血管內皮細胞層在調節白血球能否穿過血管進入組織的過程中扮演著重要的角色。在本研究中，我們模擬多形核白血球(PMN)穿出血管內皮細胞層的過程，並同時在單細胞層次記錄內皮細胞內鈣離子濃度(EC[Ca2+]i)變化。首先將人類臍帶靜脈內皮細胞培養在覆蓋著一薄層透明膠原蛋白凝膠的蓋玻片上，在加入白血球趨化物f-MLP處理一小時後，再以不含f-MLP的緩衝液沖洗，使位於一個流體室中的內皮細胞層上下之間產生f-MLP的濃度梯度；藉此吸引PMN在內皮細胞上滾動、黏著、爬行，最後穿過內皮細胞層進入膠原蛋白凝膠。在此同時記錄PMN在內皮細胞層上活動的相位差影像，及其穿過內皮細胞層時EC[Ca2+]i螢光影像的變化。我們發現當單一PMN開始穿過內皮細胞層，會引發緊鄰的內皮細胞 [Ca2+]i上升，但對其附近未直接接觸此一PMN的內皮細胞，則沒有影響。如EC[Ca2+]i訊號被阻斷，則PMN無法穿過內皮細胞層。反之，如PMN [Ca2+]i訊號被阻斷，卻不能阻止其穿過內皮細胞層。
　　其次，我們使用能專一識別內皮細胞間連接體蛋白的抗體去螢光標識VE-cadherin及PECAM-1的位置，並觀察當PMN穿過內皮細胞層時這兩種連接體蛋白移動的即時變化。我們發現在PMN穿過內皮細胞層時，這兩種連接體蛋白都不會突然消失，而是發生了漸進的相對移動--相鄰內皮細胞間原本彼此接合的VE-cadherin會沿著細胞間的界線移向PMN兩端；而PECAM-1的變化則是打開原本彼此間的鍵結，改而圍繞在PMN週邊。 PMN穿過內皮細胞層後，這些觀察到的變化均迅速恢復原狀。此外，已穿過內皮細胞層的PMN能在內皮細胞層下快速的水平移動。上述現象不僅可在培養的內皮細胞層觀察到，也會發生在臍靜脈血管的內皮細胞層上。
　　根據以上結果，我們推測PMN在移動到血管內皮細胞交界處之後，會給予後者一個訊息使EC[Ca2+]i上升，進而以不同的方式打開內皮細胞間的連結分子，使PMN得以順利通過。

　　Vascular endothelium plays an important role in regulating the transendothelial migration of polymorphonuclear leukocytes (PMNs). In this study, we examined at single-cell level the intracellular calcium ion ([Ca2+]i) signaling of endothelial cells (ECs) during PMN transmigration. Human umbilical vein ECs were cultured on a thin layer of collagen gel. The ECs were labeled with fura-2 AM, immersed in formyl-Met-Leu-Phe, and subsequently perfused with fresh buffer in order to establish a gradient of chemoattractant across the EC monolayer. The entire process of PMN rolling on, adhering to, and transmigrating across the EC monolayer was recorded under both phase-contrast and fluorescence optics. We found that 1) At high concentration (~3 x106/ml), both PMN suspension and its supernatant stimulated frequent EC [Ca2+]i elevations across the monolayer; 2) when used at lower concentration (~5 x 105/ml) to avoid the interference of soluble factors, PMN transmigration, but not rolling nor adhesion, was accompanied by EC [Ca2+]i elevation; 3) the latter EC [Ca2+]i elevation occurred simultaneously in ECs adjacent to the transmigration site, but not in those which were not in direct contact with the transmigrating PMN; 4) this EC [Ca2+]i elevations was an initial and required event for PMN transmigration; 5) BAPTA-pretreated PMNs transmigrated with accompanying EC [Ca2+]i elevation but they became much elongated in the collagen gel. In conclusion, PMNs induce adjacent EC [Ca2+]i signaling that apparently mediates the ‘gating’ step for their subsequent transmigration.

　　Most existing evidence regarding junction protein movements during transendothelial migration of leukocytes comes from taking post-fixation snap shots of the transendothelial migration process that happens on a cultured endothelial monolayer. In this study, we used junction protein-specific antibodies that did not interfere the transendothelial migration to examine the real-time movements of vascular endothelial-cadherin (VE-cadherin) and platelet/endothelial cell adhesion molecule-1 (PECAM-1) during transmigration of polymorphonuclear leukocytes (PMNs) either through a cultured endothelial monolayer or through the endothelium of a dissected human umbilical vein tissue. Both junction proteins showed relative movements, not transient disappearance, at the PMN transmigration sites under either experimental model system. VE-cadherin moved away from the transmigration site to different ends of it, whereas PECAM-1 opened to surround the periphery of a transmigrating PMN. Junction proteins usually moved back to their original positions when the PMN transmigration process was completed in less than 2 min. The relative positions of some junction proteins might rearrange to form a new inter-endothelial contour when PMNs preferentially transmigrated through multi-cellular corners. Although transmigrated PMNs maintained good mobility, they only moved laterally underneath the vascular endothelium instead of deeply into the vascular tissue. In conclusion, our results obtained from using either cultured cells or vascular tissues showed that VE-cadherin-containing adherent junctions were relocated aside, not opened or disrupted, while PECAM-1-containing junctions were opened, during PMN transendothelial migration.

