883Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 100(8): 883-887, December 2005
Evaluation of the natural killer cytotoxicity and the levels of
cytokines in rats with type I diabetes mellitus
Isil Fidan/+, Sevgi Yüksel, Ayse Kalkanci, Turgut Imir, Semra Kustimur
Department of Medical Microbiology, Faculty of Medicine, Gazi University, Besevler, Ankara 06500, Turkey
Type I diabetes mellitus (insulin-dependent DM = IDDM) is a chronic disease characterized by specific destruc-
tion of pancreatic β cells, resulting in an absolute lack of insulin. Immune mechanisms, genetic susceptibility, and
environmental factors are all implicated in the pathogenesis of Type 1 diabetes. This study was aimed at determining
the efficiency of cytokines, natural killer (NK) cells in the pathophysiology of IDDM. Therefore, we evaluated the
plasma levels of cytokines by specific enzyme-linked immunosorbent assay (ELISA) and the cytotoxicity activity of
NK cells by anti-candididal index in rats with type I diabetes. We found that the cytotoxicity activity of NK cells in
IDDM groups significantly decreased compared to the control groups. The levels of interferon-γ (IFN-γ) in IDDM
groups were slightly higher than in healthy controls. These results indicate that the changes of TH1 type cytokines
such as IFN-γ and NK cell activity can play a role in the etiology of IDDM. The data may provide new strategies for
the treatment of IDDM.
Key words: Type 1 diabetes mellitus - cytokines - natural killer cells
Type I diabetes mellitus (insulin-dependent DM =
IDDM) is a chronic disease characterized by specific de-
struction of pancreatic β-cells, resulting in an absolute
lack of insulin (Boitard 2002). Immune mechanisms, ge-
netic susceptibility, and environmental factors are all im-
plicated in the pathogenesis of Type 1 diabetes. There are
many recent evidences that the immune system may be
involved in the pathogenesis of IDDM in humans as well
as in animal models (MacKay et al. 1986). There is an
autoimmune destruction of the insulin-producing β-cells
in the pancreatic islets of Langerhans. Recent studies in
humans and animals suggest that autoimmune diabetes is
associated with an imbalance between TH1-type cytokines
and TH2-type cytokines in immune system (Lo et al. 2004).
Cytokines are small proteins which play a central role in
the regulation of host defenses. TH1 cytokines (interferon-
γ, IFN-γ, interleukin-2, IL-2, tumor necrosis factor-β, TNF-
β) primarily activate cellular immune reponses whereas
TH2 tip cytokines (IL-4, IL-6, IL-10) primarily stimulate
humoral immune responses (Hayashi et al. 1998). It is
suggested that the more production of TH1 type cytokines
by islet-infiltrating leukocytes may induce β-cell destruc-
tion whereas the more production of TH2 type cytokines
in islets may protect against β-cell destruction. Thus, TH2
type cytokines may have a protective role against the
development of diabetes (Rabinovitch 1998).
Natural killer (NK) cells are a subset of mononuclear
cells. They are nonphagocytic large granular lymphocytes
which are cytotoxic for certain tumor cell lines and virally
infected cells. NK cells play several important roles in the
host defense against certain microorganisms (Baxter &
+Corresponding author. E-mail: email@example.com,
Received 20 July 2005
Accepted 16 November 2005
Smyth 2002). They secrete multiple immunomodulatory
cytokines such as IFN-γ, TNF-α , IL-10. (Abbas & Zit-
chman 2003). Because of this property, NK cells have been
suggested to play an immunoregulatory role in organ-
specific autoimmune disorders, such as type 1 diabetes
(Johansson et al. 2004). It is considered that autoimmune
diseases result from chronic viral infections and NK cells
can inhibit disease by clearing virus (Baxter & Smyth 2002).
