Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Two major events characterize the findings in most patients with atopic asthma: production of increased levels of allergen-specific IgE and sustained eosinophilic inflammation of the airways (1). The increases in allergen-specific IgE are postulated to directly or indirectly initiate or perpetuate the clinical features of the disease. Analyses of atopic patients show a correlation between IgE serum levels and the prevalence or severity of asthma symptoms (2, 3). This finding is supported by studies in mice identifying a major role for IgE in the development of airway inflammation (4) and airway hyperresponsiveness (AHR) (5). Interleukin (IL)-4 is the main isotype switch factor for IgE production, and treatment of mice with anti-IL-4 antibody before airway challenge (6) or allergen sensitization followed by airway challenge of IL-4-deficient mice (7, 8) results in deficient IgE production, impaired eosinophil recruitment, and little AHR.

Airway inflammation characterized by eosinophil infiltration of the peribronchial regions is a constant feature in most patients with bronchial asthma (9). Increased numbers of eosinophils are found in the bronchoalveolar lavage fluid (BALF) and in bronchial biopsy specimens from asthma patients; eosinophil numbers correlate with severity of the disease (12). The importance of IL-5-mediated airway eosinophilia in allergen-sensitized mice was confirmed in two recent studies. We showed that treatment of high-IgE-responder BALB/c mice with anti-IL-5 antibody during airway sensitization completely abolished eosinophil infiltration of the airways and prevented the development of AHR despite increased levels of allergen-specific IgE and immediate cutaneous hypersensitivity (13). Similarly, sensitization and airway challenge of IL-5- deficient mice also induced antigen-specific IgE production, but did not result in increased numbers of eosinophils or altered airway reactivity unless IL-5 production in the lungs was reconstituted (14).

The purpose of the present investigation was to further define the role of B cells and/or allergen-specific IgE in the induction of eosinophil airway infiltration and the development of AHR. We utilized B-cell-deficient µMt^/ mice (15), since these mice are incapable of generating any antibody responses. µMt^/ mice were previously shown to have normal T-cell activation after sensitization with protein antigens (16). In one such previous study (19), we showed that after 10-d exposure to allergen via the airways, B-cell-deficient µMt^/ mice failed to develop AHR, as assessed in vitro, unless passively sensitized with allergen-specific IgE. In the study reported here we found that intraperitoneal sensitization plus repeated airway challenge of B-cell-deficient mice with allergen results in normal eosinophilic airway inflammation and development of AHR, both in vitro and in vivo. These data indicate that neither B cells nor allergen-specific antibodies (IgE or other isotypes) are required for the recruitment and activation of eosinophils in the airways that result in the development of AHR in sensitized and challenged airways, and contrast with our earlier findings (19) made with a different allergen-exposure protocol.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Animals

B-cell-deficient mice of a B10.BR background were generated as previously described (15, 19). Identical results were obtained in B-cell-deficient mice of a C57BL/6 background. Normal female mice from 8 to 12 wk of age were obtained from Jackson Laboratories (Bar Harbor, ME). Spleens and lymph nodes from B-cell-deficient (µMt^/) mice contained less than 3% B220⁺ cells, whereas normal (µMt^+/+) mice containted  70% B220⁺ cells in their spleens and  30% B220⁺ cells in their lymph nodes. All mice were maintained on ovalbumin (OVA)-free diets, and all mice used in the 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 groups of age- and sex-matched mice (three or four mice per group per experiment, with three or four experiments) were studied: (1) ipNeb mice. These mice were sensitized by intraperitoneal injection of 20 µg OVA (Sigma; St. Louis, MO) emulsified in 2 mg aluminum hydroxide (alum) (AlumImject; Pierce, Rockford, IL) in a total volume of 100 µl on Days 1 and 14. The mice were then challenged via the airways with OVA (1% in phosphate-buffered saline [PBS]) for 20 min on Days 28, 29, and 30 through ultrasonic nebulization (DeVilbiss, Somerset, PA), and were assessed on Day 32 (2 d after the last airway challenge with allergen) for airway responsiveness. (2) Neb mice. These control animals received only airway challenges with OVA on Days 28-30. (3) TRFK-5 mice. Anti-IL-5 antibody (50 µg TRFK-5, kindly provided by Dr. R. Coffman, DNAX Institute, Palo Alto, CA) was administered intranasally into lightly anesthetized (avertin, 2% intraperitoneally) animals 2 h before each airway challenge on Days 28-30. Control animals received rat IgG₁, which had no effect on the inflammatory responses or development of AHR. Intranasal delivery was chosen because it was shown in preliminary experiments to be superior to other protocols (intraperitoneal or intravenous) in reducing airway eosinophilia and inhibiting AHR. Administration of aluminum hydroxide alone with or without airway challenge, or of OVA/alum in the absence of airway challenge, provided results that were not different from those with control animals.

