COPD患者痰液中性粒细胞氧化吞噬功能及相关受体变化的临床意义

doi:10.3969/j.issn.1673-8640.2020.06.012

摘要/Abstract

摘要：

目的探讨慢性阻塞性肺疾病（COPD）患者中性粒细胞氧化吞噬功能及相关受体[趋化因子C-C基序受体-1（CCR1）和Toll样受体（TLR）2、TLR4]变化的临床意义。方法建立用于检测COPD患者痰液中性粒细胞氧化吞噬功能及相关受体的流式细胞术方法。采用流式细胞术检测30例COPD患者（COPD组）、30例肺炎患者（肺炎组）和30名体检健康者（正常对照组）外周血及痰液（正常对照组除外）中性粒细胞氧化吞噬功能及其表面CCR1、TLR2和TLR4的表达水平。结果肺炎组和COPD组外周血中性粒细胞氧化吞噬功能均显著低于正常对照组（P<0.05）,肺炎组与COPD组之间差异无统计学意义（P>0.05）。COPD组、肺炎组及正常对照组之间外周血中性粒细胞表面CCR1表达差异均有统计学意义（P<0.05）,而TLR2、TLR4表达3组之间差异均无统计学意义（P>0.05）。COPD组痰液中性粒细胞氧化吞噬功能显著低于肺炎组（P<0.05）,CCR1表达显著高于肺炎组（P<0.05）,TLR2和TLR4表达2个组之间差异均无统计学意义（P>0.05）。Pearson相关分析结果显示,COPD组痰液中性粒细胞氧化吞噬阳性率与CCR1表达呈负相关（r=-0.548,P<0.01）,与TLR2、TLR4表达均无相关性（r值分别为-0.206、-0.219,P>0.05）。结论 COPD患者痰液中性粒细胞氧化吞噬功能显著下降,其表面CCR1表达升高。COPD患者气道中性粒细胞的氧化吞噬功能受损可能是COPD发病的机制之一。

关键词: 趋化因子C-C基序受体-1, Toll样受体, 中性粒细胞, 氧化吞噬, 慢性阻塞性肺疾病

Abstract:

Objective To investigate the roles of neutrophil oxidative phagocytosis and related receptors [chemokine C-C receptor -1（CCR1）,Toll-like receptor（TLR） 2 and TLR4] in patients with chronic obstructive pulmonary disease（COPD）. Methods A flow cytometry method was established to determine neutrophil oxidative phagocytosis and related receptors in sputum samples of COPD patients. By flow cytometry,neutrophil oxidative phagocytosis in peripheral blood and sputum samples of 30 COPD patients（COPD group）,30 pneumonia patients（pneumonia group） and 30 healthy subjects（healthy control group） was determined. Neutrophil oxidative phagocytosis and the expression levels of CCR1,TLR2 and TLR4 on the surface were determined. Results The neutrophil oxidative phagocytosis of peripheral blood in pneumonia group and COPD group was lower than that in healthy control group（P<0.05）,and there was no statistical significance between pneumonia and COPD groups（P>0.05）. There was statistical significance in the expression of CCR1 on the surface of peripheral blood neutrophils among COPD,pneumonia and healthy control groups（P<0.05）,but there was no statistical significance for TLR2 and TLR4 expression levels（P>0.05）. The neutrophil oxidative phagocytosis in sputum samples in COPD group was lower than that in pneumonia group（P<0.05）,the expression level of CCR1 was higher than that in pneumonia group（P<0.05）,and there was no statistical significance for the expression levels of TLR2 and TLR4（P>0.05）. Pearson correlation analysis showed that the positive rate of neutrophil oxidative phagocytosis in sputum samples in COPD group was negatively correlated with the expression level of CCR1（r=-0.548,P<0.01）,and it had no correlation with the expression levels of TLR2 and TLR4（r=-0.206 and -0.219,P>0.05）. Conclusions Neutrophil oxidative phagocytosis in sputum samples of COPD patients is decreased,which can increase CCR1 expression level on the surface,suggesting that COPD patients'respiratory neutrophil oxidative phagocytosis injury may be one of the mechanisms of COPD pathogenesis.

