Kinetics of Allergen-Induced Airway Eosinophilic Cytokine Production and Airway Inflammation

Kinetics of Allergen-Induced Airway Eosinophilic Cytokine Production and Airway Inflammation GAIL M. GAUVREAU, RICK M. WATSON, and PAUL M. O BYRNE Department of Medicine, McMaster University, Hamilton, Ontario, Canada Airway eosinophilia is the hallmark of asthma exacerbation. Coordination of cytokines such as interleukin (IL)-5, eotaxin and regulated on activation, normal T-cell expressed and secreted (RANTES) seem to be necessary for eosinophil extravasation including adhesion, chemotaxis, and activation. The purpose of this study was to characterize both the kinetics of allergen-induced inflammatory cell recruitment to the airways and cytokines selective for eosinophil chemotaxis, activation, or resolution. Eight atopic asthmatic individuals demonstrating a dual response to inhaled allergen completed a diluent-controlled crossover study. The subjects showed significant allergen-induced early and late airway asthmatic responses (p 0.001), and an increase in the number of sputum eosinophils and metachromatic cells (p 0.05). The number of eosinophils immunopositive for IL-5, eotaxin, and RANTES increased 7 h after allergen inhalation (p 0.05), coincident with the peak number of activated eosinophils. Sputum cells immunopositive for IL-10 decreased significantly following allergen challenge (p 0.04), and correlated negatively with sputum eosinophils (r 0.34, p 0.02). This study shows that allergen-induced increases in sputum eosinophils are associated with the presence of cytokines specific for the activation and chemotaxis of eosinophils, and suggests that cooperation of eosinophilic cytokines may be important for the accumulation and regulation of activated eosinophils at the site of allergic inflammation. Gauvreau GM, Watson RM, O Byrne PM. Kinetics of allergen-induced airway eosinophilic cytokine production and airway inflammation. AM J RESPIR CRIT CARE MED 1999;160:640 647. Eosinophilic infiltration into affected tissue is one of the hallmarks of allergic inflammation. Airway eosinophilia has been demonstrated in asthmatic individuals as compared with normal controls (1, 2), and is enhanced in the airways of individuals with atopic asthma following allergen inhalation challenge (3 5), in association with development of a late airway asthmatic response to allergen and airway hyperresponsiveness (AHR) to methacholine (6 8). The kinetics of allergen-induced airway eosinophilia has been investigated in bronchoalveolar lavage fluid (BALF) (3), but the invasive nature of bronchoalveolar lavage restricts the number of samples obtainable. Advantages of sputum examination include the ability to obtain repeated, noninvasive samples of airway secretions, which is more appropriate for examination of inflammatory cell kinetics than is a single time-point measurement. Furthermore, increased inflammatory markers in sputum after allergen exposure, and particularly, those associated with eosinophil activation and chemotaxis (9), have been demonstrated immunocytochemically (6, 7). (Received in original form September 29, 1998 and in revised form January 19, 1999) Supported by Astra Draco and the Medical Research Council of Canada. Dr. O Byrne is a Medical Research Council of Canada Senior Scientist. Correspondence and requests for reprints should be addressed to Dr. P. M. O Byrne, Department of Medicine, Rm 3U-1 Health Sciences Center, McMaster University, 1200 Main St. West, Hamilton, ON, L8N 3Z5 Canada. Am J Respir Crit Care Med Vol 160. pp 640 647, 1999 Internet address: www.atsjournals.org Tissue eosinophilia is probably due to a combination of several rather specific and coordinated cellular processes appearing at the different stages of eosinophil extravasation including adhesion, chemotaxis, and activation (10). Several cytokines are implicated in eosinophilia. Interleukin (IL)-5 was first described as a factor important for proliferation and differentiation of bone marrow eosinophils (11), and is known to prime eosinophils (12) and prolong their survival (13). Chemokines found to act on eosinophils include eotaxin and regulated upon activation, normal T-cell expressed and secreted (RANTES), both of which induce chemotaxis as well as specifically activating eosinophils (14, 15). In vitro studies have demonstrated specificity of these eosinophil-selective cytokines for attraction and activation of eosinophils, and