IMMUNOLOGY ORIGINAL ARTICLE
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 Miyajima1 and
Ken Sugimoto1
1
First Department of Medicine, Hamamatsu
University School of Medicine, 2Department
of Health Science, Hamamatsu University
School of Medicine, 3Department of Infectious
Diseases, Hamamatsu University School of
Medicine, 4Department of Endoscopic and
Photodynamic Medicine, Hamamatsu Univer-
sity School of Medicine and 5Centre for
Clinical Research, Hamamatsu University
School of Medicine, Hamamatsu, Shizuoka,
Japan
doi:10.1111/imm.12647
Received 22 February 2016; revised 29 June
2016; accepted 1 July 2016.
Correspondence: Dr K. Sugimoto, First
Department of Medicine, Hamamatsu
University School of Medicine, 1-20-1 Han-
dayama, Higashi-ku, Hamamatsu 431-3192,
Japan. Email: sugimken@hama-med.ac.jp
(KS)
Senior author:Dr K. Sugimoto
Introduction
Macrophages are an essential component of both innate
and adaptive immunity and play a central role in host
defence and inflammation.1–3 It is well known that acti-
vated macrophages are divided into two subsets,
Abbreviations: Arg1, arginase1; IFN, interferon; IL, interleukin; iNOS, inducible nitric oxide synthase; IRF, interferon regulatory
factor; TNF, tumour necrosis factor
320
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 pro-
duction patterns as bone-marrow-derived macrophages. To address this
question, we examined the ability of peritoneal macrophages to be polar-
ized to the M1 and M2 phenotypes and determined the specific cytokine
profiles of cells with each phenotype. We showed that peritoneal macro-
phages, as well as bone-marrow-derived macrophages, were differentiated
into M1 and M2 phenotypes following stimulation with interferon-c
(IFN-c) 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-b 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 pro-
duction 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.
classically (M1) and alternatively (M2) activated
macrophages.4–6 The M1 phenotype is characterized by
the expression of high levels of pro-inflammatory cytoki-
nes, including interleukin-12 (IL-12), IL-23 and tumour
necrosis factor-a (TNF-a), as well as high nitric oxide
and reactive oxygen intermediate production.7,8 In
ª 2016 John Wiley & Sons Ltd, Immunology, 149, 320–328

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 progres-
sion.1 Recent studies showed that there are two key tran-
scriptional 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 con-
trast, IRF4 has been shown to be a crucial mediator of
M2 macrophage polarization.12,13 Jumonji domain con-
taining-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 Fizz113; 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 repro-
gramming of M1 and M2 macrophages. Interferon-c
(IFN-c) induces M1 macrophages, whereas stimulation of
macrophages with IL-4 or IL-13 induces M2 macro-
phages.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 therapeu-
tic 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; how-
ever, whether peritoneal macrophages can be polarized to
M1 and M2 phenotypes has not yet been fully addressed.
Interestingly, Hunter et al.18 showed that intraperi-
toneal injection of murine peritoneal macrophages differ-
entiated into the M2 phenotype reduced the severity of
experimental colitis in mice. They suggested that IL-10
production from M2-polarized macrophages partly con-
tributed to the attenuation of colitis. However, the pro-
files of the other cytokines and factors produced by these
cells and the functional effects on T cells were not fully
addressed.
ª 2016 John Wiley & Sons Ltd, Immunology, 149, 320–328
Regulatory role of M2 peritoneal macrophage
In this study, we found that resident peritoneal macro-
phages possess the same ability as bone-marrow-derived
macrophages, i.e. polarization to the M1 and M2 pheno-
types, and that each phenotype produced a specific cyto-
kine 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/inflam-
matory 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 supple-
mented 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 diges-
tion 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 per-
formed to obtain F4/80-positive and CD11c-negative cells.
Then, the harvested cells (0
5 9 106 to 1 9 106) 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-lm 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) tech-
nique using the CD4+ T-cell isolation kit II (Miltenyi
Biotec, Bisley, UK).
321
S. Oishi et al.
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 addi-
tion of mouse recombinant IFN-c 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 lg 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 DDCt-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 Synthe-
sis kit (Invitrogen). Expression of Ifng, Tnfa, Il4, Il10, Il6,
Il12a, Il13 and Il17a was quantified by using cDNA speci-
fic 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 9 106 cells
were differentiated into the M1 or M2 phenotype as
described above. The differentiated cells were activated
with 10 lg/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 9 105) stimulated with an anti-CD3/
CD28 antibody (Dynabeads Mouse T activator, Life
Technologies, Carlsbad, CA) and M1- or M2-polarized
macrophages (1 9 104) were co-cultured with LPS for
72 hr. T-cell proliferation was assessed by the Cell Titer
322
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 macro-
phages 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-acti-
vated 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 GalliosTM
(Beckman Coulter, Brea, CA) and analysed using FLOWJO
software (TreeStar, Ashland, OR).
Statistical analyses
Differences between samples in two groups were evalu-
ated 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-c
We investigated whether resident peritoneal macrophages
and bone-marrow-derived macrophages could be differ-
entiated into the M1 phenotype ex vivo by stimulation
with IFN-c. Irf5, a master regulator of the M1 phenotype,
was up-regulated in peritoneal macrophages isolated from
wild-type mice after stimulation with IFN-c (Fig. 1a).
Interestingly, the Irf5 expression level in bone-marrow-
derived macrophages isolated from wild-type mice after
stimulation with IFN-c was nearly the same as that in
bone-marrow-derived macrophages without IFN-c stimu-
lation (M0 status; Fig. 1a). To monitor M1 status, we
evaluated the mRNA expression of inducible nitric oxide
synthase (iNOS), a marker of M1 macrophages. Stimula-
tion with IFN-c 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. How-
ever, levels of Il12 and Tnfa were not up-regulated in
peritoneal macrophages as well as in bone-marrow-
derived macrophages after stimulation with IFN-c
(Fig. 1c,d). These data clearly showed that resident
ª 2016 John Wiley & Sons Ltd, Immunology, 149, 320–328
 Regulatory role of M2 peritoneal macrophage

