Macrophage Depletion Prevents Leukocyte Adhesion and Disease Induction in Experimental Melanin-protein Induced Uveitis

Abstract

The purpose was to study the effects of macrophage depletion with liposomal dichloromethylene-diphosphonate (Cl2MDP-lip) on inflammation and leukocyte-endothelium interaction in experimental melanin-protein induced uveitis (EMIU). Lewis rats (n=48) were immunized with melanin-associated protein in complete Freund's adjuvant and pertussis toxin. Control groups received adjuvants without the antigen (n=12) or no injection (n=6). Animals received treatment with either CL2MDP-lip or empty liposomes (empty-lip) on day −2, 1, 4, 6 and 8. Leukocytes were stained with rhodamine 6G i.v. and intravital fluorescence microscopy (IVM) was performed on day 4, 6, 8 and 10 to quantify leukocyte rolling and arrest. After IVM, the cell count and protein concentration were determined in aqueous humor and plasma levels of TNF-α and IFN-γ were measured by ELISA. In EMIU, leukocyte rolling increased on day 4 (10.0±1.2 cellsmin−1vs baseline of 5.7±0.7 cellsmin−1, mean±S.E.(M.)) and peaked on day 8 (40.8±4.2 cellsmin−1;P⩽0.05). Leukocyte arrest was increased on day 8 (175.4±18.2 cellsmm−2vs baseline of 59.7±7.1 cellsmm−2;P⩽0.05) and day 10 (371.7±30.7 cellsmm−2). CL2MDP-lip prevented leukocyte rolling (day 10: 16.6±1.8 cellsmin−1vs 30.7±2.9 cellsmin−1; CL2MDP-lip vs untreated EMIU;P⩽0.05) and arrest (day 8: 88.3±13 cellsmm−2; day 10: 128.5±12.9 cellsmm−2;P⩽0.05). Empty-lip had no effect on leukocyte rolling (day 10: 34.8±4.2 cellsmin−1) or arrest (day 8: 159.3±12.9 cellsmm−2, day 10: 421.2±41.6 cellsmm−2). CL2MDP-lip completely suppressed leukocyte emigration (11±2 cellsμ l−1vs 100±29 cellsμ l−1; CL2MDP-lip vs empty-lip;P⩽0.05) and protein extravasation into aqueous humor (2.7±0.3mgml−1vs 14.2±2.1mgml−1; CL2MDP-lip vs empty-lip;P⩽0.05), abrogated the TNF-α response (32.5±2.7pgml−1vs 954.9±216.3pgml−1; CL2MDP-lip vs untreated EMIU;P⩽0.05) and caused an attenuated and delayed elevation of IFN-γ. CL2MDP-lip prevented the inflammatory reaction of EMIU and inhibited the increase of leukocyte-endothelium interaction in iris vessels. Our findings emphasize the pivotal role macrophages play in the initiation of autoimmune disease.