We hypothesized that there are clinically relevant differences in eosinophil integrin expression and activation in patients with asthma. To evaluate this, surface densities and activation states of integrins on eosinophils in blood and bronchoalveolar lavage (BAL) of 19 asthmatic subjects were studied before and 48 h after segmental Ag challenge. At 48 h, there was increased expression of αD and the N29 epitope of activated β1 integrins on blood eosinophils and of αM, β2, and the mAb24 epitope of activated β2 integrins on airway eosinophils. Changes correlated with the late-phase fall in forced expiratory volume in 1 s (FEV1) after whole-lung inhalation of the Ag that was subsequently used in segmental challenge and were greater in subjects defined as dual responders. Increased surface densities of αM and β2 and activation of β2 on airway eosinophils correlated with the concentration of IL-5 in BAL fluid. Activation of β1 and β2 on airway eosinophils correlated with eosinophil percentage in BAL. Thus, eosinophils respond to an allergic stimulus by activation of integrins in a sequence that likely promotes eosinophilic inflammation of the airway. Before challenge, β1 and β2 integrins of circulating eosinophils are in low-activation conformations and αDβ2 surface expression is low. After Ag challenge, circulating eosinophils adopt a phenotype with activated β1 integrins and up-regulated αDβ2, changes that are predicted to facilitate eosinophil arrest on VCAM-1 in bronchial vessels. Finally, eosinophils present in IL-5-rich airway fluid have a hyperadhesive phenotype associated with increased surface expression of αMβ2 and activation of β2 integrins.

The contribution of eosinophils to certain aspects of asthma, such as airway hyperresponsiveness, remains controversial; nevertheless, there is evidence that eosinophil recruitment to the airway contributes to asthma exacerbations and the chronic character of asthma by regulating airway inflammation and remodeling (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15). Thus, the study of how eosinophils traffic from blood to airway is of considerable importance. Integrins, which are versatile cellular adhesion receptors (16, 17), are likely determinants of how eosinophils roll and arrest on lung endothelium, extravasate and migrate through the endothelium, underlying basement membrane, and tissue to the airway wall, and traverse the bronchial epithelium to the airway lumen (18, 19, 20).

Human eosinophils express seven integrin heterodimers: α4β1 (CD49d/CD29), α6β1 (CD49f/CD29), αLβ2 (CD11a/CD18), αMβ2 (CD11b/CD18), αXβ2 (CD11c/CD18), αDβ2 (CD11d/CD18), and α4β7 (CD49d/β7) (13, 21, 22). Each integrin heterodimer interacts with its own set of ligands, which are counterreceptors on other cells or extracellular matrix components (17, 23). The functions of an integrin on a given cell is regulated by expression level and activation state (24, 25, 26).

We hypothesized that there are clinically relevant differences in eosinophil integrin expression levels and activation states in patients with asthma. The purpose of the present study was to define changes in the expression levels and activation states of blood and airway eosinophil integrins 48 h following segmental allergen challenge in asthmatic subjects, thus reflecting the development of allergic inflammation. Segmental Ag challenge induces a strong local recruitment of eosinophils (27). We found characteristic integrin changes on blood and airway eosinophils, differences between single and dual responder asthmatics, and associations among integrin changes, magnitude of eosinophil recruitment to airway, and IL-5 concentration in bronchoalveolar lavage (BAL)3 fluid. We propose a scenario in which the following occur: 1) Ag challenge leads to activation of β1 integrins and increased surface expression of αDβ2 on blood eosinophils; 2) such activated eosinophils are more prone to arrest on VCAM-1 (CD106)-bearing endothelium in challenged segments; and 3) IL-5 and other cytokines trigger activation of β2 integrins, contributing to the hyperadhesive phenotype of airway eosinophils.

Nineteen subjects with mild asthma as diagnosed by an allergist were studied (Table I). These subjects had a history of asthma exacerbation to aeroallergen, a provocative concentration of methacholine producing a 20% fall in forced expiratory volume in 1 s (FEV1) of <8 mg/ml and/or reversibility to β-agonist of >12%. Subjects were screened as described previously with allergen skin prick tests, determination of airway hyperresponsiveness, and spirometry (28). All subjects had a positive skin prick test to one or more aeroallergens, were nonsmokers, did not have a respiratory infection within 30 days of study, and had not received antihistamines within seven days or corticosteroids within 30 days of study enrollment. The studies were reviewed and approved by the University of Wisconsin Health Sciences Human Subjects Committee (Madison, WI). Informed written consent was obtained from each subject before participation.

Table I.

Subject characteristics

Subject NoAge (years)SexaAgbScreeningWhole-Lung ChallengeSegmental Challenge
PC20 (mg/ml)cFEV1 (liters/%)dAgPD20 (CBU)eFEV1 Fall Early (%)FEV1 Fall Late (%)Eosinophils in BAL 48 h after Challenge (%)Total Eosinophils in BAL 48 h after Challenge (×106)
Single respondersf           
 1 22 RW 9.0 3.8 (90) 81 37 26 
 2 21 HDM 1.2 3.2 (85) 52 37 150 
 3 26 HDM 1.8 4.0 (99) 26 21 
 4 19 CD 0.7 4.1 (85) 85 31 ND ND 
 5 19 HDM 3.5 3.7 (105) 118 22 47 150 
 6 21 RW 0.2 4.4 (93) 115 23 26 24 
 7 20 CD 5.5 4.3 (98) 68 44 60 83 
 8 27 HDM 20 3.7 (89) 52 35 38 24 
 9 23 CD 0.4 3.0 (91) 32 11 24 10 
 10 26 CD 2.9 4.0 (109) 174 40 62 88 
 Median 22   2.4 3.9 (92)  34 37 24 
           
Dual respondersg           
 11 30 HDM 4.8 2.9 (95) 11 23 25 72 220 
 12 19 CD 3.8 4.9 (96) 37 22 17 63 230 
 13 20 RW 0.3 2.9 (87) 48 15 34 29 
 14 20 HDM 1.3 3.7 (99) 31 15 59 410 
 15 18 HDM 20 5.1 (104) 21 21 15 65 120 
 16 22 HDM 2.5 3.9 (87) 31 23 32 74 760 
 17 22 RW 20 4.7 (97) 48 35 18 66 420 
 18 20 HDM 0.4 3.4 (96) 27 31 21 67 280 
 19 23 RW 20 4.4 (106) 52 48 24 55 380 
 Median 20   3.8 3.9 (96)  31 18i 65h 280h 
Subject NoAge (years)SexaAgbScreeningWhole-Lung ChallengeSegmental Challenge
PC20 (mg/ml)cFEV1 (liters/%)dAgPD20 (CBU)eFEV1 Fall Early (%)FEV1 Fall Late (%)Eosinophils in BAL 48 h after Challenge (%)Total Eosinophils in BAL 48 h after Challenge (×106)
Single respondersf           
 1 22 RW 9.0 3.8 (90) 81 37 26 
 2 21 HDM 1.2 3.2 (85) 52 37 150 
 3 26 HDM 1.8 4.0 (99) 26 21 
 4 19 CD 0.7 4.1 (85) 85 31 ND ND 
 5 19 HDM 3.5 3.7 (105) 118 22 47 150 
 6 21 RW 0.2 4.4 (93) 115 23 26 24 
 7 20 CD 5.5 4.3 (98) 68 44 60 83 
 8 27 HDM 20 3.7 (89) 52 35 38 24 
 9 23 CD 0.4 3.0 (91) 32 11 24 10 
 10 26 CD 2.9 4.0 (109) 174 40 62 88 
 Median 22   2.4 3.9 (92)  34 37 24 
           