* P < 0·05
(a) Peritoneal BM-derived (b) Peritoneal BM-derived
Irf5 Irf5 iNOS iNOS

Relative to GAPDH

Relative to GAPDH
0·8 * 0·08 * *

0·4 0·04

0 0
IFN-γ – + – + IFN-γ – + – +

(c) Peritoneal BM-derived (d) Peritoneal BM-derived

Il12 Il12 Tnf α Tnf α

Relative to GAPDH
Relative to GAPDH

12 10

6 5

0 0
IFN-γ – + – + IFN-γ – + – +

Figure 1. Induction of the M1 phenotype in peritoneal and bone-marrow-derived macrophages by stimulation with interferon-c (IFN-c). (a)
RT-PCR assay for the expression of Irf5 mRNA in isolated peritoneal and bone-marrow-derived macrophages stimulated with IFN-c. (b) RT-
PCR assay for the expression of iNOS mRNA in isolated peritoneal and bone-marrow-derived macrophages stimulated with IFN-c. (c) RT-PCR
assay for the expression of Il12 mRNA in isolated peritoneal and bone-marrow-derived macrophages stimulated with IFN-c.(d) RT-PCR assay for
the expression of Tnfa mRNA in isolated peritoneal and bone-marrow-derived macrophages stimulated with IFN-c. Data are representative of
five independent experiments. Error bars represent SD. *P < 0
05 (Student’s t-test).

peritoneal macrophages possess the ability to differentiate
into M1-polarized macrophages following stimulation
with IFN-c ex vivo.
Peritoneal macrophages differentiate into the M2
subtype after stimulation with IL-4/IL-13
We next investigated whether resident peritoneal macro-
phages 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 mark-
ers 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.
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 stimula-
tion with LPS for 24 hr. Unexpectedly, M2-polarized
peritoneal macrophages expressed significantly higher Ifng
than M1-polarized peritoneal macrophages after stimula-
tion with LPS. Il12a, Il17a, and Tnfa were not up-regu-
lated in M2-polarized or M1-polarized peritoneal
macrophages after stimulation with LPS. The mRNA
expression of Il6 was suppressed in M2-polarized peri-
toneal macrophages compared with the levels in M1-
polarized peritoneal macrophages after stimulation with
LPS. These data clearly showed that M2-polarized peri-
toneal macrophages predominantly up-regulate Th1 and
Th2 cytokines but not Th17 and pro-inflammatory
cytokines after stimulation with LPS.
M2-polarized peritoneal macrophages predominantly
produce regulatory cytokines
We next investigated the mRNA expression of anti-inflam-
matory 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 peri-
toneal macrophages. The expression of Il10 mRNA was

ª 2016 John Wiley & Sons Ltd, Immunology, 149, 320–328 323
S. Oishi et al.

