Blood and bronchoalveolar eosinophils in allergic subjects after segmental antigen challenge: Surface phenotype, density heterogeneity, and prostanoid production

Respiratory

pathophysiologic

responses

Blood and bronchoalveolar eosinophils in allergic subjects after segmental antigen challenge: Surface phenotype, density heterogeneity, and prostanoid production Claus Kroegel, MD, PhD, Mark C. Liu, MD, Walter C. Hubbard, PhD, Lawrence M. Lichtenstein, MD, PhD, and Bruce S. Bochner, MD Baltimore,

Md.

Eosinophil infiltration into the airways has been implicated in the pathophysiology of asthma. To improve our understanding of the function of eosinophils in asthma, we have compared the phenotype and function of eosinophils obtained simultaneously from blood and bronchoalveolar lavage (BAL) of allergic subjects 19 hours after segmental lung allergen challenge. Eosinophils were purified by discontinuous density gradient centrifugation, and their distribution at various layers was quantitated. Eosinophils at the 1.080 to 1.085 gmlml interfaces from blood and BAL (purity > 70%) were analyzed by immunofluorescence and flow cytometry for several surface markers including adhesion-activation antigens. Eosinophils in BAL from antigen-challenged sites were markedly increased compared with control diluent-challenged BAL sites (0.3% k 1% vs 28.1% k 9.7%, n = 12, p < 0.002), and a greater percentage were hypodense (specific gravity < 1.080 gmlml) than in peripheral blood (51.3 +- 5.3 vs 19.0 ? 4.4, n = 15, p < 0.01). In vitro, resting and activated BAL eosinophils biosynthesized less thromboxane B, than blood eosinophils. Although both BAL and blood eosinophils expressed similar levels of Fc yRl1 (CD32), CD1 la, and CD45, resting levels of MO-I (CDllb) were upregulated on BAL eosinophils (mean fluorescence intensity, 316% ? 48% of blood eosinophils, n = 5, p < 0.05). Blood eosinophils stimulated in vitro with 1 pmol/L platelet activating factor or N-formyl-methionyl-leucyl-phenylalanine achieved levels of CDllb expression similar to those of BAL eosinophils. In contrast, CDllb expression on BAL eosinophils could not be further increased. These results confirm that antigen challenge of the lower airways promotes eosinophil infiltration in the lung. Because these eosinophils are of lower density, express maximal amounts of CDllb, and are less responsive than penbheral blood eosinophils, we conclude that eosinophils undergo activation during this recruitment process, which may be followed by a state of decreased responsiveness. (J ALLERGY Cm IMMUNOL 1994;93:725-34.) Key words: Bronchoalveolar eosinophils, phenotype, hypodense eosinophils, platelet activating factor, prostanoid generation

A characteristic feature of asthma is the presence of increased numbers of eosinophils in the circulation, sputum, and particularly in the bronchial tissue.‘.’ Such eosinophilia has been shown From Johns Hopkins Asthma and Allergy Center, Baltimore. Supported by the Bundesministerium fur Forschung und Technologie, DLR, Germany (OlKC890611) and the Kernforschungszentrum Karlsruhe, PUG, Germany (92/003/LUVA); and by grant AI27429 from the National Institutes of Health, Bethesda, Maryland. Received for publication Feb. 2, 1993; revised Sept. 28, 1993; accepted for publication Sept. 30, 1993. Reprint requests: Bruce S. Bochner, MD, Johns Hopkins Asthma and Allergy Center, Room 3A.62, 5501 Hopkins Bayview Circle, Baltimore, MD 21224-6801. Copyright 0 1994 by Mosby-Year Book, Inc. 0091-6749/94 $3.00 + 0 l/1/51804

to correlate with the severity of bronchial obstruction as measured by forced expiratory volume in 1 second (FEV,) and airway conductance,‘, 4. ’ and eosinophils inversely correlate with the sensitivity of the airways to methacholine.’ In addition, a recent study6 demonstrated that the number of eosinophils in the blood, bronchoalveolar lavage (BAL), and bronchial mucosa correlates with the degree of asthma, assessed by a clinical scoring method described by Aas.? Hence, it has been proposed that the eosinophil may be implicated in the pathogenesis of asthma.8 In agreement with this proposal, increasing evidence suggests that eosinophils represent a potent effector cell, which is equipped with a spectrum of proteolytic enzymes, cytotoxic proteins, reactive species of oxy725

