Abstract
Rationale: Mast cells have important roles in innate immunity and tissue remodeling but have remained poorly studied in inflammatory airway diseases like chronic obstructive pulmonary disease (COPD).
Objectives: To perform a detailed histological characterization of human lung mast cell populations at different severities of COPD, comparing with smoking and never-smoking control subjects.
Methods: Mast cells were analyzed in lung tissues from patients with mild to very severe COPD, GOLD I–IV (n = 25, 10 of whom were treated with corticosteroids). Never-smokers and smokers served as controls. The density, morphology, and molecular characteristics of mucosal and connective tissue mast cells (MCT and MCTC, respectively) were analyzed in several lung regions.
Measurements and Main Results: In all compartments of COPD lungs, especially at severe stages, the MCTC population increased in density, whereas the MCT population decreased. The net result was a reduction in total mast cell density. This phenomenon was paralleled by increased numbers of luminal mast cells, whereas the numbers of terminal transferase dUTP nick end labeling (TUNEL)+ apoptotic mast cells remained unchanged. In COPD lungs, the MCT and MCTC populations showed alterations in morphology and expression of CD88 (C5a-R), transforming growth factor (TGF)-β, and renin. Statistically significant correlations were found between several COPD-related mast cell alterations and lung function parameters.
Conclusions: As COPD progresses to its severe stages, the mast cell populations in the lung undergo changes in density, distribution, and molecular expression. In COPD lungs, these novel histopathological features were found to be correlated to lung function and they may thus have clinical consequences.
Mast cells are part of the resident immune system in the human lung. They have recently been ascribed roles of potential importance to chronic obstructive pulmonary disease (COPD). Yet, mast cells have only rarely been studied in lungs from COPD patients.
This study shows that the mast cell populations in the lung are altered in COPD, as exemplified by a change in the connective tissue/mucosal–mast cell balance, altered tissue distribution, and modified morphological and molecular characteristics. Collectively, our data show alterations in lung mast cells in COPD that correlate with lung function and may have significant pathophysiological consequences.
In the human lung, at healthy baseline conditions, mast cells are present in large numbers at all levels of the airway including the alveolar parenchyma (7, 8). The discovery of several roles of mast cells in, for example, innate immunity (9), blood-flow regulation (10), antigen presentation (11), and T-cell regulation (12), thus make them potentially relevant to the pathogenesis of COPD. To date, only a few COPD studies have measured mast cell density (4, 13, 14). Although these important studies confirm that there is a significant occurrence of mast cells in COPD, no general picture has emerged as to how mast cell densities change in different regions of the lungs and how mast cell density relates to disease severity. Furthermore, there is no information on the phenotypes of the different mast cell populations in COPD.
In the present study, our aim was to explore patterns of mast cell density and distribution and to perform phenotypic characterization of lung mast cells in COPD. To study the influence of disease severity, we collected human lung samples from three patient groups with different severities of COPD and from corresponding smoking and never-smoking control groups. Mast cells are highly heterogeneous cells that exist as two major subtypes, mucosal mast cells (MCT) and connective tissue mast cells (MCTC), where MCT is most common in airways (15). Among the key parameters selected was the proportion of the two subtypes, as the proportion between the two has been shown to be altered in airway diseases such as asthma (16, 17). Furthermore, because microlocalization of inflammatory cells may have functional consequences, in the present study, mast cells were analyzed at several anatomical compartments of the lung (18, 19). We have recently shown that in the healthy human lung, each of the MCT and MCTC subtypes can be further divided into site-specific populations that are specific for each anatomical compartment of the lung (8). In the present study, the activation status and morphological features of these novel subpopulations are explored in detail using histological approaches. Some of the results of this study have been previously reported in the form of abstracts (20, 21).
The present study involved 40 subjects divided into three COPD patient groups: patients with mild COPD (GOLD I, n = 6), moderate to severe COPD (GOLD II–III, n = 9), and very severe COPD (GOLD IV, n = 10). Two groups were used as controls: ex-smokers or current smokers without COPD (n = 7) and a control group with subjects who had never smoked (n = 8) (8). The patient grouping was based on GOLD classifications (22). For patient characteristics, see Table 1. Lung tissue from mild, moderate, and severe COPD was obtained in association with lung lobectomy due to suspected lung cancer, a procedure that has been used repeatedly to collect tissues from COPD patients (23–26). Only patients with solid tumors with visible borders were included in the study, and tissue was obtained as far from the tumor as possible. Smoking and never-smoking tissue was obtained from control subjects using the same procedure as for otherwise healthy nonatopic individuals. In patients with very severe COPD (GOLD IV), matching lung tissue was collected in association with lung transplantation. For all patient groups, care was taken to immerse the tissue in fixative immediately after surgical excision, and multiple large tissue blocks were prepared for histological analysis. Due to the lack of tissue from large airways from the resection material, bronchial biopsies from healthy controls were used to compare central airways in control subjects and in patients with very severe COPD (see the online supplement for patient characteristics). All subjects gave their written informed consent to participate in the study, which was approved by the local ethics committee in Lund, Sweden.
