Objectives:To study collagen phagocytosis by human extravillous trophoblast.
Methods:First-trimester extravillous trophoblastic cell lines and primary trophoblast cell preparations were cultured in vitro with collagen-coated fluorescent latex beads and fluorescent-labeled collagen. Confocal microscopy was used to demonstrate internalization of collagen and beads. The effect of cytochalasin B, temperature, metabolic inhibitors, and cytokines was studied by culturing trophoblast cells with tritated collagen. Acridine orange was used to stain for lysosomal compartments, and histochemical methods were used to demonstrate acid phosphatase in trophoblast cells.
Results:Both cell lines and primary culture cells internalize collagen and beads. Confocal microscopy unequivocally localized the phagocytosed material to the intracellular compartment. Inhibition by cytochalasin B and culture at 4C of uptake of [3H] collagen suggested that the process was phagocytosis. Cytokines and growth factors did not affect phagocytosis. Lysosomal compartments and acid phosphatase appear to colocalize.
Conclusions:The continuous remodeling and turnover of collagen which occur in a wide variety of tissues under both physiologic and pathologic conditions are thought to be mediated by two pathways: one external (involving release of proteolytic enzymes), and the other internal (involving phagocytosis). Similar remodeling events are likely to occur during trophoblast invasion. Although current views emphasize the importance of the extracellular pathway, we postulate, on the basis of our findings, that both pathways are used, with the internal pathway probably being dominant. We hypothesize that the proteolytic enzymes (extracellular pathway) disrupt collagen matrices, thereby facilitating phagocytosis. It is teleologically sound to conceive of a dominant interacellular pathway, as it allows for more precise control of the process of invasion and is economical, as the products of collagen degradation can be used as energy sources or building blocks.
1. Khong TY, Liddell HS, Robertson WB, Defective haemochorial placentation as a cause of miscarriage: A preliminary study. Br J Obstet Gynaecol1987;94:649-655.
2.
2. Brosens IA, Robertson WB, Dixon HG. The role of the spiral arteries in the pathogenesis of pre-eclampsia. Obstet Gynaecol Ann1972;1:177-191.
3.
3. Khong TY, DeWolf F, Robertson WB, Brosens I. Inadequate maternal vascular response to placentation in pregnancies complicated by pre-eclampsia and by small-for-gestational-age infants. Br J Obstet Gynaecol1986;93:1049-1059.
4.
4. Sodek K, Ferrier JM. Collagen remodelling in rat periodontal tissues: Compensation for precursor retuilization confirms rapid turnover of collagen. Collagen Rel Res1988;8:11-21.
5.
5. Everts V, Van Der Zee E, Creemers L, Beertsen W. Phagocytosis and intracellular digestion of collagen, its role in turnover and remodelling. Histochem J1996;28:229-245.
6.
6. Agelli M, Wahl SM. Cytokines and fibrosis. Clin Exp Rheum1986;4:379-388.
7.
7. Werb Z. Proteinases and matrix degradation. In: Kelly WN, Harris ED, Jr., Ruddly S, Sledge CB, eds. Textbook of rheumatology. 3rd ed.Philadelphia: WB Saunders Co., 1989:300-321.
8.
8. Perez-Tamayo R. Pathology of collagen degradation. Am J Pathol1978;92:509-566.
9.
9. Murphy G, Reynolds JJ. Extracellular matrix degradation. In: Royce PM, Steinmann B, eds. Connective tissue and its heritable disorders. New York: Wiley-Liss, 1993:287-316.
10.
10. Murphy G, Reynolds JJ. Current views on collagen degradation: Progress towards understanding the resorption of connective tissues. Bioessays1984;2:55-60.
11.
11. Murphy G, Reynolds JJ, Werb Z. Biosynthesis of tissue inhibitor of metalloproteinases by human fibroblasts in culture. Biol Chem1985;260:3079-3083.
12.
12. Circolo A, Welgus HG, Pierce GF, Kramer J, Strunk RC. Differential regulation of the expression of proteinases/antiproteinases in fibroblasts: Effects of interleukin-1 and platelet-derived growth factor. J Biol Chem1991;266:12283-12288.
13.
13. Meike MC, McGarrity AM, Thomson BM, Reynolds JJ. Bonederived growth factors modulate collagenase and TIMP (tissue inhibitor of metalloproteinases) activity and type 1 collagen degradation by mouse calvarial osteoblasts. Bone Miner1991;12:41-55.
14.
14. Richards CD, Rafferty JA, Reynolds JJ, Saklatvala J. Porcine collagenase from synovial fibroblast: cDNA sequence and modulation of expression of RNA in vitro by various cytokines. Matrix1991;11:161-167.
15.
15. Van der Zee E, Everts V, Hoeben K, Beertsen W, Interleukin-1α and EGF synergistically enhance the release of collagenase by periosteal connective tissue in vitro. Matrix1993;13:389-398.
16.
16. Fisher SJ, Leitch SM, Kantor MS, Basbaum CB, Kramer RH. Degradation of extracellular matrix by the trophoblastic cells of the first trimester placenta. J Cell Biochem1985;27:31-41.
