Inflamm. Res. (2014) 63:951–959 DOI 10.1007/s00011-014-0770-0
Inflammation Research
ORIGINAL RESEARCH PAPER
Sputum neutrophil count after bronchial allergen challenge is related to peripheral blood neutrophil chemotaxis in asthma patients Simona Lavinskiene • Ieva Bajoriuniene Kestutis Malakauskas • Jolanta Jeroch • Raimundas Sakalauskas
•
Received: 23 April 2014 / Revised: 1 August 2014 / Accepted: 23 August 2014 / Published online: 12 September 2014 Ó Springer Basel 2014
Abstract Objective The aim of this study was to estimate relations between sputum neutrophilia and the chemotactic activity of peripheral blood neutrophils after the bronchial allergen challenge in asthma patients. Materials and methods Fifteen patients with allergic asthma (AA), 13 patients with allergic rhinitis (AR), all sensitized to Dermatophagoides pteronyssinus, and 8 healthy subjects (HS) underwent bronchial challenge with D. pteronyssinus. Sputum and peripheral blood collection were performed 24 h before, 7 and 24 h after the bronchial challenge. Cell counts were determined by the MayGru¨nwald-Giemsa method. Neutrophil chemotaxis was analyzed by a flow cytometer; IL-8 levels were measured by ELISA. Results Sputum neutrophil count and peripheral blood neutrophil chemotaxis of patients with AA were greater 7 and 24 h after the challenge compared with the baseline values and patients with AR and HS (P \ 0.05). Moreover, a significant correlation was found between the neutrophil count in sputum and IL-8 levels, and the chemotactic activity of peripheral blood neutrophils 24 h after the bronchial challenge only the patients with AA (P \ 0.05). Conclusions Increased sputum neutrophil count was found to be associated with an enhanced chemotactic activity of peripheral blood neutrophils during allergen-
Responsible Editor: Andrew Roberts. S. Lavinskiene (&) I. Bajoriuniene K. Malakauskas J. Jeroch R. Sakalauskas Department of Pulmonology and Immunology, Lithuanian University of Health Sciences, Kaunas, Lithuania e-mail: [email protected]
induced late-phase airway inflammation in patients with allergic asthma. Keywords Neutrophil Chemotaxis IL-8 Allergic asthma Dermatophagoides pteronyssinus
Introduction Asthma is characterized by an airway obstructive response that is associated with chronic airway inflammation [1]. Eosinophils as well as CD4? T cells are believed to a play essential role in the pathogenesis of allergic airway inflammation through the release of inflammatory mediators [2]. Despite the fact that eosinophils are relevant in the pathogenesis of asthma, a growing body of evidence shows that neutrophils are also important cells contributing to the inflammatory process in human asthma [3]. The influx of neutrophils to the airway has been reported in severe asthma attacks [4] and after intratracheal allergen challenge in a murine asthma model [3]. Neutrophil influx to the airways is based on the reactivity of neutrophils to chemoattractive signals, a process known as chemotaxis, which is crucial for an efficient control of pathogens. During chemotaxis, cells migrate through barriers (vessel walls or epithelial layers) and tissues toward a site of infection or allergen-induced inflammation [5, 6]. The increased migration of neutrophils from blood into the airways stimulates the accumulation and abnormal activation of inflammatory cells that are largely responsible for oxidative stress and production of proteases and inflammatory cytokines contributing to lung injury and chronic inflammation [7, 8]. Interleukin (IL)-8, a member of the CXC chemokine family [9], is a cytokine that has a wide spectrum of
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biological activities on different cell types, such as neutrophils, T cells, and basophils [4]. IL-8 is a major chemoattractant of neutrophils, produced and released by neutrophils [5, 10], alveolar macrophages [11], and other activated cells. It is suggested that IL-8 plays a key role in the accumulation of neutrophils in the sites of inflammation in severe asthmatic patients [4]. However, the mechanisms that control neutrophil accumulation in the airways during allergic airway inflammation have not been completely elucidated yet. We hypothesized that the neutrophil count in sputum and neutrophil chemotaxis after bronchial allergen challenge may represent a link between neutrophil activation and neutrophilic influx to the airways in allergen-induced late-phase airway inflammation. Therefore, in the present study we aimed at investigating the neutrophil count in sputum and neutrophil chemotaxis in peripheral blood in patients with allergic asthma before and after bronchial allergen challenge with Dermatophagoides pteronyssinus (D. pteronyssinus). Study population A total of 36 nonsmoking adults (18 men and 18 women; mean age, 30 ± 4 years) were examined: 15 patients with intermittent- or mild-to-moderate persistent allergic asthma, defined according the GINA criteria [12], 13 patients with mild-to-moderate/severe persistent allergic rhinitis, defined according to the ARIA criteria [13], and 8 healthy subjects who made up the control group. The patients were recruited from the Department of Pulmonology and Immunology, Hospital of the Lithuanian University of Health Sciences, Kaunas. The study protocol was approved by the Regional Ethics Committee for Biomedical Research (work adhered to the Declaration of Helsinki), Lithuanian University of Health Sciences (protocol no. 48/2004, version 4, 2010), and each participant gave his/her informed written consent. Patients with allergic asthma and allergic rhinitis had a clinical history of the disease for C1 year, current symptoms, and positive results of the skin prick test (C3 mm) with D. pteronyssinus. All patients were instructed to refrain from using inhaled or nasal as well as oral steroids at least 1 month before visits, short-acting b2 agonists at least 12 h and long-acting b2 agonists at least 48 h before the lung function test, and antihistamines and antileukotrienes, 7 days before the skin prick test. None of the patients had a history of smoking. Baseline forced expiratory volume in 1 s (FEV1) was more than 70 % of the predicted value in all the patients. None of the patients had any clinically significant allergy to other allergens, such as pollen or cat and dog dander. All the healthy subjects were nonsmokers, without symptoms of rhinitis or asthma, with
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normal findings of spirometry, and all showed negative results of the skin prick test. Skin prick testing All the patients were screened for allergy by the skin prick test using standardized allergen extracts (Stallergenes S.A., France) for the following allergens: D. pteronyssinus, D. farinae, cat and dog dander, mixed grass pollen, birch pollen, mugwort, cockroach Alternaria, Aspergillus, and Cladosporium. Phenolated glycero-saline was used for a negative control, and histamine hydrochloride (10 mg/mL) was used for a positive control. Skin testing was read 15 min after application. The results of skin prick test were considered positive if the mean wheal diameter was C3 mm [14]. Study design On a screening visit, all subjects were informed about participation in the study, informed written consent was obtained, inclusion/exclusion criteria were verified, and also physical examination, spirometry, responsiveness to methacholine, and the skin prick test were performed. Twenty-four hours before bronchial challenge with D. pteronyssinus, spirometry was performed and peripheral blood was collected; these data were used as baseline values. Bronchial challenge with D. pteronyssinus was performed at 8:00 in the morning, and spirometry was reassessed every 10 min within the first hour and then every hour for subsequent 6 h. Physical examination and peripheral blood collection were repeated 7 and 24 h after bronchial challenge with D. pteronyssinus. Lung function testing Pulmonary function was tested using a pneumotachometric spirometer ‘‘CustovitM’’ (Custo Med, Germany). Baseline forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC), and FEV1/FVC ratio were recorded as the highest of three reproducible measurements. The results were compared with the predicted values matched for age, body height, and sex according to the standard methodology [15]. Measurement of airway responsiveness to methacholine Airway responsiveness was assessed as changes in the airway function after challenge with inhaled methacholine using a reservoir method [16]. Methacholine was nebulized into a 10-L reservoir with a pressure nebulizer (Pari Provocation I; Pari, Stanberg, Germany). Aerolized
Sputum neutrophil count after bronchial allergen
