Abstract BACKGROUND: Cytokines have been suggested to participate in the pathogenesis of infectious mononucleosis (IM) and nasopharyngeal carcinoma (NPC)

Dyah R Budiani

Sofia M Haryana

Wihaskoro Sosroseno

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Received: 29.3.2010 Accepted: 5.8.2010 
 
 
 
 
a Lecturer, Immunology Unit, Department of Pathology, School of Medicine, Sebelas Maret University, Surakarta, Indonesia. 
b Professor, Department of Histology and Cell Biology, School of Medicine, Gadjah Mada University, Yogyakarta, Indonesia. 
c Professor, School of Dentistry, AIMST University, Semeling, Bedong, Kedah, Malaysia. 
* Corresponding Author 
E-mail: wsosroseno@yahoo.com 
 
94 JRMS/ January 2011; Vol 16, No 1. 
Short Communication 
Interleukin-4 and interferon- γ levels in Epstein-Barr virus-associated infectious 
mononucleosis and nasopharyngeal carcinoma 
 
Dyah R. Budiania, Sofia M. Haryanab, Wihaskoro Sosroseno*c 
 
Abstract 
BACKGROUND : Cytokines have been suggested to participate in the pathogenesis of infectious mononucleosis (IM) and 
nasopharyngeal carcinoma (NPC). 
METHODS: Serum levels and gene expression of interleukin-4 (IL-4) and interferon-γ (IFN-γ) were assessed by immu-
nologic and PCR assays, respectively in patients with Epstein-Barr virus (EBV)-associated IM and NPC and EBV nega-
tive controls. 
RESULTS: The serum levels of IFN- γ were elevated, but those of IL-4 were decreased in IM and NPC patients as com-
pared with those of the control group (p < 0.05). 
CONCLUSIONS: These results suggest that serum levels of IFN-γ may be predominant over those of IL-4 during the 
course of IM and NPC. 
KEYWORDS: Interferon-Gamma, Interleukin-4, Infectious Mononucleosis, Nasopharyngeal Neoplasms. 
 
