Broncholaveolar lavage fluid
IL-1α and IL-1β are potent pro-inflammatory cytokines that regulate many physiological systems by binding and signaling to the same receptor termed IL-1 receptor type 1 (IL-1R1). We have investigated the role of IL-1 for pulmonary immune responses in models of allergic asthma using IL-1R1-deficient (IL-1R1–/–) mice. In a model of mild asthma, based on repeated sensitization of mice with low doses of ovalbumin in the absence of any adjuvant and multiple intranasal challenges, the pulmonary eosinophilic inflammation and goblet cell hyperplasia were strongly reduced in IL-1R1–/– as compared to control BALB/c mice. Moreover, priming of CD4+ T cells in bronchial lymph nodes and their recruitment to the lung was affected in IL-1R1–/– miceassociated with impaired antibody responses including IgG, IgE, and IgA. In contrast, sensitization of mice in the presence of alum adjuvant, a more severe asthma model, rendered the IL-1 pathway dispensable for the development of pulmonary allergic Th2 responses, as eosinophilic inflammation, antibody responses, and CD4+ T cell priming in lymph nodes were comparable between IL-1R1–/– and wild-type mice. These results suggest a critical role of IL-1/IL-1R1 for development of allergic Th2 responses, but its requirement can be overcome by using alum as adjuvant for sensitization.
Asthma is a chronic inflammatory disease characterized by airway hyperresponsiveness, goblet cell hyperplasia, epithelial destruction, smooth muscle hypertrophy, thickening of the basement membrane, and an inflammatory infiltrate mainly consisting of eosinophils and CD4+ T cells of the Th2 subset 1, 2. The latter appear to be critical regulators of asthmatic responses by secretion of IL-4, IL-5, and IL-13, which are essential for the production of IgE, eosinophils, and mucus, respectively 1, 3.
A key element in the inflammatory response is, however, the prompt production of pro-inflammatory cytokines, notably IL-1β and IL-6, that may also alter airway responses in asthma 4. Levels of IL-1, IL-6, and TNF-α are significantly higher in patients suffering status asthmaticus, which is an acute respiratory failure combining an acute bronchospastic reaction with a severe airway inflammation 5. Increased percentages of macrophages producing IL-1β in the bronchial submucosa, expression of IL-1β by bronchial epithelium, and elevated levels of IL-1β in broncholalveolar lavage fluid (BALF) as well as tracheal biopsy materials have been reported in asymptomatic and symptomatic asthmatics 6, 7. IL-1 has been shown to induce the expression of eotaxin, a potent eosinophil chemoattractant, in pulmonary epithelial cells 8, and to promote recruitment of eosinophils possibly by inducing adhesion on vascular endothelium 9. Furthermore, exogenous administration of IL-1β creates airway responsiveness similar to that elicited by incubating naive airway smooth muscle with human asthmatic serum 10.
The two cytokines IL-1α and IL-1β mediate biological activity by binding to IL-1R type I (IL-1R1) that subsequently associates with an IL-1R accessory protein (IL-1R AcP) 11. This triggers a signaling cascade ultimately resulting in the activation of NF-κB and AP1. Both cytokines also bind to IL-1 receptor type 2 (IL-1R2), a decoy receptor with a short cytoplasmic tail that does not transduce intracellular signals 12. In addition, an endogenous IL-1R antagonist (IL-1Ra) exists that blocks binding and signaling of both IL-1α and IL-1β 13. The intricate system of agonists and antagonists suggests that tight control of IL-1 biological activity is physiologically important.
IL-1α and/or IL-1β elicit a wide array of biological activities including fever, loss of appetite, acute phase protein production, chemokine production, up-regulation of adhesion molecules, vasodilation, increased hematopoiesis, and the release of matrix metalloproteinases and growth factors 14. In the immune system, IL-1 promotes the activation and cytokine secretion of T cells and NK cells 14, 15. Notably, IL-1 has been reported to specifically promote the proliferation of Th2 cells 16–18 and specific antibody responses 19–21.
