Journal of Medical Virology 67:41±46 (2002)

Cytokine Gene Expression and Protein Production in

Peripheral Blood Mononuclear Cells of Children
With Acute Dengue Virus Infections
Susan J. Gagnon,1 Masuko Mori,1 Ichiro Kurane,2 Sharone Green,1 David W. Vaughn,3
Siripen Kalayanarooj,4 Saroj Suntayakorn,5 Francis A. Ennis,1 and Alan L. Rothman1*
1
Center for Infectious Disease and Vaccine Research, University of Massachusetts Medical School,
Worcester, Massachusetts
2
First Department of Virology, National Institute of Infectious Diseases, Tokyo, Japan
3
Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand
4
Queen Sirikit National Institute of Child Health (formerly Bangkok Children's Hospital), Bangkok, Thailand
5
Kamphaeng Phet Provincial Hospital, Kamphaeng Phet, Thailand

Plasma leakage in dengue hemorrhagic fever
(DHF) is associated with elevated plasma levels of
cytokines. To de®ne further the contribution of
immune activation to DHF and the source of cyto-
kines, we analyzed the production of cytokines in
peripheral blood mononuclear cells (PBMC) ob-
tained from children with dengue, using RT-PCR
and immunostaining. Tumor necrosis factor-a
(TNF-a) and TNF-b expression was detected in all
samples by PCR and in < 50% of samples by
immunostaining. Interferon-g (IFN-g) expression
was detected in < 50% of samples by either
method. Interleukin-2 (IL-2) and IL-4 expression
was detected in a few samples by immunostain-
ing but was not detectable by PCR. We found
greater expression of TNF-a and IL-4 in DHF than
in dengue fever or other (non-dengue) febrile ill-
nesses. These results support the model of
immunopathogenesis of DHF. However, low
levels of cytokine expression in PBMC suggest
that cellular activation in tissues may contribute
to high serum cytokine levels in DHF. J. Med.
Virol. 67:41±46, 2002. ß 2002 Wiley-Liss, Inc.
KEY WORDS: RT-PCR;immunostaining;TNF-a;
IL-4; hemorrhagic fever
INTRODUCTION
Dengue virus (DV; family Flaviviridae, genus Flavi-
virus, species Dengue virus) is an important cause of
disease in many tropical and subtropical areas of the
world, with estimates of up to 100 million infections
occurring per year [Halstead, 1988]. Symptomatic
infection with DV can result in a range of clinical syn-
dromes, including a mild, undifferentiated febrile ill-
ness, classical dengue fever (DF), and the more severe
form of disease known as dengue hemorrhagic fever
(DHF) [Halstead, 1980]. DHF is characterized by in-
creased capillary permeability resulting in leakage of
plasma into the interstitial spaces, potentially culmi-
nating in hemorrhage, circulatory shock, and death.
Epidemiological studies have indicated that greater
than 90% of individuals who develop DHF are under-
going a secondary infection with a heterologous sero-
type of DV [Halstead, 1988]. Laboratory observations
indicate that patients with DHF exhibit a higher degree
of T-cell activation than do patients with DF. We
hypothesize that DHF is the immunopathological
consequence of a secondary DV infection, in which an
abnormally high degree of T-cell activation results in
the overproduction of various cytokines or chemical
mediators, which may induce the capillary leak
syndrome [Rothman and Ennis, 1999].
Previous research has indicated that patients with
DV infections have increased serum or plasma levels of
cytokines, including interferon-g (IFN-g), interleukin-2
(IL-2), tumor necrosis factor-a (TNF-a), IL-1b, and IL-6
[Kurane et al., 1991; Hober et al., 1993; Green et al.,
1999b]. However, the results of these studies have
varied, and cytokine levels have not been elevated in
Grant sponsor: National Institutes of Health; Grant numbers:
P01 AI34533, R01 AI30624; Grant sponsor: U.S. Army Medical
Research and Material Command.
Masuko Mori's present address is Fukujuji Medical Center,
Miyagi Branch of Japan Anti-tuberculosis Association, Sendai,
Japan.
David W. Vaughn's present address is Department of Virus
Diseases, Walter Reed Army Institute of Research, Silver Spring,
Maryland.
*Correspondence to: Alan L. Rothman, CIDVR/Room S5-326,
University of Massachusetts Medical School, 55 Lake Ave. North,
Worcester, MA 01655. E-mail: alan.rothman@umassmed.edu
Accepted 5 October 2001
DOI 10.1002/jmv.2190
Published online in Wiley InterScience
(www.interscience.wiley.com).

