Intrinsic AHR in IL-5 transgenic mice is dependent on CD4+ cells and CD49d-mediated signaling

Michael T. Borchers, J. Crosby, P. Justice, S. Farmer, E. Hines, J. J. Lee, and N. A. Lee

Department of Biochemistry and Molecular Biology, Mayo Clinic Scottsdale, Scottsdale, Arizona 85259


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Overexpression of interleukin (IL)-5 by the airway epithelium in mice using the rat CC10 promoter (NJ.1726 line) leads to several histopathologies characteristic of human asthma, including airway hyperreactivity (AHR). We investigated the contribution of B and T cells, as well as CD4 expression, to the development of AHR in IL-5 transgenic mice. NJ.1726 mice on a T cell or CD4 knockout background, but not on a B cell knockout background, lost intrinsic AHR. These effects occurred without decreases in IL-5 or eosinophils. We further investigated the contribution of alpha 4-integrin signaling to the development of AHR in IL-5 transgenic mice through the administration of anti-CD49d (alpha 4-integrin) antibody (PS/2). Administration of PS/2 resulted in immediate (16-h) inhibition of AHR. The inhibition of AHR was not associated with a decrease in airway eosinophils. These studies demonstrate that, despite the presence of increased levels of IL-5 and eosinophils in the lungs of NJ.1726 mice, CD4+ cells and alpha 4-integrin signaling are necessary for the intrinsic AHR that develops in IL-5 transgenic mice.

cytokines; inflammation; eosinophil; T cell; interleukin-5


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ASTHMA IS A CHRONIC INFLAMMATORY disease of the airways characterized by airway hyperreactivity (AHR) associated with infiltrating lymphocytes and eosinophils. The current paradigm of allergic inflammation suggests that CD4+ Th2 lymphocytes mediate airway pathology through the production of cytokines and chemokines that exert effects on structural cells of the lung and induce the recruitment of proinflammatory leukocytes such as eosinophils. In turn, eosinophils, through the release of additional mediators, alter the responsiveness of the airways to constrictor agonists (35). The cytokines and chemokines mediating the recruitment of eosinophils have been well studied; however, the mechanisms by which these effector cells become activated and contribute to the development of AHR are not fully understood. Several eosinophil-dependent mechanisms that contribute and/or cause pulmonary pathology and airway dysfunction have been suggested, including the release of lipid mediators, granule proteins, and reactive oxygen species (9). Eosinophil activation has been demonstrated in vitro by a number of mechanisms such as alpha 4-integrin (CD49d)-dependent binding to extracellular matrix fibronectin (29, 26), eosinophil adhesion to CD4+ T cells (5), response to CD3-stimulated Th2 cell supernatants (28), and eotaxin binding to CCR3 chemokine receptors (19). These data demonstrate that multiple and complex interactions between the eosinophil and the pulmonary microenvironment may be required for cell activation and the development of AHR.

Murine models of allergic airway inflammation have shown that pulmonary expression of Th2 cytokines [i.e., interleukin (IL)-4 (4, 8), IL-13 (10, 37), and IL-5 (6)] has pleiotropic consequences on lung structure and/or function, leading to the recruitment of eosinophils and/or lymphocytes and the development of AHR. In addition, IL-5 alone has direct effects on eosinophil maturation, survival, and activation (39, 24). Unfortunately, studies disrupting the levels or signaling abilities of these Th2 cytokines affect multiple facets of allergic inflammation. Thus the requirements of individual cells or signaling molecules for the development of AHR have been difficult to assess.

