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Am. J. Respir. Crit. Care Med., Volume 161, Number 4, April 2000, 1340-1348

Airway Inflammation Driven by Antigen-specific Resident Lung CD4+ T Cells in alpha beta -T Cell Receptor Transgenic Mice

PATRICK G. KNOTT, PAUL R. GATER, and CLAUDE P. BERTRAND

Inflammatory Diseases Unit, Roche Bioscience, Palo Alto, California


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CD4+ T cells are thought to play a major role in the initiation and perpetuation of T helper cell, type 2 (Th2)-like allergic airway inflammation. However, it is not clear whether activation of resident antigen-specific CD4+ T cells is in itself sufficient to induce such a phenotype. Using ovalbumin (OVA)-specific alpha beta -T cell receptor transgenic Balb/c DO11.10 mice, we were able to test this hypothesis. Nonsensitized DO11.10 mice but not wild-type mice responded to a primary OVA aerosol with a rapid and impressive bronchoalveolar lavage (BAL) neutrophilia followed by a smaller but significant eosinophilia. Responses in DO11.10 mice were mediated by OVA-specific activation of CD4+ T cells because in vivo depletion of CD4+ but not CD8+ T cells abrogated inflammatory cell influx. Cytokines measured in BAL fluid (BALF) after OVA aerosol exposure of DO11.10 mice were indicative of a T helper cell, type 1 (Th1)-like immune response. Further, neutralization of interferon gamma (IFN-gamma ) with antibody enhanced eosinophil influx, suggesting that IFN-gamma production was limiting the development of a Th2 response. Despite this, an increased prevalence of cells staining for mucus was seen in the bronchial epithelium, a feature more commonly associated with a Th2-immune response. Unlike what was seen in OVA-sensitized wild-type mice, multiple OVA aerosol exposures of DO11.10 mice failed to induce airway hyperresponsiveness (AHR) to inhaled methacholine. In conclusion, in vivo stimulation of resident lung CD4+ T cells with antigen caused lung inflammation with characteristics of both a Th1- and Th2-immune response but was insufficient to directly induce AHR.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

T cells and the cytokines they produce are widely believed to contribute to the development and maintenance of the chronic airway inflammation and hyperreactivity (airway hyperresponsiveness [AHR]) seen in allergic asthma (1). Allergic responses in the lung of atopic asthmatics are associated with a T helper cell, type 2 (Th2) lymphocyte phenotype (1) although T helper, type 1 (Th1) cells are also present in the asthmatic lung (4). To delineate the pathogenic mechanisms of asthma, lung immune responses have been studied extensively in animal models of allergic inflammation. Various mouse models have suggested that a number of pathways may contribute to development of the asthmatic phenotype (5). In the light of these studies, there may be a redundancy in the requirement for inflammatory pathways involving, for example, mast cells (10), B cells (11, 12), or IgE (13, 14). In contrast, there is evidence for an obligatory role for CD4+ T cells in the development of allergic immune responses in the lung (6, 15).

Animal models of allergic lung inflammation invariably require prior systemic sensitization with the antigen (18, 19). Systemic sensitization with low doses of antigen in the presence of adjuvant leads to the production of antigen-specific IgE by B cells and the clonal expansion of T cells bearing the antigen-specific T-cell receptor (TCR). Subsequent local antigen challenge to the lungs results in cross-linking of IgE on mast cells and the release of spasmogenic and proinflammatory mediators. Occurring in concert, antigen presentation to specific TCR-bearing CD4+ T cells leads to activation of cytokine gene transcription. The complexity of these immune responses makes it difficult to isolate critical elements involved in the development of AHR. Accordingly, the importance of CD4+ T-cell activation, while assumed to be crucial, has been difficult to study in vivo.

To better define the role of CD4+ T cells in the development of airway pathology after antigen challenge, we used Balb/c DO11.10 mice which are transgenic for the TCR specific for the immunodominant epitope of OVA323-339 (20). In vitro work with CD4+ T cells isolated from Balb/c DO11.10 mice has shown that stimulation with presented antigen under neutral conditions leads to T-cell activation, a loss in T-cell responsiveness to interleukin-12 (IL-12), and the develop-ment of a clear Th2-like phenotype (21, 22). It is not known if Balb/c DO11.10 mice default to a Th2-type immune response after in vivo stimulation with ovalbumin (OVA). In vivo studies performed to date using DO11.10 mice have focused on the adoptive transfer of cultured and highly polarized DO11.10 CD4+ T cells to wild-type mice (23, 24). These have been useful in characterizing the extreme cases of CD4+ T cell-mediated Th1- or Th2-like immune responses. However, whether or not the in vivo behavior of these transferred and highly polarized CD4+ T cells accurately reflects the response of endogenous CD4+ T cells is not clear. Thus, the aim of this study was to determine whether primary exposure of DO11.10 mice to OVA aerosol could activate resident lung CD4+ T cells to produce Th2-like airway inflammation and AHR. In this work, we make comparisons to the commonly used and Th2-skewed, OVA-sensitized Balb/c mouse model of allergic airway inflammation.