methacholine was inhaled through a one-way valve at 5-min intervals starting with methacholine at a dose of 15 lg dose and doubling it until a 20 % decrease in FEV1 from the baseline or a total cumulative dose of 3,870 lg was achieved. The bronchoconstricting effect of each dose of methacholine was expressed as a percentage of decrease in FEV1 from the baseline value. The provocative dose of methacholine causing a C20 % fall in FEV1 (PD20) was calculated from the log dose–response curve by the linear interpolation of two adjacent data points. Bronchial challenge with allergen Bronchial challenge was performed with a D. pteronyssinus allergen (Stallergenes SA, France) at different concentrations (0.01, 0.1, 1.0, 10, and 33.3 IR/mL) [17]. A freeze-dried lyophilized allergen was diluted with nonphenol saline. The allergen at graded doses was delivered through a Devilbiss 646 nebulizer attached to a KoKo DigiDoser (Pulmonary Data Services, Louisville, Co). The results of the challenge test were considered positive if a C20 % fall in FEV1 from the baseline was achieved. Spirometry was reassessed every 10 min within the first hour and later on every hour for subsequent 6 h for safety reasons in case of late asthmatic bronchoconstriction. Peripheral blood collection and isolation of neutrophils Peripheral blood samples for neutrophil isolation and activity measurement were collected into sterile vacutainers with ethylene diamine tetra-acetic acid (EDTA) 24 h before (at baseline) as well as 7 and 24 h after allergen challenge. Neutrophils were isolated by density gradient centrifugation. The whole blood was layered on FicollPaque PLUS (GE Healthcare, Finland) and centrifuged at 1,000g for 30 min at room temperature. The neutrophil population was separated by the hypotonic lysis of erythrocytes. Isolated neutrophils were diluted in cell culture RPMI 1640 media (Biological Industries, Israel) at a final concentration of 2 9 106/mL. The viability of neutrophils was checked by flow cytometry, and it always was [95 %. Sputum induction and processing Subjects inhaled 10 mL of sterile hypertonic saline solution (3, 4, or 5 % NaCl, Ivex Pharmaceuticals, USA) at room temperature from an ultrasonic nebulizer (DeVilbiss Health Care, USA). The duration of each inhalation was 7 min, and it was stopped after expectorationof an adequate amount of sputum. In order to detect a possible decrease in FEV1, spirometry was performed after each inhalation. Sputum was poured into a Petri dish and separated from
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saliva. A fourfold volume of freshly prepared 0.1 % dithiothreitol (DTT; Sigma-Aldrich) was added. The mixture was vortexed and placed on a bench rocker for 15 min at room temperature. Next, an equal volume of phosphatebuffered saline solution (PBS; Sigma-Aldrich) was added to DTT. The cell pellet was separated using a 40-lm cell stainer (Becton–Dickinson, USA). The mixture was centrifuged for 10 min at 4 °C; the supernatant was aspirated and stored at -70 °C for later assay. The total cell counts, percentage of epithelial cells, and cell viability were investigated using a Neubauer hemocytometer (HeinzHerenz; Germany) under a microscope (B5 Professional, Motic, China) by employing the Trypan blue exclusion method. The cytospin samples of induced sputum were prepared using a cytofuge instrument (Shandon Southern Instruments, USA). Induced sputum cell analysis Prepared sputum cytospins were stained by the MayGru¨nwald-Giemsa method for differential cell counts. Cell differentiation was determined by counting approximately 400 cells in random fields of view under a light microscope, excluding squamous epithelial cells. The cells were identified by standard morphological criteria, nuclear morphology, and cytoplasmic granulation. Cell counts were expressed as percentages of total cells and absolute values (106/L). Neutrophil chemotaxis in vitro Neutrophil chemotaxis in vitro was performed in a 10-well cell transmigration chamber (Neuro Probe, USA). The lower and upper wells of chamber were isolated by a polyvinylpyrrolidone (PVP)-treated polycarbonate track-etch membrane, containing 2 9 106 3 lm/mm2 pores (Neuro Probe, USA). The lower wells were pre-filled with isotonic Percoll (GE Healthcare, Finland) and IL-8, a chemotactic factor, at different concentrations (10, 30, or 100 ng/mL). RPMI 1640 was used as a negative control. The upper wells were filled with neutrophil culture suspension (2 9 106/ mL) and incubated for 2 h (37 °C, 5 % CO2). After the incubation, the suspensions of upper and lower wells were resuspended in tubes for flow cytometry. Nonmigrated neutrophils remained in the upper wells. The migration rate was calculated from the total number of neutrophils harvested from the lower well and expressed as percentage of the total input of neutrophils into the upper well. The number of migrated neutrophils was calculated by flow cytometry using Liquid Counting Beads (BD Biosciences, USA) according to the manufacturer’s recommendations. The amount of migrated neutrophils was expressed in percentages.
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Table 1 Demographic and clinical characteristics of the study population Characteristic
Patients with allergic asthma (n = 15)
Patients with allergic rhinitis (n = 13)
Healthy subjects (n = 8)
Age, mean ± SEM (range), years
34 ± 4 (21–50)
30 ± 4 (20–36)
31 ± 3 (22–45)
Sex (male/female), n
7/8
9/4
2/6
Wheal diameter induced by D. pteronyssinus, mean ± SEM (range), mm FEV1, mean ± SEM (range), % of predicted
6.3 ± 1.0 (4–11)
7.2 ± 0.8 (3–15)
0
94 ± 4* (82–116)
112 ± 1 1 (97–141)
108 ± 10 (79–114)
PD20, geometric mean (range)
0.3 (0.06–0.96)
–
–
Maximal decrease in FEV1 7 h after bronchial challenge with D. pteronyssinus, mean ± SEM (range), %
30 ± 10* (15–61)
6 ± 2 (2–8)
6 ± 2 (4–7)
Data are expressed as mean ± SEM * P \ 0.05 compared with healthy individuals
P \ 0.05 compared with patients with allergic rhinitis
FEV1 forced expiratory volume in the first second, PD20 provocative dose of methacholine causing a 20 % fall in FEV1
Detection of cytokine in serum and induced sputum supernatant The serum and sputum IL-8 levels were measured by an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (BioSource S.A., Belgium), The minimum detectable concentration was 2 pg/ mL. The peripheral blood cell analysis was performed on an automated hematology analyzer (Sysmex XE-5000, Japan).
assumed). Comparisons of the allergen-induced changes (before and after bronchial challenge) in dependent samples were analyzed with repeated measures analysis of variance (ANOVA). Relationships between variables were evaluated by means of Spearman rank correlation. Statistical significance was assumed at a P value of \0.05.
Results Statistical analysis Characteristics of studied subjects Statistical analysis was performed using the Statistical Package for Social Sciences, version 17.0 for Windows (SPSS 17.0). The Shapiro-Wilks test was employed to verify the normality. All the data that were normally distributed are presented as mean and standard error of the mean (SEM). The data that were not normally distributed were expressed as median and interquartile range (IQR). Due to a skewed distribution of the variable, nonparametric tests were used. The Kruskal–Wallis test was used to evaluate statistical differences between both groups of patients and the control group. Significant differences between two independent groups were determined by the Mann–Whitney U test. Changes in data before and after bronchial challenge in each group were evaluated for statistical significance at each time point with the Friedman test and by the Wilcoxon test for paired analyses. For multiple comparisons, the Bonferroni method was used to adjust P values. For normally distributed variables, differences between groups were explored using one-way ANOVA followed by a post hoc analysis using the Bonferroni test (equal variances assumed) or the Games-Howell test (equal variances not
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A total of 36 nonsmoking adults (18 men and 18 women; mean age, 30 ± 2 years) were examined: 15 patients with allergic asthma, 13 with allergic rhinitis, and 8 healthy subjects. The demographic and clinical characteristics of the study population are presented in Table 1. There were no significant age and gender differences comparing the groups. All patients were sensitized to D. pteronyssinus, which was confirmed by the skin prick test. The mean wheal diameter induced by D. pteronyssinus was similar in patients with allergic asthma and those with allergic rhinitis. Baseline FEV1 (%) was significantly lower in the patients with allergic asthma compared with patients with allergic rhinitis. Bronchial challenge with D. pteronyssinus provoked early bronchoconstriction in all 15 patients with allergic asthma. A maximum percentage fall in FEV1 after bronchial challenge was significantly greater in patients with allergic asthma than those with allergic rhinitis and healthy individuals. In patients with allergic rhinitis, bronchial challenge provoked nasal symptoms but it did not cause bronchoconstriction.
