Frequency and function of circulating invariant NKT cells in autoimmune diabetes mellitus and thyroid diseases in Colombian patients

Group of Immunovirology, University of Antioquia, Medellin, Colombia.
Human immunology (Impact Factor: 2.14). 05/2009; 70(4):262-8. DOI: 10.1016/j.humimm.2009.01.012
Source: PubMed
ABSTRACT
The frequency and functionality of peripheral blood invariant (iNKT) cells and their subsets, as well as other regulatory T-cell subsets, were evaluated in patients with type 1A diabetes mellitus (DM1), Hashimoto's disease, and Graves' disease. In addition to healthy individuals (HC), patients with type 2 diabetes mellitus (DM2) were included as controls because this disease has a different physiopathology. A similar frequency of total iNKT cells, as well as their subsets, existed among HC and the different study groups. Similar results were reported when we compared the frequency of CD4(+)/CD25(high) T cells, CD8(+)/CD28(negative) T cells, and gamma-delta T cells among HC and study groups, whereas patients with DM2 exhibited a higher frequency of CD8(+)/CD28(negative) T cells compared with HC and DM1. Also, patients with DM2 exhibited a lower frequency of CD4(negative) and CD4(+) iNKT cells expressing tumor necrosis factor-alpha (TNF-alpha) than HC. We did not observe significant differences in the frequency of iNKT cells expressing interleukin-4 or interferon-gamma among study groups and controls. Our findings support a normal frequency and function of peripheral blood iNKT cells in different endocrine autoimmune diseases, but an abnormal expression of TNF-alpha by circulating iNKT cells from patients with DM2.

Full-text

Available from: Alejandro Roman-Gonzalez, Dec 15, 2014
Frequency and function of circulating invariant NKT cells in autoimmune
diabetes mellitus and thyroid diseases in Colombian patients
Alejandro Roman-Gonzalez
a,b,
*, Maria Eugenia Moreno
a
, Juan Manuel Alfaro
b
, Federico Uribe
b
,
Guillermo Latorre-Sierra
b
, Maria Teresa Rugeles
a
, Carlos Julio Montoya
a
a
Group of Immunovirology, University of Antioquia, Medellin, Colombia
b
Endocrinology and Metabolism Group, University of Antioquia, Hospital Universitario San Vicente de Paul, Medellin, Colombia
ARTICLE INFO
Article history:
Received 18 May 2008
Accepted 16 January 2009
Available online 27 January 2009
Keywords:
iNKT cells
Autoimmune thyroid disease
Diabetes mellitus
Hashimoto’s disease
Graves’ disease
ABSTRACT
The frequency and functionality of peripheral blood invariant (iNKT) cells and their subsets, as well as other
regulatory T-cell subsets, were evaluated in patients with type 1A diabetes mellitus (DM1), Hashimoto’s
disease, and Graves’ disease. In addition to healthy individuals (HC), patients with type 2 diabetes mellitus
(DM2) were included as controls because this disease has a different physiopathology. A similar frequency of
total iNKT cells, as well as their subsets, existed among HC and the different study groups. Similar results were
reported when we compared the frequency of CD4
/CD25
high
T cells, CD8
/CD28
negative
T cells, and
-
T cells
among HC and study groups, whereas patients with DM2 exhibited a higher frequency of CD8
/CD28
negative
T cells compared with HC and DM1. Also, patients with DM2 exhibited a lower frequency of CD4
negative
and
CD4
iNKT cells expressing tumor necrosis factor-
(TNF-
) than HC. We did not observe significant
differences in the frequency of iNKT cells expressing interleukin-4 or interferon-
among study groups and
controls. Our findings support a normal frequency and function of peripheral blood iNKT cells in different
endocrine autoimmune diseases, but an abnormal expression of TNF-
by circulating iNKT cells from patients
with DM2.
2009 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights
reserved.
1. Introduction
Diabetes mellitus type 1A (DM1), Hashimoto’s disease (HD), and
Graves’ disease (GD) are organ-specific autoimmune disorders,
with development influenced by genetic and environmental factors
[1]. In DM1, pancreatic
cells are specifically destroyed, leading to
loss of insulin production, with the clinical consequences of hyper-
glycemia and complications in several organs. In HD, a lymphocyte
infiltrate destroys the thyroid gland, with hypothyroidism ensuing
at the end of the process, whereas GD is characterized by the
production of antibodies against the thyrotropin (TSH) receptor [2].
