Molecular Immunology 49 (2011) 234–238

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Molecular Immunology
journal homepage: www.elsevier.com/locate/molimm

Characterization of circulating and monocyte-derived dendritic cells in obese and

diabetic patients
Claudia Musilli a , Sara Paccosi a , Laura Pala c , Gianni Gerlini d , Fabrizio Ledda a , Alessandro Mugelli a,b ,
Carlo Maria Rotella c , Astrid Parenti a,b,∗
a
Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6-50139, Florence, Italy
b
CIMMBA, University of Florence, Florence, Italy
c
Department of Clinical Pathophysiology, University of Florence, Florence, Italy
d
Plastic Surgery Unit, Regional Melanoma Referral Center, SM Annunziata Hospital, Florence, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Dendritic cells (DCs) are suspected to be involved in the development of atherogenesis, but their role is
Received 29 June 2011 still unclear. The aim of this study was to characterize circulating DCs and monocyte-derived DCs (Mo-
Received in revised form 23 August 2011 DCs) of obese and diabetic patients (T2D), and to study their interaction with human coronary smooth
Accepted 25 August 2011
muscle cells (CASMCs). Obese post-menopausal women with or without insulin resistance were enrolled
Available online 21 September 2011
and were compared to age-matched healthy women. Myeloid circulating DCs significantly increased in
obese T2D patients compared to healthy donors and a smaller increase was observed for plasmacytoid
Keywords:
one. Mature Mo-DCs from obese T2D patients significantly decreased when compared to control, but they
Dendritic cells
Vascular wall remodeling
were significantly more capable of adhering to CASMCs compared to that from healthy controls and from
Coronary smooth muscle cells not-T2D obese subjects. Altogether these data suggest that in conditions of insulin-resistance and obesity
Obesity there is an up-regulation of myeloid DCs that might contribute to pathological vascular remodeling.
Type 2 diabetes © 2011 Elsevier Ltd. All rights reserved.

1. Introduction whose complications represent the main cause of morbidity and

Recognition that different cardiovascular risk factors often clus-
ter together has focused attention on the importance of evaluating
global cardiovascular risk in order to identify high risk patients
without clinical manifestations of atherosclerosis (Grundy et al.,
1999). Vascular remodeling, a characteristic shared by all risk fac-
tors, is a chronic multi-factorial process in which systemic or local
inflammatory pathogenetic mechanisms play a key role. Inflamma-
tion participates in atherosclerosis, from endothelial dysfunction
to end-stage thrombotic complications of the plaque (Libby, 2006).
This complex inflammatory process involves many cell types and
interactions. Dendritic cells (DCs) are localized in atherosclerotic
lesions (Waltner-Romen et al., 1998; Niessner and Weyand, 2010)
Abbreviations: BDCA1,2,3, blood dendritic cell antigen 1,2,3; BMI, body mass
index; CAD, coronary artery disease; CASMCs, human coronary smooth muscle
cells; CV, cardiovascular; CVD, cardiovascular disease; DCs, dendritic cells; FCS, Fetal
Calf Serum; Mo-DCs, monocyte-derived dendritic cells; PBMCs, peripheral blood
mononuclear cells; ␣-SMA␣, -smooth muscle actin; T2D, type 2 diabetes; VSMCs,
vascular smooth muscle cells.
∗ Corresponding author at: Department of Preclinical and Clinical Pharmacology,
University of Florence, Viale Pieraccini 6-50139, Florence, Italy.
Tel.: +39 0554271330; fax: +39 0554271280.
E-mail address: astrid.parenti@unifi.it (A. Parenti).
death in diabetic patients. The hypothesis that DCs are critical for
atherogenesis is also supported by the fact that statin therapy leads
to a lower number of DCs in atherosclerotic plaques (Yilmaz et al.,
2004). Moreover, in patients with coronary artery disease (CAD),
the number of circulating DCs is significantly decreased, while their
number in atherosclerotic plaques is increased (Yilmaz et al., 2006;
Bobryshev and Lord, 1998).
Insulin resistance is a risk factor for atherosclerosis (Reddy
et al., 2010). Most patients with type 2 diabetes mellitus (T2D)
develop cardiovascular disease (CVD) with substantial loss of life
expectancy. This is somehow related to the high prevalence of
other CVD risk factors, such as hypertension and obesity (Chilton
et al., 2011). The link between T2D and obesity is indisputable.
Clinical and preclinical data highlight the role of adipose tissue
in the development of a systemic inflammatory state through the
release of adipokines (Friedman and Halaas, 1998; Matsumoto
et al., 2002) which mediate the multiple pathogenic mechanisms in
the well-known, but poorly understood, associations between obe-
sity, cardiovascular pathology and co-morbidities such as insulin
resistance. In this context, the role of DCs in atherogenesis is poorly
understood. It has been recently demonstrated that in T2D men
there are quantitative and qualitative functional abnormalities in
circulating subpopulations of DCs, especially in those patients with
atherosclerotic complications (Corrales et al., 2007). Interestingly,

