Ovalbumin aerosols induce airway hyperreactivity in naı̈veDO11.10 T cell receptor transgenic mice without pulmonaryeosinophilia or OVA-specific antibody

Julie A. Wilder,* D. David S. Collie,† David E. Bice,† Yohannes Tesfaigzi,† C. Richard Lyons,‡

and Mary F. Lipscomb*University of New Mexico, Departments of *Pathology and ‡Internal Medicine, Albuquerque, New Mexico; and†Lovelace Respiratory Research Institute, Albuquerque, New Mexico

Abstract: The pathobiology of allergic asthma isbeing studied using murine models, most of whichuse systemic priming followed by pulmonary chal-lenges with the immunizing antigen. In general,mice develop eosinophilic pulmonary inflamma-tion, increased antigen-specific immunoglobulins,and airway hyperreactivity (AHR), all of which aredependent on antigen-specific T cell activation. Toestablish a model of allergic asthma, which did notrequire systemic priming, we exposed DO11.10 Tcell receptor transgenic mice, which have an ex-panded repertoire of ovalbumin (OVA), peptide-specific T cells, to limited aerosols of OVA protein.DO11.10 1/2 mice developed AHR in the absenceof increases in total serum IgE, OVA-specific IgG,or eosinophilia. The AHR was accompanied by pul-monary recruitment of antigen-specific T cells withdecreased expression of CD62L and CD45RB andincreased expression of CD69, a phenotype indic-ative of T cell activation. Our results support re-cent hypotheses that T cells mediate AHR directly.J. Leukoc. Biol. 69: 538–547; 2001.

Key Words: rodent z lung z allergy z T lymphocytes z inflammation

INTRODUCTION

Allergic asthma is a disease characterized by reversible epi-sodes of bronchoconstriction, chronic pulmonary inflammation,and high serum levels of allergen-specific immunoglobulin(Ig)E [1]. The disease results from the tendency of geneticallysusceptible individuals to mount an inappropriate immuneresponse to repeated inhalations of inert antigens, i.e., aller-gens. Unaffected individuals fail to respond or become tolerantto repeated inhalations of allergens. Conversely, asthmaticpatients respond to allergens by activating and expanding Thelper type 2 (Th2) cells that secrete interleukin (IL)-4 andIL-13, both critical for IgE production, and IL-5, which drivespulmonary eosinophilic inflammation. The presence of aller-gen-specific IgE in asthmatic patients allows airborne allergensto cross-link FcεR1 on mast cells and eosinophils, whichstimulate the release of compounds capable of mediating air-

way smooth-muscle contraction and airway lumen narrowing(reviewed in [2]).

Murine models of allergic asthma have been developed tostudy the pathobiology of the disease (reviewed in [3]). Most ofthese models involve systemic priming with antigen, often inadjuvant, followed by pulmonary exposure to the antigen. Theresults are varied, in part because of the strain of mouse beingtested and immunization protocol used, but generally includepulmonary inflammation, often eosinophilic in nature, highserum levels of allergen-specific IgE, and airway hyperreactiv-ity (AHR) in response to a nonspecific bronchoconstrictiveagent such as methacholine or acetylcholine. In contrast, fewmodels have been described that use aerosol exposures exclu-sively for the induction of the disease state [4]. Indeed, antigendelivery via multiple aerosol exposures has been shown toinduce tolerance in rodents as measured by their subsequentinability to respond to the same antigen when administeredwith adjuvant via an immunogenic route such as the intraperi-toneal (i.p.) one [5, 6].

We developed a murine model of asthma initiated by im-munization solely via the pulmonary route to more closelymimic the pathobiology of the human disease. Mice hemizy-gous for the DO11.10 ovalbumin (OVA)-T cell receptor trans-gene (DO11.10 1/2) [7] were exposed to an OVA or salineaerosol once/week for 3 consecutive weeks. These mice bearthe transgene on the background of a BALB/c mouse, a strainthat, once immune, requires only limited exposures to OVAaerosol to exhibit AHR [8]. Additionally, DO11.10 mice areresistant to tolerance induction by OVA given via a normallytolerogenic route [i.p. without adjuvant or intravenous (i.v.)][9]. A colony of hemizygous mice was established (DO11.101/2), in which 40% of peripheral T cells stain with theclonotypic monoclonal antibody (mAb) KJ1-26. In mice fromthis colony, the accumulation and activation status of OVA-Tcell receptor (TCR) bearing T cells in the lung and lung-

Correspondence: Julie A. Wilder, Ph.D., University of New Mexico, Depart-ment of Pathology, Albuquerque, NM 87131. E-mail: jwilder@salud.unm.edu

Current address of Dr. David Collie: Wellcome Centre for Research inComparative Respiratory Medicine, University of Edinburgh, Royal (Dick)School of Veterinary Studies, Veterinary Field Station, Easter Bush, Roslin,Midlothian EH25 9RG, Scotland.

Received July 10, 2000; revised November 30, 2000; accepted December 1,2000.

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associated lymph nodes (LALNs) could be monitored in re-sponse to OVA- or saline-aerosol exposure.

We show here that DO11.10 1/2 but not DO11.10 trans-gene-negative mice developed AHR after limited exposure toOVA aerosols. AHR was accompanied by a mild peribronchio-lar inflammatory response and accumulation of OVA-TCR1 Tcells in the lung, which expressed decreased levels of CD62Land CD45RB and increased levels of CD69, indicating thatthey were recently activated. We failed to observe significantincreases in total serum IgE or OVA-specific IgG. Nor were weable to demonstrate an increase in eosinophils in the bron-choalveolar lavage fluid or in the lung parenchyma, even whenfocusing on peribronchiolar areas. These data suggest thatAHR can be mediated by a very small number of antigen-specific T cells that have an activated phenotype.