Sputum neutrophil count after bronchial allergen
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Table 2 Cell counts in the peripheral blood of patients with allergic asthma, patients with allergic rhinitis, and healthy subjects 24 h before as well as 7 and 24 h after bronchial challenge with D. pteronyssinus Patients with allergic asthma (n = 15)
Patients with allergic rhinitis (n = 13)
Healthy subjects (n = 8)
-24 h
?7 h
?24 h
-24 h
?7 h
?24 h
-24 h
?7 h
?24 h
Eosinophils
0.25 (0.44)
0.24 (0.45)
0.38* (0.30)
0.16 (0.29)
0.21 (0.35)
0.21* (0.58)
0.16 (0.12)
0.16 (0.14)
0.14 (0.19)
Neutrophils
3.55 (1.87)
3.01 (1.67)
2.58 (1.93)
3.50 (1.34)
3.97 (0.56)
3.54 (1.79)
3.69 (1.48)
3.56 (1.00)
3.06 (1.50)
Lymphocytes
1.60 (0.55)
2.12 (0.66)
1.84 (0.21)
2.06 (0.59)
1.94 (0.86)
1.80 (0.68)
1.71 (0.60)
1.80 (0.54)
1.71 (0.55)
Cells (9109/mL)
Data are shown as median (interquartile range) * P \ 0.05 compared with healthy subjects
P \ 0.05 compared with baseline values
Table 3 Cell counts in the induced sputum of patients with allergic asthma, patients with allergic rhinitis, and healthy subjects 24 h before as well as 7 and 24 h after bronchial challenge with D. pteronyssinus Cells (9106/mL)
Patients with allergic asthma (n = 15)
Patients with allergic rhinitis (n = 13)
Healthy subjects (n = 8)
–24 h
–24 h
-24 h
?7 h
#
?24 h #
Eosinophils
0.10* (0.04) 0.52*
Neutrophils
0.86 (0.14)
Lymphocytes 0.10* (0.03) Total cells
2.67*# (0.9)
#
(0.27) 0.64*
?7 h
?24 h
?24 h
(0.31) 0.03 (0.04) 0.07* (0.02) 0.08* (0.03) 0.02 (0.02) 0.02 (0.02) 0.02 (0.02)
1.03*# (0.39) 1.26*# (0.59) 0.71 (0.23) 0.81 (0.27) #
?7 h
#
0.88 (0.39)
0.81 (0.28) 0.73 (0.11) 0.75 (0.22)
0.09 (0.08)
0.12* (0.06)
0.07 (0.05) 0.09 (0.04)
0.08 (0.03)
0.07 (0.04) 0.07 (0.03) 0.07 (0.01)
3.10*# (1.68)
3.52*# (1.51)
2.0 (0.74)
2.51 (0.69)
2.17 (0.45) 2.01 (0.58) 2.18 (0.45)
2.43 (0.52)
Data are shown as median (interquartile range) * P \ 0.05 compared with healthy subjects P \ 0.05 compared with patients with allergic rhinitis
#
P \ 0.05 compared with the values 24 h before challenge
Cell counts The eosinophil count in the peripheral blood increased significantly in the patients with allergic asthma and allergic rhinitis 24 h after bronchial challenge as compared with the baseline values (Table 2). At the baseline as well as 7 and 24 h after the bronchial challenge, the total eosinophil count in sputum was significantly higher in the patients with allergic asthma than those with allergic rhinitis and healthy subjects (Table 3). At 7 and 24 h after the bronchial challenge, the patients with allergic asthma showed a significantly higher neutrophil count compared with the patients with allergic rhinitis and healthy subjects (P \ 0.05), but there was no significant difference at the baseline. Peripheral blood neutrophil chemotaxis in vitro At the baseline neutrophil chemotaxis in the peripheral blood after the stimulation with 30 ng/mL of IL-8 caused a higher migration of neutrophils isolated from the patients with allergic asthma than those with allergic rhinitis and healthy subjects (Fig. 1). Seven and 24 h after the bronchial allergen challenge with D. pteronyssinus the greatest neutrophil chemotaxis was documented in the patients with
Fig. 1 A histogram representing the chemotaxis of peripheral blood neutrophils stimulated with 30 ng/mL of IL-8 in patients with allergic asthma, allergic rhinitis and healthy subjects 24 h before as well as 7 and 24 h after the bronchial allergen challenge with D. pteronyssinus. Data are shown as a median (IQR). AA indicates patients with allergic asthma (n = 15), AR patients with allergic rhinitis (n = 13), HS healthy subjects (n = 8). *P \ 0.05 compared with HS, #P \ 0.05 compared with AR, P \ 0.05 compared with the values 24 h before the bronchial challenge
AA, and it was significantly greater than in the patients with allergic rhinitis and healthy subjects (P \ 0.05). Also neutrophil chemotaxis was significantly higher 7 and 24 h after the bronchial allergen challenge in patients with AA
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Fig. 2 A scatter plot representing the levels of IL-8 in sputum (a) and serum (b) in patients with allergic asthma, allergic rhinitis and healthy subjects 24 h before as well as 7 and 24 h after allergen challenge with D. pteronyssinus. Data are shown as median (IQR). AA indicates patients with allergic asthma (n = 15), AR patients with allergic rhinitis (n = 13), HS healthy subjects (n = 8). *P\0.05 compared with HS, #P\0.05 compared with AR, P\0.05 compared with values 24 h before challenge
and AR, especially in AA, compared with baseline values (P \ 0.05). Bronchial allergen challenge had no significant effect on neutrophil chemotaxis in the peripheral blood of healthy subjects. IL-8 levels in induced sputum and serum There was a significant increase in the sputum IL-8 levels in patients with allergic asthma and allergic rhinitis compared with the healthy subjects at 24 h before the bronchial challenge (Fig. 2a). At 7 and 24 h after the bronchial challenge, the sputum IL-8 levels increased significantly in patients with allergic asthma and allergic rhinitis compared with the healthy subjects. Moreover, the sputum IL-8 levels at 7 and 24 h after the bronchial challenge were significantly higher in the patients with allergic asthma than those with allergic rhinitis.
