Abstract
Bronchiolitis caused by respiratory syncytial virus (RSV) infection is a major cause of hospitalization in children under 1 year of age. RSV causes common colds in older children and adults, but can cause serious disease in immunodeficient patients and the elderly. Development of effective vaccines and treatments for RSV infection is therefore a priority. Because bronchiolitis and vaccine-augmented disease are thought to be caused by exuberant T cell activation, attention has focused on the use of immunomodulators that affect T cell responses. In mice, IL-12 treatment down-regulates type 2 cytokine responses to the attachment protein G of RSV, reducing lung eosinophilia but further enhancing illness. We now show that CD8+ T cells are responsible for enhanced weight loss, whereas IL-12-activated NK cells express high levels of IFN-γ and inhibit lung eosinophilia without causing illness. Moreover, unlike immunocompetent mice, virus is detected in the mediastinal lymph nodes after elimination of both CD8+ T cells and NK cells. These studies show that innate immune responses to viral infections direct the pattern of subsequent specific immunity and are critical to the development of nonpathogenic antiviral effects. We speculate that IL-12 treatment might be beneficial and safe in T cell-deficient patients with RSV pneumonitis.
Viral bronchiolitis is the most common single cause of infantile hospitalization in the Western world. About 70% of cases of viral bronchiolitis are due to respiratory syncytial virus (RSV).3 Primary RSV infection is universal during the first 3 years of life, and reinfections cause common colds in both children and adults. There is no effective human vaccine, and formalin-inactivated vaccines cause a dramatic increase in the severity of disease, consistent with the hypothesis that viral bronchiolitis is an immunopathological disease (1). In addition, children hospitalized with bronchiolitis are more likely to suffer recurrent wheezing during childhood, and many will be subsequently diagnosed as asthmatic (2).
Viral infections trigger both innate and adaptive immune responses. The sequence of events has been well studied during infection with RSV in mice: NK cells appear by day 3, and, by day 4, are the major source of IFN-γ. By day 6, they have been largely replaced by CD4+ and CD8+ T cells. During secondary immune responses in mice primed with recombinant vaccinia viruses expressing single RSV proteins, reduced recruitment of NK cells (and IFN-γ production by them) is an early characteristic of mice that develop eosinophilia, mediated by CD4+ T cells making type 2 cytokines (3, 4)
Immunity and pathology to secondary infection depend on the viral protein(s) to which the mice are sensitized. Mice primed with the attachment protein (G) develop lung eosinophilia after intranasal RSV challenge. CD4+ T cells secreting type 2 cytokines are necessary for this response because their depletion eliminates eosinophilia (5). Eosinophilia can be avoided if CD8+ T cells are activated at the same time as CD4+ T cells (5, 6, 7). Though BALB/c mice develop lung eosinophilia after G protein priming, normal C57BL/6 mice do not. Removal of CD8+ T cells using specific Ab or by using TAP-1, β2-microglobulin (β2m), or CD8 gene knockout mice, renders C57BL/6 mice susceptible to eosinophilia (5, 8). CD8 T cells are therefore critical regulators of Th2-driven eosinophilic lung disease. This has been elegantly demonstrated by Srikiatkhachorn and Braciale (7), who showed that insertion of a nonameric CD8+ epitope into G protein expressed by recombinant vaccinia virus causes profound down-regulation of lung eosinophilia during subsequent RSV challenge. Our recent studies of 15 different strains of inbred mice show that CD4 T cell priming, class I MHC, and CD8 T cell recruitment are critical determinants of eosinophilia (8).
G-induced lung eosinophilia can also be avoided by administration of IL-12 (9). IL-12 enhances NK and CTL activity, inducing these cells to secrete IFN-γ and other cytokines (10). It also has strong effects on humoral immune responses (11, 12, 13, 14), via IFN-γ-dependent and -independent mechanisms (11, 15) and has direct effects on B cells (11). It also synergizes with IFN-α and IFN-γ to enhance Th1 development (16), but may itself be inhibited by IFN-α or -β in vitro and in vivo (17).