Dual respondersg           
 11 30 HDM 4.8 2.9 (95) 11 23 25 72 220 
 12 19 CD 3.8 4.9 (96) 37 22 17 63 230 
 13 20 RW 0.3 2.9 (87) 48 15 34 29 
 14 20 HDM 1.3 3.7 (99) 31 15 59 410 
 15 18 HDM 20 5.1 (104) 21 21 15 65 120 
 16 22 HDM 2.5 3.9 (87) 31 23 32 74 760 
 17 22 RW 20 4.7 (97) 48 35 18 66 420 
 18 20 HDM 0.4 3.4 (96) 27 31 21 67 280 
 19 23 RW 20 4.4 (106) 52 48 24 55 380 
 Median 20   3.8 3.9 (96)  31 18i 65h 280h 
a

F, Female; M, male.

b

CD, Cat dander; HDM, house dust mite; RW, ragweed.

c

PC20, Provocative concentration of methacholine producing a 20% fall in FEV1.

d

FEV1 values from the screening visit are expressed in liters (number on left) and as a percentage of the predicted value (number in parentheses).

e

CBU, Cumulative breath units.

f

Single responders are subjects with FEV1 fall 3–8 h after whole-lung Ag challenge (late-phase fall) < 15% (see Ref. 28 ).

g

Dual responders are subjects with FEV1 fall 3–8 h after whole-lung Ag challenge ≥ 15% (see Ref. 28 ).

h

p ≤ 0.01;

i

p ≤ 0.001 versus single responders.

At least 4 wk before bronchoscopy, a graded whole-lung inhaled Ag challenge was performed as described (28) to determine the provocative dose of Ag producing a 20% fall in FEV1 (AgPD20) and the magnitude of early- and late-phase responses. Briefly, baseline spirometry was performed and repeated after five breaths of saline diluent. If FEV1 remained within 10% of baseline, five breaths of allergen were inhaled and spirometry was repeated 10 min later. Consecutively greater concentrations of allergen were given until FEV1 fell by ≥20% from baseline. The maximum immediate- or early-phase (within 15–30 min) fall in FEV1 was determined and subjects were then monitored every 15 min until FEV1 returned to within 10% of baseline. Thereafter, the subjects were monitored at 1-h intervals for 8 h to determine whether a late-phase response was present (28, 29). Subjects having a FEV1 fall of ≥15% 3–8 h after the whole-lung Ag challenge were considered to have a dual response phenotype; the other subjects were considered single responders (28).

Bronchoscopy, segmental bronchoprovocation with allergen, and subsequent BAL were performed in two different bronchopulmonary segments as described (27, 28, 30). Baseline BAL at 0 h immediately before segmental bronchoprovocation was performed in two segments. Then, a total dose of 30% of the subject’s AgPD20 was administered incrementally to enhance subject safety as follows: 10% of the AgPD20 in the first segment and, when this dose was well tolerated, 20% in the second segment. Forty-eight hours later, a second bronchoscopy was performed by instilling 160 ml of sterile 0.9% NaCl warmed to 37°C in each segment. BAL fluid recovered from the two segments was pooled for analysis, and the volume of recovered fluid was measured.

Anti-αD integrin mAb 240I (31) was obtained as a gift from ICOS. Activation-sensitive β2 integrin mAb24 (32, 33) was a gift from N. Hogg (Cancer Research U.K. London Research Institute, London, U.K.). Anti-β1 mAb MAR4, anti-β2 L130, anti-β7 Fib504, anti-α4 9F10, anti-α6 GoH3, anti-αL AI111, anti-αX Bly6, PE-conjugated goat anti-mouse and anti-rat IgG, FITC-conjugated anti-CD14 and anti-CD16, the isotype controls mouse IgG1 κ (clone A112-2) and rat IgG2a κ (A110-2), and the unlabeled and biotinylated anti-IL-3, anti-IL-5, and anti-IFN-γ mAbs and corresponding recombinant protein standards for ELISA were from BD Biosciences. Anti-αM LM11 and activation-sensitive anti-β1 N29 (34) were from Chemicon. Unlabeled and biotinylated anti-GM-CSF mAb and recombinant protein standard for ELISA were from R&D Systems.

Because purification of blood eosinophils has been found to cause the activation of β1 (35), flow cytometry was done on unfractionated blood and BAL cells. Surface expressions of αM, αL, αX, αD, α4, α6, β1, β2, β7, activated β1, and activated β2 were determined. Blood was drawn routinely into lavender top standard tubes (giving a final EDTA concentration of 1.8 mg/ml) (BD Vacutainer Systems). For determination of mAb24 reactivity, blood was drawn into green top tubes (giving a final heparin concentration of 14 U.S. Pharmacopeia (USP) units/ml). Control experiments revealed that the anticoagulant had no effect on the results except, as reported, for mAb24, whose epitope is not exposed in the presence of EDTA (32). Not all samples were subjected to complete analysis due to changes in the mAb panel that were made based on ongoing analysis of results from this study and other studies. Originally, we focused on differences between blood and BAL fluid 48 h after segmental challenge. Blood from before segmental challenge was included as a routine later, when we had indications of interesting differences between blood after vs before challenge. Similarly, we originally focused on subunits of the integrins known to be able to bind VCAM-1, α4β1, α4β7, and αDβ2 (13, 21) and added αM later when we had evidence that αMβ2 is involved in the adhesion of purified airway eosinophils to VCAM-1 and other ligands (22, 36). BAL fluid cells were recovered, cytospun, and stained for differential counts as described (29, 30).

EDTA-treated blood (100 μl) was incubated with 0.5 μg of primary Ab or isotype control in 100 μl of FACS buffer (PBS with 2% BSA and 0.2% NaN3) for 30 min. For mAb24 (and its isotype control), heparin-treated blood was used because the mAb24 epitope is not exposed in EDTA (32) and was incubated with primary Ab in RPMI 1640 with 10% FBS at 37°C following the protocol especially designed for mAb24 (37). After primary Ab incubation, samples were washed with 1 ml PBS, washed with 250 μl of FACS buffer, and resuspended in 250 μl of FACS buffer containing PE-conjugated goat anti-mouse or anti-rat IgG (2 μg/ml). After incubation for 30 min, samples were washed again with PBS, resuspended in 100 μl FACS buffer with a mixture of FITC-conjugated anti-CD14 (0.125 μg) and anti-CD16 (0.625 μg) and incubated for 30 min. RBC were lysed by incubation with 2 ml of FACS lysing solution (BD Biosciences) for 10 min, followed by centrifugation. Incubations were at room temperature until after RBC lysis and then at 4°C. Samples were washed with 500 μl of FACS buffer, resuspended in 250 μl of FACS fixative (1% paraformaldehyde, 67.5 mM sodium cacodylate, and 113 mM NaCl (pH 7.2)), stored at 4°C in the dark, and washed with 1 ml of PBS and resuspended in 250 μl of FACS buffer just before data collection. Fixation did not decrease signals (data not shown). Data were collected from 30,000 - 170,000 events, using a FACSCalibur (BD Biosciences; available through the Flow Cytometry Facility, Comprehensive Cancer Center, University of Wisconsin, Madison, WI) and CellQuest software (BD Biosciences). Rainbow calibration fluorescent beads (Spherotech) were run each day to calibrate and check the performance of the instrument and channels. Data were analyzed using CellQuest and Flow Jo (Tree Star). Eosinophils were gated based both on scattering (high side scatter) and lack of staining with anti-CD14 and anti-CD16 (30, 35). Thus, the cells that were analyzed for the PE signal fit two criteria for eosinophils by being gated inside both characteristic regions in a plot of side scatter vs FITC staining and a plot of side vs forward scatter. When cells gated on side vs forward scatter were collected by cell sorting followed by cytospin, ≥96% stained for the eosinophil marker eosinophil major basic protein. To analyze circulating neutrophils and monocytes in the same data sets, these leukocyte types were gated inside characteristic regions in the two plots, neutrophils having intermediate side scatter and FITC positivity and monocytes having relatively low side scatter and FITC positivity. Data are expressed as specific geometric mean channel fluorescence (gMCF; specific gMCF = gMCF with a specific integrin mAb − gMCF with isotype control) and as percentage of positive cells (isotype control set with a marker to 2% positive cells) as before (35). For unfractionated BAL cells, flow cytometry was performed with 2 × 105 cells (in 100 μl) and the protocol was as described for blood samples, except that all incubations were at 4°C and the erythrocyte lysis step was omitted and replaced by a wash with 1 ml of PBS.