17.
17. Fisher SJ, Cui T, Zhang L, et al. Adhesive and degradative properties of human placental cytorophoblast cells in vitro. J Cell Biol1989;109:891-902.
18.
18. Bischof P, Friedgli E, Martelli M, Campana A. Expression of extracellular matrix-degrading metalloproteinases by cultured human cytotrophoblastcells: Effect of cell adhesion and immunopurification. Am J Obstet Gynecol1991;165:1791-1801.
19.
19. Shimonowitz S, Hurwitz A, Dushnik M, Anteby E, Geva-Eldar T, Yagel S. Developmental regulation of the expression of 72 and 92 kDa type IV collagenases in human trophoblast invasion. Am J Obstet Gynecol1994;171:832-838.
20.
20. Bischof P, Martelli M, Campana A, Itoh Y, Hagase H. Importance of matrix metalloproteinases in human trophoblast invasion. Early Pregnancy Biol Med1995;1:263-269.
21.
21. Beertsen W, Everts V, Van den Hooff A. Fine structure of fibroblasts in the peridontal ligament of the rat incisor and their possible role in tooth eruption. Arch Oral Biol1974;19:1087-1098.
22.
22. Everts V, Hembry RM, Reynolds JJ, Beertsen W. Metalloproteinases are not involved in the phagocytosis of collagen fibrils by fibroblast. Matrix1989;9:266-276.
23.
23. Deporter DA. Collagen phagocytosis by stimulated mouse peritoneal macrophages in vitro. J Periodont Res1979;14:323-331.
24.
24. Everts V, Beertsen W Tigchelaar-Gutter. The digestion of phagocytosed collagen is inhibited by the proteinase inhibitors leupeptin and E-64. Collagen Rel Res1985;5:315-336.
25.
25. Scherft JP, Heersche JNM. Accumulation of collagen containing vacuoles in osteoblasts after administration of colchicine. Tissue Res1975;157:353-365.
26.
26. Takahashi T, So S, Wang D, Takahashi K, Kutihara N, Kumegawa M. Phagocytosis of different matrix components by different cell types at bone forming sites in cultured mouse calvariae. Cell Tissue Res1986;245:9-17.
27.
27. Everts V, Delaisse JM, Korper W, Beertsen W. Involvement of matrix metalloproteinase and cysteine proteinases in degradation of bone matrix. In: Davidovitch Z, ed. The biological mechanisms of tooth eruption, resorption and replacement by oral implants. Birmingham: EBSCO Meda, 1994:85-97.
28.
28. Birek P, Wang HM, Brunett DM, Melcher AH. Epithelial rests of Malassez in vitro: Phagocytosis of collagen and the possible role of their lysosomal enzymes in collagen degradation. Lab Invest1980;43:61-72.
29.
29. Salonen J, Uitto VJ, Pan M, Oda D. Proliferating oral epithelial cells in culture are capable of both extracellular and intracellular degradation of interstitial collagen. Matrix1991;11:43-55.
30.
30. Everts V, Beertsen W. The cellular basis of tooth eruption: The role of collagen phagocytosis. In: Davidovitch Z, ed. The biological mechanisms of tooth eruption and root resorption. Birmingham: EBSCO Media, 1988:237-242.
31.
31. Deporter DA, Ten Cate AR. Fine structural localization of acid and alkaline phosphatase in collagen containing vesicles of fibroblasts. J Anat1973;114:457-461.
32.
32. Everts W, Aronson DC, Beertsen W. Phagocytosis of bone collagen by oesteoclasts in two cases of pycnodysostosis. Calcif Tissue Int1985;37:25-31.
33.
33. Van Noorden CJF, Everts V. Selective inhibition of cysteine proteinases by Z-Phe-AlaCH2F suppress digestion of collagen by fibroblasts and osteoclasts. Biochem Biophys Res Commun1991;178:178-184.
34.
34. Korner A. Effect of cycloheximide on protein biosynthesis in rat liver. Biochem J1996;101:627-631.
35.
35. Peterkovsky B, Tomkins GM. Evidence for the steroid-induced accumulation of tyrosine-aminotransferase messenger RNA in the absence of the protein synthesis. Proc Nat Acad Sci U S A1986;60:222-228.
36.
36. Dehm P, Prockop DJ. Synthesis and extrusion of collagen by freshly isolated cells from chick embryo tendon. Biochim Biophys Acta1971;240:358-369.
37.
37. Uitto J, Tan EM, Ryhanen L. Inhibition of collagen accumulation in fibrotic processes: Review of pharmacologic agents and new approaches with amino acids and their analogues. J Inves Dermatol1982;79(Suppl 1):113s-120s.
38.
38. Fernandez-Madrid F, Noonan S, Riddle J, Karvonen R, Sasaki D. Intracellular processing of procollagen induced by the action of colchicine. J Anat1980;130:229-241.
39.
39. Cho MI, Garant PR. Ultrastructural and cytochemical studies of the fate of unsecreted collagen precursors after administration of colchicine to mice. Acta Anat1985;121:205-215.
40.
