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Am. J. Respir. Cell Mol. Biol., Volume 21, Number 5, November 1999 586-596

Interleukin-10 Gene Transfer to the Airway Regulates Allergic Mucosal Sensitization in Mice

Martin R. Stämpfli, Monika Cwiartka, Beata U. Gajewska, David Alvarez, Stacey A. Ritz, Mark D. Inman, Zhou Xing, and Manel Jordana

Department of Pathology and Molecular Medicine, Immunology and Infection Programme, Center for Gene Therapeutics; and Department of Medicine, McMaster University, Hamilton, Ontario, Canada


    Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The objective of this study was to investigate the effect of airway gene transfer of interleukin (IL)-10, a cytokine with potent anti-inflammatory and immunoregulatory activities, on allergic mucosal sensitization. We used a recently described murine model that involves repeated exposures to aerosolized ovalbumin (OVA), daily for 10 d, in the context of granulocyte macrophage colony-stimulating factor (GM-CSF) expression in the airway environment achieved by intranasal delivery of a replication-deficient adenovirus carrying the GM-CSF transgene. The resulting inflammatory response was characterized by a T-helper 2 cytokine profile and marked airway eosinophilia. After complete resolution of the inflammatory response (Day 28), a single exposure to OVA reconstituted airway eosinophilia and induced airway hyperresponsiveness. We show that concurrent expression of IL-10 inhibited GM-CSF-driven OVA-specific inflammation in a dose-dependent manner. Specifically, IL-10 decreased the number of mononuclear cells, neutrophils, and eosinophils in the bronchoalveolar lavage fluid (BALF). Histologic evaluation of the tissue corroborated the findings in the BALF. Concurrent expression of IL-10 at the time of mucosal sensitization abrogated both the cellular and physiologic recall responses in vivo. Studies in interferon (IFN)-gamma knockout mice demonstrated that prevention of airway eosinophilia by IL-10 was IFN-gamma -independent and that expression of IL-10 was associated with decreased levels of IL-4, IL-5, and tumor necrosis factor-alpha in the BALF. Flow cytometric analysis of dispersed lung cells showed that expression of IL-10 in the airway reduced the absolute number of Class II major histocompatibility complex (MHC)+/CD11c+ (dendritic cells) and Class II MHC+/Mac-1bright (macrophages) cells expressing the costimulatory molecules B7.1 and B7.2 by 30%. However, IL-10 coexpression did not prevent expansion of CD4 and CD8 T cells or expression of the early activation marker CD69 on T cells. Thus, airway gene transfer of IL-10 altered the immune response to OVA in a way that resulted in inhibition of airway inflammation. These findings suggest that development of an immunoregulatory strategy based on IL-10, alone or in combination with GM-CSF, warrants further consideration.


    Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Despite universal exposure to aeroallergens, only a relatively small proportion of individuals develop harmful immune inflammatory airway responses after allergen exposure (1, 2). Thus, the airway-lung interface might be viewed as predisposed to immunologic homeostasis (3). Recent studies in humans have led to the hypothesis that expression of interleukin (IL)-10, and possibly other cytokines, in the airway environment may contribute to the maintenance of the normal state of allergen nonresponsiveness (7, 8). The specific objective of this study was to investigate whether expression of IL-10 in the airway could maintain immunologic homeostasis under conditions conducive to allergic mucosal sensitization.

IL-10 is a cytokine that has well-documented anti-inflammatory and immunoregulatory activities (reviewed in 9). IL-10 inhibits the synthesis of proinflammatory cytokines and chemokines by monocytes/macrophages (12), neutrophils (15, 16), and eosinophils (17). In vitro IL-10 inhibits antigen-specific proliferation of peripheral blood T cells and CD4+ T cells regardless of the subset (T-helper [Th]1, Th0, or Th2) (18, 19) and induces long-lasting anergy in human CD4+ T cells (20). In vivo, a number of reports have demonstrated powerful anti-inflammatory and immunosuppressive effects of IL-10 in endotoxemia, autoimmune diseases, immune complex-induced lung injury, antigen-induced airway inflammation, and cutaneous conditions (21). Moreover, transgenic mice expressing IL-10 under the control of the major histocompatibility complex (MHC) Class II Ea promoter fail to mount specific T- and B-cell responses to ovalbumin (OVA) and are highly susceptible to infection with intracellular pathogens (27), further illustrating the immunosuppressive activities of IL-10.

