ABSTRACT Background: Salivary gland tumors are among malignancies that have high recurrence rate. Immune responses in salivary gland tumors have not been well elucidated. T helper type 1 (Th1) and Th2 cytokines have been reported to play a role in the outcome of head and neck cancers. Objective: To evaluate the serum levels of interferon gamma (IFN-γ), as the hallmark of Th1 cytokines, and interleukin-4 (IL-4), as the hallmark of Th2 cytokines, in patients with benign and malignant salivary gland tumors in comparison with healthy controls. Methods: Fifty patients with benign and 14 patients with malignant salivary gland tumors, as well as 23 healthy individuals were recruited. Serum levels of IFN-γ and IL-4 were measured using ELISA method. Nonparametric tests were used for data analysis. Results: Serum levels of IFN-γ and IL-4 were found not to be significantly different in patients compared to the control group (0.68 ± 0.29 vs. 1.03 ± 0.57 pg/ml, p=0.58 for IFN-γ, 4.57 ± 1.57 vs. 4.41 ± 1.31 pg/ml, p=0.28 for IL-4). IFN-γ and IL-4 serum levels were also not significantly different between patients with benign and malignant salivary gland tumors (p=0.54 and p=0.86, respectively). Conclusion: The systemic levels of IL-4 and IFN-γ seem not to be associated with salivary gland tumor in our study. Investigation of other cytokines produced by Th1 and Th2 cells are warranted

Bijan Khademi

Marziyeh Tajvarpour

Mohammad Reza Haghshenas

Nasrollah Erfani

Zahra Mojtahedi

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Iran.J.Immunol. VOL.13 NO.1 March 2016                                                                                                                    9	
T-helper Type 1 and 2 Cytokine Levels in 
Patients with Benign and Malignant 
Salivary Gland Tumors 
 
 
Bijan Khademi1,2, Marziyeh Tajvarpour1, Zahra Mojtahedi1, Mohammad Reza 
Haghshenas1, Nasrollah Erfani1* 
 
1Cancer Immunology Group, Shiraz Institute for Cancer Research, School of Medicine,2Department of 
Otolaryngology, Khalili Hospital, Shiraz University of Medical Sciences, Shiraz, Iran 
 
 
ABSTRACT 
 
Background: Salivary gland tumors are among malignancies that have high recurrence 
rate. Immune responses in salivary gland tumors have not been well elucidated. T helper 
type 1 (Th1) and Th2 cytokines have been reported to play a role in the outcome of head 
and neck cancers. Objective: To evaluate the serum levels of interferon gamma (IFN-
γ), as the hallmark of Th1 cytokines, and interleukin-4 (IL-4), as the hallmark of Th2 
cytokines, in patients with benign and malignant salivary gland tumors in comparison 
with healthy controls. Methods: Fifty patients with benign and 14 patients with 
malignant salivary gland tumors, as well as 23 healthy individuals were recruited. 
Serum levels of IFN-γ and IL-4 were measured using ELISA method. Nonparametric 
tests were used for data analysis. Results: Serum levels of IFN-γ and IL-4 were found 
not to be significantly different in patients compared to the control group (0.68 ± 0.29 
vs. 1.03 ± 0.57 pg/ml, p=0.58 for IFN-γ, 4.57 ± 1.57 vs. 4.41 ± 1.31 pg/ml, p=0.28 for 
IL-4). IFN-γ and IL-4 serum levels were also not significantly different between 
patients with benign and malignant salivary gland tumors (p=0.54 and p=0.86, 
respectively). Conclusion: The systemic levels of IL-4 and IFN-γ seem not to be 
associated with salivary gland tumor in our study. Investigation of other cytokines 
produced by Th1 and Th2 cells are warranted.  
 
Khademi B, et al. Iran J Immunol. 2015; 13(1):9-15. 
 
