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1 PM 10 이 A549 Cells 에서전염증성 Cytokine 발현에미치는영향 가천대학교의과대학길병원호흡기내과김정호, 전효근, 김미경, 경선영, 안창혁, 이상표, 박정웅, 정성환 Particulate Matter from Asian Dust Storms Induces the Expression of Proinflammatory Cytokine in A549 Epithelial Cells Jung Ho Kim, M.D., Hyo Keun Jeon, M.D., Mi Kyeong Kim, M.D., Sun Yong Kyung, M.D., Chang Hyeok An, M.D., Sang Pyo Lee, M.D., Jung Woong Park, M.D., Sung Hwan Jeong, M.D. Division of Pulmonary Medicine, Department of Internal Medicine, Gachon Medical School Gil Medical Center, Incheon. Background: PM 10(Particulate matter with a diameter < 10 μm ), which is characterized by different environmental conditions, is a complex mixture of organic and inorganic compounds. The Asian dust event caused by meteorological phenomena can also produce unique particulate matter in affected areas. This study investigated the cytokine produced by A549 epithelial cells exposed to particles collected during both the Asian dust pfenomenon and ambient air particles in a non-dusty period. Method: Air samples were collected using a high volume air sampler(sibata Model HV500F) with an air flow at 500l /min for at least 6 hours. The cytokine messenger RNA(mRNA) was measured using a reverse transcriptase polymerase chain reaction(rt-pcr). The A549 cells were exposed to 10 to 500 μg / ml of a suspension containing PM 10 for 24 hours. Each was compared with those in the non-exposed control cells. Result: The mrna levels of interleukin(il)-1α, IL-Iβ, IL-8, and the granulocyte macrophage colony stimulating factor(gm-csf) increased after veing exposed to PM 10 in the ambient air particles, compared with those in the non-exposed control cells. The increase in IL-1α and IL-8 were dose dependent at a PM 10 concentration between 100 μg / ml and 500 μg / ml. The mrna level of IL-8 in the A549 epithelial cells was higher during the in the Asian dust period(500 μg / ml ) than during the non dust period. Conclusion: A549 cells exposed to the PM 10 collected during the Asian dust period produce more proinflammatory cytokine than during non-dusty period. This cytokine enhances the local inflammatory response in the airways and can also contribute to the systemic component of this inflammatory process. (Tuberc Respir Dis 2006; 60: ) Key words: Particulate matter, Asian dust, Proinflammatory cytokine. 서 대기중의먼지현상이란물이나얼음입자는거의포함되어있지않고, 주로고체입자들이대기중에서떠다니거나바람에의해서불려오르는현상을말한다. 먼지현상의한종류인 연무 (haze) 는육안으로보이지않는극히작고건조한고체입자가대기중에떠다니고있는현상이다. 특히바람에의해서지면으로부터불려올라간먼지나모래의작은입자가대기 Address for correspondence : Sung Hwan Jeong, M.D. Division of Pulmonary Medicine, Department of Internal Medicine, Gachon Medical School Gil Medical Center, Guwol-1-dong, Namdong-gu, Incheon, , Korea Phone : Fax : jsw@gilhospital.com, jwpark@gilhospital.com Received : Feb Accepted : May 론 중에떠있는것이확실할때의현상은 먼지연무 (dust haze) 라하며, 황사 는먼지연무의일종으로서주로아시아대륙의건조지대에서불려올라간다량의흙먼지가장거리수송되어하늘을덮고떠다니며서서히하강하는현상을말한다 1. 국내에서도매년봄철이면황사가주기적으로발생한다. 황사현상에서크기가큰입자들은발원지나주변에머물고국내에유입되는황사입자의크기는대개지름 10μm이하의미세먼지 (particulate matter 10; PM 10 ) 이고, 미세먼지는흡입시에하부기관지및폐의가스-교환부분까지침착하여호흡기계에손상을일으킬수있다고보고되고있다 2. 기존의미세먼지의인체내유해성에대한연구는대기오염연구에서많이이루어졌는데, 1930년벨기에의 Meuse, 1952년 12월런던에서심한대기오염으로많은사망자가발생했던것이대표적인대기오염 663

