The monocyte-derived chemokine is released in the bronchoalveolar lavage fluid of steady-state asthmatics

Authors


Dr Luis M Teran
Department of Allergy and Clinical Immunology
Instituto Nacional de enfermedades Respiratorias
Calzada Tlalpan 4502
C.P. 14080
Mexico D.F.

Abstract

Infiltration of the airways by T helper type 2 (Th2) lymphocytes is a well-recognized feature of bronchial asthma. Monocyte-derived chemokine (MDC) is a potent attractant which activates Th2 lymphocytes via the chemokine receptor CCR4. We have investigated both leukocyte recruitment and MDC release into the airways of asthmatic patients. Differential cell counts in bronchoalveolar lavage (BAL) fluid showed that numbers of lymphocytes and eosinophils were elevated in asthmatics compared with normal subjects (median, 6.1 vs. 1.0 × 103/ml, P < 0.005 and 1.4 vs. 0.24 × 103/ml, P = 0.001, respectively). By enzyme-linked immunosorbent assay it was demonstrated that MDC concentrations were significantly elevated in BAL fluid from asthmatics compared with normals (medians 282 pg/ml, range 190–780 pg/ml vs. median 29 pg/ml range 17–82 pg/ml, P < 0.001). Interestingly, there was a significant correlation between MDC levels and the bronchoconstrictive response to methacholine [PC20 forced expiratory volume (FEV)1, r = −0.78, P = 0.001], suggesting that MDC may be involved in the severity of the disease. By immunohistochemistry, MDC was localized predominantly to the bronchial epithelium in bronchial biopsies derived from stable asthmatics. Moreover, primary human airway epithelial cells were found to release MDC upon cytokine stimulation. These findings suggest that MDC may play a major role in the pathogenesis of bronchial asthma.

Infiltration of the airways by T helper type 2 (Th2) lymphocytes is a well-recognized feature of asthma. CD4 T cells are known to orchestrate the allergic inflammatory reaction through the production of cytokines such as interleukin (IL)-4, IL-5 and IL-13 (1). Interleukin-4 and IL-13 stimulates B cells to produce immunoglobulin (Ig)E while IL-5 induces activation and differentiation of eosinophils. Activated CD4 T cells have been a consistent finding in both the bronchoalveolar lavage (BAL) fluid and bronchial biopsies in stable asthma (1, 2). Furthermore, CD4 lymphocytes increase further following allergen challenge (3, 4). Lymphocyte recruitment from peripheral blood into the airways is controlled by adhesion molecules and chemokines. The chemokines are a large family of 8–14 kDa heparin-binding peptides, which have been subdivided into four subfamilies on the basis of the position of either one or two cysteine residues located near the amino terminus of the protein (CXCL, CCL, CL, and CX3CL) (5, 6).

The monocyte-derived chemokine (MDC) is a member of the CCL subfamily, which was discovered by two independent groups (7, 8). The mouse homolog of MDC was identified in activated B lymphocytes and dendritic cells and designated ABCD-1 (9). The MDC is located to human chromosome 13. Upon activation MDC is produced by a number of cellular sources including macrophages, monocytes, dendritic cells, activated T lymphocytes and epithelial cells (7–10). Original studies showed that MDC induce migration of macrophages, monocytes, natural killer (NK) cells, and activated lymphocytes (7–9). Subsequent studies however, demonstrated that MDC also attract lymphocytes of the Th2 phenotype (11, 12). CCR4 is the receptor for both MDC and thymus and activation-regulated chemokine (TARC) (12, 13). Interestingly, this receptor is preferentially expressed in Th2 but not in Th1 lymphocytes (13, 15). CCR4 is also highly expressed in skin memory T cells [these cells co-express the cutaneous lymphocyte antigen (CTAL) which functions as a homing receptor for inflamed skin] (15). Both MDC and CCR4 immunoreactivities have been found to be expressed in bronchial biopsies derived from asthmatics exposed to allergen challenge (16) but not in stable asthmatics. In the present study, we have investigated MDC in the airways of stable asthmatics.

