Immunobiology 216 (2011) 10941102

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Immunobiology
journal homepage: www.elsevier.de/imbio

Attenuated phagocytic activity of monocytes in type 2 diabetic

GotoKakizaki rats
Yuji Takeda, Mikio Marumo, Ichiro Wakabayashi
Department of Environmental and Preventive Medicine, Hyogo College of Medicine, Nishinomiya Hyogo 663-8501, Japan

a r t i c l e

i n f o

Article history:
Received 24 September 2010
Received in revised form 6 April 2011
Accepted 5 May 2011
Keywords:
CD11b/c
Lipopolysaccharide
Monocytes
Phagocytes
Type 2 diabetes

a b s t r a c t
The aim of this study was to determine whether phagocytic activity of leukocytes is altered in type 2 diabetes. GotoKakizaki (GK) rats, a genetic model for type 2 diabetes, and Wistar rats (control) were used
to analyze the immunological status of phagocytes. Direct analysis of phagocytes was performed using
peripheral whole blood. Phagocytic activity of monocytes induced by Escherichia coli BioParticles was
signicantly lower in GK rats than in the control rats, whereas no signicant differences in phagocytic
activity of granulocytes and lymphocytes were found between GK and control rats. Monocytes of GK
rats showed signicantly lower CD11b/c expression compared with that in monocytes of control rats.
However, lipopolysaccharide-stimulated activation of extracellular signal-regulated kinase and nuclear
factor-B in monocytes was not signicantly different between GK and control rats. Restriction of diet in
GK rats greatly improved their hyperglycemic status, but did not restore the levels of phagocytic activity and CD11b/c expression in monocytes of GK rats to the levels observed in control rats. The results
suggest that the phagocytic activity of monocytes is attenuated in GK rats and that this attenuation is
independent of blood glucose levels and is partly explained by a decrease in CD11b/c expression in GK
rats.
2011 Elsevier GmbH. All rights reserved.

Introduction
In patients with diabetes, immune dysfunction is an important complication that determines patient prognosis. Incidence of
infectious disease is higher in patients with diabetes than in nondiabetic people (Geerlings and Hoepelman 1999). There have been
a variety of studies of phagocytic activity of leukocytes in diabetes. Phagocytic activity of neutrophils has been reported to be
similar in diabetic and non-diabetic subjects, whereas killing of
Candida albicans by neutrophils has been found to be impaired
under conditions of hyperglycaemia and ketosis in patients with
type 2 diabetes (Wilson and Reeves 1986). Phagocytic activity of
alveolar macrophages isolated from diabetic rats has been shown
to be impaired compared to that in non-diabetic rats (Ferracini
et al. 2010). In monocytes, inammatory responses via multiple signaling pathways have been shown to be augmented under
high glucose conditions (Dasu et al. 2008; Hatanaka et al. 2006;
Shanmugam et al. 2004). Thus, the ndings for phagocytosis in diabetes are controversial. Moreover, it remains to be claried whether
the phagocytic ability of granulocytes and monocytes is altered in
animal models of diabetes.

Corresponding author. Tel.: +81 798 45 6562; fax: +81 798 45 6563.
E-mail address: wakabaya@hyo-med.ac.jp (I. Wakabayashi).
0171-2985/$ see front matter 2011 Elsevier GmbH. All rights reserved.
doi:10.1016/j.imbio.2011.05.003

Since granulocytes and monocytes in whole blood are easily

activated during preparation of the cells for experiments (Lundahl
et al. 1995; Macey et al. 1995; Watson et al. 1992), it is difcult
to prepare granulocytes and monocytes under a stable condition
without stimulation of these cells. This methodological problem
is one of the reasons for conicting ndings on phagocytic ability
of granulocytes and monocytes in diabetes. Furthermore, glucose,
ketones and triglycerides in plasma are important factors regulating leukocyte functions (Jain et al. 2007; Wilson and Reeves 1986).
We therefore attempted to solve these problems by measuring
phagocytic activity of granulocytes and monocytes using whole
blood.
Patients with diabetes mellitus are much more susceptive to
coronary heart disease and stroke (McCarron et al. 2001; Stamler
et al. 1993). Recently, the concept that atherosclerosis is a chronic
inammatory disease has gained wide acceptance in the eld of
cardiovascular research. Granulocytes and monocytes contribute to
progression of atherosclerotic plaques (Hatanaka et al. 2006; Weber
et al. 2008), and activation of monocytes has been demonstrated to
occur in both type 1 and type 2 diabetes (Cipolletta et al. 2005).
Production of reactive oxygen species in monocytes is increased in
patients with type 2 diabetes compared with that in non-diabetic
people (Chang and Shaio 1995), and expression levels of inammatory cytokines, such as tumor necrosis factor-, interleukin (IL)-1,
IL-6 and IL-8, are higher in patients with type 2 diabetes than in nondiabetic people (Gacka et al. 2008; Giulietti et al. 2007). Intracellular