 
Keywords: IFN-γ, IL-4, Salivary Gland Tumors, Serum 
 
 
 
 
 
 
--------------------------------------------------------------------------------------------------------------------------------------------------------------- 
*Corresponding author: Dr. Nasrollah Erfani, Cancer Immunology Group, Shiraz Institute for Cancer Research, School 
of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran, Tel/Fax: (+) 98 713 230 3687, e-mail: 
erfanin@sums.ac.ir 
 
Khademi B, et al. 
Iran.J.Immunol. VOL.13 NO.1 March 2016  10
INTRODUCTION 
 
Salivary gland tumors are a heterogeneous group of tumors with diverse 
clinicopathological and histological presentations that make their diagnosis and 
treatment complex (1). These tumors comprise 3-6% of all head and neck tumors. The 
average incidence rate per 100,000 populations is 4.7 for benign tumors and 0.9 for 
malignant ones per year. They can occur at any age but are more common in individuals 
older than 50 years. Approximately 80% of these tumors arise from parotid glands, but 
other major and minor glands can be affected with lower frequency (1,2). 
The antitumor activity of the immune system is often promoted by T-helper (Th1) 
lymphocytes. Interferon gamma (IFN-γ) is one of the most important cytokines secreted 
by Th1 cells which activates cellular immunity in the tumor environment. It exerts its 
antitumor effects in many different ways, such as preventing the spread of tumor 
through inhibition of angiogenesis, increasing phagocytosis of cancer cells via 
activation of macrophages, and induction of apoptosis. IFN-γ, by affecting The cells, 
can also inhibit their differentiation into Th2 cells (3). In salivary gland cells, it has been 
shown that IFN-γ induces apoptosis and results in the activation of caspase 8 as well 
asthe "death receptor" pathway (4). 
Likewise, Interleukin-4 (IL-4) is a cytokine with pleiotropic activity. IL-4, by affecting 
Th0 cells, stimulates their differentiation into Th2 cells and suppresses IFN-γ producing 
Th1 cells; therefore, antitumor immunity will be weakened (5). This cytokine increases 
the expression of MHC class II on the surface of B-lymphocytes. It also plays an 
important role in the Immunoglobulin class switching, and enhancing humeral 
immunity (6). In addition to the effects of IL-4 on the immune system, IL-4 receptor is 
detected on the surface of many solid tumors, such as breast cancer, melanoma, ovarian 
carcinoma, and carcinoma of the head and neck (7,8). 
Salivary gland tumors, among other types of head and neck tumors, have more 
diversity, complexity of treatment and relapse. Imbalances in T-cell subsets as well as 
their cytokine profiles have been reported in head and neck tumors (9,10). Our previous 
study demonstrated that the disproportion in the Th17/Treg ratio may contribute to the 
progression of salivary gland tumors (11). In addition, changes in Th1 and Th2 cytokine 
profiles can be seen in various types of cancers, Including lung, breast, kidney, liver, 
prostate and head and neck tumors, which is generally associated with an increase in 
Th2 cytokines including IL-4, and decrease in Th1 cytokines especially IFN-γ (4-6,12). 
However, expression patterns of these two main cytokines have not been investigated in 
salivary gland tumors, yet. Considering the role that the immune system plays in tumor 
formation or regression, we aimed to study and compare the serum levels of IL-4, and 
IFN-γ among patients with a benign and a malignant salivary gland tumor and normal 
population, and then checking out the mean and statistical issues in each group. 
 
 
MATERIALS AND METHODS  
 
Sixty-four patients with salivary gland tumors who referred to the Khalili hospital, 
Shiraz, Iran were included in this study after getting the definite detection, checking out 
pathology, and detecting the tumor type. Patients were divided into two groups: 50 
patients with benign salivary gland tumors, and 14 patients with malignant ones. The 
IFN-γ and IL-4 in salivary gland tumors 
Iran.J.Immunol. VOL.13 NO.1 March 2016  11
details of clinicopathological characteristics of the patients are depicted in the Table 1. 
Twenty-three healthy individuals comprised the control group. 
 
