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. 2016 Aug 9;149(3):320–328. doi: 10.1111/imm.12647

M2 polarization of murine peritoneal macrophages induces regulatory cytokine production and suppresses T‐cell proliferation

Shinji Oishi 1, Ryosuke Takano 1, Satoshi Tamura 1, Shinya Tani 1, Moriya Iwaizumi 1, Yasushi Hamaya 1, Kosuke Takagaki 1, Toshi Nagata 2, Shintaro Seto 3, Toshinobu Horii 3, Satoshi Osawa 4, Takahisa Furuta 5, Hiroaki Miyajima 1, Ken Sugimoto 1,✉
PMCID: PMC5046056  PMID: 27421990

Summary

Bone‐marrow‐derived macrophages are divided into two phenotypically and functionally distinct subsets, M1 and M2 macrophages. Recently, it was shown that adoptive transfer of M2‐polarized peritoneal macrophages reduced the severity of experimental colitis in mice. However, it is still unclear whether peritoneal macrophages possess the same ability to be polarized to cells with functionally different phenotypes and cytokine production patterns as bone‐marrow‐derived macrophages. To address this question, we examined the ability of peritoneal macrophages to be polarized to the M1 and M2 phenotypes and determined the specific cytokine profiles of cells with each phenotype. We showed that peritoneal macrophages, as well as bone‐marrow‐derived macrophages, were differentiated into M1 and M2 phenotypes following stimulation with interferon‐γ (IFN‐γ) and interleukin‐4 (IL‐4)/IL‐13, respectively. Following in vitro stimulation with lipopolysaccharide, M2‐polarized peritoneal macrophages predominantly expressed T helper type 2 (Th2) cytokines and regulatory cytokines, including IL‐4, IL‐13, transforming growth factor‐β and IL‐10, whereas M1‐polarized peritoneal macrophages expressed negligible amounts of Th1 and pro‐inflammatory cytokines. ELISA showed that M2‐polarized peritoneal macrophages produced significantly more IL‐10 than M1‐polarized peritoneal macrophages. Notably, M2‐polarized peritoneal macrophages contributed more to the suppression of T‐cell proliferation than did M1‐polarized peritoneal macrophages. The mRNA expression of Th2 cytokines, including IL‐4 and IL‐13, increased in T‐cells co‐cultured with M2‐polarized macrophages. Hence, our findings showed that M2 polarization of peritoneal macrophages induced regulatory cytokine production and suppressed T‐cell proliferation in vitro, and that resident peritoneal macrophages could be used as a new adoptive transfer therapy for autoimmune/inflammatory diseases after polarization to the regulatory phenotype ex vivo.

Keywords: adoptive transfer, M1 macrophage, M2 macrophage, peritoneal macrophage, regulatory cytokines

Abbreviations

Arg1

arginase1

IFN

interferon

IL

interleukin

iNOS

inducible nitric oxide synthase

IRF

interferon regulatory factor

TNF

tumour necrosis factor

Introduction

Macrophages are an essential component of both innate and adaptive immunity and play a central role in host defence and inflammation.1, 2, 3 It is well known that activated macrophages are divided into two subsets, classically (M1) and alternatively (M2) activated macrophages.4, 5, 6 The M1 phenotype is characterized by the expression of high levels of pro‐inflammatory cytokines, including interleukin‐12 (IL‐12), IL‐23 and tumour necrosis factor‐α (TNF‐α), as well as high nitric oxide and reactive oxygen intermediate production.7, 8 In contrast, cells of the M2 phenotype typically produce IL‐10 and IL‐1 receptor antagonist (IL‐1Ra) and have high levels of scavenger, mannose and galactose receptors.9