MATERIALS AND METHODS

Mice

DO11.10 OVA-TCR Tg 1/2 and 2/2 mice were produced at the Universityof New Mexico Animal Resources Facility (UNM ARF, Albuquerque, NM) bybreeding OVA-TCR Tg 1/2 male mice (a kind gift of Dennis Loh [7]) withBALB/c female mice. All mice were housed under specific pathogen-freeconditions and used between 8 and 18 weeks of age. UNM ARF is accreditedby the American Association for Accreditation of Laboratory Animal Care, andall animal protocols were reviewed and approved by the UNM InstitutionalAnimal Care and Use Committee.

Aerosol exposures

Mice were exposed in a nose-only aerosolization chamber (Intox, Albuquerque,NM) to pH neutral saline or 0.5% OVA (Grade V, Sigma Chemical Co., St.Louis, MO) in pH neutral saline. All mice received three aerosol exposures, 1 hin duration, delivered 7 days apart. Aerosol particles were generated using aLovelace Nebulizer such that the size was ,1 m in diameter (6 L air/min; 42psi).

Pulmonary physiology

Changes in total lung resistance (RL) were measured in anesthetized, trache-otomized, ventilated mice, which were placed in a volume-displacement ple-thysmograph as described previously [8]. Mice were anesthetized with an i.p.injection of a solution of xylazine and ketamine in sterile saline at a dose of 16mg xylazine and 80 mg ketamine/g bodyweight. A tail-vein catheter [10 cmpolyethylene (PE)-10 tubing attached to a 30-gauge needle and filled initiallywith heparinized saline] and a tracheal catheter (20-gauge needle hub) wereinserted, sealed, and secured with cyanoacrylate adhesive. Once placed in theplethysmograph, mice were ventilated at a rate of 150 breaths/min and a 0.25ml tidal vol. An opening was made in either side of the caudal chest wall byremoving a portion of a rib to equilibrate pleural-surface pressure to body-surface (box) pressure and facilitate the measurement of transpulmonarypressure. The resistance of the tracheal cannula was determined by ventilationof the plethysmograph in the absence of a mouse and the value subtracted fromall resistance measurements. Custom-designed computer software (LabView3.0.1, National Instruments, Austin, TX) was used to facilitate integration ofthe flow signal to yield volume and to derive pulmonary resistance using themethod of least-squares linear regression. Baseline measurements of RL wererecorded prior to delivery of saline and half-log increasing doses of methacho-line (.014–3.7 mg/kg) administered via the tail-vein catheter. Peak responseswere recorded and values allowed to return to within 10% of baseline beforedelivery of the next dose. Recovery was facilitated by 2–3 forced vital-capacitymaneuvers. Data are presented as the actual change in RL from baselinevalues.

Lung histology

Lungs were inflated with 10% buffered neutral formalin fixative injectedthrough PE-50 tubing inserted into the trachea. The lungs, once excised, werefixed for at least 24 h before the left lobes were sectioned longitudinally alongthe major airway and submitted to the UNM Hospital Pathology Laboratory(Albuquerque, NM) where they were embedded, sectioned, and stained withhematoxylin and eosin. Three 103 fields per lung, generally covering thewhole left lobe in total, were examined and scored separately for the percentinflammatory cell infiltration around bronchioles, peribronchial arteries, andveins, according to the following scheme as previously described [8]: Less than1% of the circumferential or longitudinal area involved with any type ofinflammation was scored as a 0; 1–5% involvement 5 0.5; 5–10% involve-ment 5 1; 10–25% involvement 5 2; 25–50% involvement 5 3; and .50%involvement 5 4. The scores of each of the three sections were averaged toobtain a peribronchiolar, periarterial, and perivenular score for each lung. Thetotal lung score represents the sum of the scores of these three areas. Twoindependent observers (J.A.W. and M.F.L.) scored each lung lobe in a blindedfashion. The average of the two observations is shown for each mouse exam-ined. Data presented are the average (6SE) of individual mice from severalexperiments. In addition, all inflamed areas of the lungs of DO11.10 1/2 miceexposed to OVA aerosols were examined under oil immersion (1003) todistinguish the types of inflammatory cells present. Monocytoid cells (a com-bination of lymphocytes and monocytes), macrophages, polymorphonuclearcells, and eosinophils were enumerated in inflamed peribronchiolar, periarte-rial, and perivenular areas.

Harvest of tissue and fluid

All mice were sacrificed by inhalation of CO2 1 day following the last aerosolexposure. Serum and tracheobronchial lavage (TBL; 0.3 ml/mouse) were col-lected as previously described [8]. Cells recovered from the TBL were countedand used for cytospin preparations (25,000 or less/slide). These cytospins werestained with the Baxter Diff-Quick kit (VWR Scientific Products, San Fran-cisco, CA), and differentials were calculated. LALN cells were dispersed intosingle-cell suspensions by gently rubbing the tissue between the frosted endsof two glass slides followed by red blood cell (RBC) lysis with ammoniumchloride. Single-cell suspensions of lung cells were prepared by mincingsaline-perfused lungs followed by a 90-min incubation with collagenase (0.7mg/ml) and DNAse (30 mg/ml) at 37°C. Lung cells were then gently pushedthrough a wire mesh and passed over a loose, nylon wool plug quickly toremove connective tissue and debris. RBCs were lysed, and the remaining cellswere spun through a layer of 30% Percoll to remove cell debris and enrich forviable cells.