In the mouse, administration of recombinant IL-12 at the time of vaccination with the RSV attachment protein G enhances IFN-γ production and decreases IL-4 and IL-5 in CD4+ and CD8+ T cells, largely preventing eosinophilia during subsequent RSV challenge (9). Similar effects are seen using formalin-inactivated RSV vaccine to induce pulmonary eosinophilia (18). Although lung eosinophilia is reduced by IL-12 treatment, clinical illness is enhanced by unknown mechanisms (9). We now report that either NK cells or CD8+ T cells can enhance RSV expulsion from the lung, but that CD8+ T cells are largely responsible for enhanced illness. Although IFN-γ production by CD8+ cells reduces lung eosinophilia, IL-12 does not depend on this mechanism for its anti-eosinophilic effects. It also acts via NK cells, which differ from CD8+ T cells in being incapable of causing enhanced disease.
Materials and Methods
Mice and virus stocks
C57BL/6 TAP-1-deficient mice were derived by L. Van Kaer in S. Tonegawa’s laboratory and CD8-α-chain deficient mice by W. P. Fung Leung in T. W. Mak’s laboratory. C57BL/6 and BALB/c mice were purchased from Harlan Olac (Bicester, U.K.). Mice were bred in pathogen-free conditions and used when 8–10 wk old. RSV and recombinant vaccinia virus expressing the attachment protein of RSV (rVV-G) or control β-galactosidase (rVV-β-gal) were grown in HEp-2 cells and assayed for infectivity as previously described (19). All stocks were free of mycoplasma infection (determined by DNA hybridization; Gen-Probe, San Diego, CA).
Mouse infection and treatment
Anesthetized BALB/c mice or C57BL/6 mice with TAP-1 or CD8α-chain gene deletions were scarified on the rump on day 0 with 3 × 106 PFU rVV-G or rVV-β-gal in a final volume of 10 μl. On day 14, mice were challenged intranasally with 3 × 106 human RSV (A2 strain, 50 μl). Mice were sacrificed on day 21 by injection of 3 mg pentobarbitone and exsanguinated via the femoral vessels. Some mice were injected with 300 ng recombinant murine IL-12 (rIL-12) or PBS control i.p. in a final volume of 500 μl daily from day −2 to day 2. IFN-γ depletion was performed by i.p. injection of 0.5 mg rat anti-mouse IFN-γ Ab (clone XMG1.2) starting 2 days before RSV challenge and then daily for 4 days (days 12–16). Two additional injections were given on days 18 and 20. For NK and CD8 T cell depletion, mice were injected i.p. with 0.5 mg anti-NK1.1 Ab (A22) or anti-CD8 mAb (YTS169.4 + YTS156.7), respectively, on alternate days from day 12 to day 16 after scarification. In some experiments, NK cell depletions were performed at vaccination and challenge. The results obtained from these mice were identical with mice depleted only at challenge. To preserve reagents, mice were therefore depleted of NK cells only at challenge. Control mice were treated identically with isotype-matched irrelevant Abs.
The efficiency of depletion was monitored by flow cytometry of appropriately stained lung cells and splenocytes. Depletion was shown to be >95%.
Cell recovery
Bronchoalveolar lavage (BAL), lung tissue, and serum were harvested on day 21 (7 days after intranasal RSV challenge) by methods described previously (20). Briefly, the lungs of each mouse were inflated six times with 1 ml of 12 mM lidocaine in Eagle’s MEM. A total of 100 μl of this BAL fluid from each mouse was retained for cytospin analysis, and the rest was immediately diluted into ice-cold RPMI containing 10% FCS, 2 mM/ml l-glutamine, 50 U/ml penicillin, and 50 mg/ml streptomycin (R10F). BAL was repeated three times with a further 1-ml volume of lidocaine, and samples were pooled from groups of mice. Pooled cells were centrifuged and resuspended at 106 cells per ml.
Enumeration of eosinophils
Eosinophils were enumerated as granulocytes by flow cytometry, using their distinctive forward and side scatter properties. Identification was confirmed by counting eosinophils in Giemsa-stained cytocentrifuge preparations.