A central, and probably common, pathogenic mechanism underlying
the beginning of the autoimmune processes could be the abnormal
frequency or malfunction of different immunoregulatory cells [2].
A unique subpopulation of regulatory T cells, known as invariant
NKT (iNKT) cells, with a potential immunoregulatory role in DM1
and autoimmune diseases has been described [3]. These T cells are
characterized by a T-cell receptor (TCR) composed of an invariant
chain with a restricted V
J
rearrangement (V
24J
18), which is
preferentially paired to the V
11 chain. This invariant TCR is re-
stricted by the nonpolymorphic major histocompatibility class I-
like molecule, CD1d, which presents glycolipid antigens mainly to
iNKT cells. The potent immunomodulatory role of iNKT cells is
based on their capacity to rapidly produce high amounts of
T-helper 1 (Th1) (interferon-
[IFN-
], tumor necrosis factor-
[TNF-
]) or T-helper 2 (Th2; interleukin-4 [IL-4]) cytokines after
activation [4,5].
Findings from investigations in the murine model of DM1, the
nonobese diabetic (NOD) mice, have indicated a central role of iNKT
cells in the pathogenesis of this disease. NOD mice have a systemic
deficiency of iNKT cells that is more remarkable in the thymus,
spleen and liver, whereas their remaining iNKT cells have a func-
tional defect in terms of cytokine production [6 –9]. Additionally,
iNKT cell activation with
-galactosylceramide (
-GalCer) in NOD
mice decreases insulitis, prevents the development of diabetes, and
improves survival [10 –12].
Human studies on iNKT cells in DM1 have yielded controversial
findings. The frequency of iNKT cells in peripheral blood has been
reported as increased, normal, or decreased compared with healthy
controls [13–20]. Also, normal and subnormal iNKT cell function, in
terms of IL-4 and IFN-
secretion, has been reported [13–20].A
different genetic background or differences in the techniques used
to evaluate iNKT cells have been proposed to explain such differ-
ences [20]. Particularly, a study detecting iNKT cells with a mono-
clonal antibody directed against the CDR3 loop of the invariant
chain (clone 6B11), which allows the most accurate detection of
* Corresponding author.
E-mail address: alejoroman@gmail.com (A. Roman-Gonzalez).
Human Immunology 70 (2009) 262-268
Contents lists available at ScienceDirect
0198-8859/09/$32.00 - see front matter 2009 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
doi:10.1016/j.humimm.2009.01.012
Page 1
NKT cells [21], reported a normal frequency of peripheral blood
iNKT cells in patients with DM1 [22] but a decreased frequency of
the CD4
iNKT cell subset and a deviated cytokine production to the
Th1 pattern [22].
The role of iNKT cells in the pathogenesis of autoimmune thy-
roid diseases is unclear. To our knowledge, there are no studies on
iNKT cells in patients with HD, whereas in GD two studies reported
a normal frequency of iNKT cells in peripheral blood [23,24] but a
decreased frequency in the thyroid gland [24]. An increased fre-
quency of iNKT cells was reported in NOD.H2
h4
mice, a murine
model of HD, suggesting that iNKT cells might have a potential
pathogenic role in this disease [25]. Also, in a murine model the
administration of
-GalCer prevents the induction of GD; however,
once the disease has developed, the therapeutic administration of
-GalCer did not represent a significant benefit [26]. These findings
suggest that iNKT cells may play a role in controlling the pathogenic
anti-TSHR immune response in GD [26].
In this study we evaluated the frequency, subsets, and cytokine
expression of peripheral blood iNKT cells in Colombian patients
with DM1, HD, and GD; samples from patients with type 2 diabetes
mellitus (DM2) were also evaluated to compare the parameters of
iNKT cells between two different physiopathological processes
(DM1 and DM2). Also, we simultaneously determined the fre-
quency of other T-cell subpopulations with immunoregulatory
roles (CD4
/CD25
high
, CD8
/CD28
negative
and
␥␦
T cells) to explore
for quantitative and qualitative alterations in regulatory T cells in
these autoimmune endocrine diseases.