Measurement of Anti-OVA Antibody and Total Ig Levels

Anti-OVA IgE and IgG₁ serum levels were measured with an enzyme-linked immunosorbent assay (ELISA) as previously described (20). The antibody titers of the samples were related to pooled standards that were generated in the laboratory. Total IgE levels were determined according to a method previously described (13). Total Ig levels were calculated by comparison with known mouse IgE standards (PharMingen, San Diego, CA). The assay limit of detection for IgE was 100 pg/ml.

Cell Preparation and Culture

Peribronchial lymph nodes (PBLN) (from four mice per experiment in three separate experiments) were harvested, and mononuclear cells (MNC) were purified by passing the tissue through stainless steel mesh followed by density-gradient centrifugation (Organon Teknika, Durham, NC). T cells were isolated by passage through nylon wool to a purity of  90% CD3⁺ cells. Cells were washed three times in PBS and resuspended in RPMI 1640 medium (GIBCO, Grand Island, NY) containing 10% fetal calf serum (FCS), 100 U/ ml penicillin, 100 µg/ml streptomycin, 5 mM glutamine, and 50 µM 2-mercaptoethanol. Antigen-presenting cells (APC) were prepared from spleens of B10.BR mice by preparing MNC, depleting MNC of T cells by incubation with antithymocyte antiserum followed by rabbit complement, and irradiating the remaining cells with 3,000 rads. T cells were plated in 96-well round-bottom plates at 200,000 cells/well and cultured in the presence or absence of OVA and mitogen at 37°C. APC were added at 400,000 cells/well. Cell-free supernates were harvested and stored at 20°C.

Proliferation Assay

PBLN T cells were cultured in the presence or absence of OVA and mitogen for 5 d. Thymidine incorporation was measured 6 h after addition of 1 µCi of [³H]thymidine (ICN, Irvine, CA).

Cytokine Production

Cytokine levels in BALF or in the supernates of cell cultures were measured as described with an ELISA after 48 h of incubation (13). Briefly, ELISA plates were coated with purified anticytokine antibodies (all reagents were obtained from PharMingen) and blocked with 10% FCS/ phosphate-buffered saline (PBS). Samples and dilution rows of purified cytokines as standards were incubated at 4°C overnight. Biotinylated anticytokine antibodies, followed by avidin-peroxidase and 2,2'-azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid substrate, were used for cytokine detection. The limits of detection were 4 pg/ml for both IL-4 and IL-5.

Bronchoalveolar Lavage and Lung Cell Isolation

Lungs were lavaged via a tracheal tube with Hanks' balanced salt solution (HBSS; three instillations of 0.5 ml each) and the cells in the lavage fluid were counted. Lung cells were isolated by enzymatic digestion as previously described (20). Cells from BALF or lungs were resuspended in HBSS and counted with a hemocytometer. Cytospin slides were stained with Leukostat (Fisher Diagnostics, Pittsburgh, PA) and differentiated 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 immunohistochemically with rabbit antimouse MBP (kindly provided by Dr. G. Gleich and Dr. J. Lee, Mayo Clinic, Rochester, MN, and Scottsdale, AZ) as previously described (13). Slides were examined in a blinded fashion with a Zeiss (Jena, Germany) microscope equipped with a fluorescein filter system. Numbers of eosinophils in the submucosal tissue around central airways were evaluated with IPLab2 software (Signal Analytics, Vienna, VA) for the Macintosh, through counting of four different sections per animal (13).