M1 macrophages have been shown to have strong anti‐bacterial and anti‐tumour effects.7, 8 In contrast, M2 macrophages are thought to have immunoregulatory functions and to be involved in parasite containment and promotion of tissue remodelling and tumour progression.1 Recent studies showed that there are two key transcriptional regulators, interferon regulatory factor 5 (IRF5) and IRF4 that polarize macrophages to the M1 and M2 phenotypes, respectively. IRF5 expression drives M1 macrophage polarization by directly inducing the expression of pro‐inflammatory cytokines, such as IL‐6, IL‐12 and IL‐23, while repressing the transcription of anti‐inflammatory cytokines such as IL‐10.10, 11 In contrast, IRF4 has been shown to be a crucial mediator of M2 macrophage polarization.12, 13 Jumonji domain containing‐3, an upstream effector of IRF4‐induced M2 polarization, and IRF4 are critically involved in IL‐4‐dependent induction of M2 marker genes, such as Arg1, Ym1 and Fizz1 13; however, the molecular mechanisms underlying the induction of M2 macrophage polarization by IRF4 are unknown.

Interestingly, the phenotype of polarized M1 and M2 macrophages can be reversed in vitro and in vivo.14, 15 Cytokines and growth factors are involved in the reprogramming of M1 and M2 macrophages. Interferon‐γ (IFN‐γ) induces M1 macrophages, whereas stimulation of macrophages with IL‐4 or IL‐13 induces M2 macrophages.16, 17 In these studies, molecular and functional analyses of murine macrophages were performed using bone‐marrow‐derived macrophages.

If we could stimulate macrophages obtained from our body to become tumoricidal macrophages (M1 polarized) or immune‐regulatory macrophages (M2 polarized) ex vivo using molecular biological methods and introduce them back into the body, these macrophages may be of therapeutic value for targeting cancer or inflammation. Although the removal of macrophages from bone marrow or spleen is invasive, it is relatively safe and easy to collect peritoneal macrophages from ascites, especially for patients with cancerous or inflammatory peritonitis. Indeed, a large number of macrophages exist in the peritoneal cavity; however, whether peritoneal macrophages can be polarized to M1 and M2 phenotypes has not yet been fully addressed.

Interestingly, Hunter et al.18 showed that intraperitoneal injection of murine peritoneal macrophages differentiated into the M2 phenotype reduced the severity of experimental colitis in mice. They suggested that IL‐10 production from M2‐polarized macrophages partly contributed to the attenuation of colitis. However, the profiles of the other cytokines and factors produced by these cells and the functional effects on T cells were not fully addressed.

In this study, we found that resident peritoneal macrophages possess the same ability as bone‐marrow‐derived macrophages, i.e. polarization to the M1 and M2 phenotypes, and that each phenotype produced a specific cytokine profile. We also found that M2‐polarized peritoneal macrophages produced regulatory cytokines and could suppress T‐cell proliferation in vitro. Based on our data, we suggest that resident peritoneal macrophages could be used in adoptive transfer therapy for autoimmune/inflammatory diseases.

Materials and methods

Mice

Female C57BL/6J mice (6–7 weeks of age) were purchased from Japan SLC (Hamamatsu, Japan). Mice were housed in an animal facility under a 12‐hr light/dark cycle and were given standard chow and water ad libitum. Mice weighing 18–22 g at 7–9 weeks of age were used for this study.

Isolation of peritoneal macrophages, bone‐marrow‐derived macrophages, and CD4+ T cells

The method used to isolate peritoneal macrophages from mice has been described previously.18 Briefly, mice were killed, and 5 ml of ice‐cold PBS was injected into the abdomen. The fluid was withdrawn, centrifuged (350 g for 5 min at 4°), and the cell pellet was resuspended in 1 ml of Dulbecco's modified Eagle's medium supplemented with 2% penicillin‐streptomycin and 10% bovine calf serum. These peritoneal exudate cells were cultured on Petri dishes (> 4 hr at 37°), non‐adherent cells were removed, and the adherent cells were detached by digestion with trypsin (0·5%).

To isolate bone‐marrow‐derived macrophages, pelvic and femoral bones were dissected, and all the tissue remaining on the bones was removed. The end of each bone was cut off, and the bone marrow was expelled. Cells from bone marrow were cultured for 7 days with 10 ng/ml macrophage colony‐stimulating factor. Adherent cells were detached by digestion with trypsin (0·5%). FACS sorting (BD Bioscience, San Jose, CA) was performed to obtain F4/80‐positive and CD11c‐negative cells. Then, the harvested cells (0·5 × 106 to 1 × 106) were cultured in six‐well plates containing complete RPMI‐1640 with 10% fetal bovine serum for 24 hr at 37°.

