DOI QR코드

DOI QR Code

수지상세포에 있어서 베로독소 수용체의 발현조절

Regulation of Gb3 Expression on Dendritic Cells

  • 임석환 (전남대학교 의과대학 소아과학교실) ;
  • 김기영 (부산대학교 의과대학 미생물학 및 면역학 교실) ;
  • 김형천 (부산대학교 의과대학 미생물학 및 면역학 교실) ;
  • 김영희 (부산대학교 의과대학 미생물학 및 면역학 교실) ;
  • 손용해 (부산대학교 의과대학 미생물학 및 면역학 교실) ;
  • 오양효 (부산대학교 의과대학 미생물학 및 면역학 교실) ;
  • 박영민 (부산대학교 의과대학 미생물학 및 면역학 교실)
  • Lim, Suk-Hwan (Department of Pediatrics, Chonnam National University Medical School) ;
  • Kim, Gi-Young (Department of Microbiology and Immunology, Pusan National University College of Medicine) ;
  • Kim, Hyung-Chun (Department of Microbiology and Immunology, Pusan National University College of Medicine) ;
  • Kim, Young-Hee (Department of Microbiology and Immunology, Pusan National University College of Medicine) ;
  • Son, Yong-Hae (Department of Microbiology and Immunology, Pusan National University College of Medicine) ;
  • Oh, Yang-Hyo (Department of Microbiology and Immunology, Pusan National University College of Medicine) ;
  • Park, Yeong-Min (Department of Microbiology and Immunology, Pusan National University College of Medicine)
  • 발행 : 2007.04.25

초록

Shiga-like toxin(SLT)을 생산하는 Esherichia coli에 의한 감염은 설사, 출혈성 대장염(hemorrhagic colitis) 및 용혈성 요독 증후군(hemolytic uremic syndrome)을 특징으로 하며, 특히 5세 이하의 소아에게서 심각한 결과를 초래한다. SLT-I의 병인으로는 다양한 숙주 매개 인자들이 알려져 있다. 본 연구에서는 E. coli 0157:H7(ATCC 43890)로 부터 정제한 SLT-I이 포유동물 세포들에 대한 세포독성과 종양괴사인자(tumor necrosis $factor-{\alpha},\;TNF-{\alpha}$)의 생산에 미치는 효과를 측정하였으며, SLT-I의 수용체인 glycolipid globotriaosylceramide(Gb3)의 발현과 SLT-I의 세포독성의 관계를 규명하고자 하였다. SLT-I 과 SLT-I B를 순수분리 정제하고 SLT-I B-FLTC 접합체를 제조하여 vero 세포, 대식세포 및 수지상세포를 대상으로 세포독성능을 측정하고 세포독성능의 차이가 SLT-I의 수용체인 Gb3의 발현과 상관관계가 있는지를 Flow cyotmetry로 분석하였다. 또한 대식세포의 종양괴사인자 생산능은 ELISA법으로 시행하였다. SLT-I은 대식세포(Raw264.7)로부터 $TNF-{\alpha}$의 생산을 증가시켰다. 연구 대상 세포 중 SLT-I에 감수성을 나타낸 Vero 세포와 수지상세포(dendritic cells)는 Gb3 발현이 각각 83%와 68%로 높았으며, 29%의 낮은 Gb3 발현을 보인 Raw264.7 세포는 감수성을 보이지 않았다. 따라서 위의 결과로부터 SLT-I에 감수성을 보이지 않은 Raw264.7세포를 대상으로 Gb3 발현 정도와 SLT-I의 세포독성의 관계를 규명하고자 Gb3의 발현을 증가시킨 후 SLT-I의 세포독성을 재차 평가하였다. 이 결과 $TNF-{\alpha}$의 처리에 의하여 6 hrs에 Gb3의 발현이 정점(43.5%)에 이르렀으며 36 hrs에 정상 수준(25.0%)으로 환원되었다. 그러나, Gb3의 발현이 증가함에도 불구하고 SLT-I의 세포독성에는 변화가 관찰되지 않았다. 따라서, SLT-I에 의한 세포독성은 세포의 종류에 따라서 다르며 또한, Gb3의 발현정도에만 의존적이지는 않을 것으로 생각된다. 이와 같은 결과는 E. coli 0157의 감염증 병인 연구에 있어 SLT-I과 Gb3의 발현의 상관관계에 대한 보다 심도 있는 연구가 필요함을 시사한다.

Infection with Shiga-like toxin (SLT)-producing Escherichia coli causes a spectrum of illnesses with high morbidity and mortality. Host mediators play an important role in the pathogenesis of SLT-I toxicity. We here investigated the effect of SLT-I on tumor necrosis $factor-{\alpha}\;(TNF-{\alpha})$ production, effect of $TNF-{\alpha}$ on glycolipid globotriaosyleramide (Gb3) expression, and relationship between Gb3 level and differential susceptibility of cells to SLT-I. In this study, we observed that detectable levels of $TNF-{\alpha}$ are produced 6 hrs after induction and continued to increase during 48 hrs by SLT-I. It was also found that Vero cells and dendritic cells expressed high levels of Gb3, 83% and 68%, respectively, and that macrophages had a low level of Gb3 (29%) and showed refractory to cytotoxicity against SLT-I. Vero cells and dendritic cells expressing high levels of Gb3 were highly susceptible to SLT-I. furthermore, macrophages showed a resistance to SLT-I cytotoxicity, despite the fact that Gb3 expression was enhanced. These results suggest that the expression of Gb3 is necessary, but not sufficient to confer sensitivity of macrophages to SLT-I and further underpin the important role of SLT-I and its receptor, Gb3, in the pathogenesis of E. coli O157 infection.