JRMS 2011; 16 (1): 94-97 
 
 
nfectious mononucleosis (IM), a syndrome 
of painful lymphodenopathy, sore throat 
and fatigue, and nasopharyngeal carci-
noma (NPC), a tumor of epidermoid origin, are 
strongly associated with Epstein-Barr Virus 
(EBV), since the EBV genome has been found 
consistently in the specimens of both disor-
ders.1 However, the precise mechanism(s) by 
which EBV alters the host immune response 
and leads to the development of EBV-
associated infections and/or malignancies re-
mains to be further elucidated. Cytokines pro-
duced by the host immunocompetent cells 
and/or EBV-infected cells may play a crucial 
role in the progression of IM and NPC. For ex-
ample, expression of T cell-derived cytokines 
such as interferon-γ (IFN-γ) and interleukin-4 
(IL-4) has been observed in the biopsies of 
NPC and IM,2 suggesting that both cytokines 
may participate during the course of EBV-
associated health disorders. Indeed, a serologi-
cal observation also revealed increased levels 
of serum IFN-γ in patients with IM3 and NPC.4 
On the other hand, the serum levels of IL-4 in 
IM patients were comparable with those of the 
healthy subjects,5 but those of NPC patients 
were slightly higher.4 Studies comparing di-
rectly the cytokine levels in patients with IM 
and NPC are, however, still lacking. The aim of 
this study was to determine the serum levels of 
IFN-γ and IL-4 and their gene expression in 
peripheral blood mononuclear cells in patients 
with EBV-associated IM and NPC. 
Methods 
After getting informed consents, peripheral 
blood and serum samples were obtained from 
32 EBV-positive NPC and 5 EBV-positive IM 
patients who were under observation and 
treatment at Dr. Sardjito's General Hospital, 
I 
Cytokines in IM and NPC Budiani et al 
JRMS/ January 2011; Vol 16, No 1. 95 
Yogyakarta, Indonesia. This study was ap-
proved by the ethical committee of the Faculty 
of Medicine, Gadjah Mada University. Ten 
EBV-seronegative subjects were used as the 
control. Serum anti-VCA and EBNA IgG as-
sessed by a commercially available kit (Panbio, 
Sinnamon Park, Queensland, Australia) and 
were used to determine the positive or nega-
tive status of EBV in serum samples. The se-
rum levels of IL-4 were determined by an ELI-
SA kit (R&D System, Minneapolis, MN, USA). 
The serum levels of IFN-γ were detected by an 
immuno-dot blot assay using diluted anti-
human IFN-γ antibodies (Boehringer Mann-
heim, Gaithersburg, Maryland, USA).6 The re-
sult of each sample subtracted from the optical 
density reading of the relevant internal control, 
was divided by 100 and expressed as densi-
tometric units (DU). 
 Peripheral blood mononuclear cells 
(PBMC) were isolated and the expression of 
IL-4 and IFN-γ mRNA was semiquantitatively 
assessed by a polymerase chain reaction 
(PCR)-colorimetric dot blot assay. Briefly, to-
tal RNA from PBMC was extracted and the 
resulting cDNA was amplified by PCR. The 
sequences of the primers used in the amplifi-
cation of human IL-4 were as follow: 5’-
CTGCAAATCGACACCTATTA-3’ and 5’-
GATCGTCTTTAGCCTTTC - 3’ (product size: 
0.44 kb). The sequences of the primers used in 
the amplification of human IFN-γ were as fol-
low: 5’-CCATGGGCCCGCAGGCGGCAGC-3’ 
and 5’-GAGGACGGAGAGCTGTTCTTCC-3’ 
(product size: 0.49 kb). The PCR products were 
then immobilized on nitrocellulose membrane 
using a modified dot blot apparatus, hybrid-
ized, visualized and read at an absorbance of 
546 nm. The result of each sample was sub-
tracted from the optical density reading of the 
relevant internal control, divided by 100 and 
expressed as densitometric units (DU). 
 Data were statistically calculated by one-
way analysis of variance followed by Fisher's 
least square difference using a statistical pack 
age (SPSS Inc., Chicago). 
Results 
The serum levels of IL-4 in patients with IM 
and NPC were significantly lower than those 
of the control (p < 0.05) (Figure 1A). No signifi-
cant difference between the serum levels of 
this cytokine in patients with IM and those 
with NPC was observed (p > 0.05) (Figure 1A). 
However, the serum levels of IFN-γ in patients 
with NPC were significantly higher than those 
in the control and patients with IM (p < 0.05) 
(Figure 1B). Slightly increased serum levels of 
IFN-γ in patients with IM as compared to those 
of the control could be detected (p < 0.05) (Fig-
ure 1B). Interestingly, the levels of IL-4 and 
IFN-γ mRNA expression in patients with IM 
and NPC were not significantly different from 
those of the control (p > 0.05) (Figure 2). 
Discussion 
The present study showed that the serum lev-
els of IL-4 in IM and NPC patients were lower, 
but the serum levels of IFN-γ in these patients 
were higher than those of the healthy subjects, 
suggesting predominant IFN-γ-driven immu-
nity in IM and NPC patients which is in 
agreement with previous reports.3-5 It has been 
believed that increased levels of IFN-γ may be 
associated with decreased levels of IL-4 during 
viral infections and vice versa.7 One may as-
sume, therefore, that IM and NPC seen in the 
present study might be predominantly associ-
ated with IFN-γ-associated immunity which 
might, in turn, down-regulate the activation of 
IL-4-associated immunity. However, this spec-
ulation remains to be further investigated. 
 Interestingly, the present study also found 
that the serum levels of IFN-γ in NPC were 
much higher than those in IM. The exact rea-
son of this finding is not well understood. 
Moreover, the previous studies have reported 
much lower levels of IFN-γ gene expression in 
infected lymphoid tissues of IM patients com-
pared to those in the tissues of NPC.8,9 Since 
Cytokines in IM and NPC Budiani et al 
96 JRMS/ January 2011; Vol 16, No 1. 
 