Y. Takeda et al. / Immunobiology 216 (2011) 10941102

signal transducers, including nuclear factor-B (NF-B) and extracellular signal-regulated kinase (ERK), are known to be involved
in production of the above cytokines in leukocytes (Ghosh and
Hayden 2008; Giraldo et al. 2010; Guha and Mackman 2001; Li and
Verma 2002; Vallabhapurapu and Karin 2009). Among a variety of
signals related to monocyte function, the PI3K/Akt signaling pathway plays a crucial role in regulation of phagocytosis. Upregulation
of Akt (protein kinase B), a downstream target of phosphatidylinositol 3-kinase (PI3K), is known to potentiate phagocytosis and
glucose metabolism, and to function as a negative regulator of Tolllike receptors (TLRs) signaling (Luyendyk et al. 2008; Shiratsuchi
and Basson 2007; Trinidad et al. 2006); however, there is only limited knowledge regarding alterations in these signal transducers in
leukocytes in diabetic patients. NF-B activation in monocytes is
augmented in patients with type 1 diabetes (Mollah et al. 2008).
However, it remains to be claried whether the intracellular signal
transducers of leukocytes are altered in type 2 diabetes.
GotoKakizaki (GK) rats, a genetic model of non-obese type
2 diabetes (Goto et al. 1976; Kitahara et al. 1978; Yagihashi
et al. 1978, 1979), are characterized by high serum glucose levels,
increased activity of hepatic enzymes (including glycogen phosphorylase, glycogen synthase, glucokinase, glucose-6-phosphatase,
and fructose-1,6-diphosphatase). GK rats show the following
characteristics: (1) non-insulin-dependent diabetes mellitus without obesity; (2) severe impairment of islet -cell response to
glucose but preservation of -cell response to non-fuel insulin secretagogues; and (3) deceased -cell mass (Ostenson et al. 1993;
Portha et al. 1991, 2010; Suzuki et al. 1997). GK rats also show
over-expression of the 2A-adrenergic receptor, encoded by the
Adra2a gene, and this abnormality is linked to impaired docking of
insulin granules at the plasma membrane, and reduced -cell exocytosis (Rosengren et al. 2009). Furthermore, a single-nucleotide
polymorphism of the Adra2a gene has been shown to be associated
with risk of overexpression of 2A-adrenergic receptor, reduced
insulin secretion, and increased type 2 diabetes risk in humans
(Rosengren et al. 2009). For these reasons, GK rats have recently
been frequently used as an experimental model of human type 2
diabetes.
We have previously demonstrated that the ERK pathway in vascular smooth muscle cell is augmented in GK rats (Takahashi et al.
2008); however, it remains to be claried whether inammatory
signals of peripheral leukocytes are altered in GK rats. Here, we
compared phagocytic activity, CD71 and CD11b/c receptor expression and the phosphorylation levels of signal transducers, ERK and
NF-B, in leukocytes in whole blood obtained from GK and control
rats.

Materials and methods

Animals
The study protocols regarding treatment of animals were in
accordance with the Guideline for Experiments Using Laboratory
Animals in Hyogo College of Medicine. Male GK rats and Wistar
rats (specic pathogen-free) aged 610 wk were purchased from
CLEA Japan (Tokyo, Japan). The rats were fed standard rat chow
(MF, Oriental Yeast Company, Tokyo, Japan) and housed under standard conditions at a temperature of 22 1 C, relative humidity
of 55% 5% and a day-night rhythm of 12 h. Water was available
ad libitum for all rats. Six-week old rats were divided into two
groups: the normal diet group received food ad libitum, while the
restricted diet group received low calorie food (50% reduction
calories compared to the normal diet). Body weight and fasting
blood glucose were measured weekly for 10 wk.