The above-mentioned studies suggest that IL-1 may significantly be involved in the development of pulmonary Th2 responses and the etiology of bronchial asthma, but its precise role remains to be established in experimental animal models of allergic asthma. We studied pulmonary immune responses in IL-1R1–/– mice. We found that IL-1 promotes asthmatic responses in a mild model of asthma, in which mice are repeatedly sensitized with low doses of ovalbumin (OVA) in the absence of adjuvant. Interestingly, IL-1 played little if any role for airway responses in a more severe model of asthma including sensitization in the presence of adjuvants.
2.1 Reduced pulmonary Th2-type inflammation in IL-1R1–/– mice
The role of IL-1α and IL-1β in the allergic inflammatory response in the lung was determined in a mouse model of asthma, which is induced by systemic sensitization with OVA followed by nasal challenges. IL-1R1–/– mice on a BALB/c background and wild-type controls were systemically sensitized by several successive intraperitoneal injections of OVA in the absence of adjuvant and exposed to inhaled OVA at four or eight successive days once a day (Fig. 1A). One day before the first and 1 day after the last nasal challenge, BALF was collected to assess the total cell number of inflammatory cell infiltrate and its composition of morphological different cell types.
The total number of cells in BALF recovered from groups of mice before inhalation of OVA was not significantly different between wild-type and IL-1R1–/– mice (5.1±0.1×104 in wild-type versus 4.97±0.2×104 in IL-1R1–/– mice); and most of these cells (>85%) were macrophages (not shown). In wild-type mice, a massive cell infiltration occurred after 4 days and 8 days challenges, when total cell numbers increased about 10-fold and 50-fold, respectively (Fig. 2). This increase primarily resulted from the infiltration of eosinophils (>50%). In contrast, cell infiltration was strikingly impaired in IL-1R1–/– mice. The number of eosinophils and lymphocytes was reduced by more than 70%, 1 day after the last exposure to allergen over 8 days.
Histological examination confirmed an extensive perivascular, peribronchial, pleural, and parenchymal inflammation in wild-type mice and few infiltrates in IL-1R1–/– mice (Fig. 3). In addition, combined alcian blue/periodic acid-Schiff staining showed a marked increase in mucus producing cells within the bronchial epithelium associated with inflammation in wild-type mice that was not present in IL-1R1–/– mice.
2.2 Reduced pulmonary migration of CD4+ but not CD8+ T cells in IL-1R1–/– mice
T cell subsets and cytokine production in the BALF were assessed by flow cytometry. The frequency of CD4+ and CD8+ T cells was reduced 20-fold and 6-fold in IL-1R1–/– as compared to wild-type mice 1 day after four challenges (Fig. 4A). Interestingly, after eight challenges only CD4+ T cells were reduced, while CD8+ T cells were augmented in IL-1R1–/– mice (Fig. 4B). At this time, the ratio of CD4+:CD8+ T cells was 10:2 versus 3:2, comparing wild-type and IL-1R1–/– mice. This indicates a specific defect in the recruitment of CD4+ T cells in the absence of IL-1R1. The effector status and polarization state of CD4+ T cells infiltrating the lung was analyzed by intracellular cytokine staining. Exposure to repeated nasal challenges with OVA allergen resulted predominantly in IL-4-producing CD4+ T cells and few IFN-γ-producing CD4+ T cells (12% versus 4% of BALF CD4+ T cells), demonstrating a preponderance of allergen-specific Th2 cells in the lung (Fig. 4c). In IL-1R1–/– mice, amongst CD4+ T cells recruited to the lung, the percentages of IL-4- and IL-5-producing cells were comparable, whereas IFN-γ-producing cells were slightly enhanced. These results suggest that IL-1 does not directly influence Th subset polarization.
We next investigated whether IL-1 directly acts on CD4+ T cells to promote lung recruitment. Therefore, IL-1R1–/– mice were crossed with OVA323–339 TCR transgenic (DO11.10) mice to obtain IL-1R1–/– DO11.10 mice. CD4+ T cells from IL-1R1–/– DO11.10 and control DO11.10 mice were purified and injected into RAG2–/– mice, which were subsequently exposed to intranasal antigen. BALF and lung parenchymal cells were recovered 24 h later and analyzed by flow cytometry for the presence of transgenic CD4+ T cells stained with KJ1.26 antibody. The frequency of IL-1R1–/– and IL-1R1+/+ OVA323–339-specific CD4+ T cells was comparable between BALF and lung tissue (Fig. 5), indicating that the absence of IL-1R1 on CD4+ T cells was not responsible for impaired pulmonary migration of CD4+ T cells in IL-1R1–/– mice.
