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Am. J. Respir. Cell Mol. Biol., Volume 20, Number 5, May 1999 992-1000

alpha 4 Integrin-Dependent Eotaxin Induction of Bronchial Hyperresponsiveness and Eosinophil Migration in Interleukin-5 Transgenic Mice

Takeshi Hisada, Paul G. Hellewell, Mauro M. Teixeira, Monika G. K. Malm, Michael Salmon, Tung-Jung Huang, and K. Fan Chung

National Heart and Lung Institute at Imperial College School of Medicine, London, United Kingdom


    Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

We investigated the roles of eosinophil infiltration and activation induced by the eosinophil-selective chemokine eotaxin, and of the expression of eosinophil alpha 4 and beta 2 integrins in causing bronchial hyperresponsiveness (BHR) in interleukin (IL)-5 CBA/Ca transgenic mice. These mice did not show BHR, despite the presence of some eosinophils in the lungs. Intratracheal mouse recombinant eotaxin (3 µg) did not induce BHR in wild-type mice. In IL-5 transgenic mice, eotaxin (3 and 5 µg) increased responsiveness at 24 h and increased eosinophils in bronchoalveolar lavage (BAL) fluid by 9.4- and 14-fold by 24 h, respectively, together with augmentation of eosinophil peroxidase activity and eosinophil infiltration in the airway submucosa. Using flow cytometry, the expression of alpha 4, CD11b, and CD18 was upregulated in BAL, but not in blood, eosinophils. A rat anti-alpha 4 antibody inhibited eotaxin-induced BHR and eosinophil migration and activation, but an anti-CD11b antibody had no significant effects on BHR. A combination of both antibodies was more effective. IL-5 and eotaxin synergize in the induction of BHR and airway eosinophilia, effects that are dependent on the induction of eosinophil alpha 4 integrin. Expression of BHR depends on the recruitment and activation of eosinophils.


    Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Bronchial hyperresponsiveness (BHR) to a variety of physical and pharmacologic stimuli is a characteristic feature of asthmatic airways. Its pathogenesis remains unclear but may be related to the chronic airway inflammatory process consisting of an infiltrate of eosinophils, T lymphocytes, and other inflammatory cells in the airway submucosal wall (1). Studies in animal models and in asthmatic human patients have indicated that eosinophils can release mediators, cytokines, and cationic proteins that may cause bronchoconstriction, damage to the airway epithelium, and ultimately BHR (4). Eosinophil migration into the lungs and airways from the circulation requires the interaction of surface-adhesion receptors on the circulating eosinophils such as the integrin heterodimers very late antigen (VLA)-4, consisting of alpha 4 and beta 1 integrin subunits, with vascular cell adhesion molecule-1 expressed on the vascular endothelium and other ligands in the extravascular tissue (7, 8) to induce eosinophil adherence to endothelium and transendothelial migration. Various eosinophil chemoattractants such as interleukin (IL)-5 and eotaxin selectively regulate eosinophil trafficking and may be involved in modulating eosinophilic inflammation (9). IL-5 regulates the growth, differentiation, and activation of eosinophils (12). IL-5 is also a pivotal cytokine in the induction of airway eosinophilia in response to allergen in sensitized mice, monkeys, and guinea pigs (18, 19). Eotaxin, a C-C chemokine, discovered as a selective chemoattractant for eosinophils in bronchoalveolar lavage fluid (BALF) obtained from an experimental model of allergen exposure of sensitized guinea pigs (9), induces selective pulmonary and intradermal eosinophil recruitment, indicating a role for eotaxin in eosinophil homing and tissue recruitment (9, 20). Both IL-5 and eotaxin have been shown to be upregulated in the airways of patients with allergic asthma (21, 22).

To study the direct contribution of eosinophil recruitment and activation into the airways to the pathogenesis of BHR, we developed a model of pure eosinophil migration into the lungs. Previous investigators have predominantly examined the more complex sensitized and allergen-exposed models in animals and sensitized humans with asthma to address this issue, with conflicting results. We used the fact that there are cooperative effects between eotaxin and IL-5 on eosinophil mobilization (11, 23, 24), with IL-5 mobilizing eosinophils from the bone marrow and locally released eotaxin in the tissues inducing homing and migration of eosinophils in the tissues. No information is available regarding the effects of this cooperation on the pathogenesis of BHR. We studied the development of BHR after instillation of eotaxin into the airways of IL-5 transgenic mice, and measured eosinophil infiltration and activation. We determined the role of the integrins alpha 4 and CD11b (Mac1) on eosinophil mobilization and BHR. Our data demonstrate a crucial role for eosinophil recruitment and activation dependent on alpha 4 expression for the establishment of BHR.

    Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Mice

CBA/Ca mice, 8 to 10 wk old, were purchased from Harlan-Olac (Bicester, UK). Age-matched CBA/Ca mice overexpressing the murine IL-5 gene (Tg1 mice) (25) were obtained from Glaxo Wellcome (Stevenage, UK). They were bred and housed in air-conditioned animal facilities at 20 ± 2°C and relative humidity of 55 ± 10%.

Special Materials

The anti-L-selectin monoclonal antibody (mAb)-producing cell line, MEL-14 (rat immunoglobulin [Ig]G2b), was purchased from ATCC (Rockville, MD), and grown in a hollow-fiber bioreactor, and the mAb was purified by ammonium sulfate precipitation. Rat IgG2b mAb antimouse alpha 4, PS/2, and rat IgG2b mAb antimouse CD11b, 5C6, were gifts of Dr. M. Robinson (Celltech, Slough, UK). Hamster IgG mAb antimouse CD18, 2E6, and purified rat IgG2b (clone 2-4A1) were gifts of Dr. B. Wolitsky (Hoffman La Roche, Nutley, NJ). Purified hamster IgG and fluorescein isothiocyanate (FITC)-conjugated goat antihamster IgG were purchased from Serotec (Oxford, UK), and FITC-conjugated goat antirat IgG and murine recombinant eotaxin were from Sigma (St. Louis, MO) and R&D Systems Ltd. (Oxford, UK), respectively. The eotaxin used contained < 0.1 ng of endotoxin per milligram.

Protocol

Mice were anesthetized with an intraperitoneal (i.p.) injection of 150 to 200 µl of anesthetic solution, containing 0.5 ml midazolam (Roche Products Ltd., Welwyn Garden City, UK) and 0.5 ml Hypnorm (0.315 mg/ml of fentanyl citrate and 10 mg/ml of fluanisone; Janssen Pharmaceuticals Ltd., Wantage, UK) in 2 ml of distilled water. Mice were intubated with an angled 18-gauge blunt needle, through which recombinant murine eotaxin (1, 3, or 5 µg dissolved in 10 µl vehicle) or 10 µl control vehicle (10 mM phosphate-buffered saline [PBS]/0.1% bovine serum albumin [BSA], pH 7.4) was instilled. For time-course experiments, measurement of lung function, BAL, and blood collection were performed at 4, 8, and 24 h after eotaxin (5 µg) was instilled into the airways of IL-5 transgenic mice.

Antibody Treatment

Anti-integrin antibodies and their respective controls were given by i.p. administration at 12 and 1 h before and at 6 h after the intratracheal instillation of 5 µl eotaxin. The dosing regimens are based on the reports by Nakajima and colleagues (26) and Chin and associates (27). IL-5 transgenic mice received 100 µg of alpha 4 mAb or control rat IgG2b and 500 µg of CD11b mAb or control rat IgG2b intraperitoneally at each time point. Combinations of both antibodies and of control IgG2b were also studied.

Airway Responsiveness to Acetylcholine

Following anesthesia, a tracheal cannula (18-gauge) was inserted into the lumen of the cervical trachea through a tracheostomy, and was connected to a mouse ventilator (Model 687; Harvard Apparatus, Kent, UK; 120 breaths/ min) with an in-line pressure transducer. Animals were ventilated at 120 breaths/min with tidal volume of 0.2 ml. Intravenous access was established via the tail vein with a 27-gauge needle. A paralytic agent (suxamethonium, 0.5 mg/kg; Antigen Pharmaceuticals Ltd., Roscrea, Ireland) was administered to eliminate spontaneous respirations. After recording a stable baseline airway pressure, acetylcholine chloride (ACh) (0.1, 0.32, 1.0, 3.3, or 10.8 µg/g; Sigma) was injected over 1 s and airway pressure was recorded for 5 min on a polygraph (Multitrace 2 Recorder 5022; Lectromed Ltd., Jersey Channel Islands, UK). Increasing half-log concentrations were administered at 5-min intervals, with one hyperinflation of the tidal volume applied 3 min after ACh administration. Final airway pressure changes were recorded as the maximum percentage of change in airway pressure from baseline. The provocative concentration of ACh in micrograms per gram that causes a 50% increase in airway pressure (logPC50ACh) was calculated.