CD4+ T cells were isolated from the spleens of wild‐type mice. Spleens were dissected from the abdominal cavity and passed through a 40‐μm nylon filter. Red cell lysis buffer was used to remove red blood cells. A single splenic cell suspension was obtained, and CD4+ T cells were isolated by a magnetic cell separation (MACS) technique using the CD4+ T‐cell isolation kit II (Miltenyi Biotec, Bisley, UK).

In vitro differentiation of macrophages

The method used to differentiate the macrophages has been described previously.18 Briefly, peritoneal and bone‐marrow‐derived macrophages were differentiated into M1‐polarized or M2‐polarized macrophages by the addition of mouse recombinant IFN‐γ or IL‐4 and IL‐13 (10 ng/ml each; Invitrogen, Carlsbad, CA) for 48 hr, respectively.

RNA extraction and quantitative real‐time PCR

RNA was obtained using TRIzol® (Invitrogen) according to the manufacturer's instructions, and complementary DNA (cDNA) was synthesized from 1 μg of total RNA using iScript® reverse transcriptase (Bio‐Rad, Hercules, CA). To detect M1 and M2 markers, real‐time PCR were performed on a LightCycler® Carousel‐based system with TaqMan® primer sets (Roche Diagnostics, Mannheim, Germany) for murine iNOS, Fizz1, Arg1, Irf4 and Irf5, and gene expression was analysed using the change‐in‐threshold ΔΔCt‐method. To detect cytokines, reverse transcription was carried out using a GeneAmp® PCR System 9700 thermal cycler (Applied Biosystems, Foster City, CA) and the SuperScript® VIVOTM cDNA Synthesis kit (Invitrogen). Expression of Ifng, Tnfa, Il4, Il10, Il6, Il12a, Il13 and Il17a was quantified by using cDNA specific TaqMan® Gene Expression assays during the second step of a two‐step RT‐PCR. Real‐time quantitative PCR after pre‐amplification was performed using the 48·48 Dynamic Array chip (BioMark®; Fluidigm, San Francisco, CA). The amplification programme consisted of one cycle at 95° for 10 min, and 40 cycles of 95° for 15 s and 60° for 1 min. Data were analysed using fluidigm real‐time pcr analysis software ver. 3.0.2. Cytokine mRNA expression levels were normalized to GAPDH.

ELISA

Peritoneal macrophages were isolated, and 2 × 106 cells were differentiated into the M1 or M2 phenotype as described above. The differentiated cells were activated with 10 μg/ml lipopolysaccharide (LPS). Twenty‐four hours later, the supernatant was collected, and IL‐10 levels were determined in duplicate series by ELISA using the Quantikine® ELISA kit (R&D Systems, Minneapolis, MN).

T‐cell proliferation assay

CD4+ T cells (1 × 105) stimulated with an anti‐CD3/CD28 antibody (Dynabeads® Mouse T activator, Life Technologies, Carlsbad, CA) and M1‐ or M2‐polarized macrophages (1 × 104) were co‐cultured with LPS for 72 hr. T‐cell proliferation was assessed by the Cell Titer 96® AQueous Non‐Radioactive Cell Proliferation Assay (Promega, Madison, WI).

Flow cytometry

CD4+ T cells isolated from the spleen of WT mice were stimulated with anti‐CD3/CD28 beads. These cells were co‐cultured with M1‐ or M2‐polarized peritoneal macrophages at a ratio of 1 : 10 (macrophages : CD4+ T cells). After co‐culturing, the floating cells were collected and stained for CD4 and F4/80 and were analysed by flow cytometry. Cells were washed once in fluorescence‐activated cell sorter (FACS) buffer (PBS/2% fetal calf serum/1 mg/ml sodium azide), incubated with anti‐CD16/CD32 blocking antibody (BD Pharmingen, San Jose, CA) for 5 min at room temperature, and stained with diluted antibodies: phycoerythrin‐labelled anti‐CD4 (BD Pharmingen), and FITC‐labelled anti‐F4/80 (Biolegend, San Diego, CA). Samples were acquired on a Gallios™ (Beckman Coulter, Brea, CA) and analysed using flowjo software (TreeStar, Ashland, OR).

