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Memórias do Instituto Oswaldo Cruz
Fundação Oswaldo Cruz, Fiocruz
ISSN: 1678-8060 EISSN: 1678-8060
Vol. 89, Num. 2, 1994, pp. 179-182
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Mem. Inst. Oswaldo Cruz, Rio de Janeiro, Vol.
89(2): 179-182, apr./jun. 1994
Cytokine Responses to Dengue Infection among Puerto Rican
Patients
Goro Kuno, Raymond E Bailey
Code Number: OC94037
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Text: 20K
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Division of Vector-Borne Infectious Diseases, National Center for
Infectious Diseases, Centers for Disease Control and Prevention,
P.O. Box 2087, Ft. Colliins, CO 80522-2087, U.S.A.
Recently, a strong correlation between high concentration of
tumor necrosis factor (TNFa) in blood and severity of dengue
hemorrhagic fever/dengue shock syndrome has been reported from
Asia and the Pacific. We wished to determine if a similar
relationship could be found in dengue patients in the Americas
where adult patients with severe syndromes have been observed
more frequently than in Asia where severe cases have been
observed mostly among children.
The concentrations of interleukin-1 (IL-1beta) in
hospistalized adult groups were significantly lower than that in
outpatient adults. In contrast, the levels of interleukin 6
(IL-6) were significantly higher in hospitalized adults and
children than in the corresponding outpatients. Levels of TNFa
were higher in hospistalized children than in outpatient children
or hospistalized adults. There was no significant difference in
the levels of these three cytokines among hospitalized patients
with or without hemorrhagic manifestations. Thus, an elevated
IL-6 level was positively associated with severity of dengue
infection in both children and adults, but IL-1beta level was
negatively associated with severity in adults.
Key words: dengue - cytokine - interleukin-1- interleukin-6 -
tumor necrosis factor
The severe forms of dengue fever, dengue hemorrhagic fever (DHF)
and dengue shock syndrome (DSS), which have been endemic for many
years primarily in some parts of southeast Asia, have recently
been more frequently observed in the Americas (Kouri et al. 1989,
Anonymous 1990). Although several hypotheses and risk factors
have been proposed to explain the pathogenicity associated with
these syndromes, thus far no consensus or definitive in
vivo evidence has been obtained.
Recently, elevated levels of tumor necrosis factor (TNFa) in
blood were found to be positively correlated with the development
of the severe dengue syndromes in Asia and Pacific (Vitarana et
al. 1991, Yadav et al. 1991, Iyingkaran et al. 1992, Hober et al.
1993). Therefore, it is important to investigate the phenomenon
among dengue patients in the Western Hemisphere.
In this study, we measured the levels of three cytokines,
interleukin-1 (IL-1beta), interleukin-6 (IL-6), and TNFa, in
Puerto Rican patients with different degrees of disease severity
and statistically analyzed the relationship between cytokine
level and disease severity.
MATERIALS AND METHODS
Patients - Two hundred thirty-three patients with
laboratory-confirmed dengue in Puerto Rico in 1991 were selected
and classified into six groups by age (children = 15 yrs and
adults), presence or absence of hemorrhagic manifestations, and
whether or not they were hospitalized (Table I). The patients
with hemorrhagic manifestations had at least one of the following
signs: hematemesis, epistaxis, petechia, purpura, ecchymosis, gum
bleeding, vaginal bleeding, frank hematuria, melena, and
prolonged bleeding from skin puncture.
Determination of cytokine levels - We determined the
concentrations of three cytokines (IL-1beta, IL-6, and TNFa) in
serum samples, collected within seven days after the onset of
illness with commercial enzyme-linked immunosorbent assay kits
(for IL-1beta and TNFa, Cistron Biotechnology, Pine Brook, NJ;
for IL-6, R&D Systems, Minneapolis, MN).* Concentrations were
calibrated with the use of recombinant cytokines. Sensitivity
limits ranged between 3.5 and 10 pg/ml among products; however,
for convenience, a cut-off limit was set at 10 pg/ml for all
cytokines.
TABLE I
Presence of detectable cytokines in Puerto Rican patients with
dengue infections by severity and age
=======================================================
Group (a) No. IL-1B (b) IL-16 (b) TNFalpha (b)
pa-
tients No. % No. % No. %
-------------------------------------------------------
A 8 4 50 4 50 5 62
B 48 25 52 23 48 36 75
C 42 14 33 22 52 25 60
D 47 22 47 21 45 27 57
E 44 19 43 10 23 16 36
F 44 27 61 4 9 25 57
=======================================================
a: abbreviations: A: children with hemorrhage (hopitalized);
B: children without hemorrhage (hopitalized); D: adults
without hemorrhage (hospitilized); E: children (outpatients);
F: adults
b (out patients).