Controls | Smokers | GOLD I | GOLD II+III | GOLD IV | |
|---|---|---|---|---|---|
| Sex (M/F, n) | 2/6, 8 | 3/4, 7 | 5/1, 6 | 7/2, 9 | 4/6, 10 |
| Age* (years) | 63 (33–76) | 56 (47–68) | 65 (56–72) | 67 (58–76) | 59 (53–66) |
| Pack years* (years) | 0 | 43 (20–80) | 43 (25–66) | 50 (34–65) | 41 (25–60) |
| Ex-smokers/current smokers | 0 | 3/4 | 2/4 | 8/1 | 10/0 |
| Inhaled GCS (y/n/unknown) | 0 | 0 | 0/6/0 | 1/8/0 | 9/0/1† |
| Oral GCS (y/n/unknown) | 0 | 0 | 0/6/0 | 0/9/0 | 1/8/1† |
| β2 agonist (y/n/unknown) | 0 | 0 | 2/4/0 | 1/8/0 | 9/0/1† |
| Anticholinergics (y/n/unknown) | 0 | 0 | 1/5/0 | 0/9/0 | 8/1/1† |
| Mucolytic (y/n/unknown) | 0 | 0 | 3/3/0 | 0/9/0 | 5/4/1† |
| Lung function | |||||
| FEV1* | 2.7 (1.7–5.1) | 2.9 (1.9–3.5) | 2.8 (1.6–3.2) | 1.8 (1.2–2.6) | 0.6 (0.4–1.0) |
| FEV1/(F)VC* | 85.9 (66–121) | 78.1 (71–88) | 67.5 (65–70) | 51.4 (41–68) | 30.2 (17–39) |
| FEV1% of pred* | 109.8 (82–141) | 97.4 (82–120) | 86.3 (78–95) | 61.8 (43–74) | 21.4 (13–27) |
Samples for immunohistochemistry were placed in 4% buffered formaldehyde, dehydrated, and embedded in paraffin. From each block, a large number of sequential sections 3 μm in thickness were generated. For electron microscopy, tissues were fixed in buffer supplemented with 1% glutaraldehyde and 3% formaldehyde, post-fixed in 1% osmium tetroxide for 1 hour, and dehydrated in graded acetone solutions and embedded. Ultrathin sections (90 nm) were cut and placed on 200-mesh copper grids. See the online supplement for details.
A double staining protocol was used for simultaneous visualization of MCTC and MCT cells (see reference [8] and the online supplement for details). Briefly, after rehydration and antigen retrieval, chymase-containing mast cells were detected with an antichymase antibody and the nonpermeable chromogen 3,3-diaminobenzidene (DAB). The remaining MCT subclass was visualized with an antitryptase antibody and Permanent Red chromogen (Table 2). The immunostaining was performed using an automated immunohistochemistry robot (Autostainer; DakoCytomation, Glostrup, Denmark) with EnVision G|2 Doublestain System (K5361, Dako).
Antibody | Species | Dilution | Clone | Origin | Secondary Antibody |
|---|---|---|---|---|---|
| Antitryptase | Mouse | 1:12 000 | G3 | Chemicon, Temecula, CA | EnVision G|2 Doublestain System |
| Antichymase | Mouse | 1:100 | CC1 | Novocastra, Newcastle upon Tyne, UK | EnVision G|2 Doublestain System |
| Antirenin | Mouse | 1:50 | Swant Scientific, Bellinzona, Switzerland | Biotinylated Horse anti-mouse IgG | |
| Anti-CD88 | Mouse | 1:500 | P12/1 | Acris Antibodies, Hiddenhausen, Germany | Biotinylated Horse anti-mouse IgG |
| Anti-TGF-β | Mouse | 1:40 | TGFB17 | Novocastra, Newcastle upon Tyne, UK | Biotinylated Horse anti-mouse IgG |
| Anticytokeratin | Mouse | 1:100 | MNF116 | Dako, Glostrup, Denmark | EnVision G|2 Doublestain System* |
Triple staining by using immunofluorescence was used to simultaneously visualize both MCTC and MCT populations and the mast cell–related molecules CD88 (the C5a receptor, recently identified as a broad activation marker on mast cells ([27]), transforming growth factor (TGF)-β (a major profibrotic growth factor), and renin (involved in vascular homeostasis and recently identified in mast cells) in paraffin sections from all patient groups. See Table 2 (8), and the online supplement for antibody references and details of the protocol. Staining was absent in control sections using isotype-matched control antibodies.
The extent of mast cell apoptosis was studied by combining antitryptase immunofluorescence immunohistochemistry (see above), a pan DNA marker (Hoechst 3332; Sigma, Stockholm), and apoptotic cell detection using the TUNEL technique (28) (ApopTag Fluorescein In Situ Apoptosis detection kit, S7110; Chemicon/Millipore, Billerica, MA). See the online supplement for details of the protocol.
