Introduction

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Bronchial asthma is a common disease that is defined by reversible bronchospasm, airway inflammation, and airway hyperresponsiveness (AHR) (1). It is now commonly accepted that the main underlying pathologic aspect of bronchial asthma is airway inflammation which correlates with severity of the disease and is responsible for the development of AHR (2, 3). The inflammatory response is characterized by the appearance of CD4⁺ T helper (Th) 2-cytokine-producing T lymphocytes, mast cells, and eosinophils in bronchial biopsies and in the bronchoalveolar lavage (BAL) fluid of asthmatic patients (4). Two cytokines, interleukin (IL)-4 and IL-5, seem to be essential to the pathogenesis of the disease. IL-4 has a central role in the regulation of atopic diseases because it is a key factor in the production of immunoglobulin (Ig) E (7, 8), induces and sustains the development of Th2 cells from precursor Th0 lymphocytes (9), and enhances airway inflammation by upregulation of chemokines, such as eotaxin, and adhesion factors, such as vascular cell adhesion molecule-1, required for the migration of eosinophils into inflamed tissue (10). IL-5, on the other hand, is the principal cytokine for the growth, differentiation, activation, and survival of eosinophils (11); levels of IL-5 are elevated in the blood, in lung tissue, and in the BAL fluid of asthmatic patients, and correlate with the degree of AHR (4, 14, 15).

Although a great deal of progress has been achieved in studies on the pathology of bronchial asthma, the mechanism(s) underlying the development of airway inflammation and AHR is still not fully defined. The question remains, which is (are) the principal pathologic event(s) initiating the inflammatory cascade? Furthermore, the basic mechanisms that link airway inflammation and AHR are not entirely clear. Recently, a number of studies using mouse models of bronchial asthma to identify the key elements involved in the regulation of airway inflammation and AHR have been reported. From these studies, a number of conflicting conclusions on the importance of specific cytokines have emerged. On the one hand, there is strong evidence for the central role of IL-5-mediated, eosinophilic inflammation as a key regulatory event in the development of AHR. Studies on IL-5-deficient mice (16) and on mice that have received anti-IL-5 treatment (17) support the importance and the requirement of IL-5 in the induction of eosinophilic inflammation and AHR. On the other hand, there are conflicting results on the role of IL-4, either showing the necessity (18) or the lack of a requirement for IL-4 (21) in the development of airway inflammation and AHR. Other studies have emphasized the importance of IgE for the activation of mast cell degranulation, enhancement of Th2 cytokine production (22), or augmentation of eosinophilic infiltration of the peribronchial regions of the lung (23). The use of different sensitization and challenge protocols, strains of mice, and read-outs of altered airway responsiveness preclude easy resolution of some of these controversies.

We have established a murine model of bronchial asthma where systemic sensitization to ovalbumin (OVA) followed by repeated allergen airway challenges induces eosinophilic airway inflammation and increased airway reactivity to inhaled methacholine (MCh) (24). Here, we used this approach to directly compare the outcome of sensitization and airway challenge in IL-4- and IL-5-deficient mice and to identify the key regulatory elements by reconstituting the deficient mice with specific cytokines as well as by passively sensitizing them with allergen-specific IgE. We show that IL-4 plays a pivotal role not only for IgE but also for IL-5 production and development of eosinophilic inflammation, and that IL-5 alone, in the absence of IgE, is capable of reconstituting IL-4-deficient mice to develop eosinophilic inflammatory responses and AHR.

	Materials and Methods

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Animals

C57BL/6-IL-4^null (IL-4-deficient mice, IL-4^/) (25) and normal C57BL/6 (B6) mice from 8 to 12 wk of age were obtained from Jackson Laboratories (Bar Harbor, ME). C57BL/6-IL-5^null (IL-5- deficient mice, IL-5^/) were obtained from the Max Planck Institut (Freiburg, Germany) (26). The mice were maintained on OVA-free diets. All experimental animals used in this study were under a protocol approved by the Institutional Animal Care and Use Committee of the National Jewish Medical and Research Center.