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Sputum neutrophil count after bronchial allergen b Fig. 3 Correlation between neutrophil chemotaxis in the peripheral
blood and the neutrophil count in sputum (a), neutrophil chemotaxis in the peripheral blood and the IL-8 level in sputum (open triangles) and serum (black squares) (b), the neutrophil count and the IL-8 level in sputum (c), and the neutrophil count in the peripheral blood and the serum IL-8 level (d) in the patients with allergic asthma 24 h after the bronchial allergen challenge
The serum IL-8 levels before the bronchial allergen challenge were significantly higher in the patients with allergic asthma than those with allergic rhinitis and the healthy subjects (P \ 0.05). However, there was no significant difference in the serum IL-8 levels comparing the patients with allergic rhinitis and the healthy subjects. At 7 and 24 h after the bronchial challenge, the serum IL-8 levels were also significantly elevated in the patients with allergic asthma compared with the patients with allergic rhinitis and the healthy subjects as well as with the baseline level (Fig. 2b). Bronchial challenge with D. pteronyssinus had no influence on the IL-8 levels in sputum and serum of healthy subjects. Relationships between activated neutrophils and neutrophil influx into the airways after bronchial allergen challenge with D. pteronyssinus in patients with allergic asthma In order to detect possible relationships between activated neutrophils and the influx of neutrophils into airways in allergen-induced late phase airway inflammation, correlation analysis was performed. This analysis revealed significant relationships only in the patients with allergic asthma at 24 h after the bronchial challenge. Neutrophil chemotaxis in the peripheral blood significantly correlated with the neutrophil count in sputum (Rs = 0.56, P = 0.02) 24 h after the bronchial challenge (Fig. 3a). Moreover, there was a significant positive correlation between neutrophil chemotaxis in the peripheral blood and IL-8 levels in sputum (Rs = 0.58, P = 0.02) and serum (Rs = 0.59, P = 0.02) (Fig. 3b) as well as the neutrophil count and the IL-8 level in sputum (Rs = 0.73, P = 0.01) (Fig. 3c), the neutrophil count in the peripheral blood and the serum IL-8 level (Rs = 0.56, P = 0.03) (Fig. 3d)
Discussion In the present study, we investigated relations between sputum neutrophilia and neutrophil chemotaxis in the peripheral blood during allergen-induced late-phase airway inflammation in asthma patients. The results showed a significant increase of sputum neutrophil count and activated peripheral blood neutrophil
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chemotaxis after the bronchial allergen challenge with D. pteronyssinus in asthma patients. Also, IL-8 levels were elevated both in sputum and serum samples. Moreover, significant relationships between the IL-8 levels, sputum neutrophilia and the chemotaxis of peripheral blood neutrophil were found in patients with allergic asthma in allergen-induced late-phase airway inflammation. Studies in humans and rodents [18, 19] have shown that airway infiltration and neutrophil activation are critical features in acute lung injury in response to endotoxin exposure. Moreover, a recent study by Fu et al. has reported that systemic inflammation was associated with airway neutrophilia in asthma, and a group of differentially expressed genes in the lung involving multiple cytokine pathways was implicated [20]. As some studies on humans found the neutrophil influx in patients with fatal or near fatal asthma [21], so firstly we aimed to evaluate the influx of cells caused by allergen exposure. We demonstrated that the total neutrophil count in sputum was significantly higher at 7 and 24 h after the bronchial challenge in the patients with allergic asthma compared with those with allergic rhinitis and the healthy subjects as well as the baseline values. These results suggest that increased neutrophilia in the lungs can be caused by allergen-induced late-phase airway inflammation. It is well known that the accumulation of neutrophils in airways, especially during chronic inflammation, for example in COPD, can be promoted by IL-8 [22]. This cytokine is a classical chemoattractant, and it is known that the expression of IL-8 in the airways of asthmatic patients is upregulated [23]. Moreover, a recent study has demonstrated that an endobronchial allergen provocation stimulated the generation of IL-8 in the bronchoalveolar lavage fluid of patients with asthma [24]. Furthermore, neutrophils stimulated by IL-8 increase the migration of eosinophils across the basement membrane, promoting their accumulation in airways [25]. Also, a study by Pelikan demonstrated that IL-8 levels in patients with allergic asthma were increased in plasma and supernatants of inflammatory cells following allergen challenge [26]. However, the mechanisms of neutrophil activity as well as the effect of IL-8 on neutrophil chemotaxis in allergeninduced late-phase airway inflammation have not been completely elucidated yet. Therefore, secondly we aimed to evaluate the chemotactic activity of peripheral blood neutrophils activated with IL-8 and the levels of IL-8 before and after allergen challenge in patients with allergic asthma, allergic rhinitis, and healthy subjects. Sackmann et al. [27] demonstrated a decrease in chemotaxis index, chemotaxis speed, and chemotaxis velocity in neutrophils in patients with allergic asthma. While, we observed that after the bronchial allergen challenge with D. pteronyssinus, especially after 24 h,
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neutrophil chemotaxis was significantly greater in the patients with allergic asthma compared with the baseline values and other groups (P \ 0.05). This suggests that the allergen challenge may lead to activation of additional pathways that may contribute to the enhanced peripheral blood neutrophil chemotaxis in patients with allergic asthma. Therefore, we found enhanced chemotaxis of blood peripheral neutrophils before allergen challenge in patients with allergic asthma, these findings show that neutrophils are already activated and can promote inflammation. Also, our study results confirmed that IL-8 levels were markedly elevated in serum samples in patients with allergic asthma 24 h before, 7 and 24 h after the bronchial challenge compared with the patients with allergic rhinitis and the healthy subjects (P \ 0.05). Moreover, we found that IL-8 levels were significantly greater not only in serum but also in sputum samples of patients with allergic asthma 24 h before, 7 and 24 h after the bronchial challenge than those with allergic rhinitis and the healthy subjects. Thus, the findings of our study demonstrate that following inhaled allergen challenge, the synthesis of IL-8 can be increased with neutrophil chemotaxis that leads to allergeninduced late-phase airway inflammation in patients with allergic asthma. Finally, we aimed to evaluate the possible relationship between neutrophil influx to the airways and activated neutrophils in allergen-induced late-phase airway inflammation. Significant correlations were observed only 24 h after the bronchial challenge and only in the patients with allergic asthma. The chemotaxis of peripheral blood neutrophils was positively correlated with the neutrophil count in sputum and the IL-8 levels both in serum and sputum. The correlation between the neutrophil count in sputum and the IL-8 levels in serum and sputum in patients with the allergic asthma 24 h after allergen exposure was documented as well. All these data confirm that IL-8 is an important cytokine in allergic airway inflammation recruiting neutrophils to the site of allergen-induced inflammation [28]. In summary, allergen-induced late-phase airway inflammation in patients with asthma is accompanied by neutrophil influx into the airways, which is related to the enhanced chemotaxis of peripheral blood neutrophils. Acknowledgments This research was funded by a grant (No. LIG18/2010) from the Research Council of Lithuania.
References 1. Bochner BS, Undem BJ, Lichtenstein LM. Immunological aspects of allergic asthma. Annu Rev Immunol. 1994;12:295–335. 2. Dworski R, Simon HU, Hoskins A, Yousefi S. Eosinophil and neutrophil extracellular DNA traps in human allergic asthmatic airways. J Allergy Clin Immunol. 2011;127(5):1260–6.
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S. Lavinskiene et al. 3. Nabe T, Hosokawa F, Matsuya K, Morishita T, Ikedo A, Fujii M, et al. Important role of neutrophils in the late asthmatic response in mice. Life Sci. 2011;88(25–26):1127–35. 4. Nakagome K, Nagata M. Pathogenesis of airway inflammation in bronchial asthma. Auris Nasus Larynx. 2011;38(5):555–63. 5. Babusyte A, Jeroch J, Stakauskas R, Stravinskaite K, Malakauskas K, Sakalauskas R. The effect of induced sputum and bronchoalveolar lavage fluid from patients with chronic obstructive pulmonary disease on neutrophil migration in vitro. Medicina. 2010;46(5):315–22. 6. Yoshikawa T, Dent G, Ward J, Angco G, Nong G, Nomura N, et al. Impaired neutrophil chemotaxis in chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2007;175(5):473–9. 7. Chung KF, Adcock IM. Multifaceted mechanisms in COPD: inflammation, immunity, and tissue repair and destruction. Eur Respir J. 2008;31(6):1334–56. 8. Rahman I, Biswas SK, Kode A. Oxidant and antioxidant balance in the airways and airway diseases. Eur J Pharmacol. 2006;533(1–3):222–39. 9. Monteseirin J, Chacon P, Vega A, El Bekay R, Alvarez M, Alba G, et al. Human neutrophils synthesize IL-8 in an IgE-mediated activation. J Leukoc Biol. 2004;76(3):692–700. 10. Willemse BW, ten Hacken NH, Rutgers B, Lesman-Leegte IG, Postma DS, Timens W. Effect of 1-year smoking cessation on airway inflammation in COPD and asymptomatic smokers. Eur Respir J. 2005;26(5):835–45. 11. Palmberg L, Larsson BM, Malmberg P, Larsson K. Induction of IL-8 production in human alveolar macrophages and human bronchial epithelial cells in vitro by swine dust. Thorax. 1998;53(4):260–4. 12. Bateman ED, Hurd SS, Barnes PJ, Bousquet J, Drazen JM, FitzGerald M, et al. Global strategy for asthma management and prevention: GINA executive summary. Eur Respir J. 2008;31(1):143–78. 13. Bousquet J, Khaltaev N, Cruz AA, Denburg J, Fokkens WJ, Togias A, et al. Allergic Rhinitis and its Impact on Asthma (ARIA) 2008 update (in collaboration with the World Health Organization, GA(2)LEN and AllerGen). Allergy. 2008;63(Suppl 86):8–160. 14. Bousquet J, Heinzerling L, Bachert C, Papadopoulos NG, Bousquet PJ, Burney PG, et al. Practical guide to skin prick tests in allergy to aeroallergens. Allergy. 2012;67(1):18–24. 15. Miller MR, Hankinson J, Brusasco V, Burgos F, Casaburi R, Coates A, et al. Standardisation of spirometry. Eur Respir J. 2005;26(2):319–38. 16. Baur X, Huber H, Degens PO, Allmers H, Ammon J. Relation between occupational asthma case history, bronchial methacholine challenge, and specific challenge test in patients with suspected occupational asthma. Am J Ind Med. 1998; 33(2):114–22. 17. Liu L, Jarjour NN, Busse WW, Kelly EA. Enhanced generation of helper T type 1 and 2 chemokines in allergen-induced asthma. Am J Respir Crit Care Med. 2004;169(10):1118–24. 18. Dillon MA, Harris B, Hernandez ML, Zou B, Reed W, Bromberg PA, et al. Enhancement of systemic and sputum granulocyte response to inhaled endotoxin in people with the GSTM1 null genotype. Occup Environ Med. 2011;68(10):783–5. 19. Wagner JG, Birmingham NP, Jackson-Humbles D, Jiang Q, Harkema JR, Peden DB. Supplementation with gamma-tocopherol attenuates endotoxin-induced airway neutrophil and mucous cell responses in rats. Free Radic Biol Med. 2013;68C:101–9. 20. Fu JJ, Baines KJ, Wood LG, Gibson PG. Systemic inflammation is associated with differential gene expression and airway neutrophilia in asthma. OMICS. 2013;17(4):187–99.