0161-5890/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.molimm.2011.08.019
 C. Musilli et al. / Molecular Immunology 49 (2011) 234–238 235

Table 1 (Miltenyi et al., 1990) according to the manufacturer’s instructions.

Clinical characteristics of the study patients.
DCs were generated by culture of CD14+ in RPMI 1640 as previously
Obese diabetic patients Obese non diabetic reported (Bellik et al., 2006).
(n = 17) patients (n = 9)

Age 70.3 ± 5.8 63 ± 13 2.4. Cell culture

BMI (kg/m2 ) 31 ± 2.5 34.5 ± 5.1
Plasma glucose (mg/dL) 123.48 ± 48 98.2 ± 13.3 Human coronary artery smooth muscle cells (CASMCs) were
HbA1c < 7% 6.38 ± 0.4 5.3 ± 0.4
purchased from Lonza Walkersville (Italy). They were grown in
HbA1c > 7% 8.18 ± 1.16 –
TOT cholesterol (mg/dL) 177.41 ± 36 179.6 ± 10.8 Basal Medium supplemented with human epidermal growth factor,
HDL-C (mg/dL) 52.41 ± 12 40.6 ± 12.8 insulin, human fibroblast growth factor, 10% Fetal Calf Serum (FCS)
LDL-C (mg/dL) 97.65 ± 37 108 ± 17.2 and antibiotics in a humidified atmosphere of 5% CO2 in air. Culture
TG (mg/dL) 148.35 ± 54 150.6 ± 59
medium was changed every 2 days. Experiments were performed
using cells of 5–10 passages.
in T2D women recruited from the ACCORD trial, there was a sig-
2.5. Adhesion assay
nificant correlation between reduction in plasmacytoid DCs, TNF-␣
production and poor glycemic control (Blank et al., 2010). However,
The adhesion of Mo-DCs to CASMCs was performed by fluo-
a through characterization of circulating and monocyte-derived
rescent assay using Vybrant Cell Adhesion Assay Kit (Molecular
DCs (Mo-DCs) in obese and T2D patients and their relationship with
Probes). Briefly, fluorescently labeled DCs were incubated with
vascular wall cells has not yet been done.
CASMC monolayer for 45 min. After cell washing, adherent cells
The aim of this paper was to measure circulating DCs and (Mo-
were immediately quantified with the use of a fluorescence multi-
DCs) in obese and T2D patients. Moreover, their ability to interact
reader (Victor 3, Perkin Elmer) (Bellik et al., 2008).
with human coronary smooth muscle cells (CASMCs) was also
investigated.
2.6. Immunofluorescence analysis