Electron Microscopy of Eosinophils

Lungs were infiltrated with 1.5% glutaraldehyde and fixed overnight with 1.5% glutaraldehyde in 0.1 M cacodylate buffer, after which they were diced into < 1-mm pieces and placed into fixative. After washing with 0.1 M cacodylate buffer, sections were stained with 3% aqueous uranyl acetate for 45 min, dehydrated with increasing concentrations of acetone, infiltrated with propylene oxide in plastic with increasing plastic concentrations, and finally embedded in pure plastic and cured for 4 d at 70°C. From each individual mouse,  10 eosinophils were analyzed for signs of degranulation and activation.

Determination of Airway Responsiveness

In vitro airway responsiveness was determined as previously described (21). Briefly, tracheal smooth-muscle segments of about 0.5 cm in length were suspended from triangular supports transducing the force of contractions and placed in Krebs-Henseleit baths. Electrical field stimulation (EFS) was delivered with increasing frequencies until peak contractile responses were reached. ES₅₀, the frequency leading to 50% of maximal contraction, was calculated from linear plots and compared for the different treatment groups.

In vivo lung resistance (RL) to methacholine (MCh) was measured as described (22), with some modifications. Mice were anesthetized, their tracheas were cannulated with an 18-gauge cannula, and they were 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 683; Harvard Apparatus, South Natick, MA) and the third port attached to one side of a differential pressure transducer. Ventilation was achieved at a rate of 160 breaths/ min, at a tidal volume (VT) of 150 µl during recording, and at a rate of 60 breaths/min and VT of 500 µl during Mch aerosol delivery. As a modification to previous work in our laboratory (23), we administered MCh (6-100 mg/ml) as an aerosol for the period of 10 breaths at each concentration via the tracheal cannula. Changes in transpulmonary pressure and volume of the plethysmograph, and in the rate of flow for each animal, were derived through digital differentiation. RL was calculated from peak values after each challenge. Data are expressed as the percent of baseline values recorded with administration of PBS.

In vivo airway responsiveness to MCh in conscious, spontaneously breathing animals was measured through barometric plethysmography (Buxco, Troy, NY) as previously described (24). Pressure differences were measured between the main chamber of the plethysmograph, containing the animal, and a reference chamber (generating a box pressure signal). Mice were challenged with aerosolized PBS or MCh (6-100 mg/ml) for 3 min, and readings were taken and averaged for 3 min after each nebulization. Data are expressed as the percent of baseline values with PBS, using the dimensionless parameter "Penh" according to the formula:

(1)

where Te is the expiratory time (in seconds); Tr is the relaxation time (in seconds) (the time of the decay of the expiratory box pressure to 36% of peak expiratory box pressure [PEP_B; PEP_B is measured in units of ml/s]); and PIP_B is the peak inspiratory box pressure (ml/s).

In measurements of airway function, there were no significant differences in baseline values of B10 and B10 µMt^/ mice, or in the different groups; baseline RL averaged 0.44 ± 0.04 cm H₂O/ml/s.

Statistical Analysis

Analysis of variance (ANOVA) was used to determine the levels of difference between all groups. Pairs of groups were compared by use of Student's t test. Comparisons for all pairs were performed with the Tukey-Kramer HSD test for airway responsiveness and with histology data. Significance was set at P < 0.05. Values for all measurements are expressed as the mean ± SD deviation, except for values of ES₅₀, which are presented as the mean ± SEM.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Sensitization and Challenge Increases Total and OVA-Specific IgE Levels in Normal but Not in B-Cell-Deficient Mice

Normal and B-cell-deficient (µMt^/) mice were sensitized by intraperitoneal injection of OVA emulsified in alum on Days 1 and 14, and repeated airway challenge with OVA was performed on Days 28, 29, and 30. Control animals received only airway challenges with OVA. Serum levels of OVA-specific and total immunoglobulins 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 B10.BR mice (Table 1). Sensitization did not significantly alter total IgE serum levels. In contrast to normal mice, serum levels of total and OVA-specific antibodies in µMt^/ mice were below the limit of detection before and after sensitization and airway challenge. This confirms the inability of the B-cell-deficient mice to generate antibody responses (15).