To measure cytokine concentrations, BAL fluid was concentrated 10-fold at 4°C using a low protein-binding Centriprep centrifugal filter unit (Millipore) with a molecular mass cutoff limit of 3 kDa. A sensitive two-step sandwich ELISA was used as described (38). The assay sensitivities were below 3 pg/ml for IL-5 and GM-CSF, 12 pg/ml for IL-3, and 25 pg/ml for IFN-γ. Values are presented as the concentration in recovered BAL fluid before the concentration step.

The Mann-Whitney U test was used to compare data between groups. The Spearman rank correlation test was used to analyze correlations. A level of p ≤ 0.05 was considered significant. Analyses were performed using Prism 3.0 (GraphPad).

All subjects responded to whole-lung inhaled Ag challenge with a drop in FEV1 within 15–30 min to a median value of 31% early FEV1 fall (Table I). Nine of the 19 subjects had a late-phase response (39) as defined by a FEV1 fall ≥15% from baseline 3–8 h after challenge (Table I). Using this criterion (28), the nine subjects were classified as having a dual-response phenotype. The other 10 subjects were considered single responders (Table I). There were no significant differences between the single and dual responders in regards to age, sex, airway responsiveness to methacholine, FEV1 at the screening visit, or early-phase fall in response to whole-lung Ag challenge (Table I). FEV1 48 h after segmental Ag challenge was decreased minimally compared with before segmental Ag challenge and was not significantly different between single and dual responders (medians 98 and 96% of the value before segmental challenge in single and dual responders, respectively).

Median number of BAL eosinophils after segmental challenge was >10-fold higher in the dual responders than in the single responders (Table I). Median numbers of BAL neutrophils and macrophages, in contrast, were ∼2-fold higher and not significantly different in dual vs single responders (medians in single and dual responders were 3.1 × 106 and 7.5 × 106 neutrophils and 43 × 106 and 93 × 106 macrophages; respectively). The neutrophil percentage was not significantly different and the macrophage percentage was significantly lower in dual responders due to the increased proportion of eosinophils (medians in single and dual responders were 2.3 and 3.0% neutrophils and 47 and 22% macrophages, respectively).

Sample flow cytometry histograms are shown in Fig. 1 for eosinophil expression of total β1 (A), the activation-sensitive β1 epitope for mAb N29 (B–D), total β2 (E), the activation-sensitive β2 epitope for mAb24 (F), αM (G), and αD (H). Expression distributions were homogeneous in some samples (Fig. 1, B and F) and heterogeneous and asymmetric with one or more “shoulders” in others (Fig. 1, A, C, D, E, G, and H). β1 distributions were mostly heterogeneous on blood eosinophils and uniformly homogeneous on BAL eosinophils (Fig. 1,A and Table II). N29 reactivity of blood eosinophils was variable (Fig. 1, B–D, and Table II). Some samples had two peaks (Fig. 1,C) or a “shoulder” of reactivity (Fig. 1,D). N29 reactivity of BAL eosinophils was uniformly homogeneous (Fig. 1, B–D, and Table II). β2 was heterogeneous on blood eosinophils, often with two or several distinct populations, whereas the peak of BAL eosinophils was mostly homogeneous and shifted to the right with higher fluorescence intensity compared with the most positive blood eosinophil population (Fig. 1,E and Table II). mAb24 reactivity was homogeneous and low on blood eosinophils and mostly homogeneous and shifted to the right on BAL eosinophils (Fig. 1,F and Table II). αM was mostly heterogeneous on blood eosinophils and homogeneous and shifted to the right on BAL eosinophils (Fig. 1,G and Table II). αD was mostly heterogeneous on blood eosinophils and mostly homogeneous on BAL eosinophils (Fig. 1,H and Table II). Thus, distributions on BAL eosinophils were typically homogeneous whereas distributions of β1, β2, αM, and αD on blood eosinophils were mostly heterogeneous.

FIGURE 1.

Integrin expression on blood and BAL eosinophils before and after segmental Ag challenge. Representative flow cytometry histograms of expression of total β1 integrin (A), activation-sensitive β1 integrin mAb N29 epitope (B–D), total β2 integrin (E), activation-sensitive β2 integrin mAb24 epitope (F), αM integrin (G), or αD integrin (H) on blood eosinophils before segmental Ag challenge (green), blood eosinophils 48 h after segmental Ag challenge (blue), or BAL eosinophils 48 h after segmental Ag challenge (red), or isotype control (brown). For N29 epitope expression, three examples are shown: one homogeneous, symmetric peak on blood eosinophils before and after challenge and higher mean expression level after challenge than before (B); one peak on blood eosinophils before challenge and two peaks after challenge, and higher mean expression level after challenge (C), and an asymmetric peak with a “shoulder” to the right on blood eosinophils before and after challenge, and decrease in the size of the “shoulder” and lower mean expression level after challenge (D). The numbers of individuals with the various patterns are summarized in Table II.

FIGURE 1.

Integrin expression on blood and BAL eosinophils before and after segmental Ag challenge. Representative flow cytometry histograms of expression of total β1 integrin (A), activation-sensitive β1 integrin mAb N29 epitope (B–D), total β2 integrin (E), activation-sensitive β2 integrin mAb24 epitope (F), αM integrin (G), or αD integrin (H) on blood eosinophils before segmental Ag challenge (green), blood eosinophils 48 h after segmental Ag challenge (blue), or BAL eosinophils 48 h after segmental Ag challenge (red), or isotype control (brown). For N29 epitope expression, three examples are shown: one homogeneous, symmetric peak on blood eosinophils before and after challenge and higher mean expression level after challenge than before (B); one peak on blood eosinophils before challenge and two peaks after challenge, and higher mean expression level after challenge (C), and an asymmetric peak with a “shoulder” to the right on blood eosinophils before and after challenge, and decrease in the size of the “shoulder” and lower mean expression level after challenge (D). The numbers of individuals with the various patterns are summarized in Table II.

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Table II.

