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. 2001 Nov;104(3):355–360. doi: 10.1046/j.1365-2567.2001.01318.x

Suppressive effect of locally produced interleukin-10 on respiratory syncytial virus infection

Yan Ruan *, Yoshitaka Okamoto *, Zensei Matsuzaki *, Shuichiro Endo *, Tomokazu Matsuoka *, Tadashi Kohno *, Hideaki Chazono *, Ito Eiko †, Kazuo Tsubota ‡, Ichiro Saito §
PMCID: PMC1783310  PMID: 11722651

Abstract

Interleukin (IL)-10 is known to be a multifunctional cytokine. This study was designed to evaluate the role of IL-10 during respiratory syncytial virus (RSV) infection using a C57BL/6 transgenic (TG) mouse model in which the expression of murine IL-10 cDNA was regulated by a human salivary amylase promoter (IL-10 TG mice). These mice expressed a large amount of IL-10 in the nasal mucosa and in salivary glands. Viral replication in the respiratory tract after intranasal infection with RSV was suppressed significantly in IL-10 TG mice compared to non-transgenic controls. This suppression was IL-10 specific, because it was prevented by treating mice with neutralizing anti-IL-10 antibodies. We also found that IL-10-stimulated T cells displayed cytotoxic activity against infected murine nasal epithelial cells. Previous data indicated that IL-10 induces Fas ligand (L) expression on mouse T cells. Taken together, these data suggest that Fas/Fas L mediated cytotoxicity is involved in the suppression of RSV replication observed in IL-10 TG mice after intranasal infection.

Introduction

Interleukin (IL)-10 was initially considered an anti-inflammatory peptide on the basis of its ability to suppress the production of proinflammatory cytokines and the function of T cells by inhibiting accessory CD28/B7-1 receptor interaction.1–3 However, the effects of IL-10 are known to be wide-ranging with respect to the inflammatory process. IL-10 can induce cell adhesion molecules on endothelial cells in vitro, the level of expression of which is comparable to that induced by IL-1β and tumour necrosis factor-α (TNF-α).4 IL-10 thereby promotes the adhesion of non-specifically activated inflammatory cells to local vessels and migration into inflamed tissues. Furthermore, transgenic expression of IL-10 in the pancreas accelerates the rate of onset of diabetes, indicating that IL-10 is not a general inhibitor of inflammatory diseases in vivo.5 IL-10 also acts as a costimulating factor for B cells that are activated through a differentiation factor promoting the secretion of immunoglobulin A (IgA), IgM, and IgG.6 Thus, IL-10 may play an important role in mediating many immune and inflammatory processes.

Respiratory syncytial virus (RSV) causes serious respiratory infection, particularly in young children. Although RSV has been recognized as a priority for vaccine development, the results of various trials have been disappointing in part because of the potential vaccine-enhancement of inflammatory processes in the lung.7,8 The mechanisms underlying development of inflammation include synthesis of cytokines and other inflammatory mediators and the participation of cell adhesion molecules in the migration of inflammatory cells.

The present study examines the roles of IL-10 in RSV infection using IL-10 transgenic (TG) mice and depletion of IL-10 in vivo. Our results show that IL-10 contributes to the antiviral defence in this model, and suggests mechanisms for these effects.

Materials and Methods

Animals

A line of mice of the C57BL/6 background transgenic for murine IL-10 cDNA regulated by a human salivary amylase promoter was created as previously described.9 The use of the laboratory animals was approved by the local Animal Ethics Committee (Yamanashi Medical University), and experiments were conducted in conformity with their guidelines.

Transgene expression was detected by Northern blot analysis and organ culture. Total RNA was isolated from all organs including the nasal mucosa by guanidine isothiocyanate extraction.10 Twenty µg/lane of RNA was separated on a 1·2% agarose gel and transferred to a nylon membrane. Hybridization was carried out using a 32P-labelled cDNA probe for mouse IL-10. The nasal mucosa for the organ culture was isolated from five IL-10 TG animals and five controls. Nasal mucosa from each mouse was cultured in a single well of a 96-well plate in RPMI-1640 supplemented with 20 mm of HEPES, 2 mm of l-glutamine, 100 µg/ml of penicillin, 100 µg of streptomycin, and 10% fetal calf serum (FCS). IL-10 production was measured after 72 hr of culturing. The level in the culture supernatant was assayed using an ELISA Kit (PharMingen, San Diego, CA).

