Bronchial asthma is a chronic inflammatory disorder

Increased Expression of the Chemoattractant Cytokines Eotaxin, Monocyte Chemotactic Protein-4, and Interleukin-16 in Induced Sputum in Asthmatic Patients* Rame A. Taha, MD; Sophie Laberge, MD; Qutayba Hamid, MD; and Ronald Olivenstein, MD Background: Induced sputum from asthmatic patients has been recently used to assess inflammatory cells. We have previously reported an increased expression of Th-2 type cytokines in induced sputum of asthmatic patients. C-C chemokines, particularly eotaxin and monocyte chemotactic protein (MCP)-4, are associated with eosinophilic infiltration. Interleukin (IL)-16 is associated with chemotactic activity for CD4 cells. Chemokine expression in BAL and bronchial biopsy specimens has been demonstrated in asthmatic airways, but not in induced sputum. Methods: We examined whether eotaxin, MCP-4, and IL-16 expression could be detected in induced sputum of asthmatic patients (n 10), and whether the expression was increased compared to normal control subjects (n 9). Eotaxin, MCP-4, and IL-16 immunoreactivity were determined by immunocytochemistry. In addition, inflammatory cells were investigated using markers for T cells (CD3), eosinophils (major basic protein [MBP]), macrophages (CD68), neutrophils (elastase), and epithelial cells (cytokeratin). Results: Our results showed that there was a significant difference in the percentages of MBP-positive and epithelial cells between asthmatic patients and normal control subjects (p < 0.05). However, there was no difference between these two groups in the percentage of CD3-, elastase-, and CD68-positive cells. Immunoreactivity for eotaxin, MCP-4, and IL-16 was expressed in the induced sputum of all asthmatic patients, and expression of these chemotactic cytokines was significantly greater than in control subjects (p < 0.001, p < 0.005, and p < 0.001, respectively). Conclusions: This study showed that induced sputum could be used to detect chemokines in patients with bronchial asthma, and that the upregulation of chemotactic cytokines in the airways can be seen using noninvasive techniques. (CHEST 2001; 120:595 601) Key words: asthma; chemokines; induced sputum Abbreviations: APAAP alkaline phosphatase antialkaline phosphatase; IL interleukin; MBP major basic protein; MCP monocyte chemotactic protein; PC 20 provocative concentration of methacholine causing a 20% fall in FEV 1 *From the Meakins-Christie Laboratories (Dr. Taha) and Montreal Chest Research Institute (Drs. Laberge, Hamid, and Olivenstein), McGill University, and Ste-Justine Hospital, University of Montreal, Montreal, Quebec, Canada. Supported by the J. T. Costello Memorial Research Fund, the Medical Research Council of Canada, and Inspiraplex. Manuscript received August 17, 2000; revision accepted February 1, 2001. Correspondence to: Ronald Olivenstein, MD, Montreal Chest Institute, McGill University, 3650 St. Urbain St, Montreal, Quebec, Canada H2X 2P4; e-mail: rolive3@po-box.mcgill.ca Bronchial asthma is a chronic inflammatory disorder characterized by airway inflammation and infiltration by eosinophils and activated T cells. 1,2 The mechanisms that regulate the selective accumulation, activation, and retention of airway eosinophils and T cells are areas of active investigation. Present evidence suggests that recruitment of eosinophils and T cells into sites of inflammation is a multistep process involving leukocyte-endothelial interactions through adhesion molecules and local generation of chemotactic agents that direct cell migration from the vascular compartment into the inflamed airways. 3 Chemokines are a superfamily of 8-kd to 10-kd proteins that are potent chemoattractants for leukocytes. 4,5 The C-C chemokines eotaxin and monocyte chemotactic protein (MCP)-4 act on a common CC chemokine receptor 3 receptor and CHEST / 120 / 2/ AUGUST, 2001 595