Sensitization and Airway Challenge

The following basic groups of age- and sex-matched mice (three to four mice/group/experiment) were studied: ipNeb: normal (B6-ipN) and cytokine-deficient (IL-4-ipN, IL-5-ipN) mice were sensitized by an intraperitoneal injection of 20 µg OVA (Sigma, St. Louis, MO) emulsified in 2 mg aluminum hydroxide (AlumImject; Pierce, Rockford, IL) in a total volume of 100 µl on Days 1 and 14. Mice were then challenged via the airways with OVA (1% in phosphate-buffered saline [PBS]) for 20 min on Days 28, 29, and 30 by ultrasonic nebulization (DeVilbiss, Somerset, PA), and lung function was assessed on Day 32. Neb: nonsensitized control animals that only received OVA airway challenges on Days 28 through 30.

Reconstitution Experiments

Mice were passively sensitized three times before the first allergen challenge via the airways by an intravenous injection of anti-OVA-specific IgE (27) (4 µg in 20 µl PBS) on Days 26, 27, and 28 of the sensitization protocol. Passive sensitization resulted in cutaneous and systemic anaphylactic reactions in IL-4-deficient mice after intradermal or intravenous injections of allergen, respectively (data not shown).

Cytokine production was restored in IL-4- and IL-5-deficient mice by intranasal instillation of recombinant adenovirus vectors (AV) expressing the complementary DNA (cDNA) for murine IL-4 or IL-5 (AV-IL-4, AV-IL-5) (28, 29). Control animals were nonsensitized, IL-4-deficient mice receiving AV-IL-4 or AV-IL-5 before airway challenge, or sensitized, IL-4- and IL-5-deficient mice receiving a control (empty) AV. Each mouse received 5 × 10⁸ pfu of the respective vector.

Measurement of Anti-OVA Antibody and Total Ig Levels

Anti-OVA IgE and IgG1 serum levels were measured by enzyme-linked immunosorbent assay (ELISA) as previously described (17). The antibody titers of the samples were related to pooled standards that were generated in the laboratory. Total IgE levels were determined using the same method as previously described. Total Ig levels were calculated by comparison with known mouse IgE standards (PharMingen, San Diego, CA). The limit of detection was 100 pg/ml for IgE.

In Situ Hybridization of Cytokine Messenger RNA

Cytokine messenger RNA (mRNA) was detected in the lung tissue using radiolabeled RNA probes and in situ hybridization (ISH). Briefly, lung tissue was fixed in freshly prepared 4% paraformaldehyde for 3 h, then washed twice with 15% sucrose in PBS. Tissue samples were mounted, embedded in optimal cutting temperature compound, snap-frozen, and stored at 70°C. Labeling of riboprobes and ISH was performed as described (30, 31).

BAL and Lung Digestion

Lungs were lavaged via a tracheal tube with Hanks' balanced salt solution (HBSS; 3 × 0.5 ml), and the cells in the lavage fluid were counted. Lung cells were isolated by enzymatic digestion as previously described (17). Cells from BAL or lungs were resuspended in HBSS and counted with a hemocytometer. Cytospin slides were stained with Leukostat (Fisher Diagnostics, Pittsburgh, PA) and analyzed in a blinded fashion by counting at least 300 cells under light microscopy.

Immunohistochemistry

After perfusion via the right ventricle, lungs were inflated through the tracheas with 10% formalin and fixed in 10% formalin for 48 h. Major basic protein (MBP) in lung sections was localized by immunohistochemistry with rabbit-antimouse MBP (kindly provided by Drs. G. Gleich and J. J. Lee, Mayo Clinic, Rochester, NY and Scottsdale, AZ, respectively) as previously described (17). Slides were examined in a blinded fashion with a Nikon (Tokyo, Japan) microscope equipped with a fluorescein filter system. Numbers of eosinophils in the submucosal tissue around central airways were evaluated using the IPLab2 software (Signal Analytics, Vienna, VA) for the Macintosh computer, counting four different sections per animal (17).