For quantification of mast cell densities, mast cell-related molecules, and morphometric parameters, large (>4 cm2) paraffin-embedded tissue blocks from three separate regions of the lung were analyzed from each patient. In each block, mast cells were analyzed in the following anatomical structures. Small airways: bronchioles, average 5 per section, defined by absence of cartilage and diameter less than 2 mm. In small airways, mast cells were quantified in the wall (epithelium, lamina propria, smooth muscle, and adventitia) and the lumen. Pulmonary vessels: mid-size or large pulmonary arteries in the broncho/bronchiovascular axis or with an intraacinar localization (7 per block on average). Mast cells in pulmonary vessels were quantified in all distinct layers (i.e., intima, media, and adventitia). Alveolar parenchyma: four randomly selected 0.5 mm2 alveolar regions were analyzed in each block. Bronchial mast cells: densities in large airway tissue were analyzed in separate biopsies and lung resections from never-smoking controls and from patients with very severe COPD (see online supplement and Table E3). Bronchial biopsies were taken from the first and second bronchial division from the lower and upper right lobe (generation 3–4) and central airways (lung resections) were defined as airways with a diameter of 3 to 6 mm (generation 3–5) surrounded by cartilage.
In sections double-stained for MCTC and MCT, the density of each population was quantified manually in blind sections and related to the area of each type of tissue analyzed (8), which was determined using a computerized image analysis algorithm that excluded any luminal spaces. The same approach was also applied to the alveolar parenchyma (Image-Pro Plus; MediaCybernetics, Silver Springs, MD; and NIS-Elements, Nikon, Kanagawa, Japan). The proportion of the MCTC subtype was calculated according to (MCTC / [MCTC + MCT]) × 100.
In triple stained sections, MCTC and MCT were counted manually as described above. All tryptase- and chymase-positive cells were counted in four randomly selected small airway walls, four randomly selected pulmonary vessel walls, and four randomly selected 0.5-mm2 alveolar regions per section. By subsequently dividing the number of tryptase and/or chymase cells that were copositive for CD88, renin, and TGF-β, respectively, by the total numbers of MCT and MCTC, the proportion (%) of each subtype and the total number of mast cells expressing each mast cell-related molecule was obtained.
Lung samples were processed for electron microscopy using a standard protocol (29) and a Philips CM-10 TEM microscope (Philips, Eindhoven, The Netherlands). Tissue from never-smoking controls and patients with very severe COPD (GOLD IV) were used for ultrastructural analysis of lung mast cells. Apart from exploring the general ultrastructure, each individual mast cell was assessed for signs of degranulation (see the online supplement for definitions of categories) according to previously described criteria (30), and the percentage (per patient) of degranulated mast cells was calculated.
Using double-stained sections, the numbers of intraluminal MCT and MCTC were determined in all patient groups (2–3 tissue blocks per patient). Briefly, MCT and MCTC were counted in each individual airway and related to the luminal area. The airway lumen area was measured by manual cursor tracing and digital image analysis (Image-Pro Plus, Media Cybernetics, Silver Springs, MD) and intra-luminal mast cells were expressed as cells per mm2 lumen area. In never-smoking controls and in patients with very severe COPD intraluminal epithelial cells were quantified in the same way after immunohistochemical staining with the pan-epithelial cell marker cytokeratin (see Table 2) and detected using the EnVision G|2 Doublestain System (K5361; Dako). In these patient groups, the leukocyte profile of the small airway lumen was evaluated in consecutive sections stained for neutrophils, monocytes/macrophages, eosinophils, CD4+ positive T-lymphocytes, CD8+ positive T-lymphocytes, and B-lymphocytes (for protocol details and markers, see Table E2).
Paraffin sections from never-smoking controls and patients with very severe COPD were analyzed using fluorescence microscopy with triple-band UV filters, and image analysis allowed all individual tryptase-positive mast cells in a lung section to be categorized as either terminal transferase dUTP nick end labeling (TUNEL)+ or TUNEL−.
Data were analyzed using Kruskal-Wallis test with Bonferroni's multiple comparisons test for comparison among three groups or more (for mast cell densities, proportions, and molecular expression) and Mann-Whitney rank sum test was used for comparison between two groups (for mast cell densities in central airways and TUNEL) using GraphPad Prism v. 5 (GraphPad Software, Inc., La Jolla, CA). Differences between groups were considered significant at P ≤ 0.05. The Spearman rank correlation test (two-tailed) was used to study the correlation between lung function values and mast cell parameters, or correlations between intraluminal mast cells and luminal epithelial cells or leukocytes, Results were considered significant at P ≤ 0.05. To compensate for multiple testing, the false discovery rate procedure was applied to the correlation analysis, which guaranties that less than 5% of all positive results are false positive (31).
Mast cells were identified in small airways (range of 0.44–1.98 mm in diameter), pulmonary vessels (range of 0.42–4.21 mm in diameter), and alveolar parenchyma in sections from all patient groups by immunohistochemical double staining. In both control and COPD subjects, mast cells were present in all anatomical compartments of the lung. In small airways, a significant reduction in total mast cell density was found in patients with very severe COPD (GOLD IV) relative to the control groups (never-smokers and smokers) (Figure 1A, Table E3). The reduction was also significant in patients with moderate to severe COPD (GOLD II–III) relative to never-smoking controls (Figure 1A, Table E3). The total density of mast cells in pulmonary vessel walls was significantly reduced in patients with very severe COPD when compared with never-smokers, smokers, and patients with GOLD I–III COPD (Figure 1D, Table E3). No difference in total numbers was found in the alveolar septa of the parenchyma (Figure 1G, Table E3).