Statistical analyses

Differences between samples in two groups were evaluated by Student's t‐test. Values are expressed as mean ± SD. P values < 0·05 were considered significant.

Results

Peritoneal macrophages differentiate into the M1 phenotype after stimulation with IFN‐γ

We investigated whether resident peritoneal macrophages and bone‐marrow‐derived macrophages could be differentiated into the M1 phenotype ex vivo by stimulation with IFN‐γ. Irf5, a master regulator of the M1 phenotype, was up‐regulated in peritoneal macrophages isolated from wild‐type mice after stimulation with IFN‐γ (Fig. 1a). Interestingly, the Irf5 expression level in bone‐marrow‐derived macrophages isolated from wild‐type mice after stimulation with IFN‐γ was nearly the same as that in bone‐marrow‐derived macrophages without IFN‐γ stimulation (M0 status; Fig. 1a). To monitor M1 status, we evaluated the mRNA expression of inducible nitric oxide synthase (iNOS), a marker of M1 macrophages. Stimulation with IFN‐γ strongly induced the expression of iNOS in peritoneal macrophages as well as bone‐marrow‐derived macrophages (Fig. 1b). We also evaluated the mRNA expression levels of Il12 and Tnfa which are related but not specific markers of M1 phenotype. However, levels of Il12 and Tnfa were not up‐regulated in peritoneal macrophages as well as in bone‐marrow‐derived macrophages after stimulation with IFN‐γ (Fig. 1c,d). These data clearly showed that resident peritoneal macrophages possess the ability to differentiate into M1‐polarized macrophages following stimulation with IFN‐γ ex vivo.

Figure 1.

Figure 1

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Induction of the M1 phenotype in peritoneal and bone‐marrow‐derived macrophages by stimulation with interferon‐γ (IFN‐γ). (a) RT‐PCR assay for the expression of Irf5 mRNA in isolated peritoneal and bone‐marrow‐derived macrophages stimulated with IFN‐γ. (b) RT‐PCR assay for the expression of iNOS mRNA in isolated peritoneal and bone‐marrow‐derived macrophages stimulated with IFN‐γ. (c) RT‐PCR assay for the expression of Il12 mRNA in isolated peritoneal and bone‐marrow‐derived macrophages stimulated with IFN‐γ.(d) RT‐PCR assay for the expression of Tnfa mRNA in isolated peritoneal and bone‐marrow‐derived macrophages stimulated with IFN‐γ. Data are representative of five independent experiments. Error bars represent SD. *P < 0·05 (Student's t‐test).

Peritoneal macrophages differentiate into the M2 subtype after stimulation with IL‐4/IL‐13

We next investigated whether resident peritoneal macrophages could differentiate into the M2 subtype following stimulation with IL‐4 and IL‐13 ex vivo. Irf4, a master regulator of the M2 phenotype, was strongly up‐regulated in both peritoneal macrophages and bone‐marrow‐derived macrophages isolated from wild‐type mice (Fig. 2a). To monitor M2 status, we evaluated the mRNA expression level of arginase1 (Arg1) and Fizz1, two markers of the M2 phenotype. Stimulation with IL‐4 and IL‐13 induced the expression of Arg1 (Fig. 2b) and Fizz1 (Fig. 2c) in both peritoneal and bone‐marrow‐derived macrophages. These data clearly showed that resident peritoneal macrophages possess the ability to differentiate into M2‐polarized macrophages following stimulation with IL‐4 and IL‐13 ex vivo.

Figure 2.