키워드

참고문헌

  1. Barrett, T. J., M. E. Potter and N. A Strockbine. 1990. Evidence for participation of the macrophage in Shiga-like toxin II-induced lethality in mice. Microb. Pathog. 9, 95-103 https://doi.org/10.1016/0882-4010(90)90083-3
  2. Bergstein, J. M., M. Riley and N. U. Bang. 1992. Role of plasminogen-activator inhibitor type 1 in the pathogenesis and outcome of the hemolytic uremic syndrome. N. Engl. J. Med. 327, 755-759 https://doi.org/10.1056/NEJM199209103271102
  3. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254 https://doi.org/10.1016/0003-2697(76)90527-3
  4. Cohen A., G. E. Hannigan, B. R. G. Williams and C. A. Lingwood. 1987. Roles of globotriosyl- and galabiosylceramide in Verotoxin binding and high affinity interferon receptor. J. BioI. Chem. 262, 17088-17095
  5. Fong J. S., J. P. de Chadarevian and B. S. Kaplan. Hemolytic-uremic syndrome. 1982. Current concepts and management. Pediatr. Clin. North. Am. 29, 835-856 https://doi.org/10.1016/S0031-3955(16)34216-X
  6. Furutani M, K. Kashiwagi, K. Ito, Y. Endo and K. Igarashi. 1992. Comparison of the modes of action of a Verotoxin (a Shiga-like toxin) from Escherichia coli, of ricin, and of alpha-sarcin. Arch. Biochem. Biophys. 293, 140-146 https://doi.org/10.1016/0003-9861(92)90376-8
  7. Hansen M. B., S. E. Nielsen and K. Berg. 1989. Reexamination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J. Immunol. Methods. 119, 203-210 https://doi.org/10.1016/0022-1759(89)90397-9
  8. Imberechts H., H. De Greve and P. Lintermans. 1992. The pathogenesis of edema disease in pigs. Vet. Microbiol. 221-233
  9. Inward C. D., J. Willias, I. Chant, J. Crocker, D. V. Milford and P. E. Rose. 1995. Verotoxin-1 induces apoptosis in Vero cells. J. Infect. 30, 213-218 https://doi.org/10.1016/S0163-4453(95)90693-2
  10. Jacewicz M. S., M. Mobassaleh, S. K. Gross, K. A. Balasubramanian, P. F. Daniel and S. Raghavan. 1994. Pathogenesis of Shigella diarrhea:XVII. A mammalian cell membrane glycolipid, Gb3, is required but not sufficient to confer sensitivity to Shiga toxin. J. Infect. Dis. 169, 538-546 https://doi.org/10.1093/infdis/169.3.538
  11. Jackson M. P. 1990. Structure-function analyses of Shiga toxin and Shiga-like toxins. Microb. Pathog. 8, 235-242 https://doi.org/10.1016/0882-4010(90)90050-Z
  12. Johnton R. B. J., 1987. Current concepts. Immunology. Monocytes and macrophages. N. Engl. J. Med. 318, 747-752
  13. Karmali M. A., M. Petric, C. Lim, R. Cheung and G. S. Arbus. 1985. Sensitive method for detecting low numbers of Vero toxin-producing Escherichia coli in mixed cultures by use of colony sweeps and polymyxin extraction of Verotoxin. J. Clin. Microbiol. 22, 614-619
  14. Kiarash A., B. Boyd and C. A. Lingwood. 1994. Glycosphingolipid receptor function is modified by fatty acid content. J. Biol. Chem. 209, 11138-11146
  15. Klimetzek V. and H. G. Remold. 1980. The murine bone marrow macrophage, a sensitive indicator cells for murine migration inhibitory factor and a new method for their harvest. Cell. Immunol. 53, 257-266 https://doi.org/10.1016/0008-8749(80)90327-5
  16. Konowalchuk J., J. I. Speirs and S. Stavric. 1977. Vero response to a cytotoxin of Escherichia coli. Infect. Immun. 18, 775-779
  17. Laemmli U. K. 1976. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. J. Biol. Chem. 227, 880-885
  18. Lingwood C. A., H. Law, S. Richardson, M. Petri, J. L. Brunton and S. D. Grandis. 1987. Glycolipid binding of purified and recombinant Escherichia coli produced Verotoxin in vitro. J. Biol. Chem. 262, 8834-8843
  19. Lingwood C. A. and S. C. K. Yiu. 1992. Glycolipid modification of interferon binding: Sequence similarity between interferon receptor and the Verotoxin (Shiga-like toxin) B-subunit. Biochem. J. 282, 25-32 https://doi.org/10.1042/bj2820025
  20. Lingwood C. A. 1991. Glycolipids as receptors. Adv. Lipid. Res. 1, 39-45