Earlier, we reported the generation and characterization of transgenic mice expressing IL-5 from the airway epithelium (24). These mice (NJ.1726 line) exhibit intrinsic AHR (i.e., no antigenic stimuli) accompanied by increased numbers of eosinophils and lymphocytes in the lung tissue. In this study, we used NJ.1726 mice to further resolve the mechanisms involved in IL-5-mediated AHR. Specifically, we investigated the dependence of AHR on lymphocyte populations (i.e., T cells, CD4+ cells, and B lymphocytes) and alpha 4-integrin signaling among resident pulmonary leukocytes. The elevated IL-5 levels and eosinophil accumulation in the lung of NJ.1726 mice were unaffected in the absence of T cells, CD4+ cells, or B lymphocytes. However, depletion of T cells or CD4+ cells, but not of B lymphocytes, abolished the AHR in IL-5 transgenic mice. The data presented demonstrate that even in the absence of antigenic stimuli, CD4+ cells are necessary, whereas eosinophils and/or IL-5 alone are not sufficient for the development of AHR. Moreover, these effects occurred independent of IL-4, IL-13, and eotaxin expression. We further investigated the importance of integrin-mediated activation of resident pulmonary leukocytes in the IL-5 transgenic mice by inhibiting the alpha 4-heterodimeric integrins with a neutralizing antibody (PS/2). These studies demonstrate that integrin signaling by resident airway leukocytes is required and represents an important mechanism for leukocyte activation and the persistence of AHR.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mice. Transgenic mice (NJ.1726) constitutively expressing murine IL-5 from the lung epithelium were generated as previously described (24) and were maintained by continual backcross to C57BL/6J mice. NJ.1726 mice were bred to C57BL/6J-Tcrbtm1Mom Tcrdtm1Mom, C57BL/6J-Cd4tm1Knw, and C57BL/6J-lgh-6tm1Cgn mice (Jackson Laboratories, Bar Harbor, ME), generating IL-5 transgenic mice deficient of T, CD4+, and B cells, respectively. The genotype/phenotype of offspring from these crosses was confirmed by a two-step process separately assessing the presence of the IL-5 transgenes and the absence of the specific lymphocyte subpopulations under question. The presence of the IL-5 transgenes was determined by PCR of tail DNA as previously described (24). Furthermore, the presence of these transgenes was confirmed phenotypically by the demonstration of a peripheral blood eosinophilia in naive IL-5 transgenic animals. The presence and/or absence of specific lymphocyte subpopulations in these mice was determined by flow cytometry of peripheral blood samples using fluorochrome-conjugated antibodies against CD3, CD4, and B220 (see below). Control C57BL/6J mice were also obtained from Jackson Laboratories. All procedures were conducted on mice 8-12 wk of age that were maintained in microisolator cages housed in a specific pathogen-free animal facility. The sentinel cages within this animal colony were negative for viral antibodies and the presence of known mouse pathogens. Protocols and studies involving animals were conducted in accordance with National Institutes of Health and Mayo Clinic Foundation guidelines.

Lung cell isolation and flow cytometry. Leukocytes within the lung parenchyma were assessed by collagenase digestion of perfused lungs. Isolation of lung cells was performed as previously described (14). Briefly, perfused lungs were removed and diced into pieces <300 µl in volume. Four milliliters of Hanks' balanced salt solution (HBSS; GIBCO BRL, Gaithersburg, MD) containing 175 U/ml collagenase (Sigma, St. Louis, MO), 10% FCS (GIBCO BRL), 100 U/ml penicillin, and 100 µg/ml streptomycin were added to the tissue and incubated for 60 min at 37°C in an orbital shaker. The digested lungs were sheared with a 20-gauge needle and filtered through 45- and 20-µm filters. Cells were washed three times and resuspended in HBSS before being counted with a hemocytometer. Lymphocyte populations in the lung were subsequently identified and are expressed as the product of the total cell count and the percentage of total cells analyzed (1 × 105) by flow cytometery. CD3+, CD4+, CD3+/CD4+ double-positive, and B cells were identified and quantified by staining with the following conjugated antibodies: phycoerythrin-anti-mouse CD3 (Caltag, Burlingame, CA), FITC-anti-mouse CD4 (PharMingen, San Diego, CA), and phycoerythrin-anti-mouse B220 (PharMingen). Flow cytometry was performed on a FACScan cytofluorometer (Becton Dickinson, Franklin Lakes, NJ). Data acquisition and analysis were performed using CellQuest software (Becton Dickinson).

Ovalbumin sensitization and challenge. Wild-type mice were sensitized with an intraperitoneal injection (100 µl) of 20 µg of chicken ovalbumin (Ova; Sigma) emulsified in 2 mg of Imject Alum [Al(OH)3/Mg(OH)2; Pierce, Rockfield, IL] on days 0 and 14. Mice were subsequently challenged with an aerosol generated from 1% Ova or saline alone for 20 min by ultrasonic nebulization (DeVilbiss, Somerset, PA) on days 24, 25, and 26. Assessments of bronchoalveolar lavage (BAL) fluid IL-5 levels, tissue eosinophils, and AHR were made on day 28.