2. Patients and methods
2.1. Patients
Twenty-six obese post-menopausal women, (to avoid poten-
tially confounding factors related to menstrual cycle-related
fluctuation in plasma estrogen concentration), were recruited from
the Clinic of Metabolic Disorders and Diseases of the University
of Florence, AUO-Careggi. Seventeen of them had T2D and were
being treated with oral drugs; all were evaluated for their lipidic
and glycemic profile (fasting plasma glucose). They had a median
age of 70 years with a BMI > 30 (Table 1). Exclusion criteria included:
presence of hepatic, cardiac or renal insufficiency, cancer, insulin
therapy and any previous cardiovascular event. The control group
consisted of twelve healthy post-menopausal women. For each
participant 3 samples of about 5 mL of fresh peripheral blood in
test-tubes with EDTA were collected. Written consent was obtained
from each participant to obtain a sample of peripheral blood. The
protocol was approved by the local Ethical Committee (prot. N.
0011762).
2.2. Blood sampling and analysis of circulating DCs
EDTA venous blood samples were obtained in the morning. Iden-
tification and quantification of circulating DCs were performed
on peripheral blood mononuclear cells (PBMCs) using a direct
immunofluorescence, single-platform flow cytometry technique,
as previously described (Miltenyi et al., 1990). Briefly, PBMCs
were stained immediately while the remaining cells were used
for monocyte isolation (CD14+ ). Monoclonal antibodies that rec-
ognize the different lineage markers: BDCA-2 FITC for lymphoid
DCs, BDCA-1 PE and BDCA-3 FITC for two subsets of myeloid DCs
(BDCA1,2,3 Miltenyi Biotec) were used. Negative lineage mark-
ers (Lin1-FITC, containing antibodies against CD3-14-16-19-20-56;
BD-Pharmingen; San Diego, CA) and BDCA+ cells were considered
as DCs.
2.3. Ex vivo generation of Mo-DCs
CD14+ cells were separated by performing a positive selection
with CD14 micromagnetic beads by magnetic cell sorting (MACS)
Following Mo-DC adhesion to CASMCs, cells were fixed in
2% phosphate-buffered formaldehyde for 10 min, washed with
PBS and quenched for 20 min with 2% BSA. These cells were
labeled overnight at 4 ◦ C at manufacturer-recommended concen-
trations with anti-human ␣-SMA monoclonal antibody (Dako A/S
Denmark.) and then with an Alexa Fluor 594 conjugated goat-anti
mouse secondary antibody (Molecular Probes, OR, USA) for 1 h at
room temperature. Then cells were labeled with an anti-human
HLADR-FITC antibody (BD Pharmigen, MA, USA) for 2 h at room
temperature, followed by the addition of an anti-FITC-Alexa Fluor
488 antibody for 90 min at room temperature. Samples were exam-
ined with a fluorescence microscope.
2.7. Statistical evaluation
Data are reported as means ± SEM. Statistical analysis was per-
formed using Student’s t-test for unpaired data and ANOVA test
(Bonferroni’s and Dunnett’s Multiple Comparison Test). P < 0.05
was considered significant.
3. Results
3.1. Characterization of circulating DCs in obese and T2D patients
Circulating myeloid BDCA1+ DCs were slightly increased in
obese patients compared to healthy subjects, while BDCA2+ and
BDCA3+ were not significantly changed (Fig. 1A). Myeloid DCs,
either BDCA1+ or BDCA3+ , were significantly increased in obese and
diabetic patients (obese T2D). BDCA1+ -DCs, indeed, represented
2.59 ± 0.59% of the total cell population (twofold increase compared
to healthy control P < 0.01) and BDCA3+ -DCs were twice as high
when compared to healthy subjects (0.79 ± 0.18 vs. 0.38 ± 0.08,
P < 0.05). Plasmacytoid BDCA2+ -DCs increased when compared
to healthy subjects, although this difference was not significant
(1.30 ± 0.43 vs. 0.6 ± 0.09) (Fig. 1A). Based on these results, myeloid
DCs, either BDCA1+ or BDCA3+ , in obese T2D patients, were com-
pared to their glycemic control. However, myeloid DCs of T2D
patients with poor glycemic control did not differ from those of
T2D patients with good glycemic control (Fig. 1B). The analysis of
mature myeloid DCs (Mo-DCs) obtained from circulating precur-
sors showed no differences in the yield of Mo-DCs obtained from
236 C. Musilli et al. / Molecular Immunology 49 (2011) 234–238

Fig. 1. Analysis of circulating-DCs and phenotypical characterization of mature Mo-DCs in obese T2D patients. (A) Flow cytometric analysis of circulating DC subsets was
performed on PBMCs isolated from obese patients (obese), obese and diabetic patients (obese T2D) and healthy women (ctr). Means ± SE of positive cells. *P < 0.05, **P < 0.01
vs. healthy control (ctr). (B) Flow cytometric analysis of BDCA1+ and BDCA3+ DCs in obese T2D patients with poor and good glycemic control. (C) Flow cytometric analysis
of mature Mo-DCs obtained ex vivo from healthy (ctr), obese (obese) and obese and diabetic (obese T2D) patients. Mean ± SE of positive cells. *P < 0.05, **P < 0.01 vs. healthy
control (ctr).