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Abbreviations

ANOVA: BAL: FEV,

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used

Analysis of variance

Bronchoalveolar lavage Forced expiratory volume in 1 second FMLP: N-formyl-methionyl-leucylphenylalanine LT: Leukotriene mAb: Monoclonal antibody PAF: Platelet activating factor PE: Phycoerythrin PG: Prostaglandin PMA: Phorbol myristic acid

gen, and bioactive lipid mediators.” Indeed, in asthma damaged ciliated and other epithelial cells, desquamating epithelium, and bronchial plugging have been found in conjunction with an accumulation of eosinophils or deposits of eosinophil products in the airway wall.“-” The mature eosinophil is primarily a tissuedwelling cell, which is normally released at a low rate from the bone marrow.“’ I3 However, in inflammatory diseases such as asthma, eosinopoiesis may be increased and migration of eosinophils into tissue may be redirected, enabling the cells to actively migrate to the site of ongoing inflammation. Although the eosinophil is considered to be a prime effector cell in asthmatic inflammation, little is known about the cellular basis of eosinophil recruitment and the activation status of the eosinophils. Efforts to distinguish activated eosinophils of patients with hypereosinophilic diseases from eosinophils obtained from normal individuals have revealed morphologic,‘“, ” metabolic,‘” and functional differences.“, ” In addition, eosinophils from subjects with asthma comprised a higher portion of hypodense cells when these cells were compared with eosinophils from healthy subjects”, ” and exhibited a diminished capacity for synthesis of leukotriene C, (LTC4)” and chemiluminescence when stimulated with opsonized zymosan.“’ *3 Other investigators have reported that hypodense cells show an increased cytotoxic activity,“’ greater numbers of immunoglobulin and complement receptors,‘+ ” and enhanced release of inflammatory mediators after stimulation21, 26-28when compared with normodense eosinophils. Thus the pathogenetic significance of eosinophil heterogeneity in disease is far from understood and remains to be elucidated. In view of this lack of conclusive information, experiments were designed to examine the phenotypic and functional

CLIN IMMUNOL APRIL 1994

properties of eosinophils from blood and BAL obtained from allergic subjects before and after antigen challenge. METHODS Subjects Thirteen male and female allergic volunteers with asthmaor allergic rhinitis between the agesof 21 and 35 with no smokinghistorieswho had given informed consentparticipated in this study. Subjectswith asthma had a history of asthma,appropriate positive prick test resultsand an increasedmethacholinesensitivity. Subjects with allergicrhinitis had appropriate histories,had positive skin test results, and were unresponsive to methacholine. Every subject was free of symptomsat the time of the study, and none were receiving medication. Reagents Dextran, Ficoll-Paque, and Percoll were purchased from Pharmacia Fine Chemicals(Uppsala, Sweden). Platelet activating factor (PAF) and LTB, were purchased from Biomol Research Labs, Inc. (Plymouth Meeting, Pa.). Phorbol myristic acetate (PMA), Nformyl-methionyl-leucyl-phenyalanine (FMLP), and lyso-PAF were obtained from SigmaChemicalCo. (St. Louis, MO.). Unconjugated monoclonal antibodies (mAbs) recognizing CDllb (H5A4) and CDlla (MHM24) were provided by Dr. JamesHildreth (Johns Hopkins University, Baltimore, Md.) and mAb IV.3 recognizing CD32 was provided by Dr. Paul Guyre (Dartmouth Medical School, Hanover, N.H.). Leu M5 antibody, recognizingCDllc, waspurchasedfrom Becton-Dickinson (Mountainview, Calif.), HLA class I from Accurate Chemical Co. (Westbury, N.Y.), antiplatelet gpIIb (CD4lb, SZ.22) from AMAC Inc. (Westbrook, Maine), and CD23 (B6) and CD45 (KC56) from Coulter Corporation (Hialeah, Fla.). Phycoerythrin (PE)-conjugated goat anti-mouseIgG antibodieswere purchasedfrom Tago (Burlingame, Calif.). Airway