have been implicated in the pathogenesis of asthma by their ability to promote both the migration and activation of eosinophils. 6 8 Interleukin (IL)-16 is another cytokine with in vitro chemotactic activity for CD4 cells, including CD4-bearing eosinophils. 9,10 The potential role of eotaxin, MCP-4, and IL-16 in the development of allergic inflammation is supported by findings of predominant expression of these specific cytokines in BAL and in bronchial biopsy specimens of atopic asthmatic patients, compared to normal control subjects. 3,8,11,12 Sputum induction by hypertonic saline solution represents a safe noninvasive tool to investigate the pathology of airway inflammation. 13,14 Recently, we have shown 15 elevated expression of Th2-type cytokines messenger RNA for IL-4 and IL-5 in induced sputum of asthmatic patients compared to control subjects, demonstrating the feasibility of this technique to detect cellular associated cytokine expression. Furthermore, this latter study confirmed the finding in other studies of an increased percentage of eosinophils in induced sputum of asthmatic patients compared to nonasthmatic control subjects. 13 Therefore, the principal objectives of this study were to determine and compare the expression of cytokines with chemoattractant properties for eosinophils and T cells in induced sputum of asthmatic patients and in normal control subjects using immunocytochemistry. Specifically, we determined and compared the expression of MCP-4, eotaxin, and IL-16. Since airway epithelial cells are a major cellular source of MCP-4, eotaxin, and IL-16, we compared immunoreactive protein for these cytokines in both epithelial and nonepithelial cells. Subjects Materials and Methods Subjects included 10 patients fulfilling the American Thoracic Society criteria for asthma and classified as having mild-tomoderate asthma who were recruited from the Asthma Clinic at the Montreal Chest Institute, McGill University, and 9 nonasthmatic nonatopic nonsmoker control subjects. Asthmatic patients had documented airway reversibility to inhaled 2 agonists or an increased airway responsiveness to methacholine (provocative concentration of methacholine causing a 20% fall in FEV 1 [PC 20 ] of 8 mg/ml). Asthmatic medications included only inhaled short-acting selective 2 -agonists. Atopy was based on skin-test reactivity to a series of common allergens. Clinical characteristics of the subjects are given in Table 1. Asthmatic patients were nonsmokers or ex-smokers who had stopped smoking for at least 12 months and had smoked 5 pack-years in their lifetime. Exclusion criteria included use of inhaled or oral corticosteroids, nonsteroidal anti-inflammatory medications (cromolyn, ketotifen), theophylline, long-acting B 2 -agonists, leukotriene antagonists, or antihistamines within the 3 months prior to the study, and occurrence of respiratory tract infection within the previous 6 weeks or immunotherapy within the previous 12 months. Table 1 Clinical Characteristics of Asthmatic Patients and Control Subjects Characteristics Study Design The Ethics Review Board of the Montreal Chest Institute approved the study, and all subjects gave written informed consent. The patients and control subjects were screened using a questionnaire, spirometry, and a methacholine inhalation test (if FEV 1 was 70%). Allergy skin prick tests were performed, and atopy was defined as 3-mm skin wheal response to one or more of 13 common allergens. Blood was drawn for CBC count and differential and total serum IgE. Sputum was induced in the laboratory at least 48 h after methacholine bronchoprovocation. The difference in FEV 1 on the two test days did not exceed 10%. Sputum Induction Asthmatic Patients Control Subjects Participants, No. 10 9 Age, yr Mean 30.4 34.6 Range 21 60 27 51 Sex Male 6 3 Female 4 6 FEV 1, % predicted Mean 89 105.2 Range 71 118 91 109 PC 20, mg/ml Median 2.0 16 Range 0.16 6.0 Total serum IgE, U/mL Median 67.2 36.2 Range 32.7 4240 32.7 96.5 Eosinophils, 10 6 /L Mean 206 70 Range 47 570 45 119 Sputum induction was performed using a modification of the method of Pin and coworkers. 16 Subjects were administered salbutamol, 200 g, to inhibit possible bronchoconstriction during sputum induction followed by increasing concentrations (3%, 4%, and 5%) of hypertonic saline solution generated by an ultrasonic nebulizer (DeVilbiss Ultra-Neb 99; Sunrise Medical; Somerset, PA). The procedure was interrupted every 2 min to measure peak expiratory flow. Subjects were asked to rinse the mouth and blow the nose to minimize contamination with saliva and postnasal drip and also instructed to cough sputum into a sterile container. These procedures were repeated sequentially for 8-min periods at each concentration unless a fall in peak expiratory flow 10% occurred, in which case the procedure was terminated. Sputum Examination Sputum sample volumes were recorded. Sputum that was macroscopically free of salivary contamination was selected to minimize squamous cell contamination and processed within 2 h by a modification of the technique described by Pizzichini and coworkers. 13 Briefly, sputum was treated by adding 2 ml of Hanks balanced salt solution containing 0.1% dithiothreitol, with vortexes for 2 to 3 min to homogenize the sample, 2 ml of 596 Laboratory and Animal Investigations