Flow cytometric analysis of intracellular and cell surface Ags
To detect intracellular cytokines, 106 cells per ml were incubated with 50 ng/ml PMA (Sigma, St. Louis, MO), 500 ng/ml ionomycin (Calbiochem, La Jolla, CA), and 10 mg/ml brefeldin A (Sigma) for 4 h at 37°C. Cells were stained with either Quantum-red-conjugated Abs to CD3, CD4, or CD8 (Sigma) or PE-conjugated anti-mouse NK1.1 (PharMingen, San Diego, CA) for 30 min on ice and then fixed for 20 min at room temperature with 2% formaldehyde. All samples were then permeabilized with 0.5% saponin in PBS containing 1% BSA and 0.1% azide for 10 min. CD4- and CD8-stained samples were incubated with PE-conjugated anti-mouse IL-5 (TRFK-5; PharMingen) and FITC-conjugated anti-IFN-γ (XMG1.2; PharMingen). NK1.1-stained cells were incubated with FITC-conjugated anti-mouse IFN-γ. After 30 min, all samples were washed with PBS containing 1% BSA and 0.1% sodium azide and analyzed on a Coulter (Palo Alto, CA) EPICS Elite flow cytometer collecting data on at least 40,000 lymphocytes.
Lung virus titer
Clearance of RSV was assessed in lung homogenates at days 2 and 4 after virus challenge. Lungs were removed from four mice per group and homogenized. After centrifugation at 4000 rpm for 4 min, supernatant was titrated in doubling dilutions on HEp-2 cell monolayers in 96-well flat-bottom plates. Twenty-four hours later, monolayers were washed and incubated with peroxidase-conjugated goat anti-RSV Ab (Biogenesis, Poole U.K.). Infected cells were detected using 3-amino-9-ethylcarbazole, infectious units being enumerated by light microscopy.
Enzyme-linked immunospot (ELISPOT) for murine cytokines
Multiscreen 96-well filtration plates (IP sterile plate, 0.45-μm hydrophobic, high protein binding immobilon-P membrane; Millipore, Bedford, MA) were coated with rat Abs to mouse IL-4, IL-5, and IFN-γ (all from PharMingen; clone numbers BVD4-1011, TRFK5, and R4-6A2, respectively) in 0.1 M carbonate/bicarbonate buffer pH 9.6 and left overnight at 4°C. Plates were washed six times with 200-μl sterile PBS and blocked with 200 μl R10F at 37°C for 2–4 h. A total of 100 μl of cell suspension in doubling dilutions (from 105 to 1.25 × 104 cells per well) and 100 μl of UV-inactivated RSV (1 PFU/cell) or control material (a lysate of HEp-2 cells) was added to each well and incubated overnight at 37°C. Cells were then removed and wells were incubated with 100 μl of biotinylated anti-cytokine Ab (BVD6–24G2, TRFK4, and XMG1.1 for IL-4, IL-5, and IFN-γ, respectively; all from PharMingen) for 2–4 h followed by 100 μl of streptavidin-alkaline phosphatase for 1–2 h (Sigma, Poole, U.K.; 1/1000 in PBS). Bound Ab was visualized by incubating plates with alkaline phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium according to the manufacturer’s instructions (Sigma). Color development was stopped by gently washing the plates in water. Between each addition, the plates were washed six times with sterile PBS. Spots were enumerated per well after air drying on an inverted microscope. The results are expressed as spots per million.
Results
Role of NK, CD8+ T cells, and IFN-γ on the anti-eosinophilic effects of IL-12
To study the mechanisms responsible for reduced pulmonary eosinophilia after IL-12 treatment, rVV-G primed BALB/c mice were depleted of CD8+ T cells or IFN-γ at the time of RSV challenge. Lung lavage was performed 7 days after RSV challenge. As in previous studies, IL-12 treatment reduced lung eosinophilia in normal, rVV-G-primed BALB/c mice. This effect was also observed in BALB/c mice depleted of CD8+ T cells, showing that the reduced eosinophilia can be mediated by other cell types.
We have previously shown that immunocompetent C57BL/6 mice do not develop G-induced eosinophilia, but do so when CD8+ T cells are impaired (such as in CD8, β2m, and TAP-1 knockout mice) (8). To confirm that the reduction of eosinophilia by IL-12 does not require CD8+ T cells, C57BL/6 mice with CD8 and TAP-1 gene deletions were examined. Similar to anti-CD8-treated BALB/c mice, IL-12 treatment resulted in a reduction of lung eosinophilia (TAP−/− and CD8−/− control in Fig. 1⇓). Therefore, CD8+ T cells are not necessary for the reduction of lung eosinophilia by IL-12, and this effect does not depend on the background of the mouse strain.
