Abstract
We have investigated whether mast cells are associated with bronchodilator responsiveness and airway vascular changes in chronic obstructive pulmonary disease (COPD) airways. We have previously shown that the reticular basement membrane is hypervascular and the lamina propria is hypovascular in COPD.
Bronchial biopsies from 32 COPD subjects, 15 smokers with normal lung function and 17 controls, were immunostained for factor VIII, mast cell tryptase and chymase antibodies. Mast cells in the airway smooth muscle, the reticular basement membrane and the underlying lamina propria were quantitated.
41% of COPD subjects had significant bronchodilator responsiveness, but this was not related to smooth muscle mast cell numbers. The reticular basement membrane had greater mast cell density in all groups compared with controls (p<0.01). In this compartment, perivascular mast cell density was related to hypervascularity. Lamina propria mast cell density was increased only in COPD (p<0.05). Perivascular mast cell density in the lamina propria was not related to its decreased vessel density.
COPD中的支气管扩张剂反应性与两种类型的大气道平滑肌肥大细胞无关。COPD患者的网状地下膜和固有层肥大细胞都增加了,血管周围肥大细胞可能参与网状基底膜的血管生成增加。
肥大细胞是分泌细胞,含有颗粒,其中包含多种生物活性剂,包括促炎,促纤维化,促纤维化,促血管生成(e.g.vascular endothelial growth factor (VEGF)) and anti-angiogenic factors [1]。
Mast cells are thought to contribute to bronchoconstriction, mucus secretion, mucosal oedema, bronchial hyperreactivity (BHR), inflammation, angiogenesis and airway remodelling in asthma [2–5]。In particular, an increase in the number of airway smooth muscle (ASM) layer mast cells has been suggested to be related to asthmatic BHR [3]。支气管扩张剂反应性(BDR)已显示与BHR相关[6),可能是基于类似的基础工作anisms.
It is generally held that, by definition, airway obstruction in chronic obstructive pulmonary disease (COPD) is irreversible. However, significant BDR is in fact present in a large subgroup of patients with COPD, although they are mainly screened out from therapeutic studies [7,8]。Some investigators have suggested that this BDR feature in COPD is related to “asthma-like” pathology,i.e.重叠综合征[9–11]。A substantial number of COPD subjects have been shown to have BHR [12,13] with a significant correlation between BDR and BHR [14]。Therefore, given the relationships between both BHR and BDR in asthma and COPD, it is relevant to examine the smooth muscle layer in COPD airways for the number and type of mast cells, and its relationship to BDR in this setting.
Most cellular mediators can only effectively function close to the cells of origin to effect nearby target cells [3,15]。We have previously reported hypervascularity in the reticular basement membrane (Rbm) and hypovascularity of the lamina propria (LP) in the airways of current smokers with COPD (S-COPD) and to some extent smokers with normal lung function (S-N) [16]。We questioned whether these vascular changes could be associated with alterations in the number of mast cells in these compartments, especially in close apposition to vessels. We hypothesised that 1) patients with COPD who show BDR have higher mast cell numbers in the ASM layer; and 2) mast cells are increased in the Rbm and decreased in the LP, especially around blood vessels, in current smokers.
METHODS
我们分析了来自64名受试者,包括17名S-COPD,15名具有COPD的前吸烟者(ES-COPD),15 s-N和17个健康的非吸烟对照(H-N),检查气道壁血管性和肥大细胞密度,分析了包括17名S-COPD,15个S-s-Smoker(ES-COPD),15 s-N和17个健康的非吸烟对照(H-N)的支气管呼吸道活检。全球慢性阻塞性肺疾病指南的倡议用于定义COPD [17]。All COPD subjects were on relief anticholinergics only. Subjects with other types of respiratory diseases, those with recent exacerbations or who had used systemic or inhalational steroids during the last 12 weeks were excluded. Study subjects had little if any chronic bronchitis.