Determination of Airway Responsiveness

In vivo lung resistance (RL) to MCh was measured as described (32). Briefly, the mice were anesthesized, and tracheas were cannulated and placed in a pressure plethysmograph. A four-way connector was attached to the tracheostomy tube, with two ports connected to the inspiratory and expiratory sides of the ventilator (model no. 683; Harvard Apparatus, South Natick, MA) and the third port attached to one side of a differential pressure transducer. Ventilation was achieved with a rate of 160 breaths/min, tidal volume of 150 µl during recording, and with a rate of 60 breaths/min, tidal volume of 500 µl during MCh aerosol delivery. MCh (6 to 100 mg/ml) was administered as an aerosol for each concentration via the tracheal cannula. Changes in transpulmonary pressure and volume of the plethysmograph and the animal flow were derived with digital differentiation. RL was calculated from peak values after each challenge. Data are expressed as the percent of PBS baseline values.

Statistical Analysis

Analysis of variance was used to determine the levels of difference between all groups. Pairs of groups were compared by Student's t test. Comparisons for all pairs were performed by Tukey-Kramer HSD test for airway responsiveness and histology data. Values of P for significance were set to 0.05. Values for all measurements are expressed as the mean ± standard deviation (SD) except values for RL, which are presented as the mean ± standard error of the mean (SEM), and MCh doses for 100% increase, which are presented as mean values (upper and lower 95% confidence limits).

	Results

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IL-4-Deficient Mice Fail to Produce IgE but Do Produce Allergen-Specific IgG2a

Normal and cytokine-deficient B6 mice were sensitized by an intraperitoneal injection of OVA emulsified in alum on Days 1 and 14 followed by daily airway challenges with OVA performed on Days 28, 29, and 30. Control mice received airway challenges alone. Serum levels of OVA-specific and total Igs were measured 2 d after the last airway challenge, on Day 32. Sensitization and challenge with OVA resulted in significantly increased serum levels of anti-OVA IgE and IgG1 and of total IgE in normal (Table 1) as well as in IL-5-deficient mice. Sensitization did not significantly alter total IgG serum levels. In contrast, serum levels of total and OVA-specific IgE antibodies were below the limit of detection before (data not shown) and after sensitization and airway challenge in IL-4-deficient mice, whereas this strain of mice, in contrast to  normal or IL-5-deficient mice, produced measurable amounts of allergen-specific IgG2a.

IL-4- and IL-5-Deficient Mice Produce Little or No IL-5

To assess local cytokine production at the site of allergen challenge, ISH of cytokine mRNA in lung tissue was performed. The results of IL-4 and IL-5 mRNA hybridization in all groups, expressed as the number of positive cells/ mm², are shown in Figure 1. Sensitization and challenge significantly enhanced IL-4 mRNA (by 18-fold) and IL-5 mRNA (by > 11-fold) production in lung tissue of normal B6 mice compared with nonsensitized animals. In IL-5- deficient mice, sensitization and airway challenge induced an even higher increase in numbers of IL-4 mRNA-positive cells compared with normal B6 mice, whereas IL-5- positive cells predictably were absent. In contrast, IL-4- deficient mice not only showed an impaired ability of IL-4 production but also displayed a significantly reduced number of IL-5-positive cells in lung tissue compared with sensitized and challenged normal B6 mice (< 3-fold increase compared with nonsensitized control mice). Nonsensitized, IL-4- and IL-5-deficient mice showed little or no IL-4- or IL-5-positive cells and thus were not significantly different from nonsensitized, normal control mice (data not shown). These data indicate that sensitization and airway challenge with OVA induces a Th2-like cytokine profile with induction of IL-4 and IL-5 mRNA-positive cells at the site of local allergen challenge in the lung tissue. Moreover, the results indicate that the increase in IL-5- positive cells is not seen in the IL-4-deficient mice.

View larger version (19K): [in this window] [in a new window]   Figure 1.   IL-4 and IL-5 production in lung tissue increase after sensitization and challenge. Normal ( filled columns) (B6 ipNeb; n = 16), IL-4-deficient (wide-striped columns) (IL-4-ipNeb; n = 12), and IL-5-deficient (closely striped columns) (IL-5-ipNeb; n = 12) mice were sensitized by intraperitoneal injection of OVA/alum on Days 1 and 14 followed by airway challenges with OVA on Days 28, 29, and 30. Nonsensitized but challenged mice serve as normal controls (open columns) (B6 Neb; n = 12). Two days after the last challenge, lungs were harvested and prepared for ISH for IL-4 and IL-5 mRNA. Numbers of IL-4- and IL-5-positive cells per mm2 lung tissue were counted in a blinded fashion. Values expressed are the mean ± SD (of positive cells) from three independent experiments. *P < 0.01 compared with controls.

