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A Low Interleukin-10 Tumor Necrosis Factor-a Ratio Is Associated with
Malaria Anemia in Children Residing in a Holoendemic Malaria Region in
Western Kenya
Caroline Othoro, Altaf A. Lal, Bernard Nahlen,
Davy Koech, Alloys S. S. Orago,
and Venkatachalam Udhayakumar
Vector Biology and Control Research Center, Kenya Medical Research
Institute, Kisumu, and Department of Zoology, Kenyatta University,
Nairobi, Kenya; Division of Parasitic Diseases, National Center for
Infectious Diseases, Centers for Disease Control and Prevention,
Atlanta, Georgia
The balance between Th1 cytokines (tumor necrosis factor [TNF]-a, interferon [IFN]-g)
and Th2 cytokines (interleukin [IL]-10, -4) may be critical in the development of severe falciparum malaria. Therefore, plasma concentrations of these cytokines were determined in
children with various manifestations of malaria. Plasma levels of IFN-g and IL-4 were undetectable in most children. However, TNF-a and IL-10 were significantly elevated in children
with high-density parasitemia and malaria anemia compared with children in control groups.
In children with mild malaria, IL-10, but not TNF-a, was significantly elevated. While the
highest concentrations of TNF-a were found in children with malaria anemia, IL-10 levels
were highest in children with high-density uncomplicated malaria. The mean ratio of IL-10
to TNF-a was significantly higher in children with mild and high-density parasitemia (4.64,
P ! .005) than in children with malaria anemia (1.77). Thus, higher levels of IL-10 over TNFa may prevent development of malaria anemia by controlling the excessive inflammatory
activities of TNF-a.
An estimated 1–2 million children die annually in Africa from
falciparum malaria [1], usually from complications due to cerebral malaria or severe anemia [2]. Although the pathologic basis
for the development of cerebral malaria and malaria anemia is
not well understood, it is apparent that cytokines play a significant role. Inflammatory cytokines such as tumor necrosis
factor (TNF)-a, interleukin (IL)-1, interferon (IFN)-g, and IL6 are highly elevated in acute Plasmodium falciparum infections
[3–5]. TNF-a in particular has been associated with cerebral
malaria and death in children [4, 5]. Subsequently, it was shown
that elevation of TNF-a was not exclusively associated with
cerebral malaria but was also associated with anemia and highdensity P. falciparum infections [6]. These data notwithstanding,
it is important to recognize that not all malaria-infected children
with higher levels of TNF-a develop severe malaria. Thus, we
reasoned that the cytokine network as a whole, rather than a
Received 16 March 1998; revised 19 August 1998.
Presented in part: American Society of Tropical Medicine and Hygiene
annual meeting, Orlando, Florida, 7–11 December 1997 (abstract 411).
This work was done in accordance with US Department of Health and
Human Services and Kenya Medical Research Institute regulations governing the protection of human subjects in medical research. The ethical review
boards of both institutions approved the study. Parents of study children
provided written informed consent.
Grant support: USAID (BST-0453-P-HC-2086-07, HRN-6001-A-003018-00).
Reprints or correspondence: Dr. V. Udhayakumar, Mail Stop F-12, 4770
Buford Highway, Atlanta, GA 30341 ([email protected]).
The Journal of Infectious Diseases 1999; 179:279–82
q 1999 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/99/7901-0041$02.00
single cytokine, must be carefully investigated to better understand the interactive role of various cytokines in the severe
disease manifestations of malaria.
Experimental studies with rodent malaria models have shown
that the up-regulation of Th2 cytokines prevents the development of cerebral malaria and that Th1 cytokines promote disease [7]. However, it is unclear whether Th2 cytokines assist in
preventing severe disease in humans. IL-10, a Th2 cytokine,
regulates the functional activity and the production of a Th1
cytokine, TNF-a. IL-10 can inhibit P. falciparum–induced
TNF-a production [8]. Preliminary studies have shown that IL10 levels are elevated during malaria infection as are TNF-a,
IFN-g, and other cytokines [9]. Thus, in this study, we investigated the hypothesis that a higher level of Th2 cytokines (IL4, -10, or both) than Th1 cytokines (TNF-a, IFN-g, or both)
may prevent development of malaria anemia in an area in which
malaria is highly endemic by down-regulating the inflammatory
activities of Th1 cytokines.