1095

Reagents and antibodies

The following reagents and antibodies were used: RPMI
1640 medium and lipopolysaccharide (LPS) from Escherichia coli
serotype 055:B5 (Sigma, St. Louis, MO, USA); uorescein isothiocyanate (FITC)- and pHrodo-conjugated E. coli BioParticles
(invitrogen, Eugene, OR, USA); BD Phosow Lyse/Fix Buffer and
BD Pharmingen Stain Buffer (BD Biosciences, (San Jose, CA, USA);
rabbit anti-phospho-ERK1/ERK2 (T202/Y204) antibody (R&D systems, Minneapolis, MN, USA); rabbit anti-p44/42 MAPK (ERK1/2)
antibody, rabbit anti-NF-B p65 (C22B4) monoclonal antibody
(mAb), rabbit anti-phospho-NF-B p65 (Ser536) (93H1) mAb,
rabbit anti-Akt (pan) (C67E7) mAb, and rabbi anti-Phospho-Akt
(Ser473) (D9E) XP mAb (Cell Signaling Technology, Danvers, MA,
USA); AlexaFluor488-conjugated donkey anti-rabbit IgG antibodies (Invitrogen), FITC-conjugated mouse anti-rat CD71 (OX-26)
mAb, FITC-conjugated mouse anti-rat CD45 (OX-1) mAb, FITCconjugated mouse anti-rat CD11b (WT.5) mAb, and phycoerythrin
(PE)-conjugated mouse anti-rat granulocytes (RP-1) mAb (BD
Biosciences); Allophycocyanine (APC)-conjugated mouse anti-rat
CD11b/c (OX-42) mAb (BioLegend, San Diego, CA, USA); FITCconjugated mouse anti-rat CD36 (JC63.1) mAb, rabbit anti-rat CD32
(EP888Y) mAb, and rabbit anti-Scavenging Receptor SR-BI polyclonal Ab (abcam, Cambridge, UK).
Blood collection
Rats were anesthetized with diethyl ether and then subjected
to cardiac puncture, and arterial blood was collected into a
syringe containing low-molecular-weight heparin (approximately
5 U/mL).
Blood glucose measurement
Blood was sampled from the tail vein, and fasting blood glucose
levels were measured immediately by using a glucose test meter
(MediSense Precision Xtra, Abbott Laboratories, Abbott Park, IL,
USA).
Cell surface staining of whole blood
Blood was aliquoted into microtubes (50 L/tube) and reacted
with antibodies on ice for 60 min. The blood was then mixed
with Lyse/Fix buffer for 10 min at 37 C. Red blood cells that were
lysed by the buffer during xation were removed by washing with
phosphate-buffered saline (PBS). The remaining leukocytes were
suspended in PBS and their uorescence intensity was measured
by ow cytometry using a BD FACSCalibur ow cytometer (BD Biosciences). The data for individual cells were then gated, using the
forward and side scatter values, to separate the data for the various cell population (granulocytes, monocytes, and lymphocytes).
The uorescence intensity of each population was analyzed using
CellQuest Pro software version 4.0.2 (BD Biosciences).
Because phagocytosis of FITC-BioParticles was inhibited by antiCD11b/c antibodies, expression levels of these receptors were
measured by ow cytometry using specic antibodies in the
absence of FITC-BioParticles. Therefore, the results of these experiments were independent of phagocytosis.
Measurement of phagocytosis in each leukocyte fractions in whole
blood
Because molecules in red blood cells are known to strongly
inhibit phagocytosis (Baranji et al. 1994), hemolytic blood was
not used for phagocytosis assays in this study. Whole blood in
microtubes was incubated with FITC-labeled E. coli BioParticles

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Y. Takeda et al. / Immunobiology 216 (2011) 10941102

50

FITC
C- BioParticle
e MFI

Lymphocytes

Monocytes

1000

Granulocytes

8
6
4
2

40

800

30

600

20

400

10

0
0

10

30

*
0

10

30

Wistar
GK

200
0

10

30 (min)

Fig. 1. Phagocytosis of E. coli BioParticles by monocytes in the peripheral blood. Whole blood from Wistar rats (open circle: control) and GK rats (closed circle) was incubated
with E. coli BioParticles (0.05 mg/mL) for 030 min at 37 C. The blood was then immediately mixed with Fix/Lyse buffer. The geometric mean of uorescence intensity (MFI)
on each leukocyte fraction in the blood was calculated using ow cytometry. *, P < 0.05, GK rats vs Wistar rats; n = 89.

(0.05 mg/mL) and incubated for 530 min at 37 C. Subsequently,

50 L of blood was transferred into 1 mL of Lyse/Fix buffer and incubated at 37 C for 10 min. In several experiments, we used E. coli
BioParticles labeled with pHrodo dye (pHrodo-labeled BioParticles), according to the manufacturers instructions. Lysed red blood
cells were removed by washing with PBS, and the mean of uorescence intensity (MFI) of each leukocyte population was measured
by ow cytometry.
Preparation of mononuclear leukocytes
Blood (5 mL) was diluted with an equal volume of PBS and
layered over 5 mL Ficoll-Hypaque in a 15-mL tube. The tube was
centrifuged at 320 g for 30 min at 25 C. Mononuclear leukocytes
(MNLs) in the middle layer were collected, washed twice with
PBS, and then suspended in Wistar-rat plasma. The suspension was
maintained on ice until the incubation with BioParticles.
ERK, NF-B, and Akt activity of monocytes in whole blood
To evaluate the phagocytic activity of leukocytes, we measured
the number of E. coli BioParticles that were engulfed by the cells.
In contrast, to evaluate the cell signaling involved in phagocytosis,
E. coli LPS, rather than E. coli BioParticles, was used as the stimulant because signals such as ERK, NF-kB, and Akt, measured in