Sensitization followed by Challenge Induces Antigen-Specific T-Cell Responsiveness and Cytokine Production in Normal and in B-Cell-Deficient Mice

To assess antigen-specific proliferative responses of T cells after sensitization and challenge with OVA, we cultured PBLN T cells for 5 d in the absence or presence of OVA. Sensitization and challenge of normal B10.BR mice to OVA resulted in dose-dependent OVA-specific responses in T cells prepared from PBLN (Figure 1). Little response to OVA was observed in mice sensitized only or challenged only, or in control mice (data not shown). Sensitization and challenge of µMt^/ mice resulted in OVA-specific T-cell responses similar to those observed in the wild-type animals.

View larger version (13K): [in this window] [in a new window]   Figure 1.   Proliferative responses of PBLN T cells. Normal (n = 12) and B-cell-deficient (n = 12) mice were sensitized by intraperitoneal injection of OVA in alum on Days 1 and 14, followed by airway challenges with OVA on Days 28, 29, and 30. Two days after the last challenge, PBLN T cells (2 × 105 cells per well) were cultured in triplicate in the absence or presence of OVA together with APC (4 × 105 cells per well) for 72 h. Thymidine uptake was measured after pulsing the cells with 1 µCi [3H]thymidine for 6 h. Data points represent the mean cpm ± SD of two independent experiments (P > 0.2 for B10 versus B10 µMt/ mice). Control cultures in the absence of OVA produced less than 300 cpm.

To evaluate the importance of B cells and allergen-specific antibodies in the induction of Th2-type T-cell responses following sensitization and challenge, we measured the production of OVA-induced IL-4 and IL-5 levels in cultured PBLN T cells after 48 h. Sensitization and challenge significantly enhanced antigen-specific IL-4 and IL-5 production by PBLN T cells as compared with T cells of nonsensitized animals (Figure 2A). In µMt^/ mice, the production of IL-4 and IL-5 after airway sensitization was increased to an extent similar to that in normal mice. IL-4 and IL-5 protein levels were measured in the supernates of the BALF from sensitized and challenged normal and B-cell-deficient mice, and showed similar increases in both sensitized groups (Figure 2B). These data indicate that sensitization with OVA induces antigen-specific T-cell activation (proliferation and cytokine production) in both B-cell-deficient and B-cell-sufficient mice.

View larger version (28K): [in this window] [in a new window]   Figure 2.   IL-4 and IL-5 production after OVA sensitization. Mice were sensitized and challenged as described in Figure 1. Two days after the last challenge, BALF was harvested and PBLN T cells from sensitized, challenged normal (n = 12) and B-cell-deficient (n = 12) mice were cultured for 24 h (2 × 105 cells per well) in the absence or presence of OVA (50 µg/ml) together with APC (4 × 105 per well). IL-4 (A) and IL-5 (B) protein levels in supernates and in BALF were determined through ELISA. Expressed are the mean ± SD value (pg/ml) from three independent experiments. Mice that had nebulization only had < 25 pg/ml of IL-4 and IL-5 in their BALF and supernates.

Sensitization and Repeated Airway Challenge Induces Eosinophil Infiltration of the Lung in Normal and in B-Cell-Deficient Mice

To evaluate the importance of B cells in the development of allergic inflammation after airway sensitization and challenge, we compared total leukocyte and differential cell counts in isolated lung cells and in the BALF of individual mice. Numbers of total leukocytes, lymphocytes, and neutrophils were increased and numbers of macrophages were decreased after allergen sensitization and airway challenge. The number of eosinophils was significantly increased in lung cell preparations from sensitized and challenged normal B10 and B-cell-deficient B10 mice, by ~ 12- and ~ 17-fold, respectively (Figure 3A). Sensitization plus airway challenge also resulted in more than a 3-fold increase in total cell numbers in the BALF of normal and µMt^/ mice, with the predominant cell type being eosinophils (Figure 3B). Sensitization and challenge of B10 µMt^/ mice resulted in even higher numbers of eosinophils (256,000 ± 89,000/mm³ versus 600 ± 450/mm³ in nonsensitized µMt^/ mice) than in normal B10 mice (234,000 ± 70,000/mm³ versus 650 ± 350/mm³ in nonsensitized B10 mice). These data indicate that eosinophil infiltration of lung tissue and eosinophil accumulation in BALF does not depend on the presence of antigen-specific antibodies or B cells after sensitization and airway challenge.

