Allergen-induced Asthmatic Responses Relationship Between Increases in Airway Responsiveness and Increases in Circulating Eosinophils, Basophils, and Their Progenitors 1 - 3

PETER G. GIBSON,4 PATRICK J. MANNING, PAUL M. O'BYRNE, ADELE GIRGIS-GABARDO, JERRY DOLOVICH, JUDAH A. DENBURG, and FREDERICK E. HARGREAVE

Introduction

Allergen inhalation causes airway inflammation (1, 2) and increased airway responsiveness (3), both of which are important characteristics of clinical asthma (4-6). The airway inflammatory response to allergen inhalation is characterized by an initial, temporary influx of neutrophils (7) followed by a sustained accumulation of eosinophils (Eo) (1, 2, 7) and basophils (B)(8).The eosinophilia after allergen inhalation corresponds to that seen in clinical asthma (6), and the sustained nature of this eosinophilic response suggests increased production of these cells. Eosinophils and B arise from a common progenitor cell, called the Eo-B colony-forming unit (Eo/B-CFU) (9). This cell circulates in increased numbers in allergic individuals (10)and fluctuates with exacerbation and resolution of clinical asthma (11). The eosinophilia and basophilia associated with allergen inhalation and clinical asthma could be reflected by an increasein progenitor cells committed for Eo/B differentiation. In this study we evaluated circulating granulocyte progenitors and peripheral blood Eo and B in allergicasthmatic subjects before and after allergen inhalation and related these to the pattern of response in airway function. The changes in airway function that occur after allergen inhalation include an immediate fall in FEV t (early asthmatic response), a late fall in FEV t beginning 3 to 4 h after inhalation, and an increase in histamine airway responsiveness at 24 h. As it is this latter aspect that is characteristic of symptomatic clinical asthma, we have related the peripheral blood changes to the occurrence of increased histamine airway responsiveness after allergen inhalation.

SUMMARY The Inflammatory response during allergen-Induced asthma was a_ _d using serial measures of peripheral blood eoslnophlls (Eo), basophlls (8), and Eo/8 progenitor cells (Eo/&CFU). A group of 14 stable asthmatic Individuals Oh-agonlsts only 88 needed) had Inhalation provocation tests with allergen (18 tests In total) .nd with diluent. Serl.1 blood sampl.s were taken before and 1 and 24 h after the tests; methylcellulose cultures for Eo/B-CFU.nd granulocyt.macrophage (GM-eFU) were scored .t 14 d8Y8. Clrcul.tlng Eo, 8, and Eo/B-CFU were Incre88ed at 24 h after allergen Inhalation when this resulted In Increased histamine airway responsiveness (n 13). In the 5 subjects with Isol.ted early asthm.tlc responses the Eo, 8, and Eo/B-CFU counts did not change. There was no change In the GM-eFU after .lIerg.n. The ratio ch.nge In circulating Eo/&0.8,P < 0.05). CFUwas negatively correlated with baselln. histamine airway responsiveness (r Four subjects who had an Isolated early response and no blood chang.s to one all.rgen developed an Incre.se In histamine airway responsiveness and an Increase In Eo, 8, .nd Eo/8 progenlto... after Inh.latlon of. second different ....rgen. The results Indlc.te that In subjects with an allergenInduced Increase In histamine airway responsiveness, an Inflammatory response occu ... that Includes an Increase In the number of Eo/8 progenlto.... This response, possibly mediated by Eo/8 growth and dlfferentl.tlon facto..., could lead to the accumulation of these c.lIs In the .Irway .nd contribute to the airways Inflammation present In asthma. AM REV RESPIR DIS 1991; 143:331-335

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Methods Subjects A group of 14 atopic subjects with current or seasonal asthma were studied at a time when the asthma was mild, stable, and treated by inhaled bronchodilator alone. All had a baseline FEV 1 > 70070 of the predicted normal on all study days. None were currently exposed to allergens to which they were sensitized, with the exception of the housedust mite. The study was approved by the Research Committee of McMaster Health Sciences Centre, and each subject gave written informed consent. Study Design and Procedures Documentation at entry included subject characteristics, skin prick test sensitivity to allergen extracts, and baseline airway responsiveness to histamine, expressed as the provocation concentration that caused a 20070 fall in FEV 1 (PC 20 ) (12). A control inhalation test with phosphate-buffered saline in 1.4070 benzyl alcohol, the diluent of the allergen extract, was carried out, and a histamine inhalation test was repeated 24 h after this. Extracts of Dermatophagoides pteronyssinus (100,000 allergy units/ml) were obtained from Pharma-

cia (Dorval, Quebec). The remaining extracts (1:20 wt/vol) were obtained from Bencard (Mississauga, Ontario). Extracts were stored in aliquots at - 20 0 C and diluted for skin tests or allergen inhalation on the day they were to be used. For each allergen test, subjects attended the laboratory on 3 consecutive days. Inhaled P2agonists werewithheld for at least 8 h. A histamine inhalation test was performed on the first day. An allergen inhalation test (3, 13)

