Fetal bovine serum (FBS), RPMI-1640 medium, sodium pyruvate solution, MEM non-essential amino acids solution, l-glutamine, penicillin, and streptomycin were purchased from Gibco Life Technologies (Carlsbad, CA, USA). Lymphoprep was from Axis-Shield PoC AS (Oslo, Norway). Recombinant human IFN-γ (rhIFN-γ), recombinant human IL-10 (rhIL-10), and recombinant human IL-4 (rhIL-4) were purchased from PeproTech (Rocky Hill, NJ, USA). Carboxy-H2DFFDA and carboxyfluorescein succinimidyl ester (CFSE) were from Molecular Probes by Life Technologies (Eugene, OR, USA). Sulfo-NHS-Biotin was from Thermo Scientific (Waltham, MA, USA); streptavidin was from Calbiochem (San Diego, CA, USA), and bovine serum albumin (BSA) was from Sigma (St. Louis, MO, USA). All culture media were supplemented with 10% heat-inactivated FBS and 1 mM sodium pyruvate, 0.1 mM non-essential amino acids solution, 0.1 mM l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (complete media). Cultures were maintained in a humidified atmosphere at 37°C with 5% CO₂. Murine monoclonal anti-hCD13 (clone 452, IgG1) was purified in our laboratory from culture supernatants of the hybridoma, kindly donated by Dr. Meenhard Herlyn (Wistar Institute of Anatomy and Biology, Philadelphia, PA, USA). Murine monoclonal IgG1 anti-human FcγRI (clone 32.2) and murine monoclonal IgG2a anti-human FcγRII (clone IV.3) mAbs were purified in our laboratory from supernatants of the corresponding hybridomas obtained from American Type Culture Collection. Fab fragments of the antibodies were prepared with immobilized ficin (Pierce, Rockford, IL, USA) following the manufacturer’s instructions.

Biotin-F(ab′)2 fragments of goat anti-mouse IgG (H + L) were from Zymed (Invitrogen) and from Life Technologies (Eugene, OR, USA). Goat anti-mouse-FITC, used as a secondary antibody for immunostaining, was from Zymed (Invitrogen). Monoclonal mouse anti-human CD14 (clone RM032, IgG2a) was from Beckman Coulter Company (CA, USA). Monoclonal mouse anti-human CD80 (clone 2D10, IgG1) was from BioLegend (San Diego, CA, USA). Monoclonal mouse anti-human CD209 (clone DCN 47.5, IgG1) was from Miltenyi Biotec (Bergisch Gladbach, Germany). Monoclonal mouse anti-human CD11b (clone ICRF44, IgG1), CD11c (clone B-ly6, IgG1, κ), CD86 (clone 2331 [FUN-1], IgG1), CD206 (clone 19.2, IgG1), CD163 (clone GHI/61, IgG1, κ), and CD16 (clone 3G8, IgG1, κ) were all from BD Pharmingen (San Diego, CA, USA). Trizol, Turbo DNA-free kit, Oligo (dT) 12–18 primers and dNTP Mix 10 nM were from Invitrogen. Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV-RT) was from Promega. SYBR Green PCR Master Mix was from Applied Biosystems.

Buffy coats from healthy male donors were obtained from the Central Blood Bank of the Centro Médico Nacional Siglo XXI, IMSS, which also approved of their use for these experiments. All experiments carried out with cells from human donors were performed following the Ethical Guidelines of the Instituto de Investigaciones Biomédicas, UNAM, Ciudad de México, México. PBMCs were isolated from buffy coats by gradient centrifugation with Lymphoprep. PBMCs were washed three times with PBS, pH 7.4, and were seeded (8–10 × 10⁷ PBMCs/plate) in 100 mm × 20-mm cell culture-treated polystyrene culture dishes (Corning 430167, New York, NY, USA), in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated autologous plasma-derived serum, 1 mM sodium pyruvate solution, 2 mM MEM non-essential amino acid solution, 0.1 mM l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin for 1 h at 37°C in a humidified atmosphere with 5% CO₂, to allow monocytes to adhere to the plastic plate. Non-adherent cells were eliminated by washing, and adherent cells, enriched for monocytes (≥95% purity, as determined by flow cytometry by use of CD14 as a marker of the monocytic population), were cultured for 6 days for differentiation into macrophages, in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated FBS, 1 mM sodium pyruvate solution, 2 mM MEM non-essential amino acid solution, 0.1 mM l-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and recombinant human (rh) M-CSF at 5 ng/mL, at 37°C in a humidified atmosphere with 5% CO₂. The resulting hMDMs were polarized by incubation with rhIFN-γ (30 ng/mL), or rhIL-4 (50 ng/mL), or rhIL-10 (20 ng/mL) for 48 h. The concentration of the cytokines was established in dose–response experiments. For experiments, polarized or non-polarized macrophages were harvested by gentle pipetting. Less than 1% cell death was observed in all conditions. Macrophages from each different donor were polarized in independent experiments. Non-polarized macrophages are referred to as M0 macrophages, and hMDMs polarized with IFN-γ, IL-4, or IL-10 are referred to as Mϕ-IFN-γ, Mϕ-IL-4, or Mϕ-IL-10, respectively.