View larger version (29K): [in this window] [in a new window]   Figure 3.   Sensitization plus airway challenge increases numbers of eosinophils in lung digests and BALF of normal and B-cell- deficient mice. Normal (n = 12) and B-cell-deficient (n = 12) mice were sensitized and challenged as described in Figure 1. Control animals received only OVA airway challenges (n = 8). Numbers of total cells and eosinophils in lung digests (A) and in BALF (B) prepared 2 d after the last airway challenge were determined. The means ± SD of the percentage of the different cell types from three independent experiments are shown. (*P < 0.01 versus PBS.)

Sensitization and Repeated Airway Challenge Induces Peribronchial Eosinophil Infiltration in Normal and in B-Cell-Deficient Mice

To localize eosinophils in the lung tissue, we performed immunohistochemistry with anti-MBP antibody on formalin-fixed lung sections. The number of MBP-positive cells was measured in the peribronchial tissue of central airways through a computer-assisted analysis, and was compared for the different groups of mice. In normal B10 as well as in B10 µMt^/ mice, sensitization plus airway challenge significantly increased the numbers of peribronchial eosinophils by ~ 15-fold (Figure 4). There were no differences in the distribution of eosinophils in relation to the epithelial layer when normal B10 and B-cell-deficient B10 mice were compared (Figures 5A to 5C). Sensitization plus airway challenge thus induces peribronchial eosinophil infiltration to a similar degree and at a similar location in both groups of mice.

View larger version (16K): [in this window] [in a new window]   Figure 4.   Peribronchial eosinophil accumulation after airway sensitization and challenge with OVA. Normal (n = 12) and B-cell-deficient (n = 12) mice were sensitized and challenged as described in Figure 1. Control animals received only OVA airway challenges (n = 8). Sections of peribronchial tissue were stained with rabbit antimouse MBP antibodies. Four sections per lung, from around central airways of similar size, were chosen randomly, and the number of eosinophils in the connective tissue was counted with a computer-assisted program. Histogram shows mean ± SD numbers of eosinophils per mm2 of lung tissue from three independent experiments. (*P < 0.01 versus PBS.)

View larger version (58K): [in this window] [in a new window]   Figure 5.   In situ eosinophil airway infiltration after sensitization plus airway challenge with allergen. Lung sections were prepared and stained for eosinophils with anti-MBP antibody as described in MATERIALS AND METHODS. Shown are central airways (magnification: ×400) of (A) a nonsensitized control, (B) a sensitized, challenged normal mouse, and (C) a sensitized, challenged, B-cell- deficient B10 mouse. Note the marked peribronchial eosinophilia in B and C.

Sensitization and Repeated Airway Challenge Induces Eosinophil Activation in Normal and in B-Cell-Deficient Mice

To assess the state of activation of the eosinophils, we performed electron microscopy on fixed lung sections from sensitized and challenged normal and B-cell-deficient mice. The extent of activation was assessed by evaluating degranulation, granule deformation (loss of the central core, granule irregularity), cell irregularity (pseudopods, nonrounded shape), and hypodensity. At least 10 eosinophils from each mouse were evaluated. The degree of activation was defined as low (< 20% of granules showing degranulation/granule deformation), medium (20 to 50% of granules affected), and high (> 50% of granules affected). After sensitization plus repeated airway challenge with allergen, more than 90% of the eosinophils from normal B10 mice were shown to be activated (Figure 6A). In B-cell-deficient B10 mice, the percentage of highly activated eosinophils after sensitization plus airway challenge was similar to that in normal mice (Figure 6B). These data indicate that the presence of B cells and/or allergen-specific antibodies is not required for activation of eosinophils.

View larger version (90K): [in this window] [in a new window]   Figure 6.   Eosinophil activation and degranulation. Lung sections were prepared as described in MATERIALS AND METHODS. Shown are eosinophils from (A) a sensitized, challenged normal mouse and (B) a sensitized, challenged, B-cell-deficient mouse. Arrows mark granule deformation and degranulation (loss of the central core, hypodensity). Notice the development of pseudopods in both cells.

