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Phagocytosis of Silicon Oil or the Partially Fluorinated Alkane F6H8 by Isolated Human Mononuclear Leukocytes
Kociok N., Joussen A. M., Gavranic C., Kirchhof B.,
Universität zu Köln, Zentrum für Augenheilkunde, Abteilung für Netzhaut- und Glaskörperchirurgie (Köln)
Purpose: It is unclear whether stimulation of recurrent membrane formation during intraocular tamponades may be induced significantly by emulsification of the used tamponades. It has been hypothesized that macrophage activation might be supported by emulsified silicone oil droplets which might get phagocytosed at a certain size. Therefore, we analyzed the ability of human macrophages to phagocyte intraocular tamponade (silicon oil or F6H8) vesicles in vitro and analysed the cell activation status.
Method: Homogeneous sized vesicles of silicon oil (5000s and 1000s) and partially fluorinated alkanes (F6H8) were produced using a hand-driven, mini-extrusion apparatus by extrusion through polycarbonate membranes with pore sizes of 100 nm. Human mononuclear lymphocytes were isolated from blood donors by centrifugation through a solution of polysucrose and sodium diatrizoate with a density of 1.077 g/ml. Cultivation of lymphocytes with vesicle preparation occurred for 18 hours under cell culture conditions. Phagocytized vesicles were monitored by a photo microscope. Activation of lymphocytes were measured by a myeloperoxidase ELISA.
Results: Extruding silicon oil as well as F6H8 through polycarbonate membranes with pore sizes of 100 nm into water resulted in the production of stable vesicles. Whereas F6H8-vesicles disappeared after 24h, silicon oil vesicles remained stable after that time. The mean vesicle diameter was similar (F6H8: 6.54±1.47 µm, silicon oil 1000s: 6.03±2.8 µm, silicon oil 5000s: 5.03±2.3 µm). The size distribution of the produced vesicles was different in F6H8-vesicles or silicon oil vesicles. Extruding F6H8 through a 100 nm membrane resulted in vesicle diameter from 1.25 to 7.25 µm. Silicon oil extrusion resulted in 1.75 to 16.76 µm or 1 to 12.79 µm diameter vesicles when using 1000s or 5000s oil, respectively. After incubation of isolated human mononuclear lymphocytes with F6H8-vesicles or silicon oil vesicles for 18 hours in different concentrations we detected only in a small number of F6H8-vesicle treated cells phagocytized vesicles and an even lower percentage of phagocytized vesicles in silicon oil-treated cells.
Conclusions: Extruding silicon oil or F6H8 through membranes is a quick and easy method to produce vesicles of relatively homogeneous size for further analysis. Vesicles with the used size distribution are not an ideal subject for phagocytosis by mononuclear lymphocytes. This short term observations data suggest that in vitro phagocytosis of silicone oil or F6H8 vesicles is not a prominent feature.
Supported by DFG Jo 324/4-1 and Kämpgen Stiftung