Materials and Methods
Study area. This cross-sectional study was conducted in the
population living in Kisumu Town, Nyanza Province, western
Kenya. Malaria is transmitted perennially in this area and P. falciparum malaria accounts for ∼97% of malarial infections. Residents experience 100–300 infective mosquito bites per year [10].
Study population. Children aged 3–11 years admitted to the
Kisumu District Hospital were enrolled in the study. More than
90% of the population in this area are of the Luo ethnic group.
Study participants were categorized into six groups: mild malaria,
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Othoro et al.
high-density uncomplicated malaria, malaria anemia, hospital controls, asymptomatic, and aparasitemic. Group characteristics are
summarized in table 1. Aparasitemic and asymptomatic children
were recruited from the same rural areas in which the hospitalized
children lived; they were recruited by cross-sectional surveys conducted in the same season and year. Children were excluded from
the study if they were severely immunosuppressed, malnourished,
or had other concurrent infections (based on clinical signs determined by a clinical officer) that may cause fever, as were children
admitted for malaria who received medication 1 4 h.
Blood sample collection. All children had a physical examination. The presence of malarial parasites was determined using a
fingerprick Giemsa-stained thick blood smear, and parasitemia was
determined by the number of asexual stage parasites per 300 white
blood cells. The parasitemia density per microliter of blood was
estimated by the formula: (number of parasites/300) 3 8000 (the
mean number of white blood cells per microliter of blood). Hemoglobin levels were determined by spectrophotometer (Hb;
HemoCue, Helsingborg, Sweden). Eligible children were enrolled
in the study, and 5 mL of venous blood was collected into sterile
heparinized vacutainers from each enrolled child (Becton Dickinson, Rutherford, NJ). Plasma was immediately separated and frozen in liquid nitrogen to avoid denaturation of cytokines in the
plasma.
Cytokine ELISA. A two-site sandwich ELISA was used to
determine cytokine levels in plasma. TNF-a, IFN-g, IL-4, and IL10 levels were estimated using optimal concentrations of antibodies
and cytokine standards according to the manufacturer’s instructions. Cytokine concentrations in the test samples were determined
from a standard curve with various concentrations of cytokines
(3–3000 pg/mL; R&D Systems, Minneapolis).
Statistical analysis. Differences between the mean concentra-
JID 1999;179 (January)
tion of cytokines in various groups were compared using the nonparametric Kruskal Wallis test. The ratio of IL-10 to TNF-a was
calculated for each study participant. The statistical difference in
the mean ratios between the mild and severe disease groups was
tested using the Wilcoxon test. P ! .05 was considered statistically
significant.
Results
TNF-a levels. Mean plasma TNF-a levels in children with
different clinical symptoms were compared. Results are presented in table 1. The mean TNF-a concentration was lowest
in the healthy aparasitemic children and did not vary significantly in the asymptomatic and hospital control children. In
children with mild malaria, the TNF-a levels were not different
from the control groups. In the high-density uncomplicated
malaria group, TNF-a levels were ∼4-fold higher than in the
mild malaria group (P ! .001 ). Malaria-anemic children had the
highest levels of TNF-a, which was highly significant when
compared with any of the control groups (P ! .001).
IL-10 levels. The mean plasma concentrations of IL-10 in
various patient groups are shown in table 1. Lowest levels of
IL-10 were found in the aparasitemic children and asymptomatic children with malaria parasitemia. For children with mild
malaria, although TNF-a levels were not elevated when compared with the control groups, IL-10 levels were significantly
higher than in control asymptomatic children (P ! .004 ). In
contrast to TNF-a levels, mean IL-10 levels were higher in the
high-density uncomplicated malaria group than in the malaria
Table 1. Cytokine response in different groups of children.