2 JH Kim et al. : PM10 induce proinflammatory cytokine in bronchial epithelial cells 사례로잘알려져있다 3. 임상적으로는대기오염에의하여천식과만성폐쇄성폐질환과같은호흡기질환의유병률증가및급성악화가일어날수있다는것이여러연구에서증명되었으며 4-8, 대기오염에포함된미세먼지중에서말초호흡기로침투가가능한크기인 PM 10 이천식발작및만성호흡기질환의악화에많이기여하는것으로알려져있다 국내에서시행된연구에서도대기오염의위해성은임상적, 실험적으로밝혀져있으며 12-13, 특수한자연적대기오염의한형태인황사현상의경우이기간중에대기중의 PM 10 이크게증가하며이에따라기관지천식환자의호흡기증상악화를유발할수있음이밝혀진바있다 14. 미세먼지는유기물과무기물의복합체로발생시기와장소에따라물리적특성과구성성분이매우다양하고, 생물학적으로영향을줄수있는금속성분도함유하고있다. PM 10 이폐장내에흡입시폐포대식세포와폐상피세포가 PM 10 에직접적으로노출이되고, 특히폐상피세포에서 interleukin(il)-1, IL-6, IL-8, monocyte chemotactic protein(mcp)-1, tumor necrosis factor(tnf)-α, granulocyte macrophage colony stimulating factor(gm-csf) 과같은염증성 cytokine의발현이증가하는것으로알려져있으며, 이로인해호흡기질환의악화가일어나는것으로추측되고있다 하지만황사의 PM 10 이폐상피세포에서전신반응을일으킬수있는염증성물질의생성에관한연구는아직많지않다. 본연구에서는황사에포함된 PM 10 이 A549 cells에작용하여전염증성 cytokine 및 cytokine messenger RNA(mRNA) 의발현에어떤영향이있는지를관찰하여만성천식등호흡기질환의증상악화기전에미치는역할을규명하고자하였다. 대상및방법 1. 미세먼지의포집공기포집은 3일간의황사기간과 10일간의비황사기간에시행되었다. 황사기간의공기포집은 2004년 3월 10일, 3월 30일, 4월 23일시행하였고비황사기간의공기포집은 2004년 4월 21일, 4월 24일, 5월 9 일, 5월 22일, 5월 26일, 6월 3일, 6월 26일, 7월 7일, 7월 31일, 8월 14일시행하었다. 2. PM 10 준비공기포집기 (HV500F, Sibata, Japan) 을이용하여도심지인인천시 ( 인천시남동구구월동가천의대길병원응급센터옥상 ) 에서분당 500 liter로하루 6시간씩 ( 오전 10시부터오후 4시까지 ) 실외의장소에서대기분진을 pore size 0.25μm필터 (Prefilter AP, 124mm, Milipore, Bedford, MA, USA) 에포집하였다. 포집한필터를조각내어 phosphate buffered saline(pbs) buffer 10ml를넣은튜브 (tube) 에넣었다. 틈틈이소용돌이혼합기 (vortex mixer) 를사용하면서 30분간실온에서배양했다. 필터를건져낸후 10μm pore의필터 (Mitex TM membrane filters, Milipore, Bedford, MA, USA) 로여과하였다. 그후약 1분간 3번초음파파쇠 (sonication) 하였다. 1.5ml튜브로나누어담아무게를측정하고 121 에서 15분간멸균한후사용전까지 -20 에보관하였다. 3. 세포배양폐암상피세포주인 A549 cells( 한국세포은행주 ) 을 fetal bovine serum(fbs) 이 10% 포함된 RPMI-1640 media(welgene, Daegu, Korea) 에서배양하였다. 세포가 % 로배양되었을때황사와비황사시기에포집한 PM 10 을각각 10, 100, 500μg / ml의농도로첨가해주었다. 그리고각각을처리 24시간후 RNA 를추출하였다. 대조군은세포배양만시행하고 PM 10 처리를하지않은것으로하였다. 4. RNA 추출과역전사중합효소연쇄반응 (reverse transcriptase polymerase chain reaction; RT-PCR) 배지를제거한후 PBS buffer로 2번세척하였다. 664

3 Tuberculosis and Respiratory Diseases Vol. 60. No.6, Jun Table 1. RT-PCR 에사용한 primer IL-1α IL-1β IL-8 GM-CSF 5'-GTC TCT GAA TCA GAA ATC CTT CTA TC -3' 5'-CAT GTC AAA TTT CAC TGC TTC ATC C -3' (420bp) 5'-AAA CAG ATG AAG TGC TCC TTC CAG G -3' 5'-TGG AGA ACA CCA CTT GTT GCT CCA -3' (388bp) 5'- ATG ACT TCC AAG CTG GCC GTG GCT -3' 5'-TCT CAG CCC TCT TCA AAA ACT TCT C-3' (289bp) 5'- ACA CTG CTG AGA TGA ATG AAA CAG TAG -3' 5'- TGG ACT GGC TCC CAG CAG TCA AAG GGG ATG-3' (172bp) 5'-GTG GAT ATT GTT GCC ATC AAT GAC C-3' GAPDH 5'-GCC CCA GCC TTC TCC ATG GTG GT-3' (248bp) GM-CSF; granulocyte macrophage colony stimulating factor, IL; interleukin, RT-PCR; reverse transcriptase polymerase chain reaction Trizol(Invitrogen, Life Technologies, Inc., Carlsbad, CA, USA) 를넣고세포를넣은후 1.5ml튜브로옮겼다. 실온에서 5분간배양한후 chloroform을첨가하였고 inverting 15초를하여다시실온에서 3분간배양하였다 g 4 에서 15분간원심분리한후 75% 에탄올 (ethanol) 로 2번세척하였으며침전물 (pellet) 을건조시킨후 diethyl pyrocarbonate(depc) 로처리된증류수를넣어정량하였다. 