Sensitization and Repeated Airway Challenge Induces Altered Airway Function In Vitro and In Vivo in Normal and in B-Cell-Deficient Mice

To assess the importance of B cells and allergen-specific antibodies in the development of AHR after sensitization and airway challenge with OVA, we measured in vitro and in vivo responses through three different methods. The response of tracheal smooth-muscle segments to EFS was measured in normal B10 and B-cell-deficient B10 mice, and compared with that of nonsensitized controls. EFS assesses altered neural function of tracheal smooth muscle from sensitized mice caused by the increased release of acetylcholine as a result of muscarinic (M₂) autoreceptor dysfunction (25). As illustrated in Figure 7A, ES₅₀ values were decreased (a decrease indicates increased smooth-muscle reactivity to EFS) after sensitization and challenge in both normal and B-cell-deficient mice (B10: 2.2 ± 0.4 Hz; B10 µMt^/: 2.3 ± 0.5 Hz) as compared with nonsensitized controls (B10: 3.7 ± 0.2 Hz; B10 µMt^/: 3.8 ± 0.4 Hz; P < 0.02).

View larger version (10K): [in this window] [in a new window]   View larger version (17K): [in this window] [in a new window]   View larger version (16K): [in this window] [in a new window]   View larger version (19K): [in this window] [in a new window]   Figure 7.   Development of AHR in normal and B-cell-deficient mice. Normal and B-cell-deficient mice were sensitized and challenged as described in Figure 1 (ipNeb). Control animals received only OVA airway challenges (Neb). (A) In vitro airway responsiveness of tracheal smooth-muscle segments to EFS. The frequencies causing 50% maximal contraction (ES50 ± SEM in Hz) in sensitized, challenged mice were compared with those causing such contraction in nonsensitized, nonchallenged mice. ES50 values were 3.7 ± 0.2 Hz for B10 and 3.8 ± 0.4 Hz for B10 µMt/ mice, respectively, and were taken as 100%. Expressed are the data from two independent experiments (*P < 0.02 versus controls). (B) In vivo airway responsiveness to aerosolized MCh was measured in unrestrained, conscious mice, using barometric plethysmography. Mice were placed into the main chamber of the body box and first received nebulized PBS, followed by increasing doses (6-100 mg/ml) of MCh for 3 min for each nebulization. Readings of breathing parameters were made for 3 min after each nebulization. Penh values were determined as described in MATERIALS AND METHODS. Expressed are the means ± SEM of Penh as a percentage of baseline (PBS) values from three independent experiments (*P < 0.01 versus nonsensitized controls). (C) In vivo airway responsiveness to aerosolized MCh was measured in anesthetized, tracheostomized, and ventilated mice. Aerosolized PBS and MCh were administered via the tracheostomy. Pulmonary resistance was calculated as RL = P (difference in tracheal pressure)/ (flow change) from peak values after each challenge. Expressed are the means ± SEM of RL as percents of PBS baseline values from two independent experiments (*P < 0.01 versus nonsensitized controls). (D) Effect of anti-IL-5 antibody. Anti-IL-5 antibody (50 µg) was administered intranasally 2 h before airway challenge with allergen to sensitized normal B10 (n = 6) and B-cell-deficient B10 µMt/ mice (n = 6). Normal IpN mice received rat IgG1 as a control. Measurements and expression of data are as in Figure 7B (*P < 0.05 versus B10 ipN treated with anti-IL-5 antibody).

In vivo airway responsiveness to aerosolized MCh in spontaneously breathing, conscious B10 mice was assessed through barometric plethysmography (24). This method detects AHR after sensitization and airway challenge in mice, and the results obtained with this technique correlate closely with results of invasive measurements of intrapleural pressure and RL (24). Sensitization plus airway challenge of B10 mice significantly increased airway reactivity over that of nonsensitized but challenged controls (P < 0.02). The dose-response curve for MCh was both increased and shifted to the left (Figure 7B). Similarly, in B10 µMt^/ mice, in vivo airway reactivity after systemic sensitization plus airway challenge was significantly increased over that of control animals (Figure 7B).

To verify the results obtained with barometric plethysmography, we monitored RL upon administration of aerosolized MCh in normal and B-cell-deficient mice. As seen with barometric plethysmography, sensitization and repeated airway challenge with allergen increased in vivo airway responsiveness to aerosolized MCh, with a left shift in the dose-response curve in both normal and B-cell- deficient mice (Figure 7C). These data indicate that development of hyperresponsiveness to MCh after systemic sensitization and airway challenge is not dependent on B cells or antigen-specific antibodies.