Expression of surface markers on hMDMs was analyzed by flow cytometry (Attune Acoustic Focusing Flow Cytometer, Applied Biosystem, Foster City, CA, USA). Fluorochrome-labeled monoclonal antibodies specific for CD14, CD11b, CD11c, CD80, CD86, CD206, CD209, CD163, CD64, CD32, CD16, and CD13 were used. Equivalent concentrations of matched isotype controls were included. Before staining, Fc receptors were blocked with 10% autologous human serum. Cells were fixed in 1% paraformaldehyde and analyzed by flow cytometry. The surface expression levels of each marker were measured on polarized and non-polarized macrophages of each individual donor. The panel of surface molecules was selected based on the reports of human cells (22–31), as well as potential involvement of specific molecules in macrophage activation. Data were analyzed with Attune^® Cytometric Software version 1.2.5, compatible with both Blue/Violet and Blue/Red configurations. Values are expressed as the mean fluorescence intensity (MFI) of the marker of interest and as the ratio of the MFI of the marker over the MFI of the same marker on non-polarized cells from the same donor.

Polarized or non-polarized macrophages (3 × 10⁶) were harvested and lysed in TRIZOL (Invitrogen). Total RNA was extracted according to the manufacturer’s protocol. The precipitated RNA was dissolved in RNase-free water. The quality of the RNA was assessed by measuring the ratio of absorbance at 260 and 280 nm and by visualization of the integrity of the 28S and 18S bands in agarose gels. RNA samples were treated with DNase to remove contaminating DNA. Briefly, 10 μg total RNA was treated with TURBO DNase for 30 min at 37°C. Digestion was stopped by addition of DNase inactivation reagent, for 5 min at room temperature. The samples were centrifuged, and the supernatant containing RNA was recovered. For first-strand synthesis of cDNA from RNA molecules, 1 μg RNA was incubated with oligo-dT 12–18 primer for 5 min at 70°C, and dNTPs and M-MLV-RT were added. The mixture was incubated for 60 min at 37°C and for 15 min at 75°C.

Quantitative real-time PCR was performed using gene-specific primers designed using Primer Express (Applied Biosystems). The primers are shown in Table 1. Quantitative RT-PCR (qRT-PCR) analyses were set up using 1.0 μL cDNA, 5 μL of SYBR^®Green PCR Master Mix (Life Technologies), 0.25 μL of each forward and reverse primer (250 nM), 0.2 μL of uracil-N-glycosylase (Applied Biosystems), and 3.5 μL of injectable water, totalizing a final volume of 10 μL. Reactions were run in a 7500 Fast Real-Time PCR system (Applied Biosystems) under the following conditions: 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 1 min. Melting curve analysis was carried out at the end of each PCR to confirm the specificity of PCR products.

Results were analyzed using the 7500 software (7500/7500 Fast Real-time PCR System) and were normalized using the endogenous gene HPTR-1 and the ΔΔcycle threshold method. Data are expressed in terms of relative mRNA levels in polarized macrophages to mRNA levels in non-polarized cells (M0). Ten replicates per experimental condition were performed, and differences were assessed with one-way ANOVA test with Tukey post hoc test.

To analyze the cytokine secretion profile of non-stimulated polarized macrophages, hMDMs were treated or not treated with rhIFN-γ, rhIL-4, or rhIL-10 for 48 h. The cells were collected and washed three times with PBS, pH 7.4; fresh media was added, and the cells were incubated for additional 24 h. The cell-free culture supernatants were collected and used to quantitatively measure IL-8, IL-1β, IL-6, IL-10, TNF-α, and IL-12p70 protein levels using the Cytometric Bead Array (CBA) Human Inflammatory Cytokines Kit. To determine the cytokine secretion by stimulation, polarized macrophages were stimulated for 24 h with LPS, a ligand for cell surface TLR4. The cell-free culture supernatants were used to quantitatively measure IL-8, IL-1β, IL-6, IL-10, TNF-α, and IL-12p70 protein levels by CBA. The assays were performed according to the manufacturer’s instructions and analyzed using flow cytometry. The amount of each cytokine in the supernatant was extrapolated using a standard curve based of the known amounts of the recombinant cytokine. The concentrations of the standards ranged from 20 to 5,000 pg/mL.