imply their participation in the specific recruitment of eosinophils to sites of allergic inflammation. In contrast, IL-10 is a cytokine known to inhibit the production of proinflammatory cytokines and chemokines by monocytes (16), T-cells (17), neutrophils (18) and eosinophils (19). Inhibition of the effector functions of inflammatory cells suggests that IL-10 has potent antiinflammatory activities. Through downregulation of chemokine production, IL-10 may regulate recruitment and activation of eosinophils at sites of allergic inflammation. Understanding the mechanisms of eosinophil recruitment to the airways, and targeting pathways critical for regulation of eosinophil chemotaxis and activation, will aid in the development of new antiinflammatory agents for the treatment of asthma. The purpose of this study was to characterize the kinetics of inflammatory cell recruitment to the airways following allergen inhalation in atopic asthmatic subjects, with par-

Gauvreau, Watson, and O Byrne: Allergen-Induced Airway Inflammation 641 ticular attention to the involvement of cytokines selective for eosinophil chemotaxis and activation, and to a cytokine known to regulate the inflammatory response. METHODS Subjects We selected eight subjects with which to examine the kinetics of allergen-induced airway inflammation. The sample size was considered sufficient for examining the kinetics of airway inflammation, since previous studies have shown that a group of eight or more subjects can demonstrate allergen-induced airway eosinophilia assessed with induced sputum, through the same methodology used in this study (6, 7). Subjects were nonsmokers with mild atopic asthma, and were selected because they demonstrated an allergen-induced early and late asthmatic response of at least a 15% reduction in FEV 1. Subjects gave signed informed consent to participate in the study, which was approved by the Ethics Committee of McMaster University Health Sciences Center. Subjects were not exposed to sensitizing allergens and did not have asthma exacerbations or respiratory tract infections for at least 4 wk before allergen challenges. All subjects had stable asthma, with an FEV 1 above 70% predicted on all study days before challenge to the airways, and used no regular medication other than infrequent ( twice weekly) inhaled 2 -agonist as required to treat their symptoms. All medications were withheld for at least 8 h before each visit, and subjects were instructed to refrain from rigorous exercise, tea, or coffee in the morning before visits to the laboratory. Study Design The eight subjects completed two study periods, consisting of an allergen and a diluent (0.9% saline) inhalation challenge. Each study period consisted of six visits to the laboratory. Baseline measurements of FEV 1, the provocative concentration of methacholine causing a 20% decrease in FEV 1 (PC 20 ), and inflammatory cell counts in induced sputum were made on the day before the inhalation challenge. The inhalation challenge was done the following morning, and the FEV 1 was measured at intervals for 7 h after challenge. Sputum samples were obtained at 7 h, during the late asthmatic response. Sputum could not be induced earlier than 7 h because of the requirement for pretreatment with inhaled 2 -agonists before sputum induction, which would have interfered with subsequent measures of FEV 1. Methacholine PC 20 and sputum samples were obtained again at 24 h, 2 d, 4 d, and 7 d after challenge. Laboratory Procedures Methacholine inhalation test. Methacholine inhalation challenge was performed as described by Cockcroft (20). Subjects inhaled normal saline, followed by doubling concentrations of methacholine phosphate from a Wright nebulizer (Roxon Medi-Tech, Montreal, PQ, Canada) for 2 min. FEV 1 was measured at 30, 90, 180, and 300 s after each inhalation. Spirometry was done with a Collins water-sealed spirometer (Warren E. Collins, Braintree, MA) and kymograph. The test was terminated when a decrease in FEV 1 of 20% of the baseline value occurred, and the methacholine PC 20 was calculated. Allergen and diluent inhalation challenge. Allergen challenge was performed as described by O Byrne and colleagues (8). The allergen producing the largest-diameter skin wheal was diluted in 0.9% saline for inhalation. The concentration of allergen extract for inhalation was determined from a formula described by Cockcroft and coworkers (21), using the results of