40. Klaus GC. Cytochalasin B. Dissociation of pinocytosis and phagocytosis by peritoneal macrophages. Exp Cell Res1973;79:73-78.
41.
41. Davies P, Allison AC. Effect of cytochalasin B on endocytosis and exocytosis. In: Tanenbaum SW, ed. Cytochalasins: Biochemical and cell biological aspects. Amsterdam: Elsevier Biomedical Press, 1978;143-160.
42.
42. Berg RA, Schwartz ML, Crystal RG. Regulation of the production of secretory proteins: Intracellular degradation of newly synthesised defective collagen. Proc Natl Acad Sci U S A1980;77:4746-4750.
43.
43. Ten Cate AR, Freeman E. Collagen remodeling by fibroblast in wound repair: Preliminary observations. Anat Rec1974;179:543-546.
44.
44. Choy MY, Manyonda IT. The phagocytic activity of human first trimester extravillous trophoblast. Hum Reprod1998;13:2941-2949.
45.
45. Loke YW, Burland K. Human trophoblast cells cultured in modified medium and supported by extracellular matrix. Placenta1988;9:173-182.
46.
46. Burrows TD, King A, Loke YW. Expression of adhesion molecules by endovascular trophoblast and decidual endothelial cells: Implications for vascular invasion during implantation. Placenta1994;15:21-33.
47.
47. Aplin JD. Expression of integrin alpha 6 beta 4 in human trophoblast and its loss from extravillous cells. Placenta1993;14:203-215.
48.
48. O'Grady RL, Nethery A, Hunter E. A fluorescent screening assay for collagenase using collagen labeled with 2-methoxy-2,4-diphenyl-3(2H)-furnanone. Anal Biochem1984;140:490-494.
49.
49. Enders AC, Schlafke S. Cytological aspects of trophoblast uterine interaction in early implantation. Am J Anat1969;125:1-30.
50.
50. Smith MS, Wilson IB. Histochemical observations on early implantation in the mouse. J Embryol Exp Morphol1971;25:165-174.
51.
51. Knoth M, Larsen JF. Ultrastructure of a human implantation site. Acta Obstet Scand1972;51:385-393.
52.
52. Loke YW, Eremin O, Ashby J, Day S. Characterization of the phagocytic cells isolated from the human placenta. J Reticuloendothel Soc1982;31:317-324.
53.
53. Melcher AH, Chan J. Phagocytosis and digestion of collagen by gingival fibroblasts in vivo: A study of serial sections. J Ultrastr Res1981;77:1-36.
54.
54. Ten Cate AR. Morphological studies of fibrocytes in connective tissue undergoing rapid remodeling. J Anat1972;112:401-414.
55.
55. Van der Zee E, Everts V, Hoeben K, Beertsen W. Cytokines modulate contraction of periosteal explants from explants from rabbit calvariae. Connect Tissue Res1995;31:141-151.
56.
56. Mohamed-Ali H, Scholz P, Merker HJ. Intracellular collagen-like material in mouse embryonic mesenchymal cells incubated with TNF-a and IL-la in organoid culture. Cell Tissue Res1996;284:509-515.
57.
57. Athanassakis I, Bleackley RC, Paetkau V, Guilbert L, Barr PJ, Wegmann TG. The immuno-stimulatory effects of T cells and T cell lymphokines on murine fetally derived placental cells. J Immunol1987;138:37-44.
58.
58. Armstrong DTA, Chaouat G. Effects of lymphokines and immune complexes on murine placental cell growth in vitro. Biol Reprod1989;40:400-406.
59.
59. Chaouat G, Menu E, Clark DA, Dy M, Minkowski M, Wegmann TG. Control of fetal survival in CBX × DBA/2 mice by lymphokine therapy. J Reprod Fertil1990;89:447-458.
60.
60. Raghupathy R. Th1-type immunity is incompatible with successful pregnancy. Immunol Today1997;18:478-482.
61.
61. Piccinni MP, Beloni L, Livi C, Maggi E, Scarselli G, Romagnani S. Defective production of both leukemia inhibitory factor and type 2 T-helper cytokines by decidual T cells in unexplained recurrent abortions. Nat Med1998;4:10020-10024.
62.
62. Librach CL, Feigenbaum SL, Bass KE, et al. Interleukin-1 beta regulates human cytotrophoblast metalloproteinase activity and invasion in vitro. J Biol Chem1994;269:17125-17131.
63.
63. Bass KE, Morrish D, Roth I, et al. Human cytotrophoblast invasion it up-regulated by epidermal growth factor: Evidence that paracrine factors modify this process. Dev Biol1994;164:550-561.
64.
64. Librach CL, Werb Z, Fitzgerald ML, et al. 92kDa type IV collagenase mediates invasion of human cytotrophoblasts. J Cell Biol1991;113:437-449.
65.
65. Yagel S, Parhar RS, Jeffrey JJ, Lala PK. Normal non-metastatic human trophoblast cells share in vitro properties of malignant cells. J Cell Physiol1988;136:455-462.
66.
66. Graham CH, Lala PK. Mechanism of control of trophoblast invasion in situ. J Cell Physiol1991;148:228-234.