Integrin expression on blood and BAL eosinophils as distribution patterns and percentage of positive cells before and 48 h after segmental Ag challenge

Integrin (mAb)Blood Eosinophils before Segmental Ag ChallengeBlood Eosinophils 48 h after Segmental Ag ChallengeBAL Eosinophils 48 h after Segmental Ag Challenge
Homogenous/TotalaPercentage PositivebHomogenous/TotalaPercentage PositivebHomogenous/TotalaPercentage Positiveb
All tested subjects       
 Total β1 (MAR4) 2/9 82 ± 4 6/16 80 ± 5 17/17 85 ± 6 
 Activated β1 (N29) 8/9 54 ± 7 11/16 57 ± 7 15/15 55 ± 8 
 Total β2 (L130) 1/10 91 ± 2 1/17 93 ± 2 12/14 97 ± 1fh 
 Activated β2 (mAb24) 3/3 23 ± 17 3/3 18 ± 11 10/13 65 ± 11 
 αM 1/4 92 ± 5 2/12 95 ± 3 8/9 96 ± 3 
 αD 2/9 79 ± 2 4/15 87 ± 6g 13/16 81 ± 8 
       
Single respondersc       
 Total β1 (MAR4) 2/5 85 ± 7 5/9 79 ± 8 9/9 80 ± 9 
 Activated β1 (N29) 4/5 53 ± 11 5/9 48 ± 10 8/8 49 ± 10 
 Total β2 (L130) 1/5 94 ± 2 0/9 92 ± 4 6/7 95 ± 3 
 Activated β2 (mAb24)d 2/2 30 ± 27 2/2 26 ± 14 6/6 42 ± 19 
 αM 1/2 100 ± 0 1/7 93 ± 4 4/5 95 ± 5 
 αD 2/5 82 ± 4 3/8 80 ± 12 7/8 77 ± 11 
       
Dual responderse       
 Total β1 (MAR4) 0/4 78 ± 3 1/7 83 ± 5 8/8 90 ± 7f 
 Activated β1 (N29) 4/4 56 ± 8 6/7 70 ± 7 7/7 61 ± 14 
 Total β2 (L130) 0/5 88 ± 4 1/8 94 ± 2 6/7 99 ± 0f 
 Activated β2 (mAb24) 1/1 1/1 4/7 85 ± 4 
 αM 0/2 84 ± 4 1/5 97 ± 1 4/4 98 ± 2 
 αD 0/4 76 ± 1i 1/7 95 ± 2g 6/8 85 ± 11f 
Integrin (mAb)Blood Eosinophils before Segmental Ag ChallengeBlood Eosinophils 48 h after Segmental Ag ChallengeBAL Eosinophils 48 h after Segmental Ag Challenge
Homogenous/TotalaPercentage PositivebHomogenous/TotalaPercentage PositivebHomogenous/TotalaPercentage Positiveb
All tested subjects       
 Total β1 (MAR4) 2/9 82 ± 4 6/16 80 ± 5 17/17 85 ± 6 
 Activated β1 (N29) 8/9 54 ± 7 11/16 57 ± 7 15/15 55 ± 8 
 Total β2 (L130) 1/10 91 ± 2 1/17 93 ± 2 12/14 97 ± 1fh 
 Activated β2 (mAb24) 3/3 23 ± 17 3/3 18 ± 11 10/13 65 ± 11 
 αM 1/4 92 ± 5 2/12 95 ± 3 8/9 96 ± 3 
 αD 2/9 79 ± 2 4/15 87 ± 6g 13/16 81 ± 8 
       
Single respondersc       
 Total β1 (MAR4) 2/5 85 ± 7 5/9 79 ± 8 9/9 80 ± 9 
 Activated β1 (N29) 4/5 53 ± 11 5/9 48 ± 10 8/8 49 ± 10 
 Total β2 (L130) 1/5 94 ± 2 0/9 92 ± 4 6/7 95 ± 3 
 Activated β2 (mAb24)d 2/2 30 ± 27 2/2 26 ± 14 6/6 42 ± 19 
 αM 1/2 100 ± 0 1/7 93 ± 4 4/5 95 ± 5 
 αD 2/5 82 ± 4 3/8 80 ± 12 7/8 77 ± 11 
       
Dual responderse       
 Total β1 (MAR4) 0/4 78 ± 3 1/7 83 ± 5 8/8 90 ± 7f 
 Activated β1 (N29) 4/4 56 ± 8 6/7 70 ± 7 7/7 61 ± 14 
 Total β2 (L130) 0/5 88 ± 4 1/8 94 ± 2 6/7 99 ± 0f 
 Activated β2 (mAb24) 1/1 1/1 4/7 85 ± 4 
 αM 0/2 84 ± 4 1/5 97 ± 1 4/4 98 ± 2 
 αD 0/4 76 ± 1i 1/7 95 ± 2g 6/8 85 ± 11f 
a

Number of subjects with homogeneous expression distribution out of the total number of subjects.

b

Expression as percentage of positive cells (mean ± SEM).

c

Single responders are subjects with FEV1 fall 3–8 h after whole-lung Ag challenge < 15% (see Ref. 28 ).

d

Data with n < 3 are within the ranges of our overall data on blood eosinophils from unchallenged donors.

e

Dual responders are subjects with FEV1 fall 3–8 h after whole-lung Ag challenge ≥ 15% (see Ref. 28 ).

f

p ≤ 0.05 and

g

p ≤ 0.01 versus blood eosinophils before segmental challenge;

h

p ≤ 0.05 versus blood eosinophils after segmental challenge;

i

p ≤ 0.05 versus single responders.

Expression data were scored either as percentage of positive cells or as expression level (specific gMCF) (Table II and Fig. 2). Fig. 3 shows a comparison between the results with these two scoring methods on blood eosinophils for N29 (Fig. 3,A) and αD (Fig. 3,B). At lower fluorescence intensity the percentage of positive cells and the expression level correlate, whereas at higher intensity the percentage of positive cells plateaus (Fig. 3, A and B). Because of its greater dynamic range, specific gMCF was chosen as the more informative measure of integrin expression and activation (Fig. 2). It should be emphasized that for the samples displaying heterogeneous bimodal or multimodal distributions of the β2 integrin subunits, neither gMCF nor percentage of positive cells fully captures the complexity of the distributions.

FIGURE 2.

Integrin expression on blood and BAL eosinophils before and 48 h after segmental Ag challenge. Expression of total β1 integrin (A), activation-sensitive β1 integrin mAb N29 epitope (B), total β2 integrin (C), activation-sensitive β2 integrin mAb24 epitope (D), αM integrin (E), and αD integrin (F) on blood eosinophils before segmental Ag challenge (light gray), blood eosinophils 48 h after segmental Ag challenge (medium gray), and BAL eosinophils 48 h after segmental Ag challenge (dark gray) in all tested subjects, single responders, and dual responders. Values shown are specific gMCF (mean ± SEM). For n values and expression given as percentage of positive cells, see Table II. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001 vs blood eosinophils before segmental challenge; †, p ≤ 0.05; †††, p ≤ 0.001 vs blood eosinophils after segmental challenge; ‡, p ≤ 0.05 vs single responders.

FIGURE 2.

Integrin expression on blood and BAL eosinophils before and 48 h after segmental Ag challenge. Expression of total β1 integrin (A), activation-sensitive β1 integrin mAb N29 epitope (B), total β2 integrin (C), activation-sensitive β2 integrin mAb24 epitope (D), αM integrin (E), and αD integrin (F) on blood eosinophils before segmental Ag challenge (light gray), blood eosinophils 48 h after segmental Ag challenge (medium gray), and BAL eosinophils 48 h after segmental Ag challenge (dark gray) in all tested subjects, single responders, and dual responders. Values shown are specific gMCF (mean ± SEM). For n values and expression given as percentage of positive cells, see Table II. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001 vs blood eosinophils before segmental challenge; †, p ≤ 0.05; †††, p ≤ 0.001 vs blood eosinophils after segmental challenge; ‡, p ≤ 0.05 vs single responders.

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FIGURE 3.

Integrin expression on blood eosinophils as the percentage of positive cells and the expression level. Blood eosinophil expression of activation-sensitive β1 integrin mAb N29 epitope (A) or αD integrin (B) before (open symbols) and 48 h after (filled symbols) segmental Ag challenge, expressed as the percentage of positive cells (y axes) or as expression level (specific gMCF) (x axes).

FIGURE 3.