Measurements of AHR. AHR was determined by inducing airflow obstruction with a methacholine aerosol. Total pulmonary airflow in unrestrained conscious mice was estimated using a whole body plethysmograph (Buxco Electronics, Troy, NY). Pressure differences between a chamber containing the mice and a reference chamber were used to extrapolate minute volume, tidal volume, breathing frequency, and enhanced pause (Penh). Penh is a dimensionless parameter that is a function of total pulmonary airflow in mice during each respiratory cycle. This parameter closely correlates with airway resistance as measured by traditional invasive techniques using ventilated mice (15).

BAL, eosinophil counts, and cytokine level determinations. Lungs were lavaged three times with 0.5 ml of HBSS containing 2% FCS. Individual BAL returns were pooled and centrifuged, generating a BAL cell pellet and a cell-free supernatant. Total cell counts were determined with a hemocytometer, and eosinophil counts were performed on Wright-stained cytospin slides (Cytospin 3; Shandon Scientific, Pittsburgh, PA) by counting >= 300 cells. Cell-free lavage fluid was frozen on dry ice and stored at -80°C until used. Murine IL-4, IL-5, IL-13, and eotaxin-1 levels were measured using ELISA kits as described by the manufacturer (PharMingen, San Diego, CA).

Immunohistochemical detection of lung eosinophils. Immunohistochemistry was performed using a rabbit polyclonal antibody against murine major basic protein (MBP). MBP antigen-antibody complexes were detected in 4-µm sections of formalin-fixed, paraffin-embedded sections of mouse lungs (n = 5/group) with the VECTASTAIN ABC Peroxidase Elite goat IgG kit (Vector Laboratories, Burlingame, CA). Endogenous peroxidase was blocked with 3% hydrogen peroxide in methanol for 20 min at 25°C. Sections were trypsin digested (0.1% trypsin in 0.02 M Tris · HCl, pH 7.8) for 30 min at 25°C and washed in PBS (GIBCO BRL) for 15 min. Sections were blocked with PBS containing 1.0% normal goat serum and 1% BSA for 30 min at 25°C before incubation with primary antibody (1:2,000 dilution) for 60 min at 25°C. Antibody-bound slides were washed in PBS-1.0% BSA and incubated (30 min, 25°C) with biotinylated goat anti-rabbit antibody (1:500 dilution). Sections were incubated first with avidin-biotin-peroxidase complex (30 min, 25°C) and then diaminobenzidine (3 min, 25°C) for the development of a colored reaction product before being counterstained with methyl green. Tissue eosinophils were quantitated by counting the total number of MBP-positive cells in six randomly selected high-power fields and are expressed as eosinophils per square millimeter of lung tissue (n = 4-5 mice/group).

PS/2 administration in IL-5 transgenic mice. NJ.1726 and C57BL/6J mice received rat anti-mouse CD49d monoclonal antibody (PS/2; ATCC, Manassas, VA) administered via the tail vein (200 µg in 50 µl of saline). Genotype-matched control groups received isotype-matched rat IgG (Sigma). AHR and BAL eosinophil numbers were determined 12-16 h after the administration of antibodies.

Statistical analysis. Data are presented as means ± SE. Statistical analysis was performed on parametric data using t-tests, with differences between means considered significant at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Transgenic expression of IL-5 in the airways leads to the expansion of T cell, CD4+ cell, and B lymphocyte populations in the lung. In addition to the increase in tissue and airway eosinophils observed in naive NJ.1726 mice (24), increases in lung T cells, CD4+ cells, and B lymphocytes (compared with littermate controls) also occurred (Table 1). IL-5 transgenic mice demonstrate a 15-fold increase in CD3+ cells (i.e., T lymphocytes), the majority of which are also CD4+. More strikingly, NJ.1726 mice exhibit a 58-fold increase in the number of B cells in the lung compared with littermate controls.

                              
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Table 1.   CC10 IL-5 transgenic mice exhibit increased CD4+ cells and B cells

Intrinsic AHR is abolished in T cell- and CD4-deficient mice. As previously reported (24), NJ.1726 mice exhibited increased airway reactivity to methacholine provocation (compared with nontransgenic littermate controls) in the absence of antigen sensitization and challenge. Compound IL-5 transgenic/gene knockout mice deficient of either T or CD4+ cells failed to display the increased reactivity to methacholine observed in NJ.1726 mice (Fig. 1). In contrast, IL-5 transgenic mice on a B lymphocyte-deficient background still displayed the intrinsic increased reactivity to methacholine associated with naive NJ.1726 mice (Fig. 1). Baseline Penh values were not different between any of the groups tested [C57BL/6J = 0.43 ± 0.05 , NJ.1726 = 0.49 ± 0.08, NJ.1726/T cell(-/-) = 0.50 ± 0.09, NJ.1726/CD4(-/-) = 0.57 ± 0.10, and NJ.1726/B cell(-/-) = 0.57 ± 0.15].


