Bone Marrow-Derived and Elicited Peritoneal Macrophages Are Not Created Equal: The Questions Asked Dictate the Cell Type Used

Abstract

Macrophages are a heterogeneous and plastic population of cells whose phenotype changes in response to their environment. Macrophage biologists utilize peritoneal (pMAC) and bone marrow-derived macrophages (BMDM) for in vitro studies. Given that pMACs mature in vivo while BMDM are ex vivo differentiated from stem cells, it is likely that their responses differ under experimental conditions. Surprisingly little is known about how BMDM and pMACs responses compare under the same experimental conditionals. While morphologically similar with respect to forward and side scatter by flow cytometry, reports in the literature suggest that pMACs are more mature than their BMDM counterparts. Given the dearth of information comparing BMDM and pMACs, this work was undertaken to test the hypothesis that elicited pMACs are more responsive to defined conditions, including phagocytosis, respiratory burst, polarization, and cytokine and chemokine release. In all cases, our hypothesis was disproved. At steady state, BMDM are more phagocytic (both rate and extent) than elicited pMACs. In response to polarization, they upregulate chemokine and cytokine gene expression and release more cytokines. The results demonstrate that BMDM are generally more responsive and poised to respond to their environment, while pMAC responses are, in comparison, less pronounced. BMDM responses are a function of intrinsic differences, while pMAC responses reflect their differentiation in the context of the whole animal. This distinction may be important in knockout animals, where the pMAC phenotype may be influenced by the absence of the gene of interest.

Keywords: bone marrow-derived macrophages; cytokines; flow cytometry; gene expression; peritoneal macrophages; phagocytosis; polarization.

Figure 1

Bone marrow-derived macrophages exist as two distinct populations. Bone marrow was extracted and differentiated in L cell media as described in Methods. Adherent cells were collected 7 days post-harvesting and analyzed by flow cytometry (representative of BMDMs from 10 animals). (A) Virtually all (98 ± 2%) of the live singlets were CD11b⁺F4/80⁺. (B) After gating out dead cells/debris and selecting for singlets, two populations were identified: a minor (15.8 ± 3.4%) population of high forward and side scatter (large) cells and a major population that is smaller with lower side scatter. The large population had significantly higher expression of both F4/80 and CD11b (p < 0.01, n = 10, paired t-test).

Figure 2

The size and granularity of bone marrow-derived and peritoneal macrophages are similar, but not identical. (A) Harvested peritoneal cells contain a population of small, moderately granular cells (purple arrow) that are reduced upon adhesion and not found in preparations of BMDMs. (B,C) Harvested peritoneal cells have a minor (11 ± 4.4%) population of Siglec F⁺ cells that is substantively removed upon adhesion (2.1 ± 1.1% post-adhesion) that co-localizes with the small, granular population. (D) BMDMs and adherent pMACs are similar with respect to size (FSC) and granularity (SSC). Representative of 10 preparations each of bone marrow and peritoneal macrophages.

Figure 3

Expression of surface molecules by steady state BMDMs and pMACs. BMDMs and adherent pMACs were stained for the indicated molecules and their expression quantified by flow cytometry. Results of one BMDM and one pMAC preparation, stained on the same day, are presented. Data is presented as histograms with compensated fluorescence of the indicated antigen on the x-axis. Representative of BMDM and pMACs from 7 mice. (A) Antigens whose expression was not significantly different between BMDMs (black) and pMACs (red). (B) Antigens significantly upregulated in BMDM compared with pMACs. Table: unpaired t-test (n = 7 BMDM and 7 pMACs preparations) p-values for differences in antigen expression; green shading highlights significant differences, with expression in BMDMs significantly higher than pMACs. N.D. Not determined.

Figure 4

BMDMs are more phagocytic than pMACs. BMDMs and pMACs were subjected to synchronized phagocytosis using pHrodo E. coli ± IgG (50:1 MOI) (A), Zymosan 488 ± IgG (5:1 MOI) (B), or BIgG (20:1 target to cell ratio) (C). (A) Phagocytosis was stopped at the indicated times by dislodging bound targets, diluting the sample in cold buffer, and analyzing by flow cytometry. (B) The fluorescence of extracellular zymosan was quenched with trypan blue immediately before analysis (n = 3 animals, 1 × 10⁵ cells collected/sample). (C) For BIgG, cells were fixed and incomplete phagosomes were detected by the addition of Alexa 488-conjugated goat anti-rabbit IgG (Invitrogen) to label the IgG on the exposed targets. The number of fully internalized targets was quantified microscopically and the phagocytic index (PI) calculated. PI = (# internal beads/# cells counted) × 100. (n = 3 animals, 30–40 cells per animal). (A–C) *p < 0.05; **p < 0.01; ***p < 0.001, unpaired t-test. (D) Composite data from 3 each pMAC and BMDM preparations reporting the rate of phagocytosis (slope of the line) and determining statistical significance using an unpaired t-test. Because internalization of zymosan plateaus by 30 min, an initial rate of phagocytosis was calculated using the 5–15 min timepoints (dashed line). BMDM are more phagocytic for all targets, regardless of the receptor used or the method used to quantify phagocytosis.