Integrin expression on blood eosinophils as the percentage of positive cells and the expression level. Blood eosinophil expression of activation-sensitive β1 integrin mAb N29 epitope (A) or αD integrin (B) before (open symbols) and 48 h after (filled symbols) segmental Ag challenge, expressed as the percentage of positive cells (y axes) or as expression level (specific gMCF) (x axes).

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On circulating eosinophils of dual responders the expression level of the epitope for activation-sensitive anti-β1 mAb N29 (34, 40, 41) was higher 48 h after Ag segmental challenge as compared with the level before challenge (Fig. 2,B). Circulating eosinophils also had significantly higher expression of the αD integrin subunit compared with before segmental challenge (Fig. 2 F). αD and N29 epitope expression on blood eosinophils 48 h after segmental challenge correlated with each other (rs (Spearman rank correlation coefficient) = 0.73; p = 0.002). Thus, the results demonstrate that segmental Ag challenge causes significantly increased surface expression of the αD subunit of β2 integrins and, in dual responders, activation of β1 integrins at 48 h on blood eosinophils.

Eosinophils in BAL 48 h after segmental Ag challenge had significantly higher β2 and αM subunit expression than eosinophils in blood obtained before or 48 h after segmental challenge (Fig. 2, C and E). The expression of the epitope for the activation-sensitive anti-β2 mAb24 (32, 33) was significantly increased on BAL eosinophils; the binding of mAb24 to blood eosinophils was low in blood both before and after segmental challenge (Fig. 2,D). αD expression was significantly increased on BAL eosinophils as compared with blood eosinophils before challenge but not as compared with blood also sampled at 48 h (Fig. 2,F). In dual responders, BAL eosinophils had significantly more total and activated β1 (as ascertained by N29) than blood eosinophils before challenge (Fig. 2, A and B). α6 expression was lower on BAL eosinophils than on blood eosinophils after challenge (specific gMCF = 560 ± 140 (mean ± SEM) vs 920 ± 110, p ≤ 0.05)(data not shown). No differences were found in the expression of αL, αX, α4, or β7 (data not shown). Overall, the results indicate that αMβ2 is up-regulated on BAL eosinophils and that BAL eosinophil β2 integrins are in a conformationally altered and activated state compared with β2 integrins on blood eosinophils.

To examine the specificity of the changes on blood eosinophils, circulating neutrophils and monocytes were also analyzed. Before segmental challenge, neutrophils had higher N29 reactivity than eosinophils and monocytes had even higher N29 reactivity (Figs. 4,B and 5,B; compare with Fig. 2,B). At 48 h after segmental Ag challenge, circulating neutrophils and monocytes, like eosinophils, had significantly higher αD expression compared with before challenge (Figs. 4,F and 5,F). Monocytes from dual responders had higher αD after segmental challenge than those from single responders (Fig. 5,F). N29 reactivity of neutrophils (as with eosinophils) increased significantly in dual responders upon segmental challenge, although to a lesser degree (1.4-fold for neutrophils, 1.7-fold for eosinophils) (Fig. 4,B; compare with Fig. 2,B), whereas N29 reactivity of monocytes did not increase significantly (Fig. 5,B). Furthermore, αM on blood neutrophils and monocytes and β2 on blood monocytes increased significantly upon segmental challenge (Figs. 4,E and 5, C and E) in contrast to blood eosinophil αM and β2, which did not (Fig. 2 C,E).

FIGURE 4.

Integrin expression on blood neutrophils before and 48 h after segmental Ag challenge. Expression of total β1 integrin (A), activation-sensitive β1 integrin mAb N29 epitope (B), total β2 integrin (C), activation-sensitive β2 integrin mAb24 epitope (D), αM integrin (E), and αD integrin (F) on blood neutrophils before segmental Ag challenge (light gray) and 48 h after segmental Ag challenge (medium gray) in all tested subjects, single responders, and dual responders. Values shown are specific gMCF (mean ± SEM). *, p ≤ 0.05 vs before segmental challenge.

FIGURE 4.

Integrin expression on blood neutrophils before and 48 h after segmental Ag challenge. Expression of total β1 integrin (A), activation-sensitive β1 integrin mAb N29 epitope (B), total β2 integrin (C), activation-sensitive β2 integrin mAb24 epitope (D), αM integrin (E), and αD integrin (F) on blood neutrophils before segmental Ag challenge (light gray) and 48 h after segmental Ag challenge (medium gray) in all tested subjects, single responders, and dual responders. Values shown are specific gMCF (mean ± SEM). *, p ≤ 0.05 vs before segmental challenge.

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FIGURE 5.

Integrin expression on blood monocytes before and 48 h after segmental Ag challenge. Expression of total β1 integrin (A), activation-sensitive β1 integrin mAb N29 epitope (B), total β2 integrin (C), activation-sensitive β2 integrin mAb24 epitope (D), αM integrin (E), and αD integrin (F) on blood monocytes before segmental Ag challenge (light gray) and 48 h after segmental Ag challenge (medium gray) in all tested subjects, single responders, and dual responders. Values shown are specific gMCF (mean ± SEM). *, p ≤ 0.05; **, p ≤ 0.01 vs before segmental challenge; ‡, p ≤ 0.05; ‡‡, p ≤ 0.01 vs single responders.

FIGURE 5.

Integrin expression on blood monocytes before and 48 h after segmental Ag challenge. Expression of total β1 integrin (A), activation-sensitive β1 integrin mAb N29 epitope (B), total β2 integrin (C), activation-sensitive β2 integrin mAb24 epitope (D), αM integrin (E), and αD integrin (F) on blood monocytes before segmental Ag challenge (light gray) and 48 h after segmental Ag challenge (medium gray) in all tested subjects, single responders, and dual responders. Values shown are specific gMCF (mean ± SEM). *, p ≤ 0.05; **, p ≤ 0.01 vs before segmental challenge; ‡, p ≤ 0.05; ‡‡, p ≤ 0.01 vs single responders.

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Thus, we determined the following: 1) αD is up-regulated on all three leukocyte populations, not just eosinophils; 2) β1 at baseline is more activated on neutrophils and monocytes compared with eosinophils; 3) in dual responders we found an increased activation state of β1 on neutrophils despite the high baseline activation state; and 4) surface expression of αM is up-regulated on monocytes and neutrophils, but not on blood eosinophils.

As with the variability in eosinophil numbers in BAL after segmental Ag challenge in the 19 subjects (Table I), there was considerable variability in integrin activation or expression on BAL eosinophils (Fig. 2). BAL eosinophils from dual responders had significantly higher N29 epitope and total β2 expression than BAL eosinophils from single responders (Figs. 2 and 6). Grouping all subjects together, the reactivity of BAL eosinophils with the activation-sensitive mAbs, N29 against β1 and mAb24 against β2, correlated significantly with the percentage of eosinophils in BAL (Fig. 7). Surface expression of other integrin subunits on BAL eosinophils did not correlate with BAL eosinophil percentage (data not shown). These results indicate that activation of both β1 and β2 integrins are associated with eosinophil recruitment to the airway.

FIGURE 6.

Differences in integrin activation and expression on BAL eosinophils between single and dual responders. BAL eosinophil expression 48 h after segmental Ag challenge of activation-sensitive β1 integrin mAb N29 epitope (A) or total β2 integrin (B) in single and dual responders. Bar, mean; *. p ≤ 0.05 vs single responders.

FIGURE 6.

Differences in integrin activation and expression on BAL eosinophils between single and dual responders. BAL eosinophil expression 48 h after segmental Ag challenge of activation-sensitive β1 integrin mAb N29 epitope (A) or total β2 integrin (B) in single and dual responders. Bar, mean; *. p ≤ 0.05 vs single responders.