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Fig. 1.   The intrinsic airway hyperreactivity (AHR) displayed by naive NJ.1726 mice is dependent on T and/or CD4+ cells. Airway reactivity [enhanced pause (Penh)] of each experimental group (n = 10-12 mice/group) is plotted as a function of increasing doses of inhaled methacholine. Values presented are means ± SE.

Depletion of T cells, CD4+ cells, or B lymphocytes does not affect BAL fluid IL-5 levels in NJ.1726 mice. IL-5 levels (ng/ml) in the BAL fluid of transgenic mice were significantly elevated compared with those in wild-type mice sensitized and challenged with Ova (258 ± 61 vs. 0.21 ± 0.06, respectively; Fig. 2). These BAL fluid IL-5 levels were not altered in the absence of T cells, CD4+ cells, or B lymphocytes (299 ± 42, 265 ± 27, and 333 ± 47 ng/ml, respectively). BAL fluid IL-5 levels were at or below the limit of detection (<5 pg/ml) in wild-type mice challenged with saline alone. BAL fluid levels of IL-4, IL-13, and eotaxin were detectable in wild-type Ova-challenged mice (53 ± 30, 75 ± 39, and 28 ± 23 pg/ml, respectively) but were undetectable in wild-type saline-challenged mice and naive IL-5 transgenic mice, including all crosses on knockout backgrounds.


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Fig. 2.   Bronchoalveolar lavage (BAL) fluid interleukin (IL)-5 levels remain unaltered in naive compound NJ.1726/lymphocyte-deficient mice. Values presented are means ± SE; n = 5-6 mice/group. ND, not detected. *Significantly different (P < 0.05) from wild-type saline (WT SAL). **Significantly different (P < 0.05) from wild-type ovalbumin-treated (WT Ova) mice.

Pulmonary eosinophilia in naive NJ.1726 mice are unaffected in the absence of lymphocyte subtypes. Ova sensitization and challenge of wild-type mice led to the differential recruitment and accumulation of eosinophils in the perivascular/peribronchial compartments of the lung (Fig. 3, A and B). A similar, but significantly higher, pulmonary eosinophilia was observed in NJ.1726 mice without antigen provocation. These levels of tissue eosinophils were not significantly affected by the depletion of T cells, CD4+ cells, or B lymphocytes (Figs. 3, C-F, and 4).


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Fig. 3.   Localization of eosinophils within specific compartments of naive NJ.1726 lungs is unaffected in the absence of either T or B lymphocytes. Eosinophils were identified in the peribronchial regions of the lung by immunohistochemistry using a rabbit anti-mouse eosinophil major basic protein polyclonal antisera. Photomicrographs are representative of 5 mice/group. A: WT SAL; B: WT Ova; C: NJ.1726; D: NJ.1726/T cell(-/-); E: NJ.1726/B cell(-/-); and F: NJ.1726/CD4(-/-). Scale bar = 50 µm.



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Fig. 4.   Pulmonary eosinophilia associated with naive NJ.1726 mice occurs independent of either T or B lymphocytes. Eosinophils within the lung parenchyma were quantified as a function of tissue area. Values are means ± SE derived from multiple sections/mouse; n = 5-6 mice/group. *Significantly different (P < 0.05) from wild-type saline. **Significantly different (P < 0.05) from wild-type Ova.

Anti-CD49d antibody abolishes AHR in naive NJ.1726 mice. Previous studies with the monoclonal antibody PS/2 showed that this neutralizing reagent completely inhibited AHR in Ova-sensitized and challenged mice (2, 17). A single administration of PS/2 also completely abrogated AHR in NJ.1726 mice within 16 h (Fig. 5) without a concomitant decrease in airway eosinophils (Fig. 6). Administration of a control rat IgG had no effect on AHR or airway eosinophil accumulation in NJ.1726 or C57BL/6J mice.