phosphate-buffered solution was added to stop the action of dithiothreitol, and then the suspension was centrifuged at 300g for 10 min. The cell pellet was resuspended in phosphatebuffered solution, and total cell counts of leukocytes and cell viability were determined. For immunocytochemistry, coded cytospins were briefly fixed in a solution of acetone:methanol (40:60), air dried, and stored at 20 C until further use. Immunocytochemistry Specific immunohistochemical markers for eosinophils (antimajor basic protein [MBP]; a gift from Dr. R. Moqbel, University of Alberta, Canada), T lymphocytes (anti-cd3; Becton-Dickinson; Mississauga, Ontario, Canada), neutrophils (antielastase; Becton-Dickinson), macrophages (anti-cd68; Dako Diagnostic; Mississauga, Ontario, Canada) and epithelial cells (anticytokeratin, CAM-5.2, is a murine monoclonal antibody) were used to detect the phenotype of inflammatory cells recovered by sputum induction. Immunocytochemistry was performed using a modified alkaline phosphatase antialkaline phosphatase (APAAP) method as previously reported. 15 To detect eotaxin, MCP-4, and IL-16 immunoreactivity recovered by sputum induction, we used specific polyclonal antibodies raised against human eotaxin and MCP-4 (supplied by Dr. A. Luster) and a monoclonal antibody against IL-16 (clone 14.1, a gift of Dr. D. M. Center). Briefly, slides were incubated with the specific antibodies overnight at 4 C. After washing in Tris-buffered saline solution, the secondary antibodies were applied and the slides were washed in Trisbuffered saline solution and incubated with the APAAP complex. The reaction was visualized by alkaline-phosphatase substrate added to fast red TR (Sigma Chemical; St. Louis, MO). All the slides were counterstained with hematoxylin and mounted before examination. Negative control experiments were performed in a similar manner but in the absence of the primary antibody; the primary antibody was replaced by nonspecific mouse Ig or Tris-buffered saline solution. Data Analysis Sputum samples containing 20% of contaminating squamous cells were considered suitable for analysis. The numbers of positive cell markers of cellular phenotypes were counted per 1,000 total cells excluding squamous cells with the help of a phase contrast lens. The numbers of cells with immunoreactivity for eotaxin, MCP-4, or IL-16 were classified as epithelial (cytokeratin positive) or nonepithelial in origin, then were counted per 500 total cells excluding squamous cells. Positive cells were reported as the mean (range). To avoid observer bias, slides were coded prior to analysis and read in a blinded fashion. At least two cytospins were counted for each immunocytochemical marker, and the mean value of these slides was reported. Significant differences were detected by analysis of variance and Mann- Whitney test. Correlation coefficients were determined by using the Spearman correlation test. Significance was accepted at the level of 95%. Results The total volume of sputum in asthmatic patients was 4.2 ml (range, 1.7 to 8.6 ml) compared to 2.1 ml (range, 1.1 to 4.3 ml) in control subjects (p 0.05). Quantification of Inflammatory Cells in Induced Sputum The total cell count in asthmatic subjects was 4.4 10 6 /L (range, 2.4 to 11.5 10 6 /L), compared to 2.3 10 6 /L (range, 1.1 to 4.3 10 6 /L) in control subjects. Cell viability was 90% in all samples. The percentage of epithelial cells in asthmatic patients was significantly increased (9.5%; range, 5.0 to 14.0%) compared to nonasthmatic subjects (0.95%; range, 0.4 to 1.5%; p 0.05; Table 2). Among nonepithelial cells, the percentage of MBP-positive cells in asthmatic patients (5.06%; range, 1.8 to 9.6%) was significantly greater than in nonasthmatic