Cell phenotyping

Lung and LALN cells (0.5–13106) were stained with antibodies of interest ina volume of 0.06 ml staining buffer [phosphate-buffered saline (PBS)11% fetalcalf serum (FCS)] for 30 min on ice. Antibodies included KJ1-26-biotin [10](prepared from hybridoma supernatants by sodium ammonium sulfate precip-itation and biotinylated), CD4-fluorescein isothiocyanate (FITC) or CD4-allo-phycocyanin (APC), CD62L-PE, CD45RB-PE, and CD69-FITC (all fromPharmingen, San Diego, CA). Incubation with these primary antibodies wasfollowed by three washes in staining buffer and fixation with 0.5% parafor-maldehyde at 4°C or in the case of KJ1-26-biotin-stained cells, incubation withStreptavidin-PerCP (Becton-Dickinson, San Jose, CA) for 30 min on ice andsubsequent washing and fixation. All cells were analyzed on a Becton-Dick-inson FACScan or FACSCalibur and analyzed using PCLYSIS or CELLQUESTsoftware (both from Becton-Dickinson), respectively.

Immunoglobulin enzyme-linked immunosorbentassays (ELISAs)

OVA-specific IgG in the sera was measured as previously described [8] usingOVA-coated polyvinylchloride (PVC) plates (Falcon brand, Fisher Scientific,Pittsburgh, PA; 0.1 mg/ml OVA). Sera was diluted in PBS containing 0.25%bovine serum albumin (BSA) and 0.05% Tween 20 (blocking buffer). Threedilutions of sera/mouse were added to the plates in duplicate, and incubationproceeded overnight at 4°C. OVA-specific IgG was detected using horseradishperoxidase (HRP)-coupled goat anti-mouse IgG1 or IgG2a (Southern Biotech-

Wilder et al. Ovalbumin aerosol-induced AHR in DO11.10 mice 539

nologies, Birmingham, AL) diluted in blocking buffer, incubated 2 h at roomtemperature (RT), followed by addition of HRP substrate (ABTS, Sigma). Colordevelopment proceeded at RT until the least dilute-serum sample approachedan OD405 of 1.0–2.0. Plates were read on a Dynatech ELISA reader (Bio-TekInstruments, Inc., Winooski, VT). The OD405 of each serum dilution wasmultiplied by its dilution factor to give an arbitrary unit. For each mouse, theunits of IgG anti-OVA are calculated from the three serum dilutions and anaverage presented. Total IgE levels in the serum were determined by asandwich ELISA using PVC plates coated with rat anti-mouse IgE (clone R35,Pharmingen; 2 mg/ml) and detected using rat anti-mouse IgE-HRP (SouthernBiotechnologies). Coating, serum dilutions, washing, and development proce-dures were carried out as described for the OVA-specific IgG ELISA. A knownamount of monoclonal IgE was used to construct a standard curve in eachassay, and values are shown as ng/ml.

Production and analysis of cytokines

Lung cell cultures and cytokine analysis were performed as described previ-ously with minor modifications [11]. Lung cells were incubated on plastictissue culture dishes for 2 h at 37°C, and nonadherent cells were harvested,washed, and resuspended at 5 3 106/ml in culture medium [RPMI 1640supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin/streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 1 mM nonessentialamino acids (all from Gibco BRL Life Technologies, Grand Island, NY), 5 31025 M 2-mercaptoethanol (Eastman Kodak, Rochester, NY), 1 mg/ml indo-methacin (Sigma), and 250 units/ml catalase (Worthington Biochem, Freehoff,NJ)]. Anti-IL-4 receptor (1 mg/ml; Genzyme, Cambridge, MA) was included inall cultures to prevent uptake of secreted IL-4 and facilitate its assessment byELISA. Cells were left unstimulated or stimulated with OVA protein (100 mM),OVA peptide (323–339, 4 mM; Research Genetics, Huntsville, AL), or keyholelimpet hemocyanin (KLH; 5 mg/ml; Calbiochem Novabiochem, La Jolla, CA) induplicate or triplicate for 48 h, after which supernatants were collected andanalyzed for cytokine content by sandwich ELISA. ELISA plates (Nunc Max-isorb Immunoassay, VWR) were coated with capture antibodies diluted in 0.1M Na2HPO4 overnight at 4°C, washed, and blocked with 1% BSA in PBS.Samples were added to the plates after subsequent washes and incubatedovernight at 4°C. Detection proceeded the following day by adding biotinylatedmAbs, streptavidin-HRP (1 mg/ml), and ABTS substrate, and the OD405 wasread as described above. mAb pairs for the cytokines were purchased fromPharmingen in the case of IL-4 (11B11 and biotin-BVD6-24G2), IL-5 (TRFK5and biotin-TRFK4), and interferon (IFN)-g (R46A2 and biotin-XMG1.2).Antibodies specific for IL-13 (38213.11 and biotin-BAF413) were purchasedfrom R&D Systems, Minneapolis, MN. All cytokines were quantified bycomparison to standard curves generated using recombinant cytokines (Pha-mingen). Detection limits for each cytokine assay were assigned as the lowestconcentration in the linear portion of the standard curve, generally between 16and 250 pg/ml.

Statistics

Differences in RL responses to methacholine between groups were analyzed byrepeated measures analysis of variance (ANOVA). RL responses betweengroups at individual doses were compared using unpaired t-tests to determinesignificance. Differences in all other measured variables were analyzed usingANOVA statistics using the Bonferroni-Dunn post-hoc test when four groupswere being compared or unpaired two-tailed t-tests when two groups werebeing compared. Values of P , .05 were considered significant for allcomparisons.