We have recently reported a novel murine model of respiratory mucosal sensitization that involves repeated aerosolizations of antigen (OVA) in the context of a granulocyte macrophage colony-stimulating factor (GM-CSF) airway milieu (28). The ensuing inflammatory response is characterized by airway eosinophilia, expression of a distinct Th2 cytokine profile, antigen-specific immunoglobulin (Ig)E and the development of long-term antigen-specific memory. In the present study, we used an adenoviral (Ad)-mediated gene transfer approach to coexpress IL-10 in the airway, and investigated acute and remote (in vivo recall) cellular and physiologic responses to aerosolized antigen.

    Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Animals

Female BALB/c mice aged 6 to 8 wk were purchased from Harlan (Indianapolis, IN). Interferon (IFN)-gamma knockout (KO) mice on a BALB/c background were obtained from Jackson Laboratories (Bar Harbor, ME). Mice were housed in a specific pathogen-free environment following a 12-h light-dark cycle. All experiments described in this study were approved by the Animal Research Ethics Board of McMaster University.

Aerosolization Protocol

Mice were exposed as previously described (28) for 20 min daily to aerosolized OVA (1% wt/vol in 0.9% saline) over a period of 10 consecutive days (Days 0 to 9) in a Plexiglas chamber (10 × 15 × 25 cm). The OVA aerosol was generated by a Bennet/Twin nebulizer at a flow rate of 10 liters/ min. For the recall challenge experiments, mice were exposed to a 1% OVA aerosol for a 1-h period, twice, 4 h apart, after resolution of the acute inflammatory response at the indicated time points.

Administration of Ad Constructs

Replication-deficient human type 5 Ad (RDA) constructs carrying the transgenes for murine GM-CSF (29) or murine IL-10 (22) in the E1 region of the viral genome were delivered intranasally. As a control, we included an E1- deleted RDA construct carrying no transgene (30). All Ad constructs were delivered in a total volume of 30 µl of phosphate-buffered saline (PBS) vehicle (two 15-µl administrations, 5 min apart) into anesthetized animals.

Collection and Measurement of Specimens

Mice were killed at various time points during and after the aerosolization protocol. Immediately before the animals' death, blood was collected by retro-orbital bleeding. Blood smears were prepared and total white blood cell number in peripheral blood was counted after lysis of red blood cells (RBCs) using RBC lysis buffer. Serum was obtained by centrifugation after incubating whole blood for 30 min at 37°C. Bronchoalveolar lavage was performed according to a standard protocol (31). Briefly, the lungs were dissected and the trachea was cannulated with a polyethylene tube (Becton Dickinson, Sparks, MD). The lungs were lavaged twice with PBS (0.25 ml followed by 0.2 ml); approximately 0.3 ml of the instilled fluid was consistently recovered. Total cell counts were determined from BAL fluid (BALF) using a hemocytometer. After centrifugation, supernatants were collected and stored at -20°C for cytokine measurements by enzyme-linked immunosorbent assay (ELISA). The cell pellets were resuspended in PBS and smears were prepared by cytocentrifugation (Shandon, Inc., Pittsburgh, PA) at 300 rpm for 2 min. All smears, BALF, and peripheral blood, were stained with Diff-Quik (Baxter, McGaw Park, IL). Differential counts of BALF cells and peripheral blood were determined from at least 500 and 200 leukocytes, respectively, using standard hemocytologic criteria to classify the cells as neutrophils, eosinophils, or mononuclear cells. Finally, lung tissue was fixed in 10% formalin and embedded in paraffin. Sections 3 µm thick were stained with hematoxylin and eosin (H&E) or Congo red to identify eosinophils further.

Cytokine and OVA-Specific IgE Measurement

ELISA kits for IL-4, IL-10, GM-CSF, and tumor necrosis factor (TNF)-alpha were purchased from R&D Systems (Minneapolis, MN) and the kit for IL-5 was obtained from Amersham (Buckinghamshire, UK). Each of these assays has a threshold of detection of 5 pg/ml. Serum levels of OVA-specific mouse IgE were measured using a previously described antigen-capture ELISA method (31). Units of OVA-specific IgE were calculated according to a standard serum derived from intraperitoneally sensitized mice 3 d after antigen challenge (31). The amount of 1,000 U/ml corresponds to undiluted standard serum.