Reduction of eosinophilia by IL-12 treatment in CD8 or TAP-1 knockout C57BL/6 mice is caused by IFN-γ-producing NK cells. Normal or knockout mice were scarified with rVV expressing RSV G-protein and treated with PBS (−, •) or IL-12 (+, ○) daily from day −2 to day +2 of sensitization. Two weeks later, all mice were challenged with RSV intranasally. Some mice were depleted of IFN-γ or NK cells before and during RSV challenge. Percent lung eosinophilia (y-axis) in BAL was assessed on cytocentrifuge preparations 7 days after challenge. Results from individual mice are shown. ∗, Data that is significantly different from control treated mice in the same group (p < 0.01).
IL-12 treatment enhances Th1 and Tc1 but inhibits Th2 cell development. We have previously shown that IFN-γ depletion induces eosinophilia to the G protein of RSV in immunocompetent C57BL/6 mice that are otherwise resistant. CD8+ T cells are the most potent subset producing IFN-γ in this model. To determine whether the IL-12 reduced eosinophilia in the absence of CD8 T cells depends on IFN-γ, BALB/c, and C57BL/6 CD8 knockout mice were additionally depleted of IFN-γ. With IFN-γ depletion, IL-12 did not reduce G-induced lung eosinophilia (Fig. 1⇑). Identical results were observed in TAP-1 knockout mice and C57BL/6 mice treated with anti-CD8 Abs (data not shown), showing that, although CD8 T cells are not necessary for inhibition of eosinophilia by IL-12, IFN-γ production is essential.
Because IFN-γ production was necessary for reduction of eosinophilia by IL-12, we sought to determine the role of a second major source of IFN-γ, the NK cell. Unlike the role played by CD8+ T cells in C57BL/6 mice, depletion of NK cells from immunocompetent C57BL/6 mice does not result in lung eosinophilia and is consequently not affected by treatment with IL-12 (C57BL/6 + anti-NK in Fig. 1⇑). When NK and CD8+ T cells were absent (anti-NK1.1-treated CD8 knockout mice), IL-12 treatment had no effect on eosinophilia. We were unable to perform similar studies in BALB/c mice because the DX5 Ab that recognizes NK cells in this strain does not deplete NK cells in vivo, and anti-asialo-GM1 treatment caused complications in that (as previously reported (21)) it also removed some CD45RB low activated CD8+ T cells (data not shown).
These results show that IL-12-activated NK cells inhibit lung eosinophilia and that the mechanism of inhibition requires IFN-γ. They also show that IL-12 has no effect on eosinophilia in the absence of NK and CD8 T cells, and therefore does not operate via any other cell population (such as CD4+ T or B cells).
Role of in NK, CD8+ T cells, and IFN-γ in IL-12-enhanced illness
We have previously shown that IL-12 treatment enhances illness in G-primed, RSV-challenged BALB/c mice. This effect is seen regardless of the timing of IL-12 administration, but is most pronounced if IL-12 is given at the time of initial priming. Doses higher than 300 ng do not cause significantly greater weight loss, whereas lower doses, though causing less weight loss, do not significantly reduce lung eosinophilia (20). Therefore, we chose this fixed dose for the current studies. IL-12 treatment alone, in the absence of virus infection, has no effect on weight loss, showing that the IL-12 treatment itself is not causing a septic condition (data not shown).
To confirm the mechanism of IL-12-enhanced weight loss, BALB/c mice were depleted of CD8+ T cells using specific Ab. Removal of CD8+ T cells prevented IL-12-enhanced weight loss (Fig. 2⇓A). Depletion of IFN-γ using specific Ab produced a similar effect (Fig. 2⇓A). These data implicate CD8+ T cells and IFN-γ in causing enhanced weight loss in IL-12-treated mice. Depletion of CD8+ T cells or IFN-γ also prevented the more mild G-induced weight loss that occurred in the absence of IL-12 (data not shown).