Histological analysis

Freshly frozen sections of nasal mucosa were stained using the avidin–biotin immunoperoxidase complex (ABC) method with anti-mouse IL-10 (PharMingen). Frozen sections were fixed in a peroxidase–lysine–paraformaldehyde solution for 15 min at 4°, rinsed in phosphate-buffered saline (PBS) containing 0·01 m HEPES and 0·1% saponin, and incubated with a blocking agent (Vector Laboratories, Inc., Burlingame, CA) for 20 min. They were then incubated for 1 hr with a monoclonal rat anti-IL-10 antibody (1 : 100 dilution; PharMingen), followed by incubation with a biotin-labelled rabbit anti-rat antibody (PharMingen) for 30 min. After incubation with the ABC reagent (Vector Laboratories) for 30 min, the sections were treated with a freshly prepared solution of 0·05% 3,3,-diaminobenzidine and 0·005% H2O2 in Tris–HCL buffer (0·05 m, pH 7·6) for 5 min, washed with distilled water, and counterstained with haematoxylin–eosin. All controls treated with normal rat IgG2a (PharMingen) instead of the first antibody and blocked with recombinant mouse IL-10 (PharMingen) gave negative results.

Experimental infection with RSV

The long strain of RSV (prototype RSV group A strain) was grown in HEp-2 cells in minimal essential medium (MEM) supplemented with 2% FCS, 2 mm l-glutamine, and antibiotics. RSV was partially purified by polyethylene glycol precipitation, followed by centrifugation in a 35–65% discontinuous sucrose gradient as described elsewhere.11

RSV (1 × 106 plaque-forming units (PFU)) in 20 µl were administered intranasally to IL-10 TG mice and control mice. Four days later, these mice were killed by CO2 overdose and exsanguinated; blood, nasal washings, lung tissue, and nasal mucosa were collected. For nasal washings, mice were pinned onto a support tilted to elevate the head. Two hundred µl of Hanks' balanced salt solution without Ca2+ or Mg2+ (HBSS) was injected into the larynx and toward the nose. Nasal washings were collected in a tube as they exited the nares. The fluids were separated by low-speed centrifugation and stored at −70° until used for the determination of cytokines. TNF-α and interferon-γ (IFN-γ) were analysed with an enzyme-linked immunosorbent assay (ELISA) based on the manufacturer's manual (Genzyme, Cambridge, MA).

Lung and nasal mucosa were weighed and homogenized in MEM containing 2%-FCS and stored at −70° until the assays. RSV was assayed using the plaque method with Hep-2 cells in 24-well microplates. The overlay for the plaque assay consisted of MEM supplemented with 2% FCS, antibiotics, and 1% methylcellulose. Plates were incubated for 7 days at 37°. After the methylcellulose was removed, plaques were fixed with 10% formaldehyde and stained with 0·1% crystal violet.

Treatment with a neutralizing IL-10 antibody

The preventive effect of treatment with a neutralizing anti-IL-10 antibody in vivo was investigated. A rat neutralizing monoclonal antibody (mAb) for mouse IL-10 (JES 2A5, IgG1: PharMingen) and isotype mouse control IgG1 mAb (PharMingen) were used. Antibodies were injected intraperitoneally twice a week at a dose of 0·1 mg for IL-10 TG mice (n = 5) and for control non-transgenic mice (n = 5) for 2 weeks before RSV inoculation, and the antibodies were administered intranasally for 4 consecutive days after inoculation. RSV replication in the lungs and nasal mucosa of these mice 4 days after inoculation was examined in the same manner as described previously.

In situ TdT-mediated biotin–dUtP nick-end labelling (TUNEL) staining

On the 4th day after RSV inoculation the heads of the mice were removed along the line between the upper and lower jaws; they were then fixed in formalin and decalcified. The frontal section of the nasal cavity at the front of the eyeball was examined and processed for paraffin sectioning. The sections were mounted onto microscope slides and incubated overnight at 55°. Sections were then deparaffinized for 5 min in xylene, 5 min in ethanol, 3 min in 95% ethanol, 3 min in 70% ethanol, and 5 min in PBS. After washing in distilled water, endogenous peroxidase was quenched with 2% H2O2 for 5 min at room temperature and sections were washed twice in PBS. Labelling of 3′-ΟΗ fragmented DNA ends was performed with an in situ apoptosis detection kit (Apoptag, Oncor, Gaithersburg, MD) following the manufacturer's instructions. Detection of labelled ends was done with an anti-digoxigenin–peroxidase antibody and a diaminobenzidine (DAB) substrate kit (Vector Laboratories).