challenge and BAL

Late responsesto the instillation of antigen into the peripheral airways were examined in six subjectswith allergic rhinitis and sevensubjectswith asthma.Before bronchoscopy,spirometry was performed, and FEV, wasgreater than 75% of predicted value. Bronchoscopy was performed as previously described.” After premedication and induction of local anesthesia,a sham challenge was performed by instilling 5 or 10 ml of

normal saline solution into a subsegment of the lingula or right middle lobe. At a separatesite, allergen challenge (5 ml of ragweed,timothy grass,or Dermatophagoides farinae antigen diluted in normal saline solution to a concentration of 20 to 100 protein nitrogen units/ml) was performed. Subjectswere observedovernight in the Clinical ResearchCenter. After 17 to 22 hours (mean, 19 hours), a secondbronchoscopy was performed with BAL at the sitesof previoussalineand

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TABLE

I. Cell counts

Kroegel

in BAL approximately

19 hours

after segmental

antigen Late

Control

4.5 f 2.7 k 0.4 k 1.4 ? 0.1 i 0.3 t 2.5 L 1.3 ?

Total cell number (X 106) Macrophages( x 10h) Lymphocytes(x 10h) Neutrophils (X 10”) Eosinophils( x 10h) Epithelial cells (X 105) Mast cells (X 109) Basophils(X 104)

et al.

provocation

727

--

response Antiaen

0.8 0.4 0.1 0.5 0.0 0.0 0.1 0.1

29.3 t3.9 ‘3.1 t 5.8 t 16.7 + 0.2 + 15.3 t 38.1 ?

8.1” 1.4 03* 1.7 5.81 0.1 0.6$ l.Ol

Allergic subjects with rhinitis and/or asthma were challenged with antigen and physiologic saline solution at different sites as described in the Methods section. Thirteen subjects were challenged and observed overnight, and BAL was performed 17 to 22 hours later. *I, i 0.01. tp < 0.002. $p i 0.o.i.

antigenchallenges.Blood sampleswere collected at the time of each bronchoscopyfor differential cell counts and separationof eosinophils. Purification

of eosinophils

Discontinuous Pet-call density gradients. Eosinophils obtained from both blood and BAL fluid were separatedin parallel by meansof the following purification steps.Heparinized venousblood and BAL fluid recovered at the time of the late response(17 to 22 hours after challenge) from 13 patients with allergic asthmawas sedimentedwith 6% dextran for 90 minutes, and the buffy coat wascollected and washedtwice in Ca”+- and Mg’ ’ -free Hanks’ balanced salt solution. The cellswere suspendedin 2 ml Percoll (Pharmacia), density 1.065gmiml, supplementedwith 5% heat-inactivated fetal calf serum and layered on an &step discontinuousPercoll density gradient consistingof 1.5 ml 1.100 gm/ml, 2 ml 1.095 gmiml, 2 ml 1.090 gmiml, 2 ml 1.085gm/ml, 2 ml 1.080gmiml, 2 ml 1.075gm/ml, and 2 ml 1.070gmiml in a 15ml polyethylene tube. The gradientswere centrifuged at 15” C for 20 minutes,and eight density fractions were collected. Platelets were usually found in the eosinophil-poor 1.070 gm/ml to 1.080 gm/ml density fractions in decreasingnumbers. Although platelets could not be detected in eosinophilrich fractions ( > 1.080gm/ml), potentially contaminating platelets were hypotonically lysed, and cells were washedthree times in ice-cold Hanks’ balanced salt solution. The distribution of the leukocytes at each density interface and the removal of platelets was determined by counting in a hemocytometer with Kimura’s