the two groups of patients compared to the control group, repre-
senting nearly 2% of recovered PBMCs.
The analysis of the mature Mo-DCs showed that these cells
were significantly lower in obese and T2D patients, compared with
healthy and obese subjects (Fig. 1C). There was a 38% (P < 0.01),
40% (P < 0.05) and 14% (P < 0.05) reduction in CD83, (the DC matu-
ration marker), CD80 and CD86, (the DC’s costimulatory molecules),
respectively, compared to healthy controls. With regard to the
number of Mo-DCs obtained from obese not T2D patients, the per-
centage of CD83+ , CD80+ , CD86+ DCs was the same as that of healthy
individuals (Fig. 1C).
3.2. Mo-DCs adhesion to human coronary smooth muscle cells
Since it has been reported that DCs accumulate in the atheroscle-
rotic lesion (Bobryshev and Lord, 1998), suggesting their role in its
pathogenesis and/or its complication, we investigated the ability
of mature Mo-DCs, generated ex vivo from circulating precursors of
obese and T2D patients, to adhere to a monolayer of human coro-
nary smooth muscle cells (CASMCs). Mo-DCs obtained from obese,
obese T2D patients and controls were calcein-labeled and then
incubated for 45 min with CASMC monolayers. Mo-DCs obtained
from obese T2D patients were significantly more capable of adher-
ing to CASMCs compared to healthy DCs (+81% over control, P < 0.05,
Fig. 2A). This adhesion is documented in the bottom panels of Fig. 2
in which adherent HLADR-FITC labeled Mo-DCs on ␣-SMA-labeled
CASMCs are reported.
Moreover, the adhesion of Mo-DCs obtained from obese and
diabetic patients was significantly higher than that of Mo-DCs
obtained from obese non-diabetic ones (P < 0.01 vs. obese), whose
adhesion was superimposable on that of controls (Fig. 2A).
4. Discussion
Our study demonstrates that circulating myeloid DCs show
quantitative abnormalities in obese-diabetic patients (obese T2D)
with no previous major cardiovascular events compared to obese
or healthy subjects. Moreover, the most interesting finding of the
present paper is that the mature Mo-DCs from obese and T2D
patients significantly increased their adhesion to human coronary
smooth muscle cells compared to either obese patients or con-
trols. This adhesion is confirmed by means of immunofluorescence
analysis of ␣-SMA+ smooth muscle cells and HLADR+ Mo-DCs.
This enhanced adhesiveness to smooth muscle cells, which has
not been demonstrated so far, may suggest increased DC adhesion
molecules and or other receptors involved in this process. Inflam-
mation is a key process in pathological vascular remodeling. It has
been demonstrated that diabetic conditions stimulate binding of
monocytes to vascular smooth muscle cells (VSMCs), thus suggest-
ing that vascular wall inflammation, present in diabetic patients,
may accelerate atherosclerosis (Meng et al., 2010). It is well known
that obesity and insulin resistance are associated with a low-grade
inflammation which can drive vascular wall remodeling (Chilton
et al., 2011; Andersson et al., 2008). Obesity exacerbates or causes a
number of debilitating, chronic medical conditions, including T2D.
The link between T2D and overweight/obesity is indisputable. The
risk of developing diabetes escalates with the degree of excess
weight (Ford et al., 1997). Epidemiological evidence suggests that
CV risk begins to develop many years before the diagnosis of T2D.
Insulin resistance is itself associated with an increased risk for CVD
and is commonly comorbid with hypertension, dyslipidemia, obe-
sity, and a prothrombotic state (Chilton et al., 2011; Ishizaka et al.,
2003). Endothelial cell dysfunction, perhaps promoted by insulin
resistance has been associated with deleterious changes in the vas-
cular wall, changes that have been involved in the development of
atherosclerosis and hypertension (Ishizaka et al., 2003; Ausk et al.,
2010). Altered endothelial function has a significant impact on DC
adhesion to the endothelium (Weis et al., 2002), although no infor-
mation has been reported, to our knowledge, on the interaction
between VSMCs and DCs in a condition of insulin resistance and
compromised immune functions. Although DCs are demonstrated
to reside in the media layer of a vessel wall between smooth mus-
cle cells (␣-SMA+ ) (Bobryshev and Lord, 1998), they are mainly
localized in the neointima together with other inflammatory cells.
However, it is noteworthy that smooth muscle cells are also res-
ident in the neointima when endothelial dysfunction occurs and
 C. Musilli et al. / Molecular Immunology 49 (2011) 234–238 237

