CD30 활성화에 의한 호산구 자멸사에서 caspase-9의 매개작용
CD30 Activation Induced Eosinophil Apoptosis is Mediated by Caspase-9
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목적:CD30은 자멸사가 진행중인 호산구에서 발현이 더 증가되는 것으로 알려져 있으나 실제 CD30 활성화가 어떻게 호산구 자멸사를 일으키는지는 명확하지 않다. 본 연구에서는 인체세포 자멸사의 중요한 요소인 caspase가 CD30 활성화에 의한 호산구 자멸사에 미치는 역할을 규명하고자 한다. 방 법:건강한 성인의 말초혈액 90 mL를 채취하여 혈액을 Percoll 밀도차와 MACS magnetic system으로 호산구를 분리한 후 호산구 표면 CD30 발현율과 자멸사율을 flow cytometry 로 측정하였다. 호산구 배양은 CD30 단클론항체와 동형의 immunoglobulin G1을 대조항체로 하여 24 well plates에 미리 코팅하고 고정한 후 호산구를 분주하고 interleukin (IL)-5, Dexamethasone을 첨가하여 4시간, 24시간 배양한 후 자멸사 정도를 각각 비교하였다. 또한 CD30에 의한 호산구 자멸사에서 caspase의 역할을 규명하기 위해 caspase-9 억제제를 첨가한 후 배양하여 자멸사율 변화를 측정하고 Western blotting을 이용하여 procaspase-9 단백을 검출하였다. 결 과:호산구 표면 CD30 발현은 배양시간이 연장됨에 따라 점점 증가하였다. 호산구 자멸사율은 항CD30 단클론항체에서 4시간, 24시간 배양 시 29.1±6.1%와 47.3±4.7 %으로 대조항체의 17.1±6.7%와 29.4±9.2%에 비해 유의하게 증가하였다(P<0.05). 항CD30 항체투여군의 자멸사율은 스테로이드 투여군에 비해 빠르게 중가하였으며 IL-5투여군의 호산구 생존능을 현저히 감소시켰다. 또한 항CD30 항체투여군에 caspase-9 억제제를 첨가하면 18시간과 36시간 배양 후 자멸사율이 첨가전의 54.8±16.9%와 71.5±11.6%에서 24.5±6.0%와 47.8±11.4%로 각각 유의하게 감소하였으며(P<0.05) 항CD30 항체투여군의 procaspase-9 단백의 발현이 IL-5 투여군과 대조항체군에 비해 저하됨을 Western blot을 통해 관찰하였다. 결 론:본 연구에서 CD30 활성화는 호산구 자멸사를 촉진하며 caspase-9 활성화가 자멸사 과정에서 중요한 역할을 할 것으로 보인다.
Abstract
Purpose:Although CD30 is known to be expressed more on eosinophils undergoing apoptosis, it is still not known how CD30 activation leads to eosinophil apoptosis. In this study, we evaluated whether ligation of CD30 incites apoptosis and investigated whether the mechanisms of CD30 induced eosinophil apoptosis are dependent on caspase activation. Methods:We drew 90 mL of peripheral blood from healthy donors and then purified eosinophils using a MACS system. Expression of CD30 on eosinophils was measured, and eosinophils were cultured in wells pretreated with anti-CD30 mAb, isotype control immunoglobulin G1, interleukin (IL)-5, and dexamethasone in Roswell Park Memorial Institute 1640 media supplemented with 10% fetal bovine serum. Their rates of apoptosis were then compared using flow cytometry. To evaluate whether caspase-9 is involved in CD30-induced eosinophil apoptosis, the apoptotic rate was evaluated after the addition of caspase-9 inhibitor. The expression of procaspase-9 was also measured using Western blot. Results:Expression of CD30 molecules on eosinophils increased steadily as the culture time lapse. The apoptotic rates of eosinophils cultured in the presence of anti-CD30 mAb were significantly increased to 29.1±6.1% and 47.3±4.7% compared to 17.1±6.7% and 29.4±9.2% of the control at 4 and 24 hours, respectively (both P<0.05). The apoptotic rates of eosinophils treated with anti-CD30 mAb were even faster than those of eosinophils treated with dexamethasone, and the mAb also suppressed the IL-5-induced enhancing effect of eosinophil survival. Caspase-9 inhibitor suppressed mAb induced eosinophil apoptosis from 54.8±6.9% and 71.5±11.6% to 24.5± 6.0% and 47.8±11.4% at 18 and 36 hours, respectively (both P<0.001). We also demonstrated that the expression of procaspase-9 with mAb was diminished compared to that of the control and of IL-5. Conclusion:This study showed CD30 activation enhances eosinophil apoptosis, and the effect is mediated by caspase-9 activation.