stain.3” Adhesion

of platelets

to eosinophils

was further excluded by staining of cells with mAb against the (Ychain of IIbiIIIa (CD41) expressedby platelets and analysisof transmissionelectron micrographs.” In parallel, the meanviability of the recovered cells was assessed by trypan blue exclusion and calculated as 98.3%. Dextran sedimentation did not alter

eosinophil phenotype and function as measured by surfacemarker expressionand thromboxaneB, release (data not shown). Immunomagnetic separation. When prostanoid production was to be determined, additional purification was achieved with a modification of the indirect immunomagneticseparationtechnique describedelsewhere.” In brief, cell suspensions (1 to 4 x 10’) in 400 ~1were incubated with 100~1 of the mAb CD16 (1: 10 dilution; Medarex Inc., West Lebanon, N.H.) for 45 minutes at 4” C on a rotary mixer, and washedthree times in RPM1 medium (Gibco, Grand Island, N.Y.) containing 10% fetal calf serum.Cellswere then incubated with washedmagneticbeadscoated with sheep anti-mouseIgG (Dynal Corp., Rochester,N.Y.) (2 to 4 x 108/ml)at 4” C for 45 minutes (bead:target cell ratio 10 to 2O:l) on a rotary mixer. RPM1 with 10% fetal calf serumwasaddedto bring the volume up to 10 ml, and CD16-positive cells (neutrophils) adhering to the beadswere removed with three consecutivemagnetic extractions, leaving CD16-negativecells (eosinophils) in the solution. The remaining cell suspension was centrifuged and resuspendedin Hanks’ balanced salt solution. The purity of eosinophilsafter immunomagneticseparationwasgreater than 98%. Incubation

procedure

Purified eosinophilswere resuspendedin piperazine-N, N’-bis[2-ethanesulfonic acid]-buffered saline containing 0.003% human serum albumin, 0.1% Dglucose,1 mmol/L CaCl,, and 1 mmol/L MgCl,) at a concentration of 3.3 x 10’ eosinophilsiml,prewarmed for 5 minutes, and dispensedin 90 l.~laliquots. Stimulation wasinitiated by addition of buffer or stimulus(10 l.rl) asindicated and incubation for 5 minutesat 37” C. Reactionswere terminated by the addition of 1 ml of ice-cold acetone.Sampleswere processedfor measurement of prostanoid releaseby combined gaschromatography/massspectrometry.

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0

cl.065

CLIN IMMUNOL APRIL 1994

Blood (n = 14)

1.065

1.070

1.075 1.080

Density

1.085

1.090

1.095

1.100

(g/ml)

FIG. 1. Density distribution of eosinophils in blood and BAL fluid 19 hours after antigen challenge. Centrifugation of both blood and BAL eosinophils on Percoll yielded nine fractions with densities ranging from 1.065 to 1.100 gm/ml. When compared with blood cells the density distribution of BAL eosinophils showed a leftward shift toward lower densities. The percentage of hypodense eosinophils (defined as cells with a density ~1.080 gm/ml) was 19% k 4% for blood cells and 52% t 2% for BAL cells (p c 0.01). Data shown in the figure represent mean ?SEM.

Gas chromatography/mass spectrometry Sample preparation and derivatization for analysis by combined capillary gaschromatography/massspectrometry wasperformed with modificationsof a previously published procedure.33Mass spectrometric analysis of derivatized prostanoidswas performed with a Finnigan MAT TSQ-700 GC/MS/MS/DS (Finnigan MAT (USA), San Jose, Calif.), operated as a single quadrupole massspectrometer. Prostanoid identification and quantification were basedon retention times of endogenousspeciesrelative to tetradeuterated analogsof prostaglandin(PG)F,,, PGD,, thromboxaneB,, and 6-keto-PGF,, used asinternal standards.The concentration of each prostanoid was calculated as the integral area under the peak. Flow cytometric

