Pediatr Infect Vaccine Vol.24, No.2, 2017 PEDIATRIC 4,5). 가와사키병은소아의후천심장질환을일으키는가장 중요한원인으로, 적절하게치료받지않은환아의 20% 25% 에서관상동맥병변 (coronary arterial lesion [CA

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1 PEDIATRIC ORIGINAL ARTICLE pissn eissn Pediatr Infect Vaccine 2017;24: DOI: 김주현외 5인 : 패혈증과가와사키병의관상동맥 PEDIATRIC 병변 패혈증마우스모델에서가와사키병의관상동맥병변관찰 : 예비연구와문헌고찰 김주현 1 ᆞ 김효진 1 ᆞ 신정하 2 ᆞ 최의윤 1 ᆞ 이수영 1 ᆞ 한지환 1 가톨릭대학교의과대학소아과학교실 1, 병리학교실 2 Coronary Arterial Lesions of Kawasaki Disease Observed in a Mouse Model of Sepsis: A Pilot Study and a Review of the Literature Joo-Hyun Kim 1, Hyo-Jin Kim 1, Jung-Ha Shin 2, Ui-Yoon Choi 1, Soo-Young Lee 1, Ji-Whan Han 1 Departments of 1 Pediatrics and 2 Pathology, College of Medicine, The Catholic University of Korea, Seoul, the Republic of Korea Purpose: Coronary arterial lesions (CALs) were reported to have developed in children with systemic inflammatory diseases, as well as those with Kawasaki disease (KD). The purpose of this study was to confirm that the CAL development in children with KD occurs in a mouse model of sepsis presenting typical systemic inflammatory response syndrome (SIRS). Methods: To induce the sepsis mouse model with SIRS, 6-week-old C57BL/6 mice were intraperitoneally injected with endotoxin. We compared histological findings of the major organs between the control and the sepsis groups and examined CAL in the heart of the septic mice. Results: Infiltrating inflammatory cells were relatively increased in the heart, liver, and kidneys of the sepsis group, compared with those of the control group. We confirmed lymphocytic infiltration in the myocardium (myocarditis) and the pericardial soft tissue of the heart. Furthermore, coronary artery of the septic mouse was identified, but CAL was not observed. Conclusions: In this study, we failed to confirm the existence of CAL in a mouse model of sepsis. However, it is well-known that CALs are seen in many kinds of diseases that cause SIRS. Our findings suggest further investigation into the clinical significance of CAL in various systemic inflammatory diseases, including KD. Key Words: Coronary aneurysm; Mucocutaneous lymph node syndrome; Sepsis; Systemic inflammatory response syndrome 서론 패혈증 (sepsis) 은감염원인이의심되거나증명된대표 * 이연구는가톨릭중앙의료원성의장학학술연구비지원을받음접수 : 2016 년 9 월 17 일수정 : 2016 년 10 월 19 일승인 : 2016 년 10 월 24 일 책임저자 : 이수영가톨릭대학교의과대학성바오로병원소아청소년과 Tel: 02) , Fax: 02) sylee@catholic.ac.kr 적인소아기전신염증반응증후군 (systemic inflammatory response syndrome [SIRS]) 으로적절히치료하지않으면중증패혈증, 패혈증쇼크, 다발기관부전으로악화될수있다 1,2). 내독소동물모델 (animal models of endotoxin) 을이용하여패혈증의중요한병리기전들이밝혀졌다 1,3). 가와사키병 (Kawasaki disease) 은원인미상의급성열성전신혈관염 (systemic vasculitis) 으로, 주로영유아에게발생한다 4). 5일이상발열과양측결막염, 입술과구강점막의변화, 부정형피부발진, 손발의염증성변화, 경부림프절종대의비특이적임상소견들이함께나타나는것이특징으로, 이는현재의진단기준으로사용되고있다 102