2.3 IL-1 promotes the activation of CD4+ T cells
To assess the role of IL-1 for the priming of CD4+ T cells in lymphoid organs in response to inhaled allergen, we measured the proliferation of antigen-specific CD4+ T cells in lung draining lymph nodes after restimulation with OVA together with irradiated splenocytes in vitro. One day after the last of four nasal allergen exposures, CD4+ T cells obtained from IL-1R1–/– mice showed a reduced proliferation as compared to wild-type controls (Fig. 6). A similar result was observed 1 day after the last of eight exposures (not shown).
2.4 IL-1 is required for efficient antibody responses
It was previously reported that IL-1α β–/– mice mounted reduced antibody responses to sheep red blood cells 21, whereas antibody responses were normal in IL-1R1–/– mice immunized with TNP-keyhole limpet hemocyanin (TNP-KLH) 22, 23. We examined whether IL-1 was involved in antibody production in response to an immunization protocol resulting in allergic airway responses including high IgE antibody levels. To differentiate local and systemic antibody levels, we collected both BALF and serum 1 day after the final airway challenge. OVA-specific antibody levels of all isotypes including IgM, IgG, and IgE were considerably reduced in both the lung (Fig. 7A) and the serum (Fig. 7B) of IL-1R1–/– mice. These results show that IL-1 is required for efficient Th cell-independent (i.e. IgM) and Th cell-dependent (IgG, IgE) antibody responses.
2.5 Normal immune responses in IL-1R1–/– mice in a more severe model of asthma
The experiments described above were performed in a relatively mild model of airway inflammation, in which animals are repeatedly sensitized to low doses of OVA without adjuvant before exposure to aeroallergen. However, in the protocol used most commonly, animals are allergen-sensitized in the presence of alum as adjuvant before exposure to aeroallergen. This protocol results in a model with stronger responses and appears to represent a more severe type of an inflammatory response, because blood IgE levels are much higher. Interestingly, it has been described that the various models respond differently to modulation 24–26.
To investigate the role of IL-1 in a more "severe" immunization protocol including adjuvants, mice were sensitized with OVA in alum i.p. at day 0 and challenged intranasally from day 10 to 13 once per day. At day 1 after the final challenge, BALF was recovered and assessed by differential cell counting and by flow cytometry. The inflammatory infiltrate including mainly eosinophils was not significantly different between wild-type and IL-1R1–/– mice (Fig. 8A), although in the latter CD4+ T cells were slightly decreased and CD8+ T cells slightly increased (Fig. 8B). Antigen-specific CD4+ T cells of lung draining lymph nodes of IL-1R1–/– mice sensitized with OVA in alum proliferated normally after in vitro restimulation with OVA (Fig. 9). Last not least, levels of OVA-specific antibodies including all isotypes were unaffected in BALF and serum of IL-1R1–/– mice sensitized with OVA together with alum (Fig. 10). Together, these results demonstrate that IL-1 is dispensable for pulmonary Th2 responses mediating asthma when mice are sensitized in the presence of alum as adjuvant.
A body of literature demonstrated that exaggerated IL-6 and IL-1 production is a characteristic feature of asthmatic disease, implicating that these pro-inflammatory cytokines contribute to airway inflammation. However, studies in IL-6-deficient mice recently demonstrated that IL-6 inhibits rather than promotes pulmonary Th2-type inflammatory responses after exposure to aeroallergen 27. This prompted us to investigate asthmatic responses in IL-1R1–/– mice. Our results demonstrate that IL-1 promotes eosinophilic airway inflammation and goblet cell hyperplasia dependent on the severity of sensitization. In a mild asthma model, in which mice are repeatedly sensitized with low doses of OVA in the absence of adjuvants, IL-1R1–/– mice showed reduced pulmonary antibody responses, eosinophilia, and goblet cell mucus production, which are mediated by the Th2 cytokines IL-4, IL-5, and IL-13 1. Thus our results may argue that the reduced asthmatic responses resulted from impaired Th2 cell development in the absence of IL-1R1.