Lung Cell Isolation and Flow Cytometric Analysis

Lungs were cut into small (~ 2 mm diameter) pieces and agitated at 37°C for 1 h in 15 ml 150 U/ml collagenase III (Worthington Biochemical Corp., Freehold, NJ) in Hanks' balanced salt solution (HBSS). By means of a plunger from a 5-cc syringe, the lung pieces were triturated through a metal screen into HBSS, and the resulting cell suspension was filtered through nylon mesh. After lysing red blood cells with ACK lysis buffer (0.5 M NH4Cl, 10 mM KHCO3, and 0.1 nM Na2-ethylenediaminetraacetic acid at pH 7.2 to 7.4), cells were washed once and mononuclear cells were isolated by density centrifugation in 30% Percoll. Cells were washed twice and stained for flow cytometric analysis according to a standard protocol (28). Briefly, for each antibody combination, 106 cells were incubated with 0.5 µg Fc Block (CD16/CD32; PharMingen, Mississauga, ON, Canada) at 0 to 4°C for 10 min and then with first-stage monoclonal antibodies at 0 to 4°C for 30 min. Cells were then washed and treated with second-stage reagents. Data were collected using a FACScan and analyzed using PC-LYSYS software (Becton Dickinson, Sunnyvale, CA). The following antibodies and reagents were used: anti-MHC Class II, fluorescein isothiocyanate (FITC)-conjugated M5/114.152 (kindly provided by Dr. D. Snider, McMaster University, Hamilton, ON, Canada); anti-B7.1, biotin-conjugated 16-10A1 (PharMingen); anti-B7.2, biotin-conjugated GL1 (PharMingen); anti-CD11b (Mac-1alpha ), phycoerythrin (PE)-conjugated M1/70 (PharMingen); anti-CD11c, PE-conjugated HL3 (PharMingen); anti-CD3, biotin-conjugated 145-2C11 (PharMingen); anti-CD4, FITC-conjugated L3T4 (PharMingen); anti-CD8alpha , FITC-conjugated Ly-2 (PharMingen); anti-CD69, PE-conjugated H1 2F3 (PharMingen); and Streptavidin PerCP (Becton Dickinson, San Jose, CA). Titration was used to determine the optimal concentration of each antibody.

Measurement of Airway Hyperresponsiveness

Airway responsiveness was measured on the basis of the response of total respiratory system resistance (RRS) to increasing intravenous (internal jugular vein) doses of methacholine (MCh) as previously described (32). Briefly, mice were anesthetized with tribromoethanol (287 mg/kg intraperitoneally), prepared according to a standard protocol (33).

The trachea was exposed and cannulated, and a constant inspiratory flow was delivered by mechanical ventilation (RV5; Voltek Enterprises Inc., Toronto, ON, Canada). Heart rate and oxygen saturation were monitored via infrared pulse oxymetry (Biox 3700; Ohmeda, Boulder, CO), using a standard ear probe placed over the proximal portion of the mouse's hind limb. Paralysis was achieved using pancuronium (0.03 mg/kg intravenously) to prevent respiratory effort during measurement. RRS was measured after consecutive intravenous injections of saline, then 10, 33, 100, 330, and 1,000 µg/kg of MCh (ACIC [Can], Brantford, ON, Canada), each delivered as a 0.2-ml bolus. During each MCh dosing, the mouth-pressure signal from the ventilator was converted to a digital signal (Dash 16; Metrabyte, Staughton, MA) and recorded at 400 Hz on a PC computer. RRS was calculated as described previously (32). Evaluation of airway responsiveness was based on the peak RRS measured in the 30 s after the saline and MCh challenges.

Data Analysis

Data are expressed as means ± standard error of the mean (SEM). Statistical interpretation of results is indicated in figure captions. Differences were considered statistically significant when P < 0.05.

    Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Levels of IL-10 and GM-CSF Expression in the BALF

First, experiments were carried out to determine whether concurrent delivery of Ad/IL-10 and Ad/GM-CSF alters GM-CSF levels in the BALF. Mice were infected intranasally with 3 × 107 plaque-forming units (pfu) Ad/GM-CSF alone or in combination with 3 × 107 pfu Ad/IL-10 or empty control virus (RDA). Administration of Ad/GM-CSF resulted in approximately 160 and 70 pg/ml of GM-CSF in the BALF at Days 5 and 8, respectively (Figure 1, left panel). Coexpression of IL-10 or delivery of an empty control virus did not significantly alter the GM-CSF content in the BALF. In addition, note that IL-10 levels were detected only in mice receiving the Ad/IL-10 construct, with expression of approximately 80 and 60 pg/ml in the BALF at Days 5 and 8, respectively (Figure 1, right panel). Neither IL-10 nor GM-CSF was detected in the serum of any of the groups, confirming previous observations that transgene expression is, at these doses, compartmentalized within the airways (34).