    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Aerosol Exposure and Bronchoalveolar Lavage

Homozygous, naive alpha beta -TCR transgenic Balb/c DO11.10 mice were bred in house; mice ranging in age from 8 to 12 wk and of either sex were used. Male wild-type Balb/c mice (8 to 12 wk; Charles River Laboratories, Wilmington, MA) were sensitized to OVA (10 µg in 0.2 ml 2% Al(OH)3; intraperitoneally; Sigma Chemical Co., St. Louis, MO) on Day 0 and Day 14 before aerosol exposure to OVA on Day 21. For OVA aerosol exposure, mice were placed in a Plexiglass box and exposed for 20 min to an aerosol of a 5% solution generated by a Pari Star nebulizer (Pari-Werk GmbH, Starnberg, Germany). At various times after aerosol exposure, mice were killed (urethane; 1 g kg-1; intraperitoneally) and bronchoalveolar lavage (BAL) performed on their lungs with phosphate-buffered saline (PBS) (4 × 0.3 ml). If present, red blood cells were lysed and the total number of leukocytes in an aliquot of the BAL fluid (BALF) was determined using a Coulter Counter (Coulter Electronics, Hialeah, FL). Differential leukocyte counts were made by counting 300 cells on stained (Diff-Quik; Dade Diagnostics, Aguada, PR) cytospin preparations by light microscopy using standard morphologic criteria.

Histologic Detection of Mucins

At 24 h after PBS or OVA aerosol, groups of 5 to 6 mice were killed, perfused through the right ventricle with heparinized saline and then 0.4 ml of 4% paraformaldehyde (Sigma Chemical Co.) was introduced to the lungs via a tracheal cannula. Paraffin-embedded tissues were sectioned at 5 µm, stained with alcian blue/periodic acid-Schiff (AB/PAS) to detect both neutral and acidic mucins and a light hematoxylin counterstain applied. Light microscopy was used to qualitatively assess positive staining for mucins.

Determination of Cytokine Protein Levels in BALF

It has been shown that the peak elevations in BALF cytokines seen after OVA aerosol exposure of OVA-sensitized wild-type mice occurred at 24 h postexposure (25). Thus, we measured BALF cytokine concentrations in samples from mice taken 24 h after OVA or PBS aerosol exposure. BALF was centrifuged at 1,500 rpm for 10 min at 4° C to pellet cells, and supernatants removed and stored at -80° C. IL-4, IL-13, and interferon gamma (IFN-gamma ) levels were determined in BALF using commercially available ELISA kits for murine cytokines (IL-4, Pharmingen, San Diego, CA; IL-13 and IFN-gamma , R&D Systems, Minneapolis, MN).

Measurement of Total IgE in Serum

Serum was obtained from blood taken by cardiac puncture before BAL and stored at -80° C before use. An IgE-specific ELISA was performed using IgE capture and detection antibodies and purified mouse IgE standards (Pharmingen). Sera samples were tested at several dilutions in duplicate and the optical density measured at 450 nm using a microplate reader (Molecular Devices, Mountain View, CA). Sample IgE concentrations were calculated with reference to the optical density readings in the standard curve (1 to 200 ng ml-1).

Administration of IFN-gamma Neutralizing Antibody

A neutralizing antibody directed against mouse IFN-gamma was used to determine the influence of this cytokine on the influx of inflammatory cells into the lungs of DO11.10 mice after OVA aerosol. At 30 min before 5% OVA aerosol exposure, mice were lightly sedated by halothane anesthesia and 50 µg of either rat anti-mouse IFN-gamma (XMG1.2; Pharmingen) or isotype control (rat IgG1; Pharmingen) were administered intranasally in a volume of 50 µl of PBS vehicle. BAL was performed 96 h after the 5% OVA aerosol and cells enumerated as already described.