2 Pediatr Infect Vaccine Vol.24, No.2, 2017 PEDIATRIC 4,5). 가와사키병은소아의후천심장질환을일으키는가장 중요한원인으로, 적절하게치료받지않은환아의 20% 25% 에서관상동맥병변 (coronary arterial lesion [CAL]) 이발생한다 5-7). 최근가와사키병뿐만아니라, 심한전신염증소견을보 이는다른질병에서 CAL 의존재가보고되었다 8-10). 지금까 지가와사키병의동물모델에서 CAL 의발생빈도는 10% 20% 정도로알려져있지만 7), 그외다른질병의동물모델 에서 CAL 의발생을관찰한연구는없었다. 패혈증과마찬 가지로, 가와사키병은소아기 SIRS 를유발할수있는중요 한선행질병이다 11). SIRS 를유발하는선행질병들이악화 될때, 다발기관부전이라는공통적현상을보이기도한 다. 이와마찬가지로, CAL 역시가와사키병에국한된병변 이라기보다는, 선행하는원인질병의종류와무관하게그 질병의염증의정도가심해져서 SIRS 가발생할때, 나타날 수있는 비특이적염증지표 (nonspecific inflammatory markers) 중하나라고가정해보았다. 본연구에서는 SIRS 의대표적인원인질병인패혈증에 가와사키병환아에게관찰되는 CAL 이발생할수있는지 확인하기위해서, 패혈증마우스모델을활용하였고 SIRS 를유발하는다양한원인질병에서 CAL 의존재를보고한 문헌에대하여고찰하였다 (Table 1). 방법 1. 실험동물 생후 6 주 C57BL/6 마우스 (H-2 b, OrientBio Inc., Seongnam, Korea) 를이용하였다. 총 20 마리마우스를대상으로 하였으며, 이중 15 마리는실험군, 나머지 5 마리는대조군 으로하였다. 특정병원체가없는 (a specific pathogenfree) 조건하에서멸균수와 γ- 선이조사된멸균고형식 으로사육하였으며, 실험시작전 1 주일동안적응기간을 거쳐실험에이용하였다. 전체연구계획은연구시작전에 가톨릭대학교인천성모병원동물실험윤리위원회의승인 을받았다 ( 승인번호 : CIMH ). 2. 패혈증모델유도 잘알려진내독소인지질다당질 (lipopolysaccharide [LPS], Sigma-Aldrich Inc., St Louis, MO, USA) 을마우스 의복강내주사 (intraperitoneal injection) 하여패혈증모델 을유도하였다 12,13). 마우스당 100 μg (5.0 mg/kg) 의 LPS 를 0, 2, 5, 7, 9 일에총 5 회복강내주사하였고, 10 일째안 락사시켰다. 대조군마우스에는같은날짜에 LPS 대신, 동량의인산염버퍼용액을주사하였다. 3. 조직학적소견의관찰대조군과패혈증마우스를안락사후얻은주요장기들 ( 심장, 간, 콩팥 ) 은 10% 포르말린으로옮긴후에파라핀고정하였고, H&E 염색을하여광학현미경으로조직학적소견을관찰하였다. 추가로, 심장을 100 μm 간격으로수평절단한별도의슬라이드를제작하여, SIRS가발생한패혈증마우스에서가와사키병환아에게관찰되는심장병변 ( 심근염, CAL 등 ) 이발생할수있는지를확인하였다. 4. 염증정도의평가와 CAL의정의주요장기의염증정도는세포의침윤과조직의괴사정도에따라서 5단계로나누었는데, 0점은병변이없는정상소견, 1점은병변이 25% 이하, 2점은병변이 25% 50%, 3 점은병변이 50% 75%, 4점은병변이 75% 100% 에서관찰되는것으로하였다 14). CAL의발생은혈관의내층 (internal lamina) 과외층 (external lamina) 의파괴여부와무관하게혈관의전층에걸쳐염증세포가침윤된것으로정의하였고, 세포의침윤이혈관내층에국한되거나혈관주위최소한적침윤 (perivascular minimal cell infiltration) 은 CAL이아닌것으로판정하기로하였다 15). 결과 1. 주요장기의조직학적변화장기별조직소견비교에서, 패혈증마우스의심장, 간, 콩팥에대조군에서관찰할수없었던중등도 (1/4 2/4 점 ) 의염증반응을관찰하였다. 전체 15마리패혈증마우스중 2마리심장조직의심근 (myocardium) 과심장막의연조직 (pericardial soft tissue) 에증가된림프구침윤 (1/4 2/4점) 을확인하였다 (Fig. 1A, B). 간조직소견의비교에서도, 패혈증마우스 2마리에염증성변화 (2/4 점 ) 를확인하였고, 그중한마리의간조직에서는미세농양 (microabscess) 을관찰하였다 (Fig. 1C, D). 또한, 패혈증마우스 1마리의콩팥조직에서콩팥주위지방 (perirenal fat) 의림프구침윤 (1/4점) 을확인하였다 (Fig. 1E, F). 103