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  		Figure 1.   Levels of GM-CSF and IL-10 expression in the BALF. BALB/c mice were treated intranasally with Ad/GM-CSF alone, Ad/GM-CSF and Ad/IL-10, or Ad/GM-CSF and control virus (RDA). All constructs were delivered at 3 × 107 pfu. Data presented are GM-CSF and IL-10 protein levels in the BALF at Days 5, 8, and 10 (means ± SEM, n = 6).

Effect of IL-10 on OVA-Specific, GM-CSF-Driven Airway Eosinophilia

To investigate whether IL-10 can inhibit GM-CSF-driven OVA-specific airway inflammation, we concurrently administered 3 × 107 pfu Ad/IL-10 and 3 × 107 pfu Ad/GM-CSF intranasally 24 h before daily exposures to OVA over a period of 10 d. Mice were killed 48 h after the last exposure and the cellular profile in the BALF was assessed (Figure 2). Exposure to OVA in the context of GM-CSF resulted in marked eosinophilic airway inflammation in accordance with our previous observations (28). Concurrent expression of IL-10 inhibited this inflammatory response. In addition to eosinophils, expression of IL-10 markedly prevented the increase in neutrophils and mononuclear cells. No significant changes in the BALF cellular profile and, specifically, no significant inhibition of airway eosinophilia was observed in mice that had concurrently received 3 × 107 pfu of an empty control virus (RDA). Figure 3 shows that IL-10 inhibited airway eosinophilia in a dose-dependent manner with maximal effect at 3 × 108 pfu.


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  		Figure 2.   Effect of IL-10 on the inflammatory response in the BALF of mice exposed to OVA in the context of GM-CSF. Ad/IL-10 and Ad/GM-CSF were administered concurrently (intranasally) 24 h before 10 daily exposures to OVA. Mice were killed 48 h after the last exposure. Bars represent, from left to right, untreated mice (naive), mice exposed to OVA in the context of GM-CSF (NoRx), GM-CSF and control virus (RDA), and GM-CSF and IL-10. Results are expressed as means ± SEM (naive n = 6; OVA/GM-CSF: NoRx n = 9, RDA n = 6, IL-10 n = 10). Statistical analysis was performed using analysis of variance (ANOVA) with Fisher's PLSD post hoc test; *P < 0.05.


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  		Figure 3.   Dose-dependent inhibition of GM-CSF-driven OVA-specific airway eosinophilia by IL-10. Ad/GM-CSF (3 × 107 pfu) and increasing doses of Ad/IL-10 were delivered concurrently intranasally 24 h before 10 daily exposures to OVA. Mice were killed 48 h after the last exposure. Results are expressed as means ± SEM (naive n = 6; OVA/GM-CSF: NoRx n = 9; 3 × 106 Ad/IL-10 n = 4; 3 × 107 Ad/IL-10 n = 10; 3 × 108 Ad/IL-10 n = 3). Statistical analysis was performed using ANOVA with Fisher's PLSD post hoc test; *P < 0.05.

We conducted histologic evaluations to corroborate the BALF findings. Figure 4A shows that mice exposed to OVA in the context of GM-CSF developed extensive peribronchial and perivascular inflammation, primarily eosinophilic in nature (Figure 4B). Whereas concurrent delivery of an empty control virus (3 × 107 pfu) altered neither the extent (Figure 4C) nor the nature (Figure 4D) of this inflammatory response, expression of IL-10 (3 × 107 pfu) markedly reduced the inflammatory (Figure 4E), and in particular the eosinophilic (Figure 4F), response in the tissue.


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  		Figure 4.   Light photomicrograph of paraffin-embedded sections of lung tissues. Ad/IL-10 or an empty control virus (RDA) were administered concurrently with Ad/GM-CSF intranasally 24 h before daily exposures to OVA over a period of 10 d. All tissues were obtained 48 h after the last OVA exposure. Panels A, C, and E show sections stained with H&E; the sections in panels B, D, and F were stained with Congo red. Panels represent exposure to OVA in the context of GM-CSF alone (A, B), GM-CSF/RDA (C, D), or GM-CSF/IL-10 (E, F). Original magnification of panels: A, C, E, ×200; B, D, F, ×640.

Peripheral Blood Eosinophilia and OVA-Specific IgE Production

Table 1 shows that mice exposed to OVA in the context of GM-CSF developed peripheral blood eosinophilia, and that this was prevented by concurrent expression of IL-10 (3 × 107 pfu) in the airway microenvironment. In fact, the eosinophil count in the blood of mice exposed to OVA concurrently expressing IL-10 and GM-CSF was similar to that in naive mice.