Administration of Anti-CD4 and Anti-CD8 Antibodies

Mice were given an intraperitoneal injection of either rat anti-mouse CD4 (250 µg; Clone GK1.5) or rat anti-mouse CD8 (125 µg; Clone 53-6.7) or the same amount of the respective isotype control antibody (Rat IgG2a or Rat IgG2b; all antibodies from Pharmingen) at 48 h and 24 h before 5% OVA aerosol. These doses of antibody have been shown to deplete CD4+ and CD8+ cells in vivo (26). BAL was performed at 6 h and 96 h after OVA aerosol exposure and cells were counted as described previously. In these experiments, CD4 and CD8 depletion was confirmed using fluorescensce-activated cell sorter (FACS) analysis based on the cell surface expression of CD4 and CD8. Briefly, peribronchial lymph nodes were excised into cold Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). After homogenization and separation over a Lympholyte gradient (Cedarlane, Hornby, ON, Canada), cells were stained with fluorescent-conjugated antibodies for mouse Thy 1.2 in combination with either antibodies for mouse CD4 or mouse CD8 (Pharmingen). Analysis of the lymphocyte population was performed with a FACS Scan flow cytometer (Becton Dickinson, Palo Alto, CA) and these confirmed that selective depletion of either CD4+ or CD8+ cells was obtained under the appropriate conditions.

Airway Reactivity to Inhaled Methacholine

DO11.10 mice or sensitized wild-type mice were exposed once or on 4 consecutive days with PBS or OVA aerosol as described previously, and airway reactivity measurements were taken 24 h or 96 h after the final exposure. Bronchoconstriction in response to inhaled methacholine was determined from changes in enhanced pause (Penh) that were measured by barometric plethysmography in conscious mice as previously described by other workers (27). Mice were placed in whole body plethysmographs (Buxco, Troy, NY), exposed to PBS aerosol for 45 s and the average Penh value was calculated during the next 5 min. After a 10-min recovery period, mice were challenged with increasing concentrations of methacholine (2.5 to 20 mg ml-1; Sigma Chemical Co.) by aerosol for 45 s at intervals of 20 min. The average Penh value for the 5 min after challenge was calculated. Approximately 1 h after the completion of the dose-response curve for methacholine, BALF was collected and cells were analyzed as already described.

Data Analysis

Data are expressed as mean ± SEM of n observations. Significant differences between treatment groups were tested with analysis of variance (ANOVA) in conjunction with the Dunnett's modified t statistic (28). Differences were considered significant if p < 0.05.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Leukocytes in BALF after OVA Aerosol Exposure

The infiltration of leukocytes into BALF was assessed over a 1-wk period after aerosol exposure (Figure 1). In PBS aerosol-exposed mice, regardless of sensitization status or presence of the transgene, the predominant cell type seen in BAL was the alveolar macrophage. As expected, sensitization was important in wild-type mice because exposure of nonsensitized wild-type mice to 5% OVA aerosol resulted in no inflammatory cell influx into the lung at any time point (data not shown). Upon sensitization, wild-type mice responded to 5% OVA aerosol with a significant BAL eosinophilia which was evident at 24 h and continued for at least 1 wk. Exposure of DO11.10 mice to a 5% bovine serum albumin (BSA) aerosol failed to alter the cellular composition of BALF at any time point tested (data not shown). In contrast, exposure of DO11.10 mice to the specific antigen (5% OVA) caused an influx of eosinophils into BALF, an effect which was relatively small compared with that seen in sensitized wild-type mice (Figure 1B). Neutrophils were found in BALF from wild-type mice at 6 h and 24 h after OVA aerosol exposure (Figure 1C). However, at 6 h after aerosol exposure in DO11.10 mice, OVA caused a 15-fold higher neutrophil influx. Neutrophils were still present in BALF from the DO11.10 mice at 96 h after OVA aerosol but not in BALF from wild-type mice. Significant numbers of lymphocytes were found in BALF from DO11.10 mice as early as 6 h and the levels continued to increase until 168 h after OVA aerosol exposure. Lymphocytes were not seen in BALF from wild-type mice until 24 h after OVA aerosol exposure by which time the numbers were about 5-fold less than in DO11.10 mice (Figure 1D).


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  		Figure 1.   Time course of (A) macrophage, (B) eosinophil, (C ) neutrophil, and (D) lymphocyte recovery in BALF from sensitized Balb/c wild-type (open columns) and nonsensitized DO11.10 (filled columns) mice after a single 5% OVA aerosol exposure. PBS data from each of these groups were collected 24 h after a single PBS aerosol exposure. Results are expressed as mean ± SEM of data from 6 to 8 mice.