* P < 0·05 and M2-polarized peritoneal macrophages. These data

(a) Peritoneal BM-derived showed that M2-polarized macrophages produce higher
Irf4 Irf4 amounts of anti-inflammatory cytokines, including trans-
* * forming growth factor-b and IL-10, than M1-polarized
Relative to GAPDH

0·8 peritoneal macrophages.

M2-polarized peritoneal macrophages suppress T-cell

0·4
proliferation
To evaluate the functional effect of M1-polarized or M2-
0 polarized peritoneal macrophages on T cells, a T-cell
IL-4 + IL-13 – + – +
macrophage co-culture proliferation assay was performed.
(b) Peritoneal BM-derived M0-status (control), M1-polarized and M2-polarized
Arg1 Arg1
peritoneal macrophages were cultured with CD4+ T cells
1·0 isolated from the spleen in the presence of an anti-CD3/
* *
CD28 antibody. T cells exhibited high levels of prolifera-
Relative to GAPDH

tion in the presence of M0-status peritoneal macrophages

and M1-polarized peritoneal macrophages (Fig. 5a). In
0·5
contrast, M2-polarized peritoneal macrophages markedly
suppressed the proliferation of CD4+ T cells in vitro
(Fig. 5a). Further, to address whether peritoneal macro-
phages modulate T-cell proliferation by acting directly on
0
IL-4 + IL-13 – + – + T cells or whether this inhibition is mediated by soluble
factors, we stimulated T cells in a transwell tissue culture
(c) Peritoneal BM-derived system; however, no inhibitory activity could be detected
Fizz1 Fizz1 in M2-polarized peritoneal macrophage co-cultures (data
8·0
* *
not shown). These results indicate that M2-polarized
peritoneal macrophage-mediated suppression of T-cell
Relative to GAPDH

proliferation is cell–cell contact dependent.

4·0
0
IL-4 + IL-13 – + – +
Figure 2. 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 stimu-
lated 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).
significantly higher in M2-polarized peritoneal macro-
phages 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 macro-
phages than in M1-polarized peritoneal macrophages after
stimulation with LPS (Fig. 4b). However, there was no sig-
nificant difference in IL-10 production between M0-status
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).
Interestingly, CD4+ T cells co-cultured with M2-polar-
ized peritoneal macrophages strongly expressed Th2 cytoki-
nes, 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 macro-
phages (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

324 ª 2016 John Wiley & Sons Ltd, Immunology, 149, 320–328
 Regulatory role of M2 peritoneal macrophage

* P < 0·05
(a) LPS (–)
* * *
45
*
40

35

Relative to GAPDH
30

25

20

15

10

0
M0 M1 M2 M0 M1 M2 M0 M1 M2 M0 M1 M2 M0 M1 M2 M0 M1 M2 M0 M1 M2

Tnfα Ifn γ Il4 Il6 Il12a Il13 Il17a

* P < 0·05
(b) LPS (+)
160 * * * * *

140 * *

120
Relative to GAPDH

100

80

60

40

20

0
M0 M1 M2 M0 M1 M2 M0 M1 M2 M0 M1 M2 M0 M1 M2 M0 M1 M2 M0 M1 M2
Tnfα Ifn γ Il4 Il6 Il12a Il13 Il17a

Figure 3. 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).

function of M1- and M2-polarized murine peritoneal
macrophages. (i) Similar to bone-marrow-derived macro-
phages, resident peritoneal macrophages possess the abil-
ity to differentiate into M1- and M2-polarized
macrophages ex vivo, (ii) M2-polarized but not M1-polar-
ized 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 sup-
press T-cell proliferation ex vivo, (v) CD4+ T cells co-cul-
tured 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.19–22 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 macro-
phages have anti-inflammatory effects,1 macrophages
could be developed as a new cell-based therapy for cancer
and/or inflammatory diseases. However, most basic stud-
ies 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 dis-
eases could be differentiated into an anti-tumour pheno-
type for the treatment of cancer or into an anti-
inflammatory phenotype for the treatment of inflamma-
tory diseases, and then these cells could be transferred
back into patients; hence, peritoneal macrophages could

ª 2016 John Wiley & Sons Ltd, Immunology, 149, 320–328 325
S. Oishi et al.