                              
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TABLE 1
Peripheral blood cell counts and serum levels of OVA-specific IgE

To investigate whether IL-10 inhibited IgE synthesis in vivo we measured serum levels of OVA-specific IgE. We detected approximately 50 U/ml OVA-specific IgE in the serum of mice exposed to OVA in the context of GM-CSF (Table 1). IL-10 (3 × 107 pfu) significantly suppressed IgE synthesis by 50%. No significant inhibition was observed in mice that had received 3 × 107 pfu of an empty control virus (RDA).

In Vivo Rechallenge of Mice Exposed to OVA in the Context of GM-CSF and IL-10

Next, we examined whether expression of IL-10 at the time of the induction of allergic mucosal sensitization prevented the development of Th2-polarized OVA-specific memory. We have demonstrated previously that the acute inflammatory response in the airway is resolved at around Day 28 (28). In the current experiment, mice were re- exposed to aerosolized OVA at Day 65 (i.e., well after resolution of the acute inflammatory response) and the cellular profile in the BALF was assessed 3 d later. Figure 5 shows that mice initially exposed to OVA in the context of GM-CSF readily developed eosinophilic airway inflammation. In contrast, mice that had been concurrently exposed to IL-10 (3 × 107 pfu) did not develop airway inflammation. Inhibition was significant for mononuclear cells, and almost complete for neutrophils and eosinophils (over 90%). Moreover, a similar inhibition of airway inflammation was observed in mice challenged 6 mo after resolution of the acute inflammatory response (data not shown). It is of interest that delivery of an empty control virus (3 × 107 pfu) alone moderately prevented the increase in total cell number and eosinophils at Day 65. 


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  		Figure 5.   Effect of IL-10 on OVA recall response in vivo. Ad/IL-10 was administered concurrently with Ad/GM-CSF intranasally 24 h before 10 daily exposures to OVA. At Day 65 of the protocol, mice were re-exposed to aerosolized OVA and killed 3 d later. Data represent mice exposed to OVA in the context of GM-CSF (NoRx), GM-CSF and empty control virus (RDA), or GM-CSF and IL-10. Results are expressed as means ± SEM (OVA/GM-CSF: NoRx n = 4, RDA n = 4, IL-10 n = 4). Statistical analysis was performed using ANOVA with Fisher's PLSD post hoc test; *P < 0.05.

Airway Hyperresponsiveness

Figure 6 illustrates the response of RRS to intravenous MCh challenge. Six months after resolution of the acute inflammatory response, mice were re-exposed to aerosolized OVA and the airway responsiveness to MCh was assessed 2 d later. Mice that had been exposed to OVA in the context of GM-CSF or GM-CSF plus empty control virus (3 × 107 pfu RDA) were hyperresponsive relative to naive controls, as indicated by significantly higher resistance at individual MCh doses. Responsiveness to MCh in IL-10-treated mice was similar to that in naive mice.


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  		Figure 6.   Effect of IL-10 on MCh-induced increases in RRS. Ad/IL-10 was administered concurrently with Ad/GM-CSF intranasally 24 h before 10 daily exposures to OVA. At 6 mo later mice were re-exposed to aerosolized OVA, and airway function was assessed 48 h later. Results are expressed as means ± SEM (naive n = 9; OVA/GM-CSF: NoRx n = 5, RDA n = 4, IL-10 n = 4). *RRS was significantly greater in OVA/GM-CSF mice than in naive control mice (P < 0.05). Small square: RRS was significantly greater in OVA/GM-CSF/RDA mice than in OVA/GM-CSF mice (P < 0.01). RRS in IL-10-treated mice was significantly less than that measured in the OVA/GM-CSF (dagger) or OVA/GM-CSF/RDA (double dagger) groups (P < 0.01). Statistical analysis was performed using ANOVA with Newman-Keuls post hoc test.

Effect of IL-10 on Allergic Airway Inflammation in IFN-gamma KO Mice

To ascertain whether Ad/IL-10-mediated inhibition of airway eosinophilia was directly due to expression of IL-10 in the airway, and was not an artefact of IFN-gamma induced by the Ad infection, we recapitulated our experimental protocol in IFN-gamma KO mice. Figure 7 shows that expression of IL-10 (3 × 107 pfu) fully prevented the development of allergic airway inflammation in these mice. The degree of inhibition in IFN-gamma KO mice was similar to that in wild-type BALB/c mice (Figure 2), and attenuated mononuclear cells and neutrophils as well as eosinophil accumulation. Moreover, IL-10 expression under these conditions remarkably inhibited IL-4 and IL-5 as well as TNF-alpha levels in the BALF (Figure 8). We should point out that for these experiments, mice were killed at Day 9 rather than Day 11 inasmuch as cytokines are optimally detected in the BALF at this time point. A similar inhibition of IL-4, IL-5, and TNF-alpha levels was observed in the BALF of wild-type mice concurrently expressing IL-10 (data not shown).