该研究得到了人类研究伦理委员会(澳大利亚塔斯马尼亚州)网络的批准。所有受试者均提供了书面知情同意书。
Pulmonary function tests were performed according to European Respiratory Society/American Thoracic Society (ATS) guidelines [18]。根据ATS标准定义了BDR,i.e.1 s的基线强迫呼气量的增加≥12%和200 mL(FEV1)在200μg吸入的salbutamol [7]。
之前已经描述了支气管镜和进行支气管调查活检的细节[16]。从每个个体中获得了右下叶中节段支气管的八个活检。没有遇到并发症。
Tissue processing
在活检中,有四个在4%中性缓冲福尔马林中固定2小时,然后使用Leica(Leica Microsystems,Wetzlar,德国)ASP 200组织处理器通过分级的酒精和二甲苯在石蜡中加工成石蜡。从单个石蜡块中切开3μm切片,用鞘久蛋白和曙红染色,并在形态学上评估了适合免疫染色的能力,被选中以最大程度地减少上皮的切向切片,并提供最大的上皮细胞。在每张载玻片上收集了两个与此类块最少50μm分离的3μm切片。在通过降级乙醇等级去除蜡和水合后,使用单克隆小鼠抗人类肥大细胞胰蛋白酶抗体(抗AA1,Dako M7052(Dako M7052)(Dako,cambridge,UK,UK,1,500),使用单克隆小鼠抗人类肥大细胞胰蛋白酶抗体对肥大细胞进行免疫染色,1,500°C)或单克隆小鼠抗人类芝士酶(ABCAM AB2377克隆CC1(ABCAM,ABCAM,剑桥,马萨诸塞州,美国,美国)在1/100稀释1小时,在22°C下);(使用Dako稀释剂S0809稀释所有抗体)。串行切片用小鼠免疫球蛋白G1同种型对照(Dako X0931在同等浓度和条件下)处理。所有免疫染色都包括阳性组织控制。在添加原代抗体之前,使用3%的水中溶解内源性过氧化物酶在水中淬火15分钟,并用0.025%VIII型蛋白酶(Sigma P-5380; Sigma-Aldrich,Sigma-Aldrich,St Louis,St Louis,St louis,St louis,st louis,st louis,st louis,st louis,st louis,st louis,stt Louis,st louis,stt louis,st louis,st louis,st louis,st louis,st louis,st)美国MO)在PBS中在37°C下持续3分钟以暴露抗原。在初级抗体之后,将Dako Invision+(Dako K4001)应用于切片30分钟。 The sections were then incubated with diaminobenzidine (Dako) as chromogen for 10 min. For sections stained for mast cell tryptase, immunostaining with monoclonal mouse anti-human von Willebrand factor (Dako M0616; 1/50 for 2 h at 22°C) was undertaken for vessel staining. This antibody was elaborated using a “Real™” (Dako 5005) biotinylated link followed by “Real™” (Dako K5355) streptavidin-labelled alkaline phosphatase for 20 min each. Antibodies were visualised using permanent red (Dako K0640). Mayer’s haematoxylin was used to elaborate nuclei and eosin counterstaining was used to discriminate the smooth muscle fibres from surrounding tissue in the immunostained slides.
Measurements
Sections were randomised and coded independently of the histologist who examined them, blinded to diagnosis and order. Tissue examination was performed using a computer-assisted image analysis tool (Image-Pro version 5.1; Media Cybernetics, Bethesda, MD, USA) at ×400 magnification. As many nonoverlapping pictures as possible were taken from each slide, and eight of these separate fields were randomly chosen for enumeration. Only tissue samples with intact epithelium and basement membrane were selected for examination of the Rbm and LP (如图。1)。As prospective criteria, developed after pilot studies on our material, the amount of smooth muscle available for mast cell analysis had to be sufficient to constitute 30% of the length immediately below the LP, and to have definite muscle bundles present. In general, this constituted ∼0.03 mm of smooth muscle per slide, which is less than described by Brightlinget al. [3], although the specifics given were somewhat ambivalent.
The number and cross-sectional area of vessels in the Rbm, and separately up to 150 μm from the antilumenal border of the Rbm in the LP (图2和3), were quantitated and normalised to the length of the Rbm and the area of LP, respectively [16]。由于结果证实了先前的观察[16],i.e.hypervascularity in the Rbm and hypovascularity in the LP, these data are not reported. The numbers of tryptase-positive mast cells (MCT) in the Rbm (如图。3) and the LP (图2和3]) were counted and normalised to the area of the Rbm and LP to calculate the density of mast cells per square millimetre of tissue. The number of MCT and chymase-positive mast cells (MCTC) in the ASM (如图。4)数,除以面积compartment examined. Using an image analysis tool, a perimeter with a width of 10 μm was drawn around the margin of each vessel in the Rbm and LP, and the density of mast cells per square millimetre of tissue in this perimeter compartment was measured. Given the relative size of mast cells (usually 8–10 μm in diameter, but up to 20 μm) to the perivascular area of interest, any mast cell that was even partially located within this perimeter or in contact with the perimeter, was counted as being perivascular.