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Fig. 5.   Administration of anti-CD49d antibody (PS/2) inhibits intrinsic AHR in naive NJ.1726 mice. Airway reactivity (%increase in baseline Penh) to increasing doses of inhaled methacholine was determined 16 h after a single intravenous (iv) injection of PS/2 or control (con) rat IgG in naive NJ.1726 and C57BL/6J mice. Values presented are means ± SE; n = 10-12 mice/group.



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Fig. 6.   Accumulation of airway eosinophils in naive NJ.1726 mice is attenuated as a consequence of repeated administration of PS/2. Total recovered eosinophils were enumerated from BAL performed 16 h after a single iv injection of PS/2 or control rat IgG in naive NJ.1726 and C57BL/6J mice. Values presented are means ± SE; n = 8 mice/group.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IL-5 is a pluripotent cytokine known to exert effects on resident and infiltrating cells of the lung, including increased proliferation of B cells (32), activation and increased survival of eosinophils (3), and increased reactivity of airway smooth muscle (11, 12). Furthermore, the importance of IL-5 as a causative agent leading to allergic pulmonary inflammation and AHR has been demonstrated in animal models through the use of recombinant protein/neutralizing antibodies (34) and the generation of IL-5 knockout (6) and IL-5 transgenic mice (20). These experimental strategies have each demonstrated a link between IL-5 expression and the development of AHR; however, the demonstration of a causative relationship has remained elusive. Specifically, it is not understood whether IL-5 induces AHR through direct effects on airway smooth muscle or indirectly via eosinophil recruitment and execution of effector functions or if the role of IL-5 is much more complex, involving a multitude of inflammatory mediators and cellular activators.

The dependence of IL-5-induced AHR in naive NJ.1726 mice on T cells or CD4+ cells, but not on B lymphocytes, parallels the cell-type requirements of antigen-induced AHR in wild-type mice (4, 7, 16). The discourse between the intrinsic AHR of naive NJ.1726 mice and the presence or absence of B lymphocytes was not unexpected given that AHR in this model occurs without antigen sensitization and challenge. This observation, along with previous studies of Ova-sensitized and challenged mice demonstrating that AHR occurs independent of B cells (16, 22, 25), suggests that, although IL-5 has significant agonist activities on B lymphocytes, these activities are not required for the development of AHR.

The necessity of T cells and/or CD4+ cells for the development of the intrinsic AHR in NJ.1726 mice in the absence of antigen provocation was surprising since we have presumably bypassed the requirement of CD4+ T cell production of IL-5 through the ectopic expression of this cytokine in the airway epithelium. Previous studies in mice using CD4+ cell-depleting antibodies demonstrated an abrogation of both AHR (7) and pulmonary eosinophilia (29). Moreover, expression of the Th2 cytokines IL-4 (4) and IL-13 (10, 36) and the chemokine eotaxin (21) has been shown to be important for AHR and eosinophil accumulation. The implicit conclusion was that AHR is a pathophysiological consequence of eosinophil effector function and that Th2 cytokine/chemokine expression, including the release of IL-5, by CD4+ cells was required for the production, recruitment, and/or activation of pulmonary eosinophils. However, the development of AHR in naive NJ.1726 mice occurs without concurrent increases in pulmonary expression of IL-4, IL-13, or eotaxin-1. In addition, the inhibition of AHR in compound IL-5 transgenic/T (or CD4+) cell-deficient mice is not associated with changes in IL-5 levels, pulmonary eosinophil numbers, or their location within the parenchyma. These observations suggest that an additional, as yet unknown function of pulmonary T and/or CD4+ cells is required for the development of AHR. The potential release by T and/or CD4+ cells of a previously unidentified factor(s) is a likely mechanism, although evidence suggests that cell-cell interactions may also lead to AHR. For example, activated T cells adhere to airway smooth muscle via integrins and other cell adhesion molecules (CAMs), inducing smooth muscle DNA synthesis (23). Furthermore, CD4+ T cells use CAMs to bind eosinophils, inducing activation of these cells as determined by leukotriene production (5). Regardless of the specific mechanism utilized by T and/or CD4+ cells, the dependence of the intrinsic AHR associated with naive NJ.1726 mice suggests that potential direct IL-5 agonist activities on airway smooth muscle alone are not sufficient to elicit AHR.