the skin test and methacholine PC 20 measurement. The starting concentration of allergen extract for inhalation was two doubling concentrations below that predicted to cause a 20% early decrease in FEV 1, and doubling concentrations of allergen were given until a 15% early decrease in FEV 1 was reached. The FEV 1 was measured at 10 min and again at 20, 30, 45, 60, 90, and 120 min after allergen inhalation, followed by measurement at each hour until 7 h after allergen inhalation. The early asthmatic response was taken to be the largest decrease in FEV 1 within 2 h after allergen inhalation, and the late asthmatic response was taken to be the largest decrease in FEV 1 between 3 h and 7 h after allergen inhalation. Only subjects who developed a late decrease in FEV 1 of at least 15% were used in the study. The area under the curve was determined during the early (0 to 2 h) and late (3 to 7 h) responses by plotting the response with graphics software (Fig P; Fig P Software Corporation, Durham, NC), that calculated the area of the FEV 1 time response. The diluent challenge was similar to the allergen challenge, with 0.9% saline inhaled rather than allergen. Sputum analysis. Sputum was induced and processed according to the method described by Pizzichini and coworkers (22). Subjects inhaled 200 g albuterol followed 10 min later by 3%, 4%, and 5% saline for 7 min each. The induction was stopped when an adequate sample was obtained or if the FEV 1 dropped 20% from baseline. Cell plugs with few or no squamous epithelial cells were selected from the sample, separated from saliva, and weighed. Samples were digested with four times their volume of 0.1% dithiothreitol (Sputolysin; Calbiochem Corp., San Diego, CA) on a bench rocker for 15 min, after which four times their volume of Dulbecco s phosphate buffered saline (DPBS) (Life Technologies Inc., Grand Island, NY) was added and mixed on a bench rocker for an additional 5 min. The cell suspension was filtered through a 52- m nylon gauze (BNSH Thompson, Scarborough, ON, Canada) to remove debris, and the filtrate was centrifuged at 1,500 rpm for 10 min. The total cell count was made with a Neubauer hemocytometer (Hausser Scientific, Blue Bell, PA) and was expressed as the number of cells per milliliter of sputum. Cells were resuspended in DPBS at 0.75 to 1.0 10 6 /ml, and cytospins were prepared on glass slides, using 50 l of cell suspension and a Shandon III cytocentrifuge (Shandon Southern Instruments, Sewickley, PA) at 300 rpm for 5 min. Differential cell counts were obtained from the means of two slides stained with Diff-Quik (American Scientific Products, McGaw Park, IL), with 400 cells counted per slide. The same observer counted all study slides, and achieved a high reproducibility of the cell counts using the methods described (22). Metachromatic cell (MCC) (mast cells and basophils) counts on slides stained with toluidine blue were obtained from the mean of two slides, with 1,500 cells observed on each slide. Cytospins were also prepared on Aptex (Sigma Chemical Co., St. Louis, MO) coated slides, and were fixed for 10 min in periodate lysine paraformaldehyde for immunocytochemical staining. Slides were stained with murine monoclonal antibodies to the activated form of human eosinophil cationic protein (ECP) at 1.0 g/ml (EG2) (Kabi Pharmacia, Uppsala, Sweden); to IL-5 and RANTES at 30.0 g/ml each; and to eotaxin at 100 g/ml (R&D Systems, Minneapolis, MN); and with a rat monoclonal antibody to human IL-10 at 30 g/ml (Pharmingen, San Diego, CA). All antibodies were diluted in 1.0% bovine serum albumin (Sigma) in wash buffer made up of DPBS, 0.01M 4-(2-hydroxyethyl)-1-piperazine-N -2-ethanesulfonic acid (Hepes) buffer (Life Technologies) and 0.01% saponin (Sigma). Briefly, slides were incubated overnight with antihuman monoclonal primary antibodies, and protein was detected on the following day with the alkaline phosphatase antialkaline phosphatase (APAAP) method (23), using rabbit antimurine (IL-10; rabbit antirat) secondary antibodies and monoclonal mouse (IL-10; monoclonal rat) APAAP tertiary antibodies (DAKO, Glostrup, Denmark). Nonspecific staining with the primary and secondary antibodies was blocked by incubation with 75% human AB serum and 25% normal rabbit serum (Sigma) diluted in wash buffer. Negative isotype controls (murine IgG 1 and rat IgG 2a ; Sigma) for each slide were included at the same concentration as the primary antibody. Calcium