analysis

cein isothiocyanate-conjugatedanti-CD16 to differentiate eosinophils(CD16-negative) from contaminating neutrophils (CD16-positive).‘* After washing,eosinophils were fixed in 1% paraformaldehydeand analyzed with a Coulter EPICS Profile flow cytometer. Three log decadehistogramsof PE fluorescencewere generated after appropriate gating and analysisof 5000 or more cells. Statistical analysis Unless otherwise stated, the data in the text and figures are expressedas mean ? SEM. All data were analyzed nonparametrically with the Wilcoxon matched-pairsignedrank test or the Mann-Whitney U test for paired and unpaired variates, respectively. When more than two groups of related data were compared, statistical evaluation was assessedby Kruskal-Wallace analysisof variance (ANOVA). Significance was accepted for p values of lessthan 0.05.

Aliquots of cells (1 to 4 x lo5 in phosphate-buffered saline/bovineserum albumin buffer) were labeled for RESULTS 30 minutes at 4” C with saturating concentrations of Cell recovery and differential cell counts unconjugated mAbs recognizing various surface molecules.Eosinophilswere washedand then resuspended After 17 to 22 hours (mean, 19 hours), total cell in saturating concentrations of PE-conjugated goat recovery of the antigen site increased by more anti-mouseIgG (30 minutes; 4” C). Negative controls than six times from 4.5 ? 0.8 x lo6 in the control consistedof cells labeled with isotype-matchedirrelsegment to 29.3 +- 8.1 x lo6 in the antigen-chalevant mAbs. For positive controls, antibody to HLA class I or CD45 antigen was used. In experiments lenged site (Table I). In addition, there were marked increases in the number of eosinophils examining CDllb expressionon eosinophilsat lower (p <: 0.002), lymphocytes, basophils (p < O.Ol), purity before and after stimulation, cellswere doubleand mast cells @ < 0.05). Neutrophils also inlabeled with PE-conjugated anti-CDllb and fluores-

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60

q

Blood

c

0.0001

Jo1

0.01 PM

0.1

1.0

10

WJ)

FIG. 2. A, Thromboxane B, biosynthesis from peripheral blood and BAL eosinophils stimulated with various stimuli in vitro. Highly purified ceils (obtained after density gradient centrifugation and immunomagnetic separation) were incubated with buffer (n = 6), 1 +mol/L PAF (n = 6), 1 hmol/L LTB, (n = 6), 10 rig/ml PMA (n = 4), or 1 kmol/L calcimycin (n = 6) for 5 minutes at 37” C. Cells were solubilized by the addition of ice-cold acetone, and the concentration of the mediator was estimated by means of gas chromatography/mass spectrometry. The results represent mean * SEM (Kruskal-Wallace ANOVA). *p I 0.05, **p < 0.01. 6, Concentration response curve of thromboxane B, biosynthesis from peripheral blood and BAL eosinophils stimulated with PAF in vitro. Highly purified cells were incubated with increasing concentrations of PAF for 5 minutes at 37” C. Cells were solubilized by the addition of ice-cold acetone, and the concentration of the mediator was estimated by means of gas chromatography/mass spectrometry. The results represent means r SEM obtained from three independent experiments. The differences were significant at all concentrations tested (p < 0.05; Kruskal-Wallace ANOVA).

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podense cells constitute a significantly higher proportion. The data shown in Fig. 1 suggest that eosinophils may undergo a density shift during recruitment from the blood or that there is preferential recruitment of hypodense eosinophils.

