Characterization of murine macrophages from bone marrow, spleen and peritoneum

Abstract

Background: Macrophages have heterogeneous phenotypes and complex functions within both innate and adaptive immune responses. To date, most experimental studies have been performed on macrophages derived from bone marrow, spleen and peritoneum. However, differences among macrophages from these particular sources remain unclear. In this study, the features of murine macrophages from bone marrow, spleen and peritoneum were compared.

Results: We found that peritoneal macrophages (PMs) appear to be more mature than bone marrow derived macrophages (BMs) and splenic macrophages (SPMs) based on their morphology and surface molecular characteristics. BMs showed the strongest capacity for both proliferation and phagocytosis among the three populations of macrophage. Under resting conditions, SPMs maintained high levels of pro-inflammatory cytokines expression (IL-6, IL-12 and TNF-α), whereas BMs produced high levels of suppressive cytokines (IL-10 and TGF-β). However, SPMs activated with LPS not only maintained higher levels of (IL-6, IL-12 and TNF-α) than BMs or PMs, but also maintained higher levels of IL-10 and TGF-β.

Conclusions: Our results show that BMs, SPMs and PMs are distinct populations with different biological functions, providing clues to guide their further experimental or therapeutic use.

Figure 1

Morphological characteristics of cultured macrophages derived from spleen (A, D), bone marrow (B, E) and peritoneal cavity (C, F), and their cell size assessment (G). All cells were cultured in complete RPMI1640 on 6-well plates, and after removal of supernatant, cells were then stained with Giemsa-wright dye (A, B, C) and to demonstrate lysosome, anti-LAMP1 (D, E, F) (original magnification x400). Cell size was assessed by flow cytometry analysis (G).

Figure 2

Expression of surface molecules on resting SPM, BM and PM was determined by flow cytometry. Red solid lines, staining with (A) anti-CD115, anti-CD206, anti-Gr-1, (B) anti-CD80, anti-CD86, anti-MHC II, (C) anti-B7-H1, anti-B7-H2, anti-B7-H3 and anti-B7-H4; grey filled , staining with the relevant isotype controls. The percentage positivity is shown at the upper right of each histogram. Data are representative of 5 separate experiments of each macrophage preparation. D: summary data of surface molecules expression. Data are mean ± SEM. *p < 0.05, **p < 0.01.

Figure 3

Macrophage growth rate treated with different M-CSF concentrations. BM, SPM and PM were cultured with M-CSF in concentrations of 2 ng/ml (A) or 10 ng/ml (B) for 0, 4, 7 and 14 days. The numbers of macrophages were quantified. Images are representative of 3 separate experiments. Data are mean ± SEM. *p < 0.05, **p < 0.01.

Figure 4

FITC-dextran uptake assay of macrophages from the three different sources. (A) Purified macrophages were incubated with FITC-dextran at 37°C for 45 min, and then washed extensively to remove excess FITC-dextran, followed by FACS analysis. Representative histograms are shown. Solid grey histograms represent control groups; solid red lines represent the percentage of phagocytic macrophages. (B) Group histograms showing both population and median fluorescence intensity (MFI) values. Data are the mean ± SEM from five separate experiments. *p < 0.05.

Figure 5

Stimulation of CD4+ T cells by macrophages presenting OVA in [3H]-thymidine incorporation assays. Isolated macrophages and dendritic cells (DCs) were loaded with OVA (10 μg/ml) and irradiated; then co-cultured with DO11.10 CD4+ T cells for 48 hours. Cultures were then pulsed with [3H]-thymidine, and incorporated counts determined. DCs were used as positive control. Data are the mean ± SEM from three separate experiments.

Figure 6

Cytokine mRNA expression profiles of the three populations (SPMs, BMs and PMs) with and without activation with LPS. mRNA levels of IL-10, TGF-β, IL-6, IL-12 and TNF-α in SPMs, BMs and PMs were measured by real time PCR with β-actin as the housekeeping gene; (n = 5). Values are expressed as 10x (gene of interest vs β-actin). *p < 0.05, **p < 0.01, ***p < 0.001.