ionophore-stimulated human peripheral blood eosinophils and lipopolysaccharide-stimulated human peripheral blood mononuclear cells were included as positive controls. The percentage of positively immunoreactive cells was determined from a count of 400 cells under light microscopy. To demonstrate specific staining of eosinophils on slides stained for eotaxin, IL-5, and RANTES, these slides were double stained with fluorescein isothiocyanate (FITC) (Sigma), a stain specific for eosinophils, for 10 min at a concentration of 10 g/ml. Statistical Analysis Summary statistics are expressed as mean and SEM, with the exception of methacholine PC 20 measurements, which are expressed as geometric mean and geometric standard error of the mean (GSEM). Methacholine PC 20 measurements were made by linear interpolation of log dose response curves. Methacholine PC 20 and sputum MCC values were log-transformed to fit a normal distribution prior to anal-

642 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 160 1999 Figure 1. Methacholine PC 20 and maximum percent late fall in FEV 1 (top panel), sputum eosinophils (middle panel) and metachromatic cells (bottom panel) in response to diluent (open bars) and allergen (stippled bars) inhalation challenge. All data are expressed as mean and SEM, except for methacholine PC 20 which is expressed as geometric mean and geometric SEM. ysis. The allergen-induced shifts (change from baseline) in PC 20 and in sputum inflammatory cells were analyzed with repeated measures analysis of variance (ANOVA) (24). The allergen-induced early and late asthmatic responses were compared with the response to diluent challenge using Student s paired t test. Pearson s correlation coefficient was used to test for relationships between physiologic and cellular variables. All correlations involving sputum cells were examined with cells expressed as a percentage of the total number of cells rather than as absolute numbers of cells per milliliter of sputum, since comparisons of absolute numbers of cells are always positively correlated owing to the high variability between sputum samples in the total cell count. RESULTS Airway Responses All subjects had a dual response to allergen, with a mean maximal decrease in FEV 1 of 36.1 3.2 % during the early asthmatic response (p 0.001) and 26.3 2.0% during the late asthmatic response (p 0.001). There was a significant change in methacholine airway responsiveness as measured by a reduction in the methacholine PC 20 for 7 d after allergen exposure as compared with exposure to diluent (p 0.003) (Figure 1). Airway Inflammation There was an allergen-induced increase in the number of sputum eosinophils and activated eosinophils (EG2-positive) per milliliter of sputum. Eosinophils remained increased for 7 d (p 0.02) (Figure 1), and EG2-positive cells remained significantly increased for 2 d, from 13.8 7.5 10 4 /ml at baseline to 94.3 24.6 10 4 /ml at 7 h, 73.2 14.8 10 4 /ml at 24 h, and 51.6 12.6 10 4 /ml at 2 d, and 39.3 19.3 10 4 /ml at 4 d, and 24.8 7.1 10 4 /ml at 7 d after allergen challenge (p 0.02). There was an allergen-induced increase in the number of sputum MCC (p 0.01), which was maximal at 7 h (range: TABLE 1 TOTAL SPUTUM CELL COUNT AND PERCENT SPUTUM INFLAMMATORY CELLS AT BASELINE AND AFTER ALLERGEN AND DILUENT CHALLENGES Baseline 7 h 24 h 2 d 4 d 7 d Ag Dil Ag Dil Ag Dil Ag Dil Ag Dil Ag Dil TCC, 10 6 /ml 4.2 1.9 1.6 0.3 8.2 2.7 1.8 0.6 7.2 3.5 1.9 0.3 4.4 1.3 3.2 0.4 2.4 0.7 2.0 0.2 4.0 1.6 1.8 0.2 Eos, % 4.9 1.7 4.0 1.3 21.6 4.0* 4.2 1.5 25.4 2.9* 8.1 2.8 21.3 3.9* 6.4 3.0 18.5 6.3* 6.9 2.3 8.2 2.0* 3.7 1.3 MCC, % 0.05 0.03 0.04 0.02 1.11 0.43* 0.09 0.06 0.40 0.09 0.28 0.02 0.23 0.04 0.18 0.14 0.15 0.04 0.13 0.07 0.14 0.06 0.06 0.02 Neut, % 62.0 5.5 40.7 8.5 61.1 6.2* 66.2 7.4 59.5 8.6* 53.8 8.3 36.8 9.7* 46.6 7.8 41.1 8.6* 61.1 4.2 55.6 9.0* 58.4 5.3 Macro, % 32.7 4.5 54.3 7.9 19.1 3.7* 29.3 7.4 23.3 5.9* 29.3 8.0 40.3 8.0* 46.4 8.4 40.0 4.8* 32.7 3.9 35.5 7.6* 37.0 5.1 Squam, % 5.4 2.3 10.2 3.4 7.2 5.1 8.4 4.2 8.2 3.9 7.2 2.9 6.0 2.2 6.2 2.2 11.0 3.2 11.7 4.2 8.3 2.8 6.7 1.4 Viability, % 83.4 1.9 72.0 4.1 81.9 4.5 75.0 8.1 75.5 7.6 86.4 3.8 80.2 6.8 79.0 7.7 75.5 2.5 83.2 3.5 74.6 6.1 77.9 3.2 Definition of abbreviations: Ag allergen challenge; Dil diluent challenge; Eos eosinophils; Macro macrophages; MCC metachromatic cells; Neut neutrophils; Squam squamous cells; TCC total sputum cell count. *p 0.05 Ag versus Dil change from baseline.