(Received in original form April 2, 1990 and in revised form August 27, 1990) 1 From the Asthma Research Group, Departments of Medicine and Pediatrics, McMaster University and St. Joseph's Hospital, Hamilton, Ontario, Canada. 2 Supported by the Medical Research Council of Canada. 3 Correspondence and requests for reprints should be addressed to Dr. F. E. Hargreave, Firestone Regional Chest & Allergy Unit, St. Joseph's Hospital, 50 Charlton AvenueEast, Hamilton, Ontario, Canada L8N 4A6. .. Boehringer Research Fellow.

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GIBSON, MANNING, O'BYRNE, GIRGIS-GABARDO, DOLOVICH, DENBURG, AND HARGREAVE

was performed in the morning of the second day, and a histamine inhalation test was repeated on the third day. A subject was considered to have a definite late asthmatic response if there was a fall in FEV! of 15'10 or more between 3 and 7 h after allergen inhalation (3, 13) and an increase in histamine airway responsiveness if there was a 1.5-fold or greater decrease in histamine PC20 after 24 h. To examine whether the effects were dependent on the allergen used, four subjects with an isolated early asthmatic response consented to an inhalation test with a second different allergen to which they were sensitized. These repeat allergen tests were performed at least 14 days after the first. Blood was sampled immediately before and then 24 h after the baseline histamine inhalation test in nine subjects, and before and at 1 and 24 h after each allergen inhalation. The baseline and 24 h samples were collected each morning, at the same time of day, to control for the diurnal variation in cell counts.

Procedures Cellcounts and methylcellulose cultures. All counts were performed using coded slides, with the investigator blind to the clinical characteristics of the subject. Peripheral blood Eo and B counts were obtained for each subject from a white cell count (Coulter) and 1,000 cell differential counts of two smears stained with May-GriinwaldOiemsa (MOG). Methylcellulose cultures were performed using washed, low-density nonad-

herent peripheral blood cells that were cultured [l X 106 per 35 x 10 mm tissue culture dish (Falcon Plastics, Oxnard, CAl] in supplemented Iscove's modified Dulbecco's medium (with 1% penicillin-streptomycin and 2-mercaptoethanol), 200/0 fetal calf serum, 0.90/0 methylcellulose, and 50/0 vol/vol conditioned medium from a human T cell line (MO) (9, 10). Day 14 granulocyte colonies of ~ 40 cells were enumerated and classified as either Eo/B type (tight, compact, round refractile cell aggregations) or as neutrophilmacrophage type (OM; loose and irregular under inverted microscopy) (9, 10). For each culture of cells from a given subject, six Eo/Btype and six OM-type colonies were picked at random from replicate dishes, placed on slides, and stained with MOO for morphologic evaluation and differential counting. When possible an additional six Eo/B-type colonies were placed in 100 III phosphatebuffered saline for histamine assay. A proportion of Eo/B colonies have been demonstrated to consist almost entirely of basophilic cells, and this is corroborated by the amount of histamine present in the colony (9, 10). The precursor of such a colony has been operationally defined as a B colonyforming unit (CFU-B) (9, 10). A colony was considered B positive if a count of up to 200 cells demonstrated ~ 10010 basophilic granulecontaining polymorphonuclear leukocytes (9, 10). The metachromatic nature of the granules in cells in Eo/B colonies was confirmed using 0.50/0 toluidine blue at pH 0.5, and the

probable relationship of these cells to the blood B has been shown by immunophenotypic analysis (14). Histamine assay was performed using isotopic radioenzyme conversion, with the lower limit of sensitivity 0.1 ng/colony. A colony was considered histamine positive if it contained ~ 0.20 ng after adjustment for dilution (9, 10).