참고문헌 (31)
- Gleich GJ, "The eosinophil and bronchial asthma: current understanding", J Allergy Clin Immunol, 85, pp.422~436 (1990)
- Gleich GJ, "Mechanisms of eosinophil-associated inflammation", J Allergy Clin Immunol, 105, pp.651~663 (2000)
- Sanderson CJ, "Interleukin-5: an eosinophil growth and activation factor", Dev Biol Stand, 69, pp.23~29 (1988)
- Kerr JF, "Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics", Br J Cancer, 26, pp.239~257 (1972)
- Allen RT, "Morphological and biochemical characterization and analysis of apoptosis", J Pharmacol Toxicol Methods, 37, pp.215~228 (1997)
- Alnemri ES, "Human ICE", CED-3 protease nomenclature, 1996, pp.87, 171~
- Matsumoto K, "Induction of apoptosis in human eosinophils by anti-Fas antibody treatment in vitro", Blood, 86, pp.1437~1443 (1995)
- Walsh GM, "Ligation of CD69 induces apoptosis and cell death in human eosinophils cultured with granulocyte-macrophage colony-stimulating factor", Blood, 87, pp.2815~2821 (1996)
- Blaylock MG, "Ligation of CD45 and the isoforms CD45RA and CD45RB accelerates the rate of constitutive apoptosis in human eosinophils", J Allergy Clin Immunol, 104, pp.1244~1250 (1999)
- Dürkop H, "Molecular cloning and expression of a new member of the nerve growth factor receptor family that is characteristic for Hodgkin's disease", Cell, 68, pp.421~427 (1992)
- Stein H, "The expression of the Hodgkin's disease associated antigen Ki- 1 in reactive and neoplastic lymphoid tissue: evidence that Reed-Sternberg cells and histiocytic malignancies are derived from activated lymphoid cells", Blood, 66, pp.848~858 (1985)
- Jung W, "Opposite effects of the CD30 ligand are not due to CD30 mutations: results from cDNA cloning and sequence comparison of the CD30 antigen from different sources", Mol Immunol, 31, pp.1329~1334 (1994)
- Masuda M, "Dual action of CD30 antigen: anti-CD30 antibody induced apoptosis and interleukin-8 secretion in Ki-1 lymphoma cells", Int J Hematol, 67, pp.257~265 (1998)
- Mir SS, "Differential effects of CD30 activation in anaplastic large cell lymphoma and Hodgkin disease cells", Blood, 96, pp.4307~4312 (2000)
- Gruss HJ, "Pleiotropic effects of the CD30 ligand on CD30-expressing cells and lymphoma cell lines", Blood, 83, pp.2045~2056 (1994)
- Muta H, "CD30 signals integrate expression of cytotoxic effector molecules, lymphocyte trafficking signals, and signals for proliferation and apoptosis", J Immunol, 165, pp.5105~5111 (2000)
- Pinto A, "Human eosinophils express functional CD30 ligand and stimulate proliferation of a Hodgkin's disease cell line", Blood, 88, pp.3299~3305 (1996)
- Chiarle R, "CD30 in normal and neoplastic cells", Clin Immunol, 90, pp.157~164 (1999)
- Berro AI, "Increased expression and activation of CD30 induce apoptosis in human blood eosinophils", J Immunol, 173, pp.2174~2183 (2004)
- Matsumoto K, "Extremely rapid and intense induction of apoptosis in human eosinophils by anti-CD30 antibody treatment in vitro", J Immunol, 172, pp.2186~2193 (2004)
- Hansel TT, "An improved immunomagnetic procedure for the isolation of highly purified human blood eosinophils", J Immunol Methods, 145, pp.105~110 (1991)
- Simon HU, "Direct demonstration of delayed eosinophil apoptosis as a mechanism causing tissue eosinophilia", J Immunol, 158, pp.3902~3908 (1997)
- Leckie MJ, "Effects of an interleukin-5 blocking monoclonal antibody on eosinophils, airway hyper-responsiveness, and the late asthmatic response", Lancet, 356, pp.2144~2148 (2000)
- Flood-Page PT, "Eosinophil's role remains uncertain as anti-interleukin-5 only partially depletes numbers in asthmatic airway", Am J Respir Crit Care Med, 167, pp.199~204 (2003)
- Matsumoto K, "Rapid and strong induction of apoptosis in human eosinophils by anti-CD30 mAb-coated microspheres and phagocytosis by macrophages", Arch Allergy Immunol, 143(Suppl 1), pp.60~67 (2007)
- Gedrich RW, "CD30 contains two binding sites with different specificities for members of the tumor necrosis factor receptor-associated factor family of signal transducing proteins", J Biol Chem, 271, pp.12852~12858 (1996)
- Horie R, "A novel domain in the CD30 cytoplasmic tail mediates NFkappaB activation", Int Immunol, 10, pp.203~210 (1998)
- Dempsey PW, "The signaling adaptors and pathways activated by TNF superfamily", Cytokine Growth Factor Rev, 14, pp.193~209 (2003)
- Muzio M, "An induced proximity model for caspase-8 activation", J Biol Chem, 273, pp.2926~2930 (1998)
- Zou H, "Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c- dependent activation of caspase-3", Cell, 90, pp.405~413 (1997)
- Matsumoto K, "Analysis of signal transduction pathways involved in anti-CD30 mAb-induced human eosinophil apoptosis", Int Arch Allergy Immunol, 152(Suppl 1), pp.2~8 (2010)