Figure 1.
Determination of the absolute number of B-1 cell subsets in vivo and in vitro.
Peritoneal cells from P. acnes-, PS- or saline-treated mice were analyzed 24 h after treatment (in vivo). The non-adherent population of 5-day-old B-1b enriched cultures was also analyzed (in vitro). The cells were stained with mAbs to identify the B-1 cell subsets. (A to C) The small (R1) and large granular (R2) cells from the SSC-H×FSC-H dot plots are shown, followed by the CD23×CD19 analysis from R1 and R2 and the CD5×CD11b analysis from R3 and R4. (D and E) The absolute numbers of B-1b, B-1a and B-1c lymphocytes from mice treated with saline (control group), P. acnes and PS. The absolute numbers of the B-1a and B-1b lymphocytes are the sum of R1 and R2, and the B-1c values are derived from R1. The absolute cell number is the mean of two independent experiments with similar results. * p<0.05 compared to the control group. # p<0.05 between the P. acnes and PS treated groups.
Figure 2.
Analysis of the activation status of B-1b lymphocytes in vivo.
Cells from P. acnes-, PS- or saline- (control group) treated mice were analyzed 24 h after treatment to determine TLR, co-stimulatory molecule, MHC II and cytokine expression by B-1b lymphocytes. The cells were stained with mAbs to determine the absolute number (A to C) of B-1b lymphocytes that expressed each molecule and the mean fluorescence intensity (MFI) of each marker (D to F). The absolute cell number and MFI are the means of two independent experiments with similar results.* p<0.05 between the control and treated groups. #p<0.05 between the P. acnes and PS treated groups.
Figure 3.
Analysis of the activation status of B-1b lymphocytes in vitro.
The non-adherent cell population that was obtained after 5 days in a B-1b enriched culture obtained from P. acnes-, PS-, or saline- (control group) treated mice was analyzed to determine TLR, co-stimulatory molecule, MHC II and cytokine expression in B-1b lymphocytes. The cells were stained with mAbs to identify the absolute numbers (A to C) of B-1b lymphocytes that expressed each molecule and the mean fluorescence intensity (MFI) of each marker (D to F). The absolute cell number and MFI are the means of two independent experiments with similar results. * p<0.05 between the control and treated groups. # p<0.05 between the P. acnes and PS treated groups.
Figure 4.
Differentiation of B-1b cells into B-1 cell-derived phagocytes (B-1CDP).
The peritoneal cells from mice treated with P. acnes, PS or saline (control group) were cultured for 5 days. The non-adherent cells were re-cultured on cover glasses for 1 to 120 hours for phagocyte differentiation. The cells that adhered to the cover glasses were then fixed, stained with Giemsa and analyzed using optical microscopy. Magnification 500×.
Figure 5.
Myeloid and lymphoid gene expression by B-1b-enriched cells and B-1 cell-derived mononuclear phagocytes (B-1CDP).
The peritoneal cells from P. acnes-, PS- or saline-treated mice were cultured for 5 days. The non-adherent population (B-1b-enriched cells) was re-cultured for 5 more days for phagocyte differentiation (B-1CDP). After each of these periods, the lymphoid and myeloid gene expression by B-1b cells (before re-culture) and B-1CDP (after re-culture) was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). The PCR products were amplified using primers that detected the indicated transcripts. The PCR products were visualized on agarose gels by ethidium bromide staining. The amount of input cDNA was normalized by analyzing the control GAPDH transcripts. IL-7Rα: alpha subunit for interleukin 7 receptor, E2A: E box protein, Pax5: paired box 5, VpreB: surrogate light chain, EBF: early B-cell factor, LYS: Lysozyme; M-CSFR: macrophage colony-stimulating factor receptor, G-CSFR: granulocyte colony-stimulating factor receptor, GAPDH: glyceraldehyde 3-phosphate dehydrogenase.
Figure 6.
Evaluation of B-1CDP cell phagocytic activity.
B-1b cells from the supernatant of 5 day-old cultures from the saline (control group), P. acnes and PS groups were re-cultured for 24 or 120 hours to differentiate into phagocytes (B-1CDP). Saccharomyces cerevisiae cells were added to the B-1CDP cells after each time point, and the phagocytic function was determined from the phagocytic index (bars) and the percentage of phagocytic cells (lines). * p<0.05 between the control and PS groups at 120 hours (percentage of phagocytic cells).