RESULTS

Mice hemizygous for the OVA-TCR transgene (DO11.10 1/2)and their nontransgenic littermates (DO11.10 2/2) were ex-posed to 3 weekly aerosols of OVA or saline 1 h in duration.AHR was measured 1 day after the third aerosol. DO11.101/2 mice exposed to OVA aerosols exhibited a significantlyincreased change in pulmonary resistance in response tomethacholine (Fig. 1), whereas the three control groups of

mice did not (DO11.10 1/2 mice exposed to three salineaerosols or nontransgenic littermates exposed to three OVA orthree saline aerosols). Concurrent with AHR expression, thelungs of DO11.10 1/2 mice exposed to OVA aerosols hadsignificantly increased levels of inflammation compared withthe other three control groups as assessed histologically (Figs.2 and 3A). Significant inflammation was apparent around thebronchioles and venules (Fig. 3, B and D). Although there wasa trend to greater inflammation around arterioles, this differ-ence did not reach statistical significance when comparing thefour groups of mice. When the inflamed areas of OVA- andsaline-exposed DO11.10 1/2 mice were examined underhigh-power magnification (1003), monocytoid cells and mac-rophages were the most prevalent cell types in all anatomicalareas scored, and eosinophils were the least abundant cell typeidentified (Figs. 2B and 3E). A subset of lungs from each groupwas also stained with Alcian Blue and Periodic Acid Schiff formucus glycoproteins and positive-staining cells in all airwayswere scored. There were fewer than 20 positive-staining cellsin all lungs, with most lungs being completely devoid of anymucus-containing cells. No significant difference betweengroups was observed (unpublished results).

Inflammation was characterized further by enumerating totallung and TBL cells and assessing numbers of macrophages,lymphocytes, neutrophils, and eosinophils in Wright-Giemsa-stained cytospin preparations. Consistent with an increase ininflammation graded histologically, exposure of DO11.10 1/2mice to three OVA aerosols resulted in a significant increase in

Fig. 1. DO11.10 1/2 mice exhibit AHR after 3 weekly OVA-aerosol expo-sures. All mice received 3 weekly, 1-h aerosols of saline or OVA as describedin Materials and Methods. RL with increasing doses of intravenously deliveredmethacholine was measured in anesthetized, mechanically ventilated mice.Repeated measures ANOVA revealed that DO11.10 1/2 mice receiving threeOVA aerosols responded to methacholine in a manner that was statisticallydifferent than the other three control groups (P,05). *, Doses of methacholineat which OVA-exposed DO11.10 1/2 mice responded differently than allother groups of mice as measured by unpaired t-tests (P,.05). Data representthe mean change from baseline resistance in cm H2O/ml/sec for individualmice 6 SE.

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total lung-cell numbers (Fig. 4A), macrophages, and lympho-cytes recoverable from collagenase-digested, minced-lung tis-sue (Fig. 4B). Although no increase in total TBL cellularity wasapparent in DO11.10 1/2 mice exposed to OVA (Fig. 4C), asignificant increase in polymorphonuclear neutrophil (PMN)cells in the lavage fluid was observed (Fig. 4D). Similarly, weobserved a significant increase in LALN cellularity inDO11.10 1/2 mice exposed to OVA (Fig. 4E).

Further, we asked if a limited number of OVA-aerosolexposures were sufficient to induce accumulation and activa-tion of OVA-specific T cells in the lungs of DO11.10 1/2 or2/2 mice. Antibodies were used to identify helper T cells(CD41), T cells bearing the OVA-TCR (KJ1-261), naı̈ve Tcells (CD62Lhi and CD45RBhi), and activated/memory T cells(CD62Llo/medium, CD45RBlo/medium, and CD69). The cellsstained with various mAbs were analyzed by three- or four-color flow cytometry. Figure 5 represents the type of dataobtained when lung cells from naı̈ve, saline-aerosol-exposed,or OVA-aerosol-exposed mice were stained with antibodies toKJ1-26, CD4, and CD62L, showing the percentages of cellsfalling within relevant gates. Data such as these revealed thatDO11.10 1/2 mice exposed to three OVA aerosols recruitedsignificantly increased numbers of CD41 cells that bore theOVA-TCR receptor (KJ1-261) cells to their lungs when com-pared with control mice (Fig. 6A). DO11.10 1/2 mice re-ceiving OVA aerosols had more than double the number ofOVA-TCR1 cells in the lung than DO11.10 1/2 mice re-ceiving saline aerosols (1.633106 vs. 0.83106 cells, respec-tively), whereas DO11.10 2/2 mice receiving three OVAaerosols had only background levels of OVA-TCR1 cells

(6.53104). The numbers of OVA-TCR1 cells in the lungs ofDO11.10 1/2 mice receiving saline aerosols were not signif-icantly different than those found in lungs of naı̈ve DO11.101/2 mice (0.553106). When we assessed the activation statusof these cells in the lungs of OVA-exposed DO11.10 1/2mice, we observed a significant increase in the absolute num-bers of OVA-TCR1 cells, which expressed low-to-mediumlevels of CD62L or CD45RB compared with DO11.10 1/2mice exposed to saline or naı̈ve 1/2 mice, suggesting thatthese cells were memory or activated cells (Figs. 5 and 6B) [12,13]. Further evidence of their recent activation was providedby the observation that significantly more OVA-TCR1 cells inthe lungs of DO11.10 1/2 mice exposed to OVA aerosolsexpressed CD69 compared with their saline-exposed or naı̈vecontrols (Fig. 6B) [14]. The majority of CD691 OVA-TCR1cells co-expressed low levels of CD62L or CD45RB (92.4 and68.1%, respectively).

To assess whether the activated OVA-TCR1 T cells in thelungs of OVA-exposed DO11.10 1/2 mice might be secretingincreased levels of cytokines, we cultured nonadherent lungcells from naı̈ve and saline- and OVA-exposed mice withmedia alone, OVA peptide (323-339), OVA protein, or KLH asa nonspecific antigenic stimulus. Supernatants were collectedafter 48 h of culture, and cytokine content was measured byELISA as described in Materials and Methods. Because OVA-TCR1 cells are present in the lungs of all three groups of miceexamined (naı̈ve, saline-exposed, and OVA-exposed; see Figs.5 and 6A), we were looking for increases in cytokine secretionas a result of the OVA-aerosol exposure and compared theselevels with those observed from saline-exposed mice specifi-

Fig. 2. Representative pulmonary pathology after OVA (A and B)- orsaline (C)-aerosol exposure of DO11.10 1/2 mice. All mice receivedthree aerosol exposures delivered once weekly for 60 min. (A) Arrowindicates an area of inflammation that is magnified further in B. Thesection depicted in C shows the average inflammatory pattern of DO11.101/2 mice exposed to three aerosols of saline and is the same as DO11.102/2 mice exposed to three aerosols of saline or OVA. (A and C) Originalbar 5 100 m; (B) original bar 5 25 m.