the present study, are known to be activated during the process of

phagocytosis and to be stimulated by LPS (Blander and Medzhitov
2006; Blander 2007; Underhill and Ozinsky 2002).
Blood was aliquoted into microtubes and pre-incubated in a heat
block for 10 min at 37 C. Blood was treated with LPS (1 g/mL) for
0 to 10 min at 37 C, and immediately xed with Lyse/Fix buffer at
37 C for 10 min. Intracellular staining was performed according to
the procedure recommended by cell signaling technology. In brief,
xed cells were permeabilized in 90% methanol (pre-chilled at
20 C) for 30 min on ice. The cells were washed with BD Pharmingen Stain Buffer to block non-specic antibody reactions, and then
reacted with aliquots of the optimal concentration of antibodies
(anti-phospho-ERK1/ERK2 Ab, anti-p44/42 MAPK Ab, anti-NF-B
p65 mAb, anti-phospho-NF-B p65 mAb, anti-Akt (pan) mAb, and
rabbi anti-Phospho-Akt mAb) at room temperature for 30 min in
the dark. After the cells were washed with PBS containing 0.4%
bovine serum albumin, the cells were reacted with AlexaFluor488conjugated donkey anti-rabbit IgG antibodies (1:800) for 30 min at
room temperature in the dark. The cells were then washed with
PBS, re-suspended in PBS and analyzed by ow cytometry.
Statistics
The data are displayed as the mean standard error (SE). Comparison in data groups was performed by analysis of variance

Fig. 2. Ingestion and phagocytosis of BioParticles by monocytes. (A) Ingestion of pHrodo-labeled BioParticles by monocytes in the peripheral blood. Whole blood from Wistar
rats (open circles) and GK rats (closed circles) was incubated with pHrodo-labeled BioParticles (0.2 mg/mL) for 0, 5, 10 or 30 min at 37 C. The geometric mean of uorescence
intensity (MFI) of the monocyte fraction in the leukocytes was calculated using ow cytometry, and displayed as Ingestive index (y-axis). *, signicant difference between
GK and Wistar rats (P < 0.05, n = 6). (B) Phagocytosis of FITC-labeled BioParticles by monocytes in mononuclear cells (MNLs). MNLs were puried from Wistar rats (open
columns) and GK rats (closed columns). FITC-labeled BioParticles were opsonized by incubation with plasma (plasma:PBS, 1:1) obtained from Wistar rats for 15 min at 37 C.
MNLs (5 106 cells/mL) were mixed with the opsonized BioParticles (0.25 mg/mL) for 60 min on ice (binding) or for 30 min at 37 C (total). The cells were then immediately
xed by 10% formaldehydePBS on ice. The MFI of the monocyte fraction was measured using ow cytometry. Net phagocytosis was calculated as the difference of total
and binding values. *, signicant difference between GK and Wistar rats (P < 0.05, n = 4).