IL-4- and IL-5-Deficient Mice Have Little or No Signs of Airway Inflammation

To evaluate the importance of IL-4 and IL-5 in the development of allergic inflammation after sensitization and challenge, total leukocyte and differential cell counts in isolated lung cells and in the BAL fluid of individual mice were compared. Numbers of total leukocytes, lymphocytes, and neutrophils were increased, and numbers of macrophages were decreased after allergen sensitization and airway challenge (data not shown). Sensitization and airway challenge also resulted in a more than 3-fold increase in total cell numbers in the BAL fluid of normal B6 mice (data not shown), and the predominant cells were eosinophils with a 70-fold increase in number (Table 2). The number of eosinophils was also significantly increased in lung cell preparations from sensitized and challenged normal B6 mice to approximately 13-fold compared with nonsensitized control mice. In contrast, sensitization and allergen challenge of IL-4-deficient mice resulted in a significantly lower increase in eosinophil numbers (2-fold in lung digests, 10-fold in BAL fluid) when compared with nonsensitized, control mice and when compared with sensitized and challenged normal B6 mice. In IL-5-deficient mice, few eosinophils were detected among the cells from lung digests or in BAL fluid after sensitization and airway challenge.

To localize eosinophils in the lung tissue, immunohistochemistry with anti-MBP antibody was performed on formalin-fixed lung sections. The number of MBP-positive cells was measured in the peribronchial tissue of central airways using computer-assisted analysis (17). In normal B6 mice, sensitization and airway challenge significantly increased the numbers of eosinophils by approximately 17-fold (Table 2). Again, both IL-4- and IL-5-deficient mice showed markedly decreased (IL-4^/) or no (IL-5^/) eosinophil infiltration of the airways after allergen sensitization and challenge. These data indicate that eosinophil infiltration of lung tissue and their accumulation in BAL fluid is virtually dependent on the presence of IL-5 and, to a lesser degree, on IL-4.

IL-4- and IL-5-Deficient Mice Fail to Develop Increased Airway Responsiveness

To assess the importance of IL-4 and IL-5 in the development of AHR after sensitization and airway challenge with OVA, we monitored RL after challenge with aerosolized MCh. Sensitization and repeated airway challenge with allergen of normal B6 mice increased airway responsiveness to aerosolized MCh with a left shift in the dose-response curve (100% increase of RL with 17.8 mg/ml [range, 14.6; 23.5] of MCh compared with 54.7 mg/ml [range, 44.6; 68.4] of MCh in nonsensitized controls) and increased RL (507 ± 42% increase after 100 mg/ml of MCh compared with 138 ± 11% in nonsensitized controls) (Figure 2). In contrast, neither IL-4- nor IL-5-deficient mice showed any evidence of increased airway responsiveness after sensitization and airway challenge: the dose-response curves were similar to those of nonsensitized control mice and there was no increase in RL above control values. These data imply that development of AHR after systemic sensitization and airway challenge is dependent on IL-4 and IL-5 production.

View larger version (16K): [in this window] [in a new window]   Figure 2.   Absence of AHR in IL-4- and IL-5-deficient mice. Groups and sensitization are as in Figure 1. Two days after the last airway challenge, airway responsiveness to aerosolized MCh was assessed as described in MATERIALS AND METHODS. Aerosolized PBS and MCh were administered via the tracheostomy. Pulmonary resistance was calculated as RL = difference in tracheal pressure/flow change from peak values after each challenge. Values expressed are the means ± SEM of RL as % of PBS baseline values from two independent experiments. *P < 0.01 versus nonsensitized controls. Baseline resistance values were not significantly different between groups: B6, 0.42 ± 0.06; IL-4/, 0.43 ± 0.08; IL-5/, 0.44 ± 0.12 cm H2O ml1 s.