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Fig. 3. DO11.10 1/2 mice exhibit pulmonaryinflammation after 3 weekly OVA-aerosol expo-sures. All mice received 3 weekly aerosols of sa-line or OVA, and the total lung (A), peribronchial(B), periarterial (C), and perivenular (D) inflamma-tory scores were determined as described in Ma-terials and Methods. Two-way ANOVA revealedthat DO11.10 1/2 mice had statistically differentlevels of total, peribronchial, and perivenular in-flammation compared with the other three controlgroups of mice (*, p,.05). Data represent themean (6SE) inflammatory score of 10–13 mice ineach group. (E) The sum of the number and typesof inflammatory cells enumerated under high-power magnification in OVA- and saline-exposedlungs from DO11.10 1/2 mice. All inflamed areas[peribronchiolar (PB), perivenular (PV), and peri-

arterial (PA)] were examined from the left lobe of 13 OVA-exposed mice with an average of 8.5 areas examined/lung (4.5 PB, 2 PV, and 2 PA) and 10 saline-exposedmice with an average of 2.6 areas examined/lung (1.5 PB, 0.9 PV, and 0.2 PA). Two-way ANOVA revealed that DO11.10 1/2 mice exposed to OVA aerosolshad significantly more monocytoid cells and macrophages in perivenular and/or peribronchiolar areas compared with mice exposed to saline aerosols, as indicatedby * (P,.05).

Fig. 4. DO11.10 1/2 mice exhibit significantincreases in lung cell and LALN cell numbersafter OVA-aerosol exposure. All mice received 3weekly aerosols of saline or OVA. Numbers oftotal lung cells (from collagenase-digested lungtissue), total TBL cells, total LALN cells, andindividual types of lung and TBL cells wereassessed as described in Materials and Methods.Two-way ANOVA revealed differences in totallung cell numbers (A) and increases in macro-phages and lymphocytes (B) in the lungs ofDO11.10 1/2 mice exposed to three OVA aero-sols. Although OVA-exposed DO11.10 1/2mice failed to recruit significantly more cells to

the TBL (C), a significant increase in airway neutrophils was observed in these mice (D). DO11.10 1/2 mice also had a significant increase in LALN cell numberafter three OVA-aerosol exposures (E). Data represent the mean cell number (6SE) of 20–23 mice in each group.

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cally. Figure 7 shows that lung cells from DO11.10 1/2 micereceiving three OVA aerosols secrete significantly more IL-4and IL-13 than their saline-exposed controls in response toOVA protein and/or OVA peptide. We also observed a signif-icant increase in spontaneous secretion of IL-5 by OVA-exposed DO11.10 1/2 lung cells, which was not enhanced byOVA protein or peptide exposure in vitro. In contrast, noincrease in IFN-g secretion above that seen in cultures ofsaline-exposed DO11.10 lung cells could be demonstrated. Forreasons that are unclear to us, secretion of IL-5 by naı̈veDO11.10 1/2 lung cells in response to OVA protein and OVApeptide was also increased significantly compared with thatsecreted by similarly treated lung cells from saline-exposedmice. This was not the case for any other cytokine measured. Itis important that cytokine secretion in response to KLH, anonspecific antigenic stimulus, was never significantly greaterthan that observed in media alone, regardless of aerosol expo-sure.

We next asked whether OVA-TCR Tg 1/2 mice, which hadreceived three OVA-aerosol exposures, also exhibited evi-dence of humoral immunity to OVA. Serum was collected 1 dayfollowing the last aerosol exposure, and levels of total IgE and

OVA-specific IgG were measured by ELISA. Neither DO11.101/2 mice exposed to OVA aerosols nor any of the threecontrol groups of mice mounted an IgG1- or IgG2a-anti-OVAresponse or increased their total serum IgE above that found innormal BALB/c mice (Table 1).

DISCUSSION

We exposed DO11.10 Tg 1/2 and Tg 2/2 littermates to threeOVA aerosols, delivered once weekly for 1 h each, to deter-mine if naı̈ve mice with an expanded repertoire of antigen-specific T cells would develop AHR when exposed to a limitednumber of pulmonary antigenic exposures in the absence ofsystemic priming with antigen. DO11.10 1/2, but notDO11.10 transgene-negative, mice developed AHR after ex-posure to 3 weekly, 1-h OVA aerosols. AHR was accompaniedby a mild peribronchiolar inflammatory response, increases inlung and LALN cellularity, increased TBL neutrophilia, and anearly twofold increase in OVA-TCR1 T cells in the lung.Exposure to three OVA aerosols also resulted in an increase innumbers of OVA-TCR1 cells in the lung, which had a de-creased expression of CD62L or CD45RB and increased ex-pression of CD69, suggesting these T cells were either recentlyactivated or memory T cells [12–14]. Lung cells from OVA-exposed DO11.10 1/2 mice also secreted increased levels ofIL-4, IL-13, and IL-5 in culture, spontaneously (IL-5 only) orin response to stimulation with OVA protein and/or OVApeptide. Although OVA-exposed DO11.10 1/2 mice devel-oped AHR, they failed to develop a humoral-immune response(no increases in serum IgG1 or IgG2a anti-OVA or total IgE).Further, no significant increases in pulmonary eosinophiliawere observed.