PEDIATRIC 김주현외 5 인 : 패혈증과가와사키병의관상동맥병변 2.

(myocarditis) 을패혈증마우스 2마리의심장에서관찰하였다 (Fig. 1B).

을관찰하였고, 그중한마리에서관상동맥과그주위에소수의염증세포침윤을확인하였으나 (Fig.

고찰 CAL은가와사키병뿐만아니라, SIRS를유발하는다른질병들에서도발생할수있다는가설하에본연구를계획하였다.

에부합하는병변을확인하지는못하였다. 내독소로유도한패혈증동물모델의결과를그대로사 A B C D E F Fig. 1.

compared between the control and the sepsis groups.

(B) Heart, sepsis: lymphocytic infiltration in the myocardium

3 PEDIATRIC 김주현외 5 인 : 패혈증과가와사키병의관상동맥병변 2. 가와사키병환아에게관찰되는심장병변 ( 심근염, CAL 등 ) 의확인급성기가와사키병에흔하게관찰되는심근염 (myocarditis) 을패혈증마우스 2마리의심장에서관찰하였다 (Fig. 1B). CAL의발생을확인하기위해서별도로제작한심장조직에서패혈증마우스중 4마리의관상동맥 (coronary artery) 을관찰하였고, 그중한마리에서관상동맥과그주위에소수의염증세포침윤을확인하였으나 (Fig. 2), CAL의정의 15) 에부합하는병변은아니었다. 심장막염 (pericarditis) 혹은판막염 (valvulitis) 과같은기능적평가가뒷받침되어야하는심장병변은본연구에 서확인할수없었다. 고찰 CAL은가와사키병뿐만아니라, SIRS를유발하는다른질병들에서도발생할수있다는가설하에본연구를계획하였다. 패혈증마우스에서가와사키병환아에게가장흔하게발생하는심장합병증인심근염은관찰하였지만, CAL 의정의 15) 에부합하는병변을확인하지는못하였다. 내독소로유도한패혈증동물모델의결과를그대로사 A B C D E F Fig. 1. Histologic findings (H&E stain) of the major organs are compared between the control and the sepsis groups. (A) Heart, control: unremarkable ( 100). (B) Heart, sepsis: lymphocytic infiltration in the myocardium and the pericardial soft tissue ( 40; inset, 200). (C) Liver, control: unremarkable ( 100). (D) Liver, sepsis: a few microabscesses ( 100; inset, 200). (E) Kidney, control: unremarkable ( 100). (F) Kidney, sepsis: lymphocytic infiltration in the perirenal soft tissue ( 100). 104