IL-5 but Not IgE Reconstitutes Airway Inflammation and AHR in IL-4- and IL-5-Deficient Mice

To further delineate the roles of IL-4 and IL-5 in the development of AHR in this model, and to further distinguish whether the decrease in IgE production and/or the marked decrease in IL-5 production was responsible for the reduced inflammation and airway response in IL-4-deficient mice, we reconstituted animals with allergen-specific IgE, IL-4, and IL-5. Allergen-sensitized, IL-4-deficient mice were passively sensitized with anti-OVA IgE three times (two days and one day before, and on the day of the first allergen airway challenge). In different groups of mice, allergen-sensitized IL-4- or IL-5-deficient mice received a single intranasal dose of AV expressing IL-4 or IL-5 cDNA, respectively, 48 h before the first airway challenge with allergen. Control animals were nonsensitized IL-4- or IL-5- deficient mice receiving AV-IL-4 or AV-IL-5 before airway challenge, or sensitized IL-4- and IL-5-deficient mice receiving control AV.

Passive sensitization of IL-4-deficient mice with allergen-specific IgE generated measurable amounts of allergen-specific IgE serum levels, comparable to the levels detected in sensitized/challenged, normal B6 mice (Table 1), and induced anaphylactic reactions after intradermal or intravenous injection of allergen (data not shown). However, passive sensitization with anti-OVA IgE failed to influence eosinophil numbers in the BAL, lung digests, or tissue (Table 2), or airway responsiveness to inhaled MCh (Figure 3). In contrast, reconstitution of allergen-sensitized IL-4-deficient mice with either IL-4 or IL-5 before airway challenge with OVA fully restored eosinophilic inflammation of the lungs and airways (Table 2) and resulted in the development of AHR, indistinguishable from the normal B6 mice (Figure 3). Reconstitution of sensitized IL-5-deficient mice with IL-5, but not with IL-4, restored eosinophil airway infiltration (Table 2) and development of increased airway responsiveness (data not shown). Sensitized, IL-4-deficient mice treated with control AV or nonsensitized, IL-4-deficient mice treated only with IL-4 or IL-5 AV had no significant airway inflammation and no increases in airway responsiveness (data not shown).

View larger version (19K): [in this window] [in a new window]   Figure 3.   IL-5 but not IgE reconstitutes AHR in IL-4-deficient mice. Groups and sensitization are as in Figure 1. Measurement of RL is the same as described in Figure 2. Groups: nonsensitized but challenged, normal mice (B6-Neb; n = 12); sensitized and challenged, normal mice (B6-ipNeb; n = 16); sensitized and challenged, IL-4-deficient mice (IL-4-ipNeb, n = 12); sensitized and challenged, IL-4-deficient mice passively sensitized with anti-OVA IgE (IL-4-IgE; n = 12); sensitized and challenged, IL-4- deficient mice treated with IL-4-transfected AV (IL-4-AV4; n = 12); and sensitized and challenged, IL-4-deficient mice treated with IL-5-transfected AV (IL-4-AV5; n = 12). *P < 0.01 versus nonsensitized controls. Control animals: sensitized and challenged, IL-4-deficient mice treated with sham AV; nonsensitized, IL-4-deficient mice treated with IL-4 or IL-5 AV had airway responses similar to nonsensitized, normal B6 mice.

	Discussion

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In the present study, we investigated the effects of systemic sensitization with allergen followed by repeated airway challenge on inflammatory responses and the development of AHR in normal and in IL-4- and IL-5-deficient mice. Systemic sensitization induced a marked increase in allergen-specific IgE and IgG1 in normal and in IL-5-deficient mice. In contrast, IL-4-deficient mice failed to produce measurable IgE levels but did increase production of allergen-specific IgG2a. The increase in IgE production after sensitization and challenge was paralleled by increases in IL-4 mRNA⁺ cells in the lung tissue in normal and in IL-5- deficient mice. In addition to IL-4, we also observed a significant increase in local IL-5 mRNA⁺ cells after repeated airway challenge of sensitized, normal mice. Associated with the increases in IL-5 production, normal mice developed infiltration of eosinophils in the airways, mostly located in the peribronchial regions of the smaller and middle-sized airways identified by immunohistochemistry and accumulation in the BAL fluid. Sensitized, IL-4- and IL-5- deficient mice showed significantly lower (IL-4-deficient) or absent (IL-5-deficient) IL-5 production in the lung tissue after repeated airway challenges compared with normal sensitized and challenged mice. This resulted in significantly lower or absent eosinophilic airway inflammation. In parallel, only normal mice developed AHR to inhaled MCh after systemic sensitization and airway allergen challenges, whereas both IL-4- and IL-5-deficient mice developed airway responses comparable to those of nonsensitized control mice.

