pissn: 2288-42 eissn: 2288-41 2(5):332-336, November 214 http://dx.doi.org/1.4168/aard.214.2.5.332 ORIGINAL ARTICLE 소아천식환아의호기농축시료에서염증매개물질측정의의의 한윤기, 박준영, 김미나, 김윤희, 박현빈, 김민정, 이경은, 이희선, 허원일, 홍정연, 김경원, 손명현, 김규언 연세대학교의과대학소아과학교실및알레르기연구소 Increased inflammatory mediator in exhaled breath condensate from asthmatic children Yoon Ki Han, Jun Young Park, Mi Na Kim, Yoon Hee Kim, Hyun Bin Park, Min Jung Kim, Kyung Eun Lee, Hee Seon Lee, Won Il Heo, Jeong Yeon Hong, Kyung Won Kim, Myung Hyun Sohn, Kyu-Earn Kim Department of Pediatrics, Severance Hospital, Institute of Allergy, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea Purpose: There has recently been increasing interest in the use of exhaled breath condensate (EBC) as a simple noninvasive means for understanding the physiology of asthma. The aim of this study was to evaluate the levels of leukotriene B4 (LTB4) and eosinophil cationic protein (ECP) in the EBC of asthmatic children. Methods: We measured LTB4 and ECP levels in EBC from children aged 6 14 years, including healthy children (n = 25) and asthmatic children (n = 25). We also measured serum LTB4 and serum ECP. Pulmonary function tests and methacholine challenge tests were performed on all subjects. Results: Exhaled LTB4 levels were increased significantly in patients with asthma compared to normal subjects (7.1 ± 3.7 pg/ml vs. 2.2 ± 1.7 pg/ml, P<.5). Serum LTB4 levels were not significantly different in patients with asthma compared to normal subjects (674.7 ± 484.1 pg/ml vs. 487.1 ± 272. pg/ml, P=.156,) and no significant correlations were found between exhaled and serum LTB4 concentrations in children with asthma (r=.52, P=.758). Exhaled ECP levels were not significantly different in patients with asthma compared to normal subjects (P=.419). Serum ECP levels were significantly increased in patients with asthma compared to normal subjects (44.37± 32.14 μg/l vs. 16.4± 13.23 μg/l, P=.1). Conclusion: We found significantly elevated LTB4 levels in the EBC of asthmatic children. Our results suggest that EBC may be one of the supportive tools to measure airway inflammation in children with asthma. ( 214;2:332-336) Keywords:, Exhaled breath condensate, Leukotriene B4, Eosinophil cationic protein 서론천식은반복적인천명, 호흡곤란또는만성기침등의증상을보이면서만성적인기도폐쇄및기도손상과기도개형을특징으로하는염증성폐질환이다. 1-3) 이러한천식의병태생리에는비만세포와호산구에의해생성되는염증성매개물질 (leukotriene B 4 [LTB 4], leukotriene C 4 [LTC 4], leukotriene D 4 [LTD 4], leukotriene E 4 [LTE 4]), Th2 세포관련사이토카인 (interferon-γ [IFN-γ], tumor necrosis factor-α [TNF-α], interleukin [IL] 2, IL-4, IL-5, IL-1), 중성구등의다른종류의다양한세포와염증성매개물질들이관여한다. 