  21. Lingwood C. A. 1993. Verotoxins and their glycolipid receptors. Adv Lipid Res. 25, 189-196
  22. Louise C. B. and T. G. Obrig. 1991. Shiga toxin-associated hemolytic-uremic syndrome: combined cytotoxic effects of Shiga toxin, interleukin-1 beta, and tumor necrosis factor alpha on human vascular endothelial cells in vitro. Infect. Immun. 59, 4173-4179
  23. Mahan J. D., C. McAllister and M. Karmali. 1996. Verotoxin-1 induction of apoptosis in human glomerular capillary endothelial cells (GCEC) in vitro is dependent on cytokines, cell confluence, and cell cycle. J. Am. Soc. Nephrol. 7, 1661
  24. Mulvey G., R. Vanmaele, M. Marzek, M. Cahill and G. D. Armstrong. 1998. Affinity purification of Shiga-like toxin I and Shiga-like toxin II. J. Micobiol. Meth. 32, 247-252 https://doi.org/10.1016/S0167-7012(98)00028-1
  25. Nacilla H., B. Emmanuelle, B. Sophie, A. Olivier, W. Guy and L. Valie. 2002. The B subunit of shiga toxin fused to a tumor antigen elicits CTL and targets dendritic cells to allow MHC class I-restricted presentation of peptides derived from exogenous antigens. J. Immunol. 165, 3301-3308
  26. O'Brien A. D. and R. K. Holmes. 1987. Shiga and shiga-like toxins. Microbiol. Rev. 51, 206-220
  27. O'Brien A. D., G. D. LaVeck, M. R. Thompson and S. B. Formal. 1982. Production of Shigella dysenteriae type 1-like cytotoxin by Escherichia coli. J. Infect. Dis. 146, 763-769 https://doi.org/10.1093/infdis/146.6.763
  28. O'Brien A. D., V. L. Tesh, A. Donohue-Rolfe, M. P. Jackson, S. Olsnes and K. Sandvig. 1992. Shiga toxin: biochemistry, genetics, mode of action, and role in pathogenesis. Curr. Top. Microbiol. Immunol.180, 65-95
  29. Ramegowda B. and V. L. Tesh. 1996. Differentiation-associated toxin receptor modulation, cytokine production, and sensitivityof Shiga-like toxin in human monocytes and monocytic cell lines. Infect. Immun. 64, 1173-1180
  30. Richardson S. E., M. A. Karmali, L. E. Becker and D. R. Smith. 1988. The histopathlogy of the hemolytic uremic syndrome associated with Verocyto-toxin-producing Escherichia coli infections. Hum. Pathol. 19, 1102-1108 https://doi.org/10.1016/S0046-8177(88)80093-5
  31. Sandvig K., M. Ryd, O. Garred, E. Schweda, P. K. Holm and B. van Deurs. 1994. Retrogradetransport from the Golgi complex to the ER of both Shiga toxin and the nontoxic Shiga B-fragment is regulated by butyric acid and cAMP. J. Cell. Biol. 126, 53-64 https://doi.org/10.1083/jcb.126.1.53
  32. Tarr P. I. 1995. Escherichia coli O157:H7. Clinical, diagnostic, and epidemiological aspects of human infection. Clin. Infect. Dis. 20, 1-10 https://doi.org/10.1093/clinids/20.1.1
  33. Tesh V. L. and A. D. O''Brien. 1991. The pathogenic mechanisms of Shiga toxin and the Shiga-like toxins. Mol. Microbiol. 5, 1871-1922
  34. Tesh V. L., B. Ramegowda and J. E. Samuel. 1994. Purified Shiga-like toxins induce expression of proinflammatory cytokines from murine peritoneal macrophages. Infect. Immun. 62, 5085-5094
  35. Tyrrell G. j., K. Ramotar, B. Toye. B. Boyd, C. A. Lingwood and J. L. Brunton. 1992. Alteration of the glycolipid specificity of VTB by site-directed mutagenesis. Proc. Natl. Acad. Sci. 89, 524-529 https://doi.org/10.1073/pnas.89.2.524
  36. Van de Kar N., L. Monnens, M. Karmali and V. van Hinsbergh. 1992. Tumor necrosis factor and interleukin-1 induce expression of the Verocytotoxin receptor globotriaosylceramide on human endothelial cells: implications for the pathogenesis of the hemolytic uremic syndrome. Blood. 180, 2755-2764
  37. Van Setten P., L. Monnens, R. Verstraten, L. van den Heuvel and van Hinsbergh. 1996. Effects of Verocytotoxin-1 on Nonadherent Human Monocytes: Binding Characteristics, Protein Synthesis, and Induction of Cytokine Release. Blood. 88, 174-183
  38. Vernon L. T., R. Belakere and E. S. James. 1994. Purified shiga-like toxins induce expression of proinflammatory cytokines from murine peritoneal macrophages. Infect. Immun. 62, 5085-5094