These data demonstrate that the pathologic mechanisms underlying allergic inflammation are largely dependent on the presence of IL-4. In these experiments, IL-4-deficient mice not only failed to produce IgE but mounted marginal airway inflammatory responses and no increase in airway reactivity to MCh. This confirms and extends previous studies in IL-4-deficient mice that pointed to the requirement for IL-4 in inflammatory responses and subsequent development of AHR (19). These experiments also support previous experimental approaches that targeted IL-4 (33, 34). IL-4 is the main cytokine involved in the regulation of IgE production by inducing isotype switching to the  -heavy chain (7). IgE binds to the high affinity receptor expressed on mast cells and mediates the activation, production, and degranulation of these cells releasing cytokines, histamine, and other proinflammatory mediators (35). Indeed, it is attractive to suggest that this role of IgE is a major trigger for the induction of tissue inflammation and tissue injury leading ultimately to the development of AHR (22, 23).

However, the role of IgE in this and other models may be more limited. It has been demonstrated in IgE-deficient mice that anaphylactic reactions after allergen sensitization can occur independently of IgE (38), possibly mediated by allergen-specific IgG1 (27). Moreover, airway inflammation and AHR develop in mast cell-deficient mice to a similar degree as in normal littermates (32), ruling out a major contribution of this cell in the induction of airway sensitization in mice. This would also explain the findings that mast cells are not increased in lung tissue or BAL fluid of sensitized and challenged mice (16). Other investigators have proposed a role for IgE in the induction of Th2 cytokine production (22). They demonstrated that anti-IgE antibody treatment of sensitized mice before airway challenge inhibited eosinophil accumulation in the lungs and suggested that IgE enhances T-cell function in a B cell-mediated fashion via the low affinity IgE receptor CD23. We have previously demonstrated that CD23 genetically deficient mice show similar degrees of eosinophilic infiltration of the airways and development of AHR when compared with normal littermates after a sensitization and challenge protocol as performed in this study (39). In our own studies using anti-IgE antibody, we did not find any inhibiting activity of the antibody on airway inflammation and AHR despite its ability to prevent anaphylactic reactions (40). A possible role for high IgE serum levels for the enhancement of eosinophil trafficking into the airway lumen after airway challenge of sensitized mice has been proposed (23). We have examined the effects of sensitization and airway challenge in B cell-deficient mice and detected no differences in the lung or BAL inflammatory responses, including the localization and degranulation of eosinophils in the lung tissue, and the development of AHR when compared with normal mice (41).

To complete the assessment of the role of IgE production in airway inflammation and AHR in the present study, we reconstituted sensitized, IL-4-deficient mice with allergen-specific IgE before allergen airway challenge. We previously demonstrated that passive sensitization in this way induces systemic and cutaneous anaphylactic reactions, and, in combination with three airway challenges, mild eosinophil airway infiltration and AHR in normal BALB/c mice (27). In contrast, this mode of sensitization and airway challenge was not able to generate inflammatory responses or increased airway reactivity in T cell-deficient (nude) BALB/c mice unless they were treated with IL-5 before airway challenges (42). These findings were confirmed in the present study using a different approach: passive sensitization of IL-4-deficient mice with allergen-specific IgE before airway allergen challenge did not induce significant increases in eosinophil numbers in lung tissue or BAL fluid, or increases in airway reactivity to MCh despite the appearance of anaphylactic reactions after intradermal or intravenous injections of allergen (data not shown). Cumulatively, these results demonstrate the independence of airway inflammation and AHR from (allergen-specific) IgE in mice sensitized systemically and challenged via the airways.

