Summary
Clodronate (dichloromethylene bisphosphonate) inhibits the activity of osteoclasts, thereby preventing bone resorption in disorders characterized by excessive bone loss. Intravenously injected clodronate encapsulated in liposomes is also known to inactivate phagocytic cells in spleen and liverin vivo. The macrophage suppressive effect of clodronate is of interest in autoimmune diseases, like rheumatoid arthritis, in which phagocytic cells are involved in inflammatory processes, but knowledge of the interaction of clodronate with phagocytic cells is scarce. We have studied the uptake of clodronate, both free and encapsulated in negatively charged liposomes, by the macrophage-like cell line RAW 264 and by other types of cell lines. The uptake was assessed by a growth inhibition assay. The liposome-encapsulated lodronate was 50 and 350 times more potent than free drug for RAW 264 and CVI-P, respectively. Cell lines with a lower endocytotic capacity were insensitive to liposome-mediated delivery of the drug. The action of free clodronate seemed to be extracellular in all cell lines studied. Calcium and/or iron have been suggested to be involved in the intracellular uptake and action of clodronate in phagocytic cells. We found that the uptake of free clodronate by RAW 264 cells was indeed mediated by calcium and iron, while the uptake of liposomal drugs was only slightly affected by calcium. The increased intracellular calcium concentration in macrophages did not significantly affect the growth-inhibitory properties of clodronate, whereas iron loading of the cells partially restored the cell growth. The data do not support the role of calcium chelation as a mechanism of action of clodronate, but suggest that intracellular iron is, at least partially involved.
Similar content being viewed by others
References
Fleisch H (1991) Bisphosphonates. Pharmacology and use in the treatment of tumor-induced hypercalcaemia and metastatic bone disease. Drugs 42:919–942
Mönkkönen J, Koponen H-M, Ylitalo P (1990) Comparison of distribution of three bisphosphonates in mice. Pharmacol Toxicol 66:294–298
Mönkkönen J, Urtti A, Paronen P, Elo HA, Ylitalo P (1989) The uptake of clodronate (dichloromethylene bisphosphonate) by macrophages in vivo and in vitro. Drug Metab Dispos 17:690–693
Mönkkönen J, Ylitalo P (1990) The tissue distribution of clodronate (dichloromethylene bisphosphonate) in mice. The effects of vehicle and the route of administration. Eur J Drug Metab Pharmacokin 15:239–243
Van Rooijen N (1992) Liposome-mediated elimination of macrophages. Res Immunol 143:215–219
Flanagan AM, Chambers TJ (1989) Dichloromethylene bisphosphonate (Cl2MBP) inhibits bone resorption through injury to osteoclasts that resorb Cl2-MBP-coated bone. Bone Miner 6:33–43
Carano A, Teitelbaum SLO, Konsek JD, Schlesinger PH, Blair HC (1990) Bisphosphonates directly inhibit the bone resorption activity of isolated avian osteoclasts in vitro. JClin Invest 85:456–461
Chambers TJ (1980) Diphosphonates inhibit bone resorption by macrophages in vitro. J Pathol 132:255–262
Van Rooijen N (1989) The liposome-mediated macrophage “suicide” technique. J Immunol Methods 124:1–6
Van Rooijen N, Van Nieuwmegen R (1984) Elimination of phagocytic cells in the spleen after intravenous injection of liposome-encapsulated dichloromethylene disphosphonate. An enzyme-histochemical study. Cell Tissue Res 238:355–358
Mönkkönen J, Van Rooijen N, Ylitalo P (1991) Effects of clodronate and pamidronate on splenic and hepatic phagocytic cells of mice. Pharmacol Toxicol 68:284–286
Van Rooijen N (1991) High and low cytosolic Ca2+ induced macrophage death? Hypothesis. Cell Calcium 12:381–384
Van Lent PLEM, Van den Hoek A, Van den Bersselaar, Dijkstra CD, Van Rooijen N, Van den Berg WB (1992) Role of synovial macrophages in experimental arthritis. Res Immunol 143:229–234
Bresnihan B (1992) The synovial lining cells in chronic arthritis. Br J Rheumatol 31:433–436
Szoka FC, Papahadjopoulos D (1978) Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation. Proc Natl Acad Sci USA 75:4194–4198
Heath TD, Brown CS (1990) Liposome dependent delivery of N-(phosphonacetyl)-L-aspartic acid to cells in vitro. J Lipos Res 1:315–329
Claassen E, Van Rooijen N (1986) Preparation and characteristics of dichloromethylene diphosphonate-containing liposomes. J Microencapsul 3:109–114
Bartlett GR (1959) Phosphorus assay in column chromatography. J Biol Chem 234:466–468