Methods

Subjects

A group of 17 symptom-free asthmatics (14 female, median age 36 years, range 18–49) treated with inhaled salbutamol alone (given as required and no more than three times a day) and 13 healthy subjects (eight male, median age 30 years, range 18–55) volunteered to participate in the study. Clinical characteristics of both asthmatic and normal subjects are shown in Table 1. According to the GINA guidelines patients were considered to suffer either intermittent (n = 3) or mild-persistent asthma (n = 14). Their atopic status was confirmed by skin prick testing (atopy was defined as a positive skin prick test wheal >3 mm) with a series of common inhalant allergens including Dermatophagoides pteronyssinus and farinae, mixed grass, tree pollen, cat and dog dander and cockroach (Alk Bello, Roun Rock, TX). For at least 2 months before the bronchoscopy none of the patients received any treatment with corticosteroids, sodium cromoglycate or nedocromil sodium. The Hospital's Ethic Committee approved the study, and patients gave their written informed consent.

Table 1.  Clinical characteristics of subjects
 SexAge (years)FEV1%PC-20
Asthmatics14 F3693.81.8
3 M(15–49)(80–146)(0.2–9.5)
Control5 F3598.832
8 M(18–55)(88–138) 

Fiber-optic bronchoscopy

Bronchoscopy was undertaken according to National Institutes of Health guidelines (17). After topical anesthesia of the upper airways (lignocaine) the bronchoscope (Olympus IT20D, Olympus America, Mexico) was passed through the nose or mouth and wedged into the medial segment. Bronchoalveolar lavage was performed with 150 ml of 0.9% saline solution (8 × 20 ml aliquots) at room temperature through the biopsy channel. On completion of the procedure, subjects were observed for 3 h and further nebulized salbutamol given as necessary.

The recovered BAL fluid was pooled and centrifuged at 400 g for 15 min at 4°C, the cells were separated and the supernatant stored at −70°C. A 100-μl aliquot of cell was subjected to cytocentrifugation (Logan, UT), air-dried and stained with a Diff-Quick Stain kit (Dade Behring, Newark, DE). Four hundred cells per cytospin were counted.

Measurements of MDC

Measurement of immunoreactive MDC in both BAL fluid and airway epithelial culture supernatant was performed in duplicate samples using a specific sandwich ELISA following the manufacturer's protocol (R & D Systems, Minneapolis, MN). Briefly, ELISA plates (Costar, Cambridge, MA) were coated with 100 μl of a monoclonal mouse antihuman MDC antibody (4 μg/ml) in carbonate/bicarbonate buffer (pH 9.6). The antibody was allowed to bind overnight at 4°C, and the plates were then washed four times with phosphate-buffered saline (PBS) containing 0.05% Tween 20 before the addition of 100 μl of either samples to be tested or standard, consisting of serial dilutions from 1 to 0.007 ng/ml of recombinant human MDC (R & D Systems). Blank wells containing no MDC were routinely included in each assay. Following overnight incubation, the plates were washed four times with PBS-Tween 20, and 100 μl of the biotinylated anti-MDC antibody (200 ng/ml) were added to each well. Plates were incubated for 2 h at room temperature before washing and adding 100 μl/well of StreptoAvidin Peroxidase Conjugate (Sigma, St Louis, MO) at 1 : 500 dilution and incubation at room temperature for 30 min. The plates were washed again with PBS Tween and the substrate added (Sigma). After 15-min incubation, the reaction was stopped by the addition of 50 μl of 2 M H2SO4, and the absorbance of each well was measured at 450 nm in micro-ELISA plate reader (Bio-Rad, CA). Concentrations of MDC in samples were calculated from the standard curve. The lower limit of detection was 10 pg/ml, and the interassay coefficient variation was 5%.

