Figure 1.
Identification of resident peritoneal MØ subsets.
PC from C57BL/6 were harvested and stained with fluorochrome-labeled antibodies directed against F4/80, CD19, CD11c and IAb for flow cytometry analysis. (A) Doublet cells were excluded according to forward scatter profiles (FSC-A and FSC-H). Subsequently, (B) CD19high cells and (C) CD11chigh cells were also excluded, and (D) F4/80+ cells were selected. (E) F4/80 and IAb expression defined three populations: LPM (F4/80highIAb-neg), SPM (F4/80lowIAb-high) and granulocytes (F4/80lowIAb-neg). These three subpopulations were purified by cell sorting on a FACS Vantage, and their morphology was evaluated ex vivo from cytospin slides (F), or after in vitro culture in chamber slides for 12 h (G). Slides were stained with hematoxylin and eosin (H&E) and analyzed by optical microscopy (40×).
Table 1.
Phenotypic analysis of resident peritoneal MØ subsets.
Figure 2.
Zymosan and T. cruzi injection alters the MØ compartment of PerC.
C57BL/6 mice were injected i.p. with zymosan (1 mg/mouse) or T. cruzi (106 parasites/mouse) and at 30 min (A) or 48 h (B) after stimulation, PC from naive and injected mice were harvested and stained as described in M&M. Sequential gates were made as shown in Fig. S1. Plots show the frequencies of each subpopulation. (C) F4/80lowMHCIIint cells present within PerC 48 h after injections were evaluated according the expression of Ly6C. Gray lines represent FMO [23], [25], the black lines show F4/80lowMHCIIint cells from zymosan- (hairline) or T. cruzi-(bold line)-exposed PerC. (D) Total numbers of SPM, LPM and monocytes 48 h after zymosan or T. cruzi exposure (within MØ gate) are shown in panel. Data are representative of more than 3 independent experiments.
Figure 3.
Zymosan and T. cruzi inoculation leads to PerC cellular renewal and improves local cell effector activities.
C57BL/6 mice were injected i.p. with zymosan (1 mg/mouse) or T. cruzi (106 parasites/mouse) and 48 h later PC from naive and injected mice were harvested and cultured for 4 h. Non-adherent cells were removed by sequential washes. (A) β-Gal staining was performed as described in M&M. Slides were analyzed by optical microscopy (40×) and pictures were captured by Sony Camera (zoon 3.0). (B and C) Adherent PCs cells from 48 h-exposed mice and controls were stimulated in vitro with media or LPS (1 µg/mL). (B) Nitrite concentrations in culture supernatants were determined by the Griess reaction after 48 h of in vitro culture. Numbers represent the mean ± SD of triplicate samples. ***p<0.001 in relation to non-stimulated group and &&& p<0.001 when compared to the control group. (C) Frequencies of IL-12-producing F4/80+ cells were evaluated by intracellular staining as described in M&M. Bars indicate the standard deviation (SD). *** p<0.001 when compared to the control group. Data are representative of more than 3 independent experiments.
Figure 4.
SPM are more responsive than LPM and monocytes to infectious stimuli.
C57BL/6 mice were injected i.p. with zymosan (1 mg/mouse) or T. cruzi (106 parasites/mouse) and PCs were harvested 30 min or 48 h after stimulation. (A) SPM and LPM from C57BL/6 zymosan-exposed mice were FACS-sorted 30 min after injection and the presence of internalized zymosan particles was observed by optical microscopy. Slides were made with 105 cells from purified MØ subsets, stained with H&E and analyzed by optical microscopy (40×). (B) Numbers represent the mean ±SD of internalized zymosan particles per cell in each MØ subset. *** p<0.001 when compared to the LPM group. (C) PC from control or 48 h-exposed mice were cultured for 6 h in the presence of brefeldin A with or without LPS (1 µg/ml) plus rIFN-γ (5 ng/ml). Titles above plots indicate in vivo - in vitro stimulations. Values inside gates represent the frequencies of IL-12-producing cells in each subpopulation. Data are representative of more than 3 independent experiments.