NK cells may play both pathological and protective roles
in type 1 diabetes (Johansson et al 2004). Defects in NK
cell numbers and their function have been identified in
animal models of autoimmunity. In order to determine cer-
tain roles of NK cells and cytokines profiles in type 1
diabetes may provide new strategies for the treatment of
The present study was aimed at determining the effi-
ciency of cytokines such as TNF-α, IL-1β, IL-2, IFN-γ, and
NK cells in the pathophysiology of IDDM. Therefore, we
evaluated the plasma levels of cytokines and the cytotox-
icity activity of NK cells in rats with type I diabetes.
MATERIALS AND METHODS
Animals - Eight female Wistar albino rats weigting 200-
250 g were used in this study. All rats were 6-8 weeks old.
Streptozotocin-induced diabetic rats - The animal ex-
periments were conducted according to the Guide for the
Care and Use of Laboratory Animals of the Gazi Univer-
sity School of Medicine. Local Ethical Committee permis-
sions were obtained. The experimental group (n = 8) was
made diabetic by a single intraperitoneal injection of
freshly dissolved streptozotosin (STZ, Sigma) (45 mg/kg
body weight in citrate buffer 0.01 M, pH 4.5) into over-
night fasted rats. The control group (n = 8) was injected
with buffer only. Forthy-eight hours after injection of
STZ, the plasma levels of glucose were assessed. The
plasma glucose levels were determined using a glucometer.
Rats were defined as diabetic if their plasma glucose con-
centration was above 400 mg/ml and these rats were used
in the experiment (Zhang & Tan 2000).
884 NK cytotoxicity and levels of cytokines • Isil Fidan et al.
Isolation of lymphocytes for NK cytotoxicity assay
(effector cell preparation) - Heparinized blood was col-
lected from tail vein. Peripheral blood mononuclear cells
(PBMCs) were isolated from blood by sedimentation on a
Ficoll-hypaque gradient (Sigma). PBMCs were washed in
phosphate buffered saline (PBS) three times and resus-
pended in RPMI 1640 medium containing 2 mmol/l
glutamine, 200 U/ml penicillin, 100 mg/ml streptomycin,
and supplemented with 10% foetal calf serum. The sus-
pension of PBMC was poured into petri dishes sensitized
autologous serum and was incubated 1 h at 37°C. After
incubation, non-adherent cells were gently harvested and
same procedure was applied second time. The non-adher-
ent cells washed in RPMI-1640 and used as the effector
cells (Gülay & Imir 1996). The relative percentage of mono-
cytes was less than 5% as determined by morphological
examination following Giemsa staining. Effector cell vi-
ability was 95% by the trypan blue exclusion test.
Target cell preparation for NK cytotoxicity assay -
Candida species (Candida albicans) used in this
study were isolated from clinical specimens in our labo-
ratory. Candida was grown on a blood agar base for 18
h. Yeast cells were resuspended in RPMI 1640 medium
and adjusted to the concentration of 4 × 103 viable or-
NK cytotoxicity assays (colony forming inhibition
assay = CFI) - NK cytotoxic activity was assessed by
CFI assay. Yeast cells used as target cells were incubated
with effector cells in 96-well U-bottomed microtitre plates
at different target:effector (T:E) ratios (1:3 and 1:30) for 2 h
at 37°C in a CO2 incubator. Control wells contained target
cells in RPMI 1640 medium. After incubation, aliquots of
25 µl from each well were plated on petri dishes contain-
ing Saburoud dextrose agar. Cultures were then incubated
for 48 h at 37°C and colonies were counted. The percent-
age reduction in colony forming unit was determined as
the anti-candidial index (ACI) according to the following
ACI = (1– experimental colony forming unit/control colony
forming unit )×100
Each experiment was performed in quadruplicate.
Cytokine assays - For cytokines assay, blood was col-
lected from tail vein. After centrifugation, the plasma was
collected and stored at –80°C until analyzed. Levels of
cytokines such as TNF-α, IL-1, IL-2, IL-6, IL-10, IFN-γ in
plasma samples were determined by specific enzyme-linked
immunosorbent assay (ELISA) techniques according to
the manifacturer’s instructions (Biosource, California, US).