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FIGURE 7.

Correlations between integrin activation on BAL eosinophils and BAL eosinophil percentage. Correlations between BAL eosinophil expression of activation-sensitive β1 integrin mAb N29 epitope (A) or activation-sensitive β2 integrin mAb24 epitope (B) and the percentage of eosinophils in BAL 48 h after segmental Ag challenge. rs, Spearman rank correlation coefficient.

FIGURE 7.

Correlations between integrin activation on BAL eosinophils and BAL eosinophil percentage. Correlations between BAL eosinophil expression of activation-sensitive β1 integrin mAb N29 epitope (A) or activation-sensitive β2 integrin mAb24 epitope (B) and the percentage of eosinophils in BAL 48 h after segmental Ag challenge. rs, Spearman rank correlation coefficient.

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We also analyzed for possible correlations between eosinophil integrins after segmental challenge and the magnitude of the maximum fall in FEV1 3–8 h after whole-lung allergen challenge (late-phase fall). αD expression and reactivity with N29 of blood eosinophils 48 h after segmental challenge each correlated with the magnitude of the late-phase fall in FEV1 after whole-lung challenge (Fig. 8, A and B). β2 expression and reactivity with mAb24 of BAL eosinophils 48 h after segmental challenge also correlated with the magnitude of the late-phase FEV1 fall (Fig. 8, C and D), as did αM expression of BAL eosinophils (rs = 0.83, p = 0.008) (data not shown). In addition, the late-phase FEV1 fall correlated with the percentage of eosinophils in BAL 48 h after segmental challenge (rs = 0.70, p = 0.001) (data not shown). Thus, the results indicate that greater αD up-regulation and β1 activation on blood eosinophils and greater αMβ2 up-regulation and β2 activation on BAL eosinophils after segmental challenge in a subject are associated with a greater late-phase FEV1 fall in response to whole-lung challenge.

FIGURE 8.

Correlations between integrin activation and expression on blood and BAL eosinophils and the magnitude of the fall in FEV1 3–8 h after whole-lung Ag challenge. Correlations between blood eosinophil expression of activation-sensitive β1 integrin mAb N29 epitope (A) or αD integrin (B) or BAL eosinophil expression of total β2 integrin (C) or activation-sensitive β2 integrin mAb24 epitope (D) 48 h after segmental Ag challenge and the magnitude of the late-phase fall in FEV1 3–8 h after whole-lung Ag challenge. rs, Spearman rank correlation coefficient.

FIGURE 8.

Correlations between integrin activation and expression on blood and BAL eosinophils and the magnitude of the fall in FEV1 3–8 h after whole-lung Ag challenge. Correlations between blood eosinophil expression of activation-sensitive β1 integrin mAb N29 epitope (A) or αD integrin (B) or BAL eosinophil expression of total β2 integrin (C) or activation-sensitive β2 integrin mAb24 epitope (D) 48 h after segmental Ag challenge and the magnitude of the late-phase fall in FEV1 3–8 h after whole-lung Ag challenge. rs, Spearman rank correlation coefficient.

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To identify factor(s) possibly responsible for the changes in integrins observed following segmental Ag challenge, the concentrations of IL-5, GM-CSF, IL-3, and IFN-γ in BAL fluid were measured (Fig. 9). The concentration of all four cytokines was significantly higher 48 h after segmental challenge than in samples obtained immediately before segmental challenge (Fig. 9). There was a highly significant difference in BAL fluid IL-5 after segmental challenge between single and dual responders; median IL-5 concentration in BAL fluid from dual responders was ∼40-fold greater than in BAL fluid from single responders (Fig. 9). Also IL-3 and IFN-γ were significantly different between single and dual responders; medians from dual responders were ∼10- and ∼3-fold those from single responders for IL-3 and IFN-γ, respectively (Fig. 9). GM-CSF was not different between single and dual responders (Fig. 9).

FIGURE 9.

Cytokine concentrations in BAL fluid before and 48 h after segmental Ag challenge. Concentrations of IL-5 (A), GM-CSF (B), IL-3 (C), and IFN-γ (D) in BAL fluid before (light gray bars) and 48 h after (medium gray bars) segmental Ag challenge from all tested subjects (n = 19), single responders (n = 10), or dual responders (n = 9). Values shown are medians with 25th and 75th percentiles of concentrations in recovered, unconcentrated BAL fluid. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001 vs before segmental challenge; ‡, p ≤ 0.05; ‡‡‡, p ≤ 0.001 vs single responders.

FIGURE 9.

Cytokine concentrations in BAL fluid before and 48 h after segmental Ag challenge. Concentrations of IL-5 (A), GM-CSF (B), IL-3 (C), and IFN-γ (D) in BAL fluid before (light gray bars) and 48 h after (medium gray bars) segmental Ag challenge from all tested subjects (n = 19), single responders (n = 10), or dual responders (n = 9). Values shown are medians with 25th and 75th percentiles of concentrations in recovered, unconcentrated BAL fluid. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001 vs before segmental challenge; ‡, p ≤ 0.05; ‡‡‡, p ≤ 0.001 vs single responders.

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The concentration of BAL fluid IL-5, but not that of the other cytokines, correlated significantly and strongly with the percentage of eosinophils in BAL 48 h after segmental Ag challenge (rs = 0.69, p = 0.001 for IL-5; rs = 0.23, p = 0.36 for GM-CSF; rs = 0.32, p = 0.19 for IL-3; and rs = 0.39, p = 0.11 for IFN-γ). It is possible that one or both of the other IL-5 family cytokines would correlate with the BAL eosinophil percentage if a higher number of subjects were studied. IL-5 also correlated inversely with the percentage of macrophages (data not shown), reflecting the decreased macrophage percentage as eosinophil percentage increases. Further, BAL fluid IL-5 after segmental challenge correlated strongly with the magnitude of the late-phase fall in FEV1 after whole-lung challenge (rs = 0.79, p < 0.0001). IL-3 also correlated with late-phase FEV1 fall, but less well (rs = 0.51, p = 0.03). GM-CSF (rs = 0.37, p = 0.12) and IFN-γ (rs = 0.36, p = 0.13) did not correlate with late-phase FEV1 fall.

Levels of β2 and reactivity with mAb24 of BAL eosinophils correlated significantly with the concentration of IL-5 in BAL fluid 48 h after segmental Ag challenge (Fig. 10), as did level of αM (rs = 0.70, p = 0.04) (data not shown). These integrins did not correlate with concentrations of the other cytokines (data not shown). There was no correlation with BAL fluid IL-5 before segmental challenge (data not shown). Integrins of blood eosinophils did not correlate with cytokine concentrations in BAL fluid 48 h after segmental Ag challenge (data not shown). Thus, a greater IL-5 concentration in BAL fluid in a subject is associated with greater αMβ2 up-regulation and β2 activation on BAL eosinophils after segmental challenge.

FIGURE 10.

Correlations between integrin activation and expression on BAL eosinophils and IL-5 in BAL fluid. Correlations between blood eosinophil expression of total β2 integrin (A) or activation-sensitive β2 integrin mAb24 epitope (B) and the concentration of IL-5 in BAL fluid 48 h after segmental Ag challenge. rs, Spearman rank correlation coefficient.

FIGURE 10.

Correlations between integrin activation and expression on BAL eosinophils and IL-5 in BAL fluid. Correlations between blood eosinophil expression of total β2 integrin (A) or activation-sensitive β2 integrin mAb24 epitope (B) and the concentration of IL-5 in BAL fluid 48 h after segmental Ag challenge. rs, Spearman rank correlation coefficient.