IL-12-augmented weight loss does not occur in the absence of CD8+ T cells or IFN-γ. Mice were primed and challenged as described in Fig. 1⇑, and treated with IL-12 or placebo. Day zero represents a pool of the weights recorded up to 2 days before the experiment. The percent weight loss (y-axis) is then determined from this original basal weight of the mice. The mean and SDs of at least four mice per group are shown. Similar results were obtained in two to three replicate experiments. A, Results from IL-12-treated BALB/c mice with or without anti-CD8 or anti-IFN-γ treatment. B, Weight loss in IL-12-treated C57BL/6 mice with or without gene deletions for TAP-1 or CD8 or anti NK1.1 treatment. C, Weight loss in immunocompetent C57BL/6 mice treated with anti-NK1.1 or anti- IFN-γ but not IL-12.
The effect of IL-12 has not been described in other mouse strains. Therefore, we tested the effect of IL-12 treatment on illness in G-primed C57BL/6 mice, with or without CD8 or TAP-1 gene deletions. As in BALB/c mice, C57BL/6 mice show enhanced weight loss when treated with IL-12 (Fig. 2⇑B, □); this was further enhanced when IL-12 was administered at both vaccination and challenge (data not shown). However, IL-12-enhanced weight loss was not observed in CD8 or TAP-1 knockout animals (Fig. 2⇑B, ○ and •, respectively), showing that CD8 T cells are required for enhanced weight loss to occur.
Because NK cells also produce IFN-γ, we examined whether they contributed to enhanced weight loss. Depletion of NK cells from IL-12-treated immunocompetent C57BL/6 mice (i.e., with CD8 cells) did not prevent the enhanced weight loss (Fig. 2⇑B, ▪). CD8 or TAP-1 knockout mice also depleted of NK cells showed a similar weight loss profile to nondepleted knockout animals (data not shown). Immunocompetent G-primed C57BL/6 mice also display mild weight loss after RSV challenge even without IL-12 treatment (○ in Fig. 2⇑C). Depletion of IFN-γ (•) or CD8+ T cells but not NK cells (▪ in 2C) also abrogated this illness (Fig. 2⇑C). Collectively, these data indicate that CD8+ T cells and IFN-γ are responsible for IL-12-induced weight loss, whereas IL-12-enhanced NK cell responses are nonpathogenic. Regardless of immune status or IL-12 treatment, all mice had recovered by day 9 or 10 after RSV challenge.
Antiviral effects of NK, CD8+ T cells, and IFN-γ
As in previous studies (20), IL-12 treatment did not affect vaccinia virus replication during G protein priming. Mice were infected with recombinant vaccinia expressing β-gal, and a colorimetric assay was used to show that IL-12 did not affect expression of galactosidase in lesions form C57BL/6 or knockout mice (data not shown). In G-primed mice, RSV is cleared from the lung by day 4 after intranasal challenge. Spread outside the lung has not been observed. Fig. 3⇓ shows that the presence of NK cells (in C57BL/6, TAP-1−/−, and CD8−/− mice) or CD8+ T cells (C57BL/6 and C57BL/6 + anti-NK1.1) results in elimination of RSV from the lung with similar kinetics to that seen in immunocompetent mice. If both subsets are absent (TAP-1 −/− or CD8 −/− mice treated with anti-NK1.1), virus persists in the lungs and can also be recovered from mediastinal lymph nodes. This phenomenon occurs irrespective of IL-12 treatment. Untreated animals still failed to clear the virus in the absence of both CD8 and NK cells (data not shown). Either CD8 or NK cells are therefore sufficient to eliminate virus infection.
Combined depletion of NK cells and CD8+ T cells allow RSV persistence in the lung and viral spread to mediastinal nodes. Mice were primed and challenged as described in Fig. 1⇑ and treated with IL-12. Lungs and mediastinal nodes were removed at days 2, 4, and 7 after RSV challenge for virus titration. Mice treated with anti-NK1.1 Ab are shown by the open symbols and those treated with isotype control by closed symbols. The y-axis represents the PFUs recovered from the whole lung (top) or mediastinal lymph node (bottom). Normal C57BL/6 mice (⋄) cleared virus, whereas mice with TAP-1 (▴) or CD8α-chain (▪, □) genetic deletions failed to clear virus if also depleted of NK cells. The SD of five to six mice are shown.