4 Pediatr Infect Vaccine Vol.24, No.2, 2017 PEDIATRIC Atrium CA Fig. 2. Histologic findings (H&E stain) of the heart are shown ( 200). There is lymphocytes aggregation near coronary artery (CA) of the septic mouse. Table 1. Reported Cases of Coronary Artery Lesion and Their Pre-existing Diseases Infectious diseases 8,17-23) Epstein-Barr virus infection Cytomegalovirus infection Rabies infection Escherichia coli sepsis Staphylococcal toxic shock syndrome Streptococcal toxic shock syndrome Rheumatic fever Viral myocarditis Rheumatic diseases 9,10,24-29) Systemic onset juvenile idiopathic arthritis Systemic lupus erythematosus Takayasu arteritis Behcet disease Crohn disease Ulcerative colitis Familial Mediterranean fever Sarcoidosis 람에게적용할수는없지만, 이모델은지금까지알려진 패혈증의병리기전연구에유용하게활용되었다. 내독소 동물모델은대장균과같은그람음성세균을직접주입하거 나정제된내독소를주사하여패혈증을유도하는방법이 다 1,3). 본연구에서는실험방법이비교적간단한내독소 복강내주사방법을이용하였다. 내독소에자극받은숙주 의면역체계는여러종류의면역매개물질들을분비한다. 종양괴사인자 (tumor necrosis factor) 는핵심매개물질로 써, 내피세포와조직을손상해이차적으로다른매개물질들을생성시키고, 결국에는연속되는 (cascade) 전신염증반응을개시하는역할을한다 2,3). 가와사키병의심혈관증상은장기적인이환율및사망률에중요한원인이된다 4). 해부학적으로심장의여러부위, 즉심낭, 심근, 판막, 그리고관상동맥등에가역적혹은비가역적병변을유발할수있다. 가와사키병에서관찰되는 CAL을포함한심장병변들은유발인자 (triggering factor) 라고여겨지는미생물, 이들과반응하는면역세포, 그리고손상된숙주의조직에서부수적으로생성된물질 (substances) 에의해형성될것이라고추정된다 16). CAL은가와사키병의중요한특징이라고여겨져서, 고전적진단기준을충족하지못하는불완전가와사키병진단에유용하게활용되고있다 4-6). 임상적으로다양한감염질환에서 CAL의동반을확인함으로써, 최종적으로불완전또는비정형 (incomplete or atypical) 가와사키병을진단하게된흥미로운증례들이보고되었다 8,17-23). 하지만, CAL은가와사키병진단기준에포함되지않으며환아의약 1/4 정도에나타나는낮은민감도 (sensitivity) 의진단보조적제한된역할을한다는관점에서, 이들증례의최종진단은 특정감염병으로오인되었던가와사키병 보다는, 심장초음파에서 CAL이확인된, 심한전신염증소견을보였던특정감염병 일수있다는의문이들었다. 실제로 CAL은가와사키병에국한하여발생하는것이아니다. 가와사키병외다른전신염증질병 (systemic inflammatory disease) 에서 CAL이발생한다는것이이미많은문헌에보고되었다 9,10,24-29). 즉, CAL은 SIRS 를유발할수있는다양한감염혹은비감염원인의질병에서발생하는것이다 (Table 1). CAL의발생률은선행하는원인질병에따라서차이를보이기도하였다. 예를들어, 바이러스심근염 (viral myocarditis) 환아의 64%, 그리고전신형소아류마티스관절염 (systemic-onset juvenile idiopathic arthritis) 환아의 41% 에서 CAL이발생하였다고보고되었는데 8,9), 이는가와사키병환아의발생률 (20% 25%) 보다휠씬높은것이다. 이러한발생빈도의차이는 CAL이선행질병의고유한특징이라기보다는, 그질병에동반된염증의중증도 (the severity of the inflammation) 의차이에서기인하는것으로추정해보았다. 본연구결과에서 CAL을확인하지못한근본적인이유는소규모예비연구 (pilot study) 로써의한계점과저자들의동물실험에대한경험의부족때문이라고생각한다. 또한, 연구방법에서간과하지말아야하는중요한제한점 105

