Investigation of the chemotactic activities of the HL-60 cells by time-lapse microscopy

Abstract

목적: 본 실험에서는 전골수구세포 단계인 HL-60 세포의 chemokine 자극에 의한 화학주성을 새로이 개발된 저속도촬영 현미경과 KK microchamber을 이용해 실시간 관찰해 보고자 하였다. 재료 및 방법: HL-60 세포를 dimethly formamide (DMF)나 phorbol myristate acetate(PMA)를 첨가하여 RPMI 1640 배지에 배양하며 분화를 유도하였다. 배양4일 후 이 세포들을 형태학적으로 관찰하고 유세포 분석을 시행하였으며 chemokine에 대한 화학주성을 관찰하였고 비교를 위하여 사람 말초혈액 과립구를 정제하였다. 결과: DMF를 첨가한 경우, 40-50% 세포가 과립구로 분화하였으며 CD11c, CD18 발현이 증가되었다. PMA를 첨가한 세포들은 대부분 단구로 분화하였으며 CD14의 발현이 증가되었다. PMA로 분화시킨 세포들은 CCR2, CCR3, CCR9, CXCR2, CXCR4 발현이 증가되었으나 DMF를 첨가한 세포에서는 수용체 발현의 증가는 없었다. Transwell 분석에서는 DMF나 PMA로 분화되거나 분화되지 않은 HL-60세포 모두에서 SDF-1α, IL-8 자극에 의해 세포 이동이 증가하였다. 그러나 KK microchamber를 이용한 저속촬영에서는 DMF로 분화시킨 세포에서만 SDF-1α, IL-8, MIP-1α, RANTES 등의 chemokine 자극에 의해 방향성 운동이 관찰되었다. 또한 세포의 이동속도와 이동한 세포의 비율은 chemokine에 의해 증가되었다. 비록 이동 속도가 이용한 chemokine의 종류에 따라 차이가 있었으나 DMF를 첨가하여 분화시킨 HL-60 세포의 이동속도는 사람 말초혈액 과립구 속도(0.21 ± 0.04 ㎛/sec)의 2 분의 1또는 3분의 1 정도로 측정되었다. 이런 방향성 이동은 백일해 독소에 의해 저해되는 것으로 보아 Gα단백을 이용한 신호전달이 관여함을 확인할 수 있었다. 결론: KK microchamber를 이용한 저속촬영 방법을 이용하여 DMF로 분화된HL-60 세포에서 chemokine자극에 의한 방향성 이동을 관찰하였으며 이러한 방법은 세포의 화학주성을 연구하는데 많은 도움이 될 것이다.;PURPOSE: In this study, real-time movement of the promyelocytic cell line, HL-60, in chemotactic response has been investigated by time-lapse microscopic observation using a KK microchamber. METHODS: HL-60 cells were cultured in RPMI 1640 medium and stimulated with 0.8% dimethyl formamide (DMF) or phorbol myristate acetate (PMA). After 4 days the cells were harvested for the analysis of morphology, flow cytometry and chemotactic response by chemokines. Human peripheral blood granulocytes were also purified for the comparison. RESULTS: After stimulation with DMF, about 40 - 50% cells showed granulocytic differentiation and increased the expression of CD11c and CD18. After stimulation with PMA, almost all cells showed monocytic differentiation and increased the expression of CD14. Stimulation with PMA induced increased expression of CCR2, CCR3, CCR9, CXCR2 and CXCR4, whereas that with DMF did not. Chemotactic activity was analyzed by time-lapse microscopic observation of the real-time movement of the cells in a small microchamber using a special equipment, EZ-TAXIScan, as well as by conventional method using transwells. For comparison, human granulocytes purified from adult peripheral bloods were also analyzed. Transwell assay showed that stromal derived factor-1α (SDF-1α) and interleukin-8 (IL-8) increased the random migration of both unstimulated and stimulated HL-60 cells with DMF or PMA. But time-lapse microscopic observation showed that only HL-60 cells stimulated with DMF migrated directionally in response to the chemokines tested, SDF-1α, IL-8, macrophage inflammatory protein-1α (MIP-1α) and regulated on activation, normal T cells, expressed and secreted (RANTES). The speed as well as the proportion of migrating cells was increased by stimulation with chemokine, and the speed of migration was different according to the type of chemokine. HL-60 cells stimulated with DMF migrated with a speed from one third to half of that of human granulocytes that migrated randomly at a speed of 0.21 ± 0.04 ㎛/sec (n=3) without chemokine stimulation. The directional migration of HL-60 cells stimulated with DMF was inhibited by pretreatment of the cells with pertussis toxin, and that implies an involvement of Gα-mediated signal transduction. CONCLUSION: Time-lapse microscopic observation using KK microchamber provides more information on the chemotactic response of the cells and showed that only HL-60 cells stimulated with DMF migrated directionally in response to chemokine stimulation.