2. Subjects and methods
2.1. Study population
Fifty-eight Hispanic adult patients with endocrine diseases
were recruited from the Endocrinology Outpatient Service of the
Hospital Universitario San Vicente de Paul in Medellin, Colombia
(demographic information is provided in Table 1). Patients with
long-term DM1 (n 15) or long-term DM2 (n 15) were diagnosed
according to the criteria from the American Diabetes Association
[27]. All patients with DM1 had positive antiglutamic acid decar-
boxylase antibodies (mean 41.13 IU/ml; positive value if above 5
IU/ml). Patients with DM2 presented with other comorbidities,
such as hypertension (100%), high LDL levels, or therapy with st-
atins (100%) and neuropathy was determined by screening with
monofilament (90%). Patients with HD (n 15) were diagnosed by
abnormal hormonal levels of TSH and free T
4
plus positive titers of
antimicrosomal antibodies (Table 1); in some patients who pre-
sented with thyroid nodules, thyroid biopsy was required to rule
out thyroid malignancies and confirm the diagnosis of HD. Patients
with GD (n 13) were diagnosed by the presence of suppressed
levels of TSH plus elevated levels of free T
4
and free T
3
and the
presence of scintigraphic findings of diffuse hyperthyroidism, with
or without Graves’s ophthalmopathy. Finally, this study also in-
cluded 16 healthy controls without a first-degree family history of
autoimmune disease. This study was approved by the ethical com-
mittee of the Center of Medical Investigations (University of Antio-
quia). A clear explanation of the objectives and the implications of
the results were given to each participant; subsequently, each
participant signed an institution-approved informed consent.
2.2. Antibodies and reagents
The following monoclonal antibodies (mAb) against human
molecules were used: clone 6B11 (a mAb against the CDR3 loop of
the
chain from the invariant TCR), anti-CD3, anti-CD4, anti-CD8,
anti-CD25, anti-CD28, anti-CD95, anti-
␥␦
TCR, anti-TNF-
, anti-
IL-4, and anti-IFN-
and the corresponding isotype controls. All
antibodies were from Becton Dickinson–Pharmingen (San Jose,
CA). The Fc
-receptor blocking reagent was from Miltenyi Biotec
(Bergisch Gladbach, Germany).
2.3. Isolation and stimulation of peripheral blood
mononuclear cells (PBMC)
Heparinized whole-blood samples were diluted 1:2 with fresh
sterile phosphate-buffered saline (PBS); PBMC were isolated by
density gradient centrifugation using lymphocyte separation me-
dium (Cambrex Bio Science, Walkersville, MD, USA) and washed
twice with PBS. Viability and number of PBMC were determined by
0.5% trypan blue exclusion; all samples were processed within 2
hours of collection. PBMC were suspended in complete culture
medium, including RPMI 1640 (BioWhittaker, Walkersville, MD)
supplemented with 10% heat-inactivated fetal bovine serum (Gem-
Cell, Woodland, CA) and 2 mM of L-glutamine (Sigma, St. Louis, MO)
and incubated in six-well plates for 4 hours at 37
o
C and 5% CO
2
in
the presence of phorbol myristate acetate (PMA; 20 ng/ml) and
ionomycin (500 ng/ml); unstimulated PBMC were incubated simi-
larly to determine the basal functional activity. The protein secre-
tion inhibitor brefeldin A (Becton Dickinson, 10
g/ml) was added
simultaneously to allow the intracellular accumulation of newly
synthesized proteins during PBMC sample incubation.
2.4. Flow cytometry
The frequency and subsets in whole blood of iNKT cells (6B11
/
CD3
lymphocytes) and the frequency of other regulatory T-cell
subpopulations (
␥␦
T cells, CD4
/CD25
high
T cells, and CD8
/
CD28
negative
T cells) were determined by four-color flow cytometry.
For cell surface staining, 100
l of anticoagulated peripheral blood
was incubated with appropriate fluorescence-labeled mAb for 20
min at room temperature in the dark. The erythrocytes were lysed
by incubation for 10 min with 2 ml of 1 FACS lysing solution
(Becton Dickinson). The cell suspension was centrifuged for 5 min
at 250g, the supernatant was discarded, and the cells were washed
twice with 2 ml of cold PBS at 250g for 5 minutes. Finally, cells were
fixed with 250
l of 2% formaldehyde.
For detection of intracellular cytokines into iNKT cells, after
stimulation of PBMC the nonspecific antibody binding to Fc recep-
tors was blocked by preincubation of the cells with Fc
-receptor
blocking reagent (Miltenyi Biotec) for 20 minutes at 4
o
C. Then,
PBMC were washed and surface staining was performed as de-
scribed above. After being washed, PBMC were incubated with BD
Cytofix/Cytoperm solution for 20 minutes at 4
o
C. Cells were
washed twice with BD Perm/Wash buffer and stained with intra-
cellular staining monoclonal antibodies for 30 minutes. Finally, the
samples were washed twice in Perm/Wash buffer, fixed with 1%
formaldehyde, and stored at 4
o
C until analysis.