The concentration of cytokines was determined spectro-
photometrically. The absorbance was read at 450 nm. We
constructed a standard curve using cytokines standards.
The cytokine concentrations for unknown samples were
calculated according to standard curve.
Statistical methods - The NK cytotoxicity and the
cytokine concentrations from the rats with diabetes were
compared with those from healthy controls using the
Mann-Whitney U test; p < 0.05 was considered to be
The percentage of inhibition of colony formation (ACI)
found was 7, 12, 14, 9, 11, 14; 8 and 15% for T:E ratios of
1:3 in 8 rats with diabetes and 23, 32, 24, 25, 32, 29; 34, 26%
for T:E ratios of 1:30 in 8 rats with diabetes. The difference
between T:E ratios of 1:3 and 1:30 in ACI was statistically
The percentage of ACI found was 18, 29, 25, 27, 24,
37; 16 and 23% for T:E ratios of 1:3 in control groups and
32, 50, 48, 57, 53, 54; 56, 63% for T:E ratios of 1:30 in con-
The comparative results of ACI for T:E ratios of 1:3
and 1:30 in diabetic and control groups were shown in
Figs 1 and 2, respectively. There was a significant differ-
ence between diabetic groups and control groups (p <
Fig. 2: the comparative results of anti-candicial index (ACI) for
target:efector ratios of 1:30 in diabetic and control groups (%).
Fig. 1: the comparative results of anti-candicial index (ACI) for
target:efector ratios of 1:3 in diabetic and control groups (%).
In this study we could not demostrate that diabetic
groups differ in serum concentrations of TNF-α, IL-1, IL-
2, IL-6, IL-10 from controls (Fig. 3) (p > 0.05). However,
IFN-γ plasma levels in diabetic rats were significantly
higher than in healthy controls (Fig. 4) (p < 0.05) (Table I).
Type 1 DM is a immune-mediated disease character-
ized by specific destruction of insulin-producing β cells
885Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 100(8), December 2005
of the pancreatic islets, resulting in the loss of glucose
homeostasis (Poirot et al. 2004). The most important aim
of the research on DM is to have an understanding of the
genetics and immunopathological basis of the disease
because it may provide new therapy strategies. Recent
evidence from animal models of autoimmune diabetes
indicates that there is an autoimmune attack directed
against β-cells in IDDM. This effect may be due to im-
paired immunoregulatory balance (Rapoport et al. 1998).
Thus, we investigated the roles of NK cells and cytokines
in diabetic rats. Therefore, the plasma levels of cytokines
and the cytotoxicity activity of NK cells in rats with type
I diabetes were studied and compared with those of con-
In order to determine NK cytotoxic activity, we used
CFI assay. In this procedure, target cells (T = Candida)
were incubated with effector cells (E = rat lymphocytes)
at different T:E ratios (1:3 and 1:30) and ACI was deter-
mined. There was an increase in the ACI; both ratios in
diabetic groups were compared with controls and the re-
sult was significant (Figs 1, 2) (p < 0.05). Accordingly, we
found significant decrease in the cytotoxicity activity of
NK cells in IDDM groups compared with control groups.