The contribution of IL-1 to Th2 cell polarization is controversial. Some reports suggested that IL-1 promotes proliferation and differentiation of Th2 cells 16–18, whereas another study demonstrated exaggerated Th2 responses in IL-1R1–/– mice 23. We found that the frequency of IL-4, IL-5, and IFN-γ producers amongst the pulmonary CD4+ T cells was not affected in IL-1R1–/– mice suggesting that IL-1 did not regulate Th subset development per se. Nevertheless, the total number of Th2 cells present in the lung was reduced, because the total number of pulmonary CD4+ T cells was diminished in IL-1R1–/– mice.
In contrast, numbers of CD8+ T cells were slightly augmented after nasal challenges suggesting that IL-1 selectively promotes the recruitment of CD4+ T cells into the lung after aeroallergen exposure. IL-1 may promote pulmonary CD4+ T cell recruitment by induction of chemokine(s), up-regulation of adhesion molecule(s) on lung endothelium, or by a direct signal through the IL-1R1 facilitating CD4+ T cell migration. We excluded the latter possibility by reconstitution of RAG2–/– mice with IL-1R1–/– and IL-1R1+/+ OVA323–339-specific TCR transgenic CD4+ T cells, which showed a comparable pulmonary migration after OVA inhalation. Although IL-1 has been shown to up-regulate expression of several adhesion molecules including vascular cell adhesion molecule-1 (VCAM-1), ICAM-1, E-selectin, and P-selectin on human umbilical vein endothelial cells, we do not think that this mechanism accounts for reduced CD4+ T cells and augmented CD8+ T cell number in the lung of IL-1R1–/– mice, because none of this adhesion molecules is known to differentially regulate CD4+ and CD8+ T cell extravasation.
The reduction of pulmonary CD4+ T cells probably reflects diminished priming of CD4+ T cells in bronchial lymph nodes of IL-1R1–/– mice as shown in Fig. 6. This is consistent with a recent publication showing that IL-1 production by APC promotes T cell priming by up-regulation of CD40L and OX40 21. Furthermore, we have demonstrated that IL-1R1–/– mice are protected from autoimmune myocarditis, because of impaired CD4+ T cell priming by dendritic cells. In fact, we found that IL-1R1 was essential for efficient activation of dendritic cells 28. Similarly, defective dendritic cell activation may explain impaired CD4+ T cell activation and protection from asthma in IL-1R1–/– mice. Furthermore, reduced OVA-specific antibody responses, including all isotypes (i.e. IgM, IgG, IgA, and IgE) in the lung and in the serum are consistent with impaired CD4+ T cell help in IL-1R1–/– mice after allergen exposure.
Interestingly, we found that the absence of IL-1R1 did not affect allergic immune responses in a more severe model of asthma including the potent Th2 adjuvant alum for sensitization of mice. IL-1R1–/– mice did not show significant differences in the number of eosinophils and CD4+ T cells in the lung. Moreover, CD4+ T cell priming and antibody responses were comparable between IL-1R1–/– and control mice after sensitization with OVA/alum and repeated intranasal challenges. Using a similar sensitization regimen with OVA/alum, another report recently described reduced pulmonary eosinophilia in IL-1R1–/– mice, but did not investigate any other parameter of the asthma immune response 9. We cannot explain the discrepancy with regard to lung eosinophilia, but it should be noted that we have used IL-1R1–/– mice backcrossed to BALB/c, a background that is highly susceptible to asthma, whereas the other study was done with hybrid C57BL/6 × 129J mice, a genetic background that is more resistant to development of asthmatic responses.
Our study demonstrates that usage of alum adjuvant bypasses the requirement of IL-1 for development of immune responses leading to airway inflammation typical for asthma. Of note, previous reports showed that specific antibody responses were normal or even enhanced in IL-1R1–/– mice after immunization with TNP-KLH/alum or TNP-KLH/CFA 22, 23, whereas specific antibody responses were reduced in IL-1R1–/– mice after immunization with sheep red blood cells 21, and it is conceivable that the differences reflect usage of adjuvant.