For all experiments, appropriate isotype-matched control anti-
bodies were included. Based on the low frequency of iNKT cells in
peripheral blood, 5 10
5
total cells were analyzed. Dead cells were
gated out by forward and side scatter. Flow cytometry was per-
Table 1
Demographic data of studied patients
Characteristic HC
(n 16)
DM2
(n 15)
DM1
(n 15)
HD
(n 15)
GD
(n 13)
Age (mean) 27.81 58.93 19.13 33.00 29.55
Female/male 10/6 12/3 10/5 14/1 12/1
All patients have long-term evolution of their endocrine disease (more than 6 month after
diagnosis) and all were under therapy and medical follow-up with an endocrinologist. All
patients with DM1 had positive antiglutamic acid decarboxylase antibodies. Patients with
DM2 also were affected by hypertension (100%) higher LDL levels or were taking a statin
(100%), or had neuropathy determined by screening with monofilament (90%).
DM1 type 1A diabetes mellitus; DM2 type 2 diabetes mellitus; HC healthy control;
HD Hashimoto’s disease; GD Graves’s disease.
A. Roman-Gonzalez et al. / Human Immunology 70 (2009) 262-268 263
Page 2
formed using the Becton Dickinson FACSORT instrument and ana-
lyzed using CellQuest software.
2.5. Statistical analysis
Results are presented as the median, 25th and 75th percentiles,
and the range. Statistical comparisons among three or more differ-
ent study groups were performed using the analysis of variance
nonparametric Kruskal–Wallis test; Dunn’s posttest was per-
formed to compare the different groups by pairs. Differences were
considered significant when p 0.05. All statistical tests were
performed using GraphPad Software, version 5.00 (La Jolla, CA).
3. Results
3.1. Frequency of iNKT cells and other regulatory T cells
in peripheral blood
Compared with the healthy and DM2 control groups, there were
no significant differences in the frequency of total iNKT cells in
patients with DM1, HD, and GD (Fig. 1B). Similarly, we did not
observe any significant difference in the frequency of the iNKT
subpopulations CD4
, CD8
, double positive (CD4
/CD8
), and
double negative (CD4
negative
/CD8
negative
, DN) among controls and
patients with the autoimmune diseases evaluated (Fig. 1C).
HC DM2 DM1 HD GD
0.0
0.5
1.0
1.5
2.0
2.5
% iNKT cells
A
B
0
20
40
60
80
CD4+ CD8+ DN
DP
HC
DM2
DM1
HD
GD
% iNKT cells
C
Fig. 1. Frequency of total iNKT cells and their subsets in peripheral blood. The iNKT cells were specifically detected by whole blood flow cytometry in the region of lymphocytes
(R1) using monoclonal antibodies against the CDR3 loop of the invariant
chain (clone 6 B11) and against CD3 (lymphocytes CD3
/6B11
, R2); the iNKT subpopulations were
defined in the CD3
/6B11
cells using antibodies agains CD4 and CD8 molecules. Representative dot plots are presented (A) as a demonstration of the technical proficiency.
The total frequency (B) and subsets (C) of iNKT cells were determined in whole blood samples from healthy controls (n 16) and patients with DM2 (n 15), DM1 (n 15),
HD (n 15), and GD (n 13). Results are presented as medians, 25th and 75th percentiles, and range. Statistical comparison among the groups was performed using the analysis
of variance (ANOVA) nonparametric Kruskal–Wallis test, with a confidence level of 95%. Dunn’s posttest was performed to compare the different groups by pairs. DM1 type 1A
diabetes mellitus; DM2 type 2 diabetes mellitus; HC healthy controls; HD Hashimoto’s disease; GD Graves’s disease; DN double negative; DP double positive.
A. Roman-Gonzalez et al. / Human Immunology 70 (2009) 262-268264
Page 3
We wanted to explore the frequency of iNKT cells in relation to
the frequency of other well-known immunoregulatory T cells to
achieve a broader understanding of the immune dysregulation
process associated with these autoimmune endocrine diseases.