2μg의 RNA에 random hexamer(promega, Madison, WI, USA) 를넣어 94 에 5분, 얼음에 3분간배 Figure 1. IL-1α mrna expression in A549 cells after 24 hours incubation with PM 10. A549 cells were exposed to 10 to 500 μg / ml of a suspension of ambient particulate matter with a diameter of less than 10 μm (PM 10) for 24 hours. Results shown in the box plot graph are the percentage from control(medium alone) values(*p<0.05, p<0.1) compared with control. The black lines in boxes show median value of IL-1a mrna expression. 1A. The expression of IL-1α mrna measured in the supernatant collected at 24 hours increased and were significantly higher than those in the control nonexposed cells (10 to 500 μg / ml ) 1B. The mrna Levels of IL-1α were increased after exposed to PM 10 collected during Asian dust period, compared to those in non-dusty period(500 μg / ml ) and the control(500 μg / ml ) IL; interleukin, mrna; cytokine messenger RNA 665

4 JH Kim et al. : PM10 induce proinflammatory cytokine in bronchial epithelial cells 양했다. M-MLV(Progma, Madison, WI, USA), RNasin(Progma, Madison, WI, USA), dntps(takara, Otsu, Japan) 을넣어역전사반응을했다 (37, 1시간 ). 이 cdna를주형 DNA로이용하고 interleukin(il)-1α, IL-1β, IL-8, granulocyte macrophage colony stimulating factor(gm-csf) 의 primer set들을이용하여 RT-PCR을시행하였다. 그리고 2% agarose gel에전기영동후각각의밴드를농도계측기 (densitometry) 을이용하여측정하였다 (Table 1). 5. 분석방법모든결과는평균 ± 표준편차로표시하였다. 두군간의차이는 Wilcoxon signed ranks test로검정하였다. 분석은 SPSS(Version 13.0) 프로그램을이용하였고 p<0.05인경우에통계학적으로유의하다고판단하였다. 결과인천지역에서 PM 10 의연평균농도는 2001년까지는감소하는추세였으나그이후에는약간의증가추세를보이고있고환경부의보고에따르면연구가수행된 2004년도에는연평균 62μg / m3, 황사기간을제외하면 59μg / m3를보이고있었다. 본연구에서공기포집이시행된 2004년도에인천지역의황사기간은 6일이었다. 인천보건환경연구원자료에의하면황사기간의 PM 10 의평균농도는 ±55.54μg / m3이었고한시간 PM 10 최대값은 ±164.07μg / m3이었다. 1. PM 10 이 A549 cells 에서 IL-1α mrna 발현에미치는영향 A549 cells을황사기간과비황사기간을포함한전 Figure 2. IL-1β mrna expression in A549 cells after 24 hours incubation with PM 10. A549 cells were exposed to 10 to 500 μg / ml of a suspension of ambient particulate matter with a diameter of less than 10 μm (PM 10) for 24 hours. Results shown in the box plot graph are the percentage from control(medium alone) values(*p<0.05, p<0.1) compared with control. The black lines in boxes show median value of IL-1a mrna expression. 2A. The expression of IL-1β mrna measured in the supernatant collected at 24 hours increased and were significantly higher than those in the control nonexposed cells(10 to 500 μg / ml ) 2B. The mrna Levels of IL-1β were increased after exposed to PM 10 collected during Asian dust period, compared to those in non-dusty period(500 μg / ml ) and the control(100 μg / ml, 500 μg / ml ). IL-1β mrna expression increased after 24 hours of exposure to PM 10 in a dose-dependent manner. IL; interleukin, mrna; cytokine messenger RNA 666

5 Tuberculosis and Respiratory Diseases Vol. 60. No.6, Jun 체 PM 10 으로처리하고 24시간후에세포배양상층액으로 RT-PCR을시행한결과를보면 10, 100, 500μg / ml의각농도에서대조군보다의미있게 IL-1α mrna 발현이증가하였다 (PM 10 10μg / ml ±88.49% of control, p=0.039, PM μg / ml ±106.00% of control, p=0.015, PM μg / ml ±105.95% of control, p=0.002). PM 10 10μg / ml보다 100μg / ml에서발현이증가하는경향을보였고 (p=0.098), 500μg / ml에서는유의하게증가하였다 (p=0.016)(figure 1A). 100 μg / ml에서는황사기간의 PM 10 에서는대조군보다의미있게 mrna 의발현이증가하지않았지만 (141.55± % of control, p=0.400), 비황사기간의 PM 10 에서는의미있게 IL-1α mrna발현이증가하였다 ( ±113.34% of control, p=0.035). 