Gauvreau, Watson, and O Byrne: Allergen-Induced Airway Inflammation 643 0.4 10 4 /ml to 62.8 10 4 /ml) and remained higher than that with diluent for 7 d (Figure 1). There was no significant difference in the number of neutrophils (p 0.11) or mononuclear cells (p 0.59) after diluent or allergen challenge. The total cell count in sputum did not increase after allergen challenge as compared with diluent (p 0.76). With the sputum cells expressed as a percentage of the total cells, the allergen-induced changes in sputum eosinophils (p 0.02) and MCC (p 0.01) persisted (Table 1). In addition, there was a significant increase in the percent neutrophils after diluent challenge and a significant decrease in the percent macrophages after allergen challenge (Table 1). There was no effect of allergen or repeated methacholine challenges or of sputum induction on sputum cell viability or squamous cell contamination (Table 1). There was a significant negative relationship between the allergen-induced percent eosinophils and percent activated eosinophils in sputum and the change in measured methacholine PC 20 after allergen challenge (r 0.41, p 0.009 and r 0.33, p 0.04, respectively) (Table 2). These relationships between AHR and sputum eosinophils were not statistically significant when comparisons were made at individual time points after allergen challenge, or when comparisons were made with individual subjects throughout the course of the allergen challenge study period. There was a weak negative correlation between the percentage of sputum MCC and the change in methacholine PC 20 measured after allergen challenge (r 0.38, p 0.02), but there were no other significant relationships observed between measurements of airway physiology and sputum cell differential counts (Table 2). Airway Cytokines Granulocytes (eosinophils and neutrophils) were immunopositive for eotaxin, IL-5, and RANTES, and mononuclear cells (macrophages/monocytes and lymphocytes) were immunopositive for eotaxin and IL-5 (Figure 2). The total number of sputum cells immunopositive for eotaxin, IL-5, and RANTES was increased after allergen inhalation, but this change did not reach statistical significance (Figure 3). There was, however, a significant allergen-induced increase in the number of sputum eosinophils immunopositive for eotaxin at 7 h (p 0.0002), for IL-5 at 7 h and 24 h (p 0.01), and for RANTES at 7 h (p 0.0002). Peak immunopositivity for eotaxin, IL-5, and RANTES coincided with the maximal number of EG2-positive eosinophils, which occurred 7 h after allergen challenge; however, there was no significant correlation between the percent eosinophils or percent EG2-positive cells and the percent sputum cells immunopositive for eotaxin, IL-5, or RANTES (r 0.24, p 0.10) (Table 2). There was a weak negative relationship between FEV 1 and percent cells immunopositive for RANTES (r 0.30, p 0.04) and between the allergen-induced change in methacholine PC 20 and percent cells immunopositive for IL-5 (r 0.38, p 0.03) and for RANTES (r 0.37, p 0.04). A significant allergen-induced reduction in the number of sputum cells immunopositive for IL-10, of 53 44 10 4 /ml, occurred by 24 h after allergen challenge, as compared with an increase of 25 18 10 4 /ml at 24 h after diluent challenge, and these reduced numbers remained significantly lower than baseline values for 7 d after allergen challenge (p 0.04) (Figure 4). Cells staining positively for IL-10 included granulocytes and mononuclear cells, but the percent cells immunopositive for IL-10 did not correlate with the percent neutrophils or percent macrophages in sputum (Table 2). There was an inverse relationship between the percent cells immunopositive for IL-10 and the percent eosinophils (r 0.34, p 0.02) and percent cells immunopositive for EG2 (r 0.29, p 0.04) (Table 2) measured after allergen challenge, but there was no relationship between IL-10 and sputum cells immunopositive for IL-5, eotaxin, or RANTES. DISCUSSION This study demonstrated that the maximal numbers of activated eosinophils, MCC, and eosinophils immunopositive for eotaxin, RANTES, and IL-5 occur at 7 h after allergen inhalation, during the late airway asthmatic response; however, persisting eosinophilic airway inflammation and AHR remained for 7 d after allergen inhalation. Also, allergen inhalation reduced the numbers of IL-10-positive sputum cells that persisted for 7 d after allergen inhalation, as it also did with the duration of change in airway eosinophils and AHR. The study also confirmed that repeated methacholine challenges and/or sputum inductions have no effect on sputum cell viability or the number of inflammatory cells in induced sputum, permitting measurements and comparisons of airway physiology and airway inflammation at regular intervals after allergen challenge. These repeated measurements permit the