Sputum neutrophil count after bronchial allergen 21. Tonnel AB, Gosset P, Tillie-Leblond I. Characteristics of the inflammatory response in bronchial lavage fluids from patients with status asthmaticus. Int Arch Allergy Immunol. 2001;124(1–3):267–71. 22. Nadigel J, Audusseau S, Baglole CJ, Eidelman DH, Hamid Q. IL8 production in response to cigarette smoke is decreased in epithelial cells from COPD patients. Pulm Pharmacol Ther. 2013;26(5):596–602. 23. Shannon J, Ernst P, Yamauchi Y, Olivenstein R, Lemiere C, Foley S, et al. Differences in airway cytokine profile in severe asthma compared to moderate asthma. Chest. 2008;133(2):420–6. 24. Hoskins A, Reiss S, Wu P, Chen N, Han W, Do RH, et al. Asthmatic airway neutrophilia after allergen challenge is associated with the glutathione S-transferase M1 genotype. Am J Respir Crit Care Med. 2013;187(1):34–41.
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Inflammation Research
ORIGINAL RESEARCH PAPER
Sputum neutrophil count after bronchial allergen challenge is related to peripheral blood neutrophil chemotaxis in asthma patients Simona Lavinskiene • Ieva Bajoriuniene Kestutis Malakauskas • Jolanta Jeroch • Raimundas Sakalauskas
•
Received: 23 April 2014 / Revised: 1 August 2014 / Accepted: 23 August 2014 / Published online: 12 September 2014 Ó Springer Basel 2014
Abstract Objective The aim of this study was to estimate relations between sputum neutrophilia and the chemotactic activity of peripheral blood neutrophils after the bronchial allergen challenge in asthma patients. Materials and methods Fifteen patients with allergic asthma (AA), 13 patients with allergic rhinitis (AR), all sensitized to Dermatophagoides pteronyssinus, and 8 healthy subjects (HS) underwent bronchial challenge with D. pteronyssinus. Sputum and peripheral blood collection were performed 24 h before, 7 and 24 h after the bronchial challenge. Cell counts were determined by the MayGru¨nwald-Giemsa method. Neutrophil chemotaxis was analyzed by a flow cytometer; IL-8 levels were measured by ELISA. Results Sputum neutrophil count and peripheral blood neutrophil chemotaxis of patients with AA were greater 7 and 24 h after the challenge compared with the baseline values and patients with AR and HS (P \ 0.05). Moreover, a significant correlation was found between the neutrophil count in sputum and IL-8 levels, and the chemotactic activity of peripheral blood neutrophils 24 h after the bronchial challenge only the patients with AA (P \ 0.05). Conclusions Increased sputum neutrophil count was found to be associated with an enhanced chemotactic activity of peripheral blood neutrophils during allergen-
Responsible Editor: Andrew Roberts. S. Lavinskiene (&) I. Bajoriuniene K. Malakauskas J. Jeroch R. Sakalauskas Department of Pulmonology and Immunology, Lithuanian University of Health Sciences, Kaunas, Lithuania e-mail: [email protected]
induced late-phase airway inflammation in patients with allergic asthma. Keywords Neutrophil Chemotaxis IL-8 Allergic asthma Dermatophagoides pteronyssinus
Introduction Asthma is characterized by an airway obstructive response that is associated with chronic airway inflammation [1]. Eosinophils as well as CD4? T cells are believed to a play essential role in the pathogenesis of allergic airway inflammation through the release of inflammatory mediators [2]. Despite the fact that eosinophils are relevant in the pathogenesis of asthma, a growing body of evidence shows that neutrophils are also important cells contributing to the inflammatory process in human asthma [3]. The influx of neutrophils to the airway has been reported in severe asthma attacks [4] and after intratracheal allergen challenge in a murine asthma model [3]. Neutrophil influx to the airways is based on the reactivity of neutrophils to chemoattractive signals, a process known as chemotaxis, which is crucial for an efficient control of pathogens. During chemotaxis, cells migrate through barriers (vessel walls or epithelial layers) and tissues toward a site of infection or allergen-induced inflammation [5, 6]. The increased migration of neutrophils from blood into the airways stimulates the accumulation and abnormal activation of inflammatory cells that are largely responsible for oxidative stress and production of proteases and inflammatory cytokines contributing to lung injury and chronic inflammation [7, 8]. Interleukin (IL)-8, a member of the CXC chemokine family [9], is a cytokine that has a wide spectrum of
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biological activities on different cell types, such as neutrophils, T cells, and basophils [4]. IL-8 is a major chemoattractant of neutrophils, produced and released by neutrophils [5, 10], alveolar macrophages [11], and other activated cells. It is suggested that IL-8 plays a key role in the accumulation of neutrophils in the sites of inflammation in severe asthmatic patients [4]. However, the mechanisms that control neutrophil accumulation in the airways during allergic airway inflammation have not been completely elucidated yet. We hypothesized that the neutrophil count in sputum and neutrophil chemotaxis after bronchial allergen challenge may represent a link between neutrophil activation and neutrophilic influx to the airways in allergen-induced late-phase airway inflammation. Therefore, in the present study we aimed at investigating the neutrophil count in sputum and neutrophil chemotaxis in peripheral blood in patients with allergic asthma before and after bronchial allergen challenge with Dermatophagoides pteronyssinus (D. pteronyssinus). Study population A total of 36 nonsmoking adults (18 men and 18 women; mean age, 30 ± 4 years) were examined: 15 patients with intermittent- or mild-to-moderate persistent allergic asthma, defined according the GINA criteria [12], 13 patients with mild-to-moderate/severe persistent allergic rhinitis, defined according to the ARIA criteria [13], and 8 healthy subjects who made up the control group. The patients were recruited from the Department of Pulmonology and Immunology, Hospital of the Lithuanian University of Health Sciences, Kaunas. The study protocol was approved by the Regional Ethics Committee for Biomedical Research (work adhered to the Declaration of Helsinki), Lithuanian University of Health Sciences (protocol no. 48/2004, version 4, 2010), and each participant gave his/her informed written consent. Patients with allergic asthma and allergic rhinitis had a clinical history of the disease for C1 year, current symptoms, and positive results of the skin prick test (C3 mm) with D. pteronyssinus. All patients were instructed to refrain from using inhaled or nasal as well as oral steroids at least 1 month before visits, short-acting b2 agonists at least 12 h and long-acting b2 agonists at least 48 h before the lung function test, and antihistamines and antileukotrienes, 7 days before the skin prick test. None of the patients had a history of smoking. Baseline forced expiratory volume in 1 s (FEV1) was more than 70 % of the predicted value in all the patients. None of the patients had any clinically significant allergy to other allergens, such as pollen or cat and dog dander. All the healthy subjects were nonsmokers, without symptoms of rhinitis or asthma, with