비황사기간의 PM μg / ml에서대조군보다의미있게 IL-1α mrna 발현이증가하였고 (156.80±88.39% of control, p=0.025), 황사시기의 PM μg / ml에서도대조군보다의미있게 IL-1α mrna발현이증가하였다 (205.19±124.73% of control, p=0.012)(figure 1B). 2. PM 10 이 A549 cells 에서 IL-1β mrna 발현에미치는영향 A549 cells을 PM 10 으로황사기간과비황사기간을포함한전체처리하고 24시간후에세포배양상층액으로 RT-PCR을시행한결과를보면 10, 100, 500μg / ml의각농도에서대조군보다의미있게 IL-1β mrna발현이증가하였다 (PM 10 10μg / ml ± % of control, p=0.028, PM μg / ml ± 92.19% of control, p=0.008, PM μg / ml ± 87.47% of control, p=0.009)(figure 2A). 그러나농도에따른의미있는차이는보이고있지않았다. 황사기간의 PM μg / ml와 500μg / ml에서는대조군보다 IL-1β mrna의발현이증가하는경향을보였다 (174.02±126.79% of control, p=0.068, ±116.64% of control, p=0.068). 비황사기간의 PM μg / ml에 Figure 3. IL-8 mrna expression in A549 cells after 24 hours incubation with PM 10. A549 cells were exposed to 10 to 500 μg / ml of a suspension of ambient particulate matter with a diameter of less than 10 μm (PM 10) for 24 hours. Results shown in the box plot graph are the percentage from control(medium alone) values(*p<0.05, p<0.1) compared with control. The black lines in boxes show median value of IL-1a mrna expression. 3A. The expression of IL-8 mrna measured in the supernatant collected at 24 hours increased and were significantly higher than those in the control nonexposed cells(10 to 500 μg / ml, 100 to 500 μg / ml ). 3B. The mrna Levels of IL-8 were increased after exposed to PM 10 collected during Asian dust period, compared to those in non-dusty period(100 μg / ml ) and the control(100 μg / ml, 500 μg / ml ) IL; interleukin, mrna; cytokine messenger RNA 667

6 JH Kim et al. : PM10 induce proinflammatory cytokine in bronchial epithelial cells 서도대조군보다 IL-1β mrna의발현이증가하는경향을보였다 (186.11±115.54% of control, p=0.068). 또한황사시기의 PM 10 에서는 10μg / ml에서보다 100 μg / ml와 500μg / ml에서 mrna발현이증가하는경향을보였다 (PM 10 10μg / ml ±119.22% of control, PM μg / ml ±126.79% of control, p=0.068, PM μg / ml ±116.64% of control, p=0.068) (Figure 2B). 3. PM 10 이 A549 cells 에서 IL-8 mrna 발현에미치는영향 A549 cells을황사기간과비황사기간을포함한전체 PM 10 으로처리하고 24시간후에세포배양상층액으로 RT-PCR을시행한결과를보면 10, 500μg / ml의각농도에서대조군보다의미있게 IL-8 mrna발현이증가하였다 (PM 10 10μg / ml ±105.12% of control, p=0.008, PM μg / ml ±132.46% of control, p=0.006)(figure 3A). PM μg / ml에서는대조군보다증가하는경향을보이고있다 (PM μg / ml ±125.79% of control, p=0.091). PM 10 10μg / ml보다 500μg / ml에서의미있게 IL-8 mrna발현이증가하였고 (p=0.041), PM μg / ml보다 500μg / ml에서도의미있게 mrna 발현이증가하였다 (p=0.041) (Figure 3A). 비황사기간의 PM 10 에서는 10, 500μg / ml에서 (PM 10 10μg / ml ±128.59% of control, p=0.068, PM μg / ml ±143.94% of control, p=0.068), 황사기간의 PM 10 에서는 100μg / ml, 500μg / ml에서 (PM μg / ml ±148.63% of control, p=0.068, PM μg / ml ±183.14% of control, p=0.068) 대조군보다 IL-8 mrna의발현이증가하는경향을보였다 (p<0.1)(figure 3B). 그리고비황사기간보다황사기간의 PM μg / ml에서 mrna 의발현이증가하는경향을보였다 (177.47±156.04% of control, ±148.63% of control, p=0.068)(figure 3B). Figure 4. GM-CSF mrna expression in A549 cells after 24 hours incubation with PM 10. A549 cells were exposed to 10 to 500 μg / ml of a suspension of ambient particulate matter with a diameter of less than 10 μm (PM 10) for 24 hours. Results shown in the box plot graph are the percentage from control(medium alone) values(*p<0.05, p<0.1) compared with control. The black lines in boxes show median value of IL-1a mrna expression. 