Anti-IL-5 Treatment before Airway Challenge Prevents Eosinophil Infiltration into the Airway and Development of AHR

To delineate the role of IL-5-mediated eosinophilia in the development of AHR in the model used in our study, sensitized normal B10 and B-cell-deficient B10 mice were given anti-IL-5 antibody intranasally 2 h before each of the three airway challenges with allergen. Treatment with 50 µg of anti-IL-5 antibody, but not with control rat IgG₁ (> 300 eosinophils/mm² in both groups), virtually eliminated the influx of eosinophils into the lungs, and no peribronchial eosinophil infiltration was observed (< 20 eosinophils/mm² in both groups). In association with this decrease in eosinophil accumulation was the failure of sensitized mice receiving anti-IL-5 antibody before each airway allergen challenge to develop increased responsiveness to aerosolized MCh (Figure 7D). On the other hand, treatment with anti-IL-5 antibody did not alter B-cell (Ig production; Table 1) or T-cell function (cytokine production, proliferation; data not shown).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We investigated the requirement for B cells and allergen-specific antibodies in T-cell activation, airway infiltration by eosinophils, and development of altered airway responsiveness after allergic sensitization and airway challenge. For this approach, we compared the responses of normal and B-cell-deficient mice and analyzed two different mouse strains, B10.BR and C57BL/6. Intraperitoneal sensitization followed by repeated airway challenge induced allergen-specific T-cell responses and eosinophil infiltration into the airway in both normal and µMt^/ mice. Only the normal mice developed allergen-specific IgE and IgG₁ production. After sensitization and challenge, increased numbers of eosinophils were detected in BALF and in lung digests. Most of the eosinophils in both groups of mice were located in the peribronchial regions of the airways and exhibited signs of activation/degranulation. Despite the absence of specific antibody production or B cells, sensitization and repeated airway challenge with allergen induced changes in lung function: the development of in vitro airway responsiveness of tracheal smooth-muscle preparations to EFS, and in vivo responsiveness to aerosolized MCh, indicated increased airway responsiveness in B-cell-deficient mice to an extent comparable to that in their B-cell-sufficient counterparts.

Recent observations made on µMt^/ mice indicated that B cells are not required for the induction of T-cell activation (16), peripheral T-cell tolerance (26), or long-lasting T-cell memory (18). We previously reported that after adjuvant-free airway sensitization, allergen-specific T-cell responses and cytokine production were similarly intact in B-cell-deficient mice (19). In the present study, after systemic sensitization with adjuvant and repeated airway challenges, we found no differences between normal and µMt^/ mice in the magnitude of T-cell allergen-specific responses and Th2-type cytokine production (IL-4, IL-5). These data confirm that neither B cells nor allergen-specific IgE (or other Igs) are required for T-cell activation after allergen sensitization.

The role of IgE in the development of eosinophilic inflammation and AHR after allergen sensitization requires careful analysis of the protocols and measurements of airway function that are utilized to assess it. One potential link might be the production of IL-4, which is increased in atopic asthmatic individuals (27) and which induces production of Th2-type cytokines (28) and IgE (29). The central role of IL-4 in allergic sensitization and development of AHR has been emphasized in a number of studies. Corry and colleagues (6) showed that treatment of sensitized mice with anti-IL-4 antibody during allergen sensitization decreased serum IgE levels and eosinophil infiltration of the airway, and inhibited development of AHR. We made similar findings in an adjuvant-free system, using either anti-IL-4 antibody or soluble IL-4 receptor (30). Development of eosinophil accumulation and AHR is impaired in IL-4-deficient mice (7, 8). Data from the present study support this concept: allergen sensitization plus airway challenge induced IL-4 production by PBLN, airway infiltration of eosinophils, and development of AHR to MCh to a similar extent in normal and B-cell-deficient mice, but these effects appear to be independent of IgE or IgG antibody production, or of B cells.