The fact that DO11.10 1/2 mice responded to a limitednumber of OVA-aerosol exposures by developing AHR sug-gests that an expanded repertoire of OVA-specific T cells issufficient for mice to manifest AHR in response to this minimalpulmonary stimulus. These data help explain why most murinemodels of allergic asthma, which include AHR as a manifes-tation of antigen exposure, require a systemic priming step withantigen prior to pulmonary-antigenic challenge [15–21]. Sys-temic priming serves to expand the pool of antigen-specific Tcells. In naı̈ve DO11.10 1/2 mice, however, 40% of the cellsin the T cell repertoire bear the OVA-TCR receptor by virtueof the transgene expression, which eliminates a systemic prim-ing requirement. However, DO11.10 2/2 littermates, whichhave a normal T cell repertoire and numbers of OVA-TCR1cells similar to normal BALB/c mice, were unable to respondto limited pulmonary exposure to OVA by exhibiting AHR. Theonly other study of normal mice developing AHR in responseto pulmonary antigen exposure in the absence of systemicpriming or adoptive transfer of previously primed cells re-quired a more prolonged aerosol exposure protocol (daily for 10days) [4]. These latter mice did not develop pulmonary inflam-mation but developed high levels of circulating OVA-specificIgE and IgG.

It was recently shown that homozygous, transgenic DO11.10mice exposed to a single whole-body OVA aerosol (20 min induration) or four OVA aerosol exposures delivered on consec-

Fig. 5. Representative phenotype of lung cells from naı̈ve DO11.10 1/2mice as well as 1/2 and 2/2 mice after 3 weekly aerosols of saline or OVA.Lung cells were stained with KJ1-26 antibody and anti-CD4 and anti-CD62Lantibodies as described in Materials and Methods. Plots represent profiles oflung cells from individual mice after gating on the lymphocyte subpopulation(small, nongranular cells). Numbers shown in quadrants represent the percent-ages of gated lung cells that fall in that quadrant.

Wilder et al. Ovalbumin aerosol-induced AHR in DO11.10 mice 543

utive days failed to develop AHR, as measured by increases inenhanced pause (PenH) to aerosolized methacholine [22].These data suggest that the timing of OVA aerosol exposure(acute vs. more chronic), the mode of OVA aerosol delivery(whole-body or nose-only), the number of transgenic T cells(presumably more in homozygous vs. hemizygous DO11.10mice), or the methodology used to measure AHR (Buxco boxvs. whole-body volume-displacement plethysmography) is crit-ical in inducing AHR in DO11.10 transgene-positive mice. Itis interesting that, in agreement with our current studies, theseauthors also described TBL neutrophilia, little eosinophilia,and a failure to mount a humoral immune response after asingle exposure of DO11.10 1/1 mice to OVA by the aerosolroute. However, in contrast to our data, Knott et al. [22]describe the appearance of mucins in the bronchial epithelium

in response to OVA aerosol exposure, whereas we never foundevidence of mucous-cell metaplasia (unpublished results).They also describe that a single OVA aerosol exposure induceda skewing toward a Type 1 cytokine milieu in the lung asevidenced by the appearance of IFN-g but no IL-4 or IL-13 inthe BAL. Conversely, we show a clear skewing toward a Type2 immune response in the lung, as evidenced by increases inIL-4, IL-5, and IL-13 cytokine secretion by OVA-exposedDO11.10 1/2 lung cells ex vivo. Again, the differences inthese results may be because of many factors, such as the strainof mice, the mode of OVA exposure, and the protocols used tomeasure secreted cytokines.

In DO11.10 Tg 1/2 mice, AHR developed in the absenceof eosinophilia and IgE. Eosinophils have been shown to playimportant roles in the manifestation of AHR in some murinemodels of allergic asthma [23–26], whereas other studies havesuggested that they are neither required nor sufficient for AHR[15, 17–19]. Similarly, IgE has been shown to be essential forthe development of AHR [27] in some studies while appearingunnecessary in others [18, 25, 28, 29]. Recently, we showedthat neither eosinophilia nor elevated serum levels of IgE weregood predictors of AHR [8]. In these studies, we used threedifferent inbred strains of mice and a more classical, systemicpriming event with OVA-alum followed by a limited pulmonarychallenge with aerosolized OVA (two aerosol exposures deliv-ered in a single day). In these studies, BALB/c mice exhibitedAHR in the absence of eosinophilia, whereas C57BL/6 andBDF1 mice had eosinophilia and elevated IgE levels and failedto develop AHR.

Although controversy remains about the precise immunemechanisms that lead to AHR in murine asthma models, a rolefor T cells is undisputed. If CD41 T cells are depleted [20] ortheir activation is inhibited [21], AHR fails to develop. Fur-thermore, exogenous OVA-specific Th2 cells, when transferredto naı̈ve mice, mediate AHR in response to pulmonary chal-lenge with OVA [30]. Our data support the critical role thatactivated T cells play in manifesting AHR in that we can showaccumulation of OVA-specific T cells in the lungs of DO11.101/2 mice after OVA-aerosol exposure, which has decreasedL-selectin and CD45RB expression and increased CD69 ex-pression, a phenotype indicating that the T cells were activatedrecently or are of the memory phenotype [12–14]. Further, wehave shown that the accumulation of these cells in the lungs ofOVA-exposed DO11.10 1/2 mice results in increased IL-4,IL-13, and IL-5 secretion by OVA-stimulated, nonadherentlung cells in vitro.