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  		Figure 7.   Effect of IL-10 on airway eosinophilic inflammation in IFN-gamma KO mice. Ad/IL-10 and Ad/GM-CSF were administered concurrently (intranasally) 24 h before 10 daily exposures to OVA. Mice were killed 48 h after the last exposure. Bars represent naive mice and mice exposed to OVA in the context of GM-CSF (NoRx), GM-CSF and control virus (RDA), and GM-CSF and IL-10. Results are expressed as means ± SEM (naive n = 2; OVA/GM-CSF: NoRx n = 4, RDA n = 4, IL-10 n = 4). Statistical analysis was performed using ANOVA with Fisher's PLSD post hoc test; *P < 0.05.


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  		Figure 8.   Cytokine content in BALF of IFN-gamma KO mice. Ad/GM-CSF, Ad/GM-CSF/RDA, or Ad/GM-CSF/ Ad/IL-10 were administered 24 h before daily exposures to aerosolized OVA. Cytokine levels were assessed in BALF at Day 9 of the aerosolization protocol. Values represent means ± SEM (n = 4).

Flow Cytometric Analysis of Costimulatory Molecule Expression on Antigen-Presenting Cells and Expression of Early Activation Markers on T Cells

To investigate the effect of IL-10 expression in the airway on antigen presentation, we examined Class II MHC+, CD-11c+ (dendritic cells) (35) and Class II MHC+, Mac-1bright (macrophages) (36) populations and measured expression of the costimulatory molecules B7.1 and B7.2 on these cells at Day 7. Table 2 shows increased numbers of dendritic cells and macrophages expressing B7.1 and B7.2 in mice exposed to OVA in the context of GM-CSF when compared with naive mice. Coexpression of IL-10 reduced the absolute number of both macrophages and dendritic cells by approximately 30% in two independent experiments. However, IL-10 did not reduce the proportion of macrophages and dendritic cells expressing B7.1 and B7.2.

                              
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TABLE 2
Expression of B7.1 and B7.2 on dendritic cells and macrophages

Table 3 shows that exposure to OVA in the context of GM-CSF increases the numbers of CD4 and CD8 T cells in the lung over numbers seen in naive mice. A qualitatively similar expansion of the CD4 and CD8 T-cell compartment was observed in mice concurrently expressing IL-10 or receiving an empty control virus (RDA). Whereas only a small proportion of CD4 and CD8 T cells expressed the early activation marker CD69 in naive mice, exposure to OVA in the context of GM-CSF markedly increased the proportion of CD69+ T cells regardless of the treatment.

                              
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TABLE 3
Expression of CD69 on CD4+ and CD8+ T cells

    Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The objective of this study was to investigate whether establishment of an IL-10 airway microenvironment could modulate allergic mucosal sensitization, a process fundamental to the pathogenesis of asthma. Borish and colleagues (7) studied BALF and mononuclear cells from patients with asthma and reported that asthmatics had a comparatively reduced ability to produce IL-10, a cytokine with well documented anti-inflammatory and immunoregulatory activities (9). In a subsequent report, Hobbs and associates (8) documented polymorphisms, putatively associated with defective transcription, in the promoter region of the IL-10 gene in patients with allergies and asthma. These associations led to the speculation that constitutive expression of IL-10 in the airway might contribute to maintain the normal state of allergen nonresponsiveness. Hypothetically, then, deficient production of IL-10, and possibly of other molecules such as transforming growth factor-beta , could allow allergic sensitization.

Murine models of experimental asthmatic inflammation have traditionally involved the intraperitoneal route to deliver antigen in association with complex adjuvants such as aluminum hydroxide (25, 31, 34). Both the route of delivery and the use of adjuvants in these models sharply contrast with aeroallergen sensitization in humans and establish an awkward pattern of antigen trafficking and presentation to the appropriate immune cells. Although mucosal exposure to OVA alone induces inhalation tolerance (3), we have recently demonstrated that expression of GM-CSF in the airway overrides this process (28). Under these conditions, the features of the ensuing immune-inflammatory response are consistent with an asthma-like process. The advantage of using an adenovirus-mediated gene-transfer approach to express GM-CSF in the airway environment, over delivery of recombinant cytokine, is that the transgene can be expressed in a dose-dependent manner in a given compartment for an extended, yet transient, period of time (22, 29, 34). In the present study we investigated whether concurrent expression of IL-10 in the airway environment could modulate GM-CSF-driven OVA-specific immune-inflammatory responses. As shown in Figures 2-4, expression of IL-10 in the airway inhibited the inflammatory response elicited by repeated OVA exposures in the context of GM-CSF in a dose-dependent manner. The inhibitory effect was not restricted to eosinophils but similarly affected neutrophils and mononuclear cells. In addition, the eosinophil number in the peripheral blood of IL-10-treated mice was reduced to the level observed in naive mice (Table 1), suggesting that inhibition of airway eosinophilia was likely the consequence of reduced bone-marrow eosinopoiesis rather than of mechanisms primarily connected with the migration of blood-borne eosinophils into the tissue. In addition, expression of IL-10 in the airway at the time of initial antigen exposure inhibited airway eosinophilia after recall challenge in vivo up to 6 mo, i.e., at a time when the acute inflammatory response had long since resolved.