Analysis Results are reported as arithmetic mean with standard error of the mean (SEM) in parentheses, except PC 20 values, which were logarithmically transformed and are expressed as geometric mean and geometric standard error of the mean. Repeated measures analysis of variance and Newman-Keuls test were used to examine for differences in cell counts, with significance accepted at the 95 % level.

Results

Three patterns of airway response to allergen inhalation were observed (table 1). First, an isolated early asthmatic response with no change in airway responsiveness to histamine and no definite late asthmatic response occurred on five occasions (Allergen Tests 1through 5). Second, an early asthmatic response with an increase in airway responsiveness to histamine and no definite late asthmatic response occurred after four allergen tests (6a, 7a, 9a, and 11). Third, an early asth-

TABLE 1 SUBJECT CHARACTERISTICS AND ALLERGEN-INDUCED AIRWAY RESPONSES·

Allergen Test Isolated early response 1 2 3 4 5 Mean SEM Late response and/or increased responsiveness 6a 7a 8a 9a 10 11 12 13 14 15 16 17 18 Mean SEM

Age (Gender)

Allergen Inhaled (Dilution)

25 (F) 34 (F) 23 (M) 26 (M) 37(M)

D. farinae Ragweed Ragweed D. farinae D. farinae

25 (F) 34 (F) 23(M) 26 (M)

Cat (1:128) D. farinae (1:64) D. farinae (1:32) Cat (1:8) Grass (1:64) Ragweed (1: 128) D. farinae (1:32) Ragweed (1: 16) D. farinae (1:8) D. farinae (1: 16) D. farinae (1:256) Ragweed (1:128) Dog (1:16)

30 (F) 23 37 23 23 37 46 27 22

(M)

(F) (M) (F) (M) (F) (M) (M)

(1:128) (1: 128) (1:32) (1:64) (1:1,024)

PC 20 Histamine

EAR

20 11 18 18 14 16.2 1.6 18 22 22 24 36 42 20 37 23 28 15 11 20 24.46 2.5

LAR

Before Allergen

After Allergen

14 9 8 1 14 9.2 2.3

1.45 7.36 6.50 6.47 0.31 2.69 1.85

1.77 6.95 10.00 6.40 0.36 3.09 1.85

11 13 21 11 25 6 34 17 38 51

1.85 6.80 7.20 8.00

0.47 3.90 1.72 3.35 11.50 0.62 1.23 2.16 0.87 0.77 0.72 6.70

29 20 27 23.31 3.50

> 32 10.00 5.28 6.06 11.20 5.30 1.69 > 32 0.21 7.24 1.29

1.72t 1.34

Definition of abbreviations: EAR = magnitude of the early asthmatic response, % fall in FEV1; LAR = rnaqmtude of the late asthmatic response, fall in FEV1; PC20 = provocation concentration of histamine causing a 20°1i> fall in FEV1. * Subjects of Allergen Tests 1 through 4 underwent challenge on a second occasion with a different allergen (Allergen Tests 6a through 9a). t p < 0.05 versus PC20 before allergen.

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PROGENITORS IN ALLERGEN-INDUCED ASTHMA 70

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Fig. 1. Circulating eosinophillbasophil progenitors (Eo/B-CFU , left) and neutrophil-macrophage (GM-GFU,right) expressed as colony-forming unitsl1O" nonadherent mononuclear cells before and 24 h after allergen inhalation in subjects with allergen-induced increases in histamine airway responsiveness. Assays for progen itors were not performed during two allergen tests. *p < 0.05. Solid squares represent mean values.

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Fig. 3. (Left) Basophil-positive (open bars, n ~ 8) and histamine-pos itive (hatched bars, n = 5) colonies expressed as colony-form ing units/10" nonadherent mononuclear cells before and 24 h after allergen inhalation in subjects with allergen-induced increases in histamine airway responsiveness. *p < 0.05. Mean ± SEM. (Right) Mean (± SEM) metachromatic cells per colony (open bars, n ~ 8) and histamine content per colony (hatched bars, n = 5) in SUbjects with allergen-induced increases in histamine airway responsiveness. *p < 0.05.