Histologic Detection of Mucin Staining in the Bronchial Epithelium

The bronchial epithelium of OVA-sensitized but not nonsensitized wild-type mice stained positive for mucins at 24 h after OVA aerosol exposure (Figures 2A and 2B). PBS aerosol exposure of DO11.10 mice (Figure 2C) induced no increase in mucin staining whereas exposure to OVA aerosol induced positive staining in the bronchial epithelium as early as 6 h after OVA aerosol exposure (data not shown) and this was more evident at 24 h after OVA aerosol challenge (Figure 2D). Mucus occluding the bronchial lumen was occasionally seen 24 h after OVA aerosol exposure in both sensitized wild-type and DO11.10 mice.


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  		Figure 2.   Effect of PBS and OVA aerosol exposure (tissue harvested at 24 h postaerosol) on staining for mucins in the peripheral lung of wild-type or DO11.10 mice. Nonsensitized wild-type mouse exposed to OVA aerosol (A). OVA-sensitized wild-type mouse exposed to OVA aerosol (B). Nonsensitized DO11.10 mice exposed to PBS (C ) or OVA (D) aerosol. Tissues were sectioned and slides stained with AB/PAS and counterstained with hematoxylin. The dark blue staining (positive for mucins) was evident only in the bronchial epithelium and lumen of OVA aerosol-exposed, sensitized wild-type and nonsensitized DO11.10 mice. These fields are representative of those obtained from groups of 5 to 6 mice. Magnification is ×50.

BALF Cytokines after OVA Aerosol Exposure

IL-4, IL-13, and IFN-gamma concentrations were measured in BALF of sensitized wild-type and DO11.10 Balb/c mice 24 h after either PBS or OVA aerosol exposure (Figure 3). In PBS aerosol-exposed wild-type and DO11.10 mice, IL-4, IL-13, and IFN-gamma concentrations in BALF were very low or undetectable. Exposure of sensitized wild-type mice to 5% OVA aerosol caused a significant increase in IL-4 and IL-13 concentrations without affecting IFN-gamma concentrations. In contrast, exposure of DO11.10 mice to OVA aerosol increased IFN-gamma concentrations but not IL-4 and IL-13 (IL-13 levels were actually significantly decreased).


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  		Figure 3.   Effect of OVA and PBS aerosol exposures on BALF cytokine levels in sensitized wild type and nonsensitized DO11.10 mice. IL-4 (A), IL-13 (B), and IFN-gamma (C ) levels were measured by ELISA in BALF taken 24 h after either PBS (open columns) or OVA (filled columns) aerosol exposure. In both PBS- and OVA-exposed sensitized wild-type mice, IFN-gamma was below the level of detection (bd ). Results are expressed as mean ± SEM of data from five mice. Significance (asterisk) was determined by Student's t test and indicates OVA aerosol treatment significantly different from PBS aerosol-exposed mice (p < 0.05).

Serum Total IgE in Wild-type and DO11.10 Mice

Serum total IgE was measured to determine if there was significant humoral immunity present in DO11.10 mice after both PBS aerosol or 5% OVA aerosol exposure. As a comparison, measurements were made using serum from similarly exposed nonsensitized and OVA-sensitized wild-type mice. Low levels of total IgE were detected in serum from PBS-exposed DO11.10 mice, and these were not altered at 96 h after a single OVA aerosol exposure (Figure 4). As expected, nonsensitized, PBS aerosol-exposed wild-type mice had low circulating IgE and these levels were not altered by a single OVA aerosol exposure. In contrast, high levels of circulating IgE were induced in wild-type mice by OVA sensitization and these were not affected by local antigen challenge to the lung (Figure 4).


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  		Figure 4.   Determination of serum total IgE after PBS or OVA aerosol challenge in nonsensitized wild-type and DO11.10 mice and in sensitized wild-type mice. Total IgE was measured by ELISA in serum taken at 96 h after PBS or 5% OVA aerosol challenge of nonsensitized wild-type or DO11.10 mice. Additionally, serum total IgE was measured 96 h after PBS or 5% OVA aerosol exposure of OVA-sensitized wild-type mice. Results are expressed as mean ± SEM of data from 4 to 8 mice. Significance (asterisk) was determined by ANOVA in conjunction with the Dunnett's modified t statistic and indicates OVA-sensitized wild-type mice significantly different from nonsensitized wild-type mice (p < 0.05).