We have previously reported that Ad infection per se can inhibit allergic airway inflammation in mice. This inhibition requires Ad doses substantially higher (20-fold) than those used in these experiments, and is IFN-gamma -dependent (37). In the studies reported here, we carefully controlled for the effect of the Ad infection by systematically including in all experiments a control group that received an equivalent dose of an empty-cassette Ad vector. However, to establish more conclusively that the inhibition of airway inflammation was indeed due to IL-10, we carried out a number of experiments in IFN-gamma KO mice. As shown in Figure 7, administration of the Ad/IL-10 construct to IFN-gamma KO mice inhibited GM-CSF-driven, OVA-induced airway inflammation to a degree similar to that in wild-type mice, and this was associated with dramatically reduced levels of IL-4, IL-5, and TNF-alpha in the BALF at Day 9 (Figure 8).

Airway responsiveness to MCh was examined at a number of time points in the course of our experimental protocol. RRS was measured during the acute inflammatory response, i.e., at 24, 48, and 72 h after the last aerosolization. Only at 48 h, RRS was slightly increased in mice exposed to OVA in the context of GM-CSF when compared with the RRS observed in naive mice (data not shown). In contrast, at a number of time points after resolution of the acute inflammatory response, recall challenge in vivo significantly increased RRS in mice that had been initially exposed to OVA expressing GM-CSF compared was naive mice. Even after 6 mo, OVA recall challenge induced airway hyperresponsiveness (AHR) (Figure 6), indicating that allergic mucosal sensitization led to long-lasting physiologic changes. Responsiveness to MCh in mice in which we had expressed IL-10 at the time of allergic mucosal sensitization was very similar to that observed in naive mice. Responsiveness to MCh in mice that had been exposed to RDA was not attenuated but, interestingly, significantly greater compared with that in GM-CSF-treated mice. The latter demonstrates that the abrogation of AHR in Ad/IL-10-treated mice was the consequence of IL-10 expression in the airway.

It is generally accepted that T cells are the main source of IL-5 in experimental models of asthmatic inflammation (38). That IL-10 inhibited T cell-dependent cytokine synthesis as well as the recall response in vivo strongly suggests that IL-10 prevented the generation of a productive, type 2-polarized T-cell response. However, in contrast to IL-5, both IL-4 and TNF-alpha can be produced by a variety of cell types during allergic inflammatory reactions (39). We wish to draw attention to the documented IgE-dependent release of TNF-alpha and IL-4 by mast cells (39, 40). Although OVA-specific IgE synthesis was only partially inhibited in mice treated with the Ad/IL-10 construct, the levels of these cytokines in the BALF of IL-10-treated mice were remarkably reduced, suggesting that IL-10 directly affected the ability of cells to produce cytokines. Indeed, we and others have shown that recombinant IL-10 markedly inhibits production of a number of cytokines by mast cells (43), eosinophils (17), and monocyte/macrophages (12, 13) in vitro.

