Cell Tissue Res (1995) 280:189-196 Cell&Tissue Research 9 Springer-Verlag 1995 Macrophage depletion in the rat after intraperitoneal administration of liposome-encapsulated clodronate: depletion kinetics and accelerated repopulation of peritoneal and omental macrophages by administration of Freund's adjuvant Jeike Biewenga, Marja B. van der Ende, Lambert F.G. Krist, Annemarie Borst, Mohammed Ghufron*, Nico van Rooijen Department of Cell Biology and Immunology, Medical Faculty, Vrije Universiteit, Van der Boechorststraat 7, NL-1081 BT Amsterdam, The Netherlands Received: 18 April 1994 / Accepted: 12 October 1994 Abstract. The purpose of this study was to develop a method for the depletion of macrophages from the peri- toneal cavity and the omentum of the rat. Rats received two intraperitoneal injections (at days 0 and 3) with lipo- some-encapsulated clodronate (dichloromethylene bis- phosphonate: C12MBP-liposomes). This treatment result- ed in complete elimination of mature tissue macro- phages (ED2-positive macrophages) from the peritoneal cavity and the omentum within 2 days. The elimination included the strongly ED2-positive spindle-shaped cells of the omental membrane. Repopulation of the omental ED2-positive macrophages was not seen within the next 23 days. Whereas ED2-positive macrophages were com- pletely depleted, few EDl-positive cells remained and repopulation of EDl-positive cells was faster. The treat- ment further depleted macrophages from the spleen, es- pecially from the red pulp, parathymic lymph nodes and liver. Freund's incomplete adjuvant administered one day after the last injection of C12MBP-liposomes consid- erably accelerated repopulation in the omentum. The protocol described might be used to investigate the con- tribution of mature tissue macrophages to the induction of immune responses, drug metabolism and the elimina- tion of intestinal tumours. Key words: Macrophage - Peritoneal cavity - Omentum - Depletion - Repopulation - Freund's adjuvant - Rat (Wistar) Introduction In animal studies, the peritoneal cavity is an important site for the injection of antigens, drugs or tumour cells. Macrophages, which form 70%-90% of peritoneal cells in man and laboratory animals (Bos et a1.1988; Plasman * Present address: Department of Histology, Medical Faculty, Gadjah Mada University, Yogyakarta, Indonesia Correspondence to: J. Biewenga and Vray 1993), probably play a major role in the pro- cessing and transportation of injected foreign materials. The omentum has a direct contact with the peritoneal cavity through wide gaps in the mesothelial lining over- lying the omental milky spots (Hodel 1970; Liebermann- Meffert 1983). As a consequence, intraperitoneally (i.p.) injected materials can easily enter the omentum, so that omental macrophages may also contribute to the pro- cessing and transportation of these materials. Moreover, the omental milky spots contain an abundance of macro- phages and macrophage precursors (Beelen et al.1980a; Wijffels et al. 1992) and could be the major source of peritoneal macrophages, especially in the case of inflam- mation. As peritoneal macrophages might play a role in im- mune responses against i.p. injected antigens, the ques- tions arise whether and for what period of time rats can be depleted of peritoneal macrophages, in order to ex- plore the effect of macrophages on i.p. induced immune responses. In previous studies, macrophage depletion has been obtained using liposome-encapsulated clodron- ate (dichloromethylene bisphosphonate: C12MBP-lipo- somes) (Delemarre et al. 1991). C12MBP-liposomes are ingested by macrophages, into which the C12MBP is subsequently released intracellularly after disruption of the liposomal phospholipid bilayer under the influence of phospholipases. Almost complete and long-lasting de- pletion (1-3 months) of peripheral lymph nodes has been obtained by a single subcutaneous (s.c.) injection of C12MBP-liposomes (Delemarre et al. 1990). In the spleen, macrophages have been eliminated by a single intravenous (i.v.) injection of C12MBP-liposomes. Re- population occurs between 10 days for macrophages of the red pulp and 2 months for macrophages of the mar- ginal zone (Van Rooijen et al. 1989, 1990). In a pilot study, involving the use of a single i.p. in- jection of C12MBP-liposomes, we have shown that peri- toneal macrophages are not completely depleted and that repopulation starts within 3-4 days (Soesatyo et al. 1991). As macrophages that repopulate the peritoneal cavity might immigrate from the omentum, the aims of