BAL and Cell Counting

Mice were given a lethal dose of pentobarbital (60 mg/kg intraperitoneally, Sagatal; May & Baker Ltd., Dagenham, UK), and the lungs were lavaged six times with 500-µl aliquots of PBS solution through the tracheostomy. The lavage fluid was centrifuged (300 × g for 10 min at 4°C), and the supernatant was stored at -20°C before analysis of eosinophil peroxidase (EPO) activity. The cell pellet was resuspended in 1 ml PBS with 0.1% BSA and 0.01% sodium azide. Total cell counts were performed by adding 10 µl of the cell suspension to 90 µl of Kimura stain, and by using a Neubauer chamber (American Optical Corp., Southbridge, MA). Differential cell counts were made on cytospin preparations, prepared by centrifuging at 300 rpm for 6 min and staining with May-Grunwald stain. Cells were identified as macrophages, neutrophils, eosinophils, lymphocytes, and epithelial cells according to standard morphology. Five hundred cells were counted under ×400 magnification. The rest of the cell suspension was used for flow cytometry.

Quantitation of Blood Eosinophils

Blood was collected into heparinized syringes by cardiac puncture after the injection of a lethal dose of pentobarbital. Total leukocyte counts were performed using Kimura stain, and differential cell counts were made on blood smear following staining with May-Grunwald-Giemsa. Under these conditions, eosinophils were distinguished from neutrophils and mononuclear cells.

BALF EPO Activity

The EPO activity in the supernatant of the cell-free BALF was measured according to the method of Strath and coworkers (28), based on the oxidation of o-phenylenediamine (OPD) by EPO in the presence of hydrogen peroxide (H2O2). The substrate solution consisted of 10 mM OPD (Sigma) and 0.1% Triton X 100 (BDH, Poole, UK) in 0.05 M Tris-buffer (pH 8) and 4 mM H2O2 (Sigma). Substrate solution (100 µl) was added to BAL cell supernatant samples (50 µl) in a 96-well microplate and incubated at room temperature for 30 min before stopping the reaction with 50 µl of 4 M sulfuric acid. Absorbance was then measured at 492 nm using a spectrophotometer. Duplicate incubations were carried out in the absence and presence of the EPO inhibitor 3-amino-1,2,4-triazole (AMT) (2 mmol/liter; Sigma). Blanks were cell-free BALF samples (50 µl) incubated with Tris-HCl buffer. Serial dilutions of horseradish peroxidase (400 ng/ml; Sigma) were used to quantitate the amount of peroxidase in the samples. Results are expressed as nanograms per milliliter of peroxidase activity and were corrected for the activity of other peroxidases, which were not inhibitable by AMT.

Flow Cytometric Analysis

We evaluated the level of expression of adhesion molecules of CD11b, L-selectin, alpha 4, and CD18 on circulating blood eosinophils and BAL eosinophils by fluorescence-activated cell sorter (FACS). For circulating blood cells, red blood cells were lysed with FACS lysis fluid (Becton Dickinson, San Jose, CA) and approximately 107 cells in 100 µl PBS (containing 0.1% BSA and 0.01% sodium azide) were prepared for analysis. For BAL cells, about 1 × 105 cells in 100 µl PBS were prepared for the analysis. The cells were incubated with rat IgG, hamster IgG, 5C6, MEL-14, PS/2, or 2E6 at each optimum concentration for 15 min at 4°C. The cells were then washed twice with PBS, goat antirat (for cells treated with 5C6, MEL-14, and PS/2) or goat antihamster (for cells treated with 2E6) IgG antibody conjugated with FITC was added, and the cells were incubated for 30 min at 4°C. Cells were then washed three times with PBS and then resuspended in PBS plus 2% paraformaldehyde. Fluorescences of all cell preparations were determined on a FACScan flow cytometer (Becton Dickinson, Oxford, UK) and analyzed using CELLQuest software. Granulocyte, lymphocyte, and monocyte (or macrophage) populations were delineated using forward- and side-scattering properties of the three cell populations. Mean specific fluorescence values were determined from the histograms of the gated populations, and each mean fluorescence was obtained by subtracting the measured control IgG fluorescence from the mean fluorescence for the population.

