Apoptotic cell phagocytosis is increased in CD68⁺F4/80⁺ RP macrophages in the absence of MZMs. (A) Eight- to 12-week-old female B6 mice were injected intravenously with 167 μg of CLs or an equivalent amount of PBSLs. Forty-eight hours later 10⁷ PKH26-labeled apoptotic thymocytes were transferred intravenously, and at 30 minutes and 2 hours after apoptotic cell injection CD68⁺F/480⁺ macrophages were assessed for cell uptake. Histograms are representative for 5 mice/group enumerated in panel B. (C) Relative PKH26 fluorescence intensity for the CD68⁺F/480⁺PKH26⁺ macrophage populations depicted in panel A 2 hours after apoptotic thymocyte administration. MFI indicates mean fluorescence intensity; n = 5 mice/group. (D) Relative expression of the surface antigens CD86, MHCII, and CCR7 was examined in CD68⁺F4/80⁺ macrophages by FACS analysis in mice treated as described in panel A at 2 hours after apoptotic cell injection. Values were parsed on the basis of PKH26 fluorescence, which was indicative of apoptotic cell phagocytosis. Bars represent the mean value of 5 samples ± SD. (E) Spleens from mice treated as in panel A were collected at 2 hours after apoptotic cell injection. Frozen sections (5 μm) were stained with antibodies against the macrophage marker MOMA2 and examined for localization of macrophages and apoptotic material by fluorescent microscopy. Arrow highlights area of apoptotic cell capture in the MZ zone in PBSL control spleens which is absent in CL-treated splenic sections. Images are representative for 3 mice/group. Image magnification 20×. All experiments were repeated multiple times with similar results. *P < .05 and **P < .01 as determined by the unpaired Student t test.