ß 2002 WILEY-LISS, INC.

42
all subjects with severe dengue disease. This ®nding
could result from the dif®culties inherent in inter-
preting cytokine levels measured in the peripheral
blood, such as the short half-life of most cytokines and
the rapid binding of these molecules to either cellular
or soluble receptors, which may preclude their detec-
tion. Examination of cytokine production at the cel-
lular level might provide a more accurate picture of
cytokines produced in response to an immunological
challenge [Andersson and Andersson, 1993; Mori et al.,
1999].
We used reverse transcription-polymerase chain
reaction (RT-PCR) and immunocytochemical staining
to corroborate and extend previous data, indicating that
there is a detectable level of cytokine-producing cells
in the peripheral blood mononuclear cells (PBMC) of DV-
infected individuals during acute infection. We found
that the level of expression of TNF-a mRNA and the
frequencies of PBMC producing TNF-a and IL-4 were
higher in patients with DHF than in those with DF or
non-dengue febrile illnesses.
MATERIALS AND METHODS
Clinical Study Design
Thai children between the ages of 6 months and 14
years were enrolled in the Dengue Hemorrhagic Fever
Project, as previously reported [Kalayanarooj et al.,
1997]. Brie¯y, children were enrolled with undiffer-
entiated fever of < 72-hr duration. A venous blood
sample was drawn daily until 1 day after defervescence,
and a convalescent blood sample was drawn between
study days 8 and 11. Informed consent was obtained
from the parent or guardian of all participants. Human
experimentation guidelines of the U.S. Department of
Health and Human Services were followed in the
conduct of this research. The study protocol was
approved by the Institutional Review Boards of the
Ministry of Public Health, Thailand, the U.S. Army
Of®ce of the Surgeon General, and the University of
Massachusetts Medical School (UMMS).
Clinical diagnoses of DF and DHF were assigned
according to World Health Organization (WHO) cri-
teria [Anonymous, 1997]. DV infections were con®rmed
by serology and virus isolation [Vaughn et al., 1997].
Cases with negative dengue serology and no virus iso-
lated were categorized as other febrile illnesses (OFI).
Sample Processing
Samples for PCR analysis were prepared as pre-
viously described [Gagnon et al., 2001]. For immuno-
cytochemical staining, PBMC were cryopreserved and
stored in liquid nitrogen. Samples were shipped from
Thailand to UMMS on dry ice. Samples were analyzed
under code at UMMS.
PCR Analysis of Cytokine Gene Expression
Total cellular RNA was isolated by the acid guanidi-
nium-phenol-chloroform method [Chomczynski and
Gagnon et al.
Sacchi, 1987]. PCR for cytokines and b-actin was per-
formed using the primers and ampli®cation conditions
previously reported [Caceres-Dittmar et al., 1993].
cDNA was resuspended in 200 ml of 1/20 TE, and the
reaction was carried out using 10 ml of cDNA solution in
a ®nal volume of 25 ml, containing 0.3 mM each of 50 and
30 primers (synthesized at the DNA Synthesis Facility,
UMMS), 1.5 mM MgCl2 (Promega, Madison, WI),
0.2 mM each dNTP (Pharmacia Biotech), and 0.63 units
of Taq polymerase (Promega). For semiquantitative
analysis, b-actin and the cytokine of interest were
ampli®ed in the same reaction. 30 primers were end-
labeled with 32P, using T4 polynucleotide kinase
(Promega) and puri®ed using Chroma Spin-10 columns
(Clontech Laboratories, Palo Alto, CA). A volume of
labeled 30 primer equivalent to 105 cpm was used in
each reaction. PCR was performed using the hot-start
method [Nuovo et al., 1991].
After PCR, total reaction mixtures were electro-
phoresed on a 5% polyacrylamide gel. The radio-
activity of each ampli®ed fragment was examined
with a Betascope (Betagen, Mountain View, CA) for
100 min, and the ratio (cpm cytokine)/(cpm b-actin)
was calculated. Speci®city of the PCR products was
con®rmed in preliminary experiments by Southern
blot.
Immunocytochemical Staining
Cells were thawed and washed once with RPMI 1640
(Life Technologies) without FCS followed by a wash in
N-2-hydroxyethyl-piperazine-N0 -2 ethane sulfonic acid
(HEPES) (Life Technologies) buffered Hank's balanced
salt solution (HBSS) containing Ca2‡ and Mg2‡. Cells
(2±3  105) were applied to coated slides (Shandon,
Pittsburgh, PA) by cytospin (Shandon) at 700 rpm for
10 min. Cells on cytospin slides were then ®xed with 2%
paraformaldehyde (Kodak, Rochester, NY) in phos-
phate-buffered saline (PBS) at 48C for 15 min. After
washing 3 times in HBSS containing 1% saponin
(Sigma), slides were stored at 708C until examination.
Immunocytochemical staining was performed as
previously described [Mori et al., 1997]. After incuba-
tion with antibodies, slides were washed and then
incubated with horseradish peroxidase (HRP)-conju-
gated streptavidin (Vector Laboratories) for 10 min at
room temperature. After several washes, color was
developed with 0.03% 30 -3-diaminobenzidine tetra-
hydrochloride (Dojin, Kumamoto, Japan) containing
0.006% H2O2. The slides were counterstained with
methyl green (Sigma) and mounted for light micro-
scopy. Immunoabsorption tests for the speci®city of
cytokine staining were performed as previously
described [Mori et al., 1997]. Cytokine-producing cells
were counted under a Nikon Labophot-2 microscope
(Nikon, Garden City, NY). All cell areas were examined
under  400 magni®cation to enumerate positively
staining cells. Cell frequencies were calculated using
as the denominator the total number of cells applied to
the slide.
Cytokine Production in Dengue 43