Y. Takeda et al. / Immunobiology 216 (2011) 10941102

Results
Atteunation of phagocytic activity of monocytes in GK rats
To estimate the phagocytic activity of various sub-populations
of leukocytes, whole blood was incubated with FITC-labeled E. coli
BioParticles and subjected to ow cytometry analysis. The uorescence intensity estimate, MFI, was used as an indicator of the
number of BioParticles taken up by the cells. In granulocytes and
monocytes, the MFI gradually increased with time of incubation,
whereas in lymphocytes the MFI increased for the rst 5 min of
incubation and then plateaued (Fig. 1). The phagocytic activity of
granulocytes was more than ten times higher than the phagocytic activity of monocytes (Fig. 1). The phagocytic activity at 10
and 30 min of incubation was signicantly lower in the monocyte
population from GK rats than in that from Wistar rats (Fig. 1, middle panel). In contrast, the phagocytic activity of lymphocytes and
granulocytes were not signicantly different between GK rats and
Wistar rats (Fig. 1, left and right panels).
Atteunation of ingestive and binding activities of monocytes in
GK rats
We used pHrodo-labeled BioParticles, which have recently been
developed as a sensitive method to detect formation of intracellular phagosomes. The pH inside phagosomes is known to be acidic,
which results in dramatic increases in the uorescence intensity
of pHrodo dye. As shown in Fig. 2A, ingestion of pHrodo-labeled
BioParticles measured at 10 and 30 min of incubation was signicantly lower in monocytes from GK rats than in monocytes from
Wistar rats. These results agree with our original ndings for pHindependent FITC-labeled BioParticles shown in Fig. 1.
To discriminate BioParticles bound on the cell surface and those
internalized into the cells, we used a method of chilling on ice,
which is known to stop the process of endocytosis. Because chilling
of whole blood also inhibits opsonization and activates platelets, we
rst prepared opsonized FITC-BioParticles, and then used puried
mononuclear leukocytes (MNL) to examine the effects of chilling
on phagocytic activity in MNL. As shown in Fig. 2B, uorescence
intensity of chilled MNL was signicantly lower in the GK rat
group than in the Wistar rat group. This indicates that surface binding of BioParticles was signicantly decreased in GK rats. The net
increase corresponding to phagocytosis (the difference between
total uorescence intensity and the intensity under chilling) was
then calculated, and GK rats showed a signicantly lower net
increase in uorescence intensity of the BioParticles compared to
Wistar rats (Fig. 2B). Taken together, these results indicate that
both ingestion of BioParticles and surface binding of BioParticles
by monocytes is attenuated in GK rats compared to Wistar rats.
Effects of plasma-exchange on phogocytic activity of monocytes
To determine whether the attenuation of phagocytic activity
of monocytes from GK rats was related to plasma factor(s), we
isolated mononuclear leukocytes (MNLs) and suspended them in
plasma obtained from Wistar rats. Because repetitive washing of
rat whole blood induces hemolysis, we used isolated MNL for this
plasma-exchange experiment. At 10 min, phagocytosis of monocytes was comparable between the Wistar and GK rat groups, but
at 30 min it was signicantly lower in the GK rat group than in the

50

FITC-BioP
Particle MFI

(ANOVA), the Mann Whitney test or the Wilcoxon test. Data analysis was performed using the Prism Software version 5.03 (GraphPad
Software, San Diego, CA, USA). P-values less than 0.05 were considered signicant.

1097

MNL
Wistar

40
30
20

GK

10
0
0

10

30

(min)

Fig. 3. Effect of plasma exchange on monocyte phagocytosis. MNLs were puried

from the blood of Wistar rats (open circles) and GK rats (closed circles). The cells
were washed with PBS and suspended in plsma (plasma:PBS 1:1 v/v) obtained from
Wistar rats. The cells (5 106 cells/mL) were mixed with FITC-labeled BioParticles
(0.05 mg/mL) for 0, 5, 10 or 30 min at 37 C, and then immediately xed by 10%
formaldehydePBS on ice. The geometric mean of uorescence intensity (MFI) of
the monocyte fraction was measured using ow cytometry. *, Signicant difference
between GK and Wistar rats (P < 0.05, n = 4).

Wistar rat group (Fig. 3). Therefore, the monocyte itself is responsible for the attenuation of phagocytosis found in this study. The
reason for the absence of a difference in phagocytosis at 10 min is
not known; however, one possible explanation is the inuence of
plasma exchange on the phagocytic receptors of monocytes from
GK rats (Figs. 1 and 3).
Decrease in CD11b/c expression of GK rats
We analyzed representative phagocytic receptors of monocyte/macrophages such as CD32 (Fc receptors), CD36 and SR-BI
(scavenger receptor), and CD11b and CD11b/c (complement receptor). While, CD71 (transferrin receptor), which is not involved in
phagocytosis, was used as a reference. As shown in Fig. 4, levels of
CD36, CD32 and SR-BI tended to be lower in monocytes of GK rats
than in monocytes of Wistar rats, although these differences were
not statistically signicant. In contrast, the percentages of CD11b
and CD11b/c-positive cells were signicantly lower in GK rats than
in Wistar rat (Fig. 4E and F). In the whole blood cell-staining method
used in the present study, an activation marker of granulocytes (RP1) was at a trace level both in Wistar rats and in GK rats (data not
shown).
Levels of ERK, NF-B and Akt activation
Up-regulation of ERK, NF-B and Akt signaling has been shown
to be involved in phagocytosis (Agrawal et al. 2007) and to be
induced by high glucose conditions (Dasu et al. 2008; Shanmugam
et al. 2004) or stimulation with LPS. To examine the possibility of
alterations in the above signals in monocytes of GK rats, levels of
ERK, NF-B and Akt activation were evaluated as shown in Fig. 5.
The rate of ERK phospholyration after 5 min of LPS stimulation
was signicantly higher than the basal level in GK and Wistar
rats (Fig. 6A). The rate of NF-Bp65 phosphorylation also significantly increased after 5 min of LPS stimulation (Fig. 6B). There
were no signicant differences in the rates of ERK and NF-Bp65
phosphorylation between GK and Wistar rats (Fig. 6A and B). The
phosphorylation rate of Akt in monocytes was not affected by LPS
stimulation and was comparable between the GK and Wistar rat
groups at each time point during phagocytosis (Fig. 6C). Therefore,
the PI3K/Akt signaling pathway is also not responsible for the difference in the phagocytic activity of monocytes in GK and Wistar
rats.