subjects (0.6%; range, 0.1 to 1.2; p 0.05; Table 2). There was no significant difference between asthmatic patients and normal control subjects in the percentage of CD3-, CD68-, or elastase-positive cells (Table 2). Cytokine Immunoreactivity in Induced Sputum Immunoreactivity for eotaxin, MCP-4, and IL-16 was present in all asthmatic patients. Eotaxin, MCP-4, and IL-16 immunoreactivity were examined in both epithelial and nonepithelial cells (Fig 1). Omission of the primary antibody or addition of an irrelevant antibody did not result in any specific immunostaining. For eotaxin, the percentage of immunoreactive cells in asthmatic patients was 17 1.5%, of which 10.3 0.9% were epithelial cells and 6.7 0.6% were nonepithelial cells (Fig 2, top, A). Similarly, the percentage of IL-16 immunoreactive cells was 10 2.1%, of which 4 0.9% were epithelial cells and 6 1.2% were nonepithelial cells (Fig 2, middle, B). Lastly, the percentage of MCP-4 immunoreactive cells was 21.3 2.8%, of which 13 1.6% were epithelial and 8.3 1.2% were nonepithelial cells (Fig 2, bottom, C). There was a significant increase in the percentage of both epithelial and nonepithelial cells demonstrating eotaxin, MCP-4, and IL-16 expression in induced sputum of asthmatic patients compared to control subjects (Fig 2). There was a significant correlation between the percentage of eosinophils and the percentage of epithelial cells (r 2 0.68; p 0.05) expressing eotaxin immunoreactivity in asthmatic patients. There was also a significant correlation between eosinophils and the percentage of nonepithelial cells (r 2 0.6; p 0.05) expressing eotaxin immunoreactivity in asthmatic patients. In contrast, there was no significant correlation between eosinophils and the expression of MCP-4 or IL-16 in epithelial or nonepithelial cells in asthmatic patients. Also, there was no significant correlation of CD3 cells to the expression of IL-16 in asthmatic patients. There was CHEST / 120 / 2/ AUGUST, 2001 597

Table 2 Percentage of Inflammatory Cells in Induced Sputum From Asthmatic Patients and Control Subjects* Variables Eosinophils Neutrophils Macrophages Epithelial cells T Lymphocytes Asthmatic patients 1 1.4 36 37 13 8 2 2.3 37 29 8 2 3 9.6 51 35 12 5 4 7.1 49 36 9 4 5 4.2 53 30 8 2 6 8.1 42 41 7 7 7 2.3 44 40 7 5 8 2.7 40 26 5 4 9 5.1 50 27 14 3 10 8.2 53 33 12 6 Median (range) 5.06 (1.2 9.6) 45.5 (37 53) 33.4 (26 44) 9.5 (5 14) 4.6 (2 8) Control subjects 1 0.1 48 44 1.3 3 2 0.9 43 28 0.5 4 3 1.1 39 35 0.8 1 4 0.8 45 41 1.5 5 5 0.4 49 43 0.9 2 6 0.1 43 39 1.1 3 7 1.2 47 38 0.6 1 8 0.3 45 37 0.7 3 9 0.5 39 44 1.1 4 Median (range) 0.6 (0.1 1.2) 44.2 (39 49) 38.7 (28 44) 0.9 (0.4 1.5) 2.8 (1 5) *There was a significant difference in the percentages of MBP-positive and epithelial cells between asthmatic patients and control subjects (p 0.05). no significant correlation between the percentage of eosinophils and the percentage of epithelial and nonepithelial cells expressing eotaxin in normal control subjects. There was no significant correlation between the number of cells expressing eotaxin, MCP-4, and IL-16 to PC 20 in asthmatic patients. Discussion In this study, we have investigated airway inflammation in asthmatic patients using the noninvasive sampling technique of induced sputum. We have demonstrated the ability to detect eotaxin, MCP-4, and IL-16 immunoreactive protein in inflammatory cells recovered from induced sputum. To our knowledge, these results demonstrate for the first time that cytokines with chemoattractant activity, such as eotaxin, MCP-4, and IL-16, can be detected in induced sputum in both epithelial and nonepithelial cells of asthmatic patients. Furthermore, the numbers of both epithelial and nonepithelial cells expressing those cytokines were increased in asthmatic patients compared with normal control subjects, extending our previous findings in bronchial biopsy specimens and BAL. 8,11,12 In addition, we demonstrated that eotaxin immunoreactivity in epithelial and nonepithelial cells correlated significantly with the percentage of eosinophils in induced sputum, supporting the important role for eotaxin in eosinophil migration into the airways in asthma. The study of the mechanisms responsible for the recruitment of inflammatory cells into the airways is of primordial importance to our understanding