3-5) 특히천식기도의염증은점막부종, 상피탈락, 기저막아래의섬유화의과정을거치며이과정에서호산구와 T 세포의침윤, 비만세포수의증가등이두드러진역할을하는것은잘알려져있고, 천식발작과중증천식에서기도내중성구또한활성화되고수의증가도관찰된다. 5) 천식가이드라인에서는천식증상의빈도, 천식악화빈도및 1 Correspondence to: Kyung Won Kim Department of Pediatrics, Severance Hospital, Institute of Allergy, Brain Korea 21 Plus Project for Medical Science, Yonsei University College of Medicine, 5 Yonsei-ro, Seodaemun-gu, Seoul 12-752, Korea Tel: +82-2-2228-25, Fax: +82-2-393-9118, E-mail: kwkim@yumc.yonsei.ac.kr Received: June 5, 214 Revised: August 3, 214 Accepted: September 2, 214 214 The Korean Academy of Pediatric Allergy and Respiratory Disease The Korean Academy of, Allergy and Clinical Immunology This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3./). 332 http://www.aard.or.kr
한윤기외 소아천식에서의호기농축시료 초간노력성호기량 (forced expiratory volume, FEV 1) 등의폐기능을근거로중증도를평가하고치료반응을추적관찰하도록제시하고있고, 6) 실제임상에서도많이이용되고있다. 그러나천식증상이나폐기능이직접적으로만성적인기도염증을반영하지못하므로이러한기도염증을측정하는방법들이제시되고있다. 이러한방법으로는기관지경을이용한기관지생검을통하여직접적인염증을확인하는방법, 간접적인방법으로폐포세척술, 유도객담검사를통하여얻어진시료를통하여염증을확인하는방법및호기산화질소농도 (exhaled nitric oxide) 를측정하는방법등이있다. 호기농축시료 (exhaled breath condensate, EBC) 는기도를감싸고있는에어로졸화된액체와휘발성물질을포함하고있어폐의염증활성도를측정할수있는것으로알려져있다. 1,2) 또한호기농축시료를이용한기도염증평가방법은침습적인방법들과는달리어린나이에서도쉽게반복적으로측정할수있는장점이있으며부작용도없어단순하고안전한방법이라는장점이있다. 이전연구에서성인천식환자에서의호기농축시료분석결과대조군보다 LTE 4, LTB 4 의수치가높았었다. 7) LTB 4 는비만세포, 대식세포, 중성구, 호산구에서생성되며평활근의수축에직접적으로관여하지는않고효과도미미하지만중성구의강한화학주성인자이고또한중성구의활성도를증가시켜국소부종과점막분비물을증가시켜기도를좁게만드는역할을한다. 5,8) LTB 4 는소아천식환아의폐포세척액에서증가되었다는연구는있지만 9) 소아의호기농축시료에서는연구된바없었다. 혈청 eosinophil cationic protein (ECP) 는호산구에서유리되는단백질로강한세포독성을갖고있어기도상피에손상을주며천식의염증반응시에증가하여중증도를평가하는표지자로이용되어왔다. 1) 천식환자의유도객담에서의 ECP 증가에대한연구는있었지만 11,12) 소아의호기농축시료에서는연구된바없었다. 본연구에서는소아천식환아에서호기농축시료를이용하여간접적으로기도염증을평가할수있는 LTB 4 와 ECP를측정하여대조군과비교해봄으로써호기농축시료측정의임상적인의의와유용성에대해알아보고자하였다. 대상및방법 1. 연구대상세브란스병원소아청소년과에내원한만 6세에서 14세사이의 5명환아를대상으로하였다. 천식군은기침, 천명, 호흡곤란등천식이의심되어내원하여천식으로처음진단받은 25명을대상으로하였고, 이들은최근 4주이내에전신스테로이드, 흡입스테로이드또는류코트리엔조절제등의약물치료력이없음을확인하였다. 천식의진단은미국흉부학회 (American Thoracic Society) 지침에따라최근 12개월동안감기와동반되지않은기침이나천명, 호흡 곤란등의전형적인증상을보이면서메타콜린기관지유발검사에서 1초간노력성호기량이 2% 감소되는농도 (provocation concentration causing a 2% fall in FEV 1) 가 16 mg/ml 미만또는기관지확장제투여후 1초간노력성호기량이투여전보다 12% 이상증가를보인경우로정의하였다. 12) 정상대조군은정기적인신체검진이나예방접종을위하여내원한환아중쌕쌕거림이나반복적, 만성적염증질환이없는환아를선별하였고이들은적어도최근 2주간호흡기감염의병력이없었다. 전체대상군에서호기농축시료가수집되었고, 폐기능검사및메타콜린기관지유발검사를시행하였으며, 말초혈액호산구수및혈청총 IgE가측정되었다. 전체대상자중 38명에서혈청 LTB 4 를측정하였고, 42명에서혈청 ECP를측정하였다. 본연구는대상아들의보호자들에게연구의목적과방법을설명하고동의서를받은후시행되었다. 2. 연구방법 1) 호기농축시료의수집호기농축시료는일회용 collection kit (RTube, Respiratory Research, Charlottesville, VA, USA) 를사용하였다. RTube는 polypropylene으로만들어진수집관으로호기농축시료수집전에 2 C 로냉각시킨알루미늄관으로덮게되며, one-way vlave를통해흡입구와연결되어있다. 