Marginal macrophage depletion alters apoptotic cell localization without disrupting the marginal sinus. (A) Eight- to 12-week-old female B6 mice were injected intravenously with 167 μg of CLs or an equivalent amount of PBSLs in 200 μL final volume. Forty-eight hours later the mice were injected intravenously with 108 PKH26-labeled apoptotic thymocytes. Two hours after apoptotic cell injection the spleen was removed, and 5-μm frozen sections were stained with antibodies against MARCO. The sections were examined by microscopy for localization of the apoptotic cells within the spleen. RP indicates red pulp; and PALS, periarteriolar lymphoid sheath. CL-N = 7 mice, PBSL-N = 4 mice. (B) Image analysis of PHK26 fluorescence in the PALS of MZM-depleted and control mice. Y-axis values for size are image pixels and are representative for relative signal size. Bars represent the mean values for CL-N = 7 mice, PBSL-N = 4 mice. **P < .01 as determined by the unpaired Student t test. (C) B6 mice, as in panel A, were injected with the indicated amounts of CLs or an amount of PBSLs equivalent to the highest dose of CLs administered intravenously. Forty-eight hours later spleens were collected, and 10-μm frozen sections were stained with antibodies against MadCAM-1 (red) and B220 (green). Images are representative of 5 mice/group. (D) B6 mouse splenic section (n = 3 mice/group) 2 hours after injection with 5 × 108 fluorescent microparticles administered as described in “Immunofluorescence and immunohistochemistry” and stained with antibodies against MARCO (red) and F4/80 (blue). (E) B6 mice (n = 3 mice/group) were depleted of MZMs, and microparticles were injected as described in panel D. Two hours after injection spleens were collected, and sections were examined for particle localization and stained with antibodies against B220 (red) to visualize the follicle. All images are 2-μm confocal pictures captured as described in “Immunofluorescence and immunohistochemistry” and are representative for each group. Image magnification 20×. These experiments were repeated at least twice with similar results.
