton Dickinson, Mountain View, Calif.). A lymphocyte gate was used to select at least 20,000 events.

To detect intracellular cytokines, 10⁶ lung cells per ml was stimulated with 50 ng of phorbol myristate acetate (PMA) per ml (Sigma) and 500 ng of ionomycin per ml (Calbiochem) in the presence of 10 μg of brefeldin A per ml (Sigma) for 5 h at 37°C. Cells were washed with FACS buffer and stained with QR-conjugated CD4 and FITC-conjugated CD8 for 30 min on ice and then fixed for 30 min at room temperature with 2% paraformaldehyde in PBS. Samples were permeabilized with 0.5% saponin in FACS buffer for 10 min. PE-conjugated anti-mouse IFN-γ (clone XMG1.2; Pharmingen) was added for a further 30 min at room temperature and the cells were washed once with 0.5% saponin in FACS buffer and twice in FACS buffer alone. Cells were analyzed on a Becton Dickinson flow cytometer collecting data on at least 20,000 lymphocytes.

Nitrite determination.Measurement of nitrite in BAL fluids provides an indirect indication of the amount of nitric oxide (NO) production in the lung. Nitrite was measured colorimetrically at 540 nm by mixing 100 μl of BAL fluid with freshly prepared Greiss' reagent as described elsewhere (16). Each sample was assayed in duplicate, and sodium nitrite was used to generate standard curves.

Virulence of VV lacking C12L (vΔC12L).VV strain vΔC12L was attenuated in a murine model of infection (54). To investigate the basis for this phenotype, groups of mice were infected with wild-type (vC12L), deletion mutant (vΔC12L), or revertant (vC12L-rev) virus and monitored for weight loss (Fig. 1A), signs of illness (Fig. 1B), and infectious virus titers in lungs, spleen, and brain on days 3, 7, and 10 p.i. (Fig. 2). Levels of weight loss were similar between all infected groups on days 1 to 7; however, thereafter, mice infected with vΔC12L recovered more rapidly than controls. Signs of illness were also milder in animals infected with vΔC12L. Virus titers in lung homogenates were similar between all groups at day 3 p.i.; however, mice infected with vC12L or vC12L-rev virus retained significantly higher titers of virus at days 7 and 10 (Fig. 2).

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FIG. 1.

Virulence of recombinant VVs in the murine intranasal model. Groups of five BALB/c mice were mock-infected (⋄) or infected with 10⁴ PFU of vC12L (▪), vΔC12L (○), or vC12L-rev (▴). (A) Mice were weighed daily, and the results are expressed as the mean percent weight loss of each group ± standard error compared with the weight immediately prior to infection. Data are from five mice, except for the group infected with vC12L-rev, in which one mouse was sacrificed at day 9 and another at day 10. P values were determined by using Student's t test and indicate mean percent weight changes of mice infected with vΔC12L that were significantly different from those of mice infected with vC12L or vC12L-rev. (B) Animals were monitored daily for signs of illness, which was scored from 1 to 4 as described previously (2). Data from each day are expressed as the mean ± standard error from five mice, except for the group infected with C12L-rev, in which 1 mouse was sacrificed at day 9 and another at day 10. P values were determined by using Student's t test and indicate mean signs of illness of mice infected with vΔC12L that were significantly different from those of mice infected with vC12L or vC12L-rev.

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FIG. 2.

Replication and spread of recombinant VVs in vivo. Mice were infected with 10⁴ PFU of vC12L (▪), vΔC12L (○), or vC12L-rev (▴). On the indicated day p.i., five animals infected with each virus were sacrificed, and the titer of infectious virus in the lungs, brains, and spleens was determined by plaque assay on BS-C-1 cells. Virus titers are expressed as PFU per organ. The broken line indicates the minimum detection limit of the plaque assay.

In the intranasal model, inoculum doses of VV WR of ≥10⁴ PFU are accompanied by extensive respiratory infection and virus dissemination (59, 60). To examine the ability of the three viruses to spread in vivo, homogenates of brain and spleen were assayed for infectious virus. At day 3 p.i., infectious virus was recovered from the brains of some animals and from the spleens of all animals infected with the recombinant VVs (Fig. 2). No differences in virus titer were observed between groups at this time. At day 7, less virus was detected in brain and spleen samples from vΔC12L-infected mice, and by day 10, virus had been cleared from the brains and spleens of all vC12L- or vC12L-rev-infected mice, but only three of five mice infected with either vC12L or vC12L-rev. In a second experiment, virus was recovered from the brains and spleens of none of five vΔC12L-infected mice, and two of five and three of five mice infected with vC12L or vC12L-rev, respectively, 10 days after infection. These findings indicate that the vΔC12L virus is capable of in vivo spread; however, it is cleared more rapidly than vC12L and vC12L-rev.