CDlla

Prostanoid generation CD45

CD32

Fluorescence intensity FIG. 3. Surface expression of blood (gray lines) and BAL (black lines) eosinophils obtained 19 hours after challenge. Aliquots of eosinophils isolated without immunomagnetic separation were labeled with antibodies recognizing CD1 la (MHM24), CD1 lb (H5A4), CD32 (IV.3) or CD45 (KC56) and were analyzed by flow cytometry. The dashed vertical line represents maximal background nonspecific binding of irrelevant IgG antibody (control). The results are from a single experiment representative of three independent experiments.

creased from 1.4 1 0.5 x lO“/ml in the control segment to 5.8 ? 1.2 x 104/ml after antigen challenge, but the difference was not statistically significant. No difference was observed for the number of macrophages or epithelial cells in the lavage fluid, nor were any differences observed between subjects with allergic rhinitis and those with allergic asthma. The results clearly demonstrate that antigen challenge of airways in allergic subjects induces an inflammatory cell recruitment that is numerically predominated by eosinophils. Density distribution

profiles

Cell suspensions obtained from blood and BAL 17 to 22 hours after bronchoalveolar antigen challenge were fractionated by means of Percoll gradients with densities ranging from 1.065 gm/ml to 1.100 gm/ml. Cells were removed from each density interface and counted in a hemocytometer with Kimura’s stain. As demonstrated in Fig. 1, comparison of the density distribution revealed a slight leftward shift of the BAL eosinophils toward lower densities. The percentage of hypodense eosinophils (defined as cells with a density < 1.080 gm/ml) was 19% f 4% for blood eosinophils and 52% -+ 2% for BAL cells @ < 0.01; Kruskal-Wallace ANOVA). These data demonstrate that among the BAL eosinophils obtained during the late-phase response, hy-

It has been previously demonstrated that human blood eosinophils obtained from allergic subjects generate a spectrum of prostanoids.‘S To characterize the functional properties of eosinophils obtained from blood and BAL, platelet-free eosinophils were incubated for 5 minutes at 37” C with vehicle, PAF (1 PmoliL), LTB, (1 lJ,mol/L), PMA (10 ngiml), or calcimycin (1 pmol/L). Lipids were extracted as described in the Methods section and processed for prostanoid analysis. There was a significant difference in thromboxane B, biosynthesis between eosinophils obtained from blood and those obtained from BAL (Fig. 2, A). Both spontaneous and stimulated thromboxane B, synthesis were significantly lower in BAL eosinophils than in blood eosinophils obtained in parallel (control, 0.5 5 0.1 vs 8.7 + 1.1 pg/lO” cells; PAF, 8.1 ? 0.9 vs 28.9 I- 3.4 pg/lO’ cells; LTB,, 4.4 t 1.3 vs 10.2 -+ 3.5 pg/lO” cells; PMA, 4.9 ? 1.6 vs 13.9 ‘-t 4.7 pg/lO’ cells; and calcimytin, 21.8 2 5.9 vs 50.4 ? 8.6 pgilO^ cells). The results indicate that BAL and blood eosinophils differ in their spontaneous and agonist-induced releasability as measured by thromboxane B, synthesis. To further define the reduced responsiveness of the BAL cells, eosinophils obtained from the blood and BAL approximately 19 hours after segmental antigen challenge were incubated with increasing concentrations of PAF (100 pmol/L to 10 pmol/L). As shown in Fig. 2, B, diminished thromboxane B, production by BAL eosinophils occurred at all concentrations tested. These data support the results mentioned above and indicate that the hyporesponsiveness represents a decrease in cellular sensitivity to stimulation with agonists. Phenotype

of blood and BAL eosinophils

Aliquots of normodense eosinophils were incubated with a panel of antibodies recognizing CDlla, CDllb, CDllc, CD23 (FceRII), CD32, and CD45 and labeled cells detected by flow cytometry as described in the Methods section. As shown in Fig. 3, blood eosinophils expressed CDlla, CDllb, CD32, and CD45 epitopes on the membrane surface. In contrast, expression of CDllc and CD23 (FccRII) could not be detected (not shown). When compared with blood cells,

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TABLE

II. Effect of PMA, FMLP, and PAF on blood Experiment

and BAL eosinophil

1

Experiment

expression

of CD1 1 b

2

Experiment

3

Stimulus

BAL

Blood

BAL

Blood

BAL

Blood

None PMA ( 10 @ml) FMLP (1 kmol/L) PAF (i Fmol/L) Eosinophil purity (%)

22 23 27 25 63

9 31 16 23 5

58 61 60 60 85

27 41 32 49 78

29 28 31 31 99

13 20 19 31 3x

Eosinophils recovered from blood and BAL of subjects with asthma 19 hours after challenge were incubated with the indicated stimuli for 10 minutes at 37” C before labeling for CDllb expression. Data shown represent the results from three independent experiments. Values are expressed as mean fluorescence intensity.