4A. The expression of GM-CSF mrna measured in the supernatant collected at 24 hours increased and were significantly higher than those in the control nonexposed cells(10 to 500 μg / ml ). 4B. The mrna Levels of GM-CSF were increased after exposed to PM 10 collected during Asian dust period, compared to those in the control(100 μg / ml, 500 μg / ml ). GM-CSF mrna expression increased after 24hours of exposure to PM 10 in a dose-dependent manner. GM-CSF; granulocyte macrophage colony stimulating factor, mrna; cytokine messenger RNA 668

7 Tuberculosis and Respiratory Diseases Vol. 60. No.6, Jun PM 10 이 A549 cells 에서 GM-CSF mrna 발현에미치는영향 A549 cells을황사기간과비황사기간을포함한전체 PM 10 으로처리하고 24시간후에세포배양상층액으로 RT-PCR을시행한결과를보면 10, 100, 500μg / ml의각농도에서대조군보다의미있게 GM-CSF mrna 발현이증가하였다 (PM 10 10μg / ml ± % of control, p=0.008, PM μg / ml ± % of control, p=0.046, PM μg / ml ± % of control, p=0.008)(figure 4A). 그러나농도에따른의미있는차이는보이고있지않았다. 비황사기간의 PM 10 10μg / ml, 500μg / ml과 (PM 10 10μg / ml ±146.03% of control, p=0.066, PM μg / ml ±143.02% of control, p=0.066) 황사기간의 PM 10 10μg / ml, 100μg / ml, 500μg / ml에서 (PM 10 10μg / ml ±140.60% of control, p=0.066, PM μg / ml ±135.45% of control, p=0.066, PM μg / ml ±170.49% of control, p=0.066) 대조군보다 GM-CSF mrna발현이증가하는경향을보였다 (p<0.1). 그리고비황사기간에는 PM 10 10μg / ml보다 100μg / ml에서 GM-CSF mrna발현이증가하는경향이있었다 (PM 10 10μg / ml ±146.03% of control, PM μg / ml ±161.74% of control, p=0.066)(figure 4B). 고찰본연구에서황사기간과비황사기간을포함한전체 PM 10 은 A549 cells에서 IL-1α, IL-1β, IL-8, GM-CSF mrna의발현을증가시켰다. IL-1α 에서는 500μg / ml에서 10μg / ml보다 cytokine의발현이증가하였고 IL-8에서는 10μg / ml와 100μg / ml보다 500μg / ml에서 cytokine의발현이증가한것을확인할수있었다. 또한 IL-1α에서는 500μg / ml에서황사시기의 PM 10 이비황사시기의 PM 10 보다이러한염증성 cytokine 의발현을더많이유도하고있었다. IL-1, IL-8, GM-CSF는모두골수에서백혈구의방출을촉진하고간에서급성기반응물질 (acute phase reactant) 를생산하고혈관내피세포를자극할수있는 cytokine들이다 IL-1은호중구성및호산구성염증반응에서다른종류의 cytokine의발현및합성을증가시키는것을통해중요한역할을한다 18. IL-8은호중구의활성화와화학주성에가장중요한역할을하며 22, 알레르기질환에서도중요한역할을할수있다. GM-CSF는골수에서과립백혈구 (granulocyte) 와대식세포 (macrophage) 의회전 (turnover) 과유리 (release) 에결정적인역할을한다 23,24. 이 cytokine들은폐내국소적인염증반응을야기할수있으며, 전신순환을하게될경우전신적염증반응도유발할수있는것으로보고되고있다 이로인해기관지천식이나만성폐쇄성폐질환같은기도질환의악화와나아가심장질환의악화도유발할수있을것으로생각된다. 본연구에서황사기간과비황사기간을포함한전체 PM 10 은 IL-8의 100 μg / ml의농도를제외하고는 10, 100, 500μg / ml의농도에서 IL-1α, IL-1β, IL-8, GM-CSF mrna의발현을대조군에비하여유의하게증가시켰다. 이러한결과는기존의대기오염물질에서 PM 10 이전염증성 cytokine의발현을유발하고 PM 10 에의해 IL-1, IL-8, GM-CSF의생성이증가한다는결과와유사하다 15,28,29. 하지만 IL-8의 100μg / ml의농도에서 mrna발현이의미있게증가하지않은이유는정확히알수는없었고추가연구가필요하다고사료된다. 본연구에서는황사기간과비황사기간을포함한전체 PM 10 을 A549 cells에처리를해주었을때고농도에서는저농도에서보다 IL-1α와 IL-8에서 mrna 의발현이의미있게증가하는양상을보여주고있으나 IL-1β, GM-CSF 에서는의미있는차이는보여주고있지않았다. PM 10 에의한 cytokine의증가기전에대해서는아직명확히규명된것은없으나, PM 10 입자자체의물리적영향이나내독소 (endotoxin) 에의해전염증성 cytokine의발현이증가될수있으며 30,31 최근 PM 10 에함유된철 32, 바나듐 15, 구리 16 같은금속성분들이잠재적으로산화-환원반응에관여하면서전염증성 cytokine을증가시킬수있다고보고된바있다. 또한 Adamson 들은 PM 10 에포함된수용성금속성분이쥐의폐에염증반응을증가시켜 669