Figure 1. (A, D, and G) Total mast densities and densities of each subtype, (B, E, and H) mucosal mast cells (MCT) and (C, F, and I) connective tissue mast cells (MCTC), in lung tissue compartments of never-smoking and smoking control subjects and patients with mild to very severe chronic obstructive pulmonary disease (COPD) presented as mast cells per mm2 lung tissue. Data are presented for (A–C) small airways (SA), (D–F) pulmonary vessels (PV), and (G–I) alveolar parenchyma (AP), and are expressed as scatter plots where the line denotes median. Statistical analyses were performed using Kruskal-Wallis test with Bonferroni's multiple comparisons test. Overall significance is shown in each picture. Asterisks show statistical difference when compared with very severe COPD (GOLD IV) where *P < 0.05, **P < 0.01, and ***P < 0.001. Significant differences between controls and moderate to severe COPD (GOLD II–III) are shown as #P < 0.05.
[More] [Minimize]Differentiation of MCT and MCTC populations revealed that in all anatomical compartments of the lung there was a gradual and significant reduction in MCT density (Figure 1B, 1E, 1H, and Table E3). In contrast, in the walls of small airways and in alveolar parenchyma, the density of MCTC in patients with very severe COPD was significantly higher than in controls (Figures 1C, 1I, 2A, 2D, 3A−3F, and Table E3). In pulmonary vessels, the density of both MCT and MCTC populations dropped significantly in patients with very severe COPD (Figures 1E-1F, 3G–3H and Table E3). Calculation of the MCTC percentage of the total mast cell population revealed that in lungs from patients with very severe COPD there was a several-fold increase in the proportion of MCTC cells in small airways, small airway epithelium, pulmonary vessels, and alveolar parenchyma (Figure 2A−2D, Table E3). In lung tissue from patients with very severe COPD, some larger airways of the peripheral lung containing submucosal glands were analyzed. Notably in these structures, MCTC comprised 95% of the total mast cell density (41 [28–56] mast cells per mm2, median and range).
Figure 2. The proportion of connective tissue mast cells (MCTC) and mucosal mast cells (MCT), expressed as the percentage of MCTC mast cells, in anatomical lung compartments in never-smoking and smoking controls and patients with mild to very severe chronic obstructive pulmonary disease (COPD) (GOLD I–IV). Data are presented for (A) small airways, (B) small airway epithelium, (C) pulmonary vessels, and (D) alveolar parenchyma. Data are expressed as scatter plots, where the line denotes median. Statistical analyses were performed using Kruskal-Wallis test with Bonferroni's multiple comparison test. Overall significance is shown in each panel. Asterisks show levels of statistical difference when compared with very severe COPD (GOLD IV) where *P < 0.05, **P < 0.01, and ***P < 0.001.
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Figure 3. Representative micrographs of immunohistochemical double staining of tryptase-positive mucosal mast cells (MCT: permanent red) and chymase-positive connective tissue mast cells (MCTC: DAB-brown) in different anatomical compartments of the lung. (A and B) Representative small airways are shown at low magnification from (A) a healthy control subject and (B) a patient with very severe chronic obstructive pulmonary disease (COPD). Inset in B represents a close-up image (600×) of neighboring MCTC and MCT cells. (C and D) Small airway mast cells are shown at higher magnification in a (C) control subject and (D) a patient with very severe COPD. Note the higher proportion of brown chymase-positive MCTC in COPD. The alveolar parenchyma is shown from (E) a control and (F) from a patient with very severe COPD; the increased proportion of MCTC in severe COPD is highlighted in the insets. (G and H) Show mast cells in pulmonary vessels from (G) a control subject and from (H) a patient with very severe COPD. Scale bars (A and C–H) = 100 μm; (B) = 200 μm. SA = small airways; v = pulmonary vessel; ep = small airway epithelium; sm = airway smooth muscle layer; lu = small airway lumen; and alv = alveolar parenchyma.
[More] [Minimize]Bronchial mast cell densities in large airway tissue were analyzed in separate biopsies and lung resections from never-smoking controls and patients with very severe COPD. As for the small airways, a reduction in the total numbers of mast cells and an increased proportion of MCTC was also apparent in the central airways in patients with very severe COPD relative to the controls (see online supplement and Table E4).
The distribution of mast cells was analyzed in more detail in subanatomical compartments within the small airways and pulmonary vessels. In small airways from COPD patients, a shift in relative mast cell densities from the outer to the inner wall layers (epithelium and lamina propria) was observed (Figure 4A−4B). For both MCT and MCTC, the percentage of intraepithelial mast cells increased significantly in patients with very severe COPD, whereas the proportion of both subtypes decreased in the smooth muscle and adventitia layers (Figure 4A−4B). A reduction in airway smooth muscle–associated mast cells was also apparent in the smoking non-COPD group (Figure 4A−4B). No significant differences were found around pulmonary vessels (data not shown).