What is the molecular mechanism for allergic airway responses induced in the mild and in the more severe model of asthma? More recently, it has been shown that low doses (0.1 μg) of lipopolysaccharide (LPS) and Toll-like receptor 4 (TLR-4) are essential for development of pulmonary allergic Th2 responses after antigen (OVA) inhalation in a protocol abstaining from sensitization with alum adjuvant 29. In contrast, TLR-4 was not required when mice were sensitized with OVA together with alum adjuvant. In the mild model of asthma used here, Th2 cell priming and pulmonary Th2 responses in wild-type BALB/c mice may be explained by contamination of OVA with LPS, and IL-1R1–/– mice may be defective in low-dose LPS-induced Th2 responses, which we are currently investigating. The absence of allergic airway responses in TLR-4–/– mice sensitized with OVA and low doses of LPS has been suggested to result from impaired dendritic cell (DC) activation 29. In keeping with this, we have previously shown that DC maturation in response to LPS is impaired in IL-1R1–/– mice 28.
Taken together, our results underscore a key role of IL-1 in the development of Th2-type allergic airway responses to inhaled antigen in the absence of alum as adjuvant for sensitization. Our data also demonstrate that usage of alum adjuvant in animal models may mask the essential requirement of inflammatory signals driving asthmatic responses.
4 Materials and methods
4.1 Animals and immunization regimen
BALB/c wild-type mice were purchased by IFFA Credo (France). IL-1R1–/– mice 23 (originally kindly provided by M. Labow, Roche, Nutley) were backcrossed for eight generations to BALB/c and were maintained at the Basel Institute for Immunology (Basel, Switzerland) in a facility free of specific pathogens. Mice were used for experiments at the age of 8 to 12 weeks. For immunization, we used two protocols which have been referred to as a mild and a more severe asthma model 26. In the mild model of asthma, mice were i.p. immunized with 10 μg OVA (grade V; Sigma, St. Louis, MO) in 200 μl PBS/mouse at eight successive days, once per day. Ten days after the last OVA injection, mice were intranasally challenged with 100 μg OVAin 50 μl PBS for four or eight successive days, once per day. One day after each challenge period, mice were sacrificed for examinations. In a more severe model of asthma, mice were i.p. immunized with OVA in alum (50 μg/0.2 ml PBS) once at day 0. Ten days after OVA sensitization, mice were intranasally challenged with 100 μg OVA in 50 μl PBS for 4 days, once per day. One and 4 days after the last challenge, mice were sacrificed for examinations.
4.2 Bronchoalveolar lavage
OVA-immunized mice were sacrificed by CO2 at the specified time points after OVA challenge. The tracheae of individual mice were cannulated with a 22-G needle surrounded by plastic tubing. PBS (0.3 ml) was repeatedly injected and withdrawn with a syringe until a final volume of 1.2 ml BALF was collected. BALF cells were harvested by centrifugation. Total cell numbers per BALF were determined with a Coulter Counter (IG Instrumenten Gesellschaft AG, Basel, Switzerland), and cells were processed for differential cell counts and flow cytometry analysis. BALF was used to measure levels of OVA-specific antibodies of different isotypes by ELISA.
4.3 Morphological differentiation of BALF cells
BALF cells of individual mice were spun (600×g, 10 min) onto glass slides using a cytospin centrifuge and fixed with methanol for 2.5 min before staining with undiluted May-Grünwald solution (Fluka, Buchs, Switzerland) for 3 min. Thereafter the staining was performed in a 50% May-Grünwald solution for further 3 min. In a last step cells were stained in 7% Giemsa solution (Fluka) for12 min. Slides were rinsed with tap water and air-dried overnight. Dried cells were embedded in Eukit solution under glass cover slips. The relative frequencies (%) of eosinophils, lymphocytes, macrophages and neutrophils per 200 cells were determined and the total number of each cell type per BALF was calculated from the total number of BALF cells (100%).