Figure 1. Serum levels of IL-4 (A) and IFN-γ 
(B) in patients with infectious mononucleosis 
(IM) and nasopharyngeal carcinoma (NPC).  
* P < 0.05 is considered as significant; C: controls. 
 
increased levels of IFN-γ are indicators of a 
more active cellular immunity in NPC,10 a pos-
sibility that IFN-γ-driven cellular immune re-
sponse in NPC may be more intense than that 
in IM cannot be ruled out. 
 Surprisingly, IL-4 and IFN-γ mRNA levels  
in PBMC of IM and NPC patients were compa-
rable with those of the control. One of the pos-
sibilities to explain this result is that the pre-
sent study has used unstimulated PBMC from 
these patients, making detection of altered cy-
tokine gene expression difficult. Support 
for this notion is the fact that elevated 
 
Figure 2. The levels of IL-4 (A) and IFN-γ (B) 
mRNA in the unstimulated peripheral blood 
mononuclear cells of patients with infectious 
mononucleosis (IM) and nasopharyngeal car-
cinoma (NPC). C: controls. 
 
cytokine production by peripheral blood cells 
from IM patients has been observed only after 
activation of the cells by mitogens11 or T cell re-
ceptor ligation.12 It seems plausible, therefore, 
that unstimulated PBMC from IM and NPC pa-
tients may not be the preferable source of cells 
to detect EBV-altered cytokine gene expression. 
If so, one may speculate further that detectable 
serum level of IL-4 and IFN-γ in both IM and 
NPC patients seen in the present study might 
be driven from the EBV-infected tissues. 
 The extrapolation of the present study in 
the management of IM and NPC remains 
Cytokines in IM and NPC Budiani et al 
JRMS/ January 2011; Vol 16, No 1. 97 
speculative. If increased serum levels of cyto-
kines seen in the present study originate from 
the infected or tumor tissues, it would seem 
that the host immune response in both IM and 
NPC is predominated by IFN-γ-driven cellular 
immunity which is much more intense in the 
latter than the former disorder. Subsequently, 
it remains to be further investigated whether 
the serum levels of IFN-γ may be used as one 
of the markers to assess the management of IM 
and NPC. The fact that the serum level of this 
cytokine has been used in IM patients to moni-
tor the progression of disease and follow the 
treatment 3,5 suggests that monitoring of IFN-γ 
serum levels may be worthy to detect the pro-
gression of NPC as well. 
Conclusions 
The present study shows that the serum levels 
of IFN-γ, but not IL-4, elevate in IM and par-
ticularly NPC patients. However, the gene ex-
pression of both cytokines in unstimulated 
PBMC of IM and NPC patients is similar to 
those of the controls. These results suggest that 
the levels of IFN-γ may be predominant over 
those of IL-4 during the course of IM and NPC. 
Acknowledgements 
This work was supported by DCRG-URGE 
(Grant batch III, 1996/1997), from The Ministry 
of National Education, The Indonesian Gov-
ernment. The authors gratefully thank Drs M. 
Tjokronagoro and T. Saleh for their assistance. 
 
Conflict of Interests 
Authors have no conflict of interests. 
Author's Contributions 
DRB and SMH designed, conducted and analyzed the study. WS designed and analyzed the study 
and prepared the paper. All authors have read and approved the content of the manuscript. 
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Short Communication Interleukin-4 and interferon-γ levels in Epstein-Barr virus-associated infectious mononucleosis and nasopharyngeal carcinoma

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Short Communication Interleukin-4 and interferon-γ levels in Epstein-Barr virus-associated infectious mononucleosis and nasopharyngeal carcinoma

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