Key words: Chemokine C-C receptor-1, Toll-like receptor, Neutrophil, Oxidative phagocytosis, Chronic obstructive pulmonary disease

中图分类号:

R446.1

闫佩毅, 张骥, 涂焕平, 张立, 仇越, 张锋英, 胡奕雯, 金姝. COPD患者痰液中性粒细胞氧化吞噬功能及相关受体变化的临床意义[J]. 检验医学, 2020, 35(6): 570-577.

YAN Peiyi, ZHANG Ji, TU Huanping, ZHANG Li, QIU Yue, ZHANG Fengying, HU Yiwen, JIN Shu. Changes of neutrophil oxidative phagocytosis and related receptors in sputum samples of COPD patients[J]. Laboratory Medicine, 2020, 35(6): 570-577.

图/表 8

图1 中性粒细胞氧化吞噬能力检测示意图

图2 不同样本的流式散点图注：（a）痰液样本;（b）外周血样本;R1为中性粒细胞群

表1 COPD组、肺炎组及正常对照组外周血中性粒细胞氧化吞噬百分率及相关受体阳性率比较 %,$\bar{x}±s$

组别	例数	氧化吞噬百分率	TLR2	TLR4	CCR1
正常对照组	30	98.27± 2.58	59.62±19.32	48.70±27.57	61.77±25.89
肺炎组	30	93.05±9.24^*	54.33±32.86	40.62±22.93	29.04±28.16^*
COPD组	30	96.22± 3.28^*	45.58±34.31	42.45±28.62	40.97±34.58^#

图3 COPD组、肺炎组及正常对照组外周血中性粒细胞氧化吞噬功能的流式直方图注：（a）正常对照组;（b）肺炎组;（c）COPD组

图4 COPD组、肺炎组及正常对照组外周血中性粒细胞表面CCR1、TLR2及TLR4表达的流式直方图注：（a）~（d）分别为同型对照、正常对照组、肺炎组、COPD组的CD282（TLR2）检测结果;（e）~（h）分别为同型对照、正常对照组、肺炎组、COPD组的CD284（TLR4）检测结果;（i）~（l）分别为同型对照、正常对照组、肺炎组、COPD组的CD191（CCR1）检测结果

表2 COPD组和肺炎组痰液中性粒细胞氧化吞噬百分率及相关受体的阳性率 %,$\bar{x}±s$

组别	例数	氧化吞噬百分率	TLR2	TLR4	CCR1
肺炎组	30	51.57±36.50	10.26±9.24	20.86±18.39	15.08±15.03
COPD组	30	33.60±31.73^*	12.64±12.13	20.30±19.15	44.99±34.11^*

图5 肺炎组和COPD组痰液中性粒细胞氧化吞噬功能的流式直方图注：（a）肺炎组;（b）COPD组

图6 肺炎组和COPD组痰液中性粒细胞表面受体的流式直方图注：（a）~（c）分别为肺炎组痰液TLR2、TLR4、CCR1的流式直方图;（d）~（f）分别为COPD组痰液TLR2、TLR4、CCR1的流式直方图