4.4 Characterization of BALF T cells
BALF CD4+ and CD8+ T cells were analyzed at indicated time points after the last OVA challenge. Isolated BALF cells were incubated with anti-CD32/CD16 mAb in PBS/0.1% BSA for30 min at 4°C to block unspecific binding of antibodies to Fc receptors. After blocking, cells were washed with PBS/0.1% BSA followed by staining with FITC-labeled anti-α βTCR, APC-labeled anti-CD4 and PE-labeled anti-CD8 mAb (BD PharMingen) in PBS/0.1% BSA for 30 min at 4°C. Subsequently, cells were washed with PBS/0.1% BSA and resuspended in PBS/1% BSA for analysis by flow cytometry.
4.5 Expression of intracellular cytokines
Cytokine production by T cells in the lung was assessed essentially as described 30. BALF cells (106/mouse) were stimulated with PMA (10–7 M) and ionomycin(1 μg/ml) for 4 h at 37°C in RPMI medium. Two hours prior to harvesting, Brefeldin A (10 mg/ml) was added to cultures to retain cytokines in the cytoplasm. Thereafter, cells were washed with PBS/0.1% BSA and incubated with anti-CD32/CD16 mAb for 30 min at 4°C to block Fc binding. After another washing step, cells were stained with APC-labeled anti-CD4 mAb (BD PharMingen) for 30 min at 4°C. Subsequently, cells were washed with PBS/0.1% BSA, fixed with 2% paraformaldehyde for 30 min at room temperature followed by intracellular staining using FITC-labeled anti-IFN-γ and PE-labeledanti-IL-4 or PE-labeled anti-IL-5 mAb (BD PharMingen) diluted in permeabilization buffer (0.5% saponin/PBS/1% BSA). After a final washing step, cells were resuspended in PBS/1% BSA and analyzed by flow cytometry (FACSCalibur; Becton Dickinson) and the Cell Quest Pro software.
4.6 Proliferation of CD4+ T cells
OVA-immunized mice were sacrificed at indicated time points after the last aerosol challenge. CD4+ T cells were positively purified from mediastinal lymph nodes of individual mic by MACS® (purity >95%), according to the instructions of the supplier (Miltenyi Biotec). CD4+ T cells (2×105) were restimulated in Iscove's modified Dulbecco's medium/10% FCS with 2×105 of irradiated splenic APC from the same genetic background. APC were pulsed with OVA in threefold dilution steps, starting with 200 μM. At day 3 of restimulation, cells were pulsed with [3H]thymidine for 12 h before [3H]thymidine incorporation (cpm) was measured.
4.7 Detection of antibodies by ELISA
At indicated time points after the last OVA challenge, BALF of mice was analyzed for OVA-specific IgG, IgE, IgM, and IgA antibodies. 96-well plates (Maxisorp; Nunc) were coated with OVA in a concentration of 50 μg/ml in 50 μl PBS overnight at 4°C. Between all following steps plates were washed five times with PBS. Coated plates were blocked with PBS/1% BSA for 2 h at room temperature. BALF from individual mice, serially diluted in PBS/0.1% BSA, was then added with a starting dilution of 1:100 followed by incubation overnight at 4°C. Thereafter, alkaline phosphatase-labeled goat anti-mouse antibodies to IgG, IgE, IgM, and IgA (Southern Biotechnology Associates, Inc.) were added at room temperature for 2 h followed by addition of the substrate p-nitrophenyl phosphate (Sigma-Aldrich) before reading the optical density (OD) at 405 nm.
4.8 Adoptive transfer experiment
CD4+ T cells of IL-1R1–/– DO11.10 and IL-1R1+/+ DO11.10 mice were enriched by elimination of CD8+ and MHC class II+ cells using antibody-coupled magnetic Dynabeads and injected into RAG2–/– mice (107 CD4+ cells/mouse). Within the next 2 days after transfer, RAG2–/– recipient mice were intranasally challengedwith 100 μg OVA in PBS twice per day separated by 8 h. Twelve hours after the last challenge, BALF was harvested, and the lung was removed and single cells were prepared after collagenase treatment. Cells were surface-stained with PE-labeled anti-α βTCR mAb in combination with a FITC-labeled anti-KJ1.26 mAb specific for the OVA-transgenic TCR.