Role of NK, CD8+ T cells, and IFN-γ in cell recruitment in IL-12-treated mice
Previous studies show that IL-12 treatment enhances cell recruitment in G-primed immunocompetent BALB/c mice challenged with RSV (20). This enhancement also occurred in C57BL/6 mice, and those depleted of NK cells (Table I⇓). However, cell recruitment was unaffected or even reduced by IL-12 treatment in CD8 or TAP-1 knockout mice (Table II⇓) or immunocompetent mice depleted of IFN-γ (see Table I⇓). Depletion of IFN-γ or NK cells reduced total cell recruitment to the lung still further in untreated knockout mice, an effect even more evident after IL-12 treatment (Table II⇓). These results suggest that the enhanced cell recruitment induced by IL-12 depends on the presence of CD8+ T cells.
Cellular recruitment and cytokine production in mice treated with IL-12a
Cellular recruitment and cytokine production in immunodeficient mice treated with IL-12a
The effect of IL-12 treatment on T cell recruitment and cytokine production
We have previously shown that IL-12 treatment of G-primed BALB/c mice enhances CD4+ T cell recruitment and intracellular IFN-γ expression (20). We observed similar enhancement of IFN-γ production in the present studies using ELISPOT and intracellular cytokine staining. In addition, we found that untreated C57BL/6 mice generally produced more IFN-γ-secreting cells than BALB/c mice (p = 0.043), particularly after IL-12 treatment (p = 0.023). This enhancement of IFN-γ expression by CD4+ T cells was not affected if C57BL/6 mice were depleted of NK cells (Table I⇑).
We wished to determine whether the enhanced disease caused by IL-12-activated CD8+ T cells was accompanied by enhanced cell recruitment to the lung. In the absence of CD8+ T cells (CD8 and TAP knockout mice), IL-12 did not cause an increase in IFN-γ production by CD4 T cells as shown by intracellular staining (not significant) (Table II⇑). The marginal increase in IFN-γ observed by ELISPOT probably represents IL-12-boosted production by NK cells, although there were only 5–10% NK cells in the BAL at the time when samples were harvested. However, by intracellular cytokine staining, we observed that more NK cells from CD8 knockout animals expressed intracellular IFN-γ after IL-12 treatment (12% ± 1.9 and 32% ± 4.3 in untreated or IL-12 treated CD8 knockout mice, respectively). As expected, depletion of IFN-γ from either immunocompetent (Table I⇑) or CD8 knockout animals (Table II⇑) almost abolished the capacity of the remaining cells to produce IFN-γ (p = 0.008 comparing IFN-γ production in CD8 knockouts to those additionally depleted of IFN-γ). Depletion of NK cells in CD8 knockout mice further reduced, but did not abolish, the number of cells producing IFN-γ (p = 0.027 comparing CD8−/− to CD8−/− + anti-NK1.1). IL-12 treatment reduced the frequency of IL-4- and IL-5-producing cells in all mice, except CD8 knockouts depleted of IFN-γ or NK cells (Table II⇑). Collectively, these results show that IL-12 treatment works through IFN-γ in reducing Th2-associated cytokines, and that NK cells are a significant source of IFN-γ in CD8 knockout animals.
Discussion
Our results show that IL-12-induced IFN-γ production by either NK or CD8+ cells inhibits lung eosinophilia, and that either cell type alone mediates viral clearance. The role of NK and CD8 cells is, however, quite different with respect to disease augmentation: although IL-12-stimulated CD8+ T cells enhance the severity of weight loss during RSV challenge, IL-12-stimulated NK cells are purely protective. CD4+ T cells also produce IFN-γ but do not appear to contribute to enhanced illness. This is supported by the observation that IL-12-treated mice do not experience weight loss when CD8+ T cells and/or NK cells are absent.