Patients with DM1, HD, or GD did not exhibit significant differences
in the frequency of peripheral blood CD4
/CD25
high
T cells (Fig. 2A),
CD8
/CD28
negative
T cells (Fig. 2B), and
␥␦
T cells (Fig. 2C) compared
with healthy controls. Patients with DM2 exhibited a similar fre-
quency of CD4
/CD25
high
T cells than HC and study groups and a
lower but not significantly different frequency of
␥␦
T cells (Figs 2A
and C, respectively); however, DM2 patients presented with a sig-
nificantly higher frequency of CD8
/CD28
negative
T cells when com-
pared with controls (p 0.001) or DM1 patients (p 0.01; Fig. 2C).
In almost all patients evaluated, most of the CD8
/CD28
negative
T cells were CD95
; consequently, the frequency of CD8
/
CD28
negative
/CD95
T cells was similar among the different groups
evaluated, but was significantly higher in DM2 patients compared
with HC or DM1 patients (data not shown).
3.2. Intracellular cytokine expression by iNKT cells
The pattern of cytokine production by total iNKT cells and their
main subsets (CD4
and CD4
negative
cells) was evaluated after stim-
ulation of PBMC with PMA and ionomycin; glycolipids presented by
the CD1d molecule (such as
-GalCer) represent the natural TCR-
mediated activators of iNKT cells, but this activation leads to the
downregulation of the surface CD3/invariant TCR complex, limiting
the detection of iNKT cells by flow cytometry [21].
There were no significant differences in the net percentage of
iNKT cells expressing IL-4 (the percentage of IL-4
cells in PMA/
ionomycin-stimulated culture minus the percentage of IL-4
cells
in unstimulated culture) among the different subgroups of patients
evaluated (Fig. 3B); the same finding was observed for the
CD4
negative
or CD4
subsets of iNKT cells (Figs 3C and D, respec-
tively). Similarly, there were no significant differences in the net
frequency of IFN-
iNKT cells (total, CD4
negative
or CD4
) when the
different subgroups of individuals were compared (Figs 4B, C, and
D, respectively).
Compared with HC, patients with DM1, HD, or GD did not dis-
play significant differences in the net percentage of total iNKT (Fig.
5B), CD4
negative
iNKT (Fig. 5C), or CD4
iNKT cells (Fig. 5D) express-
ing TNF-
. Patients with DM2 exhibit a lower, but not significant,
frequency of total iNKT cells expressing TNF-
(Fig. 5B); however,
the frequency of CD4
negative
or CD4
iNKT cell subsets expressing
TNF-
was significantly lower in DM2 patients than in HC (p 0.01,
Fig. 5C, and p 0.05, Fig. 5D, respectively).
4. Discussion
Studies in murine models have demonstrated that iNKT cells are
involved in the establishment of tolerance and the control of
several types of immune responses, such as antitumoral, anti-
infectious, and autoimmune responses [10,11,28 –30]. In particu-
lar, the role of iNKT cells in the prevention of autoimmune diabetes
has been clearly established [10–12,31]. However, the actual role of
iNKT cells in the development of human autoimmune diseases has
not been elucidated yet. In human DM1, the evidence of such a role
is contradictory, and evidence in other diseases, such as autoim-
mune thyroiditis, is lacking.
In this study, we observed a normal frequency in peripheral
blood of total iNKT cells and their subsets in Colombian patients
with DM1. Previous studies of human DM1 patients have been
controversial, reporting different frequencies of iNKT cells in pe-
ripheral blood with an increased, normal, or subnormal function
[13–15,19]. Differences in technical and methodological ap-
proaches to characterize iNKT cells, as well as the distinct racial
backgrounds, have been suggested to explain these differences.
As reported recently by us and others, detection of the invariant
TCR is the most accurate approach to characterize iNKT cells, even
improving the characterization achieved using CD1d tetramers
[21,32]. To specifically detect the iNKT cells, we used the mAb clone
6B11, which bind to epitopes from the invariant TCR-
chain. A
recent investigation in human DM1 using the same approach re-
Fig. 2. Frequency of regulatory T cells in peripheral blood. The frequency of different
subpopulations of regulatory T cells, (A) CD4
/CD25
high
T cells, (B) CD8
/
CD28
negative
T cells, and (C)
␥␦
T cells, was determined by flow cytometry in whole
blood samples from healthy controls (n 16) and patients with DM2 (n 15), DM1
(n 15), HD (n 15), and GD (n 13) and the surface expression of the following
molecules was evaluated: CD3, CD4, CD25, CD8, CD28, and the
␥␦
TCR. Results are
presented as medians, 25th and 75th percentiles, and range. Statistical comparison
among the groups was performed using the ANOVA nonparametric Kruskal–Wallis
test, with a confidence level of 95%. Dunn’s posttest was performed to compare the
different groups by pairs. DM1 type 1A diabetes mellitus; DM2 type 2 diabetes
mellitus; HC healthy controls; HD Hashimoto’s disease; GD Graves’s disease.