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Y. Takeda et al. / Immunobiology 216 (2011) 10941102

Fig. 4. Expression of a transferrin receptor (CD71) and phagocytic receptors (CD32, CD36, SR-BI, CD11b and CD11b/c) in monocytes. Whole peripheral blood from control
Wistar rats (open columns) and GK rats (closed columns) was stained separately with specic antibodies. Because CD36 is expressed not only on leukocytes but also
on platelets and erythrocytes, anti-CD36 antibody binding was measured in mononuclear leukocytes after their separation from whole blood. For each receptor type, the
percentage of receptor-positive monocytes was measured using ow cytometry. *, signicant difference between GK and Wistar rats (P < 0.05, n = 4).

Fig. 5. Representative results of ow cytometry analysis of signaling molecules in monocytes. Whole blood from Wistar rats (upper) and GK rats (lower) was incubated with
LPS (1 g/mL) for 0 or 5 min at 37 C. After the incubation, cells were immediately treated with a Fix/Lysis buffer, and then stained with anti-ERK1/2, anti-phosphorylated
ERK1/2, anti-NF-Bp65 or anti-phosphorylated NF-Bp65 antibodies (see Materials and Methods). The uorescence intensity of the stained cells was measured by ow
cytometery. The mean of uorescence intensity (MFI) in monocytes was obtained from the results of whole blood by gating with FSC and SSC parameters. The results
displayed are representative of more than six independent experiments.

Y. Takeda et al. / Immunobiology 216 (2011) 10941102

5
**

4
3

**

2
1
0

(C)

5
***
4
*
3
*
2
1
0

10

10

Phosspho:non-phosp
pho-Akt

( )
(B)
Phospho
o:non-phospho--NF-Bp65

Phospho
o:non-phospho--ERK1/2

((A))

1099

1.0
0.8
0.6
0.4

Wistar
GK

0.2
0.0
0

10
((min))

Fig. 6. Phosphorylation rate of ERK1/2, NF-Bp65, and Akt after lipopolysaccharide stimulation. Whole blood from Wistar rats (open circles) and GK rats (closed circles) was
incubated with LPS (1 g/mL) for 0, 1, 5 or 10 min at 37 C. After treatment with a Fix/Lysis buffer, the cells were stained with anti-ERK1/2, anti-phosphorylated ERK1/2, antiNF-Bp65, anti-phosphorylated NF-Bp65, anti-Akt or anti-phosphorylated Akt antibodies (see Materials and Methods). The geometric mean of uorescence intensity (MFI)
in monocytes was measured using ow cytometry and used to calculate the ratios of phosphorylated:non-phosphorylated ERK1/2, phosphorylated:non-phosphorylated NFBp65 and phosphorylated:non-phosphorylated Akt. *, P < 0.05, **, P < 0.01, ***, P < 0.001; n = 6, signicant difference from the basal level at time 0, phospho, phosphorylated;
non-phospho, non-phosphorylated.

Effects of diet restriction on body weight and glucose levels

Rats were separated into two groups: one was fed a normal diet,
and the other was fed a restricted diet. At age 15 wk, body weight
was signicantly lower in GK rats than in Wistar rats regardless
of the diet protocol used (Fig. 7A); blood glucose was signicantly
higher in GK rats fed a normal diet than in Wistar rats fed a normal
diet (Fig. 7B); and the glucose level of GK rats fed a restricted diet
was comparable to the glucose level of Wistar rats fed a normal diet
(Fig. 7B).
Effects of diet restriction on phagocytosis and CD11b/c expression
The reduction in phagocytic activity of monocytes in GK rats
compared to Wistar rats (Fig. 1, middle panel) was not signicantly
improved by diet restriction (Fig. 8A). Similarly, there was no significant difference in the percentage of CD11b/c-positive monocytes
in GK rats with and without diet restriction (Fig. 8B).
Discussion
This is the rst study demonstrating the attenuation of phagocytic activity of monocytes in type 2 diabetes rat (GK rat).
Phagocytosis consists of multiple steps including the binding, endocytosis and killing of bacteria. These steps require activation of
a variety of molecular systems, such as the phagocytic receptor,
CD11b/c, and signal transduction pathways initiated with activation of Fc receptors (FcR) and TLRs followed by activation of NF-B
and F-actin polymerization (Blander 2007; Legate et al. 2009; Stuart
and Ezekowitz 2005). TLR signaling regulates expression of inammatory cytokine genes via both NF-B- and mitogen-activated
protein kinase (MAPK)-related pathways, as well as by regulating
type I IFN via interferon regulatory factor-3 (IRF3) (Kawai and Akira
2007, 2009).
The expression levels of CD11b/c and other phagocytic receptors
in monocytes were signicantly lower in GK rats than in control rats, whereas the activities of signaling molecules known to be
involved in phagocytosis, ERK, NF-Bp65, and Akt, were not significantly different between monocytes derived from GK and control
rats. Furthermore, the phagocytic activity of monocytes in GK rats
was attenuated in the presence of the plasma obtained from Wistar
rats. Neither attenuated phagocytic activity nor reduced CD11b/c
expression was restored in GK rats fed with a calorie-restricted