* P < 0·05
(a) Tgf β Il10 *
* *

Relative to GAPDH

Relative to GAPDH
20 15 *
15
10
10
5 5

0 0
M0 M1 M2 M0 M1 M2 M0 M1 M2 M0 M1 M2

LPS (–) LPS (+) LPS (–) LPS (+)

* P < 0·05
(b) IL-10 IL-10
* * * *
40 250

30 200

pg/ml
pg/ml

150
20
100
10
50
0 0
M0 M1 M2 M0 M1 M2
LPS (–) LPS (+)

Figure 4. 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 9 106) 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).

be of therapeutic value for cancer or inflammatory dis- proliferation than M0-status or M1-polarized peritoneal
eases. macrophages. Therefore, we believe that peritoneal
Interestingly, a recent study showed that intraperitoneal macrophages polarized to an M2 phenotype ex vivo have
injection of M2-polarized peritoneal macrophages stronger anti-inflammatory effects than M0 or M1 pheno-
reduced the severity of experimental colitis in mice in type macrophages. In our study, no significant difference
part through an IL-10-dependent mechanism.18 However, was seen in the Tgfb mRNA expression between M0 and
in this study, the anti-inflammatory effects of other M1 macrophages. However, the Tgfb mRNA expression
cytokines (besides IL-10) produced by these cells and the was higher in M0 than in M1 macrophages. In addition,
functional effects of these cells on pathogenic T cells were the protein level of IL-10 was significantly higher in M0
not clear. In our study, the expression of both Il10 and than in M1 macrophages. Hence, our observations sup-
Tgfb mRNA was significantly higher in M2-polarized port the data of Liu et al.24 that M0 macrophages have
peritoneal macrophages than in M1-polarized peritoneal the ability to suppress inflammation.
macrophages. Furthermore, Th1 and Th2 cytokines were Previous studies showed that M1-polarized bone-mar-
also significantly higher in M2-polarized peritoneal row-derived macrophages predominantly produce Th1
macrophages than in M1-polarized peritoneal macro- cytokines, including IFN-c, and pro-inflammatory cytoki-
phages. Liu et al. showed that adoptive intravenous trans- nes, including TNF-a, IL-6 and IL-12.7,8 However, unex-
fer of freshly isolated murine peritoneal cells pectedly, our study showed that peritoneal macrophages
(macrophages and B cells) attenuated colitis.24 Interest- polarized into the M1 phenotype ex vivo produced negli-
ingly, in that study, although peritoneal macrophages gible amounts of Th1 and pro-inflammatory cytokines.
were not polarized into the M1 or M2 phenotype ex vivo Indeed, macrophages isolated from various tissues mani-
before adoptive cell transfer, IL-10 and transforming fest marked differences in functional activities.25–28 These
growth factor-b levels were significantly increased in peritoneal macrophages are remarkably distinct from
macrophages isolated from the colonic tissue of colitis those in other tissues for several reasons.28,29 For exam-
mice after adoptive transfer. In our study, polarization ple, Zhu et al.30 demonstrated that peritoneal macro-
into M2 macrophages ex vivo induced significantly higher phages have higher iNOS and IL-12 expression than do
Il10 and Tgfb expression than that observed in M1 splenic macrophages. However, our data suggested that
macrophages. In addition, M2-polarized peritoneal polarization into the M1 phenotype ex vivo induced lower
macrophages more effectively suppressed T-cell production of Th1 and pro-inflammatory cytokines than

326 ª 2016 John Wiley & Sons Ltd, Immunology, 149, 320–328
 Regulatory role of M2 peritoneal macrophage