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We observed changes in integrins on eosinophils in the blood and airways of 19 subjects with mild allergic asthma after segmental bronchial allergen challenge that were related to the reactivities of subjects in the whole Ag challenge protocol. Eosinophils in blood 48 h after challenge had increased expression of the αD integrin subunit and, in asthmatics with a dual response phenotype, increased reactivity with the activation-sensitive anti-β1 integrin mAb N29 compared with before challenge. Eosinophils in BAL obtained 48 h after challenge had increased expression of β2 and αM integrin subunits and increased reactivity of activation-sensitive anti-β2 integrin mAb24 compared with eosinophils in blood. β2, αM, and mAb24 epitope expression on BAL eosinophils correlated with the concentration of IL-5 in BAL fluid obtained 48 h after segmental Ag challenge. In addition, αD expression and, in dual responders, N29 reactivity were higher on BAL eosinophils than on blood eosinophils before challenge. Integrin expression patterns on BAL eosinophils were more homogeneous and symmetric than on blood eosinophils. The observations of N29 epitope and αD expression are compatible with a scenario whereby extravasated eosinophils appearing in the airway are derived from a subpopulation of the total, more heterogeneous population of circulating eosinophils. However, the observations of αMβ2 expression, β2 activation state, and IL-5 levels in BAL fluid indicate that, after leaving the circulation, eosinophils undergo IL-5-triggered activation of β2 and up-regulation of αMβ2. Analysis of neutrophils and monocytes in the same blood samples revealed that αD expression increased on all three leukocyte types upon segmental challenge; thus, αD up-regulation may occur through a common mechanism. At baseline, N29 reactivity was higher on neutrophils than on eosinophils and even higher on monocytes. N29 reactivity increased significantly on neutrophils in dual responders upon challenge but to a relatively lesser degree than on eosinophils, whereas it did not change significantly on monocytes. The mechanisms responsible for maintaining the baseline β1 activation levels on the different leukocytes and those responsible for the elevation of activation state upon challenge are unknown. Finally, neutrophil and monocyte αM and monocyte β2 were up-regulated upon challenge, in contrast to eosinophil αM and β2, which were up-regulated in BAL but not in blood. Thus, circulating eosinophils are less sensitive than neutrophils or monocytes to increased surface expression of αMβ2.

β1 activation on blood eosinophils, assessed by reactivity with N29, has been shown to correlate inversely with FEV1 in an inhaled corticosteroid withdrawal study (35). The present results complement the steroid withdrawal study by demonstrating persistent β1 activation in dual responders with more eosinophilic inflammation in the airway. Blood eosinophils from dual responders, but not from single responders, have also been shown to have enhanced activation of FcγRII (CD32) as assessed with the phage mAb A17 6 h after challenge (42). Whether activation of β1 integrin and CD32 on circulating eosinophils are triggered by the same or different stimuli and signaling pathways is an interesting question that remains to be investigated.

The changes observed here in BAL eosinophils reproduce observations in the literature, made mostly on isolated eosinophils, that BAL eosinophils have higher αM and β2 than blood eosinophils (43, 44, 45, 46), activated αMβ as monitored by anti-active αM mAb CBRM1/5 (22, 36), and increased αMβ2-inhibitable adhesion to diverse ligands (22, 36). Our finding that dual responders have higher total numbers of cells and eosinophils and a higher proportion of eosinophils in BAL after segmental Ag challenge than single responders also is in accord with prior reports (47, 48, 49, 50). We also found that the β1 activation state and β2 surface expression were significantly higher on BAL eosinophils from dual responders than on those from single responders. Activation of β1 and β2 integrins, as assessed with the activation-sensitive mAbs N29 and mAb24, respectively, on BAL eosinophils correlated with eosinophil percentage in BAL, indicating that activation of both of these subfamilies of integrins is important for eosinophil recruitment. This idea is consistent with in vivo studies supporting the involvement of α4β1 in eosinophil appearance in the airway and with in vitro studies showing the involvement of both β1 and β2 integrins in eosinophil transendothelial migration (22, 51, 52, 53, 54, 55, 56). The evidence for involvement of both α4β1 and β2 integrins in eosinophil migration in vivo is further strengthened by a recent report that eosinophil recruitment to airway after Ag challenge was severely attenuated in both a conditional α4 integrin knockout mouse and a β2-deficient mouse (57). Finally, αD expression and β1 activation on blood eosinophils as well as β2 and αM expression and β2 activation on BAL eosinophils after segmental Ag challenge all correlated with the magnitude of the late-phase fall in FEV1 in response to whole-lung Ag challenge.

A number of mediators are increased in the airway after challenge (28, 29, 49, 58, 59). Of these, the most likely candidates to account for the up-regulation and activation of αMβ2 are the IL-5 family cytokines (36, 60, 61). Concentrations of IL-5, GM-CSF, IL-3, and IFN-γ in BAL fluid recovered 48 h after segmental Ag challenge were all increased significantly compared with those in BAL fluid recovered before challenge. IL-5 correlated with BAL eosinophil αM and β2 expression and β2 activation 48 h after segmental challenge. The median concentration of IL-5 in BAL fluid from dual responders after segmental challenge was ∼600 pg/ml, which was >20-fold the median concentration of the other cytokines. Further, the IL-5 concentration in the five BAL samples containing eosinophils with the most highly activated β2 was 100 to >1000 pg/ml. During the recovery of BAL, the volume of the fluid lining the airway epithelium in vivo is estimated to become diluted 100-fold (62). Thus, the levels of IL-5 in the epithelial lining fluid of these subjects (all of whom were dual responders) are estimated to be ∼10–100 ng/ml. Treatment of blood eosinophils in vitro with IL-5 at concentrations in this range is known to saturate the IL-5 receptor (63, 64, 65, 66, 67, 68) and lead to αMβ2 up-regulation and activation and induction of adhesion to ICAM-1 and other substrates (36, 60, 61), priming and enhanced response to chemoattractants (69), enhanced viability (70), degranulation and granule protein release (30), IL-5 receptor α down-regulation (71), and enhanced expression of certain genes (72). In contrast, median concentrations of the other IL-5 family cytokines before dilution are estimated to be ∼1–3 ng/ml. Thus, the concentration of IL-5 in the lining fluid in vivo, assuming that a significant portion of the IL-5 measured represents active IL-5, is estimated to be sufficient to cause αMβ2 up-regulation and activation of β2 integrins. These results are in accord with the earlier observation that the IL-5 receptor is down-regulated specifically compared with the GM-CSF receptor on eosinophils recovered by BAL 48 h after segmental Ag challenge (30). We also found that BAL fluid IL-5 correlated with the percentage of eosinophils in BAL and with the magnitude of the late-phase fall in FEV1. A correlation between BAL fluid IL-5 and eosinophil recruitment to the airway after segmental Ag challenge has been reported previously (38, 73, 74, 75, 76) and is consistent with the observation that anti-IL-5 therapy causes a significant decrease in sputum eosinophils (1).