diet, which was effective for reduction of body weight and blood
glucose in GK rats. Taken together, these results suggest that
attenuation of phagocytic activity in GK rats is, at least in part,
explained by low expression of receptors involved in phagocytosis,
and is not due to post-receptor signaling, plasma-derived factor(s),
or glucose intolerance.
In both GK and Wistar rats, phagocytic activity of lymphocytes
was much lower than that of monocytes and granulocytes (Fig. 1).
Thus, although there have been some papers that have reported
the phagocytic activity of lymphocytes (Koszewski et al. 1957;
Svedentsov et al. 2004), our data is consistent with the present consensus (Brown 1991; Lee et al. 2003; Underhill and Ozinsky 2002)
that monocytes and neutrophils, but not lymphocytes, play central
roles in the elimination of bacterial pathogens in vivo.
CD18/CD11b,c (2 integrin) are key molecules for bacterial
binding to monocyte (Stuart and Ezekowitz 2005). CD18/CD11b
and CD18/CD11c are also known as complement receptor (CR) 3
and CR4, respectively. In this study, CD32, CD36 and SR-BI were
also measured. These receptors, FcRII CD32 is Fc receptor II (FcRII),
and both CD36 and SR-BI are scavenger receptors (SRs). CR3, CR4,
FcRII and SRs are closely related to phagocytosis (Areschoug and
Gordon 2009; Underhill and Ozinsky 2002). In contrast, CD71, a
transferrin receptor, is not associated with phagocytosis and was
used here as a reference in analyses of receptor levels. CD45,
which is a leukocyte surface marker that is useful for discriminating cell debris and erythrocytes, was also used as a reference.
Expression of CD11b and CD11b/c was signicantly lower in the
GK rat group than in the Wistar rat group, whereas CD45 and
CD71 expression was comparable between the GK and Wistar
rat groups. Thus, expression of CD11b/c is specically attenuated
in monocytes of GK rats compared with monocytes of Wistar
rats.
CD16, CD64, SRA, MARCO and CD206 also related with phagocytosis (Areschoug and Gordon 2009; Underhill and Ozinsky 2002).
Because reliable antibodies against rat CD16, CD64, SRA, MARCO
and CD206 were not commercially available, these receptors were
not included in the analysis. The expression of CD11b/c in monocytes was signicantly different between diabetic rats and control
rats, but the size of this difference was not sufcient to account
for the large difference in the phagocytosis of BioParticles. Therefore, alteration in other mechanism(s) may also be involved in the
impaired phagocytic activity of monocytes in diabetic rats, and
these mechanism(s) need to be claried in future studies.

1100

Y. Takeda et al. / Immunobiology 216 (2011) 10941102

(A)

Wistar

Normal diet

(B)

GK

***
***

GK

200

100

NS

**

200

***
***

***

NS

Wistar

Restricted diet

***

***

300

***

Body weight (g)

**

400

Blood gluccose (mg/dl)

300

100
10

11

12

13

14

15

Weeks

10

11

12

13

14

15

Weeks

Fig. 7. Effects of diet restriction on body weight and glucose level. Body weight (A) and fasting blood glucose (B) were measured weekly until age 15 wk. The data from
rats fed a normal diet (n = 8 in each group) and fed a restricted diet (n = 8 in each group) are shown as circles and triangles, respectively. *, P < 0.05, **, P < 0.01; n = 6. NS, not
signicant.