kinetics of allergen-induced responses to be evaluated. Methacholine PC 20 is known to correlate with sputum eosinophil counts in asthmatic individuals (25), possibly as a re- TABLE 2 CORRELATION COEFFICIENTS DEMONSTRATING THE RELATIONSHIPS (r VALUES) BETWEEN MEASUREMENTS OF AIRWAY PHYSIOLOGY, PERCENT SPUTUM CELLS, AND PERCENT SPUTUM CELLS IMMUNOPOSITIVE FOR EG2, IL-5, EOTAXIN, RANTES, AND IL-10 DURING THE ALLERGEN CHALLENGE STUDY PERIOD FEV 1 PC 20 EG2-positive IL-10-positive Eotaxin-positive RANTES-positive IL-5-positive Eosinophils 0.01 0.41* 0.65* 0.34* 0.02 0.12 0.27 Neutrophils 0.18 0.23 0.41* 0.08 0.55* 0.17 0.51* Macrophages 0.27 0.04 0.10 0.28 0.65* 0.32* 0.48* MCC 0.17 0.38* 0.27 0.30 0.18 0.35* 0.25 IL-5-positive 0.03 0.38* 0.25 0.03 0.41* 0.22 RANTES-positive 0.30* 0.37* 0.24 0.22 0.10 Eotaxin-positive 0.25 0.07 0.03 0.15 IL-10-positive 0.17 0.25 0.29* EG2-positive 0.37* 0.33* PC 20 0.25 Definition of abbreviations: EG2 eosinophil cationic protein (ECP); IL-5 interleukin-5; MCC metachromatic cells; PC 20 concentration of methacholine causing a 20% decrease in FEV 1 ; RANTES regulated on activation, normal T-cell expressed and secreted. *p 0.05. p 0.10.

644 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 160 1999 Figure 2. Sputum cells immunopositive for eotaxin (red signal) 7 h following diluent (A) and allergen (B) inhalation challenge with negative isotype control (C). Bar 3.0 m. sult of damage to the airway epithelium by eosinophils (26). We were unable to demonstrate a significant relationship between measurements of airway physiology (methacholine PC 20 and FEV 1 ) and sputum eosinophils at specific time points after allergen challenge, owing to a large intersubject variability in these measurements, as well as to intra-subject variability in airway responses to allergen inhalation. However, increasing the sample size to include all measurements after allergen challenge has shown that a relationship exists between the change in methacholine PC 20 and sputum eosinophils/activated eosinophils, and between FEV 1 and activated sputum eosinophils after allergen inhalation. In addition, we observed weak yet statistically significant negative relationships between the change in PC 20 and cells immunopositive for RANTES and IL-5, and between FEV 1 and the percent RANTES-immunopositive cells. These relationships show that levels of these cytokines known to chemoattract and activate eosinophils are associated with and can be used to predict the development of AHR and bronchoconstriction. The relationship between eosinophils and airway physiology supports the hypothesis that mediator release by activated eosinophils contributes to changes in airway physiology after inhaled allergen, and we have previously observed that allergen-induced sputum levels of ECP closely reflect allergen-induced increases in sputum eosinophils (7). However, the present study indicates that eosinophils are not the only type of cell that may contribute to AHR. MCC (mast cells and basophils) also correlated significantly with the change in methacholine PC 20, suggesting that mediators released by cells other than eosinophils can contribute to AHR. That the magnitude of allergen-induced increases in numbers of sputum eosinophils and MCC is considerably less than the magnitude of the decrease in PC 20 after 7 d suggests that the effects of cell mediators may persist after these cells are no longer present. Furthermore, allergen-induced structural changes in the airway resulting in AHR may require longer to resolve than the airway inflammation itself. The number of eosinophils remained significantly increased for 7 d after allergen inhalation, demonstrating prolonged effects of the allergen challenge on eosinophil recruitment to, and/or clearance from, the airways. The number of activated eosinophils, however, remained significantly increased for only 24 h after challenge, suggesting that the activation state of eosinophils was being suppressed or that the activation stimulus was no longer present at this time. We demonstrated increased numbers of eosinophils immunopositive for IL-5 for 24 h after allergen challenge, suggesting that participation of activating cytokines, such as IL-5, may be necessary for recruited eosinophils to become activated, and/or to prolong the survival of activated eosinophils. In asthmatic individuals, EG2-positive eosinophils have been shown to express the chain of the IL-5 receptor, further supporting this concept (27). Influx of eosinophils into the airway is thought to be partly due to the release of eosinophil-attracting/activating cytokines. In our study the number of activated eosinophils peaked 7 h after allergen challenge, which was coincident with maximal immunopositivity of eosinophils for eotaxin, IL-5, and RANTES. The number of eosinophils immunopositive for eotaxin and RANTES was only significantly increased at 7 h after allergen challenge, whereas the number of eosinophils was elevated for 7 d, suggesting that eosinophil production and/or receptor binding of these cytokines is maximal during the late response but that after 24 h they are at levels too low to be detected immunocytochemically in association with sputum eosinophils. This dissociation between the presence of eosinophils and their chemoattractants/activators has been observed in BALF after segmental bronchial allergen challenge, with increased