1. Allport JR, Ding H, Collins T, Gerritsen ME, Luscinskas FW. Endothelial-dependent mechanisms regulate leukocyte transmigration: a process involving the proteasome and disruption of the vascular endothelial-cadherin complex at endothelial cell-to-cell junctions. J Exp Med;186:517-527.1997.

2. Allport JR, Muller WA, Luscinskas FW. Monocytes induce reversible focal changes in vascular endothelial cadherin complex during transendothelial migration under flow. J Cell Biol;148:203-216,2000.

3. Bianchi E, Bender JR, Blasi F, Pardi R. Through and beyond the wall: late steps in leukocyte transendothelial migration. Immunol. Today;18:586-591,1997.

4. Burns AR, Walker DC, Brown ES, et al. Neutrophil transendothelial migration is independent of tight junctions and occurs preferentially at tricellular corners. J. Immunol.;159:2893-2903,1997.

5. Cepinskas G, Noseworthy R, Kvietys PR. Transendothelial neutrophil migration: role of neutrophil-derived proteases and relationship to transendothelial protein movement. Circ Res.;81:618-626,1997.

6. Cepinskas G, Sandig M, Kvietys PR. PAF-induced elastase-dependent neutrophil transendothelial migration is associated with the mobilization of elastase to the migrating front. J Cell Sci.;112:1937-1945,1999.

7. Del Maschio A, Zanetti A, Corada M, et al. Polymorphonuclear leukocyte adhesion triggers the disorganization of endothelial cell-to-cell adherens junctions. J. Cell Biol.;135:497-510,1996.

8. English D, Anderson BR. Single step separation of red blood cells, granulocytes and mononuclear leukocytes on discontinuous density gradients of Ficoll-Hypaque. J Immunol Methods.;5:249-252,1974.

9. Feng D, Nagy JA, Pyne K, Dvorak HF, Dvorak AM. Neutrophils emigrate from venules by a transendothelial cell pathway in response to fMLP. J Exp Med.;187:903-915,1998.

10. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem.;260:3440-3450,1985.

11. Hixenbaugh EA, Goeckeler ZM, Papaiya NN, Wysolmerski RB, Silverstein SC, Huang AJ. Stimulated neutrophils induce myosin light chain phosphorylation and isometric tension in endothelial cells. Am. J. Physiol.;273:H981-H988,1997.

12. Huang AJ, Furie MB, Nicholson SC, et al. Effects of human neutrophil chemotaxis across human endothelial cell monolayers on the permeability of these monolayers to ions and macromolecules. J. Cell. Physiol.;135:355-366,1988.

13. Huang AJ, Manning JE, Bandak TM, Ratau MC, Hanser KR, Silverstein SC. Endothelial cell cytosolic free calcium regulates neutrophil migration across monolayers of endothelial cells. J. Cell Biol.;120:1371-1380,1993.

14. Huang TY, Chu TF, Chen HI, Jen CJ. Heterogeneity of [Ca2+]i signaling in intact rat aortic endothelium. FASEB J.;14:797-804,2000.

15. Jen CJ, Chen HI, Lai KC, Usami S. Changes in cytosolic calcium concentrations and cell morphology in single platelets adhered to fibrinogen-coated surface under flow. Blood.;87:3775-3782,1996.

16. Jen CJ, Chen HI, Lai KC, Usami S. Changes in cytosolic calcium concentrations and cell morphology in single platelets adhered to fibrinogen-coated surface under flow. Blood.;87:3775-3782,1996.

17. Jen CJ, Lin JS. Direct observation of platelet adhesion onto fibrinogen- and fibrin-coated surfaces. Am. J. Physiol.;261:H1457-H1463,1991.

18. Jen CJ, Tai YW. Morphological study of platelet adhesion dynamics under whole blood flow conditions. Platelets.;3:145-153,1992.

19. Kuijpers TW, Hoogerwerf M, Roos D. Neutrophil migration across monolayers of resting or cytokine-activated endothelial cells: role of intracellular calcium changes and fusion of specific granules with the plasma membrane. J. Immunol.;148:72-77,1992.