Histology

Lungs were inflated by tracheal instillation of 1 ml of optimum cutting temperature compound (OCT; Sakura, Torrance, CA) embedding medium, diluted 1:1 with PBS. The lobes were dissected and mounted over cork disks, covered with OCT compound, and snap-frozen in isopentane (BDH) cooled by liquid nitrogen. The frozen blocks were kept at -20°C before use. Sections alongside the main intrapulmonary bronchus (6 µm) were cut in a cryostat at -30°C and collected on glass slides previously coated with poly-L-lysine (Sigma), fixed in acetone (BDH) for 10 min, wrapped in aluminum foil, and kept at -20°C before staining. Hematoxylin and eosin (H&E) were used for the staining of the tissue, and cyanide-resistant EPO activity, using potassium cyanide, diaminobenzidine, and H2O2 (BDH), was used to stain the eosinophils.

Sections were coded and read in a blind fashion. Positive cells were enumerated around the bronchi (mucosal and submucosal areas) at a magnification of ×400. Counts were performed on a minimim of five randomly selected intrapulmonary bronchi and expressed per millimeter of basal lamina using computer-assisted image analysis.

Data Analysis

All values are expressed as means ± SEM. Nonparametric analysis of variance (Kruskal-Wallis method) was used to determine significance among the groups. We used the Mann-Whitney U test to analyze for significant differences between individual groups, and a value of P < 0.05 was considered significant.

    Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Effect of Eotaxin on BHR and Eosinophil Migration into the Airway

Untreated IL-5 transgenic mice showed a similar dose- dependent increase in tracheal pressure as wild-type CBA/ Ca mice (Figure 1A). Instillation of 3 µg eotaxin into wild-type CBA/Ca mice did not change airway responsiveness at 24 h. However, there was a dose-dependent increase in airway responsiveness (i.e., a reduction in logPC50ACh) with instillation of 3 and 5 µg eotaxin in IL-5 transgenic mice (Figure 1B). One microgram eotaxin was ineffective.


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  		Figure 1.   (A) Mean dose-response curves to intravenous acetylcholine. Naive IL-5 transgenic mice (solid triangles) showed responsiveness similar to that of wild-type CBA/Ca mice (solid circles). Vehicle (solid squares) or 3 µg eotaxin (solid diamonds) was instilled into CBA mice. There was no significant difference between the two groups. Three micrograms (open squares) and 5 µg (open diamonds) eotaxin-induced airway hyperresponsiveness compared significantly with the vehicle-instilled group (open circles). (B) Mean logPC50ACh. The logPC50ACh of 3 and 5 µg eotaxin-instilled mice decreased significantly (*P < 0.05, **P < 0.01 compared with vehicle-instilled mice). Results are expressed as means ± SEM. n = 5 to 7 in each group.

Recovery of BALF was ~ 90% of instilled fluid. Eotaxin caused a dose-dependent increase in BALF eosinophil counts with a mean 9.4- and 14-fold increase after 3 and 5 µg instillation, respectively, in IL-5 transgenic mice with no significant changes in other cell types (Table 1). There was no significant increase in eosinophils in wild-type mice (Figure 2A). There were significant dose-dependent increases in eosinophils around the peribronchial area and also in alveolar regions following eotaxin in IL-5 transgenic mice (Figure 2B). Only scattered eosinophils were observed predominantly within alveolar capillaries in IL-5 transgenic mice treated with vehicle, and there was no increase after eotaxin in wild-type mice. Increased dose-dependent levels of EPO were measured in BALF of eotaxin-treated IL-5 transgenic mice (Figure 2C).

                              
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TABLE 1
Total cell counts and differential cell counts in BALF


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  		Figure 2.   Effect of eotaxin on eosinophil numbers in BALF (A), eosinophil recruitment into the airways (B), and eosinophil peroxidase activity in BALF (C ). A dose-dependent increase in eosinophil counts in BALF and airways is shown for IL-5 transgenic mice (A and B), whereas no significant effect is observed in wild-type CBA/Ca mice. Similar results are shown for EPO activity (C ). Data shown are means ± SEM. *P < 0.05, **P < 0.01 compared with vehicle-instilled animals.