: IL - Interleukin; TNF-tumor necrois factor.
TABLE II
Cytokine levels (pg/ml) in the blood samples of
dengue patients in Puerto Rico
=================================================
Group (a) No. Cytokine (b) Mean SD (c)
Patients
-------------------------------------------------
A 8 IL-1beta 16.88(d) 22.13
IL-6 232.63 506.21
TNFalpha 58.75 107.69
B 48 IL-1beta 35.13 81.18
IL-6 53.17 142.29
TNFalpha 69.92 152.48
C 42 IL-1beta 28.98 59.70
IL-6 49.48 80.38
TNFalpha 26.12 53.86
D 47 IL-1beta 67.45 177.61
IL-6 80.17 184.34
TNFalpha 24.19 28.09
E 44 IL-1beta 41.41 59.92
IL-6 12.59 18.63
TNFalpha 46.18 70.24
F 44 IL-1beta 74.02 96.09
IL-6 9.16 20.62
TNFalpha 66.14 79.03
=================================================
a: for description of groups, see Table I
b: cytokines: IL = Interleukin; TNF = Tumor necrosis factor
c: standard deviation
d: values expressed in pg/ml
TABLE III
Significance of the differences in cytokine levels in
different groups of Puerto Rican patients with dengue (a)
========================================================
Groups p-Value
e compared (b) IL-beta (c) IL-6 (c) TNFalpha (e)
--------------------------------------------------------
A and B -- (d) -- --
C and D -- -- --
E and F -- -- --
A and C -- -- --
B and D -- -- --
A and E -- -- --
B and E -- 0.005 (e) 0.038 (e)
C and F 0.003 (f) <0.001 (e) --
D and F -- <0.001 (e) --
AB and CD -- -- 0.027 (e)
AC and BD -- -- --
AB and E -- 0.004 (e) 0.041 (e)
CD and F 0.005 (f) <0.001 (e) 0.035 (f)
ABCD and EF 0.024 (f) <0.001 (e) --
ABE and CDF -- -- --
========================================================
a: based on all values including negative specimens.
b: see Table I for description of groups.
c: cytokines: IL=Interleukin; TNF=Tumor necrosis factor
d: p > 0.05. P - values were determined using the Wilcoxon
(Mann-Whitney) test for comparison between two groups.
e: first group in comparison has a higher cylokine level.
f: second group in comparison has a higher cytokine level.
RESULTS
Cytokine levels in serum specimens of the patients were analyzed
qualitatively by the proportion of cytokine-positive patients and
quantitatively by the concentration of cytokine in each patient
group or a combination of groups.
In the qualitative analysis of the hospitalized children or
adults, no significant difference in cytokine positivity was
observed between the patients with and without hemorrhagic
manifestations (Table I). However, the proportion (61%) of
outpatient adults positive for IL-1beta was significantly higher
than the proportion (40%) of the hospitalized adults (x^2 5.17;
p < 0.05). On the other hand, the proportion of hospitalized
children (73%) was significantly higher for TNFa (x^2 13.65; p
< 0.001) than that of the outpatient children (36%). For both
children and adults, a significantly higher proportion of
patients with detectable IL-6 was observed in hospitalized groups
than in outpatients (for children 48 vs 23% with x^2 6.87; p <
0.01, and for adults 48% vs 9% with x^2 19.82; p < 0.001). In
all other comparisons, the differences in proportion of cytokine
positivity were not statistically significant.
The concentrations of the three cytokines in the six groups of
dengue patients are presented in Table II. The level of IL-1beta
in the hospitalized adults with hemorrhagic manifestations was
significantly lower than that in the outpatient adults (Wilcoxon
test; p < 0.01), reflecting the lower proportion described
earlier. A similar result was obtained when the IL-1beta level
in either all hospitalized adults (a combination of the patients
with and without hemorrhagic manifestations) or all hospitalized
patients (combined groups of all hospitalized adults and
children) was compared with that of outpatient adults or all
outpatients, respectively (Table III).
On the other hand, the concentrations of IL-6 were significantly
higher (p < 0.001) in the groups of hospitalized adults and
hospitalized children without a hemorrhagic manifestation than
in the corresponding outpatients. The same result was obtained
when the IL-6 levels of all hospitalized children or all
hospitalized patients were compared with those of outpatient
children or all outpatients, respectively (Table III).
Significantly higher (p < 0.05) concentrations of TNFa were
noted in the hospitalized children without hemorrhagic
manifestations (as compared with outpatient children), in all
hospitalized children (as compared with outpatient children), and
in all hospitalized children (as compared with all hospitalized
adults) (Table III). TNFa level in all hospitalized adults was
significantly (p < 0.05) lower than that of outpatient adults.