이흡입구를통해냉각된수집관안으로 1분간평상호흡을하였을때얻어지는응결된검체를수집하며, 수집된호기호흡응축액은분석전까지 8 C에즉시저장하였다. 13) 2) 혈청총 IgE 측정, 호기농축시료및혈청내 ECP 측정, 말초혈액호산구수의측정혈청총 IgE와호기농축시료및혈청내 ECP는형광효소면역분석법 (AutoCAP system, Pharmacia Diagnostics AB, Uppsala, Sweden) 에의해시행하였다. 말초혈액호산구수는 NE-8 system (Sysmex, Kobe, Japan) 을이용하여계수하였다. 3) 호기농축시료및혈청 LTB 4 농도호기농축시료및혈청 LTB 4 농도는상품화된 human LTB4 ELI- SA kit (R&D systems, Minneapolis, MN, USA) 를사용하여측정하였다. Horseradish peroxidase-labeled LTB 4 antibody가코팅된 microtitration well에서 EBC와혈청을 1시간동안 incubation하였다. 5 μl의 LTB 4 conjugate solution (R&D systems) 을첨가하고 incubation 후에 wash buffer (R&D systems) 로 4회세척한후, 2 μl의 substrate solution (R&D systems) 을첨가하고다시 incubation하였다. 5 μl의 stopping solution (.2M sulfuric acid) 을첨가하여반응을종결시키고 3분안에 microplate reader (VersaMax, Molecular Devices LLC, Sunnyvale, CA, USA) 로 optical density 45 nm을측정하였다. http://dx.doi.org/1.4168/aard.214.2.5.332 333
Han YK Exhaled breath condensate in childhood asthma 4) 폐기능검사및메타콜린기관지유발검사폐기능검사는 Jaeger MasterScreen PFT (Jaeger, Wurzburg, Germany) 을이용하여미국흉부학회지침에따라기관지확장제를투여전후로측정하여유량-용적곡선을얻었고, 메타콜린 (Sigma Chemical, St Louis, MO, USA) 흡입유발시험은표준화방법으로시행하였다. 13,14) 5) 통계분석자료의분석은 IBM SPSS Statistics ver. 2. (IBM Co., Armonk, NY, USA) 을사용하였다. 천식군과대조군간임상적지표들의평균및표준편차를구하였고독립표본 t-검정 (independent t-test) 으로통계적유의성을조사하였다. 정규성을보이지않는변수 ( 호기농축시료와혈청내의 LTB 4, 호기농축시료와혈청내의 ECP) 는상용로그값으로변환후에분석을시행하였다. 호기농축시료내의 LTB 4 와혈청내의 LTB 4 의연관성분석에는천식군과대조군을포함한전체 5명의환아를대상으로 Spearman correlation을이용하였다. P값이.5 미만일때통계적으로유의한것으로하였다. 결과 1. 대상환아의임상적특성천식군과대조군에서남녀비와나이는유의한차이를보이지않았다. 혈액내호산구수 (P =.262) 와혈청총 IgE (P =.6) 는유의한차이를보이지않았다. 폐기능검사상 1초간노력성호기량의노력성폐활량에대한비율 (FEV 1/forced vital capacity) (P =.45) 만이천식군에서대조군보다유의하게낮았다. 1초간노력성호기량 (P =.52), 노력성호기중간유량 (forced mid expiratory flow) (P =.92) 은모두천식군과대조군에서통계학적으로유의한차이를보이지않았다 (Table 1). 2. 천식군과대조군에서호기농축시료내 LTB 4 와혈청내 LTB 4 의비교 호기농축시료내 LTB 4 는천식군 (mean±sd, 7.1±3.7 pg/ml) 에서대조군 (2.2±1.7 pg/ml) 에비해의미있게높았다 (P =.3) (Fig. 1A). 혈청내 LTB 4 는천식군 (674.7±484.1 pg/ml) 과대조군 (487.1±272. pg/ml) 이유의한차이를보이지않았다 (P =.156) (Fig. 1B). 3. 혈청내 LTB 4 와호기농축시료내 LTB 4 와의관계 혈청내 LTB 4 는호기농축시료내 LTB 4 와유의한상관관계를보 이지않았다 (r =.52, P =.758) (Fig. 2). 4. 천식군과대조군에서호기농축시료내 ECP 와혈청내 ECP 의비교 호기농축시료내 ECP 는천식군과대조군이유의한차이를보이 지않았다 (P =.419) (Fig. 3A). 혈청내 ECP 는천식군 (mean±sd, 44.37±32.14 μg/l) 에서대조군 (16.4±13.23 μg/l) 에비해의미 Table 1. Clinical characteristics (n= 5) Characteristic (n= 25) (n= 25) P-value Age (yr) 9.3± 2. 