* P < 0·05 103

(a) 95·6 3·1
*

*
102
400 000 *

CD4
101

300 000
100
T-cell number

200 000 1·3 0·0

100 101 102 103

F4/80

100 000
(b) * P < 0·05
2 * * *
1·8
0 1·6

Relative to GAPDH
T T+M0 T+M1 T+M2 1·4
1·2
CD3/CD28 (+)
1
Figure 5. Suppression of T-cell proliferation by M2-polarized peri- 0·8
toneal macrophages. (a) CD4+ T cells isolated from the spleen of 0·6
wild-type mice were stimulated with anti-CD3/CD28 beads and were 0·4
either cultured alone (T cells only) or with untreated peritoneal 0·2
macrophages (M0), interferon-c (IFN-c)-treated peritoneal macro- 0
1

2
M

M
phages (M1), or interleukin-4 (IL-4)/IL-13-treated peritoneal macro-
4/

4/

4/

4/

4/

4/

4/

4/
D

D
phages (M2) at a ratio of 1 : 10 (macrophages: CD4+ T-cells). Cells
C

were collected after 4 days and analysed by the MTT assay. Bars
show the mean  SD from five separate cultures per condi-
tion.*P < 0
05 (Student’s t-test).
that observed in M0-status macrophages. Therefore,
adoptive transfer of these M1-polarized peritoneal macro-
phages may not provide anti-bacterial or anti-tumour
effects in vivo. In contrast, polarization to the M2 pheno-
type ex vivo induced much higher production of regula-
tory 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 fluo-
rescence-labelled macrophages transferred intravenously
were detected in the spleen, mesenteric lymph nodes, cae-
cum 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 observa-
tions 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.
C
Il4 Il13 Tgfβ Il10
Figure 6. 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 peri-
toneal 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 nor-
malized to GAPDH. Data are representative of five independent
experiments. Error bars represent SD. *P < 0
05 (Student’s t-test).
Therefore, these findings suggest that both intraperi-
toneally 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 resi-
dent peritoneal macrophages possess the same ability as