Gauvreau, Watson, and O Byrne: Allergen-Induced Airway Inflammation 645 Figure 3. Number of all sputum cells (left panel) and sputum eosinophils (right panel) immunopositive for eotaxin (top panel), IL-5 (middle panel) and RANTES (bottom panel) at baseline and following diluent (open bars) and allergen (stippled bars) inhalation challenge. All data are expressed as mean and SEM. eosinophil levels present for up to 15 d after challenge and no increase in IL-5 or IL-3 (28). This finding, by Shaver and coworkers, suggests that eosinophils have a long residency in the airways of asthmatic individuals after allergen challenge, possibly owing to the effects of other cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), which are known to prolong eosinophil survival. The study demonstrated an increase in sputum of total cells immunopositive for IL-5, RANTES, and eotaxin after allergen challenge, but these increases did not reach statistical significance, possibly because of a Type II error. In addition, cells known to be a major source of IL-5, such as lymphocytes, or of RANTES and eotaxin, such as airway epithelial cells, are not represented well in sputum, being present only in very low numbers. By contrast, eosinophils immunopositive for IL-5, RANTES, and eotaxin were significantly increased after allergen challenge. Because immunocytochemistry cannot distinguish intracellular protein (synthesized, stored, or phagocytosed) from extracellular protein (receptor-bound), eosinophils immunopositive for these cytokines may reflect production of the cytokine proteins by eosinophils themselves, receptorbound proteins released from other inflammatory cells in sputum, or related structural cells. IL-5 has been measured in the sputum of asthmatic individuals (29), and is increased in sputum supernatants of those with atopic asthma at 24 h after allergen challenge (30), in BALF Figure 4. Shift in the number of sputum cells immunopositive for IL-10 following diluent (open bars) and allergen (stippled bars) inhalation challenge. Data are expressed as mean and SEM.

646 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 160 1999 at 2 d after segmental allergen challenge (28), supporting allergen-induced release of IL-5 by cells in the airway, such as T lymphocytes and eosinophils. Recent work in mice, however, has proposed a role of IL-5 distal to the airway. Systemic IL-5 may contribute to local eosinophil accumulation by releasing eosinophils from the bone marrow, by priming eosinophils, and/ or by facilitating their migration through the microvascular endothelium, and may be more relevant than the IL-5 measured in airways. Although the numbers of eosinophils immunopositive for eotaxin and RANTES were significantly increased at 7 h after allergen challenge, we again did not detect an overall increase in the number of sputum cells immunopositive for these chemokines. We did not observe mononuclear cells immunopositive for RANTES, suggesting that mononuclear cells do not store or bind sufficient RANTES to be detected immunocytochemically. Known producers of chemokines include airway epithelial and endothelial cells. Because these structural cells do not commonly appear in samples of sputum, increased expression of eotaxin and/or RANTES by these cells, as detected immunocytochemically, would not be reflected in sputum. Sputum cells may not be the best medium for detecting production of IL-5, eotaxin, or RANTES, which may explain why we did not observe positive relationships between eosinophil accumulation and cytokines known to contribute to these cells accumulation. Sputum supernatants may better reflect the levels of these cytokines. Eotaxin is associated with early tissue recruitment and activation of eosinophils, in which allergen-induced expression of eotaxin by lumen-associated cells determines a tissue gradient in the airway for recruitment through the tissues and into the airway lumen. In the guinea pig, messenger RNA for eotaxin is rapidly induced in airway epithelium and alveolar macrophages, at 3 h after allergen challenge (31), suggesting that our measurements made in sputum at 7 h after challenge may have been too late to measure allergen-induced intracellular increases in eotaxin. There was a significant allergen-induced decrease in the number of sputum