Sheep red blood cells (SRBCs) were maintained in Alsever’s solution until used. Modified SRBCs were prepared as described previously (14). In brief, erythrocytes (at 1.2 × 10⁹/mL in PBS-BSA 0.1%) were stained with 10 mM CFSE. The stained SRBCs were incubated with 250 μg/mL Sulfo-NHS-biotin for 20 min at 4°C. After washing, they were coated with 35 μg/mL streptavidin for 20 min at 4°C. The biotin-streptavidin-coated erythrocytes were washed and incubated with biotinylated F(ab′)2 fragments of goat anti-mouse IgG for 30 min. SRBCs labeled with CFSE and coated with biotin, streptavidin, and fragments of biotinylated anti-IgG antibodies are henceforth designated EBS-Fab. For phagocytosis assays, 1 × 10⁶ hMDMs were incubated with 2 μg of Fab fragments of mAb452 (anti-human CD13), or 4 μg Fab fragments of mAb32.2 (anti-human FcγRI), or 4 μg Fab fragments of mAbIV.3 (anti-human FcγRII), or 4 μg IgG1 (isotype-matched control), or without treatment (control) for 30 min at 4°C, washed, and incubated with EBS-Fab at a ratio of 1 monocytic cell:20 EBS-Fab, at 37°C for 30 min. Equivalent samples were incubated at 4°C as negative controls of phagocytosis. Non-internalized erythrocytes were lysed by hypotonic shock. Phagocytosis was quantified by flow cytometry (Attune acoustic focusing flow cytometer; Applied Biosystems, Foster City, CA, USA), with addition of Trypan blue 0.02% in PBS 1× (pH 4.5), to quench extracellular fluorescence from attached but not internalized erythrocytes. Data are expressed as the percentage of CFSE-positive cells (i.e., cells that have ingested at least one erythrocyte) and as phagocytic index (PI), calculated using the following formula: PI = (% CFSE-positive cells) × (MFI of cells containing erythrocytes). Results were analyzed using Attune^® Cytometric Software version 1.2.5, compatible with both Blue/Violet and Blue/Red configurations.

Human monocyte-derived macrophages were treated or not treated with rhIFN-γ, rhIL-4, or rhIL-10 for 48 h. The hMDM suspension (1 × 10⁶ polarized or non-polarized cells) was incubated with 20 μL of fluorescein-conjugated heat-killed E. coli suspension or with 40 μg/mL of fluorescein-conjugated zymosan particles for 30 min at 37°C. Negative controls were prepared in identical conditions but incubated at 4°C. Phagocytosis was stopped by washing the suspension of hMDM and bacteria or zymosan particles with ice-cold PBS. Each sample was analyzed immediately after addition of Trypan Blue 0.02% in PBS 1× (pH 4.5), to quench extracellular fluorescence from attached but not internalized bacteria or particle. Data are expressed as the percentage of FITC-positive cells. Results were analyzed using Attune^® Cytometric Software version 1.2.5, compatible with both Blue/Violet and Blue/Red configurations.

Human monocyte-derived macrophages, non-polarized or treated with rhIFN-γ, rhIL-4, or rhIL-10, were collected and washed with HBSS. Cell suspensions (1 × 10⁶ hMDM) were incubated for 30 min at 4°C in HBSS with 2 μg of Fab fragments of mAb452 (anti-CD13), or 4 μg Fab fragments of mAb32.2 (anti-human FcγRI), or 4 μg Fab fragments of mAbIV.3 (anti-human FcγRII), or 4 μg murine IgG1 (isotype-matched control), or in HBSS alone (control No Fab). After a brief centrifugation, the supernatant was discarded, and the cells were loaded with a cell-permeable ROS-sensitive fluorescent dye, by incubating with 20 mM carboxy-H2DFFDA, for 30 min at 37°C in HBSS. After washing, the receptors FcγRI, FcγRII, and CD13 were crosslinked for 30 min at 37°C by incubation with EBS-Fab at a ratio of 1 cell:20 EBS-Fab. To maximize contact, the cells mixed with the EBS-Fab were pelleted by a brief centrifugation. The cells were resuspended and transferred to wells of black 96-well Immuno Plates (Thermo Scientific, Waltham, MA, USA). Fluorescence from oxidized carboxy-H2DFFDA was determined immediately (initial reading) and every 15 min thereafter, in a Cytation 3 Cell Imaging Multi-Mode Reader (BioTek, Winooski, VT, USA) for 60 min. For quantification of ROS production in M2 macrophages, cells were preincubated on ice with two different Fab fragments [anti-FcγRI (32.2) + anti-FcγRII (IV.3), or anti-FcγRI (32.2) + anti-CD13 (452)], or with three Fab fragments (32.2 + IV.3 + 452), or no treatment (No Fab). Subsequently, cells were washed and loaded with carboxy-H2DFFDA. Cells were mounted in Black 96-well plates and read immediately and every 30 min thereafter, for 150 min.