This independence of allergic responses from IgE production has already been described in IgE-deficient mice that can develop anaphylaxis after active sensitization and intravenous allergen challenge (31). However, the IgE- deficient mouse model did not account for allergen-specific IgG production and IgG₁-mediated activation of mast cells or other effector cells. We have reported that passive sensitization with anti-OVA IgG₁, in the absence of allergen-specific IgE and followed by intradermal, intravenous, or airway challenge with allergen, causes cutaneous or systemic anaphylaxis and AHR, respectively (20). Therefore, development of AHR in actively sensitized and challenged IgE-deficient mice may very well be the result of allergen-specific IgG₁ production and IgG-mediated cell activation (32). Indeed, it has been shown that IgG₁ but not IgE antibody causes activation and degranulation of human eosinophils (33).

On the other hand, other studies have implicated a closer relationship between IgE and eosinophilic inflammation. Coyle and colleagues (4) observed impaired eosinophil accumulation in sensitized mice treated with anti-IgE antibody prior to airway challenge, and suggested that enhanced antigen presentation by CD23⁺ B cells, mediated by IgE, induces T-cell activation. It is unclear whether the use of a different mouse strain (BALB/c), a different allergen (house dust mite) or a different challenge protocol (single intranasal exposure) accounts for the differences between these and our results. However, using the same sensitization and challenge protocol as described in this paper, we have determined that CD23-deficient mice do not exhibit any differences from normal B6 mice in IgE production, eosinophil accumulation in the airway, or development of AHR (34).

We have previously demonstrated the importance of IL-5 in the induction of eosinophil infiltration of the airway and development of AHR in a model of adjuvant-free airway sensitization of BALB/c mice (13). The seemingly discrepant results of our studies and those of other studies in which anti-IL-5 antibody was more or less effective (6, 35) might very well be explained by differences in the timing and route of administration of IL-5 and the total amount of anti-IL-5 administered. Our data are compatible with the concept that in the absence of IgE, a critical number of (activated) eosinophils (> 20-fold increase in BALF, Figure 3; > 15-fold increase in situ, Figure 4) is needed to induce AHR. In models of sensitization that induce substantially lower numbers of eosinophils, such as the 10-day protocol of exclusive airway exposure to allergen (19), epithelial damage through the release of eosinophil cationic proteins alone may not be sufficient to trigger AHR, and IgE and/or other cells may be necessary for providing additional factors for inducing AHR. In both normal and B-cell-deficient mice that are sensitized exclusively via the airways in the absence of adjuvant, this mode of sensitization induces similar T-cell activation and cytokine production but more limited eosinophil infiltration than occurs in systemically sensitized mice; eosinophil accumulation following this mode of sensitization is substantially lower (~ 4-fold in situ) (13, 19) than with systemic sensitization as described here. As a result, (exclusive) airway sensitization does not induce the development of AHR in B-cell-deficient mice unless they are passively sensitized with anti-OVA IgE (19). In the reverse setting, athymic BALB/c nu/nu mice lacking IL-5-producing T cells and passively sensitized with allergen-specific IgE do not develop AHR after repeated airway challenge unless they are treated with IL-5 (36). As a corollary, IL-5 treatment of mice plus airway challenge induced eosinophil infiltration that did not lead to AHR in the absence of passive sensitization with allergen-specific IgE (36).

In conclusion, we have shown that systemic sensitization plus repeated airway challenge with allergen induces T-cell activation, marked airway infiltration by and degranulation of eosinophils, and development of increased responsiveness to aerosolized MCh in B-cell-deficient mice. These findings suggest that B cells and/or allergen-specific IgE (or other antibody isotypes) are not essential for eosinophilic airway inflammation and induction of AHR in mice sensitized to allergen in this way. Treatment with anti-IL-5 antibody abolished both eosinophil accumulation in the airway and development of increased airway responsiveness. Cumulatively, the data for B-cell- deficient mice highlight the important role of eosinophils in the development of AHR under most conditions, and indicate that under certain sensitization and challenge conditions, eosinophils alone (in the absence of IgE and other antibody) may be sufficient to trigger AHR. These findings emphasize the need for new strategies that target eosinophil accumulation in the treatment of allergen-induced AHR. Importantly, comparison of these and previous findings (19) underscore the way in which different approaches to allergen sensitization and challenge may reveal the requirements for development of AHR.