Figure 4. Distribution patterns of mast cell subtypes within distinct small airway compartments. Bars show the mean percentage of total airway wall mast cells for (A) mucosal mast cells (MCT) and (B) connective tissue mast cells (MCTC) in each subanatomical compartment (epithelium, lamina propria, smooth muscle, and adventitia). Significant differences between controls and subjects with very severe chronic obstructive pulmonary disease (GOLD IV) are shown in each figure. asignificant differences between very severe chronic obstructive pulmonary disease subjects and smoking controls. (C–E) show the density of airway lumen mast cells for (C) total mast cells, (D) MCT, and (E) MCTC. Data are presented as mast cells per mm2 lumen area and displayed as scatter plots where the lines denote median. Statistical analyses were performed using Kruskal-Wallis test with Bonferroni's multiple comparison test. Overall significance is indicated by P values displayed above C–E. Asterisks show statistical significance compared with never-smoking controls, where * P < 0.05.
[More] [Minimize]In light of the reduction in total mast cell density in COPD, possible causes of mast cell elimination were investigated. Mast cell elimination through apoptosis was explored through TUNEL staining in combination with immunohistochemical staining for tryptase in paraffin sections from controls and patients with very severe COPD. Egression into the airway lumen and removal by the mucociliary escalator is a physiological mode of elimination for several leukocyte types that infiltrate the lung (32). The occurrence of mast cells in the small airway lumen was investigated in all patient groups.
Scattered TUNEL-positive cell nuclei were present in all groups. In patients with very severe COPD, a large proportion of the TUNEL-positive cells were identified as MPO-positive neutrophils (which were used as positive control cells; data not shown, Figure 5H). In each tissue block, an average of five small airways, seven pulmonary vessels, and alveolar parenchyma were screened for mast cells that were double positive for tryptase and nuclear TUNEL-staining. Through this approach, the screening of approximately 6,000 mast cells per section revealed that TUNEL+ mast cells were exceedingly rare. In patients with very severe COPD, the frequency of lung mast cells that were positive for TUNEL staining was 0.035 ± 0.003% (mean ± SEM). The corresponding value for never-smoking controls was 0.047 ± 0.005%, not significantly different from that for COPD (P = 0.2).
Figure 5. (A–C) Transmission electron micrographs exemplifying different levels of degranulation in mast cells in very severe chronic obstructive pulmonary disease (COPD). (A) Nondegranulated phenotype, (B) minor piecemeal degranulation, and (C) substantial piecemeal degranulation. High-power images of intact and degranulating granules are shown as insets in A and C, respectively. Scale bars in A–C = 1 μm. (D and E) Immunofluorescence images with double staining for TUNEL+ apoptotic cells and tryptase, and the neutrophil marker myeloperoxidase, respectively (Alexa F 488-green). Cell nuclei are stained blue with the pan-DNA marker Hoechst 3,332. Arrowhead in D exemplifies a TUNEL+, tryptase− cell. Apoptotic neutrophils (see also inset in E) were readily found in lung tissues; lumen of COPD patients was used as a positive control. (F) Represents a bright-field micrograph of tryptase-stained mast cells and demonstrates a mast cell-rich luminal plug in a patient with GOLD IV COPD. Scale bars D–E = 25 μm; F = 100 μm. Ep = epithelium.
[More] [Minimize]Few mast cells were present among the luminal cells and secretions that occasionally occurred in the never-smoking and smoking control groups (Table E4, Figure 4C–4E). Luminal mast cells were also rare in COPD patients in the GOLD I–III range despite the fact that there was more luminal material present. In patients with very severe COPD (GOLD IV) whose small airways frequently contained cell-rich luminal plugs, luminal mast cells were frequently observed in significantly increased numbers compared with the controls and the COPD groups with milder disease (Table E3, Figure 4C−4E). Interestingly, the luminal mast cells in patients with COPD were mostly MCT cells (77 ± 9% of the total, mean ± SEM). To determine whether luminal mast cells could emerge from epithelial sloughing, double staining was performed with a pan-cytokeratin antibody and mast cell tryptase. Exfoliated epithelial cells increased in very severe COPD (229 [15–525] cells/mm2, median and range) compared with controls (0 [0–37] cells/mm2; P = 0.008). No statistical correlation was found between luminal epithelial cells and luminal mast cells (see the online supplement). Intraluminal neutrophils, monocytes/macrophages, CD4+ and CD8+ T-lymphocytes were all increased in COPD, whereas no change was found for eosinophils and B-lymphocytes (see online supplement and Table E5). No significant correlations were found between intraluminal mast cells and any of the examined leukocytes (Table E5).