The frequency of cytokine-producing cells in PBMC was measured by immunostaining, and calculated as cytokine-containing cells per 106 PBMC. Data are expressed as median (range; number tested).
Relative cytokine mRNA expression in PBMC was measured by semiquantitative RT-PCR, and calculated as (cpm cytokine)/(cpm b-actin). Data are expressed as median (range; number tested).
TNF-a, -b, tumor necrosis factor-a, -b; IFN-g, interferon-g; IL-2, -4, interleukin-2, -4; PBMC, peripheral blood mononuclear cells; RT-PCR, reverse transcription-polymerase chain reaction; cpm,
Statistical Analysis

0 (0±7; 10)
0 (0±8; 10)

0 (0±3; 15)
0 (0±4; 13)
0 (0±0; 14)
0 (0±3; 15)
0 (0±0; 13)
0 (0±3; 14)
0 (0±0; 15)
0 (0±5; 13)
0 (0±3; 14)
0 (0±6; 15)
0 (0±6; 13)
0 (0±0; 14)
Cytokine/b-actin ratios and frequencies of cytokine-

0 (0±0; 8)
producing cells were compared among subjects with

OFI
TABLE I. Cytokine Expression in PBMC of Children With Dengue Hemorrhagic Fever (DHF), Dengue Fever (DF), or Other Febrile Illnesses (OFI)
DHF, DF, and OFI using nonparametric analyses.
P-values of  0.05 were considered statistically signi-
®cant.

Cytokine proteinb
RESULTS AND DISCUSSION

0 (0±76; 12)

0 (0±27; 9)

0 (0±10; 9)

0 (0±32; 9)
0 (0±6; 12)

0 (0±4; 15)
0 (0±2; 13)

0 (0±4; 15)
0 (0±2; 13)

0 (0±4; 15)
0 (0±7; 13)

0 (0±5; 13)
1 (0±7; 12)
Detection of Circulating

0 (0±6; 8)

0 (0±5; 7)
Cytokine-Producing Cells

DF
Table I summarizes the results of RT-PCR assays
and immunocytochemical staining. The results are
organized according to the study day in three groups.
Study days 1±2 represent the middle of the febrile
period. Study days 3±5 correspond to the end of the