Figure 2

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Induction of the M2 phenotype in peritoneal and bone‐marrow‐derived macrophages by stimulation with interleukin‐4 (IL‐4)/IL‐13. (a) RT‐PCR assay for the expression of Irf4 mRNA in isolated peritoneal and bone‐marrow‐derived macrophages stimulated with IL‐4/IL‐13. (b) RT‐PCR assay for the expression of Arg1 mRNA in isolated peritoneal and bone‐marrow‐derived macrophages stimulated with IL‐4/IL‐13. (c) RT‐PCR assay for the expression of Fizz1 mRNA in isolated peritoneal and bone‐marrow‐derived macrophages stimulated with IL‐4/IL‐13. Data are representative of five independent experiments. Error bars represent SD. *P < 0·05 (Student's t‐test).

M2‐polarized but not M1‐polarized peritoneal macrophages predominantly express Th1 and Th2 cytokines but not Th17 cytokines

We next investigated the expression of inflammatory cytokines in peritoneal macrophages polarized to the M1 and M2 phenotypes with or without LPS stimulation in vitro (Fig. 3a,b). M2‐polarized but not M1‐polarized peritoneal macrophages strongly induced T helper type 2 (Th2) cytokines, including IL‐4 and IL‐13, after stimulation with LPS for 24 hr. Unexpectedly, M2‐polarized peritoneal macrophages expressed significantly higher Ifng than M1‐polarized peritoneal macrophages after stimulation with LPS. Il12a, Il17a, and Tnfa were not up‐regulated in M2‐polarized or M1‐polarized peritoneal macrophages after stimulation with LPS. The mRNA expression of Il6 was suppressed in M2‐polarized peritoneal macrophages compared with the levels in M1‐polarized peritoneal macrophages after stimulation with LPS. These data clearly showed that M2‐polarized peritoneal macrophages predominantly up‐regulate Th1 and Th2 cytokines but not Th17 and pro‐inflammatory cytokines after stimulation with LPS.

Figure 3.

Figure 3

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The expression of inflammatory cytokines in peritoneal M1‐ and M2‐polarized macrophages. Total RNA was isolated from the M1‐ and M2‐polarized macrophages after stimulation without lipopolysaccharide (LPS) (a) or with LPS (b) and then analysed by RT‐PCR to detect inflammatory cytokines, including Tnfa, Ifng, Il4, Il6, Il12a and Il17a. Cytokine mRNA expression levels were normalized to GAPDH. Data are representative of five independent experiments. Error bars represent SD. *P < 0·05 (Student's t‐test).

M2‐polarized peritoneal macrophages predominantly produce regulatory cytokines

We next investigated the mRNA expression of anti‐inflammatory cytokines in M1‐ and M2‐polarized macrophages. Tgfb mRNA expression was significantly higher in M2‐polarized peritoneal macrophages than in M1‐polarized peritoneal macrophages (Fig. 4a). However, there was no significant difference in Tgfb mRNA expression between M0‐status peritoneal macrophages and M2‐polarized peritoneal macrophages. The expression of Il10 mRNA was significantly higher in M2‐polarized peritoneal macrophages than in M0‐status and M1‐polarized peritoneal macrophages after stimulation with LPS (Fig. 4a). To assess IL‐10 protein level, an ELISA was performed. Production of IL‐10 was significantly higher in M2‐polarized macrophages than in M1‐polarized peritoneal macrophages after stimulation with LPS (Fig. 4b). However, there was no significant difference in IL‐10 production between M0‐status and M2‐polarized peritoneal macrophages. These data showed that M2‐polarized macrophages produce higher amounts of anti‐inflammatory cytokines, including transforming growth factor‐β and IL‐10, than M1‐polarized peritoneal macrophages.

Figure 4.

Figure 4

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The expression of regulatory cytokines in peritoneal M1‐ and M2‐polarized macrophages. (a) Total RNA was isolated from M1‐ and M2‐polarized macrophages stimulated without or with lipopolysaccharide (LPS) and then analysed by RT‐PCR to detect the anti‐inflammatory cytokines Tgfb and Il10. Cytokine mRNA expression levels were normalized to GAPDH. Data are representative of five independent experiments. Error bars represent SD. (b) M1‐ and M2‐polarized peritoneal macrophages (2 × 106) were cultured for 24 hr without or with LPS. The culture supernatants were then analysed by ELISA to detect interleukin‐10 (IL‐10). Data are representative of five independent experiments. Error bars represent SD. *P < 0·05 (Student's t‐test).