The data presented here do not identify the mechanism bywhich CD62Llo/med OVA-TCR1 cells increase in numbers inthe lungs of OVA-exposed DO11.10 mice. The possibilities arethat OVA-aerosol exposure induced 1) resting, resident lungOVA-TCR1 T cells to proliferate in situ and become activatedto secrete Th2 cytokines, 2) activation and proliferation ofnaı̈ve OVA-TCR1 cells in the LALNs, which were then re-cruited to the lung, or 3) enhanced recruitment of OVA-TCR1T cells from the periphery to the lung. The first possibility isunlikely given recent data, suggesting that antigen delivery tothe lung induces inflammation that is primarily a result of Tcell recruitment rather than in situ proliferation [31]. Indeed,although OVA-aerosol exposure of DO11.10 1/2 mice results

Fig. 6. DO11.10 1/2 mice have increased numbers of CD41 OVA-TCR1cells in their lungs after 3 weekly exposures to OVA aerosol, many of whichalso express low levels of CD62L or CD45RB and increased levels of CD69.Lung cells were stained with KJ1-26 antibody in combination with anti-CD4,anti-CD69, and anti-CD45RB or anti-CD62L antibodies as described in Ma-terials and Methods. Absolute numbers of cells of particular phenotypes werecalculated by multiplying the percent-positive cells in each quadrant by thetotal lung-cell yield as assessed by cell counting using a hemacytometer.Two-way ANOVA revealed that DO11.10 1/2 mice had significantly greaternumbers of OVA-TCR1CD41 cells in their lungs after OVA-aerosol exposurethan any of the other four control groups of mice (*, p,.05; A). DO11.10 1/2mice exposed to OVA aerosols also had significant increases in OVA-TCR1cells, which coexpressed low levels of CD62L or CD45RB or increased levelsof CD69 when compared with naı̈ve DO11.10 1/2 mice or those that hadreceived three saline aerosols. Data represent the mean cell number (6SE) of(A) 8–23 mice in each group and (B) 8–18 mice.

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in accumulation of OVA-TCR1 cells in the lung, which ex-press an activated phenotype, these cells may arrive in the lungalready expressing this activated phenotype and need notnecessarily become activated in direct response to the OVAaerosols. These memory T cells could be recruited to the lungfrom the LALNs where they were initially activated or from theperiphery. Regarding the latter possibility, it is interesting tonote that many OVA-TCR1 cells in DO11.10 1/2 miceco-express a second T cell receptor through which the cellscould be activated (by recombining endogenous TCR-a chainswith transgenic b chains). Thus, these cells may arrive in thelung having already been activated via an environmental an-tigen, not OVA [32, 33]. However, activation via their alternateTCR has been shown not to preclude them from acting asOVA-specific memory cells on exposure to OVA [34, 35].Thus, upon restimulation in the lung through their OVA-TCR,OVA-specific memory T cells may have mediated AHR. Inaddition, it is possible that OVA aerosols recruited naı̈ve andmemory OVA-specific T cells to the lung and that both types ofT cells activated in situ caused AHR.

The precise mechanisms by which the activated or memoryT cells induce AHR in OVA aerosol-exposed DO11.10 1/2mice are also not known. However, it has been shown recentlythat inoculation of naı̈ve mice with IL-4 or IL-13 can causeAHR in the absence of any antigen exposure [36, 37]. There-fore, the increase in OVA-stimulated IL-4 and IL-13 secretionby OVA-exposed DO11.10 lung cells may be playing a role inmediating the AHR observed in our model, although it is clearfrom other studies that IL-4 is not required for AHR manifes-tation [18, 30].

Whether these cytokines cause AHR directly or cause itindirectly by influencing other cells to make the primary me-diators is unclear. Grunig et al. [36] showed that intranasaladministration of IL-4 or IL-13 to naı̈ve BALB/c mice inducedsignificant pulmonary eosinophilia and goblet-cell metaplasiawith mucous-cell overproduction, leaving open the possibilitythat AHR was mediated by eosinophils and their products.Neutralization of IL-13 activity by intranasal delivery of sIL-13Ra2-Fc protein also significantly reduced eosinophilia andgoblet-cell metaplasia, normally induced by intranasal OVA

Fig. 7. Lung cells from DO11.10 1/2 miceexposed to three OVA aerosols secrete in-creased levels of IL-4, IL-13, and IL-5 in cul-ture. Nonadherent lung cells from DO11.101/2 mice were placed in culture and stimu-lated with OVA peptide, OVA protein, KLH, orleft unstimulated as described in Materials andMethods. Levels of secreted IL-4 (A), IL-5 (B),IL-13 (C), and IFN-g (D) were quantified byELISA. Unpaired t-test analysis revealed thatlung cells from OVA-exposed mice secretedsignificantly greater levels (*) of IL-4 in re-sponse to OVA peptide and OVA protein, IL-13in response to OVA peptide, and IL-5 in re-sponse to media alone, OVA peptide, or OVAprotein when compared with saline-exposedmice. Data represent the mean level of cytokine(computed from an average of duplicate or trip-licate cultures) secreted by 4–19 mice/groupfor IL-4, IL-5, and IFN-g and 4–14 mice/groupfor IL-13.

TABLE 1. Humoral Immune Response of DO11.10 Mice to Aerosol Exposure

Strain of mouse Aerosol exposure IgG1 anti-OVAa IgG2a anti-OVAa IgEb

DO11.10 1/2 Saline 48.2 (6.9) 36.0 (7.1) 162.4 (34.1)DO11.10 1/2 OVA 61.3 (11.4) 29.9 (4.4) 123.9 (15.7)DO11.10 2/2 Saline 44.6 (8.2) 31.2 (4.3) 168.1 (34.7)DO11.10 2/2 OVA 33.8 (2.8) 28.8 (3.7) 148.8 (24.9)BALB/c None 58.1 (22.5) 21.0 (9.0) 246.3 (137.1)

a IgG1 and IgG2a anti-OVA are expressed as units/ml calculated as described in Materials and Methods. b IgE is expressed as ng/ml.