参考文献 21

[1]	THULBORN S J,MISTRY V,BRIGHTLING C E,et al.Neutrophil elastase as a biomarker for bacterial infection in COPD[J]. Respir Res,2019,20(1):170.
[2]	DUNNE A E,KAWAMATAWONG T,FENWICK P S,et al.Direct inhibitory effect of the PDE4 inhibitor roflumilast on neutrophil migration in chronic obstructive pulmonary disease[J]. Am J Respir Cell Mol Biol,2019,60(4):445-453.
[3]	VOGELMEIER C F,CRINER G J,MARTINEZ F J,et al.Global strategy for the diagnosis, management, and prevention of chronic obstructive lung disease 2017 report. GOLD executive summary[J]. Am J Respir Crit Care Med,2017,195(5):557-582.
[4]	SAHADEVAN A,CUSACK R,O'KELLY B,et al. The value of the combined assessment of COPD in accurate characterization of stable COPD[J]. Ir Med J,2016,109(1):338-340.
[5]	EAPEN M S,MCALINDEN K,TAN D,et al.Profiling cellular and inflammatory changes in the airway wall of mild to moderate COPD[J]. Respirology,2017,22(6):1125-1132.
[6]	HUGHES M J,SAPEY E,STOCKLEY R.Neutrophil phenotypes in chronic lung disease[J]. Expert Rev Respir Med,2019,13(10):951-967.
[7]	WANG F,HE B.CCR1 and CCR5 expression on inflammatory cells is related to cigarette smoking and chronic obstructive pulmonary disease severity[J]. Chin Med J (Engl),2012,125(23):4277-4282.
[8]	KNOBLOCH J,CHIKOSI S J,YANIK S,et al.A systemic defect in Toll-like receptor 4 signaling increases lipopolysaccharide-induced suppression of IL-2-dependent T-cell proliferation in COPD[J]. Am J Physiol Lung Cell Mol Physiol,2016,310(1):L24-L39.
[9]	HARTJES F J,VONK J M,FAIZ A,et al.Predictive value of eosinophils and neutrophils on clinical effects of ICS in COPD[J]. Respirology,2018,23(11):1023-1031.
[10]	ULLER L,PERSSON C G,ERJEFÄLT J S. Resolution of airway disease:removal of inflammatory cells through apoptosis,egression or both?[J]. Trends Pharmacol Sci,2006,27(9):461-466.
[11]	MAKRIS D,VREKOUSSIS T,IZOLDI M,et al.Increased apoptosis of neutrophils in induced sputum of COPD patients[J]. Respir Med,2009,103(8):1130-1135.
[12]	PRIETO A,REYES E,BERNSTEIN E D,et al.Defective natural killer and phagocytic activities in chronic obstructive pulmonary disease are restored by glycolphosphopeptical (inmunoferón)[J]. Am J Respir Crit Care Med,2001,163(7):1578-1583.
[13]	DRANNIK A G,POULADI M A,ROBBINS C S,et al.Impact of cigarette smoke on clearance and inflammation after Pseudomonas aeruginosa infection[J]. Am J Respir Crit Care Med,2004,170(11):1164-1171.
[14]	STRINGER K A,TOBIAS M,O'NEILL H C,et al. Cigarette smoke extract-induced suppression of caspase-3-like activity impairs human neutrophil phagocytosis[J]. Am J Physiol Lung Cell Mol Physiol,2007,292(6):L1572-L1579.
[15]	SAPEY E,STOCKLEY J A,GREENWOOD H,et al.Behavioral and structural differences in migrating peripheral neutrophils from patients with chronic obstructive pulmonary disease[J]. Am J Respir Crit Care Med,2011,183(9):1176-1186.
[16]	俞晔珩,朱文胜,王晓川. 流式细胞仪测定成人与儿童中性粒细胞功能[J]. 复旦学报(医学版),2005,32(1):101-104.
[17]	LIU J,LOUIE S,HSU W,et al.Tyrosine sulfation is prevalent in human chemokine receptors important in lung disease[J]. Am J Respir Cell Mol Biol,2008,38(6):738-743.
[18]	GLADUE R P,BROWN M F,ZWILLICH S H.CCR1 antagonists:what have we learned from clinical trials[J]. Curr Top Med Chem,2010,10(13):1268-1277.
[19]	OLIVEIRA-NASCIMENTO L,MASSARI P,WETZLER L M.The role of TLR2 in infection and immunity[J]. Front Immunol,2012,3:79.
[20]	PRINCE L R,WHYTE M K,SABROE I,et al.The role of TLRs in neutrophil activation[J]. Curr Opin Pharmacol,2011,11(4):397-403.
[21]	VON SCHEELE I,LARSSON K,DAHLÉN B,et al. Toll-like receptor expression in smokers with and without COPD[J]. Respir Med,2011,105(8):1222-1230.