Analysis
Nonparametric ANOVA (Kruskal–Wallis) tested differences between all study groups, with事后analyses for between two groups comparisons performed using Mann–Whitney U-tests when analysis revealed a significant difference according to a priori hypothesis. Spearman correlations were used to test relationships. All analyses were performed by PASW statistics 18 (formerly SPSS Inc., Chicago, IL, USA) for Windows. Two-tailed p-values <0.05 were considered significant.
RESULTS
60 out of 64 subjects had enough tissue for both the Rbm and LP measurements. 39 out of 49 subjects who were included in the analysis for ASM measurements had adequate tissue per protocol (see Methods section for details).Table 1shows the demographics of the participants. 41% of S-COPD and 40% of ES-COPD had BDR according to ATS criteria (41% overall). 47% of S-COPD and 69% of ES-COPD suffered from mild COPD and the rest from moderate COPD (Chi-squared p=0.2) [17]。BDR的COPD中有42%,无BDR的COPD中有67%患有轻度疾病,其余的患有中等疾病(卡方P = 0.2)。FEV的中间支气管缓解剂变化1was 250 mL for all COPD subjects, 340 mL for COPD with BDR and 120 mL for COPD with no BDR.
65% of MCT in the ASM layer were stained positively for chymase. The density of ASM layer MCT was not significantly different between COPD with BDR, COPD with no BDR and H-N groups (nonparametric ANOVA, p=0.8) (如图。5), and there was no difference in the density of MCTC between COPD with BDRversusCOPD with no BDR (median (range) 28 (0–69)versus47(0–142), respectively; p=0.3).
The average density of mast cells and perivascular mast cell density was significantly increased in the Rbm of the S-N, S-COPD and ES-COPD groups compared with H-N (nonparametric ANOVA test p<0.001 and p<0.01, respectively). S-N had also significantly increased perivascular mast cell density compared with ES-COPD (如图。6)。
The average density of mast cells in the LP was significantly increased in the S-COPD and ES-COPD groups compared with H-N (如图。7)。However, the density of perivascular mast cells was not significantly different between groups (如图。7)。
相关性
No correlations were found between BDR (either as absolute change or % change in FEV1) and ASM mast cell density (either MCT or MCTC) in both COPD groups (r values between -0.01 and -0.3). However, only in the non-BDR COPD group was MCT density in the ASM correlated with FEV1预测的%(r = 0.5,p <0.05)和ASM中的MCTC密度与强制生命能力(FVC)(r = 0.8,p <0.01)和预测的FVC%(r = 0.7,p = 0.7,p = 0.7,p = 0.7,p = 0.7,p = 0.7,p = 0.7,p<0.05),与FEV呈负相关1/FVC比率(r = -0.6,p <0.05)。
血管周的肥大细胞密度作为遏制疟疾correlat中ed positively with number and area of vessels in S-COPD (r=0.8, p<0.01 and r=0.7, p<0.01 for number and area of vessels, respectively), in ES-COPD (r=0.7, p<0.05 and r=0.6, p<0.05) and in the S-N groups (r=0.8, p<0.01 and r=0.9, p<0.001). Total mast cell density (but not perivascular density) in the LP correlated negatively with the area of vessels in this compartment only in the S-COPD group (r= -0.5, p<0.05). Age was on average greater in the COPD groups, although with substantial overlap with the other groups. Furthermore, there was no suggestion of a relationship between age (either within groups or for the whole dataset), and any variables of interest in our study (as examples, in both COPD groups combined, for ageversusRBM肥大细胞,r = -0.02,p = 0.9;年龄versusLP mast cells, r= -0.2, p=0.4).
DISCUSSION
Our main airway pathophysiological findings can be summarised as follows.