Immunohistochemistry

Bronchial biopsies were fixed in paraformaldehyde and paraffin embedded. Immunohistochemistry was performed by indirect immunoperoxidase staining as previously described (19). Briefly, after deparaffinization and hydration, tissue sections were incubated with 0.3% hydrogen peroxide (H2O2) in water at room temperature for 10 min to inhibit endogenous peroxidase. Subsequently, they were incubated for 30 min with 10% normal horse serum (Dako, Carpinteria, CA) to block nonspecific binding. The primary polyclonal anti-MDC antibody in a dilution of 1 : 200 (Santa Cruz Biotechnology Inc., Santa Cruz, CA) was applied and stored at 4°C for 24 h. After incubation the sections were rinsed three times for 5 min with tris-buffered saline (TBS) and the second antibody was applied (Vector Laboratories Inc., CA). After rising with TBS (3 × 5 min), streptavidin–peroxidase complex (Glostrup; Dako) diluted at 1 : 200 was overlayed for 30 min. After rinsing with TBS (3 × 5 min) 5 mg of 3,3-deaminobenzidine tetrahydrochloride substrate (Vector Laboratories Inc.) was applied for 10 min. The sections were then counterstained using Mayer's hematoxylene. For negative controls, the primary antibody was omitted.

Culture and stimulation of human airway epithelial cells

Human airway epithelial cells (HAECs) were isolated from bronchial epithelial mucosa tissue, which was obtained from patients undergoing surgery for therapeutic reasons and grown as previously described (18). Briefly, mucosal tissues were dissected into small stripes and then incubated at 37°C for 90 min in keratinocyte growth medium (KGM) (Clonetics, San Diego, CA) containing 0.1% trypsin. After filtering the cell suspension, epithelial cells were grown in 75 cm2 flasks to confluence in KGM.

For stimulation primary nasal and bronchial cells were transferred to six-well tissue culture plates (9.6 cm2/well; Falcon, Villa Hermosa, Tab., Mexico), where two wells were used for each measurement. Cells used for stimulation were second passage and cultured until they reached near 70–80% confluence (2 × 105 cells). After removal of growth medium and twice washing with PBS, cells were cultured in KGM lacking bovine pituitary extract (BPE) for 24 h and were subsequently stimulated with various cytokines including IL-4, IL-13, interferon (IFN)-γ at concentrations of 10 ng/ml. The purity and identity of the cells were checked by immunohistochemistry using the anti-cytokeratin mAb AE1/AE3 (Zymed, San Francisco, CA).

Statistical analysis

Analysis of MDC in BAL fluid and BAL cell counts was performed with the Mann–Whitney U-test for unpaired data. Correlations between MDC and infiltrating cells in BAL fluid were evaluated by the nonparametric Sperman's rank correlation coefficient test. A value of P < 0.05 was considered statistically significant. Statistical analysis was performed using SPSS 10.0 program.

Results

Clinical findings

The median age for asthmatics and controls was 35 vs 36 years, respectively. Lung function data show that asthmatics suffered from mild-persistent asthma. FEV1% predicted was lower in the asthmatics (median 93.8%, range 80–146%) than in the normal group (median 98.8%, range 88–138%).

Bronchoalveolar lavage fluid and cell population

Bronchoalveolar lavage was obtained from all 30 subjects taking part in the study. There was no significant difference in the volume of fluid recovered between asthmatic and normal subjects (68 ± 10 ml vs 60 ± 12 ml). Comparison of total and differential cell counts obtained from BAL fluid from asthmatic and normal subjects showed that lymphocytes and eosinophils numbers were significantly elevated in BAL fluid derived from asthmatics compared with normals (medians 6.1 vs 1.0 × 103/ml and 1.4 vs 0.24 × 103/ml, P = 0.001, respectively) (Table 2). Similarly, epithelial cell numbers were increased in the asthmatic group (1.3 vs 0.02, P < 0.05). There were no differences in the number of macrophages and total cell counts between asthmatic and normal subjects.

Table 2.  Differential and total cell counts in bronchoalveolar lavage fluid (×103/ml)
 AsthmaticsNormals
  1. BAL cells are expressed as median (range). * P < 0.005; ** P < 0.001; *** P < 0.05.