Ultrastructural examination of lung mast cells by transmission electron microscopy was performed on lung samples from patients with very severe COPD and from never-smoking controls. MCTC and MCT subtypes were identified by their distinct granular morphology; MCT from their scroll-rich granules and MCTC from their scroll-poor and crystalline/lattice granules (33). In controls, the MCTC and MCT were easily identified according to these criteria (Figure 5A). Mast cells in healthy subjects were primarily of a nondegranulating phenotype, that is, they displayed filled granules lacking morphological signs of anaphylactic or piecemeal degranulation. In the lungs of patients with very severe COPD, the distinction between MCTC and MCT was less clear insofar as mast cells that displayed the traditional morphology of MCTC frequently also contained scroll-filled granules. Degranulation analysis was performed on samples from eight patients with very severe COPD and five healthy controls (2–4 tissue blocks per patient, 5–15 mast cells per patient). Based on granule appearance mast cells were classified into one of the following categories: mild piecemeal degranulation, advanced piecemeal degranulation, or classical anaphylactic degranulation (for definitions, see online supplement). Mast cells showing signs of advanced piecemeal or anaphylactic degranulation were not found in control tissue or in COPD lungs. Altogether, in COPD lungs, 20 ± 3% of the mast cells displayed signs of mild to moderate piecemeal degranulation, which was a significant increase compared with the controls (4 ± 4%; P = 0.04).
The expression of mast cell-related molecules was evaluated in the small airways, pulmonary vessels, and alveolar parenchyma from all patient groups using immunofluorescence triple staining. The levels of expression of CD88, TGF-β, and renin were changed in patients with COPD compared with the controls (Figure 6). The percentage of mast cells that expressed the C5a receptor (CD88) increased significantly in all COPD groups compared with the never-smoking and smoking controls. The increase was present in both the MCTC and MCT population and occurred in all anatomical compartments examined, that is, the small airways, pulmonary vessels, and alveolar parenchyma (Figure 6A−6C). Also, there was a less clear but significant increase in the proportion of mast cells that expressed TGF-β. In patients with COPD, the most pronounced increase was observed for the MCTC subclass (Figure 6D−6F). In contrast to CD88 and TGF-β, the expression of mast cell renin was high at baseline conditions, particularly in the MCTC subtype, which displayed almost 100% expression. Also, in contrast to the expression of CD88 and TGF-β, there was a significant decrease in renin expression (Figure 6G–6I) in the COPD lungs, particularly those affected by more severe disease (GOLD II–IV). There was no evidence of a chymase-only positive mast cell population (MCC) in any of the patient groups.
Figure 6. Molecular expression patterns of (A–C) CD88, (D–F) transforming growth factor (TGF-β), (G–I) and renin, in small airways, pulmonary vessels, and alveolar parenchyma. In each graph, results are shown as the percentage of total mast cells, connective tissue mast cells (MCTC), and mucosal mast cells (MCT) subtypes that are positive for the respective mediators. Data are expressed as median with interquartile range. Statistical analyses were performed using Kruskal-Wallis test with Bonferroni's multiple comparison test, where *P < 0.05 and **P < 0.01 when compared with controls. #P < 0.05 and ##P < 0.01 show significant differences when compared with smoking controls. Overall significance (Kruskal-Wallis) is presented for total mast cells (MCtot) in each panel, except for E–F where overall significance for MCTC is presented.
[More] [Minimize]For the COPD patient groups, several statistically significant correlations were found between mast cell and lung-function parameters (FEV1/VC and FEV1% of predicted). In all anatomical compartments, there was a positive correlation between reduced densities of both total mast cells or MCT mast cells and reduced lung function (Table 3). For MCTC, there was a correlation between increased density within the small airways and the lung parenchyma on the one hand and reduced lung function on the other. Also, there was a correlation between the increased proportion of MCTC in all compartments and worsening of lung function values. No correlations were found between mast-cell parameters and pack years. Regarding the expression of CD88, TGF-β, and renin, the correlation to lung function was less clear, although several statistically assured correlations were found (Table 3). After compensation for multiple testing using the false discovery rate (FDR) procedure, several correlations were still significant and these are presented in Table 3 in bold.
FEV1/VC | FEV1% of Predicted | |||||||
|---|---|---|---|---|---|---|---|---|
| Parameter | Anatom. | Mast Cells | rs | P Value | rs | P Value | ||
| Density | SA | MCtot | 0.43 | 0.05 | 0.33 | 0.1 | ||
| Density | SA | MCT | 0.48 | 0.03 | 0.46 | 0.03 | ||
| Density | SA | MCTC | −0.13 | 0.3 | −0.17 | 0.3 | ||
| Density | Pul. Vessel | MCtot | 0.70 | 0.0003 | 0.76 | <0.0001 | ||
| Density | Pul. Vessel | MCT | 0.72 | 0.0002 | 0.75 | <0.0001 | ||
| Density | Pul. Vessel | MCTC | 0.29 | 0.1 | 0.42 | 0.03 | ||
| Density | Parenchyma | MCtot | 0.59 | 0.003 | 0.63 | 0.001 | ||
| Density | Parenchyma | MCT | 0.69 | 0.0004 | 0.74 | <0.0001 | ||
| Density | Parenchyma | MCTC | −0.75 | <0.0001 | −0.70 | 0.0002 | ||
| Proportion | SA | MCTC | −0.38 | 0.05 | −0.40 | 0.04 | ||
| Proportion | Parenchyma | MCTC | −0.80 | <0.0001 | −0.76 | <0.0001 | ||
| Proportion | Pul. Vessel | MCTC | −0.59 | 0.003 | −0.55 | 0.005 | ||
| Proportion | SA epithelium | MCTC | −0.44 | 0.04 | −0.43 | 0.04 | ||
| CD88 | SA | MCtot | 0.25 | 0.2 | −0.05 | 0.4 | ||
| CD88 | SA | MCT | −0.14 | 0.3 | −0.40 | 0.07 | ||
| CD88 | SA | MCTC | 0.59 | 0.01 | 0.36 | 0.1 | ||
| Renin | SA | MCtot | 0.50 | 0.02 | 0.53 | 0.02 | ||
| Renin | SA | MCT | 0.53 | 0.02 | 0.54 | 0.02 | ||
| Renin | SA | MCTC | 0.43 | 0.07 | 0.55 | 0.03 | ||
| TGF-β | SA | MCtot | 0.50 | 0.02 | 0.40 | 0.06 | ||
| TGF-β | SA | MCT | 0.42 | 0.05 | 0.36 | 0.08 | ||
| TGF-β | SA | MCTC | −0.38 | 0.08 | −0.42 | 0.05 | ||
The present study shows that the lung mast cell populations are altered at severe stages of COPD. The alterations include changes in density, distribution, and cell phenotype. As mast cells appear to be key players in both innate and adaptive immunity, the functional consequences of these alterations are now emerging as an important field of research.