M2‐polarized peritoneal macrophages suppress T‐cell proliferation

To evaluate the functional effect of M1‐polarized or M2‐polarized peritoneal macrophages on T cells, a T‐cell macrophage co‐culture proliferation assay was performed. M0‐status (control), M1‐polarized and M2‐polarized peritoneal macrophages were cultured with CD4+ T cells isolated from the spleen in the presence of an anti‐CD3/CD28 antibody. T cells exhibited high levels of proliferation in the presence of M0‐status peritoneal macrophages and M1‐polarized peritoneal macrophages (Fig. 5a). In contrast, M2‐polarized peritoneal macrophages markedly suppressed the proliferation of CD4+ T cells in vitro (Fig. 5a). Further, to address whether peritoneal macrophages modulate T‐cell proliferation by acting directly on T cells or whether this inhibition is mediated by soluble factors, we stimulated T cells in a transwell tissue culture system; however, no inhibitory activity could be detected in M2‐polarized peritoneal macrophage co‐cultures (data not shown). These results indicate that M2‐polarized peritoneal macrophage‐mediated suppression of T‐cell proliferation is cell–cell contact dependent.

Figure 5.

Figure 5

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Suppression of T‐cell proliferation by M2‐polarized peritoneal macrophages. (a) CD4+ T cells isolated from the spleen of wild‐type mice were stimulated with anti‐CD3/CD28 beads and were either cultured alone (T cells only) or with untreated peritoneal macrophages (M0), interferon‐γ (IFN‐γ)‐treated peritoneal macrophages (M1), or interleukin‐4 (IL‐4)/IL‐13‐treated peritoneal macrophages (M2) at a ratio of 1 : 10 (macrophages: CD4+ T‐cells). Cells were collected after 4 days and analysed by the MTT assay. Bars show the mean ± SD from five separate cultures per condition.*P < 0·05 (Student's t‐test).

Th2 cytokine production is increased in T cells co‐cultured with M2‐polarized peritoneal macrophages

We next evaluated whether the cytokine profile was altered in CD4+ T cells co‐cultured with M1‐ and M2‐polarized peritoneal macrophages. The purity of CD4+ T cells re‐isolated after co‐culture with macrophages was confirmed using flow cytometric analyses (Fig. 6a).

Figure 6.

Figure 6

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Anti‐inflammatory and T helper type 2 (Th2) cytokine mRNA expression in CD4+ T cells co‐cultured with M1‐ and M2‐polarized peritoneal macrophages. (a) CD4+ T cells isolated from the spleen of wild‐type (WT) mice were stimulated with anti‐CD3/CD28 beads. These cells were co‐cultured with M1‐ or M2‐polarized peritoneal macrophages at a ratio of 1 : 10 (macrophages: CD4+ T cells). After co‐culturing, the floating cells were collected and stained for CD4 and F4/80 and analysed by flow cytometry. (b) Total RNA was isolated from CD4+ T cells co‐cultured with M1‐ and M2‐polarized peritoneal macrophages and then analysed by RT‐PCR to detect the T helper type 2 cytokines, Il4 and Il13, and the anti‐inflammatory cytokines, Tgfb and Il10. Cytokine mRNA expression levels were normalized to GAPDH. Data are representative of five independent experiments. Error bars represent SD. *P < 0·05 (Student's t‐test).

Interestingly, CD4+ T cells co‐cultured with M2‐polarized peritoneal macrophages strongly expressed Th2 cytokines, including Il4 and Il13, compared with the levels in CD4+ T cells co‐cultured with M1‐polarized peritoneal macrophages (Fig. 6b). However, the expression levels of regulatory cytokines, such as Tgfb and Il10, were not increased in CD4+ T cells co‐cultured with M2‐polarized peritoneal macrophages compared with the levels in CD4+ T cells co‐cultured with M1‐polarized peritoneal macrophages (Fig. 6b). These results indicate that M2‐polarized peritoneal macrophages induce CD4+ T cells to produce Th2 cytokines but not regulatory cytokines.