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normal findings of spirometry, and all showed negative results of the skin prick test. Skin prick testing All the patients were screened for allergy by the skin prick test using standardized allergen extracts (Stallergenes S.A., France) for the following allergens: D. pteronyssinus, D. farinae, cat and dog dander, mixed grass pollen, birch pollen, mugwort, cockroach Alternaria, Aspergillus, and Cladosporium. Phenolated glycero-saline was used for a negative control, and histamine hydrochloride (10 mg/mL) was used for a positive control. Skin testing was read 15 min after application. The results of skin prick test were considered positive if the mean wheal diameter was C3 mm [14]. Study design On a screening visit, all subjects were informed about participation in the study, informed written consent was obtained, inclusion/exclusion criteria were verified, and also physical examination, spirometry, responsiveness to methacholine, and the skin prick test were performed. Twenty-four hours before bronchial challenge with D. pteronyssinus, spirometry was performed and peripheral blood was collected; these data were used as baseline values. Bronchial challenge with D. pteronyssinus was performed at 8:00 in the morning, and spirometry was reassessed every 10 min within the first hour and then every hour for subsequent 6 h. Physical examination and peripheral blood collection were repeated 7 and 24 h after bronchial challenge with D. pteronyssinus. Lung function testing Pulmonary function was tested using a pneumotachometric spirometer ‘‘CustovitM’’ (Custo Med, Germany). Baseline forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC), and FEV1/FVC ratio were recorded as the highest of three reproducible measurements. The results were compared with the predicted values matched for age, body height, and sex according to the standard methodology [15]. Measurement of airway responsiveness to methacholine Airway responsiveness was assessed as changes in the airway function after challenge with inhaled methacholine using a reservoir method [16]. Methacholine was nebulized into a 10-L reservoir with a pressure nebulizer (Pari Provocation I; Pari, Stanberg, Germany). Aerolized
Sputum neutrophil count after bronchial allergen
methacholine was inhaled through a one-way valve at 5-min intervals starting with methacholine at a dose of 15 lg dose and doubling it until a 20 % decrease in FEV1 from the baseline or a total cumulative dose of 3,870 lg was achieved. The bronchoconstricting effect of each dose of methacholine was expressed as a percentage of decrease in FEV1 from the baseline value. The provocative dose of methacholine causing a C20 % fall in FEV1 (PD20) was calculated from the log dose–response curve by the linear interpolation of two adjacent data points. Bronchial challenge with allergen Bronchial challenge was performed with a D. pteronyssinus allergen (Stallergenes SA, France) at different concentrations (0.01, 0.1, 1.0, 10, and 33.3 IR/mL) [17]. A freeze-dried lyophilized allergen was diluted with nonphenol saline. The allergen at graded doses was delivered through a Devilbiss 646 nebulizer attached to a KoKo DigiDoser (Pulmonary Data Services, Louisville, Co). The results of the challenge test were considered positive if a C20 % fall in FEV1 from the baseline was achieved. Spirometry was reassessed every 10 min within the first hour and later on every hour for subsequent 6 h for safety reasons in case of late asthmatic bronchoconstriction. Peripheral blood collection and isolation of neutrophils Peripheral blood samples for neutrophil isolation and activity measurement were collected into sterile vacutainers with ethylene diamine tetra-acetic acid (EDTA) 24 h before (at baseline) as well as 7 and 24 h after allergen challenge. Neutrophils were isolated by density gradient centrifugation. The whole blood was layered on FicollPaque PLUS (GE Healthcare, Finland) and centrifuged at 1,000g for 30 min at room temperature. The neutrophil population was separated by the hypotonic lysis of erythrocytes. Isolated neutrophils were diluted in cell culture RPMI 1640 media (Biological Industries, Israel) at a final concentration of 2 9 106/mL. The viability of neutrophils was checked by flow cytometry, and it always was [95 %. Sputum induction and processing Subjects inhaled 10 mL of sterile hypertonic saline solution (3, 4, or 5 % NaCl, Ivex Pharmaceuticals, USA) at room temperature from an ultrasonic nebulizer (DeVilbiss Health Care, USA). The duration of each inhalation was 7 min, and it was stopped after expectorationof an adequate amount of sputum. In order to detect a possible decrease in FEV1, spirometry was performed after each inhalation. Sputum was poured into a Petri dish and separated from
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saliva. A fourfold volume of freshly prepared 0.1 % dithiothreitol (DTT; Sigma-Aldrich) was added. The mixture was vortexed and placed on a bench rocker for 15 min at room temperature. Next, an equal volume of phosphatebuffered saline solution (PBS; Sigma-Aldrich) was added to DTT. The cell pellet was separated using a 40-lm cell stainer (Becton–Dickinson, USA). The mixture was centrifuged for 10 min at 4 °C; the supernatant was aspirated and stored at -70 °C for later assay. The total cell counts, percentage of epithelial cells, and cell viability were investigated using a Neubauer hemocytometer (HeinzHerenz; Germany) under a microscope (B5 Professional, Motic, China) by employing the Trypan blue exclusion method. The cytospin samples of induced sputum were prepared using a cytofuge instrument (Shandon Southern Instruments, USA). Induced sputum cell analysis Prepared sputum cytospins were stained by the MayGru¨nwald-Giemsa method for differential cell counts. Cell differentiation was determined by counting approximately 400 cells in random fields of view under a light microscope, excluding squamous epithelial cells. The cells were identified by standard morphological criteria, nuclear morphology, and cytoplasmic granulation. Cell counts were expressed as percentages of total cells and absolute values (106/L). Neutrophil chemotaxis in vitro Neutrophil chemotaxis in vitro was performed in a 10-well cell transmigration chamber (Neuro Probe, USA). The lower and upper wells of chamber were isolated by a polyvinylpyrrolidone (PVP)-treated polycarbonate track-etch membrane, containing 2 9 106 3 lm/mm2 pores (Neuro Probe, USA). The lower wells were pre-filled with isotonic Percoll (GE Healthcare, Finland) and IL-8, a chemotactic factor, at different concentrations (10, 30, or 100 ng/mL). RPMI 1640 was used as a negative control. The upper wells were filled with neutrophil culture suspension (2 9 106/ mL) and incubated for 2 h (37 °C, 5 % CO2). After the incubation, the suspensions of upper and lower wells were resuspended in tubes for flow cytometry. Nonmigrated neutrophils remained in the upper wells. The migration rate was calculated from the total number of neutrophils harvested from the lower well and expressed as percentage of the total input of neutrophils into the upper well. The number of migrated neutrophils was calculated by flow cytometry using Liquid Counting Beads (BD Biosciences, USA) according to the manufacturer’s recommendations. The amount of migrated neutrophils was expressed in percentages.