Effect of Neutralizing IFN-gamma Activity on BAL Cells in OVA Aerosol-exposed DO11.10 Mice

An antibody to murine IFN-gamma was administered before OVA aerosol exposure to establish whether IFN-gamma produced after OVA challenge in DO11.10 mice (see Figure 3) was inhibiting characteristics of a Th2-type immune response such as airway eosinophilia. Administration of anti-IFN-gamma 30 min before OVA aerosol exposure increased eosinophil influx seen at 96 h by approximately 4.6-fold when compared with the isotype control treatment (Figure 5; p < 0.05). No significant effects of the antibody treatment were seen on the influx of macrophages, neutrophils, or lymphocytes.


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  		Figure 5.   Effect of in vivo neutralizing antibodies for IFN-gamma on BAL cell numbers in 5% OVA aerosol-exposed DO11.10 mice. Mice were administered an intranasal dose (50 µg; 30 min before OVA aerosol) of rat anti-mouse IFN-gamma or isotype control antibody (rat IgG1). Mice were killed 96 h after OVA aerosol exposure and BAL performed. Results are expressed as mean ± SEM of data from 4 to 5 mice. Significance (asterisk) was determined by Student's t test and indicates anti-IFN-gamma treatment significantly different from isotype control treatment (p < 0.05).

Effect of Anti-CD4 and Anti-CD8 Antibodies in DO11.10 Mice

To confirm that the effects of OVA aerosol exposure in the DO11.10 mice were CD4+ T-cell-dependent, BALF cells were enumerated after pretreatment with depleting antibodies specific for either CD4+ or CD8+ T cells. The successful depletion of these cells with the respective antibody was confirmed by FACS analysis. On the basis of the time course data, two time points were chosen that coincided with the peak of neutrophil (6 h) or eosinophil (96 h) influx into BALF after OVA aerosol exposure. Treatment with anti-CD8+ had no significant effect on the cellular composition of BALF at either time point (Figures 6A and 6B). In contrast, treatment with anti-CD4+ caused a 69% reduction in the 6 h neutrophil infiltration (p < 0.05) and completely inhibited the subsequent influx of eosinophils, neutrophils, and lymphocytes into the BALF at 96 h (Figures 6C and 6D; p < 0.05).


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  		Figure 6.   Effect of CD8+ or CD4+ T-cell depletion (filled columns) on BAL macrophage (Mac), eosinophil (Eos), neutrophil (Neu), and lymphocyte (Lym) influx in Balb/c DO11.10 mice after a single OVA aerosol exposure. BAL was recovered (A) 6 h and (B) 96 h after CD8 depletion or (C ) 6 h and (D) 96 h after CD4 depletion. Control data (open columns) are for the respective isotype control antibody. At 6 h there were undetectable eosinophil and lymphocyte levels in BALF. Results are expressed as mean ± SEM of data from 4 to 6 mice. Significance (asterisk) was determined by ANOVA in conjunction with the Dunnett's modified t statistic and indicates anti-CD4 or anti-CD8 antibody treatment significantly different from isotype control treatment (p < 0.05).

Airway Reactivity to Inhaled Methacholine

Increases in Penh in response to inhaled methacholine were measured in conscious mice and used as an indicator of AHR. Single OVA aerosol exposure of both wild-type and DO11.10 mice failed to induce AHR (Table 1). However, 24 h after the last of four daily OVA aerosol exposures, AHR was demonstrated in sensitized wild-type mice (Figure 7A, Table 1) because there were significantly elevated Penh responses to methacholine at all concentrations tested. AHR in these mice did not persist, because when measurements were repeated at 96 h, there was no difference between OVA and PBS aerosol- exposed groups (Table 1). In contrast, DO11.10 mice did not develop AHR after four consecutive daily exposures to OVA aerosol (Figure 7A, Table 1). One hour after completing lung function measurements, mice exposed to four daily challenges with OVA were killed and BAL performed. There was a significantly greater (approximately 7-fold) eosinophil influx in wild-type mice compared with DO11.10 mice (Figure 7B; p < 0.05). Despite this, BALF collected from DO11.10 mice contained significantly greater numbers of neutrophils and lymphocytes relative to wild-type mice (Figure 7B; p < 0.05).