Our current understanding of the RSV murine model is that lung eosinophilia depends on effective CD4+ T cell priming. Eosinophilia is abolished if CD4+ T cells are depleted (5), a strong CD4 T cell epitope is removed (22), or T cells are skewed toward a Th1 with IL-12 therapy (9). In addition, CD8+ T cells inhibit CD4 driven lung eosinophilia via production of IFN-γ (5, 7). IFN-γ is therefore a central factor regulating eosinophilia. Our previously published studies show that NK cells appear very early and are a major source of IFN-γ on day 3 and 4 of primary RSV infection in mice, a time when CD4+ and CD8+ T cells are being first recruited. Moreover, NK cells, and particularly those producing IFN-γ, are scarce in mice with eosinophilic disease (3, 4). Indeed RSV lacking G or SH proteins show increased NK cells in the lung (23). In the current study, we show that NK cells only prevent eosinophilia when IL-12 is also administered.
The contribution of IFN-γ from NK and CD8+ T cells appeared to differ. IL-12-activated CD8+ T cells seem to cause CD4+ T cells to express more IFN-γ (Ref. 9 and this study), whereas IL-12-activated NK cells do not have this effect. Therefore, although IL-12-activated NK cells are able to reduce eosinophilia, they have no direct influence on CD4+ T cells. We believe that this may reflect: 1) the different kinetics with which each subset expands during RSV challenge; 2) local anatomical proximity of different cell subsets in the evolving infection; or 3) the relative levels of IFN-γ produced by IL-12-activated NK and CD8+ T cells. In support of this last possibility, we have observed that the mean fluorescent intensity of intracellular IFN-γ is 10-fold higher in CD8+ T cells than that in NK cells (our unpublished observations).
In IL-12-treated mice, the specificity of recruited CD8+ T cells is intriguing. Cross-reactive epitopes and bystander activation of cells specific for heterologous proteins do play a role during some viral infections. We have no evidence that such cross-reactivity exists between RSV and vaccinia virus. For example, mice primed with a vaccinia virus expressing a control protein (β-gal) display the same cellular phenotype and recruit a similar number of cells to the lung after intranasal RSV challenge compared with naive mice given RSV alone. Similarly, RSV-specific cell lines do not respond in vitro to vaccinia-infected HEp-2 cells. Therefore, we do not believe that enhanced recruitment of vaccinia-specific lymphocytes during RSV challenge is responsible for the data presented in the current study. In BALB/c mice, we believe that IL-12 at the time of G protein-priming activates CD8+ T cells in general so that when they are recruited to the lung during RSV challenge they respond more quickly. The same may be true for C57BL/6 mice, but further studies are needed to determine whether a MHC H-2b-restricted CD8 epitope is present in the G protein.
Kos and Engleman (24) have shown that NK cells are required for CD8+ T cells to mature into effector cells. Therefore, NK cells may not directly influence CD4+ T cells in our model, but may do so via the induction of activated CD8+ T cells. This may also be true of other studies where NK cells are thought to affect the differentiation of CD4+ T cells into those with a Th1 phenotype (25, 26). It is also possible that they act by enhancing Ag presentation, cognate interactions, and the expression of costimulatory molecules (B7-1, B7-2, MHC class I and II) by dendritic cells and macrophages. Alternatively, macrophages and dendritic cells are susceptible to lysis by NK cells (27), which could therefore affect eosinophilia indirectly by killing APCs. This second possibility is supported by the observation that total cell recovery from the lung was reduced in CD8 knockout animals, and that IFN-γ levels in CD4 cells were not affected.
A recent study by Ohteki et al. (28) shows potent IFN-γ production by dendritic cells after IL-12 treatment in mice lacking T, B, and NK cells. This pathway does not appear to play a significant role in our studies, because IL-12 had no effect on eosinophilia in mice depleted of both CD8 and NK cells. In the OVA challenge model of airway eosinophilia, NK cells (but not NK T cells) and, to a lesser extent, γδ T cells, are essential for the production of eosinophilia (29). By contrast, our present results showed that NK cells inhibit lung eosinophilia. The critical timing of NK cell depletion also differs in our models. In the case of OVA-induced eosinophilia, depletion of NK cells is only effective if performed at sensitization (29), whereas we found that depletion has to occur at challenge; depletion at sensitization only had minimal effects. Intriguingly, Peritt et al. (30) have shown that NK cells grown in the presence of IL-4 differentiate to IL-4 producing “NK-2” cells, whereas those grown in the presence of IL-12, IL-10, and IFN-γ become “NK-1” cells. It is not known whether these subsets exist in vivo, but IL-5-producing NK cells appear to contribute to peritoneal eosinophilic infiltration in mice (31). In our studies, NK cells isolated ex vivo express abundant IFN-γ but never IL-4 or IL-5 (demonstrable by intracellular staining), even in mice with intense lung eosinophilia. Similarly, NK cells expressing TCR (NK T cells) have been described in the mouse and man (32, 33) but were not evident in our studies (<0.5% of total NK cells were TCR+ in treated or untreated mice; data not shown). The functions of NK cells are clearly diverse, dependent on the nature of the Ag, timing, and site of encounter.