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Table 1 Demographic and clinical characteristics of the study population Characteristic
Patients with allergic asthma (n = 15)
Patients with allergic rhinitis (n = 13)
Healthy subjects (n = 8)
Age, mean ± SEM (range), years
34 ± 4 (21–50)
30 ± 4 (20–36)
31 ± 3 (22–45)
Sex (male/female), n
7/8
9/4
2/6
Wheal diameter induced by D. pteronyssinus, mean ± SEM (range), mm FEV1, mean ± SEM (range), % of predicted
6.3 ± 1.0 (4–11)
7.2 ± 0.8 (3–15)
0
94 ± 4* (82–116)
112 ± 1 1 (97–141)
108 ± 10 (79–114)
PD20, geometric mean (range)
0.3 (0.06–0.96)
–
–
Maximal decrease in FEV1 7 h after bronchial challenge with D. pteronyssinus, mean ± SEM (range), %
30 ± 10* (15–61)
6 ± 2 (2–8)
6 ± 2 (4–7)
Data are expressed as mean ± SEM * P \ 0.05 compared with healthy individuals
P \ 0.05 compared with patients with allergic rhinitis
FEV1 forced expiratory volume in the first second, PD20 provocative dose of methacholine causing a 20 % fall in FEV1
Detection of cytokine in serum and induced sputum supernatant The serum and sputum IL-8 levels were measured by an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (BioSource S.A., Belgium), The minimum detectable concentration was 2 pg/ mL. The peripheral blood cell analysis was performed on an automated hematology analyzer (Sysmex XE-5000, Japan).
assumed). Comparisons of the allergen-induced changes (before and after bronchial challenge) in dependent samples were analyzed with repeated measures analysis of variance (ANOVA). Relationships between variables were evaluated by means of Spearman rank correlation. Statistical significance was assumed at a P value of \0.05.
Results Statistical analysis Characteristics of studied subjects Statistical analysis was performed using the Statistical Package for Social Sciences, version 17.0 for Windows (SPSS 17.0). The Shapiro-Wilks test was employed to verify the normality. All the data that were normally distributed are presented as mean and standard error of the mean (SEM). The data that were not normally distributed were expressed as median and interquartile range (IQR). Due to a skewed distribution of the variable, nonparametric tests were used. The Kruskal–Wallis test was used to evaluate statistical differences between both groups of patients and the control group. Significant differences between two independent groups were determined by the Mann–Whitney U test. Changes in data before and after bronchial challenge in each group were evaluated for statistical significance at each time point with the Friedman test and by the Wilcoxon test for paired analyses. For multiple comparisons, the Bonferroni method was used to adjust P values. For normally distributed variables, differences between groups were explored using one-way ANOVA followed by a post hoc analysis using the Bonferroni test (equal variances assumed) or the Games-Howell test (equal variances not
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A total of 36 nonsmoking adults (18 men and 18 women; mean age, 30 ± 2 years) were examined: 15 patients with allergic asthma, 13 with allergic rhinitis, and 8 healthy subjects. The demographic and clinical characteristics of the study population are presented in Table 1. There were no significant age and gender differences comparing the groups. All patients were sensitized to D. pteronyssinus, which was confirmed by the skin prick test. The mean wheal diameter induced by D. pteronyssinus was similar in patients with allergic asthma and those with allergic rhinitis. Baseline FEV1 (%) was significantly lower in the patients with allergic asthma compared with patients with allergic rhinitis. Bronchial challenge with D. pteronyssinus provoked early bronchoconstriction in all 15 patients with allergic asthma. A maximum percentage fall in FEV1 after bronchial challenge was significantly greater in patients with allergic asthma than those with allergic rhinitis and healthy individuals. In patients with allergic rhinitis, bronchial challenge provoked nasal symptoms but it did not cause bronchoconstriction.
Sputum neutrophil count after bronchial allergen
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Table 2 Cell counts in the peripheral blood of patients with allergic asthma, patients with allergic rhinitis, and healthy subjects 24 h before as well as 7 and 24 h after bronchial challenge with D. pteronyssinus Patients with allergic asthma (n = 15)
Patients with allergic rhinitis (n = 13)
Healthy subjects (n = 8)
-24 h
?7 h
?24 h
-24 h
?7 h
?24 h
-24 h
?7 h
?24 h
Eosinophils
0.25 (0.44)
0.24 (0.45)
0.38* (0.30)
0.16 (0.29)
0.21 (0.35)
0.21* (0.58)
0.16 (0.12)
0.16 (0.14)
0.14 (0.19)
Neutrophils
3.55 (1.87)
3.01 (1.67)
2.58 (1.93)
3.50 (1.34)
3.97 (0.56)
3.54 (1.79)
3.69 (1.48)
3.56 (1.00)
3.06 (1.50)
Lymphocytes
1.60 (0.55)
2.12 (0.66)
1.84 (0.21)
2.06 (0.59)
1.94 (0.86)
1.80 (0.68)
1.71 (0.60)
1.80 (0.54)
1.71 (0.55)
Cells (9109/mL)
Data are shown as median (interquartile range) * P \ 0.05 compared with healthy subjects
P \ 0.05 compared with baseline values
Table 3 Cell counts in the induced sputum of patients with allergic asthma, patients with allergic rhinitis, and healthy subjects 24 h before as well as 7 and 24 h after bronchial challenge with D. pteronyssinus Cells (9106/mL)
Patients with allergic asthma (n = 15)
Patients with allergic rhinitis (n = 13)
Healthy subjects (n = 8)
–24 h
–24 h
-24 h
?7 h
#
?24 h #
Eosinophils
0.10* (0.04) 0.52*
Neutrophils
0.86 (0.14)
Lymphocytes 0.10* (0.03) Total cells
2.67*# (0.9)
#
(0.27) 0.64*
?7 h
?24 h
?24 h
(0.31) 0.03 (0.04) 0.07* (0.02) 0.08* (0.03) 0.02 (0.02) 0.02 (0.02) 0.02 (0.02)
1.03*# (0.39) 1.26*# (0.59) 0.71 (0.23) 0.81 (0.27) #
?7 h
#
0.88 (0.39)
0.81 (0.28) 0.73 (0.11) 0.75 (0.22)
0.09 (0.08)
0.12* (0.06)
0.07 (0.05) 0.09 (0.04)
0.08 (0.03)
0.07 (0.04) 0.07 (0.03) 0.07 (0.01)
3.10*# (1.68)
3.52*# (1.51)
2.0 (0.74)
2.51 (0.69)
2.17 (0.45) 2.01 (0.58) 2.18 (0.45)
2.43 (0.52)
Data are shown as median (interquartile range) * P \ 0.05 compared with healthy subjects P \ 0.05 compared with patients with allergic rhinitis
#
P \ 0.05 compared with the values 24 h before challenge
Cell counts The eosinophil count in the peripheral blood increased significantly in the patients with allergic asthma and allergic rhinitis 24 h after bronchial challenge as compared with the baseline values (Table 2). At the baseline as well as 7 and 24 h after the bronchial challenge, the total eosinophil count in sputum was significantly higher in the patients with allergic asthma than those with allergic rhinitis and healthy subjects (Table 3). At 7 and 24 h after the bronchial challenge, the patients with allergic asthma showed a significantly higher neutrophil count compared with the patients with allergic rhinitis and healthy subjects (P \ 0.05), but there was no significant difference at the baseline. Peripheral blood neutrophil chemotaxis in vitro At the baseline neutrophil chemotaxis in the peripheral blood after the stimulation with 30 ng/mL of IL-8 caused a higher migration of neutrophils isolated from the patients with allergic asthma than those with allergic rhinitis and healthy subjects (Fig. 1). Seven and 24 h after the bronchial allergen challenge with D. pteronyssinus the greatest neutrophil chemotaxis was documented in the patients with
Fig. 1 A histogram representing the chemotaxis of peripheral blood neutrophils stimulated with 30 ng/mL of IL-8 in patients with allergic asthma, allergic rhinitis and healthy subjects 24 h before as well as 7 and 24 h after the bronchial allergen challenge with D. pteronyssinus. Data are shown as a median (IQR). AA indicates patients with allergic asthma (n = 15), AR patients with allergic rhinitis (n = 13), HS healthy subjects (n = 8). *P \ 0.05 compared with HS, #P \ 0.05 compared with AR, P \ 0.05 compared with the values 24 h before the bronchial challenge
AA, and it was significantly greater than in the patients with allergic rhinitis and healthy subjects (P \ 0.05). Also neutrophil chemotaxis was significantly higher 7 and 24 h after the bronchial allergen challenge in patients with AA
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Fig. 2 A scatter plot representing the levels of IL-8 in sputum (a) and serum (b) in patients with allergic asthma, allergic rhinitis and healthy subjects 24 h before as well as 7 and 24 h after allergen challenge with D. pteronyssinus. Data are shown as median (IQR). AA indicates patients with allergic asthma (n = 15), AR patients with allergic rhinitis (n = 13), HS healthy subjects (n = 8). *P\0.05 compared with HS, #P\0.05 compared with AR, P\0.05 compared with values 24 h before challenge
and AR, especially in AA, compared with baseline values (P \ 0.05). Bronchial allergen challenge had no significant effect on neutrophil chemotaxis in the peripheral blood of healthy subjects. IL-8 levels in induced sputum and serum There was a significant increase in the sputum IL-8 levels in patients with allergic asthma and allergic rhinitis compared with the healthy subjects at 24 h before the bronchial challenge (Fig. 2a). At 7 and 24 h after the bronchial challenge, the sputum IL-8 levels increased significantly in patients with allergic asthma and allergic rhinitis compared with the healthy subjects. Moreover, the sputum IL-8 levels at 7 and 24 h after the bronchial challenge were significantly higher in the patients with allergic asthma than those with allergic rhinitis.