8 JH Kim et al. : PM10 induce proinflammatory cytokine in bronchial epithelial cells 독성을나타낼수있다고주장하였고 33, 박영만들도 A549 cells에서입자상물질의독성에금속, 특히철이중요하게관여하고있다고보고하였다 34. 본연구에서황사시기에채취한 PM 10 을비황사시기와비교하였을때 500μg / ml에서는같은농도의 PM 10 인데도불구하고 IL-1α 에서는황사시기의 PM 10 이비황사시기의 PM 10 보다염증성 cytokine의발현이유의하게증가되었다. 이처럼비황사시기의 PM 10 보다황사시기에같은장소에서포집된 PM 10 이고농도에서는일부의염증성 cytokine 의발현을증가시킨다. 이처럼일부의 cytokine 에서황사시기의 PM 10 이비황사시기의 PM 10 에비해이러한세포염증반응유발효과가큰것은앞서도언급했듯이 PM 10 이상황에따라구성성분과물리적특성이다양하기때문일것으로추측된다. 황사의주성분은광물성물질로규소계통의모래와황토, 알루미늄, 칼슘, 철등이대부분이지만 35 중국의급속한공업화로인위적인오염물까지포함되어있을것으로예상되고있다. 본연구가진행된 2004년도에는황사기간이총 6일이었고이기간동안 PM 10 의평균은 μg / m3로비황사기간의 59μg / m3보다 2배정도높았다. 구성성분을보면, 황사현상발현시토양기원원소인알루미늄 (Al), 칼슘 (Ca), 철 (Fe), 칼륨 (K), 망간 (Mn), 스트론튬 (Sr) 및바나듐 (Va) 의대기중농도는비황사시기보다 4.3배-9.1배정도 (PM 10 base) 의고농도를나타내었고, 이전의자료에의하면 PM 10 중의 Na +, Mg ++, Ca ++ 농도의황사 / 비황사비는각각 3.3배, 2.7배, 2.6배를나타내었다 36. 이처럼황사와비황사기간의 PM 10 의구성성분은다르고특히황사기간에는금속성분이많이포함되어있는것을알수있다. 따라서황사기간의 PM 10 에서 cytokine mrna의발현이증가하는원인으로구성성분의차이를생각해볼수있으며특히황사기간동안 PM 10 구성성분중에금속성분이많이포함되어있어이로인한영향이클것으로생각된다. 기존의보고에따르면 PM 10 에의한 cytokine증가에내독소가영향을준다는보고가있어 30,31, 본연구에서는내독소의영향을최소화하기위해포집된 PM 10 에대해고압멸균을시행하였다. 고압멸균을시 행하면내독소의영향을다소줄일수는있으나완전히배제할수는없으므로황사시기와비황사시기의 PM 10 에의한영향을비교함에있어서내독소에의한영향등에대한추가적인연구가필요할것으로생각된다. 본연구에서황사기간과비황사기간을포함한전체 PM 10 에노출된경우폐상피세포에서염증성 cytokine생성이증가되었고, 또한 IL-1α에서는고농도에서황사시의 PM 10 이비황사시보다이러한영향이더크게나타났다. 이는 PM 10 에의해폐내국소적염증반응이유발또는악화될수있으며, 전신적염증반응이일어날수있음을의미한다. 국내의특수한대기오염의한형태인황사시기에만성폐쇄성폐질환이나기관지천식등호흡기질환의증상악화가반복되며, 이러한증상악화를유발하는병태생리학적기전이 PM 10 에의해폐상피세포로부터유도분비되는전염증성 cytokine의증가와연관되어있을가능성이크며, 향후이에대한추가적인연구가필요하다. 요약연구배경 : 미세먼지는여러가지유기물과무기물의복합체로그구성성분이시간과장소에따라다르고모양과크기도일정하지않으며, 특히지름 10μm이하의미세먼지 (particulate matter 10; PM 10 ) 는흡입이가능한입자의크기여서하부기관지및폐의가스-교환부분까지침착하여호흡기계에손상을일으킬수있다. 본연구에서는황사에포함된 PM 10 과비황사시기에포집된 PM 10 이폐상피세포주에작용하여전염증성사이토카인 (proinflammatory cytokine) 및 cytokine messenger RNA(mRNA) 의발현에어떤영향이있는지를관찰하여기관지천식과만성폐쇄성폐질환등호흡기질환의증상악화기전에미치는역할을규명하고자하였다. 연구방법 : 공기포집기 (HV 500F, sibata model) 를이용하여황사와비황사기간에하루 6시간씩실외의장소에서대기분진을 membrane filter에포집한다음, PM 10 입자를추출하고폐암상피세포주인 A

9 Tuberculosis and Respiratory Diseases Vol. 60. No.6, Jun cells( 한국세포은행주 ) 에 PM 10 을농도에맞게 (10μg/ ml, 100μg / ml, 500μg / ml ) 노출시켰다. 각각의노출된세포로부터 interleukin(il)-1α, IL-1β, IL-8, granulocyte macrophage colony stimulating factor(gm- CSF) 의 mrna를역전사중합효소연쇄반응 (reverse transcriptase polymerase chain reaction; RT-PCR) 방법으로측정하였다. 결과 : 황사및비황사기간중포집된 PM10을가했을시가하지않은대조군에비하여 IL-1α, IL-1 β, IL-8, granulocyte macrophage colony stimulating factor (GM-CSF) 의 m`rna 와 cytokine 의발현이유의하게높았으며, 황사기간의고농도의 PM 10 에노출된세포의 IL-1α mrna는비황사기간의 PM 10 에노출된세포의 mrna보다증가되어있었다. 결론 : PM 10 은 A549 cells에서전염증성사이토카인의발현을증가시키고비황사기간보다황사기간중대기중에서채취한 PM 10 에노출된 A549 cells 에서일부의전염증성사이토카인의 mrna발현을더욱증가시키는것을알수있었다. 따라서황사기간의 PM 10 에의한일부의전염증성사이토카인의발현증가가만성호흡기질환의증상악화기전에연관되어있을가능성을시사하였다. 중심단어 ; 미세먼지, 황사, 전염증성사이토카인 참고문헌 1. Chun YS, Lim JY, Choi BC. The Features of Aerosol in Seoul by Asian Dust and Haze during Springtime from 1998 to Korean Meteorological Society. 