20. Kvietys PR, Sandig M. Neutrophil diapedesis: paracellular or transcellular? News Physiol Sci.;16:15-19,2001.

21. Lampugnani MG, Dejana E. Interendothelial junctions: structure, signaling and functional roles. Curr. Opin. Cell Biol.;9:674-682,1997.

22. Lawson MA, Maxfield FR. Ca2+ and calcineurin-dependent recycling of an integrin to the front of migrating neutrophils. Nature., 377:75-79,1995.

23. Mandeville JTH, Maxfield FR. Effects of buffering intracellular free calcium on neutrophil migration through three-dimensional matrices. J. Cell. Physiol.;171:168-178,1997.

24. Mandeville JTH, Maxifield FR. Calcium and signal transduction in granulocytes. Curr. Opin. Hematol.;3:63-70,1996.

25. Marks PW, Maxfield FR. Transient increases in cytosolic free calcium appear to be required for the migration of adherent human neutrophils. J. Cell Biol.;110:43-52,1990.

26. McDonald DM, Thurston G, Baluk P. Endothelial gaps as sites for plasma leakage in inflammation. Microcirculation.;6:7-22,1999.

27. Moll T, Dejana E, Vestweber D. In vitro degradation of endothelial catenins by a neutrophil protease. J. Cell Biol.;140:403-407,1998.

28. Muller WA, Randolph GJ. Migration of leukocytes across endothelium and beyond: molecules involved in the transmigration and fate of monocytes. J Leukoc Biol.;66:698-704,1999.

29. Muller WA, Weigl SA, Deng X, Phillips DM. PECAM-1 is required for transendothelial migration of leukocytes. J Exp Med.;178:449-460,1993.

30. Muller WA. Migration of leukocytes across endothelial junctions: some concepts and controversies. Microcirculation. 2001;8:181-193.

31. Nakada MT, Amin K, Christofidou-Solomidou M, et al. Antibodies against the first Ig-like domain of human platelet endothelial cell adhesion molecule-1 (PECAM-1) that inhibit PECAM-1-dependent homophilic adhesion block in vivo neutrophils recruitment. J Immunol.;164:452-462,2000.

32. Newman PJ. Switched at birth: a new family for PECAM-1. J Clin Invest.;103:5-9,1999.

33. Newman PJ. The biology of PECAM-1. J Clin Invest.;99:3-8,1997.

34. Niles WD, Malik AB. Endocytosis and exocytosis events regulate vesicle traffic in endothelial cells. J Membr Biol.;167:85-101,1999.

35. Pfau S, Leitenberg D, Rinder H, SmithBR, Pardi R, Bender JR. Lymphocyte adhesion-dependent calcium signaling in human endothelial cells. J. Cell Biol. 1995;128:969-978.

36. Saito H, Minamiya Y, Kitamura M, et al. Endothelial myosin light chain kinase regulates neutrophil migration across human umbilical vein endothelial cell monolayer. J Immunol.;161:1533-1540,1998.

37. Sandig M, Korvemaker ML, Ionescu CV, Negrou E, Rogers KA. Transendothelial migration of monocytes in rat aorta: distribution of F-actin, alpha-catnin, and PECAM-1. Biotech Histochem.;74:276-293,1999.

38. Sandig M, Negrou E, Rogers KA. Changes in the distribution of LFA-1, catenins, and F-actin during transendothelial migration of monocytes in culture. J. Cell Sci.;110:2807-2808,1997.

39. Sandig M, Voura EB, Kalnins VI, Siu CH. Role of cadherins in the transendothelial migration of melanoma cells in culture. Cell Motil. Cytoskel.;38:351-364,1997.

40. Schroeter D, Spiess E, Paweletz N, Benke R. A procedure for rupture-free preparation of confluently grown monolayer cells for scanning electron microscopy. J. Electron Microsc. Tech.;1:219-235,1984.

41. Shaw SK, Bamba PS, Perkins BN, Luscinskas. Real-time imaging of vascular endothelial-cadherin during leukocyte transmigration across endothelium. J Immunol.;167:2323-2330,2001.

42. Spinger, T.A.: Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration. Annu. Rev. Physiol. 57:827-872, 1995.

43. Su WH, Chen HS, Huang JP, Jen CJ. Endothelial [Ca2+]i signaling during transmigration of polymorphonuclear leukocytes. Blood.;96:3816-3822,2000.

44. Watanabe H, Takahashi R, Zhang XX, et al. An essential role of myosin light-chain kinase in the regulation of agonist- and fluid flow-stimulated Ca2+ influx in endothelial cells. FASEB J.;12:341-348,1998.

45. Ziegelstein RC, Corda S, Pili R, et al. Initial contact and subsequent adhesion of human neutrophils or monocytes to human aortic endothelial cells releases an endothelial intracellular calcium store. Circulation.;90:1899-1907,1994.