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Sputum neutrophil count after bronchial allergen b Fig. 3 Correlation between neutrophil chemotaxis in the peripheral
blood and the neutrophil count in sputum (a), neutrophil chemotaxis in the peripheral blood and the IL-8 level in sputum (open triangles) and serum (black squares) (b), the neutrophil count and the IL-8 level in sputum (c), and the neutrophil count in the peripheral blood and the serum IL-8 level (d) in the patients with allergic asthma 24 h after the bronchial allergen challenge
The serum IL-8 levels before the bronchial allergen challenge were significantly higher in the patients with allergic asthma than those with allergic rhinitis and the healthy subjects (P \ 0.05). However, there was no significant difference in the serum IL-8 levels comparing the patients with allergic rhinitis and the healthy subjects. At 7 and 24 h after the bronchial challenge, the serum IL-8 levels were also significantly elevated in the patients with allergic asthma compared with the patients with allergic rhinitis and the healthy subjects as well as with the baseline level (Fig. 2b). Bronchial challenge with D. pteronyssinus had no influence on the IL-8 levels in sputum and serum of healthy subjects. Relationships between activated neutrophils and neutrophil influx into the airways after bronchial allergen challenge with D. pteronyssinus in patients with allergic asthma In order to detect possible relationships between activated neutrophils and the influx of neutrophils into airways in allergen-induced late phase airway inflammation, correlation analysis was performed. This analysis revealed significant relationships only in the patients with allergic asthma at 24 h after the bronchial challenge. Neutrophil chemotaxis in the peripheral blood significantly correlated with the neutrophil count in sputum (Rs = 0.56, P = 0.02) 24 h after the bronchial challenge (Fig. 3a). Moreover, there was a significant positive correlation between neutrophil chemotaxis in the peripheral blood and IL-8 levels in sputum (Rs = 0.58, P = 0.02) and serum (Rs = 0.59, P = 0.02) (Fig. 3b) as well as the neutrophil count and the IL-8 level in sputum (Rs = 0.73, P = 0.01) (Fig. 3c), the neutrophil count in the peripheral blood and the serum IL-8 level (Rs = 0.56, P = 0.03) (Fig. 3d)
Discussion In the present study, we investigated relations between sputum neutrophilia and neutrophil chemotaxis in the peripheral blood during allergen-induced late-phase airway inflammation in asthma patients. The results showed a significant increase of sputum neutrophil count and activated peripheral blood neutrophil
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chemotaxis after the bronchial allergen challenge with D. pteronyssinus in asthma patients. Also, IL-8 levels were elevated both in sputum and serum samples. Moreover, significant relationships between the IL-8 levels, sputum neutrophilia and the chemotaxis of peripheral blood neutrophil were found in patients with allergic asthma in allergen-induced late-phase airway inflammation. Studies in humans and rodents [18, 19] have shown that airway infiltration and neutrophil activation are critical features in acute lung injury in response to endotoxin exposure. Moreover, a recent study by Fu et al. has reported that systemic inflammation was associated with airway neutrophilia in asthma, and a group of differentially expressed genes in the lung involving multiple cytokine pathways was implicated [20]. As some studies on humans found the neutrophil influx in patients with fatal or near fatal asthma [21], so firstly we aimed to evaluate the influx of cells caused by allergen exposure. We demonstrated that the total neutrophil count in sputum was significantly higher at 7 and 24 h after the bronchial challenge in the patients with allergic asthma compared with those with allergic rhinitis and the healthy subjects as well as the baseline values. These results suggest that increased neutrophilia in the lungs can be caused by allergen-induced late-phase airway inflammation. It is well known that the accumulation of neutrophils in airways, especially during chronic inflammation, for example in COPD, can be promoted by IL-8 [22]. This cytokine is a classical chemoattractant, and it is known that the expression of IL-8 in the airways of asthmatic patients is upregulated [23]. Moreover, a recent study has demonstrated that an endobronchial allergen provocation stimulated the generation of IL-8 in the bronchoalveolar lavage fluid of patients with asthma [24]. Furthermore, neutrophils stimulated by IL-8 increase the migration of eosinophils across the basement membrane, promoting their accumulation in airways [25]. Also, a study by Pelikan demonstrated that IL-8 levels in patients with allergic asthma were increased in plasma and supernatants of inflammatory cells following allergen challenge [26]. However, the mechanisms of neutrophil activity as well as the effect of IL-8 on neutrophil chemotaxis in allergeninduced late-phase airway inflammation have not been completely elucidated yet. Therefore, secondly we aimed to evaluate the chemotactic activity of peripheral blood neutrophils activated with IL-8 and the levels of IL-8 before and after allergen challenge in patients with allergic asthma, allergic rhinitis, and healthy subjects. Sackmann et al. [27] demonstrated a decrease in chemotaxis index, chemotaxis speed, and chemotaxis velocity in neutrophils in patients with allergic asthma. While, we observed that after the bronchial allergen challenge with D. pteronyssinus, especially after 24 h,
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neutrophil chemotaxis was significantly greater in the patients with allergic asthma compared with the baseline values and other groups (P \ 0.05). This suggests that the allergen challenge may lead to activation of additional pathways that may contribute to the enhanced peripheral blood neutrophil chemotaxis in patients with allergic asthma. Therefore, we found enhanced chemotaxis of blood peripheral neutrophils before allergen challenge in patients with allergic asthma, these findings show that neutrophils are already activated and can promote inflammation. Also, our study results confirmed that IL-8 levels were markedly elevated in serum samples in patients with allergic asthma 24 h before, 7 and 24 h after the bronchial challenge compared with the patients with allergic rhinitis and the healthy subjects (P \ 0.05). Moreover, we found that IL-8 levels were significantly greater not only in serum but also in sputum samples of patients with allergic asthma 24 h before, 7 and 24 h after the bronchial challenge than those with allergic rhinitis and the healthy subjects. Thus, the findings of our study demonstrate that following inhaled allergen challenge, the synthesis of IL-8 can be increased with neutrophil chemotaxis that leads to allergeninduced late-phase airway inflammation in patients with allergic asthma. Finally, we aimed to evaluate the possible relationship between neutrophil influx to the airways and activated neutrophils in allergen-induced late-phase airway inflammation. Significant correlations were observed only 24 h after the bronchial challenge and only in the patients with allergic asthma. The chemotaxis of peripheral blood neutrophils was positively correlated with the neutrophil count in sputum and the IL-8 levels both in serum and sputum. The correlation between the neutrophil count in sputum and the IL-8 levels in serum and sputum in patients with the allergic asthma 24 h after allergen exposure was documented as well. All these data confirm that IL-8 is an important cytokine in allergic airway inflammation recruiting neutrophils to the site of allergen-induced inflammation [28]. In summary, allergen-induced late-phase airway inflammation in patients with asthma is accompanied by neutrophil influx into the airways, which is related to the enhanced chemotaxis of peripheral blood neutrophils. Acknowledgments This research was funded by a grant (No. LIG18/2010) from the Research Council of Lithuania.
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