Statistical analysis was performed using GraphPad-Prism software (GraphPad, La Jolla, CA, USA). Data are expressed as the mean ± SD. One-way ANOVA was used to compare among M0, Mϕ-IFN-γ, Mϕ-IL-4, and Mϕ-IL-10, followed by Tukey’s post hoc test for comparisons between groups. Statistical significance was considered with p < 0.05. For analysis of the expression of surface markers using normalization of data (Figures 1D–F; Figure 2B, lower plots), ANOVA was used to compare among Mϕ-IFN-γ, Mϕ-IL-4, and Mϕ-IL-10, followed by Tukey’s post hoc test for comparisons between treatment groups.

To characterize phenotypically the macrophage populations polarized in vitro, we determined the expression of a panel of surface molecules by flow cytometry after 2 days of in vitro polarization with rhIFN-γ, rhIL-4, or rhIL-10, using non-polarized cells (M0) as a control. The cell surface molecules analyzed were CD163, CD209, CD206, CD14, CD11b, CD11c, CD80, and CD86; these markers were selected based on literature reports, and the involvement of these molecules in macrophage activation (22–33). Figures 1A–C show the MFIs for each marker observed in cells from 30 different donors, whereas Figures 1D–F show the same data expressed as the ratio of the MFI of the marker of interest on cytokine-polarized cells over the MFI of the same marker on non-polarized cells from the same donor, to show more clearly the effect of each treatment on membrane expression of the markers. Macrophages treated with IL-10 (Mϕ-IL-10) showed a specific upregulation of the expression of the scavenger receptor CD163 (mean 5.2-fold increase) (p < 0.001), as compared to non-polarized and IL-4 or IFN-γ polarized macrophages (Figures 1A,D; Figure S1 in Supplementary Material). IL-4 induced the expression of CD209 [dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN)], a C-type lectin (mean 4.9-fold increase) (p < 0.001) and an increase in the expression of CD206 (mannose receptor C-type 1) (mean 3.9-fold increase) (p < 0.001). CD209 is expressed neither in M0 macrophages nor in Mϕ-IFN-γ or Mϕ-IL-10 and therefore is a useful marker for Mϕ-IL-4 (34). Also, expression of CD11b is increased (mean 2.3-fold increase) (p < 0.001), and expression of CD14 is significantly decreased (to an average of half the value of M0 cells) (p < 0.001) in Mϕ-IL-4, compared with M0, Mϕ-IFN-γ, or Mϕ-IL-10 (Figures 1B,E; Figure S1 in Supplementary Material). Finally, Mϕ-IFN-γ displayed a robust and specific upregulation of the costimulatory molecules CD80 (mean 4.9-fold) (p < 0.001) and CD86 (mean 3.6-fold) (p < 0.001) compared to M0, Mϕ-IL-4, and Mϕ-IL-10 (Figures 1C,F; Figure S1 in Supplementary Material). We did not find significant differences in CD11c expression among the different subpopulations of macrophages (Figure S1 in Supplementary Material). In summary, human Mϕ-IL-10 are characterized by high expression of CD163; Mϕ-IL-4 specifically upregulates CD209, CD206, and CD11b and downregulate CD14; and Mϕ-IFN-γ specifically upregulates the expression of the costimulatory molecules CD80 and CD86.

Once validated the surface markers for Mϕ-IL-10, Mϕ-IL-4, and Mϕ-IFN-γ polarized in vitro, we investigated the effect of polarization on membrane expression of FcγRs and CD13. Figure 2A shows representative histograms, and Figure 2B shows plots of the MFI values, both as MFI and as relative expression normalized to the expression on non-polarized cells from the same donor. Mϕ-IL-10 showed a specific upregulation of FcγRIII (CD16) (mean 2.4-fold increase) compared to all other macrophage populations (p < 0.001). Mϕ-IL-10 also showed an increased expression of the high-affinity FcγRI (CD64) compared to M0 or Mϕ-IL-4 (mean 2.5-fold increase) (p < 0.001) (Figures 2A,B). CD64 was also significantly increased in Mϕ-IFN-γ (CD64^high) compared to all other macrophages populations (mean 4.5-fold increase with respect to M0) (Figures 2A,B). Although both IL-10 and IFN-γ induced an increased expression of CD64 in comparison to non-polarized macrophages (p < 0.001), the expression induced by IFN-γ is significantly higher than the expression induced by IL-10 (p < 0.05) (Figures 2A,B). We did not find significant differences in FcγRII (CD32) or CD13 expression among different populations of macrophages (Figure 2A). These results suggest that the expression levels of FcγRs (but not of CD13) are differently modulated by the macrophage activation phenotype.