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circulating cells were not significantly different before and 24 h after histamineinduced airway constriction (table 2; p > 0.05). In four subjects who had an isolated early response to one allergen (Tests 1 through 4), inhalation challenge with a second different allergen (Tests 6a through 9a) resulted in an increase in histamine airway responsiveness and increased circulating Eo/B-CFU 24 h after inhalation of the second allergen (figure 4; p < 0.05). The inflammatory response was more closely related to the allergen-induced change in airway responsiveness than to the late asthmatic response. The odds ratio for the association between the presence or absence of a definite late asthmatic response and the directional change in Eo/B progenitors (increased versus no change or decreased) was 3.26 compared with an odds ratio of 10.51 for the association between the development of airway hyperresponsiveness after allergen inhalation and the directional change of circulating progenitors. Discussion

The inflammatory reaction is an important characteristic of clinical asthma. ..J ..J Using allergenchallenge. wehavedemon~ 40 . 8 Cl w 20 c 0 « strated a selective increase in circulating W 0 15 z 30 .6 z Eo/B progenitors and mature Eo and B co ~ :i 0 :::Ii 10 ::: 20 when allergen results in increased hista.4 ;! 0 :J III a: u.. :;: mine airway responsiveness. The eleva:t: o 10 5 0 .2 ;! tion of Eo/B progenitors suggests that ~ w ~ 0 0 0 :::Ii increased production of Eo and B may Before aefore 24hr After contribute to the accumulation of these cells in the airway. To ensure that the observed changes were due to allergen inhalation, extraneous factors known to influence circulatmatic response with an increase in hista- sponse, were unchanged (p > 0.05). ingleukocytes, such as diurnal variation mine airway responsiveness and a defi- GM-CFU were unchanged after allergen and medication use. were specifically nite late asthmatic response occurred in inhalation (p > 0.05). controlled. In addition, the effects of airIn subjects who developed increased way constriction alone with a nonsennine of the allergen tests. For further analysis the results of those tests result- histamine responsivenessthe ratio change sitizing stimulus, histamine, were examing in increased histamine airway respon- (after to before) in circulating Eo/B-CFU ined and found not to increase the cirsiveness were combined (allergen tests 6a after allergen correlated closely with the culating leukocytes. baseline levelof histamine airway responthrough 18). The increase in Eo at 24 h in associaWhen allergen inhalation caused an in- siveness, expressed as PC10 (r = - 0.82. tion with allergen-induced late asthmatcrease in histamine airway responsive- P < 0.05), but not the ratio change in PC.o ic responses confirms the work ofothers ness, this was associated with a signifi- (p >0.05). Baseline histamine airway re- (13, 15-17). However, the observation of cant elevation in circulating Eo, B, and sponsiveness was also correlated with cir- increases in circulating Band Eo/B proEo/B-CFU at 24 h (figures 1 and 2; p < culating Eo before allergen (r = -0.69, genitors is novel, as is their closer relationship to prolonged increases in hista0.05). Basophil-positive and histamine- p < 0.05). When allergen inhalation resulted in mine airway responsiveness. positive colonies were also increased at Peripheral blood was sampled in 24 h, as were the mean percentage of an isolated early asthmatic response, no metachromatic cells per colony and the significant changes in peripheral blood preference to bone marrow for several mean histamine content per colony (fig- Eo, B, or circulating Eo/B progenitors reasons: it is less invasive, the results of ure 3; p < 0.05). Cell counts 1 h after were observed at 1 and 24 h after aller- repeated marrow sampling may be less allergen inhalation, during the early re- gen inhalation (table 2). Similarly, these reproducible owing to variable dilution ~