Effect of VV infection on numbers of cells in the BAL and lung.To examine the cellular infiltrate, the number of cells from BAL fluids was determined. By day 3 p.i., cell numbers in lung and BAL preparations from virus-infected animals had increased compared to those in mock-infected controls, and this increase was more pronounced on days 7 and 10 p.i. At days 3, 7, and 10 p.i., the number of lung or BAL cells recovered from vΔC12L-infected animals was not significantly different from those recovered from mice infected with control viruses (data not shown), indicating that deletion of C12L has little effect on the overall number of cells recruited to the lung.

Enhanced NK cell cytotoxicity in mice infected with vΔC12L.Because IL-18 induces NK cell cytotoxicity (25, 58) and the C12L protein inhibited murine IL-18 in vitro, we examined the role of C12L in NK cell activation in vivo. Cytotoxic activity was determined by lysis of YAC-1 tumor cells in a ⁵¹Cr-release assay. Effector cells were recovered from BAL, lung, spleen, and MLN at days 3 and 7; however, results from day 7 are not shown, because little NK cytotoxic activity was detected in cells from any of these sites at this time (<10% lysis at an effector-to-target [E:T] ratio of 100:1). Day 3 effector cells recovered from BAL, lung, spleen, and MLNs of vΔC12L-infected mice showed greater cytotoxicity toward YAC-1 targets than cells from vC12L- and vC12L-rev-infected mice (Fig. 3). To confirm that cytotoxicity was due to NK cells, lung cells taken 3 days p.i. with vΔC12L were depleted of CD8⁺ cells or asialo-GM₁⁺ cells in the presence of human complement (Materials and Methods). Treatment with complement alone or complement plus anti-CD8 MAb had little effect on cytotoxic activity, but treatment with complement plus anti-asialo-GM₁ removed virtually all activity (undepleted = 35%, complement alone = 33%, complement plus anti-CD8 = 26%, and complement plus anti-asialo-GM₁⁺ = 1.5% lysis, at an E:T ratio of 100:1).

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FIG. 3.

NK cytotoxicity in BAL, lung, spleen, and MLN after VV infection. Groups of six mice were mock infected (⋄) or infected with 10⁴ PFU of vC12L (▪), vΔC12L (○), or vC12L-rev (▴). At day 3, NK cytotoxicity was determined in cell suspensions prepared from the BAL, lung, spleen, and MLN of mice. Specific lysis of YAC-1 cells was assessed by ⁵¹Cr-release assay. Data are expressed as the mean ± standard error from two groups of mice (n = 3 per group). Cells were also stained for expression of the pan-NK cell marker DX5 and examined by flow cytometry with a minimum of 20,000 cells analyzed in a lymphocyte gate. Results are expressed as the percentage of DX5⁺ cells in the total viable cell population. P values were determined by Student's t test and indicate the mean percentage of DX5⁺ cells from mice infected with vΔC12L that were significantly different from those from mice infected with vC12L or vC12L-rev. *, P < 0.05.

Effector cell populations were also stained for expression of the NK cell marker DX5 to ascertain if the enhanced NK cytotoxic activity was due to greater recruitment of NK cells or increased activation of those cells present. The percentage of DX5⁺ cells detected in BAL and lung was significantly greater (approximately double) from vΔC12L-infected mice than that from controls, but the percentages were similar in spleen and MLN preparations from all VV-infected groups (Fig. 3). The elevation in DX5⁺ cells recovered from BAL of vΔC12L-infected mice is not sufficient to account for all of the enhanced NK cytotoxic activity observed: for example, compare the ability of BAL cells from vΔC12L-infected mice to lyse targets at an E:T ratio of 50:1 with those from vC12L and vC12L-rev-infected mice at an E:T ratio of 100:1. These findings suggest that the activation state of NK cells is also enhanced in the lung.