The effects of the polarizing cytokines on the levels of mRNA coding for FcγRs were evaluated by quantitative RT-PCR. Compared to the non-polarized macrophages (M0), Mϕ-IFN-γ have significantly higher FcγRI (CD64) mRNA levels (FcγRI, mean 72.5-fold increase, p < 0.001) (Figure 2C). In Mϕ-IL-10, mRNA levels of FcγRI and FcγRIII (CD16) were significantly increased (FcγRI, mean 27.1-fold increase, p < 0.01; FcγRIII, mean 39.7-fold increase, p < 0.001). The elevated mRNA level of FcγRI observed in macrophages treated with IFN-γ correlates with the increased membrane expression (Figure 2B). Likewise, the increased mRNA levels of FcγRI and FcγRIII in Mϕ-IL-10 (Figure 2C) agree with the increases in membrane expression of these receptors (Figure 2B). Thus, IFN-γ and IL-10 modulate the expression of FcγRs at the mRNA level.

All populations of macrophages express substantial levels of FcγRII on their membrane (Figure 2A). Although the histograms show no difference among the different populations, the RT-PCR analysis indicates differences in the ratio of FcγRIIa/FcγRIIb among the polarized macrophages (Figure 2C). This ratio is higher in Mϕ-IL-10 and lower in Mϕ-IL-4 and Mϕ-IFN-γ. Because the FcγRIIa isoform contains in its intracellular portion an activatory ITAM, while FcγRIIb contains an inhibitory ITIM, these differences are likely to be important in the triggering of effector functions through stimulus that engage FcγRII.

To analyze the cytokine secretion profile of the different populations of polarized macrophages, the polarizing cytokines were completely removed by washing after 48 h. Fresh media with or without LPS was added, and the cells were incubated for additional 24 h. Cell-free culture supernatants were used for determination of the production of IL-8, IL-6, IL-1β, IL-10, TNF-α, and IL-12p70. Unstimulated Mϕ-IL-10 secreted a low, yet significant amount of IL-10 (p < 0.05), whereas unstimulated Mϕ-IFN-γ produced significant levels of TNF-α compared to the other macrophage populations analyzed (p < 0.05) (Figure 3). In the absence of stimulation, Mϕ-IL-4 macrophages did not produce significant levels of any cytokine examined (Figure 3).

After stimulation with LPS for 24 h, macrophage subsets showed distinct profiles of cytokine production. In Mϕ-IFN-γ, LPS stimulation induced significant secretion of IL-8, IL-6, TNF-α, IL-1β, and IL-12p70 compared to non-polarized macrophages and macrophages treated with IL-4 or IL-10 (all p < 0.001) (Figure 3). In contrast, upon LPS stimulation, Mϕ-IL-10 and Mϕ-IL-4 secreted significantly higher amounts of IL-10 (p < 0.001 and p < 0.01, respectively) compared to M0 macrophages. Mϕ-IFN-γ macrophages also produced significant levels of IL-10 upon stimulation with LPS (p < 0.05) although to a lesser degree than Mϕ-IL-10 and Mϕ-IL-4 macrophages (Figure 3). Thus, macrophages activated to different phenotypes exhibited a specific cytokine profile in both resting state and response to LPS stimulation.

To determine whether differences in the expression of FcγRs observed among the different subpopulations of in vitro polarized macrophages (Figure 2) are reflected at the functional level, we examined phagocytosis mediated by FcγRs in non-polarized and polarized macrophages.

We used an experimental system to direct sheep erythrocytes (as the phagocytic prey) to individual receptors on the cell, as reported previously (14). Erythrocytes loaded with CFSE and labeled with biotin and streptavidin were coated with biotin-F(ab′)2 fragments of goat anti-mouse IgG (EBS-Fab). These EBS-Fabs specifically interact with the molecules on the cell surface tagged with bound Fab fragments of specific murine mAb. As the specificity of the system is based on antibodies and macrophages express FcγRs, we used Fab fragments to exclude any possible contribution of Fc fragments binding to FcγRs to the phagocytosis. Cells were preincubated on ice with Fab fragments of anti-FcγRI mAb32.2 (Fab32.2) or Fab fragments of anti-FcγRII mAb IV.3 (Fab IV.3) or no treatment (basal phagocytosis, designated as No Fab). Cells were washed, mixed with EBS-Fab, and incubated at 37 or 4°C for 30 min. After incubation, non-internalized erythrocytes were lysed by hypotonic shock. The percentage of cells with internalized erythrocytes (CFSE-positive cells) and the MFI of the CFSE-positive cells was determined by flow cytometry after quenching extracellular fluorescence with Trypan blue.