Cytotoxicity assay

Nasal epithelial cells were isolated from control C57BL/6 mice by protease treatment, as described previously.12 Excised tissues were washed with MEM supplemented with antibiotics and incubated with 0·1% protease (type 14; Sigma Chemical, St Louis, MO) in MEM for 16 hr at 4°. After incubation, the cell suspensions were filtered through Nitex filters (100 nm) and centrifuged at 300 g for 10 min at 4°. After further washing, the isolated cells were plated on collagen-coated tissue culture dishes (Falcon, Franklin Lakes, NJ) and incubated at 37° with 5% CO2. After 7 days of incubation, the dishes were treated with 0·1% trypsin–1 mm ethylenediaminetetraacetic acid (EDTA; Gibco, Grand Island, NY) and the detached cells were plated at a density of 3 × 104 cells in the culture dishes. Two days later, 5 × 105 PFU RSV were introduced into the dishes. After 2 hr of incubation, the cells were collected and labelled overnight at 37° in 5% CO2 with 300 µCi of sodium 51Cr-chromate. The nasal mucosal tissues were cut into small pieces with scissors, then were teased gently with frosted glass slides through nylon mesh, and were suspended in RPMI-1640 containing 10% FCS, penicillin (100 units/ml), and streptomycin (100 µg/ml). After washing twice with medium, CD4+ and CD8+ T cells were purified using magnetic beads (Dynal, Great Neck, NY) in 0·2 ml of RPMI-1640 and were then supplemented with 10% FCS. The number of CD8+ T cells recovered was less than 20% of the number of CD4+ T cells. Even for CD4+ T cells, because less than 105 could be isolated from each mouse, CD4+ T cells from 15 separate mice were pooled and used in each cytotoxicity assay. In addition, CD3+ T cells purified from splenocytes of control mice incubated with recombinant mouse IL-10, concanavalin A (Con A; EY Laboratories, San Matei, CA), recombinant mouse IL-2 (PharMingen), or anti-mouse Fas L neutralizing antibody (FLIM 58, MBL Co. Ltd, Japan) were also used as effector cells. Each well of the 96-well microtitre plates received, in a total volume of 200 µl, target cells, effector cells in the indicated proportions, and either medium. Microplates were centrifuged for 1 min at 600 g and incubated for 4 hr at 37°. After additional centrifugation, 100 µl aliquots of the supernatants were assayed for radioactivity. The fraction of the total radioactivity released was then calculated, and the results, averaged from triplicate experiments, were expressed as percent specific 51Cr release (percent experimental 51Cr release − percent 51Cr release from target cells alone).

Statistical analysis

Comparisons among experimental groups were evaluated with Student's test.

Results

Expression of IL-10

The expression of IL-10 in the various organs of IL-10 TG mice was examined by Northern blotting, IL-10 was found to be present in the nasal mucosa and salivary glands (Fig. 1). No expression was found in other organs including lung and pancreas. The level of IL-10 protein in organ-cultured nasal mucosa and salivary glands was markedly increased in IL-10 TG mice compared to non-transgenic controls (Fig. 2). Immunohistochemistry also revealed IL-10-positive cells in the nasal glands of IL-10 TG mice, but these cells were not found in their negative littermate controls (Fig. 3). Histological findings indicated no marked inflammatory changes in the nasal mucosa of IL-10 TG mice younger then 10 weeks old, although some 12-week-old TG mice showed very mild lymphocyte infiltration.

Figure 1.

Figure 1

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The expression of IL-10 mRNA in the various organs of IL-10 TG mice. Expression was observed in nasal mucosa and salivary glands, but not in other organs. 1, submaxillary gland; 2, parotid gland; 3, oral mucosa; 4, masseter muscle; 5, nasal mucosa; 6, lung; 7, spleen; 8, pancreas.

Figure 2.