Although CD11b/c-positive cells consisted of CD11b-positive

cells and CD11c-positive cells, the percentage of CD11b/c-positive
cells was lower than the percentage of CD11b-positive cells both
in Wistar and GK rats (Fig. 4E and F). This might be due to the
difference in the reactivity of the CD11b/c and CD11b antibodies,
because anti-CD11b/c antibody recognizes epitope that is functionally different from that recognized by anti-CD11b antibody
(Draskovic-Pavlovic et al. 1999). The level of CD11b expression was
slightly but signicantly lower in monocytes of GK rats than in
monocytes of Wistar rats (Fig. 4E), and this difference was smaller
than the difference in CD11b/c expression between the GK and
Wistar rats (Fig. 4E and F). Therefore, we speculate that expression
of CD11c is also lower in monocytes of GK rats than in monocytes
of Wistar rats.

The phagocytic activity of monocytes was impaired in GK

rats, but this was not the case for granulocytes. Since the phagocytic activity of granulocytes was more than ten times higher than
that of monocytes, impaired phagocytic activity of monocytes in
GK rats may not be a major reason for susceptibility to infection in type 2 diabetes. Ingestion of pHrodo-labeled BioParticles
was signicantly lower in monocytes from GK rats than in monocytes from Wistar rats. This suggests that the process of killing of
intracellular bacteria by monocytes is also attenuated in GK rats
compared to Wistar rats. Similar to the difference in phagocytosis
of FITC-labeled BioParticle between granulocytes and monocytes,
ingestion of pHrodo-labeled BioParticles, assayed after a 30 min
incubation, was four-to-ve times higher in granulocytes than in
monocytes both in GK and Wistar rats [Wistar rats: 98.81 23.32

Fig. 8. Effects of diet restriction on phagocytosis and CD11b/c protein expression. (A) Whole blood obtained from Wistar and GK rats fed a normal diet (open columns)
or a restricted diet (closed columns) were incubated with E. coli BioParticle (0.05 mg/mL) for 30 min at 37 C. The geometric mean of uorescence intensity (MFI) in each
leukocyte fraction in blood was measured using ow cytometry. (B) The percentage of CD11b/c-positive cells in each leukocyte fraction was measured using ow cytometry.
*, P < 0.05, GK rats (restricted diet) vs Wistar rats (restricted diet); n = 89. NS, not signicant.

Y. Takeda et al. / Immunobiology 216 (2011) 10941102

(granulocytes) vs 28.12 4.57 (monocytes); GK rats: 68.97 4.87

(granulocytes) vs 14.28 1.47 (monocytes), n = 7]. Thus, the process of killing of intracellular bacteria is substantially less efcient
in monocytes compared to granulocytes.
However, phagocytosis in monocytes and macrophages is
known to be an important process for immunoreactions (Palucka
and Banchereau 2002; Rosenberger and Finlay 2003). Antigen presentation on monocytes and macrophages is a bridging step from
the innate immune system to the adaptive immune system, and
is dependent on the phagocytic activity of the monocytes and
macrophages (Blander 2007; Jutras and Desjardins 2005). Therefore, decreased phagocytic activity of monocytes in patients with
type 2 diabetes is suggested to play a role in the higher incidence
of infectious diseases and cancer due to impaired immune function
in these patients.
The purpose of this study was to compare phagocytic activity of
each leukocyte fraction (granulocyte, lymphocyte and monocyte)
in diabetic and control rats. To perform Western blotting, isolation of each leukocyte fraction is required. The monocyte fraction
is usually obtained by its adhesion to the culture plate, and this procedure results in activation of monocytes and selects for monocytes
with high binding activity. Therefore, instead of Western blotting,
we evaluated the expression of specic proteins by ow cytometry, which provides quantitative measurements of specic protein
expression through the use of specic antibodies without separation of each leukocyte fraction. Flow cytometry is commonly used
for this purpose, and has recently been used for evaluation of signaltransduction proteins such as ERK and NF-kB in leukocyte fractions
(Bardet et al. 2006; Lafarge et al. 2007). As shown in Fig. 5, in monocyte fractions of whole blood isolated from Wistar rats and GK
rats, the number of cells showing immunoreactivity to phosphorylated ERK or phosphorylated-NF-kB was increased by stimulation
with LPS, whereas the number of cells showing immunoreactivity
to total ERK or total NF-kB was not altered by stimulation with LPS.
Thus, activation of signal transduction through ERK and NF-kB in
monocytes is detectable by ow cytometry.
In conclusion, the phagocytic activity of monocytes is attenuated in GK rats. This attenuation can be partly explained by a
decrease in CD11b/c expression of GK rats but is independent of
blood glucose levels.
Conict of interest
There are no conicts of interest.
Acknowledgements
This work was supported by a Grant-in-Aid for Scientic
Research from the Japan Society for the Promotion of Science (No.
22590560) and a Grant-in-Aid for researchers at Hyogo College of
Medicine (2009).
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