2003;39(4): Donaldson K, Stone V, Clouter A, Ren wick L, MacNee W. Ultrafine particle. Occup Environ Med 2001;58(3): Logan WP. Mortality in the London fog incident Lancet 1953;I: Saric M, Fugas M, Hrustic O. Effects of urban air pollution on school-age children. Arch Envirion Health 1981;36: Ware JH, Ferris BG, Dockery DW, Spengler JD, Stram DO, Speizer FE. Effects of ambient sulfar dioxides and suspended particles on respiratory health of preadolescent children. Am Rev Respir Dis 1986;133: Euler GL, Abbey DE, Magie AR, Hodgkin JE. Chronic obstructive pulmonary disease symptom effects of longterm cumulative exposure to ambient levels of total suspended particulates and dioxide in California seventh-day adventist resident. Arch Environ Health 1987;42: Ponka A, Virtanen M. Chronic bronchitis, emphysema, and low-level air pollution in Helsinki, Environ Res 1994;65: Cohen CA, Hudson AR, Clausen JL, Knelson JH. Respiratory symptoms, spirometry and oxidant air pollution in non-smoking adults. Am Rev Respir Dis 1972;105: Pope, C. A., III, M. J. Thun, M. M. Namboodiri, D. W. Dockery, J. S. Evans, F. E. Speizer, and C. W. Heath, Jr. Particulate air pollution as a predictor of mortality in a prospective study of U.S. adults. Am J Respir Crit Care Med 1995;151: Schwartz J, Slater D, Larson TV, Pierson WE, Koenig JQ. Particulate air pollution and hospital emergency room visits for asthma in Seattle. Am Rev Respir Dis 1993;147: Dokery DW, Pope CA III. Acute respiratory effects of particulate air pollution. Ann Rev Public Health 1994;15: Choi KU, Paek DM. Asthma and Air Pollution in Korea. Korean Journal of Epidemiology 1995;17: Leem JH, Lee JT, Kim DG, Shin DC, Roh JH. Short-term Effects of Air Pollution on Hospital Visits for Respiratory Disease in Seoul. Korean Journal of Occupational and Environmental Medicine 1998;10 (3): Park JU, Lim YH, Kyung SY, An CH, Lee SP, Jeong SH, Ju YS. Effects of Ambient Particulate Matter (PM10) on Peak Expiratory Flow and Respiratory Symptoms in Subjects with Bronchial Asthma During Yellow Sand Period. Tuberculosis and Respiratory Disease 2003;55(6): Carter, J. D., A. J. Ghio, J. M. Samet, and R. B. Devlin. Cytokine production by human airway epithelial cells after exposure to an air pollution particle is metal-dependent. Toxicol Appl Pharmacol 1997;146: Kennedy, T., A. J. Ghio, W. Reed, J. Samet, J. Zagorski, J. Quay, J. Carter, L. Dailey, J. R. Hoidal, and R. B. Devlin. Copper-dependent inflammation and nuclear factor-kappab activation by particulate air pollution. Am J Respir Cell Mol Biol 1998; 19: Becker, S., J. M. Soukup, M. I. Gilmour, and R. B. Devlin. Stimulation of human and rat alveolar 671

10 JH Kim et al. : PM10 induce proinflammatory cytokine in bronchial epithelial cells macrophages by urban air particulates: effects on oxidant radical generation and cytokine production. Toxicol Appl Pharmacol 1996;141: Mills, P. R., R. J. Davies, and J. L. Devalia. Airway epithelial cells, cytokines, and pollutants. Am J Respir Crit Care Med 1999;160:S Platzer, E. Human hemopoietic growth factors. Eur J Haematol 1989;42: Gabay, C., and I. Kushner. Acute-phase proteins and other systemic responses to inflammation N Engl J Med 1999;340: Bussolino, F., M. Ziche, J. M. Wang, D. Alessi, L. Morbidelli, O. Cremona, A. Bosia, P. C. Marchisio, and A. Mantovani. In vitro and in vivo activation of endothelial cells by colony-stimulating factors. J Clin Invest 1991;87: Keatings, V. M., P. D. Collins, D. M. Scott, and P. J. Barnes Differences in interleukin-8 and tumor necrosis factor-alpha in induced sputum from patients with chronic