Figure 1.
Confocal laser scanning microscopic analysis of macrophages co-cultured with DQ-OVA-liposome conjugates.
A, DQ-OVA was coupled to either stearoyl or oleoyl liposomes and added to the culture of cloned macrophages expressing DM-DsRed (class II) or labeled with red fluorescein (class I), as described in Materials and Methods. Two hours after the onset of the culture, macrophages were recovered and analyzed using confocal laser scanning microscopy. These optically merged images are representative of most cells examined by confocal microscopy. Yellow, co-localization of green (DQ-OVA after proteolytic degradation) and red (macrophage DM or class I); cell only, macrophages without co-culture with DQ-OVA-coupled liposomes. B, the green- and yellow-color compartments in the immunofluorescent pictures were quantified by the image analysis software MetaMorph, as described in Materials and Methods. Ratios of the yellow to green compartments are shown. Data represent the mean values ± SD of the images shown in Fig. 1A. Asterisk, significant (p<0.01) difference of samples.
Figure 2.
Uptake of liposome-coupled OVA by macrophages.
Alexa-labeled OVA was coupled to either stearoyl or oleoyl liposomes and added to the culture of cloned macrophages as described in Materials and Methods. Thirty minutes after the onset of the culture, macrophages were recovered and analyzed using flow cytometry.
Figure 3.
Influence of inhibitors for uptake of OVA coupled to oleoyl liposomes by macrophages.
Alexa- or DQ-labeled OVA was coupled to oleoyl liposomes and added to the culture of macrophages as described in Materials and Methods. Treatment of macrophages with cytochalasin B or DMA was done 60 minutes prior to the addition of OVA-liposome conjugates.
Figure 4.
Intracellular localization of liposomal antigens taken up by macrophages.
A, DQ-OVA was coupled to oleoyl liposomes and added to the culture of cloned macrophages of which endosomal marker EEA1-positive compartments, or lysosomal marker LAMP-1-positive compartments were stained as described in Materials and Methods. Two hours after the onset of the culture, macrophages were recovered and analyzed using confocal laser scanning microscopy. These optically merged images are representative of most cells examined by confocal microscopy. Yellow, co-localization of green (DQ-OVA after proteolytic degradation) and red (macrophage EEA1 or LAMP-1); cell only, macrophages without co-culture with DQ-OVA liposomes. B, the green- and yellow-color compartments in the immunofluorescent pictures were quantified by the image analysis software MetaMorph, as described in Materials and Methods. Ratios of the yellow to green compartments are shown. Data represent the mean values ± SD of the images shown in Fig, 4A. Asterisk, significant (p<0.01) difference of samples.
Figure 5.
IFN-γ production by splenic CD4/CD8+ T cells of mice immunized with OVA after co-culture with CD11c+ cells pulsed with OVA coupled to oleoyl liposomes.
Splenic CD4/CD8+ T cells were taken from mice immunized with OVA and were cultured with CD11c+ cells pulsed with OVA coupled to oleoyl liposomes with or without inhibitors as described in Materials and Methods. IFN-γ production of T cells in the supernatants in the absence of inhibitors was normalized to 100%. Data represent the mean values ± SD of triplicate culture. Asterisk, significant (p<0.01) difference as compared with the ‘no inhibitor’ group.