The modified double staining used in this study for detection of the two mast cell subpopulations (MCT and MCTC) does not detect the tryptase-negative MCC population described by Weidner and Austen in 1993 (34). Our triple staining using flourochrome-labeled antibodies and specific broad-spectrum filters would detect chymase-only positive mast cells. Notably, among the numerous individual mast cells examined in this study, not a single one was found to belong to the chymase only (tryptase−, chymase+) category. With the vast number of cells analyzed we believe that the present modified double-staining approach is adequate for detecting the MCT and MCTC cells in this study.
There are only a few studies where mast cell densities in smokers, or smokers who have developed bronchitis and COPD, have been examined. Healthy smokers have been reported to have increased numbers of bronchial mast cells compared with never-smoking controls (35). Also, in COPD, mast cells are present in high numbers and are sometimes elevated compared with controls (25, 36, 37). A recent study by Gosman and colleagues has suggested that the total mast cell density may also be reduced compared with control subjects (14). In the latter study, no distinction was made between different severities of COPD. Our results agree with those of Gosman and colleagues (14) and suggest that reduced mast cell density in the lungs of subjects with COPD is particularly associated with severe stages of the disease. Our analysis of distinct mast-cell populations revealed that, although the MCT population decreased in COPD, there was an increased density of MCTC in both the small airways and the alveolar parenchyma. The resulting shift in the balance between MCTC and MCT represents a key finding in the present study. Interestingly, a similar increased proportion of MCTC has been observed in severe asthma (17). It can be surmised that a common feature of severely inflamed lungs is the expansion of the MCTC population, whereas the normally prevailing MCT population becomes reduced by unknown mechanisms.
The process leading to altered MCTC/MCT balance in COPD is likely to be complex and multifactorial. One possible mechanism could be that the cellular inflammation and molecular milieu in COPD lungs promote differentiation of mast-cell progenitors into a MCTC phenotype. A more speculative possibility is that resident MCT start to produce chymase and thus transform into an MCTC phenotype. Such transformation has previously been observed in vitro (38, 39).
Another tentative mechanism behind the MCTC/MCT imbalance is selective elimination of lung MCT. Mast cell apoptosis, a proposed key mechanism for clearance of tissue mast cells, has been studied extensively in vitro, but little is known regarding lung–mast cell apoptosis under in vivo conditions. In the present study, we double-stained for TUNEL+ (apoptotic) cell nuclei and tryptase. Our results revealed that, in contrast to, for example, neutrophils, lung mast cells in COPD lungs exhibit the same low frequency of apoptosis as control subjects. This finding supports the general view of mast cells as being long-lived cells with a slow turnover. Although we could not find evidence of increased mast-cell apoptosis in COPD, it cannot be excluded that this mechanism has a role in the long-term regulation of cell numbers.
In light of the small number of apoptotic mast cells, we observed increased numbers of luminal mast cells in COPD, which indicates that loss of tissue mast cells to the airway lumen may have contributed to the decline in total mast cell numbers. Mast cells are regarded as tissue-dwelling cells, but they have been observed in luminal samples from subjects with asthma (41, 42). Although patients with mild asthma have increased mast cell numbers, those with severe asthma may have reduced numbers of tissue mast cells (43) and elevated numbers of luminal mast cells (44). The present study cannot establish a mechanism for how mast cells enter the lumen, but some potential processes should be considered. Active migration into the airway lumen has been proposed as a physiological mode of elimination for several airway leukocytes (32, 40). At present it cannot be excluded that mast cells also have the capacity to actively egress into luminal compartments in inflamed lungs. Alternatively, intraepithelial mast cells may be deliberated into the lumen as a result of epithelial sloughing. The relative contribution of these two alternatives in our study is hard to determine, especially because we failed to find any correlation between luminal mast cells and exfoliated epithelial cells or between mast cells and other intraluminal leukocyte populations. It is clear that more research is needed to elucidate the phenomenon of luminal mast cells in inflamed human lungs.