13 (0±219; 10)

0 (0±25; 12)

0 (0±23; 12)

4 (0±12; 12)
0 (0±9; 12)
0 (0±15; 8)
febrile phase and the period of plasma leakage for

0 (0±5; 8)
0 (0±0; 8)

0 (0±3; 8)
0 (0±0; 9)

0 (0±0; 8)
0 (0±0; 9)

0 (0±0; 8)
0 (0±5; 8)

3 (0±7; 8)
patients with DHF. Study days 8±11 correspond to the

DHF
early convalescent phase; subjects were asymptomatic
at this time.
Production of the Th1 cytokines TNF-a, TNF-b/
lymphotoxin, and IFN-g was detectable in many PBMC
samples at both the mRNA and protein levels using

0.28 (0.19±2.28; 11)

0.24 (0.11±5.16; 11)

0.19 (0.02±0.41; 12)

0.20 (0.07±0.43; 11)

RT-PCR analysis and immunostaining, respectively.

0.26 (0.13±3.70; 7)

0.19 (0.05±0.58; 7)

0 (0±0.09; 12)

0 (0±0.17; 11)
Low numbers (3±32 per 106 PBMC) of IL-2- and IL-4-

0 (0±0.15; 7)
producing cells were detected in one or more PBMC
OFI

samples from 9 of 34 and 18 of 33 subjects, respectively,

nd
nd
nd
nd
nd
nd
by immunostaining, although transcripts for these
cytokines were consistently undetectable by RT-PCR
(data not shown). PCR and immunostaining studies
were performed on different subjects, however, pre-
cluding direct correlation of the results of these two
0.28 (0.11±0.61; 10)

0.15 (0.07±0.62; 11)

0.21 (0.07±0.55; 10)
0.34 (0.10±0.85; 8)
0.29 (0.16±0.41; 6)

0.18 (0.09±0.63; 8)
Cytokine mRNAa

methods.
0 (0±0.02; 11)
0 (0±0.01; 10)
0 (0±0.02; 8)

One possible explanation for the observed differences

in the frequency of detection of speci®c cytokines using
DF

nd (not detected); mRNA for these cytokines was consistently undetectable.

nd
nd
nd
nd
nd
nd
these two methods is a difference in their sensitivity.
Alternatively, the differences in results may re¯ect
differences in the half-lives of mRNA and proteins.
Because we were interested in evaluating the sponta-
neous production of cytokines, we did not stimulate the
PBMC with antigen in vitro, as done by others [Murali-
0.84 (0.17±3.02; 9)
0.69 (0.18±1.16; 8)
0.37 (0.11±1.95; 7)
0.26 (0.04±0.56; 8)
0.21 (0.06±0.78; 9)
0.15 (0.10±0.87; 8)

Krishna et al., 1998]. While the intracellular retention

0 (0±0.20; 10)
0 (0±0.10; 9)
0 (0±0.18; 8)

of cytokines caused by treatment with brefeldin A

DHF

enhances the detection of intracellular cytokines by

ndc
nd
nd
nd
nd
nd

¯ow cytometry, this requires a period of in vitro culture.

Further, the work of others has shown that the fre-
quency of cells containing detectable cytokines in the
absence of antigenic stimulation is low using the ¯ow
cytometry method [Murali-Krishna et al., 1998]. The
immunostaining method is more sensitive than ¯ow
Study day

8±11

8±11

8±11

8±11

8±11

cytometry. We and others have shown that this method

1±2
3±5

1±2
3±5

1±2
3±5

1±2
3±5

1±2
3±5

can detect cytokine-producing cells in PBMC stimulated

in vitro with mitogen or antigen or in tissue samples
counts per minute.

from patients with other viral illnesses [Andersson and

Andersson, 1993; Mori et al., 1997, 1999].
These results support the ®ndings from previous
Cytokine

clinical studies, which have demonstrated elevated

TNF-a

TNF-b

IFN-g

levels of cytokines, including TNF-a, IFN-g, and IL-2, in

IL-2

IL-4

the plasma or serum of patients with acute DV infection

a
b
c
44 Gagnon et al.