It is remarkable that NK and CD8+ T cells influenced the outcome of G-induced eosinophilia during RSV challenge when the IL-12 was given only at sensitization. It is possible that the cytokine profile of some RSV-specific CD4+ T cells is programmed at the time of initial scarification. It is also possible that NK cells are activated by IL-12 treatment, and remain activated for at least 2 wk. This proposal is supported by the observed baseline elevation of intracellular IFN-γ in NK cells of IL-12-treated animals at the start of RSV challenge. The depletion of cell subsets at challenge would remove NK and CD8+ T cells preactivated by IL-12 treatment. Without these sources of IFN-γ, the ratio of IFN-γ:IL-4:IL-5-producing cells measured by ELISPOT showed a considerable shift in the cytokine balance away from IFN-γ in NK-depleted mice. IL-12-activated NK and/or CD8+ T cells may therefore be recruited to the lung, where they help maintain the Th1 phenotype. This increase in IFN-γ in NK cells, however, did not enhance virus clearance. In the presence of either CD8+ T cells or NK cells, all G-primed mice cleared the RSV challenge in 4 days, regardless of IL-12 treatment.
Though IL-12-activated NK cells are sufficient to prevent metastasis of B16 melanoma cells in RAG−/− mice (34), other studies imply a contribution toward pathology. For example, NK cells contribute to the heightened sensitivity to bacterial LPS in lymphocytic choriomeningitis virus-infected mice (35). NK cell depletion also prevents the cytokine (IL-2 and IL-12)-induced shock in mice (36).
The observation that activated CD8+ T cells are responsible for enhanced weight loss is interesting and reminiscent of our early studies showing that CD8 clones and lines cause severe disease enhancement in RSV-infected mice (37). A recent study by Liu et al. (38) shows that CD8+ T cells producing TNF-α cause bystander apoptosis of alveolar epithelial cells. It is possible that this mechanism, or release of other lytic granules by CD8+ T cells, plays an important role in enhanced illness after IL-12 treatment. We are currently performing studies to differentiate these possibilities.
Life-threatening RSV pneumonia is common in bone marrow or solid organ transplant recipients, and is occasionally seen in patients with AIDS or after thoracic surgery. In such patients and in premature infants, RSV infection may lead to respiratory failure and death (39). In one study of bone marrow recipients with RSV-induced pneumonia, the mortality rate was 78% (40). New therapeutic strategies are therefore needed, particularly in immunodeficient patients. The results presented in this paper suggest that IL-12 treatment may be beneficial in selected patients with RSV lung disease, and that toxicity will depend on the patient’s immune status. Indeed, IL-12 treatment might be safe and effective in patients with T cell dysfunction who suffer prolonged infection, while stimulating protective natural immunity.
Acknowledgments
We thank Dr. Mike Mulqueen (Roche Products) for kindly supplying recombinant IL-12.
Footnotes
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↵1 This work was supported by Program Grant 054797/Z/98/Z from The Wellcome Trust.
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↵2 Address correspondence and reprint requests to Dr. Tracy Hussell, Room 366, Respiratory Medicine, Imperial College School of Medicine, Norfolk Place, Paddington, London W2 1PG, U.K. E-mail address: t.hussell{at}ic.ac.uk
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3 Abbreviations used in this paper: RSV, respiratory syncytial virus; G, the attachment protein of RSV; rVV, recombinant vaccinia virus; β2m, β2-microglobulin; β-gal, β-galactosidase; BAL, bronchoalveolar lavage; ELISPOT, enzyme-linked immunospot.
- Received April 25, 2000.
- Accepted September 21, 2000.
- Copyright © 2000 by The American Association of Immunologists
References
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