DISCUSSION
Although several hypotheses and risk factors, such as
pre-existing enhancing antibody, ethnic factor, presence of
underlying unrelated illness or infection, and variation of
virulence among virus strains, have been proposed to explain the
exact pathogenetic mechanism(s) of DHF/DSS, the exact
pathophysiological changes leading to manifestation of severe
syndromes remain unresolved.
We focused our attention on the roles of three cytokines. They
are known to be involved in the development of pyretic response,
inflammatory reaction, change in vascular permeability and
hemorrhagic manifestations, hypotension, and/or shock in many
infectious diseases (Beutler & Cerami 1989, Dinarello &
Savage 1989, Van Snick 1990). These same signs and symptoms are
also characteristics of DHF/DSS.
Thus, it is important to review the recent reports from Asia and
Pacific correlating TNFa levels and severity of DHF/DSS, since
we found a significantly higher level of TNFa in the hospitalized
children than in outpatient children. In one study, the TNFa
levels in DHF patients (dengue grade unspecified) were reported
to be significantly higher than the levels of those patients with
classic dengue fever (Vitarana et al. 1991). However, our
statistical analysis by Wilcoxon 2-sample test of the original
data presented in that report revealed that the difference was
insignificant, whether or not the value of one DHF patient with
the lowest level of TNFa was removed from calculation, as
suggested by the authors. In other reports (Yadav et al. 1991,
Iyngkaran et al. 1992), concentrations of TNFa were extremely
high, often exceeding 1 ug/ml. Those values are highly unusual,
because the levels of TNFa in infectious diseases, such as
malaria and AIDS, rarely exceed 1 nanogram/ml (Reddy et al. 1988,
Kern et al. 1992). In our study, the levels were generally lower
than 500 pg/ml.
In our study, TNFa levels were higher in hospitalized children
than in outpatient children or in all hospitalized adults.
However, a positive association of TNFa levels was not observed
among adults. On the other hand, significantly higher levels of
IL-6 were associated with both age groups that were hospitalized.
In contrast to TNFa and IL-6, levels of IL-1beta were negatively
correlated with hospitalization, in particular, among adults.
Further, hemorrhagic manifestations and serum cytokine levels
were not correlated for any cytokine. Thus, our results generally
agree more with the report from Tahiti in which higher levels of
TNFa and IL-6 in children were associated with increased severity
(grades III and IV of DHF/DSS) but no such an association was
observed for IL-1beta (Hober et al. 1993).
One peculiar observation of DHF patients in the Americas, which
is markedly different from the situation in southeast Asia, is
the higher proportion of adults among DHF patients. In Asia, DHF
patients are predominantly children, although DHF/DSS cases in
adults have been documented in Asia, too (Rulukruedej 1988). For
example, in one study in Thailand (Kitayaporn et al. 1989), DHF
morbidity rates between 1983 and 1987 ranged from 198 to 1,203
per 100,000 persons for children (5-9 yrs) and from 4.3 to 31.5
per 100,000 for adults (=/> 25 yrs). On the other hand, during
the 1981 epidemic in Cuba, in one hospital study, 65% of DHF/DSS
patients were adults (=/> 15 yrs) (Guzman et al. 1984), and
in the 1989-90 DHF/DSS outbreak in Venezuela, one-third of the
fatal cases were adults (Anonymous 1990). Thus, it was of
interest to determine if cytokine levels in dengue-infected
adults in Puerto Rico were different from those in children. As
shown in the results, no significantly higher value was observed
for any cytokine among adults as compared with children.
In our study, we evaluated the presence of hemorrhagic
manifestations and hospitalization as possible indicators of the
severity of dengue infection. We chose these factors, rather than
the DHF/DSS grading criteria established by the World Health
Organization (WHO) (World Health Organization 1986), because the
number of available serum specimens from Puerto Rico representing
each of four WHO grades was insufficient. Despite the subjective
character of hospitalization as an indicator of disease severity,
it proved to be highly useful, because it was significantly
correlated with the levels of IL-6 in both age groups and of TNFa
in the hospitalized children without hemorrhage. These results
corroborate and reinforce a prior report that disease severity
is better correlated with IL-6 level rather than with TNFa level
(Dinarello 1992).
Because of the multiple functions of these cytokines and the
complexity of interaction among many cytokines and other related
molecules, including the lymphokines (Kurane et al. 1989),
neither the studies on macrophage-derived cytokines alone nor the
analyses on lymphokines alone may explain the severity of dengue
infection. Nevertheless, accumulation of data on all relevant
cytokines and related mediators may eventually facilitate the
elucidation of the mechanism(s) of the manifestation of severe
syndromes in dengue infection.
ACKNOWLEDGMENT
To Dr Jose Rigau of Dengue Branch, CDC, for his assistance in
selecting the patients for this study.
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