 
Table 1. The clinicopathological characteristics of the patients with malignant 
and benign salivary gland tumors. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Blood samples were taken with consent of the candidates. The volume of blood was 
about 5 ml, which was drawn in 15 ml disposable endotoxin free tubes. Serum was 
separated by centrifuge and then was stored at -20°C until testing. Serum levels of IL-4 
and IFN-γ were measured using sandwich ELISA technique. Measurement method was 
in a manner that samples in both controls and case group were incubated with the 
monoclonal antibody against each cytokine. After rinsing, substrate was added to each 
sample and the enzymatic reaction was performed; which, in the presence of antigen, 
resulted in the appearance of blue color. After the time required reaching the equilibrium in 
reaction, the reaction was stopped by adding stop solution. The intensity of the color was 
proportional to the amount of each cytokine in the sample. For IFN-γ, the ELISA kit 
(bioscience, Austria) with measurement sensitivity of 0.99 pg/ml, and for IL-4, the ELISA 
kit (bioscience, Austria) with measurement sensitivity of 0.66 pg/ml was used. Standard 
curve for each assay was drawn by showing the amount of absorption ratio to the amount of 
concentration on the curve based on the standard samples absorption. The concentration of 
each cytokine in samples was determined and recorded based on the standard curve.  
 Characteristics Classification Frequency (%) 
Malignant 
Tumors 
(N=14) 
   
Histological tumor type 
Mucoepidermoid carcinoma 5(35.7%) 
Adenoid cystic carcinama 2(14.3%) 
Squamous cell carcinoma 4(28.6%) 
Metastatic melanoma 1(7.1%) 
Epethelialmyoepethelial 
carcinoma 
2(14.3%) 
TNM stage 
Stage I 1(7.2%) 
Stage II 3 (21.4%) 
Stage III 4(28.6%) 
Stage IV 3(21.4%) 
unknown 3(21.4%) 
   
Metastasis 
Yes 3(21%) 
No 11(79%) 
    
Benign  
Tumors   
(N=50) 
 
Histological tumor type 
Pleomorphic adenoma 42(84%) 
Warth in tumor 7(14%) 
Basal cell adenoma 1(2%) 
Khademi B, et al. 
Iran.J.Immunol. VOL.13 NO.1 March 2016  12
Statistical Analysis. Kolmogorov-Smirnov test showed lack of normal distribution of 
variables; therefore, non-parametric analysis Kruskal-Wallis U-test using SPSS-24 
statistical software package was used.  
 
 
RESULTS 
 
The mean age of the case group was 44.0 ± 15.3 (ranging from 21 to 80) and the mean 
age of the control group was 44.3 ± 16.5 (ranging from 20 to 75). In the case group, 
45.3% of patients were men and 55.7% of them were women. These numbers in the 
controls were 47.8% and 52.2 %, respectively. 
The serum levels of IFN-γ and IL-4 are shown in the Table 2. There was no significant 
difference between the serum level of IFN-γ (0.68 ± 0.29 in the whole group patients 
and 1.03 ± 0.57 pg/ml in controls; p=0.58) and IL- 4 (4.57 ± 1.57 in patients vs. 4.41 ± 
1.31 pg/ml in control; p=0.28). 
The comparisons of serum levels of IL-4 and IFN-γ are also shown separately in both 
patients with benign or malignant tumors in Table 2. No significant difference was 
observed between serum levels of IFN-γ and IL-4 and in either patients with benign 
tumor or malignant tumor (p>0.05). 
The results of the present study showed that serum levels of IL-4 did not differ in both 
case group (p=0.39), and the controls (p=0.88) by the mean age. There was also no 
significant difference between the mean age and serum levels of IFN-γ in both case 
group (p=0.33), and the controls (p=0.51). Moreover, regarding the gender, this 
parameter was not associated with serum levels of IL-4 in both case groups (p=0.32), 
and the controls (p= 0.69). There was also no significant difference between the sex and 
serum level of IFN-γ in both case group (p=0.80), and the controls (p= 0.56). Therefore, 
patient’s gender or aging with ranging studied here did not increase or decrease the 
cytokines in their serum.  
 
 
DISCUSSION 
 
According to the results of the present study, mean serum level of IL-4 in patients and 
the controls were not statistically significant. No considerable differences in the levels 
of IFN- γ were observed, too. 
To the best of our knowledge, our results are the first publication on the serum levels of 
IL-4 and IFN-γ on salivary gland tumors. In other types of cancer, more data are 
available. In head and neck cancer and in the same ethnic background, it was found that 
serum IL-4 levels were increased in cancer patients compared to healthy individuals, but 
were not significantly associated with tumors stage (12). In a research done by 
Almatroodiet al. on serum levels of IL-4 in patients with large cell carcinoma of lung, 
there was a significant increase in serum level of IL-4 in comparison to the other non-
small cell lung cancer subjects and controls (13). In a study by Golstein et al. on 
prostate cancer, results showed that serum levels of IL-4 was higher in refractory 
patients (14). 
 