Figure 5

FcγRI is necessary and sufficient for IgG-mediated phagocytosis. BMDM from wild type (CD57BL/6), FcγRIIb knockout mice (CD32^−/−) or mice expressing only FcγRI (FcγRI only) were transduced with virus encoding PKC-ε-GFP (to visualize internalization) and phagocytosis followed by live imaging as detailed in Methods. Compared to CD57BL/6 (A); phagocytosis was unaffected by removal of CD32 (B). Adding α-CD16/32 (C) or α-CD16.2 (D) to CD32^−/− cells did not affect internalization. Blocking FcγRI with α-CD64 reduced internalization (E) but not target binding (inset), supporting a role for FcγRI in IgG-mediated phagocytosis. (F–H) Internalization by C57BL/6 and FcγRI only macrophages is similar. Quantitation of phagocytic rate from movies reveal that uptake by FcγRI only cells is equivalent to controls, arguing that FcγRI is necessary and sufficient for IgG-mediated phagocytosis. (H) Each dot represents data from one cell, statistical significance was tested using an unpaired t-test.

Figure 6

The respiratory burst is equivalent in BMDMs and pMACs. BMDMs and pMACs, untreated (A) or polarized overnight with IFN-γ or IL4/IL13 (B), were stimulated with an empirically determined amount of immune complexes (IC) in the presence of Amplex Red®, a H₂O₂ reporter. Fluorescence intensities were acquired every 5 min for 4 h and the relative rate of the burst determined from the slope of the line (C). Data is presented as mean ± SEM for pMACs and BMDM from 3 animals at the lowest dose of three doses of IC tested; two higher doses increased fluorescence but did not produce any difference in the burst. NSD = not significantly different.

Figure 7

BMDM and pMACs respond differently to polarizing cytokines: surface molecule expression. BMDM and pMACs were treated with IFN-γ or IL4/IL13 for 24 h, stained for the indicated antigens, and analyzed by flow cytometry. Each line represents a single animal's cells under the three conditions. Results are reported as the within animal deviation of the measurement from the means of that animal's cells under the three conditions. This essentially removes the animal-to-animal variance and considers the within animal response. Interactions are apparent when the pattern of the responses differs between the BMDM (black) and pMAC (red) lines. Genes are loosely grouped: (A) genes validating polarization, (B) polarization dependent gene expression, and (C) genes whose expression is independent of cell type and polarization state. Insets: Message levels for the α chains of the Fc receptors were quantified by qPCR following cytokine treatment. The data were normalized to β-actin and the fold increase over untreated cells was calculated using the ΔΔCt method. Statistical significance was determined by ANOVA. Data for α chain PCR are presented as mean ± SEM (n = 3 BMDM and 3 pMACs). Daggers indicate significance based on cell type: ^‡p < 0.05. Asterisks indicate differences based on polarization conditions: *p < 0.05, **p < 0.005. In general, BMDM responses are more robust than those of pMACs. pMACs and BMDM from n = 7 animals were analyzed.

Figure 8

BMDM and pMACs respond differently to polarization; relative gene expression. BMDM (white bars) and pMACs (red bars) were polarized with IFN-γ or IL-4/IL-13 as in Figure 7. RNA was extracted and subjected to qRT-PCR for the indicated genes. Expression for each gene was normalized to β-actin (ΔCt) and the RNA fold change determined using the ΔΔCt method (ΔCt gene after polarization-ΔCt M0 control). Genes are loosely grouped into M1 (A) and M2 (B) markers. After logarithmic transformation, the data were analyzed and statistical significance determined by linear regression. Daggers indicate significance based on cell type (^‡p < 0.05, ^‡‡p < 0.005). Asterisks denote differences based on polarization conditions: *p < 0.05, **p < 0.005).

Figure 9

BMDM and pMACs respond differently to polarization: release of cytokines and chemokines. BMDM and pMACs were treated with IFN-γ or IL4/IL13 as in Figure 7. At 24 h, the media was collected, cells and debris removed by centrifugation, and cytokine (A) and chemokine (B) concentrations in the supernatant quantified by Multiplex®. IL-1β, IL-12p70, and TNF-α were below the limits of detection. After logarithmic transformation, data were analyzed using a linear regression model and ANOVA. Data are presented as mean ± SEM (n = 4–7 animals). Statistical significance between cell types is indicated by daggers (^‡p < 0.05, ^‡‡p < 0.005); differences due to polarization conditions by asterisks; *p < 0.05, **p < 0.005.

Figure 10

BMDM and pMACs respond differently to polarization: variations in protein release with polarization. BMDM and pMACs were treated with IFN-γ or IL4/IL13 for 24 h, the media collected, and protein release quantified by Multiplex®. The data are plotted as the deviation from the average for each treatment. Each line represents a single animal's cells under the three conditions. Results are reported as the within animal deviation of the measurement from the means of that animal's cells under the three conditions. This is essentially a repeated measures ANOVA, removing the animal-to-animal variance and considering the within animal response. Interactions are apparent when the pattern of the responses differs between the BMDM (black) and pMAC (red) curves (e.g., IL10, CXCL1, and CCL2). (A) cytokines and (B) chemokines. In general, BMDM responses are more robust than those of pMACs. n = 4–7 for each cell type.

Figure 11

BMDM and pMACs respond differently to polarization: normalized protein secretion. Protein data from Figure 9, normalized to untreated control and reported as fold change. (A) cytokines and (B) chemokines. Data are presented as mean ± SEM (n = 4–7 animals per condition). After logarithmic transformation, data were analyzed using a linear regression model and ANOVA. Statistical significance between cell types is indicated by daggers (^‡‡p < 0.005); differences due to polarization indicated by asterisks; **p < 0.005.