Ponka P, Borova J, Neuwirt J, Fuchs O (1979) Mobilization of iron from reticulocytes. FEBS Lett 97:317–321
Heath TD, Lopez NG, Papahadjopoulos D (1985) The effects of liposome size and surface charge on liposome-mediated delivery of methotrexate-γ-aspartate to cells in vitro. Biochim Biophys Acta 820:74–84
Ng K, Heath TD (1989) Liposome-dependent delivery of pteridine antifolates: A two compartment growth inhibition assay for the evaluating drug leakage and metabolism. Biochim Biophys Acta 981:261–268
Heath TD, Lopez NG, Piper JR, Montgomery JA, Stern WH, Papahadjopoulos D (1986) Liposome-mediated delivery of pteridine antifolates to cells in vitro: Potency of methotrexate, and its α and γ substituents. Biochim Biophys Acta 862:72–80
Van Rooijen N, Claassen E (1988) In vivo elimination of macrophages in spleen and liver, using liposome-encapsulated drugs: Methods and applications. In: Gregoriadis G (ed) Lipo somes as drug carriers. John Wiley & Sons, Chichester, pp 131–143
Van Rooijen N, Kors N, ter Hart H, Claassen E (1988) In vitro and in vivo elimination of macrophage tumor cells using liposome-encapsulated dichloromethylene diphosphonate. Virchows Archiv B Cell Pathol 54:251–245
Claassen I, Van Rooijen N, Claassen E (1990) A new method for removal of mononuclear phagocytes from heterogeneous cell populations in vitro, using the liposome-mediated macrophage ‘suicide’ technique. J Immunol Methods 134:153–161
Van Rooijen N, Van Nieuwmegen N, Kamperdijk EWA (1985) Elimination of phagocytic cells in the spleen after intravenous injection of liposome-encapsulated dichloromethylene disphosphonate. Ultrastructural aspects of elimination of marginal zone macrophages. Virchows Arch B Cell Pathol 49:375–383
Van Rooijen N, Kors N, Kraal G (1989) Macrophage subset repopulation in the spleen: differential kinetics after liposome mediated elimination. J Leukoc Biol 45:97–104
Cecchini MG, Felix R, Fleisch H, Cooper PH (1987) Effect of bisphosphonates on proliferation and viability of mouse bone marrow derived macrophages. J Bone Miner Res 2:135–142
Cecchini MG, Fleisch H (1990) Bisphosphonates in vitro specifically inhibit, among the hematopoietic series, the development of the mouse mononuclear phagocyte lineage. J Bone Miner Res 5:1019–1027
Zimolo Z, Tanaka H, Rodan GA (1992) Alendronate (ALN) increases the membrane permeability of multinucleated osteoclast-like cells to NH4 +, H+ and Ca.2+. Bone Miner 17(Suppl 1):S17
Rao K, van Renswoude J, Kempf C, Klausner RD (1983) Separation of Fe3+ from transferrin in endocytosis. Role of the acidic endosomes. FEBS Lett 160:213–216
Hagenlocker BE, Walker BAM, Ward PA (1990) Superoxide responses of immune complex-stimulated rat alveolar macrophages. J Immunol 144:3898–3906
Landschulz W, Thesleff I, Ekblom P (1984) A lipophilic iron chelator can replace transferrin as a stimulator of cell proliferation and differentiation. J Cell Biol 98:596–601
Chitambar CR, Matthaeus WG, Antholine WE, Graff K, O'Brien JO (1988) Inhibition of leukemic HL60 cell growth by transferrin-gallium: effects on ribonucleotide reductase and demonstration of drug synergy with hydroxyurea. Blood 72:1930–1936
Lundberg JH, Chitambar CR (1990) Interaction of gallium nitrate with fludarabine and iron chelators: Effects on the proliferation of human leukemic HL60 cells. Cancer Res 50:6466–6470
Chitambar CR, Zivkovic Z (1987) Inhibition of hemoglobin production by transferrin-gallium. Blood 69:144–149
Ponka P, Schulman HM, Wilczynska A (1982) Ferric pyridoxal isonicotinoyl hydrazone can provide iron for heme synthesis in reticulocytes. Biochim Biophys Acta 718:151–156
Mönkkönen J, Brown CS, Thompson TT, Heath TD (1993) Liposome mediated delivery of gallium to macrophage-like cells in vitro: demonstration of transferrin independent route for intracellular delivery of metal ions. Pharm Res (in press)
D'Arcy Hart P, Young MR (1991) Ammonium chloride, an inhibitor of phagosome-lysosome fusion in macrophages, concurrently induces phagosome-endosome fusion, and opens a novel pathway: Studies of a pathogenic mycobacterium and nonpathogenic yeast. J Exp Med 174:881–889
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Mönkkö, J., Heath, T.D. The effects of liposome-encapsulated and free clodronate on the growth of macrophage-like cellsin vitro: The role of calcium and iron. Calcif Tissue Int 53, 139–146 (1993). https://doi.org/10.1007/BF01321893
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF01321893