9.7± 2.4.527 Male sex 15 (6) 16 (64) Eosinophil count (/μl) 462.61± 35.69 359.57± 38.83.262 Serum total IgE (U/mL) 76.15± 859.25 344.11± 544.12.6 FEV1 (% of predicted value) 94.51± 16.42 98.6± 8.84.52 FEV1/FVC 83.99± 9.41 88.67± 6.4.45 FEF25%-75% (% of predicted value) 74.58± 27.83 87.15± 2.96.92 Values are presented as mean± standard deviation or number (%). FEV1, forced expiratory volume in one second; FVC, forced vital capacity; FEF25%-75%, forced mid expiratory flow. 2 P=.3 2, P=.156 Exhaled LTB4 (pg/ml) 1 Serum LTB4 (pg/ml) 1, A B Fig. 1. (A) Comparison of concentrations of leukotriene B4 (LTB4) in exhaled breath condensate between asthma and control groups. LTB4 in asthma group (mean± standard deviation [SD], 7.1± 3.7 pg/ml) was significantly higher than control group (mean± SD, 2.2± 1.7 pg/ml) (P=.3). (B) Comparison of concentrations of serum LTB4 between asthma and control groups. There was no significant difference between two groups (P=.156). 334 http://dx.doi.org/1.4168/aard.214.2.5.332
한윤기외 소아천식에서의호기농축시료 있게높았다 (P =.1) (Fig. 3B). 고찰 본연구에서는호기농축시료의 LTB 4 농도는천식군에서대조군 에비해유의하게증가됨을확인하였다. 그러나혈청 LTB 4 는천식 군과대조군사이에차이가없었고, 호기농축시료의 LTB 4 농도와 도상관관계를보이지않았다. 반면 ECP 는혈청에서는천식군에서 의미있는증가를보였으나호기농축시료에서는두군사이에차이 가없었다. 호기농축시료는휴대용 RTube 를이용하여간편하게수집가능 Serum LTB4 (pg/ml) 2,5 2, 1,5 1, 5 r=.52, P=.758 2 4 6 8 1 12 14 Exhaled LTB4 (pg/ml) Fig. 2. Correlations between the concentrations of leukotriene B4 (LTB4) in exhaled breath condensate (EBC) and in serum. No significant correlations were shown between concentrations of LTB4 in EBC and in serum (r=.52, P=.758). 하며기도염증을반영할것이라고기대되었기때문에 leukotrienes, 8-isoprostane, prostaglandins, hydrogen peroxide, nitric oxide-derived products, hydrogen ions, 여러사이토카인의측정이시도되었으나표준화에어려움이있었다. 현재호기농축시료 ph 만이임상적인의미를부분적으로인정받고있는데이는호기농축시료에서수집되는단백질의양이적고개인차가많아여러가지염증매개물질, 특히비휘발성물질들의측정이잘안되거나표준화가어렵기때문이다. 14) 반면에혈청에서의 LTB 4 는천식군과대조군에서뚜렷한차이를보이지않았다. 이전연구에서도혈액응고가혈청 LTB 4 에영향을미쳐천식환자의염증평가방법으로는적절치않음을보고한바있다. 15) 또한그수집방법에있어서수집즉시 8 C 에저장을해야염증매개물질의정확한측정이가능하지만임상에서검사직후시행하기어려운점이있다. 13) 그러나본연구에서는호기농축시료 LTB 4 는천식군에서대조군에비해의미있게증가되어있었음을확인하였고, 이는천식환자의기관지생검, 기관지폐포세척액, 유도객담에서 LTB 4 가증가한다는이전연구와상응하는결과였다. 7) 반면에혈청에서의 LTB 4 는천식군과대조군에서뚜렷한차이를보이지않았고호기농축시료 LTB 4 와혈청 LTB 4 사이에도관련성을보이지않았다. 이는혈액응고가혈청 LTB 4 에영향을주어혈청에서는정확한농도를측정하기어렵기때문이며혈청 LTB 4 는천식환자의염증평가방법으로는적절치않음을뒷받침한다. 16) ECP는활성화된호산구의과립에서분비되는단백의한종류로호산구의활성도를간접적으로측정할수있는생물학적지표이며염증성매개물질로서천식의후기반응에중요한역할을한다. 17) 혈청 ECP는알레르기질환과천식에서진단과치료효과를평가하고질병의악화를예측하는데임상적으로유용하게사용되고있다. 17-19) 특히아토피성천식에서더높게측정되며천식의정도가심할수록높게측정된다는보고도있다. 2) 본연구에서도천식군에서대조군보다혈청 ECP 농도가유의하게높아이전보고와같은결과가나왔으나호기농축시료의 ECP 농도는천식군과대조군에서유의 1 P=.419 15 P=.1 Exhaled ECP (ug/l) 5 Serum ECP (ug/l) 1 5 A B Fig. 3. (A) Comparison of concentrations of eosinophil cationic protein (ECP) in exhaled breath condensate (EBC) between asthma and control groups. There was no significant difference between two groups (P=.419). (B) Comparison of concentrations of serum ECP between asthma and control groups. Serum concentration of ECP in asthma group (mean± standard deviation [SD], 44.37± 32.14 μg/l) was significantly higher than control group (mean± SD, 16.4± 13.23 μg/l) (P=.1). http://dx.doi.org/1.4168/aard.214.2.5.332 335
