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Depletion of macrophages with liposomal clodronate decreases leukocyte-endothelium interaction in experimental melanin-induced uveitis (EMIU)
J. Puchta, U. Pleyer, R. Reszka*, H. Baatz
Introduction: Experimental melanin-induced Uveitis (EMIU) is a model for an auto-immune-mediated iridocyclitis and choroiditis. The early inflammatory reaction is associated with increased leukocyte adhesion to the vascular endothelium. Macro-phages are known to play a central role in antigen processing and presentation. To study the role of macrophages in EMIU, we analysed changes in leukocyte ad-hesion to iris venules of living rats with EMIU after pretreatment with liposomal clodronate, which is known to selectively eliminate macrophages.
Methods: EMIU was induced in female Lewis rats (n = 66) by intraperitoneal im-munization with 250 µg bovine crude melanosomes (BMA) emulsified in complete Freund´s adjuvant (CFA) and pertussis toxin (PTX). Animals were treated with 2 ml of liposomal clodronate (DMDP-lip) on day 2; 1; 4; 6 and 8 after immunization. Controls received empty liposomes at the same time points after immunization. Leukocytes were stained with intravasal rhodamine 6G. Using intravital fluores-cence microscopy, postcapillary iris venules of rats were examined on day 8 and 10 to quantify leukocyte rolling and firm adhesion of leukocytes to the vascular endothelium. Cells and protein in aqueous humour, the leukocyte count in peripheral blood and TNF->a and IFN-g > serum levels were determined.
>Results: > Slit-lamp biomicroscopy showed no inflammatory signs in macrophage-depleted animals. Intravital fluorescence microscopy of iris vessels revealed a de-creased percentage of rolling leukocytes on day 8: 8.2±1.1 vs. 15.2>± 1.6; 5.2± 0.5 % (DMDP-lip vs. EMIU; control, mean ± SEM, ANOVA, p<0.05) and on day 10: (2.6± > 0.3 vs. 14.2±1.6; 5.9±0.7 %). Likewise, a significant decline in the number of firmly adherent leukocytes was detected on day 8 and 10: 88±13 vs. 175±18 /mm2 and 129±13 vs. 372±31 /mm2 (DMDP-lip vs. EMIU). No changes in leukocyte firm adhesion were detected in EMIU animals injected with empty liposomes. De-creased leukocyte-endothelium interaction corelated with a significant decrease in cell emigration (11±2 vs. 82±17; 100±29 cells/µl) and protein exsudation (2.7±0.3 vs. 12.5±1.7; 14.2±2.1 mg/ml) into the anterior chamber on day 10 (DMDP-lip vs. EMIU; control).
>Conclusions: > In EMIU, depletion of macrophages with liposomal clodronate ame-liorates the inflammatory reaction. In autoimmune-mediated uveitis, macrophages play a crucial role in the initiation of leukocyte-endothelium interaction, possibly by activating inflammatory cells and by the production of proinflammatory cytokines.
>Supported by Deutsche Forschungsgemeinschaft, Grant Ba 1408/1-1
Eye-Hospital Charité, Humboldt University, Augustenburger Platz 1, D-13353 Berlin and *Max Delbrück Centre for Molecular Medicine (MDC), "AG Drug Targeting", D-13125 Berlin-Buch