obstructive pulmonary disease or asthma. Am J Respir Crit Care Med 153: van Pelt, L. J., M. V. Huisman, R. S. Weening, A. E. von dem Borne, D. Roos, and R. H. van Oers. A single dose of granulocyte-macrophage colony-stimulating factor induces systemic interleukin-8 release and neutrophil activation in healthy volunteers. Blood 1996;87: Lopez, A. F., D. J. Williamson, J. R. Gamble, C. G. Begley, J. M. Harlan, S. J. Klebanoff, A. Waltersdorph, G. Wong, S. C. Clark, and M. A. Vadas. Recombinant human granulocyte-macrophage colony -stimulating factor stimulates in vitro mature human neutrophil and eosinophil function, surface receptor expression, and survival. J Clin Invest 1986;78: Terashima, T, Wiggs B, English D, Hogg JC, van Eeden SF. Phagocytosis of small carbon particles by alveolar macrophages stimulates the release of PMN from the bone marrow. Am J Respir Crit Care Med 1997;155: Tan, W. C., D. Qiu, B. L. Liam, T. P. Ng, S. H. Lee, S. F. van Eeden, Y. D Yachkova, and J. C. Hogg. The human bone marrow response to acute air pollution caused by forest fires. Am J Respir Crit Care Med 2000;161: Mukae, H., J. C. Hogg, D. English, R. Vincent, and S. F. van Eeden. Phagocytosis of particulate air pollutants by human alveolar macrophages stimulates the bone marrow. Am J Physiol 2000;279:L Takeshi Fujii, Shizu Hayashi, James C. Hogg, Renaud Vincent, Stephan F. Van Eeden. Particulate Matter Induces Cytokine Expression in Human Bronchial Epithelial Cells. Am J Respir Cell Mol Biol 2001;25: Boland, S., A. Baeza-Squiban, T. Fournier, O. Houcine, M. C. Gendron, M. Chevrier, G. Jouvenot, A. Coste, M. Aubier, and F. Marano. Diesel exhaust particles are taken up by human airway epithelial cells in vitro and alter cytokine production. Am J Physiol 1999;276:L Osornio-Vargar AR, Bonner JC, Alfaro-Moreno E, Martinez L, Garcia-Cueller C, Ponce-de-Leon Rosales S, Miranda J, Rosas I. proinflammatory and cytotoxic effects of Mexico City air pollution particulate matter in vitro are dependent on particle size and composition. Environ Health Perspect. 2003;111(10): Long CM, Suh HH, Kobzik L, Catalano PJ, Ning YY, Koutrakis P. A pilot investigation of the relative toxicity of indoor and outdoor fine particles;in vitro effects of endotoxin and other particulate properties. Environ Health Perspect. 2001;109(10): van Eeden, S. F., Y. Kitagawa, Y. Sato, and J. C. Hogg. Polymorphonuclear leukocytes released from the bone marrow and acute lung injury. Chest 1999;116(1 Suppl):S Adamson, I. Y., H. Prieditis, and R. Vincent. Pulmonary toxicity of an atmospheric particulate sample is due to the soluble fraction. Toxicol Appl Pharmacol 1999;157: Baak YM, Kim JH, Kim KA, Ro CU, Kim HJ, Lim Y. Effects of Particulate Matters on A549 and RAW Cells. Korean J Prev Med 2001;34: Chung YS, Kim HS, Park KH, Jhun JG, Chen SJ. Atmospheric Loadings, Concentrations and Visibility Associated with Yellow Sand Observed in : Satellite and Meteorological Analysis. Korean Meteorological Society. 2000;36(5): Kim MY, Cho SJ, Kim KR, Lee MH. The Behaviour of Dust Concentrations During Sand Storm in Seoul Area. Jour. Korean Earth Science Society. 2003;24 (4):