                              
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TABLE 1

AIRWAY REACTIVITY TO INHALED METHACHOLINE IN SENSITIZED WILD-TYPE AND NONSENSITIZED DO11.10 MICE AFTER OVA AEROSOL EXPOSURE*


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  		Figure 7.   Effect of four daily OVA aerosol exposures on airway reactivity (A) and BALF cells (B) in OVA-sensitized wild-type and nonsensitized DO11.10 mice. Airway reactivity measurements are separately illustrated for wild-type and DO11.10 mice and depict the effects of each of multiple PBS and OVA aerosol challenges. Penh values at baseline (Base.), after PBS aerosol challenge (PBS), and after increasing concentrations of inhaled methacholine are shown. In (A), PBS aerosol-exposed mice (open symbols) are compared with OVA aerosol-exposed mice (filled symbols). In (B), OVA aerosol-exposed, sensitized wild-type mice were compared with OVA aerosol-exposed, nonsensitized DO11.10 mice. Penh measurements and BALF assessments were made 24 h after the last aerosol exposure and results are expressed as mean ± SEM of data from 6 to 8 mice. Significance (asterisk) was determined by Student's t test and indicates in (A) that OVA aerosol treatment was different from PBS aerosol treatment and in (B) that sensitized wild-type mice were significantly different from nonsensitized DO11.10 mice (p < 0.05). Statistical analysis for all the airway reactivity data is presented in Table 1.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Activation of CD4+ T cells by specific antigen has been hypothesized to initiate a cascade of events that lead to asthma. As a model of antigen-specific CD4+ T-cell activation, T cells from Balb/c DO11.10 alpha beta -TCR transgenic mice (20) have been used to characterize the in vitro commitment of naive T cells to the Th1 or Th2 phenotype (22, 29). Adoptive transfer of DO11.10 CD4+ T cells, followed by OVA aerosol exposure of the syngenic recipient mouse, has also been used to characterize the in vivo role of CD4+ T cells in airway inflammation (23, 24, 29). Our objective was to study the consequences of antigen-specific activation of resident lung (not transferred) CD4+ T cells. These cells have not been manipulated ex vivo and in addition, since they are resident cells, their location within the lung was not dependent upon expression of the appropriate chemotactic signals which must guide transferred cells to the lung. Our data show that OVA aerosol specifically activates CD4+ T cells in the airways of Balb/c DO11.10 mice to cause an influx of inflammatory cells into the lung. The response has features that resemble a Th1 type of response such as acute neutrophilia, high BALF IFN-gamma levels, and lack of AHR, but also features resembling Th2-like inflammation such as eosinophilia and mucus production.

An appropriate control for an investigation of the effect of OVA aerosol exposure on Balb/c DO11.10 mice are similarly treated nonsensitized wild-type mice. It is clear from this and many other studies that nonsensitized Balb/c wild-type mice do not mount any inflammatory response after a primary exposure to OVA aerosol (23). In fact, it is generally accepted that multiple OVA aerosol challenges (around 10) are required to induce even a mild eosinophilic lung inflammation in Balb/c mice (32). Therefore, the lung inflammation we characterized in DO11.10 mice cannot be attributed to a nonspecific protein-induced response, especially since BSA aerosol failed to reproduce the effect seen with OVA aerosol. Since our hypothesis was that antigen-specific activation of resident CD4+ T cells in DO11.10 mice could generate a Th2-type immune response in the lung resembling human asthma, we decided that it would be useful to have a reference to the level of Th2-like inflammation caused by lung antigen challenge which was possible in the Balb/c strain. Thus, we performed similar assessments in wild-type Balb/c mice that were OVA-sensitized and subsequently exposed to OVA aerosol. In OVA-sensitized wild-type mice, there was clear induction of a Th2 phenotype with elevated serum IgE. This is consistent with data obtained by other workers using this strain (25). Also in accordance with numerous other studies, OVA aerosol induced an inflammatory profile consistent with a Th2 response, namely, impressive BAL eosinophilia, increased IL-4 and IL-13 in the BALF, mucus overproduction, and AHR after multiple OVA aerosol challenges (16, 18, 25, 33). The OVA response in DO11.10 mice displayed some of the features seen in OVA-sensitized and -challenged wild-type mice such as eosinophilia (albeit mild) and mucus overproduction. Although airway eosinophilia was enhanced by multiple OVA aerosol challenges, it did not reach the level seen after multiple challenges in sensitized wild-type mice nor did it lead to the development of AHR. In addition, there was no production of IgE by DO11.10 mice after OVA challenge, but this was not expected because there was no active sensitization employed. It is possible that the deficiency in IgE production might partly explain the lack of a Th2 phenotype in DO11.10 mice, because it has been suggested that IgE is required for induction of AHR in models in which airway eosinophilia is mild (34).