of the pathogenesis of asthma. Recent studies 3 have demonstrated the potential role of cytokines with chemoattractant activities such as C-C chemokines and IL-16 in this process. Most investigators 8,11,12 have looked at the expression of these cytokines in the airways using fiberoptic techniques to retrieve bronchial biopsy specimens and BAL fluid. More recently, we and others 13 15 have used the noninvasive technique of induced sputum to investigate the mechanisms involved in the development and persistence of airway eosinophilia in patients with asthma. IL-5, a cytokine involved in eosinophil differentiation and migration, is increased in induced sputum of asthmatic patients, and its expression correlates with parameters of lung function, demonstrating the feasibility of this technique in investigating the putative role of cytokines in airway disease. 15,17 The present study was undertaken to investigate the expression in induced sputum of other cytokines with more potent and specific chemotactic activity for eosinophils. Our findings demonstrate that increased expression of eotaxin, MCP-4, and IL-16 in induced sputum may be 598 Laboratory and Animal Investigations

Figure 1. Representative examples of immunocytochemistry of epithelial cells expressing eotaxin and MCP-4 immunoreactivity in induced sputum from asthmatic patients and normal control subjects (hematoxylin, original 200). Red cytoplasmic staining in top left, A, and top right, B, indicates eotaxin immunoreactivity in asthmatic patients and normal control subjects, respectively. Bottom left, C, and bottom right, D, show MCP-4 immunoreactivity in asthmatic patients and normal control subjects, respectively. potential markers for inflammation in asthma. Eotaxin expression in particular correlated with sputum eosinophilia. Eosinophil levels in induced sputum correlate with disease severity and with asthma exacerbations. 17 It is intriguing to hypothesize that expression of cytokines such as eotaxin might correlate with asthma disease severity. A novel finding of this study relates to the ability to detect chemoattractant cytokines in the cellular component of induced sputum. The measurement of cytokines in the fluid phase of induced sputum is less sensitive and has been associated with difficulties in reproducibility. 18,19 We were able to detect cytokine immunoreactivity in all asthmatic patients. Another potential advantage of this technique relates to the possibility of determining the cellular sources of specific cytokines. Eotaxin, MCP-4, and IL-16 were present in both bronchial epithelial and nonepithelial cells, which is consistent with previous findings in lung tissues that showed that eotaxin, MCP-4, and IL-16 immunoreactivity were colocalized mainly to epithelial cells and to a lesser extent to macrophages, T cells, and eosinophils. 12,20,21 Epithelial cells are believed to play a critical role in the inflammatory response in asthma. For instance, airway epithelial cells have been shown to synthesize RANTES (regulated upon activation, normally T-cell expressed and secreted), MCP-4, eotaxin, IL-16, IL-8, and others in response to proinflammatory mediators such as IL-1 and tumor necrosis factor-, suggesting that these cells, in part, contribute to the recruitment of inflammatory cells into the airways. 22 24 Eotaxin and MCP-4 induce eosinophil migration in vitro. 23,24 Furthermore, IL-16 has been shown to induce CD4-bearing eosinophil migration in vitro and in this regard was more potent than leukotriene C 4. 10 IL-16 may also contribute to the pathology of asthma through recruitment and activation of CD4 T cells. 9 Until recently, the biological activity of epithelial cells in patients with asthma has been mainly investigated using in vitro cell cultures from epithelial cells lines and freshly isolated epithelial cells from bronchial biopsy specimens or bronchial brushings. In addition, airway epithelial cells have been recovered using bronchial wash, BAL, and induced sputum. The percentage of bronchial epithelial cells in induced sputum is comparable to bronchial wash and is much greater than in BAL, suggesting that this latter technique may be less useful to study airway CHEST / 120 / 2/ AUGUST, 2001 599