It cannot be excluded that medical treatment contributed to the MCTC/MCT imbalance in our patients with COPD. Steroids, for example, have been demonstrated to reduce mast cell density in central airways (45) and have been shown to mainly affect the MCT population in various compartments of the respiratory system (17, 46, 47). However, several factors indicate that mechanisms other than medical treatment contribute to mast-cell abnormalities in COPD. First, reduced levels of total mast cells in the lung were observed even in patients with moderate to severe COPD, of which only one subject received steroid treatment. Also, the most advanced reduction was observed in pulmonary vessels, a compartment that during steroid inhalation receives only a fraction of the drug concentration delivered to the airway mucosa. Furthermore, the absolute numbers of MCTC cells increased, an event that is unlikely to be caused by medications. Taken together, the reduction in mucosal mast cells that we describe in very severe COPD may be partly caused by steroids but should be noted as a significant feature of late-stage—and thus extensively medicated—COPD. It should also be noted that it is unclear whether anti–mast-cell effects are solely beneficial. The potential affects of current and emerging drugs on lung mast cells should therefore be explored with regard to the destructive as well as proinflammatory capacity of mast cells and their potential beneficial role in innate immunity, lung defense mechanisms, and vascular homeostasis.
The few previous studies on mast cells in nonallergic inflammatory respiratory diseases make it difficult to speculate about the functional consequences of mast cell alterations. Gosman and colleagues (14) hypothesized that mast cells may have a protective role in COPD. In general, our data agree with this hypothesis but highlight the need to discriminate between mast-cell subtypes. For example, there was a positive correlation between increased densities of MCTC in pulmonary vessels and alveolar parenchyma on the one hand and worsening of both FEV1% predicted and FEV1/VC on the other, indicating that MCTC may have a negative role in COPD. Expansion of the MCTC population in COPD may have pathogenic implications. A study by Maryanoff and colleagues in this issue of the AJRCCM (pp.
The MCTC population in our study had a particularly high level of renin, a key hormone in vascular homeostasis that has also been identified in mast cells (52). This finding, together with the capacity of chymase to act as an angiotensin converting enzyme and angiotensin II activator, suggests that MCTC may have a role in vascular regulation in the lung with possible implications for COPD. The blood flow–modulating capacities of histamine and serotonin (53, 54) further underscore the potential of mast cells in vascular regulation of the lungs. As mast cells have been shown to regulate blood flow in several other organs (52, 55), loss of lung vascular mast cells may thus contribute to the vascular changes and perfusion imbalance observed in COPD (56). In further support of the clinical significance of reduced amounts of vascular mast cells, the loss of vascular mast cells was strongly correlated to reduced lung function in patients with COPD.
The role of mast cells in COPD is dependent not only on their numbers in the diseased tissue but also their activation status. Measurement of mast cell activation in tissues is complicated due to the lack of established parameters of activation. Electron microscopic assessment of granule alterations reliably detects ongoing degranulation (30), but the time-consuming analysis and small sample size limit its use. Despite the fact that we explored several tissue blocks per patient, in this study we failed to sample enough mast cells for detailed quantification. We believe, however, that some important conclusions can be drawn from our ultrastructural analysis. Of the more than 200 mast cells analyzed from the lungs of patients with COPD, not a single one showed signs of advanced piecemeal or anaphylactic degranulation. This observation suggests that advanced degranulation is a rare phenomenon in very severe COPD, at least outside acute exacerbations when the present surgical material was collected. In samples from patients but not from controls, we did, however, observe scattered cells (20 ± 3%) involved in mild piecemeal degranulation. Thus, we cannot exclude the possibility that in COPD also, mast cells may cause pathogenic effects through degranulation. It should be noted that nondegranulating mast cells might also contribute to inflammation through degranulation-independent chemokine release (57), eicosanoid release, or just differentiation into a more proinflammatory phenotype.
Our analysis of selected mast cell–related molecules revealed significantly increased mast cell expression of the C5a receptor (CD88) and TGF-β in COPD. This observation shows for the first time that mast cell expression profiles may be altered in COPD. It may, however, be too early to speculate about any functional significance of these alterations. Clearly, a more systematic evaluation of expression patterns of a variety of key mast cell-associated molecules is needed to define lung mast cell phenotypes at different stages of COPD.
In summary, the present study demonstrates that mast cell populations in the lungs of patients with COPD are altered on several counts. Apart from a marked decrease in numbers of mucosal mast cells, an increase in connective tissue mast cells led to a shift in the relative proportion of MCTC and MCT and each subtype showed a changed pattern of expression of mast cell–related molecules. These alterations, which were found to be correlated to altered lung function and took place in all major lung compartments, suggest that mast cells are part of the cellular inflammation in COPD. The nature of this involvement and the influence of common medications on mast cells now emerge as an important line of research.
We thank Karin Jansner and Britt-Marie Nilsson for skillful technical assistance with tissue processing, immunohistochemical stainin, and TEM procedures.
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