BAL eosinophils showed an increased expression of CDllb, whereas expression of CDlla, CD32, and CD45 remained unchanged. In five separate experiments an increase of 316% -t- 48% of control CDllb mean fluorescence intensity (p < 0.05) between blood and BAL eosinophils was demonstrated (control CDllb mean fluorescence intensity = 7 L 1). In vitro upregulation

of CD1 1b expression

To determine whether cells were capable of further upregulating expression of CDllb, eosinophils from blood and BAL obtained 19 hours after allergen challenge were incubated with PMA (10 @ml), FMLP (1 kmol/l), or PAF (1 pmol/L). As demonstrated in Table II by the data obtained from three independent experiments, stimulation of blood eosinophils resulted in upregulated CDllb expression to levels less than or equal to those seen on unstimulated BAL eosinophils. In contrast, CDllb expression on BAL eosinophils could not be further increased, suggesting maximal activation had already occurred in vivo. DISCUSSION

Allergen challenge of allergic subjects results in a characteristic inflammatory response that develops over a period of many hours. For example, histamine, PGD,, PGE,, and LTC, are elevated 19 to 48 hours after allergen challenge, although mediator levels are lower than in the early response. In addition, a substantial cellular influx of eosinophils, lymphocytes, and basophils can be found.“b-4” The present findings are in agreement with results of these studies and indicate that the influx of certain leukocytes (especially eosinophils) during the antigen-induced late response involves their preferential recruitment into the airway lumen.

Eosinophils comprise a heterogeneous population of cells when evaluated for their density. Although normodense eosinophils represent approximately 90% of the cells found in normal subjects,‘“, *‘. 4’ peripheral blood eosinophils from patients with certain eosinophil-associated diseases such as allergic rhinitis, asthma, allergic bronchopulmonary aspergillosis, and helminthic infestation show an increased proportion of hypodense cells.“‘, ‘(‘. “. 4’, 42 Assessment of the density distribution presented here shows that the percentage of hypodense eosinophils obtained by BAL 19 hours after segmental antigen challenge represents a significantly higher proportion than that in blood (52% ? 2% vs 19% t 4%). These data are in agreement with a previous study in which increases in hypodense eosinophils were seen in BAL fluids obtained 48 hours after challenge.37 However, consistently higher numbers of hypodense eosinophils have been demonstrated in BAL and pleural fluids from patients with chronic eosinophilic pneumonia and other nonallergic lung diseases.‘“. 41. 43. 44 Thus the significance of hypodense eosinophils in various diseases is not fully appreciated. Functionally, a number of differences have been demonstrated between normodense and hypodense eosinophils obtained from blood of patients with eosinophil-associated disease. These include differences in membrane receptor expression, cytotoxicity to immunoglobulin-coated targets, metabolic rate, and responsiveness on stimulation with cell activators.22. “, 2x,as.4h In another study, Sedgwick et a1.47 examined eosinophils of similar density from blood and BAL 48 hours after endobronchial allergen challenge. A number of in vitro functions, including superoxide production, adhesion to biologic surfaces, calcium responses, and survival were generally enhanced in BAL cells compared with blood cells. Other stud-