Han YK Exhaled breath condensate in childhood asthma 한차이를보이지않았다. 천식환자의기관지폐포세척액이나, 21) 유도객담에서는 ECP 수치가증가하는데반해, 14) 호기농축시료 ECP 농도는거의측정되지않았다. 아주작은양의비휘발성물질 을포함하는호기농축시료에서 ECP 농도를측정하는것은천식 환자의기도염증을측정하는방법으로유용하지않은것으로사 료된다. 본연구는대상환자수가 5 명인선행연구로임상적인유용성에 대하여결론짓기어렵다. 좀더많은환자를대상으로기도염증을 반영하는호기산화질소농도나유도객담검사등과의비교분석이 필요하다. 또한본연구에모집된천식환자들은모두유지치료를 하지않은상태로내원한경증또는중등증천식환자로천식의중 증도에따른 LTB 4 농도를분석하기에어려움이있었다. 이전연구에 서호기농축시료 lipoxin A 4 (LXA 4) 는천식의중증도가심해짐에 따라 LXA 4 수치가낮아진다고보고되어 22) 중증도에따라 LTB 4 농 도에도변화가있을가능성이있다. 추후호기농축시료 LTB 4 농도 에대한중증도, 치료후추적관찰에따른변화등에대한연구가 필요하다. 결론적으로, 호기농축시료에서측정한 LTB 4 농도가소아천식 환아에서증가되어있음을확인하였고이는간편하게수집가능한 호기농축시료에서 LTB 4 가기도염증을반영하는보조적인표지자 로서의가능성을보인선행연구로의미가있다. REFERENCES 1. Hunt J. Exhaled breath condensate: an evolving tool for noninvasive evaluation of lung disease. J Allergy Clin Immunol 22;11:28-34. 2. Rosias PP, Dompeling E, Dentener MA, Pennings HJ, Hendriks HJ, Van Iersel MP, et al. Childhood asthma: exhaled markers of airway inflammation, asthma control score, and lung function tests. Pediatr Pulmonol 24; 38:17-14. 3. Leung TF, Wong GW, Ko FW, Li CY, Yung E, Lam CW, et al. Analysis of growth factors and inflammatory cytokines in exhaled breath condensate from asthmatic children. Int Arch Allergy Immunol 25;137:66-72. 4. Zanconato S, Carraro S, Corradi M, Alinovi R, Pasquale MF, Piacentini G, et al. Leukotrienes and 8-isoprostane in exhaled breath condensate of children with stable and unstable asthma. J Allergy Clin Immunol 24;113: 257-63. 5. Csoma Z, Kharitonov SA, Balint B, Bush A, Wilson NM, Barnes PJ. Increased leukotrienes in exhaled breath condensate in childhood asthma. Am J Respir Crit Care Med 22;166:1345-9. 6. Papadopoulos NG, Arakawa H, Carlsen KH, Custovic A, Gern J, Lemanske R, et al. International consensus on (ICON) pediatric asthma. Allergy 212;67:976-97. 7. Montuschi P, Barnes PJ. Exhaled leukotrienes and prostaglandins in asthma. J Allergy Clin Immunol 22;19:615-2. 8. Kostikas K, Gaga M, Papatheodorou G, Karamanis T, Orphanidou D, Loukides S. Leukotriene B4 in exhaled breath condensate and sputum supernatant in patients with COPD and asthma. Chest 25;127:1553-9. 