A. Roman-Gonzalez et al. / Human Immunology 70 (2009) 262-268 265
Page 4
A
B
C
HC DM2 DM1 HD GD
0
15
30
45
60
Net % iNKT / CD4+ / IL-4+
HC DM2 DM1 HD GD
0
10
20
Net % iNKT / CD4 neg / IL-4+
D
HC DM2 DM1 HD GD
0
15
30
45
60
Net % total iNKT / IL-4+
Fig. 3. IL-4 expression by iNKT cells. The intracellular expression of IL-4 by iNKT
cells was specifically detected by flow cytometry after the incubation of PBMC
without or with stimulation with PMA and ionomycin; representative dot plots are
presented (A) to illustrate the expression of this cytokine into unstimulated and
stimulated 6B11
iNKT cells. The frequency of (B) total iNKT cells, (C) CD4
negative
iNKT cells, and (D) CD4 iNKT cells expressing intracellular IL-4 was determined in
healthy controls (n 16) and patients with DM2 (n 15), DM1 (n 15), HD (n 15),
and GD (n 13). The net percentage of IL-4
iNKT cells was defined as the percent-
age of IL-4
iNKT cells in PMA/ionomycin-stimulated culture minus the percentage
of IL-4
iNKT cells in unstimulated culture. Results are presented as medians, 25th
and 75th percentiles, and range. Statistical comparison among the groups was
performed using the ANOVA nonparametric Kruskal–Wallis test, with a confidence
level of 95%. Dunn’s posttest was performed to compare the different groups by
pairs. DM1 type 1A diabetes mellitus; DM2 type 2 diabetes mellitus; HC
healthy controls; HD Hashimoto’s disease; GD Graves’s disease.
A
B
D
HC DM2 DM1 HD GD
0
20
40
60
80
Net % total iNKT / IFN
γ
γ
γ
γ
+
HC DM2 DM1 HD GD
0
20
40
60
80
Net % iNKT / CD4 neg / IFN
γ
γ
γ
γ
+
C
HC DM2 DM1 HD GD
0
20
40
60
80
Net % iNKT CD4+ / IFN
γ
γ
γ
γ
+
Fig. 4. IFN-
expression by iNKT cells. The intracellular expression of IFN-
by iNKT
cells was specifically detected by flow cytometry after the incubation of PBMC
without or with stimulation with PMA and ionomycin; representative dot plots are
presented (A) to illustrate the expression of this cytokine into unstimulated and
stimulated 6B11
iNKT cells. The frequency of (B) total iNKT cells, (C) CD4
negative
iNKT cells, and (D) CD4
iNKT cells expressing intracellular IFN-
was determined in
healthy controls (n 16) and patients with DM2 (n 15), DM1 (n 15), HD (n 15),
and GD (n 13). The net percentage of IFN-
iNKT cells was defined as the
percentage of IFN-
iNKT cells in PMA/ionomycin-stimulated culture minus the
percentage of IFN-
iNKT cells in unstimulated culture. Results are presented as
medians, 25th and 75th percentiles, and range. Statistical comparison among the
groups was performed using the ANOVA nonparametric Kruskal–Wallis test, with a
confidence level of 95%. Dunn’s posttest was performed to compare the different
groups by pairs. DM1 type 1A diabetes mellitus; DM2 type 2 diabetes mellitus;
HC healthy controls; HD Hashimoto’s disease; GD Graves’s disease.
A. Roman-Gonzalez et al. / Human Immunology 70 (2009) 262-268266
Page 5
ported a normal frequency of total peripheral blood iNKT cells, but
a lower frequency of CD4
iNKT cells [22]. Our results are partially
in agreement with these recent publications. It is interesting that
the frequency of peripheral blood iNKT cells in murine models of
DM1 could be normal, whereas quantitative and qualitative alter-
ations are detected in immune tissues or target organs [33]. Unfor-
tunately, because we only evaluated the frequency and function of
this cell subpopulation in circulation, we cannot rule out abnormal-
ities within the lymphoid or target tissues. Evidence from murine
and human research indicates that numerical and functional pa-
rameters from iNKT cells may play a role in the development of
autoimmune diseases such as DM1 [11,12,22], with quantitative
and qualitative abnormalities observed in circulating and tissue
iNKT cells. However, some reports indicate that localization and
tissue distribution of iNKT cells differ among humans and other
mammals, which could affect iNKT cell functional response at these
sites and their role in autoimmune pathogenesis. Particularly, in
the genetically more complex human model, all or some of these
factors may play a role in disease progression in a case-by-case
manner. Consequently, the scope of our results is limited because
the extrapolation with regard to function and the role of iNKT cells
in human diseases cannot be determined strictly by peripheral
analysis. We limit our assumptions on the roll these cells may play
in disease based strictly on their peripheral cell function.