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ies indicate that hypodense eosinophils may also occur in a hyporeactive state. For instance, hypodense cells obtained from patients with asthma produced a lower amount of reactive oxygen metabolites in response to zymosan, as assessed by nitroblue tetrazolium reduction and chemiluminescence,“. 4x and low-density eosinophils obtained from patients with asthma produced only half the amount of LTC, compared with normodense cells.” Although the biologic implications are not yet clear, the data presented here demonstrate that BAL eosinophils show a reduced capacity to generate thromboxane B, than blood eosinophils, offering another example of diminished rather than augmented responsiveness of hypodense eosinophils. One explanation for our observation may be that an array of mediators is released during late-phase responses,“. “, 47 which may be responsible for both chemotactic attraction to the inflammatory focus and stimulation of cellular effector functions. Thus the attenuated capacity of BAL eosinophils to release prostanoids after stimulation in vitro may be due to a cellular desensitization process like that previously described for eosinophils.““, “’ Interestingly, neutrophils recruited to the lung after segmental antigen provocation have also been found to be desensitized to LTB,,” suggesting that desensitization of tissue-infiltrating cells may be a general process. If this is true, we hypothesize that hypodensity of eosinophils may not simply be associated with cellular hyperactivity and may occur both in hyper- and hyporeactive states. The functional condition of eosinophils in relation to their density may be variable, depending on factors within the microenvironment of the lung. This hypothesis could explain the conflicting data published regarding the functional state of hypodense cells. Our results demonstrate, in addition, that eosinophils, which migrate into the lung after antigen challenge, constitute not only an increased proportion of hypodense cells but also show a three-fold increase in CDllb expression, whereas expression of CDlla and CD32 did not change, which is in agreement with published reports from our laboratory and elsewhere? 47 CDllbKD18 (also known as Mac-l, MO-~, or CR3) is a member of a leukocyte surface glycoprotein family known as integrins. These surface structures are believed to mediate various cell-cell interactions and may also modulate leukocyte effector functions.- Our observation that stimulation of blood eosinophils with PAF, FMLP, or PMA in vitro

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upregulates the CDllb epitope to approximately the same levels as that observed in BAL cells but does not induce any further upregulation of CDllb on BAL eosinophils indicates that CD 1lb upregulation has already occurred. Because CDllb is not specifically upregulated on hypodense compared with normodense eosinophils,“’ altered CDllb expression may instead be a consequence of the process of transendothelial migration itself or exposure to chemotactic mediators or cytokines released during the inflarnmatory process in vivo.74. 54, 55 Thus eosinophils undergo both phenotypic and functional changes during their migration into the bronchial lumen. Taken together, our data show that BAL eosinophils obtained approximately 19 hours after segmental allergen challenge differ from blood eosinophils in several respects. BAL eosinophils constitute a high proportion of hypodense cells, show increased CDllb expression, and have an attenuated capacity to generate prostanoids. Because hypodensity and expression of CD1 lb can be induced by exposure of eosinophils to various stimuli in vitr0,‘4* 56, ” these phenotypic changes may indicate that eosinophils undergo activation during migration from the blood into airways. The reduced capacity of BAL eosinophils to generate thromboxane B, may be due to cellular desensitization after activation of the cells by mediators released during the ongoing inflammatory process, allowing the cell to remain at the site of inflammation. Although as yet undetermined, desensitization may be transitory and exposure of eosinophils to locally secreted cytokines may further reduce the density of cells and in turn increase cellular reactivity. Studies are currently under way to test this hypothesis. We thank Dr. James Hildreth for generously providing monoclonal antibodies and Ms. Bonnie Hebden for exceptional efforts in the preparation of this manuscript. REFERENCES 1. Lowell FC. Clinical aspects of eosinophilia in atopic disease. JAMA 1967;202:875-8. 2. Horn BR, Robin ED, Theodore J, Kessel AV. Total eosinophil counts in the management of bronchial asthma. N Engl J Med 1975;292:1152-5. 3. Baigelman W, Chodosh S, Pizzuto D, Cupples LA. Sputum and blood eosinophilia during corticosteroid treatment of acute exacerbations of asthma. Am J Med 1983;75:929-36. 4. Burrows B, Hasan FM, Barbee RM, Halonen M, Levowitz MD. Epidemiological observations on eosinophilia and its relation to respiratory disease. Am Rev Respir Dis 1980; 122:709-15.

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