Malaria
Parameters
No. of subjects
Mean age, years
Mean temperature, 7C
Mean parasitemia, parasites/mL
Mean hemoglobin, g/dL
TNF-a, pg/mL
IL-10, pg/mL
IFN-g, pg/mL
IL-4, pg/mL
Aparasitemic
Asymptomatic
Mild
High-density
uncomplicated
25
8.7
36.7
0
10.2
37 5 13
29 5 15
753
453
24
8.2
36.6
1926
11.4
61 5 14
30 5 10
10 5 3
251
19
6.4
38.3
1191
9.8
46 5 18
122 5 29
954
251
24
5
38.8
54,825
9.7
184 5 62
362 5 94
452
0
Anemia
Hospital control
52
4.1
38.1
28,187
5.5
271 5 31
327 5 75
653
251
7
6.2
39.1
0
11.6
64 5 43
26 5 16
44 5 38
252
NOTE. Study groups of children had following symptoms and signs: aparasitemic, healthy with no detectable parasitemia and no
anemia; asymptomatic, positive for parasitemia (range, 54–7425 parasites/mL) but no fever (temperature !377C) or other clinical
symptoms of malaria infection; mild malaria, asexual low-density Plasmodium falciparum parasitemia (!8000 parasites/mL [70th percentile
of observed densities in children in area]; parasitemia range, 53–8000/mL) with fever (temperature 137.57C) but free of symptoms of
severe malaria; high-density uncomplicated malaria, asexual high-density P. falciparum parasitemia (18000 parasites/mL; parasitemia
range, 10,400–174,400/mL) with fever (temperature 137.57C) and free of symptoms of severe malaria; malaria anemia, admitted to
pediatric ward with severe symptoms of malaria, fever (temperature 137.57C), and parasitemia (range, 80–176,800 parasites/mL) with
anemia (hemoglobin levels !8 g/dL) and free of other known infections (30 children had 5–8 g/dL of hemoglobin [moderate malaria
anemia]; 22 had !5 g/dL [severe malaria anemia]); hospital controls, required inpatient management but had no detectable malaria
parasitemia; most had severe lower respiratory tract infections.
Polyclonal goat anti-human tumor necrosis factor (TNF)-a antibody or monoclonal mouse anti-human antibodies for interferon
(IFN)-g, interleukin (IL)-4 (R&D Systems, Minneapolis), and IL-10 (PharMingen, San Diego) were used as capture reagents. Biotinylated monoclonal mouse anti-human IFN-g and IL-4 antibodies (Endogen, Cambridge, MA) were used for IFN-g and IL-4 detection.
Biotinylated rat anti-human IL-10 antibody (PharMingen) was used for IL-10 detection. Monoclonal anti-human TNF-a antibody
(R&D Systems) was used to detect TNF-a levels.
JID 1999;179 (January)
IL-10/TNF-a Ratio and Pathogenesis of Malaria
anemia group. However, this difference was not statistically
significant.
Plasma IL-10–to–TNF-a ratio. Since IL-10 regulates both
the production and function of TNF-a, we hypothesized that
children with a low IL-10–to–TNF-a ratio may be more likely
to have severe disease than children with a higher ratio. Therefore, we determined the individual IL-10–to–TNF-a ratio for
each child and compared the means (table 2). In this study, the
mild disease group comprised children with mild malaria and
high-density uncomplicated malaria; the severe disease group
included children with malaria anemia. The mean IL-10–
to–TNF-a ratio (4.64) was significantly higher (P ! .005 ) in
children with mild disease than in children with severe disease
(mean ratio, 1.77). When we compared the IL-10–to–TNF-a
ratio between the children with moderate (hemoglobin !8 g/
dL to 15 g/dL) and severe malaria anemia (hemoglobin ! 5 g/
dL), the difference was not significant (data not shown).
IFN-g and IL-4 levels. Plasma levels of IFN-g and IL-4
were low in most of the subjects tested, and there was no statistically significant difference between the control groups and
any of the symptomatic groups of children (P 1 .05; table 1).
Discussion
Our hypothesis predicted that higher plasma IL-10 concentrations over TNF-a levels might provide protection against
severe malaria anemia by down-regulating the severe pathologic
effects of TNF-a. This study showed that children in this holoendemic area with mild disease (mild or high-density uncomplicated malaria) had significantly higher IL-10–to–TNF-a ratios than children with severe disease (malaria anemia). This
finding is consistent with our hypothesis that the balance between the Th1 cytokine TNF-a and the Th2 cytokine IL-10 is
critical in the pathogenesis of severe disease in P. falciparum–infected persons who live in areas in which malaria is
endemic.
Consistent with our findings, recent studies from rodent
models have highlighted the significant role IL-10 plays in
down-regulating severe malaria. Linke et al. [11] showed that
IL-10 gene knockout mice with an intrinsic deficiency for IL10 production when infected with Plasmodium chabaudi chabaudi succumb to severe disease and higher mortality than their
heterozygote counterparts or normal mice [11]. An enhanced
Th1 response persisted throughout the course of infection in
the IL-10–deficient mice, while in control mice a Th2 response
was predominant. In another recent study, the exogenous administration of IL-10 to susceptible CBA mice protected them
from Plasmodium berghei–induced cerebral malaria while an in
vivo neutralization of IL-10 in resistant BALB/c mice induced
a neurologic syndrome [12]. Collectively, these findings support
the hypothesis that IL-10 is a critical factor in down-regulating
the pathogenesis of severe malaria.