In peripheral blood, patients with DM1 also displayed a normal
frequency of regulatory CD4
/CD25
high
T cells, CD8
/CD28
negative
T
cells, and
␥␦
T cells, suggesting that the immune dysregulation
underlying DM1 includes one or a combination of the following
factors: (i) quantitative alterations only evident in lymphoid or
specific tissues, (ii) qualitative alterations of these regulatory cells,
and (iii) abnormalities in other mechanisms involved in the main-
tenance of self-tolerance. The above information is additionally
supported by a normal expression of Th1 and Th2 cytokines by
peripheral blood iNKT cells from our DM1 patients. In our cohort of
DM2 patients, we observed an increased frequency of peripheral
blood CD8
/CD28
negative
T cells (most of which were CD95
), as
well as a decreased expression of cytokines by iNKT cells, being that
decrease statistically significant for TNF-
. Although unexpected,
these findings suggest abnormalities in immune regulation in DM2.
Although an autoimmune response against the pancreas is ap-
parently absent in DM2, the underlying process of insulin resis-
tance is associated with an abnormal inflammatory response as
expressed by high circulating levels of IL-6 and other proinflamma-
tory markers [34]. Additionally, patients with DM2 have increased
susceptibility to infections, suggesting other alterations in the in-
flammatory and immune responses. DM2 is also associated with
hypertension, central obesity, and dyslipidemia, a complex known
as the metabolic syndrome; a clear association of a proimmflam-
matory state and a prothrombotic risk have been well described in
this disorder [35]. Our patients with DM2 have the criteria estab-
lished for the metabolic syndrome according to the Third Report of
the National Cholesterol Education Program Expert Panel on Detec-
tion, Evaluation, and Treatment of High Blood Cholesterol in Adults
(ATP III) [36], suggesting that the presence of high glucose levels or
hyperinsulinemia could play a role in the alterations detected in
these patients. Further studies are required to determine the role of
these abnormalities and our findings in states of immune dysregu-
lation, such as hyperglycemia and hyperinsulinemia, and in the
physiopathology of DM2 complications and susceptibility to
infection.
A previous study in GD patients reported a normal frequency of
peripheral blood iNKT cells in autoimmune thyroiditis [23]. In this
investigation, we observed a normal percentage of different sub-
populations of regulatory T cells (total iNKT cells and their subsets,
CD4
/CD25
high
T cells, CD8
/CD28
negative
T cells, and
␥␦
T cells) in
Colombian patients with GD. To our knowledge, this is the first
A
B
D
HC DM2 DM1 HD GD
0
20
40
60
80
p<0.05
Net % iNKT / CD4+ / TNF
α
α
α
α
+
C
HC DM2 DM1 HD GD
0
20
40
60
80
p<0.01
Net % iNKT / CD4 neg / TNF
α
α
α
α
+
HC DM2 DM1 HD GD
0
20
40
60
80
Net % total iNKT / TNF
α
α
α
α
+
Fig. 5. TNF-
expression by iNKT cells. The intracellular expression of TNF-
by iNKT
cells was specifically detected by flow cytometry after the incubation of PBMC
without or with stimulation with PMA and ionomycin; representative dot plots are
presented (A) to illustrate the expression of this cytokine into unstimulated and
stimulated 6B11
iNKT cells. The frequency of (B) total iNKT cells, (C) CD4
negative
iNKT cells, and (D) CD4
iNKT cells expressing intracellular TNF-
was determined
in healthy controls (n 16) and patients with DM2 (n 15), DM1 (n 15), HD (n
15), and GD (n 13). The net percentage of TNF-
iNKT cells was defined as the
percentage of TNF-
iNKT cells in PMA/ionomycin-stimulated culture minus the
percentage of TNF-
iNKT cells in unstimulated culture. Results are presented as
medians, 25th and 75th percentiles, and range. Statistical comparison among the
groups was performed using the ANOVA nonparametric Kruskal–Wallis test, with a
confidence level of 95%. Dunn’s posttest was performed to compare the different
groups by pairs. DM1 type 1A diabetes mellitus; DM2 type 2 diabetes mellitus;
HC healthy controls; HD Hashimoto’s disease; GD Graves’s disease.