Pivotal to the generation of antigen-specific T-cell responses is the presentation of the processed antigen by antigen-presenting cells (APCs) in conjunction with expression of appropriate costimulatory molecules, specifically B7.1 and B7.2 (44). The effect of IL-10 on costimulatory molecule expression on APCs is still a controversial issue. Although Mitra and coworkers (45) and Beulens and associates (46) demonstrated that IL-10 significantly reduces dendritic cell expression of CD86 (B7.2) but not CD80 (B7.1), these findings were in conflict with those reported by Morel and colleagues (47) and Thomssen and associates (48), who reported that IL-10 did not alter B7.1 and B7.2 expression. Here, we show that IL-10 expression in vivo reduced the number but not the proportion of lung dendritic cells and macrophages expressing B7.1 and B7.2 by 30%. It is well known that dendritic cells are recruited into the respiratory tract mucosa in response to local challenge with bacterial, viral and soluble antigen (49). Whether IL-10 prevented expansion of the APC compartment by affecting recruitment or, alternatively, by inhibiting differentiation of monocytes to APCs (50, 51) remains to be elucidated. Furthermore, it has yet to be clarified whether the overall decrease in antigen-presenting capacity is the prime mechanism accounting for the dramatic inhibition of airway inflammation observed in IL-10-treated mice. The defining feature of an antigen-specific immune response is the generation of activated T cells. In this regard, our data show that the CD4 and CD8 T-cell populations in IL-10-treated mice expanded to a degree virtually identical to that observed in mice exposed to OVA-expressing GM-CSF in the airway. Further, the proportion of these cells expressing the early activation marker CD69 was also similarly increased. This suggests that while IL-10 prevented airway inflammation it did not prevent the generation of an immune response. Groux and coworkers have shown that chronic activation of human and murine CD4+ T cells in the presence of IL-10 in vitro leads to the generation of a regulatory T-cell subset, referred to as Tr1, that actively suppresses antigen-specific immune responses in vivo (52). In this light, it is plausible that expression of IL-10 in vivo during GM-CSF-driven allergic mucosal sensitization promoted the development of a T-cell subset, whose phenotype is presently unknown, that has regulatory/suppressive activities. We speculate, therefore, that IL-10 did not inhibit but rather deviated the Th2-polarized immune response. The concept of deviation from Th2 has principally been described in reference to IFN-gamma and/or IL-12. In this respect, we have recently shown that concurrent gene transfer of IL-12 in our model of GM-CSF-driven allergic mucosal sensitization markedly inhibited airway eosinophilia and production of the Th2 cytokines IL-5 and IL-4 (53). However, the impact of concurrent airway expression of IL-12 or IL-10 on an ongoing immune response is fundamentally different. Indeed, a recall challenge in vivo resulted in a mononuclear/neutrophilic airway inflammatory response in IL-12-treated mice, but in no inflammatory response in mice treated with IL-10. That the concurrent expression of IL-10 deviates the T-cell response is at present speculative. Alternatively, it could be argued that activation of T cells in the presence of IL-10 leads to clonal deletion or anergy. Experiments investigating the effect of IL-10 on T-cell development in our model of allergic mucosal sensitization are being intensively pursued in our laboratory.

To perform its vital function of gas exchange, the lung must expose itself to the external environment. Because continuing exposure to airborne antigens is essentially unavoidable, the lung must have developed active mechanisms to maintain immunologic homeostasis, i.e., mechanisms that prevent potentially harmful, and unnecessary, immune-inflammatory responses. As suggested by Borish and associates (7) and Hobbs and colleagues (8), and by the experimental data that we report here, IL-10 may be one of the molecules involved in "allergen control." We suggest that the concept of IL-10 administration alone or in combination with GM-CSF as an immune-regulatory strategy that results in allergen nonresponsiveness deserves further analysis and development.

    Footnotes

Abbreviations: adenoviral, Ad; analysis of variance, ANOVA; antigen-presenting cell, APC; bronchoalveolar lavage fluid, BALF; enzyme-linked immunosorbent assay, ELISA; fluorescein isothiocyanate, FITC; granulocyte macrophage colony-stimulating factor, GM-CSF; interferon, IFN; immunoglobulin, Ig; interleukin, IL; knockout, KO; methacholine, MCh; major histocompatibility complex, MHC; ovalbumin, OVA; phosphate-buffered saline, PBS; phycoerythrin, PE; plaque-forming units, pfu; replication-deficient human type 5 Ad, RDA; respiratory system resistance, RRS; standard error of the mean, SEM; T-helper, Th; tumor necrosis factor, TNF.

(Received in original form April 5, 1999 and in revised form May 27, 1999).

Acknowledgments: One author (M.R.S.) was a holder of a Fellowship from the Medical Research Council/Canadian Lung Association; one author (B.U.G) holds an MRC Studentship; one author (S.A.R.) holds an Ontario Graduate Scholarship and The Professional Fellowship from the Canadian Federation of University Women; and one author (Z.X.) is a Scholar of the Medical Research Council (Canada). This study was supported in part by the Medical Research Council (Canada), Merck Frosst Canada, the Hamilton Health Sciences Corporation, and St. Joseph's Hospital. The technical help of Susanna Goncharova, Duncan Chong, and Xueya Feng, and the secretarial assistance of Mary Kiriakopoulos are gratefully acknowledged.
    References
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Abstract
Introduction
Materials and Methods
Results
Discussion
References

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