Negishi et al. (1986) observed that NK cell activity
against target cells decreased in new onset Type 1 diabe-
tes patients whereas Type 2 diabetic patients displayed
similar range of NK activity compared to controls. They
also thought that the decreased NK cell activity in diabe-
tes could be due to several factors; that is, either IFN
production is deficient in Type 1 diabetes or NK cell sen-
sitivity to IFN is reduced. Hussain et al. (1987) demostrated
that Type 1 diabetic patients tended to have reduced
numbers of natural killer cells. Similarly, Lorini et al. (1994)
observed that NK cytotoxic activity decreased in IDDM
Ellerman et al. (1993) demonstrated that chronic treat-
ment of the BB/Wor diabetes-prone rats with monoclonal
antibodies that removed circulating NK cells did not pro-
tect against diabetes or delay its onset. Thus, they in-
formed that NK cells were not necessary for the develop-
ment of spontaneous diabetes in BB/Wor rats. Poultan et
al. (2001) informed that NOD (non-obese diabetic mice),
used as a spontaneous model of type 1 DM, had a relative
deficiency in peripheral NK cells and a defect in NK cell
export. They also observed deficiencies in NK cell activ-
ity. Johansson et al. (2004) found that the numbers of NK
cells were similar in NOD mice and controls. However,
they indicated a NK cell defect in cytotoxic potential in
NOD. In addition, they suggested that there was an in-
verse correlation between NK activity and diabetes inci-
MacKay et al. (1986) demonstrated that NK cell activ-
ity increased in BB/W rats acutely diabetic. But they had
no direct evidence that DM is an NK-cell mediated dis-
ease in rats. They also informed that NK cells per se might
be insuffient to cause diabetes.
In spite of these studies, the role of NK cells in type 1
Fig. 3: the levels of interleukin-6, interleukin-10 and tumour necrosis factor-α.
Fig. 4: the levels of interferon-γ (IFN-γ) (n = 8).
Mean, standart deviation (SD), standard error (SE) values for
Control groups Diabetic groups
886NK cytotoxicity and levels of cytokines • Isil Fidan et al.
diabetes is still unclear. Conficting results have been re-
ported on the role of NK cells in diabetes. The results
obtained from our study suggest that NK cells can play a
role in the pathogenesis of type 1 DM. We think that the
NK cells cytotoxic activities impair type 1 diabetes. If the
certain role of NK cells in diabetes is determined, new
therapy strategies can be improved. In the study of
Coutant et al. (1998) low-dose linomide which stimulates
NK cells, reduced the insulin needs and improved beta
cell function in patients with type 1 diabetes.
Autoimmune type 1 DM is thought to result from the
T-cell mediated immune destruction of the β-cells. Stud-
ies have shown that TH1 cytokines profiles (IL-12, IFN-γ)
drive the diabetogenic process, whereas TH2 immuno-
logical responses (IL-4, IL-10) prevent diabetes develop-
ment (Cetkovic-Cvrlje & Uckun 2005). In this study, we
found no significant difference in the serum levels of TNF-
α, IL-1, IL-2, IL-6, IL-10 in diabetic rats compared to
healthy controls (p > 0.05). On the otherhand, the levels
of IFN-γ were found to increase significantly in diabetic
rats (p < 0.05).
Schloot et al. (2002) found that serum levels of IFN-γ
were high in NOD mice compared to controls. But they
did not determine an increase in the serum levels of IL-10.
So, they informed that the ratio of IFN-γ/IL-10, reflecting
TH1/TH2 balance in serum increased significantly in dia-
betic compared to non-diabetic NOD mice. Suk et al. (2001)
found that IFN-γ could induce apoptosis in pancreatic
islet cells and play a role in diabetes. Hayashi et al. (1998)
also suggested that IFN-γ might play a role in islet inflam-
mation leading to islet cell destruction. Monetini et al.
(2004) informed that the release of IFN-γ was markedly
reduced in patients treated with oral insulin.
In our study, we determined that the levels of IFN-γ,
TH1 type cytokines increased in diabetes whereas there
was no increase in the levels of TH2 type cytokines such
as IL-6, IL-10. Therefore, we think that TH1-type cytokines
can cause destruction of the insulin-producing β-cells in
pancreatic islets of Langerhans and play a role in the patho-
physiology of diabetes.
In conclusion, the etiology of type 1 diabetes remains
unclear. The results obtained from our study indicate that
the changes of TH1 type cytokines such as IFNγ and NK
cell activity can play a role in the etiology of IDDM. The
data may provide new strategies in type 1 diabetes, such
as the administration of recombinant cytokines, cytokines
antagonists, and the drugs affecting the numbers and
functions of NK. Although these alternative treatments
may not cure type 1 diabetes, they may be effective to
correct some problems associated with type 1 diabetes.
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