In addition to a role in IgE production, IL-4 appears essential for the induction and maintenance of Th2-type immune reactions in vitro (43) and in vivo (25). Th2 cells mediate IL-4-dependent tissue inflammation (44), and depletion of CD4⁺ or CD8⁺ Th2 lymphocytes prevents airway eosinophilia and AHR (45, 46). We show here for the first time that local IL-5 production in the lung tissue of sensitized and challenged, IL-4-deficient mice is significantly reduced compared with that of normal mice. As a consequence, IL-4-deficient mice generate very reduced (albeit not absent) airway inflammation and no signs of increased airway reactivity to MCh. This appears to be due entirely to the decrease in local IL-5 production. This marked reduction, but not elimination, of eosinophil recruitment into the airway has previously been observed in IL-4^/ mice (21) and suggests that IL-5-producing cells (presumably T cells) can develop in response to allergen challenge to a limited extent in the absence of IL-4. We have shown a similar response in IL-4^/ mice exposed to respiratory syncytial virus, namely the presence of eosinophilic inflammation, but lower than that observed in IL-4^+/+ mice (47).

To clarify the importance of IL-4 in the deficiency of IL-5 and attenuation of AHR, we reconstituted local IL-5 production in sensitized, IL-4- and IL-5-deficient mice with IL-5-encoding AV before airway allergen challenge. Reconstitution of IL-5 production in IL-5-deficient, sensitized and challenged mice fully restored eosinophilic inflammatory responses and the development of AHR. This supports data from a similar study in IL-5-deficient mice (16). More importantly, restoration of local IL-5 production in sensitized, IL-4-deficient mice before airway challenge with allergen also fully restored eosinophilic inflammation of the airways and induced the development of increased airway reactivity to MCh. These data clearly demonstrate that the inability of sensitized and challenged, IL-4-deficient mice to generate a significant eosinophilic inflammatory response and to develop AHR to inhaled MCh is, to a substantial degree, due to the decrease in local IL-5 production in the lung.

These data establish IL-5 as the key factor in the development of eosinophilic inflammation and AHR. Sensitized, IL-5-deficient mice show no eosinophilic infiltration of the airways and no increase in airway reactivity after repeated airway allergen challenge despite elevated OVA-specific IgE serum levels. These data are similar to the results from studies where IL-5 was neutralized by monoclonal antibody treatment (17, 23) and underscore that eosinophil infiltration and IgE production are independently regulated. These studies fail to reconcile reports of the dissociation between IL-5/eosinophilic inflammation and AHR (20, 48, 49). Careful analysis of many of the reports identify potentially important differences in the protocols: the amount of antibody used to block IL-5-mediated eosinophil airway infiltration may be insufficient (20); eosinophil activation is not monitored and there may be discrepancies between tissue and BAL eosinophil numbers (50); the mode of sensitization and challenge and the use of adjuvant may differ and result in different cytokine patterns (49); the end-point measurement of alterations in airway reactivity are often only marginally different between experimental and control groups, for example, after intravenous MCh challenge (48). In contrast, MCh airway challenge via the airways used in the present study may maximize differences in AHR that may otherwise be unnoticed. Until such differences are reconciled or that different pathways may converge at AHR, comparison of these different approaches remains difficult.

Extrapolation of data generated in murine studies to the findings in human asthma are important to consider. As discussed previously for murine studies, human asthma is also heterogeneous. In addition, expression of IgE receptors on eosinophils, susceptibility of airway smooth muscles to transmitters or peptides, and neurogenic control of smooth muscle contraction may differ between species. Furthermore, human disease is more chronic and includes the potential for airway remodeling. Nonetheless, many basics of the fundamental issues may be shared or overlap and, at the present time, are best or can only be studied in animal models because of the availability of a broad variety of reagents and genetically altered strains.

In conclusion, these data illustrate that IL-4- and IL-4- dependent IL-5 production, but not (allergen-specific) IgE, are required for the induction of allergen-driven eosinophil airway inflammation and the subsequent increase in airway reactivity to inhaled MCh in sensitized and challenged animals. The important role of these cytokines in the induction of airway inflammation and AHR highlights their potential targeting in novel approaches to asthma therapy.