Discussion

Based on the findings described in this report, we drew the following conclusions concerning the immunological function of M1‐ and M2‐polarized murine peritoneal macrophages. (i) Similar to bone‐marrow‐derived macrophages, resident peritoneal macrophages possess the ability to differentiate into M1‐ and M2‐polarized macrophages ex vivo, (ii) M2‐polarized but not M1‐polarized macrophages predominantly express Th1 and Th2 cytokines, (iii) M2‐polarized but not M1‐polarized macrophages predominantly produce anti‐inflammatory cytokines, (iv) M2‐polarized peritoneal macrophages suppress T‐cell proliferation ex vivo, (v) CD4+ T cells co‐cultured with M2‐polarized peritoneal macrophages predominantly express Th2 cytokines but not regulatory cytokines.

Several basic and clinical immunotherapy studies have been performed with adoptive autologous cells, such as T cells, natural killer cells and dendritic cells, targeting cancer.19, 20, 21, 22 Immunotherapy studies advance not only the field of cancer but also the field of inflammatory disease. For example, a clinical trial of immunotherapy using mesenchymal stromal cells for Crohn's disease has been reported.23 Because M1 phenotype macrophages also have strong anti‐tumour effects7, 8 and M2 phenotype macrophages have anti‐inflammatory effects,1 macrophages could be developed as a new cell‐based therapy for cancer and/or inflammatory diseases. However, most basic studies on the regulation of macrophage polarization have used bone‐marrow‐derived macrophages. Removing macrophages from the bone marrow of patients for immunotherapy is invasive, but removing macrophages from the ascites in the peritoneal cavity, in which a large number of macrophages exist, seems less invasive. For example, a large number of macrophages extracted from the ascites of patients with cancer or inflammatory diseases could be differentiated into an anti‐tumour phenotype for the treatment of cancer or into an anti‐inflammatory phenotype for the treatment of inflammatory diseases, and then these cells could be transferred back into patients; hence, peritoneal macrophages could be of therapeutic value for cancer or inflammatory diseases.

Interestingly, a recent study showed that intraperitoneal injection of M2‐polarized peritoneal macrophages reduced the severity of experimental colitis in mice in part through an IL‐10‐dependent mechanism.18 However, in this study, the anti‐inflammatory effects of other cytokines (besides IL‐10) produced by these cells and the functional effects of these cells on pathogenic T cells were not clear. In our study, the expression of both Il10 and Tgfb mRNA was significantly higher in M2‐polarized peritoneal macrophages than in M1‐polarized peritoneal macrophages. Furthermore, Th1 and Th2 cytokines were also significantly higher in M2‐polarized peritoneal macrophages than in M1‐polarized peritoneal macrophages. Liu et al. showed that adoptive intravenous transfer of freshly isolated murine peritoneal cells (macrophages and B cells) attenuated colitis.24 Interestingly, in that study, although peritoneal macrophages were not polarized into the M1 or M2 phenotype ex vivo before adoptive cell transfer, IL‐10 and transforming growth factor‐β levels were significantly increased in macrophages isolated from the colonic tissue of colitis mice after adoptive transfer. In our study, polarization into M2 macrophages ex vivo induced significantly higher Il10 and Tgfb expression than that observed in M1 macrophages. In addition, M2‐polarized peritoneal macrophages more effectively suppressed T‐cell proliferation than M0‐status or M1‐polarized peritoneal macrophages. Therefore, we believe that peritoneal macrophages polarized to an M2 phenotype ex vivo have stronger anti‐inflammatory effects than M0 or M1 phenotype macrophages. In our study, no significant difference was seen in the Tgfb mRNA expression between M0 and M1 macrophages. However, the Tgfb mRNA expression was higher in M0 than in M1 macrophages. In addition, the protein level of IL‐10 was significantly higher in M0 than in M1 macrophages. Hence, our observations support the data of Liu et al.24 that M0 macrophages have the ability to suppress inflammation.