In previous studies [3–6, 9], the interrelationship between IL-
281
Table 2. Mean interleukin (IL)-10–to–tumor necrosis factor (TNF)a ratios in Kenyan children grouped by mild or severe malaria.
a
Parameter
c
Mean IL-10 to TNF-a ratio
No. of subjects
SD
Median
Minimum value
Maximum value
b
Mild disease
Severe disease
4.64
d
34
6.04
2.69
0
25
1.77
52
2.88
0.45
0
13.7
a
Mild disease group includes data from children with mild malaria and highdensity uncomplicated malaria.
b
Severe disease group represents children with moderate and severe malaria
anemia.
c
IL-10–to–TNF-a ratio was calculated individually for each patient and mean
values determined. Differences in mean ratios were highly significant (P ! .005,
Wilcoxon test).
d
There were 43 children in this group; 9 had no detectable TNF-a and were
excluded from analysis.
10 and TNF-a in severe malaria was not investigated. We addressed this issue using defined clinical groups and showed that
children with mild disease have higher IL-10–to–TNF-a ratios
than children with malaria anemia. However, it remains to be
determined whether the IL-10–to–TNF-a ratio is altered in
children with cerebral malaria compared with malaria anemia.
In the holoendemic area of Kisumu, cerebral malaria is rare.
Therefore, we could not study the IL-10–to–TNF-a ratio in
cerebral malaria patients. However, similar studies in areas with
seasonal transmission of malaria, where cerebral malaria is
common, will be necessary to address this issue.
The in vivo sequence of TNF-a and IL-10 production in
humans after a malaria infection is not clear. Since monocytes
and macrophages produce both cytokines, we speculate that
IL-10 may be produced soon after the production of TNF-a
to regulate the inflammatory activities of TNF-a. TNF-a induces fever, and elevated body temperatures can suppress parasitemia. While such TNF-a–induced mechanisms can serve the
host to control infection, prolonged exposure to TNF-a may
adversely affect the individual by inducing or promoting severe
disease. Indeed, a previous study showed that TNF-a suppresses erythropoiesis, thus contributing to anemia development [13]. Therefore, P. falciparum–infected persons who produce balanced levels of IL-10 to regulate excessive TNF-a
activity may escape from developing severe or moderate malaria
anemia. Recently, it was shown that some of the polymorphism
in the IL-10 gene is associated with decreased IL-10 production
[14]. Further studies are clearly needed to investigate if polymorphism in the IL-10 gene can contribute to pathogenesis of
malaria anemia.
Murine studies have shown that in addition to TNF-a, IFNg also plays a critical role in the pathogenesis of cerebral malaria [15]. Previous human studies have shown that in acute P.
falciparum infections, especially in persons with cerebral malaria, IFN-g production is elevated [3, 5] in addition to TNFa. However, in this study, there was no evidence for the production of IFN-g in any of the clinical malaria groups except
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Othoro et al.
in hospital control children. This finding suggests that this cytokine may not be a critical factor in the development of malaria anemia in holoendemic areas. IL-4 is a regulator of IFNg and TNF-a activities, and earlier studies showed that IL-4
negatively regulates the induction of cerebral malaria in a rodent model [7]. However, the lack of IL-4 production in malaria
patients, as found in this study, suggests that IL-4 may not be
a critical factor in the pathogenesis of malaria anemia.
In conclusion, this study showed that a lower IL-10–to–
TNF-a ratio is associated with malaria anemia in children residing in a holoendemic area. A higher IL-10–to–TNF-a ratio
was associated with mild disease. Our findings suggest that an
imbalance in the IL-10 cytokine regulatory network may contribute to the pathogenesis of malaria anemia.
Acknowledgments
We thank Charles Obonyo for assisting with sample collection; David
Anyona, Simon Kariuki, John Ongecha, and other staff members of
the Kenya Medical Research Institute (KEMRI)–CDC field station for
technical support; study volunteers for participation; and the KEMRI
director for approving the publication of this work.
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