[Kurane et al., 1991; Hober et al., 1993; Kuno and

Bailey, 1994; Green et al., 1999b]. TNF-b/lymphotoxin
production in DV-infected subjects has not been
examined previously, to our knowledge. The presence
of cytokine-producing cells in the PBMC is further
evidence of immune activation during acute DV infec-
tion, as they were consistently undetectable in PBMC
of healthy asymptomatic control subjects [Mori et al.,
1997, 1999].
The frequency of cytokine-expressing cells in the
PBMC was low, despite marked elevations in the
plasma levels of free cytokines. This difference may
indicate that a signi®cant fraction of the activated,
cytokine-producing cells are located in areas of the body Fig. 1. Expression of tumor necrosis factor-a (TNF-a) mRNA in
peripheral blood mononuclear cells (PBMC) of children with dengue
rather than circulating in the peripheral blood. Simi- hemorrhagic fever (DHF), dengue fever (DF), and other (non-dengue)
larly, we have found that the numbers of cytokine- febrile illnesses (OFI). Gene expression in PBMC was measured by
producing cells is much higher in the lungs and spleens semi-quantitative RT-PCR. Values shown are relative expression of
TNF-a mRNA calculated as the ratio (cpm cytokine)/(cpm b-actin). The
of patients with hantavirus pulmonary syndrome than expression of TNF-a mRNA was signi®cantly higher in subjects with
in the peripheral blood of the same patients (unpub- DHF than in those with DF (P ˆ 0.013 by the Mann-Whitney rank-
sum test). Study day 1 was the day of enrollment in the study.
lished data) [Ennis et al., 1999; Mori et al., 1999].
Although infection of circulating monocytes with DV
has been demonstrated [Halstead, 1980], it is not clear
what fraction of the total number of infected cells are in
the circulation. DV has been detected in various tissues
during acute infection, particularly the liver [Rosen
et al., 1989; Hall et al., 1991]. Dendritic cells have been
shown to be highly susceptible to DV infection in vitro
[Wu et al., 2000; Libraty et al., 2001], and skin Langer-
hans cells were found to display DV antigens in vivo
[Wu et al., 2000]. We hypothesize that some DV-speci®c
T cells are activated outside the circulation upon ex-
posure to DV-infected cells in these tissues. Alterna-
tively, DV-speci®c T cells that are activated within the
circulation may migrate into tissues as a result of in-
creased expression of adhesion molecules [Dailey, 1998;
Woods et al., 2000]. Support for this hypothesis comes
from comparing these results with the ®ndings of
Green et al that a large percentage of the circulating
CD8‡ T cells expressed CD69 during acute DV infection
[Green et al., 1999a]. Expression of CD69 is induced
early after T-cell activation, before the induction of
cytokine gene expression [Fulcher and Wong, 1999;
Zola, 2000]. Therefore, one explanation for the failure
to detect cytokine gene expression is that the activated
cells leave the circulation between these two events.