Integrins and CAMs have been shown to be critical components of pathways leading to the extravasation and subsequent recruitment of leukocytes to the lung as a consequence of allergic airway inflammation (31, 38). These studies have suggested that pulmonary pathology, including the development of AHR, is a result of effector activities executed by these recruited leukocytes. However, the ability of anti-CD49d antibodies to inhibit AHR in antigen-sensitized and challenged mice, independent of leukocyte recruitment to the lung (1), implicates a larger role for these receptor-ligand interactions. Furthermore, the ability of anti-CD49d antibodies to inhibit the intrinsic AHR of naive NJ.1726 mice without affecting airway eosinophil numbers provides compelling evidence that cell activation is also a critical determinant of AHR in these mice. The mechanism linking alpha 4-integrins and the abolishment of AHR is not fully understood but likely involves the ability of these integrins to mediate cell-cell interactions and cell-matrix interactions required for the activation of eosinophils and T lymphocytes in the airways. This hypothesis is supported by several independent studies demonstrating that 1) administration of anti-CD49d antibody to allergic mice inhibits Th2 cytokine production in the airways (17), 2) eosinophils (20) and T lymphocytes (23) bind to airway smooth muscle via alpha 4-integrin/vascular cell adhesion molecule (VCAM)-1, 3) alpha 4-integrin-dependent binding of primed eosinophils to fibronectin results in sufficient release of cysteinyl leukotrienes to induce airway narrowing of bronchial explants (27), and finally 4) eosinophil activation, as opposed to absolute number, is the probable determinant of AHR (1, 30).

Studies investigating the potential relationship(s) of IL-5, T cells, and eosinophils and the development of AHR have often been equivocal. For example, increased responsiveness of antigen-sensitized airway smooth muscle from atopic asthmatics has been shown to be dependent on IL-5 (11, 12). However, treatment of Ova-sensitized and challenged guinea pigs with low levels of anti-(IL-5) antibodies inhibited eosinophil infiltration in the airways without an effect on the development of AHR (26), and a report has been published showing that AHR occurs in IL-5 knockout mice in a strain-dependent fashion (18). The identical levels of IL-5 and pulmonary eosinophils in naive NJ.1726 or compound transgenic/T (or CD4+) cell-deficient mice suggest that increases in IL-5 alone or increased IL-5 and eosinophil accumulation in the airways are not sufficient for the development of AHR in the mouse. Similarly, although many antigen provocation studies suggest a link between pulmonary eosinophilia and AHR (6, 13, 33), other studies show that AHR can occur independent of these leukocytes (4, 18). In addition, because the experimentally manipulated parameter in all of these studies was IL-5 and not eosinophils, the specific conclusions regarding the role(s) of these leukocytes in antigen-mediated AHR are limited to extrapolation. The determination of the potential role(s) of eosinophils, if any, awaits the generation of a model in which eosinophils are eliminated without concomitant effects on recruited T cell populations and Th2 cytokine production.

The demonstration that T (and/or CD4+) cells are required for the intrinsic AHR of naive NJ.1726 mice, even in the presence of a significant pulmonary eosinophilia, suggests that T (or CD4+) cells are uniquely necessary, and perhaps sufficient, for the development of AHR. However, these data do not address potential mechanisms involving a corequirement of eosinophils or T cell-mediated activation of eosinophils leading to execution of effector functions. The preponderance of studies in apparent conflict regarding the role(s) of T cells vs. eosinophils argue that the development of AHR is likely to be a consequence of complex interactions and corequirements for both cell types. The presence of alpha 4-integrins and VCAM-1 on T cells/eosinophils and smooth muscle, respectively, and the dependence of AHR on these receptor-ligand interactions further suggests that this pathophysiological response is also a consequence of multiple concurrent signaling pathways that may also include airway smooth muscle.


    ACKNOWLEDGEMENTS

We thank Anita Jennings and Marv Ruona of the Mayo Clinic Scottsdale Histology Facility for technical assistance and graphic arts support. We also thank Linda Mardel for research assistance.


    FOOTNOTES

This study was supported by funds from the Mayo Clinic Foundation, by National Heart, Lung, and Blood Institute (NHLBI) individual investigator award (N. A. Lee), and by NHLBI Grants HL-60793-01S, HL-07897, HL-10361 (M. T. Borchers), and HL-10176 (J. Crosby).

Address for reprint requests and other correspondence: N. A. Lee, Dept. of Biochemistry and Molecular Biology, SCJMRB-Research, Mayo Clinic Scottsdale, 13400 E. Shea Blvd., Scottsdale, AZ 85259 (E-mail: nlee{at}mayo.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 8 February 2001; accepted in final form 25 April 2001.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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