9. Krawiec ME, Westcott JY, Chu HW, Balzar S, Trudeau JB, Schwartz LB, et al. Persistent wheezing in very young children is associated with lower respiratory inflammation. Am J Respir Crit Care Med 21;163:1338-43. 1. Tomassini M, Magrini L, De Petrillo G, Adriani E, Bonini S, Balsano F, et al. Serum levels of eosinophil cationic protein in allergic diseases and natural allergen exposure. J Allergy Clin Immunol 1996;97:135-5. 11. Cianchetti S, Bacci E, Ruocco L, Pavia T, Bartoli ML, Cardini C, et al. Are sputum eosinophil cationic protein and eosinophils differently associated with clinical and functional findings of asthma? Clin Exp Allergy 214; 44:673-8. 12. Kim KW, Jee HM, Park YH, Choi BS, Sohn MH, Kim KE. Relationship between amphiregulin and airway inflammation in children with asthma and eosinophilic bronchitis. Chest 29;136:85-1. 13. Montuschi P. Analysis of exhaled breath condensate in respiratory medicine: methodological aspects and potential clinical applications. Ther Adv Respir Dis 27;1:5-23. 14. Scheideler L, Manke HG, Schwulera U, Inacker O, Hammerle H. Detection of nonvolatile macromolecules in breath. A possible diagnostic tool? Am Rev Respir Dis 1993;148:778-84. 15. Fujimoto K, Kubo K, Matsuzawa Y, Sekiguchi M. Eosinophil cationic protein levels in induced sputum correlate with the severity of bronchial asthma. Chest 1997;112:1241-7. 16. Seggev JS, Wiessner JH, Thornton WH Jr, Edes TE. Comparison of serum and plasma leukotriene B4 levels in normal and asthmatic subjects. Ann Allergy Immunol 1995;75:365-8. 17. Gleich GJ, Adolphson CR. The eosinophilic leukocyte: structure and function. Adv Immunol 1986;39:177-253. 18. Venge P, Bystrom J, Carlson M, Hakansson L, Karawacjzyk M, Peterson C, et al. Eosinophil cationic protein (ECP): molecular and biological properties and the use of ECP as a marker of eosinophil activation in disease. Clin Exp Allergy 1999;29:1172-86. 19. Koh GC, Shek LP, Goh DY, Van Bever H, Koh DS. Eosinophil cationic protein: is it useful in asthma? A systematic review. Respir Med 27; 11:696-75. 2. Wolthers OD. Eosinophil granule proteins in the assessment of airway inflammation in pediatric bronchial asthma. Pediatr Allergy Immunol 23; 14:248-54. 21. Kim KW, Lee KE, Kim ES, Song TW, Sohn MH, Kim KE. Serum eosinophil-derived neurotoxin (EDN) in diagnosis and evaluation of severity and bronchial hyperresponsiveness in childhood asthma. Lung 27; 185:97-13. 22. Kazani S, Planaguma A, Ono E, Bonini M, Zahid M, Marigowda G, et al. Exhaled breath condensate eicosanoid levels associate with asthma and its severity. J Allergy Clin Immunol 213;132:547-53. 336 http://dx.doi.org/1.4168/aard.214.2.5.332