cells immunopositive for IL-10, which was maximal at 24 h and correlated negatively with sputum eosinophils and activated eosinophils. This decrease in IL-10 was not simply due to a decrease in the percentage of macrophages, which are known to produce IL-10, since the r value for the correlation between the percent IL-10-immunopositive cells and percent macrophages shows that only 8% of the decrease in IL-10 was attributable to the macrophage level (Table 2). The negative relationship between the percent macrophages and cells immunopositive for eotaxin, RANTES, and IL-5 is interesting and suggests that a reduction in the percent macrophages and possibly in IL-10 production may reduce regulation of these proinflammatory cytokines. We were unable to examine the relationship between IL-10 and lymphocytes in induced sputum after allergen challenge, since sputum contains very few lymphocytes. If IL-10 expression is suppressed in lymphocytes after allergen challenge, we were unable to investigate this. A limited number of studies provide evidence for a regulatory role of IL-10 in asthma. IL-10 is an antiinflammatory cytokine that inhibits the transcription of many proinflammatory cytokines and chemokines (32), including GM-CSF (16), which is known to be important for eosinophil survival. We did not find a negative relationship between cells immunopositive for IL-10 and eosinophil-attracting/activating cytokines, IL-5, RANTES, and eotaxin. This could have been due to the inability of sputum cells to reflect these cytokine levels, or to a lack of effect of IL-10 on these preeosinophilic cytokines in allergic airway disease. Crosslinking of the eosinophil surface marker CD40 also appears to be an important event leading to enhancement of eosinophil survival and cytokine production. IL-10 has been shown to downregulate eosinophil expression of CD40 (33), suggesting that IL-10 directly downregulates the inflammatory response of activated eosinophils; however, we did not measure CD40 expression on eosinophils in the present study. The number of MCC increased significantly after allergen challenge, with a much higher allergen-induced number than previously reported with use of the same method for sputum processing and staining as in our study (6, 7). Detection of MCC with the toluidine blue stain depends upon retention of granule proteins within mast cells/basophils. Because degranulation of MCC is possible after allergen challenge, staining of metachromatic granules within cells may grossly underestimate the actual number of mast cells (34). There is no consistent evidence for allergen-induced neutrophilia in the airways of asthmatic individuals (6, 5, 30), and we did not observe an effect of allergen on the number of neutrophils in sputum. If the neutrophil is involved in the airway response to inhaled allergen, it may reflect an early, nonspecific response that occurs with acute tissue injury, unlike the prolonged eosinophilia that occurs in response to specific allergic stimuli. The percent of sputum neutrophils (the ratio of neutrophils to other cells) was significantly increased above baseline after diluent challenge, which has been shown to occur following repeated sputum induction (35). This, however, may also be an effect of a low baseline level of neutrophils measured in the diluent administration period (Table 1). The absolute number of neutrophils (the load of neutrophils in the airway) remains unaffected by diluent challenge, and may be more relevant in assessing the eosinophilic response, since it does not depend upon the flux of other cells, as does the percentage. Although we observed neutrophils immunopositive for RANTES, this chemokine does not appear to recruit neutrophils to the airways. Allergen inhalation has been shown to increase the level of IL-8 in sputum without increasing the number of sputum neutrophils (9). A neutrophilic response to these potent neutrophil chemoattractants may require a costimulus, such as adhesion molecules or cytokines that prime neutrophils, but is not present in the airway following allergen challenge. The presence of neutrophils immunopositive for IL-5, eotaxin, and RANTES was a surprising finding, since neutrophils have not been shown to express these cytokines. However, endotoxin-induced inflammation in rat lung has been shown to induce a selective cytokine response in neutrophils, showing that the neutrophil can be an important source of cytokines in acute airway inflammation (36). Neutrophils as a major source of IL-5, eotaxin, and RANTES may explain the absence of any correlation between these cytokines and eosinophils and/or EG2-positive eosinophils. This study showed that allergen inhalation by asthmatic individuals induces an inflammatory response in the airway consisting of eosinophils and MCC. 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