Figure 2

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IL-10-protein in organ-cultured nasal mucosa and salivary glands was examined by ELISA. There was a marked increase in IL-10 levels (70.0 ± 2.5 pg in nasal mucosa, 162.5 ± 40.4 pg in salivary glands) for IL-10 TG mice compared to control non-transgenic mice (15.0 ± 2.5 pg in nasal mucosa, 65.0 ± 35.0 pg in salivary glands).

Figure 3.

Figure 3

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IL-10-positive cells were detected in the glands of the nasal mucosa of IL-10 TG mice in the immunohistological study (a). However, these cells were not distinguishable in the nasal glands of control mice (b).

RSV replication after intranasal inoculation

In IL-10 TG and normal control mice, after nasal inoculation with 1 × 106 PFU of RSV at 6 weeks of age, peak RSV replication in the lungs was observed on day 4 and declined until day 7 in both IL-10 TG and normal control mice. RSV was recovered from the nasal mucosa for 12 days after inoculation.

Figure 4 depicts the recovery of RSV in the respiratory tracts of the mice 4 days after inoculation. RSV replication in the lungs and nasal mucosa of IL-10 TG mice was significantly suppressed compared to that of control non-transgenic mice.

Figure 4.

Figure 4

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The recovery of infectious RSV in the nasal mucosa and lungs 4 days after nasal inoculation (mean ± SE). RSV replication in the respiratory tract of IL-10 TG mice (2.9 ± 0.5 ×102 PFU in lungs, 2.3 ± 0.5 ×103 PFU in nasal mucosa) was significantly suppressed compared to that of normal control non-transgenic mice (7.8 ± 1.1 × 102 PFU in lungs, 11.7 ± 2.5 ×103 PFU in nasal mucosa).

Significant levels of IFN-γ and TNF-α were found in the nasal washings of RSV-infected mice. However, there were no significant differences between transgenic and littermate control mice.

Treatment with the anti-IL-10 neutralizing antibody (Fig. 5)

Figure 5.

Figure 5

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The recovery of infectious RSV in the nasal mucosa after treatment with an anti-IL-10 neutralizing antibody in IL-10 TG and in non-transgenic mice (mean ± SE). There was no significant difference in RSV replication between these groups. The significant suppression was observed in IL-10 TG mice treated with control antibody.

Administration of the anti-IL-10 mAb to IL-10 TG mice before and after RSV inoculation prevented the suppression of RSV replication otherwise observed in IL-10 TG mice without anti-IL-10 treatment. The viral replication of anti-IL-10 mAb treated non-transgenic mice was not significantly affected.

TUNEL staining (Fig. 6)

Figure 6.

Figure 6

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TUNEL staining of the nasal mucosa of RSV-infected IL-10 TG mice. Significant numbers of nasal epithelial cells were positively stained. The number of these stained cells are shown in Table 1.

The numbers of nasal epithelial cells positively stained using the TUNEL method were significantly higher in RSV-infected IL-10 TG mice than in RSV-infected control non-transgenic mice (Table 1). Mild infiltration of lymphocytes was observed in some of the IL-10 TG and control mice, but there were no significant differences between them.

Table 1.

The number of positively stained RSV-infected nasal epithelial cells assessed by the TUNEL method

IL-10 TG mice 18·0 ± 9·5*
Control non-transgenic mice  5·3 ± 2·6
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The number of cells was examined in the frontal section of the nasal cavity at the front of the eyeball.n = 7.

*

P < 0·01.

Cytotoxic activity

Cytotoxicity of T cells from nasal tissue and spleen was investigated using primary culture mouse nasal epithelial cells as targets. Splenic T cells were stimulated with rIL-10 or Con A + rIL-2 for 6 hr before each cytotoxicity assay. As shown in Fig. 7, enhanced cytotoxicity was observed in nasal CD4+ T cells from IL-10 TG mice and rIL-10 or Con A + rIL-2-treated splenic T cells. This activity was almost completely inhibited by incubation with anti-mouse neutralizing Fas L antibody.

Figure 7.

Figure 7

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Cytotoxic activity of T cells on RSV-infected nasal epithelial cells. The enhanced cytotoxic activity was observed in the nasal CD4+ T cells (a) and in splenic CD3+ T cells treated with rIL-10 or Con A + rIL-2 (b). The enhancement was almost totally inhibited by incubation with anti-mouse neutralizing Fas L antibody.