首先,在COPD受试者中,ASM中的肥大细胞密度与胰酶或Chymase细胞亚型的BDR变异之间没有关系。其次,尤其是在当前吸烟者中发现的RBM中血管周围肥大细胞密度的增加与RBM高血管呈正相关。第三,与对照组相比,两组COPD组中低血压LP的平均肥大细胞密度均增加,但在正常吸烟者中却没有增加。然而,两组之间血管周围的肥大细胞密度没有差异,并且与LP中的血管数无关。第四,在没有BDR的COPD受试者中,肥大细胞密度与更好的气道功能之间存在正相关关系。
一些研究报告了COPD患者亚组的“哮喘特征”,并提出了重叠的哮喘表型[9–11]。However, in contrast to the findings in asthma suggesting localisation of mast cells in the smooth muscle [15], our study found no differential localisation of mast cells in the ASM layer in COPD, and localisation was not found specifically in those with more BDR, at least not in those large airway biopsies under investigation. In general, airway pathology is uniform throughout the airway system [19],即使在COPD中,BDR也不太可能仅仅是一个小的气道功能。我们认为这些发现确实表明,哮喘和COPD之间的BDR的病理生理学是不同的,尽管还可以放心,还可以进行涉及小气道组织的重复病理生理相关研究,尽管这当然在逻辑上很难。
哮喘中BHR和BDR之间存在密切的关系[6],它们可能是相关的实体。在COPD中也显示了这种关系,基线气道口径的变化低于哮喘[14]。Similarly, Reidet al. [8]没有发现任何特定的哮喘型嗜酸性炎性炎症特征,可以将COPD与COPD与COPD区分开,而没有BDR。我们的发现与其他研究发现COPD中ASM肥大细胞没有变化的研究[20–22] and no correlation between ASM mast cell number and BHR [22]。
It has been shown that BDR is variable over time in COPD subjects [23]。Bronchodilator reversibility and endobronchial airway biopsies were performed within 24 h in our subjects, and we feel it is reasonable to use our data from these two procedures as representing a single epoch. It is unlikely that the cellular or physiological picture would be very different over 24 h in stable subjects with no change in clinical or therapeutic status.
ASM mast cell densities, determined with both tryptase and chymase staining, were correlated positively with lung function parameters. Gosmanet al. [24] also found a similar positive correlation between ASM mast cell density in small airways and FEV1%预测,但他们还发现外周气道上皮和上皮下肥大细胞密度与FEV之间存在正相关。1/FVC比率。总体而言,这些数据表明肥大细胞对COPD中的气道有保护,或者在早期疾病中特别发现它们。
Our study revealed that perivascular mast cell density in the Rbm was positively associated with the number and area of vessels in that compartment, suggesting that mast cells may play a role in angiogenesis and hypervascularity in the bronchial Rbm in both smokers and COPD subjects. Our previous report of increased vessel-associated VEGF in the Rbm is compatible with this suggestion [16]。In the LP where vessel numbers were decreased, overall mast cell density was increased, but interestingly this was not the case in the perivascular space, where they were relatively decreased compared with the rest of the LP. We did find a negative correlation between overall mast cell density and vascular area, and future studies should investigate whether these cells may be secreting anti-angiogenic factors [1]。
之前的数据在COPD气道肥大细胞given mixed messages, but none have deliberately differentiated between Rbm and LP compartments. Grashoffet al. [25从当前]研究小航空公司- and ex-smokers with COPD and reported more mast cells in the epithelium, but not in the remainder of the airway wall. However, a group in Sweden found more mast cells in the epithelium, LP and ASM in biopsies taken from the main tracheal carina in asymptomatic smokers [26]。Others have not found an increase in airway mast cells in COPD, but used smokers with or without bronchitis as controls [27,28]。Other studies that have found no difference in mast cell numbers between S-COPD and S-N have had very small numbers of participants, with the possibility of type-2 statistical errors [29,30]。
Although our COPD subjects were generally older than those in the H-N and S-N groups, there was great overlap, and no suggestions of relationships between age and the outcomes of interest in this study, either in the whole dataset or in any subgroups. From 32 COPD subjects, only 25 had enough appropriate tissue (as per prospective protocols) for comparing ASM mast cell density between groups. This could raise a question about the power of the study, but图5makes it clear that a type-2 statistical error is quite unlikely.
Conclusions
Our study did not find the “asthma-type” picture of disproportionately more mast cell in the ASM layer in bronchial biopsies from the subgroup of COPD with BDR. Perivascular mast cell density was increased in the hypervascular Rbm in both the S-N and COPD groups, though it was predominantly a smoking-related effect. Mast cells were increased in number in the LP in COPD groups, but not in physiologically normal smokers, and were relatively fewer around the numerically depleted LP vessels. Our novel data suggest roles for mast cells in vascular airway remodelling in COPD, and potentially in preservation of airway function.
Footnotes
支持声明
This study was supported by a NHMRC Australia project grant and the Royal Hobart Hospital Research Foundation through a Sypkes Fellowship.
Statement of Interest
None declared.
- 已收到May 18, 2011.
- AcceptedOctober 12, 2011.
- ©ERS 2012