Our study has several limitations. The data sets were incomplete in that all integrins were not assayed from all samples. Sampling of blood and BAL was performed only at one time point (48 h) after segmental Ag challenge. Future experiments with blood sampling at various time points after segmental and/or whole-lung Ag challenge are required to record the time course of αD and N29 expression on blood eosinophils in dual and single responders and to learn when the values diverge. Further, the importance of the integrin expression heterogeneity observed on blood eosinophils, particularly regarding β2 and αM, is not known. Such heterogeneity, to our knowledge, has not been described or discussed before. Of note, we subjected whole blood to primary Ab incubation without previous cell isolation or centrifugation. Earlier studies on blood eosinophil integrin expression have been performed on a buffy coat preparation or purified eosinophils (43, 44, 45). Dextran sedimentation during buffy coat preparation has been shown to cause up-regulation of αM on blood eosinophils (and neutrophils) compared to that in whole, unfractionated blood (77). Eosinophils are likely similar to neutrophils, which are known to store a large proportion of αM in granules, wherefrom it is translocated to the plasma membrane after cell stimulation (78). Remarkably, eosinophils in BAL had strong homogeneous labeling for β2 and αM. One possibility is that higher reacting subpopulations of circulating leukocytes represent cells that have undergone “retrograde” migration back to the blood from tissues, as has been demonstrated for zebrafish neutrophils in vivo (79) and human neutrophils in vitro (80). Blood sampling at various time points after Ag challenge, including times beyond 48 h, may shed further light on circulating eosinophil subpopulations.

Our results and the literature are compatible with the schematic of eosinophil recruitment after segmental Ag challenge that is depicted in Fig. 11. At baseline, β1 and β2 integrins on a circulating eosinophil are in a low activation state, the level of eosinophil surface αD is low, and VCAM-1 is absent from endothelial surfaces of the bronchial circulation. Yet to be identified stimuli resulting from Ag challenge cause enhanced activation of β1 integrins and increased surface expression of αD on circulating eosinophils. Because increased expression of the β1 activation epitope occurs on most eosinophils, the activation likely takes place in the pulmonary circulation through which the eosinophils constantly pass or in response to the release of an activating substance into the circulation (Fig. 11). Such activation contrasts with the model for the recruitment of leukocytes (16), including eosinophils (20), in which integrin activation is assumed to occur locally and concurrently with rolling and tethering on endothelium. Interestingly, bone marrow-derived progenitor cells or circulating eosinophils of asthmatics have been shown to be activated upon Ag challenge also as measured by the up-regulation of IL-5 receptor α and CCR3, the activation of CD32, and greater responsiveness to chemoattractants (42, 81, 82, 83). Further, eosinophils from allergic asthmatics have been shown to have a greater capacity to adhere to and transmigrate through endothelium than eosinophils from normal donors (51, 84).

FIGURE 11.

Sequential up-regulation and activation of eosinophil integrins during recruitment to airway after exposure to allergen. Schematic relating activation of β1 and β2 integrins to eosinophil trafficking during and after segmental Ag challenge. Left, An eosinophil (oval) with unactivated β1 integrins and low surface expression of αD is shown entering the pulmonary circulation of a segment subjected to Ag challenge. A yet to be identified stimulus or stimuli (X) cause(s) activation of β1 integrins and increased surface expression of αD during transit of the eosinophil (rectangle) through the vessel. Right, Concurrently, IL-4 and other mediators are released and specifically induce surface expression of VCAM-1 (discontinuous black line) on the endothelial cells of a bronchial blood vessel in the challenged segment. An eosinophil with activated β1 and up-regulated αD (rectangle) is shown entering the bronchial circulation. The eosinophil arrests and adheres (elongated rectangle) to VCAM-1. After exposure to IL-5 the eosinophil becomes responsive to chemotactic factors, migrates (arrow), and assumes a hyperadhesive airway phenotype (hexagon) in the lumen. The airway eosinophil displays activated β1, up-regulated αD, activated β2, and up-regulated αM and β2.

FIGURE 11.

Sequential up-regulation and activation of eosinophil integrins during recruitment to airway after exposure to allergen. Schematic relating activation of β1 and β2 integrins to eosinophil trafficking during and after segmental Ag challenge. Left, An eosinophil (oval) with unactivated β1 integrins and low surface expression of αD is shown entering the pulmonary circulation of a segment subjected to Ag challenge. A yet to be identified stimulus or stimuli (X) cause(s) activation of β1 integrins and increased surface expression of αD during transit of the eosinophil (rectangle) through the vessel. Right, Concurrently, IL-4 and other mediators are released and specifically induce surface expression of VCAM-1 (discontinuous black line) on the endothelial cells of a bronchial blood vessel in the challenged segment. An eosinophil with activated β1 and up-regulated αD (rectangle) is shown entering the bronchial circulation. The eosinophil arrests and adheres (elongated rectangle) to VCAM-1. After exposure to IL-5 the eosinophil becomes responsive to chemotactic factors, migrates (arrow), and assumes a hyperadhesive airway phenotype (hexagon) in the lumen. The airway eosinophil displays activated β1, up-regulated αD, activated β2, and up-regulated αM and β2.

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The question then arises as to how activated circulating eosinophils localize to the parts of the lung subjected to Ag challenge in preference to neutrophils and monocytes. As a parallel response to Ag challenge, mediators, including IL-4 and IL-13, are elaborated and activate the bronchial endothelium to specifically synthesize and express VCAM-1. VCAM-1 has been shown to be preferentially expressed in the asthmatic lung or after Ag challenge (85, 86). Further, VCAM-1 can support the adhesion of eosinophils but not neutrophils (9, 18, 22, 87, 88, 89, 90, 91). Circulating eosinophils with activated β1 and up-regulated αD are presumed to have a higher probability of arresting on VCAM-1 on the activated endothelium of the bronchial circulation (Fig. 11), because adhesion of purified blood eosinophils to VCAM-1 is mediated by α4β1 (22, 92), with a possible contribution by αDβ2 (22, 31).

αM andβ2 are shown as being up-regulated and β2 integrins as being activated by IL-5, also elaborated in response to allergen. Eosinophils appearing in the airway lumen have a hyperadhesive phenotype that is marked by activated β1 integrins, up-regulated αDβ2, activated β2 integrins, and up-regulated αMβ2 (Fig. 11). Eosinophils, particularly after priming by IL-5 (69), responds to chemoattractants, such as eotaxin (9, 13) (Fig. 11). αMβ2 is believed to be important for eosinophil migration (22, 51, 53, 93, 94, 95). Thus, the scenario has migrating eosinophils using activated αMβ2 and possibly other integrins to interact with multiple substrates (22, 36, 92), including VCAM-1, ICAM-1, and extracellular matrix proteins, on endothelial cells, in connective tissue, and on epithelium of the bronchial wall.

We thank Mary Jo Jackson and Erin Billmeyer for patient recruitment, screening, and assistance with bronchoscopy; Lin-Ying Liu, Sarah Panzer, Rebecca Lawniczak, and Rose DeGrauw for processing BAL samples and providing blood samples; Melissa Heim and Lisa Skoggs for assistance with ELISA; Nancy Hogg for providing mAb24; ICOS for providing anti-αD mAb 240I; Kathleen Schell, Joan Batchelder, and Joel Puchalski for help with flow cytometry data collection and analysis; Michael Evans for advice on statistics; and Julie Sedgwick and Lin-Ying Liu for advice.

M.W.J., W.W.B., and D.F.M. have, with the help of the Wisconsin Alumni Research Foundation, a patent application pending on “Beta1 integrin activation as a marker for asthma,” based on the associations between N29 reactivity and FEV1 reported by Johansson et al. 2006 (Ref. 35). W.W.B. has or has had research support from Novartis, Dynavax, Wyeth, Centocor, GlaxoSmithKline, Medicinova, Pfizer, and Biowa/MedImmune. E.A.B.K. and N.N.J. have no financial conflict of interest.

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

1

This work was supported by Specialized Center of Research Grant HL56396, Program Project Grant HL88594, and General Clinical Research Center Grant M01 RR03186 from the National Institutes of Health.

3

Abbreviations used in this paper: BAL, bronchoalveolar lavage; AgPD20, provocative dose of Ag producing a 20% fall in FEV1; FEV1, forced expiratory volume in 1 s; gMCF, geometric mean channel fluorescence.

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