5 PEDIATRIC 김주현외 5 인 : 패혈증과가와사키병의관상동맥병변 은 CAL의발생에영향을주는숙주인자 (host factor) 를반영하지못한것이다. CAL 발생의감수성 (susceptibility) 또는 CAL을호발시키는유전학적요인을갖는, 즉 CAL 발생에대한낮은역치 (threshold) 를갖는숙주에게 SIRS가발생할때 CAL은좀더쉽게, 심하게, 빈번하게발생하는것으로여겨진다 30). 여러종류의전신염증질병에서 CAL이동반한다는것은가와사키병환아에게시행하는심장초음파검사의중요성을과소평가하는것이아니라, 오히려가와사키병을포함한다양한전신염증질병환아들에게심장평가가필요하다는것을강조하는것이다 8-10). CAL은가와사키병의가장중요한합병증이며진단에도유용한실마리를제공하지만, 가와사키병진단의필수조건이나충분조건은아니다. 향후가와사키병을포함한다양한전신염증질병에나타나는 CAL의임상적의미와발생기전을밝히기위한추가적인연구가필요할것이다. References 1. Kaplan SL, Vallejo JG. Bacteremia and septic shock. In: Cherry JD, Demmler-Harrison GJ, Kaplan SL, Steinbach WJ, Hotez P, editors. Feigin and Cherry s textbook of pediatric infectious diseases. 7th ed. Philadelphia: Saunders/Elsevier, 2014; Lee SY, Lee KH, Hwang HS, Jeong DC, Chung SY, Kang JH. Septic encephalopathy complicating acute appendicitis. Pediatr Crit Care Med 2009;10:e Zanotti-Cavazzoni SL, Goldfarb RD. Animal models of sepsis. Crit Care Clin 2009;25: Newburger JW, Takahashi M, Gerber MA, Gewitz MH, Tani LY, Burns JC, et al. Diagnosis, treatment, and long-term management of Kawasaki disease: a statement for health professionals from the Committee on Rheumatic Fever, Endocarditis and Kawasaki Disease, Council on Cardiovascular Disease in the Young, American Heart Association. Circulation 2004;110: Yun HW, Lee JY, Yang SI, Yu HJ, Kang MJ, Lee SY, et al. Comparison of cervical-lymph-node-first presentation of Kawasaki disease and typical Kawasaki disease. Pediatr Infect Vaccine 2016;23: Park HJ, Cho YJ, Bae EY, Choi UY, Lee SY, Jeong DC, et al. Macrophage activation syndrome as the extreme form of Kawasaki disease. Korean J Pediatr Infect Dis 2010;17: Lee SY, Han JW. Animal model of Kawasaki disease. Pediatr Clin Immunol 2012;4: Rached-D'Astous S, Boukas I, Fournier A, Raboisson MJ, Dahdah N. Coronary artery dilatation in viral myocarditis mimics coronary artery findings in Kawasaki disease. Pediatr Cardiol 2016;37: Binstadt BA, Levine JC, Nigrovic PA, Gauvreau K, Dedeoglu F, Fuhlbrigge RC, et al. Coronary artery dilation among patients presenting with systemic-onset juvenile idiopathic arthritis. Pediatrics 2005;116:e Agarwal A, Biglarian S, Lim-Stavros S, Votava-Smith JK, Ramanathan A. Pediatric systemic lupus erythe matosus presenting with coronary arteritis: a case series and review of the literature. Semin Arthritis Rheum 2015;45: Gatterre P, Oualha M, Dupic L, Iserin F, Bodemer C, Lesage F, et al. Kawasaki disease: an unexpected etiology of shock and multiple organ dysfunction syndrome. Intensive Care Med 2012;38: Vodovotz Y, Chow CC, Bartels J, Lagoa C, Prince JM, Levy RM, et al. In silico models of acute inflammation in animals. Shock 2006;26: von Drygalski A, Furlan-Freguia C, Ruf W, Griffin JH, Mosnier LO. Organ-specific protection against lipopolysaccharide-induced vascular leak is dependent on the endothelial protein C receptor. Arterioscler Thromb Vasc Biol 2013;33: Wang D, Chen Y, Jiang J, Zhou A, Pan L, Chen Q, et al. Carvedilol has stronger anti-inflammation and anti-virus effects than metoprolol in murine model with coxsackievirus B3- induced viral myocarditis. Gene 2014;547: Takahashi K, Oharaseki T, Wakayama M, Yokouchi Y, Naoe S, Murata H. Histopathological features of murine systemic vasculitis caused by Candida albicans extract: an animal model of Kawasaki disease. Inflamm Res 2004;53: Moon HS, Huh JS, Kim MK, Lambert MM. Kawasaki disease with influenza A virus and Mycoplasma pneumoniae infections: a case report and review of literature. Pediatr Infect Vaccine 2016;23: Kato S, Yoshimura K, Tanabe Y, Kimata T, Noda Y, Kawasaki H, et al. A child with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis complicated by coronary 106