Binding of EBS-Fab to the cells through FcγRI induced its efficient internalization by Mϕ-IL-10 (mean 39.5%) (p < 0.001) and by non-polarized macrophages (M0) (mean 35.8%) (p < 0.001) compared with EBS-Fab internalization by cells not treated with Fabs (basal phagocytosis) (mean 8.26%; Figures 4A,B). Mϕ-IL-4 exhibited a poor internalization of EBS-Fab through FcγRI (mean 14.5%), which was not significantly different from basal phagocytosis. Mϕ-IFN-γ showed the lowest uptake of EBS-Fab (mean 7.2%), which was similar to phagocytosis of EBS-Fab by M0 cells with no Fab (basal phagocytosis) (Figure 4B). As expected, no significant internalization was observed when identical samples were kept at 4°C (Figure 4A, lower dot plots). To compare the PIs (PI = % CFSE-positive cells multiplied by mean fluorescence intensity), we first normalized the PI values of each sample to the basal phagocytosis of EBS-Fabs (cells not treated with Fabs) of cells from the same donor (which was given a value of 1). This was done to correct from differences in CFSE labeling of erythrocytes and in basal phagocytosis by cells from each different donor. Comparison of PI of phagocytosis mediated by FcγRI showed similar results to those obtained from comparing the percentage of CFSE-positive cells (Figure 4C). However, unlike the results of percentages, the mean PI of internalization through FcγRI by Mϕ-IL-10 (19.2-fold increase) (p < 0.001) was significantly higher than the mean PI of M0 (non-polarized) macrophages (13.8-fold increase) (Figure 5C). Thus, Mϕ-IL10 showed the highest phagocytosis mediated through FcγRI compared to all other macrophage populations, followed by non-polarized macrophages that were significantly more phagocytic than Mϕ-IL-4 and Mϕ-IFN-γ.

The binding of EBS-Fab to the cells through FcγRII induced its efficient internalization by non-polarized macrophages (M0) (mean 49.4%) (p < 0.001), Mϕ-IL-10 (mean 48.4%) (p < 0.001), and Mϕ-IL-4 (mean 22.8%) (p < 0.01) compared with no treatment (basal phagocytosis) (mean 8.26%; Figures 4D,E). Mϕ-IFN-γ did not internalize EBS-Fabs through FcγRII (mean 7.2%). No significant internalization by polarized or non-polarized macrophages was observed when identical samples were kept at 4°C (Figure 4D, lower plots). When we compared the PI, we observed similar results to those from comparing the percentages of CFSE-positive cells (Figure 4F), except that the mean PI of internalization through FcγRII by Mϕ-IL-4 (mean 3.47%) was not statistically different from basal phagocytosis. Likewise, Mϕ-IFN-γ (mean 1.6%) exhibited a PI similar to the basal phagocytosis (mean 1.0%) (Figure 4F). There were no significant differences in PIs between Mϕ-IL-10 (17.6-fold increase) and non-polarized macrophages (16.8-fold increase) for FcγRII-mediated phagocytosis. Thus, similar to the phagocytosis through FcγRI, both M0 and Mϕ-IL-10 showed significantly higher phagocytosis through FcγRII than Mϕ-IL-4 and Mϕ-IFN-γ. The low phagocytosis through FcγRII of Mϕ-IL-4 and Mϕ-IFN-γ is consistent with the low ratio of FcγRIIa/FcγRIIb in this population (Figure 2C).

We also evaluated phagocytosis of IgG-opsonized SRBC (involving the participation of all FcγRs expressed by the cell) by M0, Mϕ-IFN-γ, Mϕ-IL-4, and Mϕ-IL-10. All populations of macrophages internalized both non-opsonized and IgG-opsonized SRBC, but to different extents. M0 macrophages showed percentages of phagocytosis of non-opsonized SRBCs of 12.1% and of 52.9% for phagocytosis of IgG-opsonized SRBCs (Figure S2 in Supplementary Material). As reported previously (35), treatment of hMDM with IFN-γ resulted in a significantly reduced FcγRs-mediated phagocytosis (mean 14.8%) (p < 0.001) compared with M0 macrophages and also a lower phagocytosis of non-opsonized SRBC (mean 8.1%) (Figure S2 in Supplementary Material). In contrast, Mϕ-IL-10 showed a significantly higher phagocytosis of IgG–SRBC (mean 74.6%) (p < 0.001) and of non-opsonized SRBC (mean 15.4%). Mϕ-IL-4 showed an intermediate phagocytic capacity between that of Mϕ-IL-10 and that of Mϕ-IFN-γ, with percentages of 9.4 and 32.6% for phagocytosis of non-opsonized and IgG-opsonized SRBCs, respectively (Figure S2 in Supplementary Material). Similar to the results of phagocytosis through FcγRI and through FcγRII, both M0 and Mϕ-IL-10 show high phagocytic capacity for IgG-opsonized preys, with Mϕ-IL-10 exhibiting the highest phagocytic capacity compared to all other populations.