Enhanced CTL activity in mice infected with vΔC12L.To assess the effect of VV C12L on VV-specific CTL activity, the cytotoxic activity of effector cells from BAL, lung, spleen, and MLN was examined against uninfected or WR-infected P815 cells. No VV-specific CTL activity was observed with effector cells recovered from any site at day 3 p.i. (data not shown). At day 7, VV-specific CTL activity was higher in BAL and lung cells from vΔC12L-infected mice than that in vC12L- and vC12L-rev-infected controls (Fig. 4). A more modest enhancement was also observed in spleen and MLN. All effector populations showed low cytotoxic activity against uninfected P815 cells (<10% at an E:T ratio of 100:1) (data not shown). Treatment of day 7 lung cells from vΔC12L-infected mice with complement plus anti-CD8 MAb abrogated virtually all CTL activity (undepleted = 45%, complement alone = 39%, complement plus anti-CD8 = 2% lysis, at an E:T ratio of 100:1), confirming that cytotoxic activity against VV-infected P815 cells was mediated by CD8⁺ lung cells.

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FIG. 4.

CTL activity in BAL, lung, spleen, and MLN after VV infection. Groups of six mice were mock infected (⋄) or infected with 10⁴ PFU of vC12L (▪), vΔC12L (○), or vC12L-rev (▴). At day 7, CTL activity was determined in cell suspensions prepared from BAL, lung, spleen, and MLN of mice. Specific lysis of WR-infected P815 cells was assessed by ⁵¹Cr-release assay. Data are expressed as the mean ± standard error from two groups of mice (n = 3 per group). Lysis of noninfected P815s by day 7 effector cell populations was always <10% at an E:T ratio of 100:1 and is not shown. Cells were also stained for expression of CD8 and examined by flow cytometry with a minimum of 20,000 cells analyzed in a lymphocyte gate. Results are expressed as the percentage of CD8⁺ cells in the total viable cell population. P values were determined by Student's t test and indicate the mean percentage of CD8⁺ cells from mice infected with vΔC12L that were significantly different from those from mice infected with vC12L or vC12L-rev. *, P < 0.05.

The proportion of CD8⁺ T lymphocytes present in lung and BAL fluids from vΔC12L-infected mice on day 7 was approximately twofold greater than that from vC12L and vC12L-rev controls (Fig. 4); however, again, this difference does not fully explain the enhanced cytotoxic activity from these sites (compare the lysis at an E:T ratio of 50:1 for vΔC12L to 100:1 for vC12L or vC12L-rev cells). Evidently, more CTLs are present in the lungs of vΔC12L-infected mice, and these CTLs are in a higher state of activation than cells from control-infected animals. Similar proportions of CD8⁺ lymphocytes were detected in all spleen and MLN samples tested.

VV C12L inhibits IFN-γ induction in vivo.IL-18 induces IFN-γ production by murine T cells and NK cells (39), and IFN-γ possesses antiviral activity (43). We therefore examined levels of IFN-γ in the lungs of VV-infected mice. First, an ELISA was used to determine levels of IFN-γ in BAL on days 3 and 7 p.i. Low levels of IFN-γ were detected in BAL from vC12L-, vΔC12L-, and vC12L-rev-infected mice at day 3 (Fig. 5A). By day 7, there was a 10-fold increase in BAL IFN-γ levels from vC12L (972 ± 253 pg/ml) and vC12L-rev (1,128 ± 380 pg/ml)-infected mice (Fig. 5B). Most striking, however, is the enhancement of IFN-γ observed in BAL from vΔC12L-infected animals (7,602 ± 1,786 pg/ml), indicating an important role for IL-18 in augmenting IFN-γ production in response to VV infection at this time.

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FIG. 5.

Production of IFN-γ in the lungs of VV-infected mice. Groups of four to six BALB/c mice were mock infected (⋄) or infected with 10⁴ PFU of vC12L (▪), vΔC12L (○), or vC12L-rev (▴). At days 3 and 7, mice were sacrificed, BALs were performed, and single-cell suspensions were prepared from lung tissue. (A and B) Levels of IFN-γ in BAL fluids of VV-infected mice. BAL samples were centrifuged, and the levels of IFN-γ present in the supernatant were determined by ELISA at days 3 and 7. Values for individual mice are shown. The broken line represents the minimum detection level of the assay. (C and D) Intracellular production of IFN-γ by lung lymphocytes from mice 7 days pi. Lung cells were stimulated with PMA and iomnomycin for 4 h, and brefeldin A was added to retain cytokines in the cytoplasm. Cells were stained with FITC-labeled anti-CD8, QR-labeled anti-CD4, and, after permeabilization with saponin, PE-labeled anti-IFN-γ before analysis by three-color flow cytometry. Shown is the percentage of CD4⁺ (C) or CD8⁺ (D) T lymphocytes producing IFN-γ. Values are averaged from two groups (n = 3 per group). P values were determined by Student's t test and indicate the mean percentage of CD8⁺ cells from mice infected with vΔC12L that were significantly different from those from mice infected with vC12L or vC12L-rev. *, P < 0.05; **, P < 0.02.