Macrophages20 (1.7–60)20.3 (1.8–129)
Lymphocytes6.1 (0–12.3)*1.0 (0–3.4)
Neutrophils5.8 (0–15)2.1 (0–10.8)
Eosinophils1.4 (0–8)**0.24 (0–0.8)
Epithelial cells1.3 (0–9.2)***0.02 (0–0.35)
Total cell count26 (3–100)31 (1.9–140)

Measurements of MDC in BAL fluid

Measurements of MDC by ELISA showed that levels of this cytokine were significantly elevated in BAL fluid from asthmatics compared with normals (medians 282 pg/ml, range 190–780 pg/ml vs 29 pg/ml, range 17–82 pg/ml, P < 0.001) (Fig. 1).

Figure 1.

Levels of immunoreactive MDC in the BAL fluid of either asthmatic or normal subjects. Horizontal lines represents the median value.

There was a significant correlation between MDC levels and bronchial hyperreactivity (PC20 FEV1 methacholine) in asthmatics (r = −0.78, P = 0.001; Fig. 2). In contrast, there was no significant correlation between MDC levels and any of the infiltrating BAL cells including lymphocytes, eosinophils and macrophages in both groups of subjects.

Figure 2.

Correlations between MDC and PC20 FEV1 to methacholine (bronchial hyperreactivity).

MDC immunoreactivity in bronchial biopsies

To investigate potential cellular sources of MDC the technique of immunohistochemistry was applied to bronchial biopsies. The MDC immunoreactivity was localized predominantly to the airway epithelium (Fig. 3). Scarce MDC was seen in mononuclear cells in the bronchial submucosa.

Figure 3.

(A) Localization of immunoreactive MDC to the airway epithelium. Scarce MDC immunoreactivity is seen in mononuclear cells. (B) Negative control (instead of the anti-MDC antibody the isotype goat antibody IgG 15256 was used as a control). Details of immunohistochemistry are given in methodology.

Production of MDC by HAECS

The ability of primary HAECs to produce MDC upon cytokine stimulation was investigated. Thus, following the second passage HAECs grown from bronchial tissue were stimulated with either Th1- (IFN-γ) or Th2 cytokines (IL-4 and IL-13). Measurements of MDC in the culture supernatant showed that both Th1- and Th2-type cytokines are potent inducers of MDC (Fig. 4).

Figure 4.

Levels of immunoreactive MDC in the culture supernatant derived from the cytokine-stimulated primary airway epithelial cells.

Discussion

The present study has demonstrated that MDC is released in increased concentrations in the airways of steady-state asthmatics. Levels of this cytokine in BAL fluid correlated with the severity of asthma as assessed by the bronchial response to methacholine (PC20) suggesting that MDC may play a major role in the pathogenesis of this disease. By immunohistochemistry, MDC was localized predominantly to the bronchial airway epithelium. Interestingly, both Th1- and Th2-type cytokines stimulated HAECs to release MDC in vitro.

Macrophage-derived chemokine was originally identified by two independent groups as a chemoattractant for macrophages, NK cells and activated T cells and subsequently shown to attract specifically Th2 lymphocytes (7–10). As CD4 lymphocytes play an important role in asthma this cytokine has been the focus of major attention in allergic inflammation. Studies in mouse models of allergen-induced airway inflammation have given evidence for a key role of MDC in the recruitment of Th2 lymphocytes into the lung (20, 21). In humans MDC has been associated with both atopic- and contact-allergic dermatitis (22–24). In a recent study, increased MDC immunoreactivity was found in bronchial biopsies derived from asthmatic exposed to allergen challenge, but not in biopsies obtained from sham-challenged asthmatics (16).