Time-course experiments showed that recruitment of eosinophils into BALF in response to 5 µg eotaxin in IL-5 transgenic mice was maximal at 4 h with a gradual reduction at 8 and 24 h (Figure 3A). The release of EPO was maximal at 24 h (Figure 3B), coincident with the appearance of BHR at 24 h (Figure 3C).


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  		Figure 3.   Time-course experiments showing a rapid increase of eosinophil recruitment into BALF of IL-5 transgenic mice maximal at 4 h after the instillation of 5 µg eotaxin (A). The number of eosinophils decreased gradually by 24 h, but remained significantly high. In B, the increase in EPO is shown to be maximal by 24 h. A reduction in mean logPC50ACh, indicating increased BHR, is induced at 24 h after instillation (C ). **P < 0.01 compared with naive IL-5 transgenic mice. Data are shown as means ± SEM.

Expression of Adhesion Molecules on Circulating and BAL Leukocytes

The majority of the gated granulocyte population in blood and BALF were eosinophils. On blood leukocytes, there were no differences in the expression of CD11b, L-selectin, alpha 4, and CD18 in vehicle/IL-5 mice (67.9 ± 3.2% of circulating white blood cells were eosinophils and 6.6 ± 1.0% neutrophils) compared with eotaxin/IL-5 mice (61.4 ± 4.0% eosinophils and 7.0 ± 1.0% neutrophils). However, on eosinophils in BALF of eotaxin/IL-5 mice (73.1 ± 5.7% eosinophils and 0.60 ± 0.16% neutrophils) compared with those from vehicle/IL-5 mice (6.3 ± 1.3% eosinophils and 0.63 ± 0.20% neutrophils), expression of CD11b, alpha 4, and CD18 was significantly increased from 2.5 ± 1.4 to 22 ± 1.5 (P < 0.01), 0.9 ± 0.5 to 9.3 ± 1.8 (P < 0.01), and 4.4 ± 1.7 to 13.2 ± 3.6 (P < 0.01) of mean fluorescence units, respectively. In contrast, eosinophil L-selectin, which was lower on BAL cells than on blood cells, was nonsignificantly reduced after exposure to eotaxin (1.43 ± 1.42 to 0.34 ± 0.11). A representative example of the expression of these integrins on BAL eosinophils is shown in Figure 4.


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  		Figure 4.   Representative channel fluorescence intensities for CD11b (A), L-selectin (B), alpha 4 (C ), and CD18 (D) expression in eosinophils from BALF from a mouse receiving instillation of eotaxin (5 µg) (thick continuous line) or of vehicle (thin continuous line). The dotted line shows the fluorescence for control rat or hamster IgG. There is a shift to the right of CD11b, alpha 4, and CD18 fluorescence indicating an upregulation of these integrins. However, there was no apparent effect on L-selectin.

Effect of Anti-Integrin Antibodies

In view of the upregulation of beta 2 and alpha 4 integrins on BAL eosinophils, we assessed the role of these adhesion molecules on eosinophil migration into the airway and airway hyperresponsiveness in this eotaxin-induced inflammation model. Eotaxin (5 µg) caused a significant increase in eosinophil numbers in BALF (P < 0.01) and airways (P < 0.01) and in EPO activity (P < 0.001) compared with vehicle-instilled and control rat IgG2b-injected IL-5 transgenic mice (Figure 5). Eotaxin-induced eosinophil accumulation into BALF was significantly inhibited by anti-alpha 4 mAb by 82.7 ± 5.3% and in airways by 90.3 ± 1.3%, whereas the reduction achieved by the anti-CD11b mAb (BALF: 19.6 ± 3.4%; airways: 52.3 ± 14.1% inhibition) failed to reach statistical significance. Combination of the two mAbs caused a near-complete inhibition of eosinophils in BALF (94.6 ± 1.1% inhibition) and airways (88.8 ± 5.8% inhibition) (Figures 5A and 5B). Anti-CD11b mAb, anti-alpha 4 mAb, and the combination of both antibodies significantly inhibited EPO activity by 51.2 ± 5.3%, 88.9 ± 4.5%, and 96.9 ± 1.5%, respectively (Figure 5C).