ª 2016 John Wiley & Sons Ltd, Immunology, 149, 320–328 327
S. Oishi et al.
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 suf-
ficient 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.
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.
Disclosures
The authors have no financial conflicts of interest to
report.
References
1 Sica A, Mantovani A. Macrophage plasticity and polarization: in vivo veritas. J Clin
Invest 2012; 122:787–95.
2 Gordon S, Martinez FO. Alternative activation of macrophages: mechanism and func-
tions. Immunity 2010; 28:593–604.
3 Benencia F, Courreges MC. Nitric oxide and macrophage antiviral extrinsic activity.
Immunology 1999; 98:363–70.
4 Mackman N. Lipopolysaccharide induction of gene expression in human monocytic
cells. Immunol Res 2000; 21:247–51.
5 Mantovani A. Molecular pathways linking inflammation and cancer. Curr Mol Med
2010; 10:369–73.
6 Mantovani A, Locati M. Orchestration of macrophage polarization. Blood 2009;
114:3135–6.
7 Biswas SK, Mantovani A. Macrophage plasticity and interaction with lymphocyte sub-
sets: cancer as a paradigm. Nat Immunol 2010; 11:889–96.
328
8 Benoit M, Desnues B, Mege JL. Macrophage polarization in bacterial infections. J
Immunol 2008; 181:3733–9.
9 Mantovani A, Sica A, Locati M. New vistas on macrophage differentiation and activa-
tion. Eur J Immunol 2007; 37:14–6.
10 Krausgruber T, Blazek K, Smallie T, Alzabin S, Lockstone H, Sahgal N, et al. IRF5 pro-
motes inflammatory macrophage polarization and TH1-TH17 responses. Nat Immunol
2011; 12:231–8.
11 Takaoka A, Yanai H, Kondo S, Duncan G, Negishi H, Mizutani T, et al. Integral role
of IRF-5 in the gene induction programme activated by Toll-like receptors. Nature
2005; 434:243–9.
12 Satoh T, Takeuchi O, Vandenbon A, Yasuda K, Tanaka Y, Kumagai Y, et al. The
Jmjd3-Irf4 axis regulates M2 macrophage polarization and host responses against hel-
minth infection. Nat Immunol 2010; 11:936–44.
13 El Chartouni C, Schwarzfischer L, Rehli M. Interleukin-4 induced interferon regulatory
factor (Irf) 4 participates in the regulation of alternative macrophage priming. Immuno-
biology 2010; 215:821–5.
14 Saccani A, Schioppa T, Porta C, Biswas SK, Nebuloni M, Vago L, et al. p50 nuclear fac-
tor-jB overexpression in tumor-associated macrophages inhibits M1 inflammatory
responses and antitumor resistance. Cancer Res 2006; 66:11432–40.
15 Guiducci C, Vicari AP, Sangaletti S, Trinchieri G, Colombo MP. Redirecting in vivo eli-
cited tumor infiltrating macrophages and dendritic cells towards tumor rejection. Can-
cer Res 2005; 65:3437–46.
16 Mantovani A, Sozzani S, Locati M, Allavena P, Sica A. Macrophage polarization:
tumor-associated macrophages as a paradigm for polarized M2 mononuclear phago-
cytes. Trends Immunol 2002; 23:549–55.
17 Gordon S. Alternative activation of macrophages. Nat Rev Immunol 2003; 3:23–35.
18 Hunter MM, Wang A, Parhar KS, Johnston MJ, Van Rooijen N, Beck PL, McKay DM,
et al. In vitro-derived alternatively activated macrophages reduce colonic inflammation
in mice. Gastroenterology 2010; 138:1395–405.
19 Restifo NP, Dudley ME, Rosenberg SA. Adoptive immunotherapy for cancer: harnessing
the T cell response. Nat Rev Immunol 2012; 12:269–81.
20 Palucka K, Banchereau J. Cancer immunotherapy via dendritic cells. Nat Rev Cancer
2012; 12:265–77.
21 Vanneman M, Dranoff G. Combining immunotherapy and targeted therapies in cancer
treatment. Nat Rev Cancer 2012; 12:237–51.
22 Dahlberg CI, Sarhan D, Chrobok M, Duru AD, Alici E. Natural killer cell-based thera-
pies targeting cancer: possible strategies to gain and sustain anti-tumor activity. Front
Immunol 2015; 6:605.
23 Forbes GM, Sturm MJ, Leong RW, Sparrow MP, Segarajasingam D, Cummins AG,
et al. A phase 2 study of allogeneic mesenchymal stromal cells for luminal Crohn’s dis-
ease refractory to biologic therapy. Clin Gastroenterol Hepatol 2014; 12:64–71.
24 Liu T, Ren J, Wang W, Wei XW, Shen GB, Liu YT, et al. Treatment of dextran sodium
sulfate-induced experimental colitis by adoptive transfer of peritoneal cells. Sci Rep
2015; 5:16760.
25 Davies LC, Taylor PR. Tissue-resident macrophages: then and now. Immunology 2015;
144:541–8.
26 Lepay DA, Steinman RM, Nathan CF, Murray HW, Cohn ZA. Liver macrophages in
murine listeriosis. Cell-mediated immunity is correlated with an influx of macrophages
capable of generating reactive oxygen intermediates. J Exp Med 1985; 161:1503–12.
27 Baccarini M, Kiderlen AF, Decker T, Lohmann-Matthes ML. Functional heterogeneity
of murine macrophage precursor cells from spleen and bone marrow. Cell Immunol
1986; 101:339–50.
28 Liu G, Xia XP, Gong SL, Zhao Y. The macrophage heterogeneity: difference between
mouse peritoneal exudate and splenic F4/80+ macrophages. J Cell Physiol 2006;
209:341–52.
29 Gundra UM, Girgis NM, Ruckerl D, Jenkins S, Ward LN, Kurtz ZD, et al. Alternatively
activated macrophages derived from monocytes and tissue macrophages are phenotypi-
cally and functionally distinct. Blood 2014; 123:e110–22.
30 Zhu YN, Yang YF, Ono S, Zhong XG, Feng YH, Ren YX, et al. Differential expression
of inducible nitric oxide synthase and IL-12 between peritoneal and splenic macro-
phages stimulated with LPS plus IFN-c is associated with the activation of extracellular
signal-related kinase. Int Immunol 2006; 18:981–90.
ª 2016 John Wiley & Sons Ltd, Immunology, 149, 320–328