Figure 2. Percentage of epithelial and nonepithelial cells expressing eotaxin, IL-16, and MCP-4 immunoreactivity (top, A; middle, B; and bottom, C, respectively) in induced sputum from asthmatic patients (n 10) and normal control subjects (n 9). epithelial cell biology. 25,26 The percentage of epithelial cells in induced sputum from healthy and asthmatic subjects varies considerably in studies 13,26 reported to date. The percentage of epithelial cells seen in induced sputum in this study is greater than in a previous study, 13 but overlaps with findings in another study. 26 This could be due to a difference in the technique used to detect the epithelial cells. Traditionally, epithelial cells are identified morphologically by microscopy or by flow cytometry, but cellular morphology might be more difficult to recognize in clinical situations where there is more epithelial cell damage 27 and epithelial cells may be mistakenly misread as generic mononuclear cells. Epithelial cell numbers may be significantly underestimated using those techniques. Immunocytochemical staining with cytokeratin identifies all epithelial cells, including ciliated and nonciliated columnar epithelial cells, and thus might be a more accurate method to measure epithelial cells in sputum. In this study, epithelial cells were identified with an immunocytochemistry technique using a modified APAAP method with cytokeratin as the specific immunohistochemical marker for epithelial cells recovered by sputum induction. The current study suggests that induced sputum is particularly useful to evaluate chemokine expression in bronchial epithelial cells in asthma patients. The findings also suggest that induced sputum can be used to differentiate epithelial chemokine expression in atopic asthmatic patients compared to normal subjects. We did not observe any difference in the percentage of sputum CD3 T cells in asthmatic patients and normal subjects despite increases in the percentage of IL-16 immunoreactive cells in asthmatic patients. The discrepancy of increased IL-16 expression and comparable CD3 cells percentages between the two groups suggests that sputum might not reflect changes of T-cell numbers in the bronchial mucosa. There was no significant correlation of CD3 T cells to the expression of IL-16 in induced sputum in asthmatic patients despite our previous demonstration that T cells are the major nonepithelial cellular source of IL-16 in mucosal biopsy specimens from asthmatic patients. 11 The percentage of sputum CD3 T cells was low in both asthmatic patients and normal control subjects, confirming previous findings in induced sputum in patients with asthma, 13,15,25,26 and suggests that induced sputum may not be the best medium to study T-cell related phenomena in asthma. In conclusion, our study confirms the predominant expression of eotaxin, MCP-4, and IL-16 in asthmatic airways. The positive correlation between eotaxin immunoreactivity and eosinophils numbers supports the potential important role of this cytokine for the development of airway eosinophilia in asthma. More importantly, our data demonstrate the utility of induced sputum as a noninvasive technique to document the presence of these chemoattractant cytokines in the airways in asthma. It is possible that the induced sputum technique might be useful to evaluate the therapeutic effects of a variety of antiinflammatory agents on cytokine and chemokine production by airway inflammatory cells, including bronchial epithelial cells, in asthmatic patients. ACKNOWLEDGMENT: We thank Ms. Elsa Schotman and Anna Aprile for technical assistance. References 1 Djukanovic R, Roche WR, Wilson JW, et al. Mucosal inflammation in asthma. Am Rev Respir Dis 1990; 142:434 457 2 Bradley BL, Azzawi M, Jacobson M, et al. Eosinophils, 600 Laboratory and Animal Investigations

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Am J Respir Crit Care Med 1995; 152:53 58 26 Grootendorst DC, Sont JK, Willems LN, et al. Comparison of inflammatory cell counts in asthma: induced sputum vs bronchial lavage and bronchial biopsies. Clin Exp Allergy 1997; 27:769 779 27 Tateishi K, Motojima s, Kushima A, et al. Comparison between allergen-induced and exercise-induced asthma with respect to the late asthmatic response, airway responsiveness, and Creola bodies in sputum. Ann Allergy Asthma Immunol 1996; 77:229 237 CHEST / 120 / 2/ AUGUST, 2001 601