Discussion

IL-10 has been shown to have anti-inflammatory activities. In in vitro studies, IL-10 may suppress the production of proinflammatory cytokines such as TNF-α, IL-1β and IL-6 from monocytes13 as well as IFN-γ and IL-2 from CD4+ T cells.14 Although little is known about the contribution of IL-10 to the inflammatory response in vivo, several studies implicate IL-10 in inflammatory processes.15–17 The proliferation and differentiation of human B cells is stimulated by IL-1018 and the preincubation of resting CD4+ T cells with IL-10 enhanced their ability to produce cytokines.19 In human studies, subjects receiving lipopolysaccharide (LPS) together with recombinant IL-10 showed enhanced LPS-induced IFN-γ release, as well as activation of cytotoxic T lymphocytes (CTL) and natural killer cells.20

The role of IL-10 in RSV infection is controversial. An in vitro infection study with alveolar macrophages21 found that RSV induced a higher concentration of IL-10 than an influenza virus. The authors suggested that RSV could suppressed the production of early immunoregulatory cytokines through induction of IL-10, which may be related to the induction of serious RSV infection. However, RSV induced the production of TNF, IL-6, IL-8, and PAF by human mononuclear cells and increased expression of cell adhesion molecules in respiratory epithelial cells.22,23 Studies with mouse spleen mononuclear cells primed with RSV glycoproteins showed an excess release of not only of T helper 1 (Th1), but also of Th2 cytokines.24,25 Recent work has suggested that an RSV-induced immune response, rather than the direct pathogenic effects of the virus, may be responsible for the underlying pathogenesis of RSV disease. Openshaw et al. showed that mice immunized systemically with RSV encoding F and G glycoproteins exhibited reduced lung virus titres following a subsequent challenge with RSV, but also had an increase in the severity of lung pathology.26

To clarify the role of IL-10 in respiratory infection, IL-10 TG mice were used, in which enhanced IL-10 production was observed only in the upper respiratory tract. This model was employed because systemic enhancement of IL-10 was not desirable and the half-life of IL-10 activity is very short with external administration.27

In IL-10 TG mice, RSV replication was significantly lower, both in lungs and nasal mucosa, than in control non-transgenic mice after intranasal inoculation, but this suppression of virus recovery was no longer observed in anti-IL-10-treated TG mice. These results suggest that increased levels of IL-10 in TG mice suppressed RSV replication in the respiratory tract. In the nasal mucosa of RSV-infected TG mice, apoptotic epithelial cells observed via the TUNEL method were stained more frequently than in control mice. CTL-mediated cytotoxicity is known to play a very important role in the elimination of viruses including RSV. Fas L functions as an effector in CTL and we previously found dose-dependent Fas L expression in CD3+ T cells that had been treated with IL-10.8 The present study examined whether IL-10 induced CD4+ T cell-mediated cytotoxicity against RSV-infected nasal epithelial cells in vitro. CD4+ T cells from the nasal mucosa of IL-10 TG mice showed enhanced CTL activity for primary cultured mouse nasal epithelial cells infected with RSV and the cytotoxic activity of splenic T cells from normal mice stimulated with IL-10 6 hr before a cytotoxic assay was enhanced. The perforin/granzyme and Fas/Fas L systems are two major mechanisms of CTL cytotoxicity,28 but the specific role of each seems to depend largely upon the target cells. CD4+ CTL often lack perforin and mainly use Fas L as an effector.29 IL-10 induced Fas L expression on CD4+ T cells is involved in the suppression of RSV replication observed in IL-10 TG mice. Thus, the role of IL-10 in RSV infection would seem to be complex. It is possible that massive induction of IL-10 in the upper respiratory tract may be useful as protection against RSV infection. However, enhanced Fas L expression induced by IL-10 may also cause tissue destruction and enhance inflammation. The treatment of Crohn's disease with low dose of IL-10 (5–10 µg/kg) has been found to improve the symptoms, but this is not accomplished with high dose of IL-10 (20 µg/kg).30 High-dose IL-10 therapy can be associated with undesired proinflammatory effects in vivo. Further analysis is needed regarding the role of IL-10 in RSV infection.

Acknowledgments

This work was supported by a grant from Grant-in Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.

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