Adoptive transfer of highly Th1- or Th2-polarized DO11.10 CD4+ T cells has been useful in characterizing the extreme cases of Th1 or Th2 immune deviation (23, 24). Transfer of DO11.10 Th1 cells to wild type Balb/c mice followed by OVA aerosol, has shown that antigen-specific Th1-type immune responses in the lung are characterized by neutrophil and lymphocyte recruitment and are notable for their absence of eosinophils and mucus production. Transfer of DO11.10 Th2 cells and OVA aerosol challenge is associated with impressive lung eosinophilia, mucus production, and AHR (23, 24). In the present study, OVA-specific activation of resident nonpolarized CD4+ T cells of DO11.10 mice resulted in an inflammatory profile intermediate of a clear Th1 or Th2 immune response. This response was independent of IgE production and CD4+ T-cell activation was critical, because CD4+ but not CD8+ T-cell depletion attenuated the response. An impressive increase in BAL neutrophils and IFN-gamma concentrations were the main characteristics suggesting Th1-like inflammation. However, the lesser but still significant BAL eosinophilia and increased epithelial cell mucus production were a strong indication that there was a small Th2 component to the inflammatory response. A study published during the preparation of this manuscript has also shown that the DO11.10 mouse can be used as a model of antigen-specific lung T-cell activation (35). Lee and coworkers demonstrated that exposure of DO11.10 mice to a 0.5% OVA aerosol for 2 to 4 d caused a mild lung eosinophilia and lung expression of messenger RNA (mRNA) for IL-4 and IFN-gamma . No data concerning other cell types were presented, but these findings support our demonstration of a small Th2 response in DO11.10 mice after OVA aerosol exposure.

The increased production of IFN-gamma in the lungs of DO11.10 mice but not wild-type mice after OVA aerosol challenge likely plays a role in inhibiting the development of Th2 inflammation. The enhanced lung eosinophilia (4.6-fold) seen after neutralizing IFN-gamma activity suggests that this was indeed the case. Since in vitro studies with CD4+ T cells from Balb/c-DO11.10 mice have shown that OVA stimulation under neutral conditions results in the production of IL-4 and loss in responsiveness to IL-12 (21), it has been suggested that CD4+ T cells from the Balb/c background default toward Th2 immune responses whereas those from other strains, such as the B10.D2, default toward a Th1 phenotype (22). In the context of the present study, the situation in vivo is not so clear; although IL-4 and IL-13 were detected in the BALF of Balb/c DO11.10 mice at baseline, in vivo exposure of these animals to OVA aerosol only induced the production of the Th1 cytokine, IFN-gamma . At best, there was only a small Th2 response. Therefore, at an in vivo level, it seems that Balb/c mice do not default toward a clear Th2 immune response after activation of lung antigen-specific CD4+ T cells. It is clear that in the presence of appropriate signals such as those encouraged by sensitization, the Balb/c mouse can mount a strong Th2 response. However, in the absence of these signals, it seems likely that CD4+ T-cell activation in the lungs of the Balb/c strain results in the default production of Th1 cytokines which serve to partially inhibit development of a Th2 immune response.

Although less widely acknowledged, BAL cells from asthmatic subjects have been shown to produce Th1-like cytokines (4, 36) and Th2 cytokines (3). This suggests that Th1 cells might also contribute to the pathophysiology of asthma. Our findings, in which direct in vivo activation of CD4+ T cells with antigen in the absence of prior sensitization resulted in an inflammatory response with characteristics of both the Th1 and Th2 phenotypes, are even more interesting in the context of recent studies. For instance, Randolph and colleagues (37) demonstrated that both IFN-gamma - and IL-4-positive CD4+ T cells are seen in the BALF taken from OVA-sensitized and aerosol-exposed mice. It is clear, however, that Th1-like inflammation in the adoptive transfer model has been shown to induce airway neutrophilia (23, 24). The elevated levels of IFN-gamma in BALF, in conjunction with the composition of the cell influx seen in our model, support the concept that the Th1 type of inflammation is predominantly neutrophilic. Even though an early lung neutrophil influx is seen after antigen challenge in mouse allergy (18) and neutrophilia is commonly seen in the airways of patients with acute, severe asthma (38, 39), it is unlikely that Th1 inflammation can lead to AHR. AHR is one of the defining features of asthma and is believed to result from chronic inflammation of the bronchial mucosa. In general, AHR in the mouse models of lung allergy requires a strong Th2 type inflammation and invariably significant lung eosinophilia (6, 40). In our study, multiple exposures to OVA aerosol enhanced the eosinophilia seen in the airways of DO11.10 mice but to a much lesser extent than that seen in sensitized wild-type mice. Accordingly, AHR after the multiple exposure protocol was only seen in OVA-sensitized wild-type mice.