Even though we did not find differences in CD13 expression between different populations of macrophages (Figure 2A), we evaluated whether there were differences in CD13-mediated phagocytosis of EBS–Fab. The percentages of CFSE-positive cells of Mϕ-IL-10 (mean 38.5%) and non-polarized macrophages (M0) (mean 25.6%) were statistically significant (p < 0.001) compared with basal phagocytosis of EBS-Fab (mean 8.26%) (Figures 5A,B), while CD13-mediated phagocytosis by Mϕ-IL-4 (mean 14.3%) or Mϕ-IFN-γ (mean 4.3%) was not significantly different from non-specific phagocytosis of EBS-Fab (Figures 5A,B). No significant internalization was observed when identical samples were kept at 4°C (Figure 5A, lower dot plots). With respect to the PI of specific phagocytosis through CD13, we observed a significant increase in PI for Mϕ-IL-10 (12.8-fold increase) (p < 0.001) and non-polarized macrophages (11.4-fold increase) (p < 0.001) compared to basal phagocytosis (Figure 5B), while PIs for Mϕ-IL-4 (mean 1.4%) and Mϕ-IFN-γ (mean 1.4%) (Figure 5B) were no different to basal phagocytosis. No significant differences in PIs of CD13-mediated phagocytosis were observed between Mϕ-IL-10 and M0 (non-polarized) macrophages.

To determine whether the differences found in specific phagocytosis through FcγRI, FcγRII, and CD13 are also evident in phagocytosis through other receptors, we analyzed the effect of polarization on phagocytosis of FITC-labeled E. coli and FITC-labeled zymosan, using flow cytometry. M0, Mϕ-IL-10, and Mϕ-IL-4 efficiently internalized FITC-E. coli; Mϕ-IL-10 showed the highest percentage of phagocytosis (mean 93.9%) (p < 0.001) (Figure 6A), while Mϕ-IL-4 (mean 81.3%) showed no significant difference with phagocytosis by M0 macrophages (mean 79.1%) (Figure 6A). However, the percentage or phagocytic cells in Mϕ-IFN-γ (mean 20%) (p < 0.001) was significantly lower compared to non-polarized macrophages (M0) (mean 79.1%) (Figure 6A). No significant internalization was observed when identical samples were kept at 4°C (Figure 6A, lower dot plots). The reduced phagocytosis shown by Mϕ-IFN-γ cannot be attributed to a reduced binding of bacteria, since the binding of bacteria to Mϕ-IFN-γ (measured at 4°C) was higher compared with non-polarized macrophages and other populations of polarized macrophages (data not shown).

With respect to internalization of FITC-labeled zymosan particles, Mϕ-IL-4 exhibited the highest level of phagocytic cells (mean 69.9%) (p < 0.001) (Figure 6B). Significant phagocytic activity of zymosan was observed also for non-polarized macrophages (M0) (mean 41.6%) and Mϕ-IL-10 (mean 31.3%) (Figure 6B). Mϕ-IFN-γ macrophages showed reduced uptake of zymosan particles compared with M0 and Mϕ-IL-4 macrophages (Figure 6B) (p < 0.001). No significant internalization was observed when identical samples were kept at 4°C (Figure 6B, lower dot plots).

Table 2 summarizes the results of phagocytosis mediated by FcγRI, FcγRII, and CD13, as well as phagocytosis of E. coli and zymosan, by non-polarized and the three types of polarized macrophages.