This is the first study to show that stable asthmatic patients release increased levels of MDC in BAL fluid. The failure to detect MDC immunoreactivity in bronchial biopsies derived from stable asthmatics in a previous report (16) suggests that analysis of BAL fluid constitutes an excellent alternative to study inflammatory mediators in mild asthma. Moreover, the finding that MDC concentrations correlated with the severity of the disease (response to methacholine PC20) suggests that this cytokine may be involved in the pathogenesis of bronchial hyperreactivity. Interestingly, CCR4, the receptor for MDC, has been found highly expressed in Th2 lymphocytes infiltrating the airways of asthmatics exposed to allergen challenge (16, 25). The lack of correlation between MDC levels and total lymphocyte numbers in the present study may be explained by the fact that Th2 lymphocytes represent only a small proportion of total BAL lymphocytes. We hypothesize that MDC may recruit Th2 lymphocytes into asthmatic airways which then amplify the allergic inflammatory reaction through the production of cytokines such as IL-4, IL-5 and IL-13. Both IL-4 and IL-13 stimulates B cells to produce IgE while IL-5 induces activation and differentiation of eosinophils (26, 27). These events may eventually lead to bronchial inflammation and bronchial hyperreactivity. Indeed, a previous report showing that pretreatment of sensitized mice with an anti-MDC antibody significantly inhibits allergen-induced bronchial hyperreactivity further supports our hypothesis and suggests that MDC may be directly involved in the pathogenesis of asthma. TARC and I-309 are other lymphocyte attractants which have been implicated in the allergic reaction (13–15). Indeed, increased TARC immunoreactivity has been reported in both bronchial biopsies and sputum derived from asthmatic subjects (28, 29), Thus, it is likely that MDC in concert with these chemokines may recruit Th2 lymphocytes into the asthmatic airways.

To investigate the cellular source of the MDC that we have detected in BAL fluid, we have performed immunohistochemical staining of bronchial biopsies. This has allowed us to demonstrate that MDC immunoreactivity was predominantly localized to the airway epithelium although scarce immunoreactivity was also observed to mononuclear cells in the bronchial submucosa. Panina-Bordignon et al. (16) have previously reported similar findings in bronchial biopsies derived from asthmatics exposed to allergen challenge but not in biopsies obtained from asthmatics exposed to sham challenge. Discrepancies between these two studies could be explain by the fact that in our study biopsies obtained from asthmatics were processed in paraffin while those in the former report were embedded in Tissue Teck II. Thus, the finding that MDC is expressed in biopsies derived from stable asthmatics further supports the role of MDC in steady-state asthma.

Having demonstrated MDC immunoreactivity localized to the airway epithelium we have next investigated whether primary HAEC release this chemokine in vitro upon stimulation with Th1- (stimulus IFN-γ) and Th2- type cytokines (IL-4 and IL-13). Interestingly, either of them were a potent stimulus for MDC production suggesting that this chemokine may not only be involved in Th2-type diseases such as asthma but also in Th1-type diseases including tuberculosis and lung granuloma formation. In relation to the allergic reaction, increased mRNA encoding IL-4 and IL-13 has been found in skin biopsies derived from atopic patients exposed to skin allergen challenge (30). All together these observations suggest that IL-4 and IL-13 may induce MDC production when released locally into asthmatic airways.

Because of the overwhelming evidences of the involvement of MDC/CCR4 in allergic airway inflammation it is tempting to hypothesize that neutralizing their effects may have substantial impact in allergic disease. Surprisingly, CCR4-deficient mice are still capable of developing allergic airway inflammation, which suggests an alternative mechanism of cell recruitment (31). Indeed, by transferring Th2 cells into naive mice it was demonstrated that the CCR3/eotaxin pathway is also critical in mediating the recruitment of Th2 cells after initial stimulation in vivo. However, after repeated antigen stimulation the CC3/eotaxin axis was superceded by the CCR4/MDC pathway inducing a sustained recruitment of Th2 cells (20). The finding that an anti-MDC antibody significantly inhibits allergen induced bronchial airway hyperreactivity in sensitized mice (21) suggests that development of small antagonists for CCR4 with clinical efficacy may have a substantial impact in asthma.

In summary our study has demonstrated that asthmatics release increased concentrations of MDC in BAL fluid and levels of this cytokine were found to be associated with bronchial hyperreactivity. The MDC was predominantly localized to the airway epithelium. Interestingly, upon cytokine stimulation HAECs were found to release MDC. All together these observations suggest that MDC may play a major role in the pathogenesis of asthma.

Acknowledgments

This work was partially supported by the CONACYT (grant no. 30693-M) and the Instituto Nacional de Enfermedades Respiratorias.

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