A. Roman-Gonzalez et al. / Human Immunology 70 (2009) 262-268 267
Page 6
report exploring the frequency of peripheral blood iNKT cells and
their subsets, as well as other regulatory T cells in patients with HD.
In addition, we observed no significant differences in production of
Th1 and Th2 cytokines by iNKT cells from HD and GD patients.
Again, further studies including quantitative and functional evalu-
ation of these cells in the thyroid and other tissues are required to
rule out the participation of the regulatory cell system in the patho-
genesis of these autoimmune thyroid diseases.
Autoimmune diseases are the result of genetic susceptibility
factors combined with environmental triggers, indicating that the
racial and genetic backgrounds have a crucial influence on the
variable pathogenic mechanisms observed in investigations per-
formed in different regions of the world. Our study presents new
data on the iNKT cell frequency and function in patients with
autoimmune diabetes and thyroiditis from a population with a
different racial background. Our results indicate a normal fre-
quency and functional pattern of peripheral blood iNKT cells in
Colombian DM1 patients, which contrasts with the results of inves-
tigations reported by other groups and underlines the importance
and the requirement of further studies using the same protocol to
evaluate iNKT cells to rule out the possibility that technical issues
are responsible for the variable results reported among different
human populations. Also, human studies on iNKT cell frequency
and function in organ-specific tissues and its regional lymphoid
tissues are necessary to define the actual compromise of iNKT cells
in the pathogenesis of autoimmune diseases.
Acknowledgments
This work was supported by the Committee for the Develop-
ment of Research from the University of Antioquia. The authors
thank Marta Galeano, Chair Nurse of the Diabetes program, Hospi-
tal Universitario San Vicente de Paul, for her help with the logistic
process of the study and Carlos Alfonso Builes, MD, for his help with
patients and comments on the manuscript.
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  • Source
    • "Although the role of Treg impairment in the pathogenesis of T1D still needs to be clarified, there is increasing evidence to support the importance of Treg expansion in controlling autoimmunity (Daniel and von Boehmer, 2011; Leslie, 2011). Similar to Tregs, the study of NKT cells in the initiation/progression of T1D has produced contradictory results (Wilson et al., 1998; Kukreja et al., 2002; Lee et al., 2002; Oikawa et al., 2002; Michalek et al., 2006; Tsutsumi et al., 2006; Kis et al., 2007; Montoya et al., 2007; Roman-Gonzalez et al., 2009; Berzins et al., 2011). However, their ability to promote immunomodulation for the treatment of diabetes has been demonstrated in animal models of T1D (Fletcher and Baxter, 2009 ) and their potential to prevent autoimmune diseases such as diabetes represents an important clinical target (Novak and Novakova, 2012). "
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    [Show abstract] [Hide abstract] ABSTRACT: Contradictory reports about the role of cytokines, particularly interleukins (IL) in atherosclerosis are found in the literature. This study was aimed to investigate the association between the levels of cytokines notably IL-4, IL-12, IL-18 and, the atherogenicity and glycemic control in patients with type 2 diabetes mellitus. Seventy five patients with type 2 diabetes mellitus (25 males and 50 females) attending diabetic clinic during 1st August 2008 to 30th December 2009 as well as seventy healthy subjects (38 males and 32 female) were enrolled in the study. Fasting serum lipid profile and IL-4, IL-12 and IL-18 levels were determined. The serum lipid profile of diabetic patients was significantly different from healthy subjects, favoring atherogenicity. IL 4, 12, and 18 were significantly higher in diabetic patients compared with healthy subjects. Significant association of high serum IL-18 with poor glycemic control (P < 0.001) assessed by HbA1c, long duration of diabetes and atherogenic index were observed. IL-18 can serve as a predictor for pre-clinical atherosclerosis and poor glycemic control in type 2 diabetes mellitus.
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  • Source
    • "However, none of further studies using different approaches for the detection of iNKT cells confirmed the results of Wilson and Kukreja. At least in the peripheral blood of T1DM patients, the proportion of iNKT cells is not reduced [67] [68] [69] [70] [71] [72] [73]. More importantly , Berzins and his colleagues addressed the simple question regarding the reciprocation of iNKT cell characteristics in peripheral blood versus those in visceral organs [74]. "
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