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  		Figure 5.   Effect of anti-alpha 4 and anti-CD11b mAbs on eotaxin- induced eosinophil recruitment in BALF (A) and in airway tissue (B), EPO activity in BALF (C ), and bronchial responsiveness (D) in IL-5 transgenic mice. Anti-alpha 4 mAb but not anti-CD11b mAb significantly inhibited eosinophil recruitment. The combination of both mAbs also caused significant inhibition. There was also a significant inhibition of anti-alpha 4 mAb and anti-CD11b mAb on EPO activity in BALF, with a further inhibition when the two antibodies were administered together (C ). The anti-CD11b antibody did not alter eotaxin-induced BHR, whereas the anti-alpha 4 antibody was effective. Both antibodies caused a further inhibition. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with rat IgG/ eotaxin-treated mice. Dagger P < 0.05, §P < 0.01 compared with CD11b mAb/eotaxin-treated mice. Data are shown as means ± SEM.

Eotaxin-induced BHR was blocked by anti-alpha 4 mAb (logPC50ACh = 0.47 ± 0.18, P < 0.05) but not by anti-CD11b mAb (logPC50ACh = -0.23 ± 0.36, NS), when compared with eotaxin-instilled, control rat IgG-injected mice (-0.34 ± 0.22). The combination of both antibodies completely inhibited the effect of eotaxin-induced airway hyperresponsiveness in IL-5 transgenic mice (logPC50ACh = 0.86 ± 0.09, P < 0.01, Figure 5D).

    Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Intratracheal eotaxin did not induce any significant effects when instilled into the airways of wild mice, but in transgenic IL-5 mice it caused large increases in BAL eosinophils expressing increased levels of alpha 4 and CD11b, with eosinophils localizing to peribronchial and parenchymal lung tissues. This was accompanied by large increases in BHR to acetylcholine, compared with those reported for sensitized, allergen-challenged mice. The eosinophilia and BHR were both inhibited particularly by an anti-alpha 4 antibody, indicating that the eotaxin effects were mediated through the expression of this integrin on eosinophils. Thus, the induction of BHR by eotaxin through cooperation with IL-5 is dependent on eosinophils expressing alpha 4 and, to a lesser extent, CD11b on their surfaces.

The IL-5 transgenic mouse we studied was established using a genomic fragment of the IL-5 gene coupled to the dominant central region from the gene encoding human CD2 (25). Despite the constitutive expression of high levels of serum IL-5 and of eosinophils in many organs, including the spleen, bone marrow, peritoneal exudate, and circulating blood, these mice do not develop airway or lung pathology (29, 30). In addition, anti-IL-5 antibodies are less efficacious in inhibiting airway hyperresponsiveness induced by allergen exposure in sensitized mice than the accompanying eosinophilia (31, 32), although both aspects have been inhibited in other studies (33). In our model, the additional recruitment and activation of large numbers of eosinophils to the airways by eotaxin was important for the development of BHR. Synergy between IL-5 and eotaxin may be necessary for the induction of tissue eosinophilia such that eotaxin secreted in airways and lungs initiates tissue eosinophilia, whereas IL-5 is important in mobilizing eosinophils from the bone marrow and in regulating eosinophil homing and migration in tissues in response to eotaxin and possibly other chemotactic factors (11, 24). Our data demonstrate the synergy occurring between eotaxin and IL-5 not only for the airway recruitment of eosinophils but also for BHR, and show the direct link between eosinophil recruitment and activation in the airways and the development of BHR.