Wilder et al. Ovalbumin aerosol-induced AHR in DO11.10 mice 545

challenge of OVA-immune mice, and these reductions wereaccompanied by a loss of AHR. In contrast, Wills-Karp et al.[37] showed that whereas IL-13 given to naı̈ve mice inducedearly pulmonary eosinophilia (after 1 day of intratracheal de-livery), it had resolved by the time AHR was detected (after 3days of delivery). In addition, although IL-13 induced trendstoward increased IgE and mucous-containing cells, these val-ues were not significantly different than PBS controls. Finally,these authors demonstrated that blockade of IL-13 action by i.pinjections of sIL-13Ra2-Fc protein failed to alter the increasedpulmonary eosinophilia and OVA-specific IgE induced bypulmonary challenge of OVA-immune A/J mice with OVA butdid abolish AHR, suggesting that eosinophilia and IgE wereincapable of mediating AHR in the absence of IL-13.

It is interesting that the levels of IL-4 and IL-5 produced bylung cells from DO11.10 1/2 mice after three OVA aerosolexposures are not sufficient to induce elevated serum IgE orIgG1 levels or pulmonary eosinophilia. Our results showingthat AHR can develop in the absence of increased IgE oreosinophilia support recent studies conducted in OVA-immuneIL4 2/2 mice treated with anti-IL-5 antibodies that manifestAHR but fail to display eosinophilia or increases in OVA-specific serum immunoglobulins [18].

We did note marked increases in neutrophils in the TBL,although monocytoid cells and macrophages were the predom-inant cell types observed in peribronchial and perivascularareas of inflammation, suggesting that the TBL compartmentdoes not always reflect the peribronchiolar inflammatory com-partment accurately and that certain cell types may be re-cruited selectively to the TBL over the more abundant celltypes present in the lung. The presence of neutrophils in theTBL of OVA-exposed DO11.10 1/2 mice is interesting, how-ever, given recent evidence that significant airway neutrophiliais often a characteristic of severe asthma [38] and has also beenobserved in allergic asthmatics within hours of segmentalallergen challenge [39]. BAL neutrophilia was also observed byKnott et al. [22] after exposure of DO11.10 1/1 mice to asingle OVA aerosol.

Previously, it has been shown that repeated OVA exposurevia the pulmonary route in the absence of systemic primingcauses tolerance [5] or is an immunologically null event inrodents [16]. In DO11.10 1/2 mice exposed to three OVAaerosols, however, we observed evidence of OVA-specific Tcell activation and/or recruitment of memory cells to the lungsinstead. In addition, we showed that after three OVA aerosolexposures, T cells isolated from the lungs of DO11.10 1/2mice retain the ability to secrete IL-4, IL-13, IL-5, and IFN-gin response to OVA protein or specific peptide (323–339)stimulation in vitro. The failure of OVA aerosol exposure toinduce tolerance in our studies is consistent with the observa-tion that DO11.10 mice appear somewhat resistant to theinduction of tolerance when OVA is given via normally tolero-genic routes (i.e., i.v. or i.p. in the absence of adjuvant) [9].Conversely, Lee et al. [40] demonstrated recently that lungcells from DO11.10 mice, which had received repeated OVA-aerosol exposures, failed to proliferate or secrete IL-2 in vitroin response to restimulation with OVA, specific peptide, oranti-CD3 [40]. The suppression of these activities was not aresult of intrinsic anergy or tolerance, however, because it was

alleviated by removal of F4/801 macrophages from the lung-cell population before culture. It is interesting that repeatedaerosol exposures did not suppress IFN-g, IL-4, or IL-5 pro-duction by OVA peptide-stimulated DO11.10 lung cells intheir hands. These data suggest that although proliferation isinhibited in the lung by repeated OVA aerosols, OVA-TCR1cells can retain effector function. This inhibition of prolifera-tion in DO11.10 lungs may also keep pulmonary inflammationin check, as reflected by only small increases in total lung cellnumbers in their study and our own.

In summary, our data show that small numbers of activated ormemory T cells are present in the lung after limited antigenexposure by the aerosol route. Once in the lung, these T cells ortheir products induce AHR in the absence of significant pulmo-nary eosinophilia or antigen-specific immunoglobulin. These dataare in agreement with recent studies showing that T cells or theirproducts can mediate AHR [36, 37, 41]. The observation that verylimited exposures to antigen can initiate a pulmonary immuneresponse driven by antigen-specific T cells suggests AHR may bedetected before the development of clinical symptoms of allergicasthma (episodes of reversible airway obstruction, pulmonary eo-sinophilia, and high levels of IgE). Preliminary observations in-dicate that upon more chronic OVA-aerosol exposure (6 h/day, 5days/week for up to 6 weeks), DO11.10 1/2 mice do developpulmonary eosinophilia, mucous cell hyperplasia, and high-serumOVA-specific IgG1 and IgE (unpublished results, J. A. W. andD. E. B.). These data indicate that the development of AHR maybe an important, early predictor of asthma development in genet-ically predisposed patients who are chronically exposed to aller-gens.

ACKNOWLEDGMENTS

This work was supported by the Specialized Centers of Re-search grant 5 P50 HL56384 from the National Heart, Lungand Blood Institute, the University of New Mexico ResearchAllocation Committee Cigarette Tax Interest Funds appropri-ated by the New Mexico State Legislature, the U.S. Departmentof Energy Office of Health and Environmental Research underContract No. DE-AC04-76EV01013, and by grant 5T32 8HL07733 from the National Institutes of Health. The authorsgratefully acknowledge the expert technical assistance of Bar-bara Forrister, Claudia Pertab, James White, Kenneth OlejarJr., Gwyneth Olson, Linda Izzo, Stephanie Wright, Kristi Rar-din, Laurie Allen, Susan Middleton, and Marina Martinez. Theauthors would also like to thank Chris Stidely and MarkEichinger for their assistance in the statistical analysis andMichael Grady for his assistance in preparing the figures.

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