Iran.J.Immunol. VOL.13 NO.1 March 2016                                                                                                                    13	
 
Table 2. Serum concentrations of IFN-γ and IL-4 in the patients with malignant and benign salivary gland tumors and control 
group. 
 
Cytokine 
Concentration pg/ml 
Ranges, Mean ± SEM 
P value 
(two-tailed) 
Patients with 
malignant 
tumors (n=14) 
Patients with 
benign tumors 
(n=50) 
Healthy control 
subjects (n=23) 
Global P 
value* 
Comparison between 
Malignant 
and benign** 
Comparison between 
Malignant and 
control** 
Comparison between 
Benign 
and control** 
IFN-γ 
0.00-6.21 
(0.64 ± 0.47) 
0.00-11.57 
(0.70 ± 0.35) 
0.00-8.80 
(1.04 ± 0.53) 
0.74 0.54 0.99 0.50 
IL-4 
0.00-16.16 
(2.7 ± 1.46) 
0.00-80.17 
(5.10 ± 1.97) 
0.00-15.88 
(4.41 ± 1.31) 
0.54 0.86 0.47 0.34 
*Global P value; Kruskal-wallis H-test results comparing three groups 
** Mann-Whcitney U-test results comparing two group 
 
 
 
 
 
Iran.J.Immunol. VOL.13 NO.1 March 2016                                                                                                                    14	
However, Kcvocs et al. in patients with different types of cancer found that there was 
no significant difference between serum level of IL-4 in patients and the controls, which 
is similar to the result of our study on salivary gland tumors (15). Regarding IFN-γ, Lee 
et al. have detected a significant association between serum levels of IFN-γ and tumor 
size and stage in patients with Hepatocellular carcinoma (16). Similar to our results, 
lack of association between IFN-γ and disease status (paired t-test, p<0.05) in breast 
cancer has been reported (17). Decrease in IFN-γ serum levels have also been observed 
in patients with non-small cell lung cancer, making intervention of systemic level of 
IFN-γ in cancer more complex (18). 
In other body fluids of human body like pleural effusions, results are controversial, as 
well. For example, in a study by Ghayyumi et al. a wide variation in IL-4, and IFN-γ 
levels in malignant pleural effusions was shown, and no significant difference was 
observed between levels of these cytokines compared to congestive heart failure case 
(19). 
According to the above publications, controversies exist on serum levels of IFN-γ and 
IL-4 as a prognostic factor and/oran index in determining the clinical disease process in 
most solid tumors. In our study, there was no significant association between the serum 
levels of IFN-γ-and IL-4 and clinic-pathologic factors of the salivary gland tumors. The 
discrepancies can be explained by the differences in the nature of tumors, in ethnic 
background, and in the number of patients' samples. 
In conclusion, we found that there is no significant association between salivary gland 
tumors and serum levels of IFN-γ and IL-4, the two main cytokines of Th1 and Th2 
responses. However, no definite conclusion on the role of these two cytokines in 
antitumor immunology of salivary gland scan be made only based on the current results. 
Although systemic levels of cytokines are footprint of cytokine levels in the body, the 
production of these cytokines in tumor cells are shown to play an important role in anti-
tumor immunity. On the other hand, other cytokines produced by Th1 and Th2 cells 
such as IL-13 may be involved in anti-tumor immunity of salivary gland tumors. 
 
 
ACKNOWLEDGEMENTS  
 
Current study was supported by a grant from Shiraz University of Medical Sciences 
(grant no 90-01-01-2720) and also a grant from Shiraz Institute for Cancer Research. 
This manuscript was derived from the MD thesis by Dr. Marziyeh Tajvarpourat the 
Shiraz University of Medical Sciences, Shiraz, Iran.  
 
 
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T-helper Type 1 and 2 Cytokine Levels in Patients with Benign and Malignant Salivary Gland Tumors

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T-helper Type 1 and 2 Cytokine Levels in Patients with Benign and Malignant Salivary Gland Tumors

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