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334

GIBSON, MANNING, O'BYRNE, GIRGI8-GABARDO, DOLOVICH, DENBURG, AND HARGREAVE

TABLE 2

T cell-derived cytokines could interact with progenitor cells to stimulate an increase in the number of colonies. Further elucidation of this point is being purIsolated Early Response (n = 5) Histamine Test (n = 9) sued by an examination of changes in cirBefore 1 h After 24 h After Before 24 h After culating progenitors cultured in the 242 (98) 209 (90) 191 (87) 265 (132) 350 (163) Eosinophils presence and absence of T cells. 54.2 (11.1) 31 (4.2) 42 (10.8) 52.6 (13.7) Basophils 30 (9.6) In this study the development of the 26.5 (7.7) 18.4 (3.2) Eo/B-CFUt 18.2 (9.7) 38.9 (13.7) 19.7 (9.3) inflammatory response was dependent GM-eFUt 7.5 (2.1) 5.6 (1.0) 5.9 (2.7) 7.8 (3.9) 5.8 (2.9) upon the allergen used, as previously * Mean (SEM). reported (29), and the magnitude of this t Eosinophil/basophil (Eo/B) and neutrophil-macrophage (GM) progenitors expressed as colony-forming units (CFU) per 10 response was related to the baseline level nonadherent mononuclear cells ("JAMC) plated. of airway responsiveness. As the baseline level of airway responsiveness is Fig. 4. Circulating eosinophil/basophil 60 60 closely related to the number of airway progenitors (Eo/B-CFU) expressed as 50 50 metachromatic cells and Eo (4, 5), it is colony-forming unitsl10' nonadherent :::» U. 40 possible that the Eo accumulation of in40 mononuclear cells before and 1 and 0 flammatory cells in the airway is depen24 h after allergen inhalation. Inhalation 30 30 a:a challenge with one allergen (left) resultdent on factors derived from these cells. ....... 20 20 0 ed in an isolated early response and no W Although previously characterized 10 10 significant change in Eo/B-CFU (p > chemotactic factors from metachromat0.05).Challenge with a second different 0 24hr ic cells seem unable to explain the Eo ac1hr Before allergen (right) resulted in increased After After histamine airway responsiveness and cumulation in asthma (30), the recent obelevated Eo/B-CFU at 24 h (p < 0.05). ALLERGEN 1 ALLERGEN 2 servations that metachromatic cells produce growth factors, including OM-CSF, upon IgE-dependent stimulation (31,32) by blood (18), and sampling of peripher- inflammatory cell accumulation (24), in raises the possibility that allergenal blood allows estimation of circulating addition to activating mature Eo and B induced cytokine production from airmature cells as wellas progenitors. In ad- (23, 25). Infusion of OM-CSF results in way cells leads to the inflammatory cell dition, the enumeration of circulating an increase in circulating granulocyte pro- recruitment and activation that characprogenitors may identify a population genitors (26) and an initial transient fall terize the late asthmatic response to with more direct accessto the tissues than in Eo followed by a subsequent increase allergen. those from bone marrow (19). at 24 h (27). There is already evidence The associations described allow a The early asthmatic response to aller- that circulating hypodense Eo are in- positive feedback loop, which could lead gen probably results from activation of creased following the late asthmatic re- to persistent airway inflammation (figmast cells and leukocytes resident in the sponse to allergen (28) and that this Eo ure 5). Subjects with greater airway airway (20), whereas the late response has phenotype can be induced by OM-CSF, hyperresponsiveness would have greater been considered to result from activation IL-3, and IL-5 (23, 25). These findings numbers of inflammatory cells in their of leukocytes that have been recruited to support the hypothesis that hematopoi- airways. When activated by allergen, the the airway (21). The recruitment of in- etic growth factors mediate the inflam- increased mediator released from these flammatory cells to the airway is there- matory cell accumulation and circulat- cells would cause augmented inflammafore an important difference between the ing inflammatory cell changes after ex- tory cell accumulation. This has been described following the nasal response to early and late asthmatic responses (1, 2). posure to allergen. Circulating activated T cellscould also allergen (8) and could create a vicious cyThe mechanisms of allergen-induced inflammatory cell recruitment are poorly contribute to the changes we have ob- cle leading to persistent airway inflamunderstood, but the observed increase in served. In the CFU assaysprogenitor cells mation. This is similar to the hypothesis Eo/B progenitors contributes to an un- were cultured in the presence of the pa- proposed by Cockcroft to explain perenderstanding of these mechanisms in two tient's T cells. It is therefore possible that nial allergicasthma (33), with the inflamways. First, it suggests that increased Eo/B production is a component of the Allergen inflammatory response, and second, be+ IgE cause Eo/B-CFU have an absolute dependence on hematopoietic growth factors for their subsequent growth and differentiation (22), it raises the possibility Fig. 5. Hypothesized mechanism for amplification of allergen-induced airthat growth factors may mediate the in11 ways inflammation based upon the obflammatory cell accumulation. Three / served association between inflammasuch molecules, interleukin (IL-3), IL-5, + / tory cell recruitment and the hypothem.dlat~r.:=::Xs. / and granulocyte-macrophage colony sis of Cockcroft (33). / / stimulating factor (OM-CSF), are known to influence Eo/B growth and differenINFLAMMATORY CELL tiation (22, 23). They are capable of inRECRUITMENT fluencing each of the steps involved in CELL COUNTS· BEFORE AND 24 H AFTER HISTAMINE-INDUCED BRONCHOCONSTRICTION AND ALLERGEN-INDUCED ISOLATED EARLY RESPONSES

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