Macrophages are phagocytic cells that produce and release ROS in response to phagocytosis or stimulation with various agents. ROS generation has been implicated in a variety of physiological responses and is a critical component of the antimicrobial repertoire of macrophages. Therefore, we investigated whether FcγRI-, FcγRII-, or CD13-mediated phagocytosis results in generation of ROS in each population of macrophages. Cells were incubated on ice with Fab fragments of anti-FcγRI mAb32.2 (Fab 32.2) or Fab fragments of anti-FcγRII mAb IV.3 (Fab IV.3) or Fab fragments of anti-CD13 (Fab 452) or no treatment (No Fab). After this, cells were washed and loaded with carboxy-H2DFFDA (ROS-sensitive fluorescent dye). Cells were washed, and the receptors FcγRI, FcγRII, and CD13 were crosslinked for 30 min at 37°C by incubation with EBS-Fab (without CFSE), so as to replicate the stimulation leading to phagocytosis. Cells and EBS-Fabs were transferred to black 96-well plates, and the fluorescence of oxidized carboxy-H2DFFDA was measured at different times. As a control, macrophages were also stimulated with heat-killed E. coli. In M0 macrophages, phagocytic stimulation through FcγRI, FcγRII, and CD13 as well as E. coli, induced a slow ROS response that after 150 min became significantly higher compared to ROS production by cells treated only with EBS-Fabs. In contrast, Mϕ-IFN-γ produced significant amounts of ROS after only 30 min. Figure 7 shows ROS production by the different populations of polarized macrophages after 60 min of stimulation with different stimuli. To evaluate the effect of the polarization on ROS production, the results are shown relative to the ROS production induced by each stimulus in M0 cells. Compared to non-polarized macrophages, crosslinking of FcγRI, FcγRII, or CD13 induces significantly higher production of ROS in Mϕ-IFN-γ when crosslinking was induced by a phagocytable particle (EBS-Fab) (Figure 7) [Fab 32.2 (α-FcγRI) + EBS-Fabs, mean 16.4-fold increase] (p < 0.001); [Fab 452 (α-CD13) + EBS-Fab, mean 11.13-fold increase] (p < 0.01); (Fab IV.3 [α-FcγRII] + EBS-Fab, mean 11.9-fold increase) (p < 0.01). The increase in ROS generation by phagocytic stimuli through FcγRI, FcγRII, or CD13 was similar to the generation of ROS by stimulation with E. coli (positive control) (Figure 7) (E. coli, mean 24.4-fold increase) (p < 0.001). In contrast, Mϕ-IL-4 and Mϕ-IL-10 showed no difference in ROS production compared to M0 cells, after 60 min of stimulation by FcγRI, FcγRII, or CD13 crosslinking (Figure 7).

Altogether, these results suggest that phagocytosis through FcγRI, FcγRII, and CD13 in Mϕ-IFN-γ leads to strong generation of ROS that could potentially promote the degradation of ingested material.

We observed that although Mϕ-IL-10 did not produce significant amounts of ROS after crosslinking of FcγRI, FcγRII, or CD13, these cells did show a modest but significant ROS production after stimulation with E. coli (mean 3.5-fold increase relative to M0 macrophages, Figure 7). Although anti-inflammatory M2 macrophages are usually described as non-ROS-producing cells (36), it was recently reported that stimulation with PMA induces ROS production by M2 macrophages (37). Thus, we considered the possibility that Mϕ-IL-10 could produce ROS, albeit at levels lower than those produced by Mϕ-IFN-γ, and this led us to evaluate ROS production at longer times and under stimulation by two or more receptors. For this, different subpopulation of macrophages were preincubated on ice with Fab fragments of anti-FcγRI mAb32.2 (Fab 32.2) alone, or with Fab 32.2 and Fab fragments of anti-FcγRII mAb IV.3 (Fab IV.3), or with Fab fragments 32.2 and Fab fragments of anti-CD13 (Fab 452) or with the three Fab fragments (32.2, IV.3, and 452), or no treatment (No Fab). Subsequently, cells were washed and loaded with carboxy-H2DFFDA. Cells were washed and incubated for 30 min at 37°C with EBS-Fab (without CFSE), so as stimulate phagocytosis. Cells and EBS-Fabs were transferred to black 96-well plates, and the fluorescence of oxidized carboxy-H2DFFDA was read immediately (initial reading) and for 2 h 30 min, with reading intervals every 30 min. As a positive control, macrophages were also stimulated with heat-killed E. coli. The results are shown in Figure 8. Mϕ-IFN-γ generated high levels of ROS after stimulation through FcγRs and CD13 or by E. coli, such that the fluorescence signal overflowed the detection limit of the instrument after 60 min. Mϕ-IL-10, on the other hand, produced ROS at amounts that became significant after 90 min of stimulation by E. coli or by coaggregation of two (FcγRI and FcγRII or FcγRI and CD13) or three (FcγRI + FcγRII + CD13) receptors. ROS production by crosslinking of FcγRI alone was significant only after 150 min (Figure 8), while crosslinking FcγRII or CD13 separately did not induce significant production of ROS (data not shown). The increase in ROS generation by stimulation of Mϕ-IL-10 simultaneously through more than one phagocytic receptor was similar to the generation of ROS by stimulation with E. coli (Figure 8). In contrast, Mϕ-IL-4 did not produce significant levels of ROS under any stimulation even after 150 min. These results suggest that simultaneous stimulation through Fcγ receptors and CD13 as well as stimulation with E. coli are strong signals that are able to induce ROS production in Mϕ-IL-10 macrophages, although this production is significantly lower than ROS generation in Mϕ-IFN-γ.

Table 2 summarizes the results of ROS production by non-polarized and the three types of polarized macrophages after stimulation through FcγRI, FcγRII, and CD13 separately and in combination.