Fig. 2. Mo-DC adhesion to human coronary smooth muscle cells (CASMCs). (A) Calcein-labeled Mo-DCs were allowed to adhere to CASMCs and then their fluorescence was
measured. Mean ± SE of fluorescence of adherent cells; *P < 0.05 vs. healthy control (ctr); § P < 0.01 vs. obese. (B) Immunofluorescence analysis of HLA-DR-labeled Mo-DCs
(green) adherent on ␣-SMA-labeled CASMCs (red). Double staining is indicated as merge. Phase contrast (PhC) is also reported. Magnification 400×.

then they may interact with DCs, thus contributing to vascular wall
remodeling.
In the present study we also demonstrate abnormalities in
circulating DC precursors. The quantitative abnormalities mainly
consisted of a significant increase in number of circulating myeloid
DCs (BDCA1+ or BDCA3+ ), in comparison to healthy controls.
Regarding plasmacytoid DCs, no major differences were observed
between the two patient groups. Obese patients, however, dis-
played an increase in myeloid BDCA1+ DCs, without any significant
difference compared to healthy woman. Interestingly, there were
significantly fewer mature Mo-DCs obtained ex vivo from myeloid
precursors of obese T2D patients, than from obese and healthy
women. It seems reasonable these obese T2D patients displayed
an impairment of myeloid DCs, the primary cells involved in Th1
response, which might be counterbalanced by an increased number
of circulating precursors (BDCA1+ and BDCA3+ ).
We are currently evaluating the function of DCs obtained from
obese T2D patients in order to verify this hypothesis. It is a common
clinical experience that T2D patients are susceptible to opportunis-
tic infections. The underlying reasons for this immune deficiency
are not completely understood, but one possible explanation deriv-
ing from our data indicates that it is difficult to reach a full
maturation state in Mo-DCs and this might lead to susceptibility
to pathogens. It has been recently demonstrated that the relative
and absolute frequency of both myeloid DCs and plasmacytoid
DCs was clearly diminished in diabetic men with poor metabolic
238 C. Musilli et al. / Molecular Immunology 49 (2011) 234–238
control compared to healthy controls, suggesting that a hyper-
glycemic metabolism does affect the pool of peripheral DCs
(Seifarth et al., 2008). Moreover, diabetic women having poor
glycemic control, selected from the ACCORD trial, had fewer cir-
culating plasmacytoid DCs than controls (Blank et al., 2010). Our
data are apparently in contrast to these. However, the high CV risk
patients selected by Blank et al. (2010) were selected from the mul-
ticenter, randomized and controlled ACCORD trial, in which the
relative CV benefits of intensively targeting normal glucose, blood
pressure and lipid status by means of 3 medical treatment strate-
gies was tested. The patients selected for the present study were
not with at high cardiovascular risk; they had normal lipid control
and had not experienced any clinical cardiovascular event. Fur-
thermore, we used a panel of selective monoclonal antibodies that
recognize the different lineage markers (BDCA1,2,3) for analysis
of circulating DCs, which allow detection of small and heteroge-
neous DC subpopulations, such as BDCA3, a small population of
CD123− CD11c+ circulating DCs (Dzionek et al., 2000).
In conclusion, our results show that in a condition of diabetes
and obesity there is a deregulation of myeloid DC precursors which
may be linked to compromised immune functions. Their increased
ability to adhere to CASMC may play a crucial role in pathological
vascular remodeling.
Acknowledgements
Supported by grants from the Italian Ministry of University and
Scientific and Technological Research to AP.
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