We noted increased mucin staining in the bronchial epithelium of DO11.10 mice as early as 6 h after OVA aerosol challenge and this was more pronounced by 24 h after challenge. Mucus production may even be a more sensitive indicator of Th2 inflammation, because in our study and similarly to others (18, 25) increased mucin staining in the epithelium preceded the increase in BALF eosinophils. The concept that Th2 cytokines play a major role in stimulating mucus production has recently gained favor. IL-4 and IL-13 acting through the IL-4Ralpha chain are likely causative factors for mucus production and the asthma phenotype in murine models of lung allergy (41, 43, 44). In addition, it has been shown that antigen-specific activation of Th2-polarized but not Th1-polarized CD4+ T cells from DO11.10 mice stimulates mucus production in the lungs through the IL-4R pathway (23). Although we were unable to detect elevated levels of Th2 cytokines in the BALF of DO11.10 mice after OVA challenge, it seems very unlikely that the increased goblet cell staining in the present study is a result of the release of Th1-like cytokines. In light of these other studies, we suggest that the enhanced mucus staining we saw in the bronchial epithelium of DO11.10 mice after OVA aerosol suggested some Th2 component to the immune response in this model.

It has been shown that only 3.4% of lymph node lymphocytes from DO11.10 mice expressing the transgenic OVA-specific TCR are not CD4+ T cells (31). This is consistent with our findings that CD4+ T-cell depletion but not CD8+ T-cell depletion abolishes the antigen-specific inflammatory response of DO11.10 mice. In addition, it has been shown that approximately 82% of the T cells in the lungs of DO11.10 mice express the transgenic TCR (35) and that despite repeated OVA aerosol challenges, the number of T cells expressing the receptor in the lungs does not change. Therefore, the lung inflammation generated in DO11.10 mice is derived from activation of resident lung CD4+ T cells. Further, because the DO11.10 mice used in the present study had not previously encountered OVA, the rapid and large neutrophilia seen after OVA aerosol suggested the involvement of a small population of memory T cells in the airway mucosa. This concept is supported by studies showing that some CD4+ T cells from the intestinal mucosa of Balb/c DO11.10 mice express activation markers (i.e. CD45RBlow, CD69high, L-selectinlow) due to the stimulation of a nontransgenic TCR expressed concurrently on CD4+ T cells also bearing the transgenic TCR. This results in generation of a memory phenotype and allows intestinal T cells to respond to stimulation more readily than do T cells isolated from spleen, mesenteric lymph nodes, and Peyer's Patches (45). CD4+ T cells in the human airway mucosa have also been shown to be of the memory phenotype (46), probably because they are also exposed to a range of environmental antigens. Therefore, in the physiologic state, CD4+ T cells residing in the airway mucosa are able to respond rapidly to inhaled antigen presented by dendritic cells.

It has been shown that pathways involving activation of gamma delta T cells and production of Th2 cytokines such as IL-4 may be important in the induction of allergic lung disease (47). gamma delta T cells are required to produce the elevations in circulating IgE and IgG1 which are seen after systemic antigen sensitization in mice. However, once challenged locally with antigen, gamma delta T cell-deficient mice produce normal levels of circulating IgE and IgG1 even though the cell infiltration into the airways is attenuated by approximately 50%. The experiments we conducted do not provide evidence for us to conclude whether gamma delta T cells are involved in the OVA-induced lung allergy since the DO11.10 mouse is transgenic for the alpha  and beta  chains of the TCR and, in any case, our model does not utilize active sensitization. It seems that alpha beta T cells are sufficient to produce mild lung inflammation but other pathways, possibly involving gamma delta T cells, are probably important in the development of a fully blown Th2 immune response in the lung.

In conclusion, activation of resident lung CD4+ T cells by antigen in the absence of systemic sensitization induces airway inflammation which is intermediate of a clear Th1- or Th2-type immune response. At least in the Balb/c mouse, signals in addition to activation of nonpolarized resident lung CD4+ T cells are required to induce a robust Th2 inflammatory response characterized by a strong airway eosinophilia and AHR.

    Footnotes

Correspondence and requests for reprints should be addressed to Paul R. Gater, Roche Bioscience S3-1, 3401 Hillview Avenue, Palo Alto, CA 94304. E-mail: paul. gater{at}roche.com

(Received in original form June 15, 1999 and in revised form September 27, 1999).

Acknowledgments: The authors would like to thank Dr. Ken Murphy for the Balb/c DO.11.10 mice breeders and Paul Cheung, John Satjawatcharaphong, and Irene Bailey-Healy for expert technical assistance. Also, they are grateful for the contributions of Meredith Peters and Maria Fuentes for the animal genotyping.
    References
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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