6 Pediatr Infect Vaccine Vol.24, No.2, 2017 PEDIATRIC artery lesion mimicking Kawasaki disease. J Pediatr Hematol Oncol 2013;35:e Usta Guc B, Cengiz N, Yildirim SV, Uslu Y. Cytomegalovirus infection in a patient with atypical Kawasaki disease. Rheumatol Int 2008;28: Boukas I, Dahdah N, Robitaille Y, Fournier A. Coronary artery dilatation and vasculitis in a case of rabies: similarity with Kawasaki disease? Pediatr Int 2013;55: Hasan A, McDonough KH. The effects of Escherichia coli sepsis and short-term ischemia on coronary vascular reactivity and myocardial function. Shock 1997;8: Wiesenthal AM, Todd JK. Toxic shock syndrome in children aged 10 years or less. Pediatrics 1984;74: Yim D, Ramsay J, Kothari D, Burgner D. Coronary artery dilatation in toxic shock-like syndrome: the Kawasaki disease shock syndrome. Pediatr Cardiol 2010;31: Gunal N, Baysal K, Haciomeroglu P, Belet N, Kolbakir F. Rheumatic heart disease and coronary vasculitis in children. Acta Paediatr 2006;95: Ouali S, Kacem S, Ben Fradj F, Gribaa R, Naffeti E, Remedi F, et al. Takayasu arteritis with coronary aneurysms causing acute myocardial infarction in a young man. Tex Heart Inst J 2011;38: Gurkan U, Kaya A, Tatlisu MA, Avsar S. A case report of coronary artery aneurysm in a patient with Behcet's disease. Turk Kardiyol Dern Ars 2014;42: Christou A, Patsilinakos S, Chinofoti I, Marinakos A, Nikolaou N, Spanodimos S. Acute myocardial infarction in a patient with coronary artery aneurysm and Crohn's disease. Hellenic J Cardiol 2012;53: Perez-Colon E, Dadlani GH, Wilmot I, Miller M. Mesalamineinduced myocarditis and coronary vasculitis in a pediatric ulcerative colitis patient: a case report. Case Rep Pediatr 2011;2011: Serrano R, Martinez MA, Andres A, Morales JM, Samartin R. Familial mediterranean fever and acute myocardial infarction secondary to coronary vasculitis. Histopathology 1998; 33: Ward EV, Nazari J, Edelman RR. Coronary artery vasculitis as a presentation of cardiac sarcoidosis. Circulation 2012; 125:e Kuo HC, Chang JC, Guo MM, Hsieh KS, Yeter D, Li SC, et al. Gene-gene associations with the susceptibility of Kawasaki disease and coronary artery lesions. PLoS One 2015;10: e 요약 목적 : 가와사키병의환아뿐만아니라, 다른전신염증질병의환아들에관상동맥병변 (coronary arterial lesion [CAL]) 의발생이보고되었다. 본연구에서는전형적인전신염증반응증후군 (systemic inflammatory response syndrome [SIRS]) 의소견을보이는패혈증마우스모델에서가와사키병환아에게관찰되는 CAL이발생하는지확인하고자하였다. 방법 : 생후 6주 C57BL/6 마우스에내독소를복강내주사하여 SIRS를보이는패혈증모델을유도하였다. 대조군과패혈증군의주요장기의조직학적소견을비교하였고패혈증마우스에서 CAL을찾기위한시도를하였다. 결과 : 대조군과비교하여, 염증세포의침윤이패혈증마우스의심장, 간, 신장에상대적으로증가하였다. 패혈증마우스의심장에서심근 ( 심근염 ) 과심장주위연조직에림프구침윤을확인하였다. 또한, 패혈증마우스의관상동맥을관찰하였지만, CAL을확인할수는없었다. 결론 : 본연구에서패혈증마우스모델에서 CAL의존재를확인하는것은실패하였다. 하지만, SIRS를유발하는많은종류의원인질병에서 CAL이발생한다는것은잘알려져있다. 가와사키병을포함한다양한전신염증질병에나타나는 CAL의임상적의미에대한연구가필요할것이다. 107

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( )Kju269.hwp 만성세균성전립선염모델흰쥐에서 의항염효과 Anti-inflammatory Effect of Lycopene on Chronic Bacterial Prostatitis Rat Model Cho Hwan Yang, Dong Wan Sohn, Yong-Hyun Cho From the Department of Urology, The Catholic University