To determine which cells in the lung were producing IFN-γ, we performed intracellular staining of lung lymphocytes for IFN-γ. At day 3, we examined IFN-γ production by DX5⁺ NK cells in the lung. Consistent with the low levels of IFN-γ in day 3 BAL fluids, IFN-γ was detected in less than 5% of DX5⁺ or DX5⁻ lymphocytes at this time (data not shown). At day 7, more CD4⁺ (Fig. 5C) and CD8⁺ (Fig. 5D) lung lymphocytes from VV-infected animals stained for IFN-γ than did those from mock-infected controls. The proportion of both CD4⁺ and CD8⁺ lymphocytes that stained for IFN-γ was significantly greater from vΔC12L-infected mice than that from lung cells from animals infected with control viruses. IL-4 and IL-10 were not detected (<1% of total lymphocytes) in any lung cells by intracellular staining at day 3 or 7 (data not shown).

Pulmonary nitrite levels are elevated during infection with vΔC12L virus.NO is produced by a wide range of mammalian cells and has been demonstrated to inhibit VV replication in vitro (28) and in vivo (45). The inducible nitric oxide synthase (iNOS) enzyme produces high levels of NO and is up-regulated by cytokines such as IFN-γ and tumor necrosis factor alpha (TNF-α). Given the marked elevation in IFN-γ levels in the lungs of vΔC12L-infected mice (Fig. 5), we investigated levels of nitrite in cell-free BAL fluid as a measure of NO activity. BAL nitrite levels peaked earlier (day 7) and at higher levels in vΔC12L-infected mice than in vC12L- or vC12L-rev-infected animals (Fig. 6). By day 10 p.i., BAL nitrite levels declined in vΔC12L-infected mice but remained high in C12L- or vC12L-rev-infected mice.

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FIG. 6.

Nitrite levels in the cell-free BAL of mock-infected mice or mice infected with 10⁴ PFU of vC12L, vΔC12L, or vC12L-rev. The data for each time point represent the mean nitrite level ± standard error of four to five individual mice. Columns marked with an asterisk indicate nitrite levels in BAL fluid from vΔC12L-infected mice that were statistically different from those from both vC12L- and vC12L-rev-infected animals. **, P < 0.02.

IL-18 is produced in response to VV infection in vivo and in vitro.Many orthopoxviruses encode IL-18bps, yet the ability of these viruses to induce IL-18 production has not been examined. We used an ELISA to measure IL-18 levels in BAL from mice infected with 10⁴ PFU of vC12L, vΔC12L, or vC12L-rev. Levels of IL-18 were below detection (<25 pg/ml) in BAL from mock-infected mice or from mice 3 days p.i. with VV, but could be detected at similar levels in BAL fluids 7 days p.i. with either vC12L, vΔC12L, or vC12L-rev (Fig. 7A). This showed that IL-18 is induced in response to VV infection in vivo and that expression of vIL-18bp does not affect levels of IL-18 in the lung at day 7 p.i. In a second experiment, recombinant vIL-18 bp containing a C-terminal six-histidine tag (C12L-His) was produced from recombinant baculoviruses and purified by metal chelate affinity chromatography. Addition of 0.1 μg of purified C12L-His per ml to BAL fluid taken from mice 7 days after infection with vΔC12L had no effect on the levels of IL-18 detected (data not shown), indicating that the ELISA could detect IL-18 even when bound by the vIL-18bp.

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FIG. 7.

IL-18 production in response to VV infection in vivo and in vitro. (A) IL-18 levels in BAL. Mice were infected with 10⁴ PFU of vC12L, vΔC12L, or vC12L-rev and were sacrificed 3 or 7 days later. BALs from four to five mice were pooled, and the level of total IL-18 protein was determined by ELISA. Data are expressed as the mean ± standard error from two independent experiments. (B) IL-18 release by lung macrophages. Lung macrophages were isolated from naive BALB/c mice and cultured in the presence of 10 PFU of vC12L, vΔC12L, or C12L-rev per cell. Supernatants were measured for IL-18 by ELISA. Results are expressed as the mean ± standard error of duplicate wells.

IL-18 is expressed in many different cell types, but macrophage-like cells are a major source of IL-18 production (15). Therefore, we investigated whether lung macrophages could release IL-18 in response to VV infection. We found significant amounts of IL-18 were released by lung macrophages 24 h p.i. with vC12L, vΔC12L, or vC12L-rev (Fig. 7B).