Previous studies showed that M1‐polarized bone‐marrow‐derived macrophages predominantly produce Th1 cytokines, including IFN‐γ, and pro‐inflammatory cytokines, including TNF‐α, IL‐6 and IL‐12.7, 8 However, unexpectedly, our study showed that peritoneal macrophages polarized into the M1 phenotype ex vivo produced negligible amounts of Th1 and pro‐inflammatory cytokines. Indeed, macrophages isolated from various tissues manifest marked differences in functional activities.25, 26, 27, 28 These peritoneal macrophages are remarkably distinct from those in other tissues for several reasons.28, 29 For example, Zhu et al.30 demonstrated that peritoneal macrophages have higher iNOS and IL‐12 expression than do splenic macrophages. However, our data suggested that polarization into the M1 phenotype ex vivo induced lower production of Th1 and pro‐inflammatory cytokines than that observed in M0‐status macrophages. Therefore, adoptive transfer of these M1‐polarized peritoneal macrophages may not provide anti‐bacterial or anti‐tumour effects in vivo. In contrast, polarization to the M2 phenotype ex vivo induced much higher production of regulatory cytokines, suggesting that adoptive transfer of these M2‐polarized macrophages may be well suited for the treatment of inflammatory diseases.

It is not yet clear whether, after intraperitoneal or intravenous adoptive transfer of M1‐ or M2‐polarized peritoneal macrophages, these cells can selectively migrate to lesion sites. However, Hunter et al.18 showed that fluorescence‐labelled macrophages transferred intravenously were detected in the spleen, mesenteric lymph nodes, caecum and colon of naive mice. In addition, Liu et al. showed that intraperitoneally transferred fluorescence‐labelled macrophages were detected in the mesenteric lymph nodes and colons of colitic mice.24 These observations suggest that adoptively transferred peritoneal macrophages can migrate to most of the organs where inflammation exists. Our observations not only showed that M2‐polarized macrophages produce humoral anti‐inflammatory cytokines but also that direct inhibition of T‐cell proliferation depends on cell–cell contact. Therefore, these findings suggest that both intraperitoneally and intravenously adoptively transferred M2‐polarized peritoneal macrophages could migrate to the target organs and directly suppress T‐cell proliferation at the site of inflammation. In addition, our observations showed that these M2‐polarized macrophages could induce CD4+ T cells to express Th2 cytokines. Therefore, adoptive transfer of M2‐polarized peritoneal macrophages may be more suitable for diseases involving Th1‐mediated inflammation, such as Crohn's disease, rather than Th2‐mediated inflammation, such as ulcerative colitis.

In conclusion, our findings showed that murine resident peritoneal macrophages possess the same ability as bone‐marrow‐derived macrophages, i.e. polarization to M1 and M2 phenotypes ex vivo, and these phenotypes have different cytokine production patterns. M1‐polarized macrophages unexpectedly do not produce sufficient pro‐inflammatory or Th1 cytokines, and so may not have sufficient anti‐tumour or antibacterial effects. In contrast, M2‐polarized macrophages sufficiently produce Th2 and regulatory cytokines and have been shown to inhibit T‐cell proliferation directly. Hence, our findings suggest novel therapeutic strategies for refractory autoimmune/inflammatory diseases based on the use of endogenous M2‐polarized peritoneal macrophages.

Disclosures

The authors have no financial conflicts of interest to report.

Acknowledgements

This work was supported by a Grant‐in‐Aid for Scientific Research (C) 25460948 from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. We acknowledge the late Dr Masato Uchijima and the late Kunio Tsujimura for their excellent technical support.

Author contributions

SO designed and performed experiments and analysed the data, RT, ST, and ST designed and performed experiments. MI, YH, KT, TN, SS, TH, SO, TF, and HM analysed the data and contributed to manuscript preparation. KS designed the study and experiments; acquired, analysed, and interpreted the data; and prepared the manuscript.

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