BAL eosinophils show upregulated expression of alpha 4, CD18, and CD11b, but not L-selectin, as assessed by flow cytometric analysis. Using maximal doses, the anti-alpha 4 antibody inhibited the eosinophil infiltration and activation and BHR; whereas the anti-CD11b antibody had only a partial effect, although it potentiated the effect of anti-alpha 4 in suppressing BHR. The eosinophil infiltration following eotaxin occurred mainly in the airways rather than in the parenchymal tissues. Our in vivo findings confirm the in vitro effects of eotaxin in increasing eosinophil surface molecule CD11b (36, 37) and in causing adhesion of eosinophils to human lung microvascular endothelial cells via VLA-4 or VLA-4 and CD18 in combination (38). Activation of the eosinophil, ascertained by EPO release in BALF, may lead to BHR through the release of this and other cytotoxic proteins (39, 40). Our data indicate that EPO release occurs maximally at 24 h after eotaxin administration whereas the eosinophil influx in BALF is at its highest at 4 h. Thus, evidence of eosinophil activation is coincident with the development of BHR. Eotaxin has the potential of inducing eosinophil degranulation (41) and stimulating the production of reactive oxygen species (36). VLA-4 is a known receptor for the abundant extracellular matrix protein fibronectin, to which both eosinophils and lymphocytes have been shown to adhere (42, 43). As a consequence of binding to fibronectin, a VLA-4-dependent increase in survival time of eosinophils via triggering of cytokine generation has been demonstrated (44). There may also be augmentation of stimulated degranulation of eosinophils by VLA-4-mediated adhesion to fibronectin (45).

Although the anti-CD11b antibody, to a limited extent, reduced eosinophil infiltration and activation, it had no effect on BHR, possibly a reflection of a threshold degree of inhibition of eosinophil activity needed for altering BHR. However, it increased the inhibitory effects of the anti-alpha 4 antibody on eotaxin-induced BHR and eosinophil activation, indicative of an additive effect and suggesting that ligands for both CD11b and VLA4 are necessary for the observed increases in eosinophil accumulation and bronchial responsiveness.

There have been several studies of the role of VLA-4 in eosinophil recruitment and BHR in allergen-exposed and sensitized animal models. In sensitized mice and rats, anti- VLA-4 antibody inhibited both lymphocytosis and eosinophilia (26, 46); and in sensitized guinea pigs, it reduced both hyperresponsiveness and eosinophil infiltration (47, 48). Studies of sensitized and allergen-exposed rats and sheep indicate that BHR inhibition by anti-VLA-4 is not always accompanied by a reduction in eosinophil infiltration (49). These conflicting observations may be related to species differences, doses of antibody, and routes of administration. However, the mechanisms of eosinophil accumulation and the involvement of integrin expression are likely to be complex. For example, allergen exposure is usually accompanied by activation of mast cells, T lymphocytes, eosinophils, and also neutrophils, and integrin expression may occur on subsets of T and B cells, natural killer cells, neutrophils, eosinophils, and airway smooth-muscle cells. Thus, other cell types may be targeted by anti-VLA-4 antibody following allergen challenge. By contrast, our model of eotaxin exposure appears to involve only eosinophils, and indicates that eosinophil recruitment and activation are solely important for the expression of BHR.

Our studies provide direct evidence for the crucial role for eosinophil recruitment and activation, induced by a combination of exogenous eotaxin and endogenous IL-5 and dependent on the expression of alpha 4 integrin and (to a lesser extent) of CD11b integrins, in the pathogenesis of BHR.

    Footnotes

Address correspondence to: Prof. K. Fan Chung, National Heart and Lung Institute, Imperial College School of Medicine, Dovehouse Street, London SW3 6LY, UK. E-mail: f.chung{at}ic.ac.uk

(Received in original form July 6, 1998 and in revised form September 14, 1998).

Abbreviations: acetylcholine chloride, ACh; bronchoalveolar lavage, BAL; BAL fluid, BALF; bronchial hyperresponsiveness, BHR; bovine serum albumin, BSA; eosinophil peroxidase, EPO; fluorescein isothiocyanate, FITC; hydrogen peroxide, H2O2; immunoglobulin, Ig; interleukin, IL; the provocative concentration of acetylcholine chloride in micrograms per gram that causes a 50% increase in airway pressure, logPC50ACh; monoclonal antibody, mAb; phosphate-buffered saline, PBS; very late antigen, VLA.

Acknowledgments: This work was partly supported by the National Asthma Campaign (UK). The authors thank Professor M. Mori for his help and advice.
    References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

1. Djukanovic, R., W. R. Roche, J. W. Wilson, C. R. W. Beasley, O. P. Twentyman, and P. H. Howarth. 1990. Mucosal inflammation in asthma. Am. Rev. Respir. Dis. 142: 434-457 [Medline].

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