Comparison of Cytokine Production in Patients

With DHF, DF, and OFI
There were no signi®cant differences between the
three groups (DHF, DF, and OFI) in the mRNA levels or
numbers of cytokine-producing PBMC for IL-2, IFN-g, or
TNF-b. However, we did ®nd statistically signi®cant
differences in TNF-a and IL-4 production. The TNF-a/b-
actin mRNA ratios measured by RT-PCR were signi-
®cantly higher in patients with DHF than in those with
DF (P ˆ 0.013 by the Mann-Whitney rank-sum test,
Fig. 1). In addition, the frequencies of TNF-a- and IL-4-
producing PBMC measured by immunostaining were
signi®cantly different among the three groups (DHF,
Fig. 2. Numbers of cells producing (A) tumor necrosis factor-a (TNF-
a) or (B) interleukin-4 (IL-4) in peripheral blood mononuclear cells
(PBMC) of children with dengue hemorrhagic fever (DHF), dengue
fever (DF), and other (non-dengue) febrile illnesses (OFI). Cyto-
kine production was analyzed by immunostaining. Values shown are
the numbers of cytokine-producing cells per million PBMC. The pro-
duction of TNF-a and IL-4 was signi®cantly different in the subjects
with DHF, DF, and OFI (P ˆ 0.016 and P < 0.01, respectively, by
Kruskal-Wallis ANOVA). Study day 1 was the day of enrollment in the
study.
Cytokine Production in Dengue
DF, and OFI) (P ˆ 0.016 and P < 0.01, respectively, by
Kruskal-Wallis analysis of variance [ANOVA], Fig. 2).
Pairwise comparisons showed a statistically signi®cant
difference between subjects with DHF and those with
DF in the number of IL-4-producing PBMC, while the
difference in the number of TNF-a-producing PBMC
was not statistically signi®cant.
Our results are consistent with previous studies
reporting that TNF-a levels were higher in the serum or
plasma of children with DHF than in those with DF
[Hober et al., 1993; Green et al., 1999b]. Hober et al.
[1996] demonstrated that in vitro exposure to DV anti-
gens induced the production of TNF-a from a monocytic
cell line. We have shown that DV-speci®c CD4‡ T
lymphocytes are also a source of TNF-a when stimu-
lated by DV antigens [Gagnon et al., 1999]. Studies of
the effects of in vivo administration of TNF-a suggest
that this cytokine could contribute to the increased
capillary permeability and plasma leakage character-
istic of DHF [Tracey and Cerami, 1993].
IL-4 production has not been evaluated extensively
during acute dengue virus infections. Chaturvedi et al.
[2000] reported elevated serum levels of IL-4 in pati-
ents with DHF. However, Green et al. [1999b] found no
difference in plasma IL-4 levels between subjects with
DHF and those with DF. Several groups have reported
that plasma or serum levels of IL-10 were higher in
subjects with DHF than in those with DF [Green et al.,
1999c; Chaturvedi et al., 2000]. Since IL-10 is known to
support T-helper type 2 responses preferentially, in-
creased IL-4 production in subjects with DHF, as we
observed in this study, might be expected. Production
of anti-in¯ammatory type 2 cytokines may re¯ect a
physiologic counterresponse to the proin¯ammatory
cascade in DHF.
Results of several studies have suggested that over-
activation of the immune system may underlie the
pathology of DHF [Kurane et al., 1991; Green et al.,
1999b]. In a recent study of DV-infected Thai children,
Green et al., demonstrated an increased expression of
CD69, an early activation marker, on CD8‡ T cells and
natural killer (NK) cells from children with DHF com-
pared with those with DF during the period of viremia
[Green et al., 1999a]. The data from the present study
provide direct evidence that DV infection results in
induction of cytokine expression, and that the degree of
activation correlates with disease severity.
This study is the ®rst to examine in a prospective
manner both cytokine gene expression and protein pro-
duction at the cellular level in the PBMC of DV-infected
individuals. Our results suggest that activation of
the immune system during DV infection results in the
production of various proin¯ammatory cytokines, in-
cluding TNF-a, IFN-g and TNF-b. Most interestingly,
TNF-a mRNA levels and the numbers of TNF-a- and
IL-4-producing cells were signi®cantly increased in
patients with DHF. These ®ndings support the hypoth-
esis that DHF is the product of an immunopathological
over-activation of the immune system induced by DV
infection.
45
ACKNOWLEDGMENTS
The opinions expressed are those of the authors and
should not be construed as representing the of®cial
policies of the NIH or the Department of Defense.
We thank Dr. Yuji Okamoto, Jurand Janus, Anita
Leporati, and Joyce Norman for technical assistance.
We thank Drs. Suchitra Nimmannitya and Ananda
Nisalak for supervision of the clinical study. We thank
the nurses and pediatricians at the Queen Sirkit
National Institute of Child Health and the Kamphaeng
Phet Provincial Hospital, Thailand, for volunteer
enrollment and patient management. We thank the
research nurses, technicians, technologists, and admin-
istrative personnel of the Department of Virology,
Armed Forces Research Institute of Medical Sciences,
Bangkok, Thailand, for specimen processing, data
management, and serologic and virologic analyses.
We thank Dr. Harry Towbin, Ciba-Geigy, Basel,
Switzerland, and Dr. Haruji Nakamura, Toray Indus-
tries, Otsu, Japan, for providing the antibodies used for
immunohistochemical analysis. We also thank the
patients and their families for their participation in
this study.
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