1207
Human Infection with Ascaris lumbricoides Is Associated with a Polarized
Cytokine Response
Philip J. Cooper,1 Martha E. Chico,3 Carlos Sandoval,3
Ivan Espinel,3 Angel Guevara,3 Malcolm W. Kennedy,4
Joseph F. Urban, Jr.,2 George E. Griffin,5
and Thomas B. Nutman1
1
Laboratory of Parasitic Diseases, National Institute of Allergy
and Infectious Diseases, National Institutes of Health, Bethesda,
and 2Immunology and Disease Resistance Laboratory, United States
Department of Agriculture, Beltsville, Maryland; 3Department
of Clinical Investigations, Hospital Vozandes, Quito, Ecuador;
4
Division of Infection and Immunity, Institute of Biomedical and Life
Sciences, University of Glasgow, Glasgow and 5St. George’s Hospital
Medical School, London, United Kingdom
To define the cytokine response to Ascaris lumbricoides infection, the cellular immune response to adult and larval-stage Ascaris antigens in young adults with moderate infection
intensities (n p 73) was compared with that of a group of uninfected control subjects (n p
40). A. lumbricoides–infected subjects had significantly greater lymphoproliferative responses
to adult and larval-stage antigens, compared with uninfected control subjects (P ! .01 ). The
frequencies of parasite antigen–stimulated peripheral blood mononuclear cell (PBMC)–
expressing interleukin (IL)–4 and IL-5 were significantly greater in the infected group (P !
.001), whereas the frequencies of IL-10– and interferon-g–expressing PBMC were similar in
the 2 groups studied. The ratios of Th2 to Th1 cytokine frequencies were significantly elevated
in the infected group, compared with those in uninfected subjects, as was IL-5 protein production by PBMC stimulated with adult (P ! .05 ) and L3/L4 stage (P ! .001) antigens. Analysis
of these data indicates that A. lumbricoides infections in endemic regions are associated with
a highly polarized type 2 cytokine response.
Gastrointestinal helminth infections are estimated to infect
a large proportion of humans in the developing world [1]. The
number of infections due to Ascaris lumbricoides alone is estimated at ∼1.5 billion [2], and, although such infections are
generally chronic and not fatal, they are associated with significant morbidity [2].
Helminth infections, including infections caused by gastrointestinal helminths, are characterized by mast cell hyperplasia,
eosinophilia, and markedly elevated levels of circulating IgE [3,
4]. In the case of human infections with tissue helminths such as
filariae, such immunologic features reflect a prominent and
skewed (absence of interferon [IFN]–g and interleukin [IL]–2)
cytokine response [4]. Few studies have examined human cytokine responses to gastrointestinal helminths, apart from Strongyloides stercoralis infection [5–7], and no studies have investiReceived 17 April 2000; revised 29 June 2000; electronically published 8
September 2000.
The study protocol was approved by the institutional review boards of
the National Institute of Allergy and Infectious Diseases (National Institutes
of Health, Bethesda, MD) and the Hospital Vozandes (Quito, Ecuador).
Informed verbal consent was obtained from the subject or a parent, as
appropriate.
Financial support: Wellcome Trust.
Reprints or correspondence: Dr. Philip J. Cooper, LPD, NIAID, 4 Center
Dr., Rm. 4/126, MSC 0425, NIH, Bethesda, MD 20892-0425 (pc102d@
hotmail.com).
The Journal of Infectious Diseases 2000; 182:1207–13
q 2000 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2000/18204-0026$02.00
gated parasite-specific cellular response to A. lumbricoides
infection in endemic populations; however, numerous studies in
experimental animal model systems have demonstrated the prominence of type 2 cytokine responses to gastrointestinal helminths
and the importance of IL-4–, IL-5–, IL-9–, and IL-13–associated
immune pathways in mediating parasite expulsion and resistance
to infection [8, 9].
Experimental animal [10–12] and human [13, 14] studies have
demonstrated that concurrent helminthiases may modulate the
immune responses to bystander antigens (e.g., vaccines). Because geohelminth infections such as ascariasis are highly prevalent in the less affluent regions of the world, it would be important to know the phenotype of the cellular response to A.
lumbricoides, because such geohelminths may have important
immune-modulating effects [15]. Such baseline studies would
also be important in the investigation of the mechanisms of
protective immunity against A. lumbricoides in humans.
We investigated the cytokine and antibody response to adult
and larval stages of Ascaris in infected young adults living in
an endemic area of Ecuador and compared these responses with
those of uninfected control subjects living in the same region.
In so doing, we demonstrated the polarized (and predominantly
parasite antigen–specific) systemic cellular responses associated
with intestinal helminthiasis.
Subjects and Methods
Study population. The study was conducted in Canton Pedernales in Manabi Province in Ecuador. Two study groups were
1208
Cooper et al.
recruited: teens and adults infected with A. lumbricoides, living in
rural communities in Canton Pedernales in the Manabi Province
of Ecuador that are known from previous surveys to be endemic
for A. lumbricoides; and uninfected volunteers from the town of
Pedernales. The socioeconomic circumstances of subjects in the
uninfected group were superior (better diet and access to piped
water and sanitary facilities) to those of the infected group (subsistence agriculturists living in communities with no access to clean
water or sanitary facilities). The selection criteria into the infected
group were an age of 13–20 years and the presence of 11000 eggs
per gram (epg) of A. lumbricoides on 2 sequential stool samples.
Subject sampling. Blood samples (20 mL) were collected. All
subjects were screened for the presence of geohelminth eggs or
larvae in 2 different stool samples with the modified Kato-Katz
[16] and formol-gasoline concentration [16] techniques. Nutritional
status was assessed in all subjects in the A. lumbricoides–infected
group by measurement of height, weight, triceps skinfold thickness,
and mid–upper arm circumference; all measurements were performed in duplicate. All subjects were offered treatment with 400
mg of albendazole.
Antigen preparation. Antigen for culture included pokeweed
mitogen (PWM; Gibco BRL, Gaithersburg, MD) and the nonparasite antigen, purified protein derivative (PPD; Statens Serum
Institute, Copenhagen). Ascaris antigens were prepared as follows.
A PBS-soluble extract (ASC) was prepared by lyophilization and
homogenization of adult A. lumbricoides worms collected during
a previous survey in the same area. The homogenate was resuspended in ice-cold PBS with a protease inhibitor cocktail (Sigma,
St. Louis), agitated overnight at 47C, and then centrifuged at 20,000
g for 30 min. The supernatant was passed through a low protein–
binding 0.2 mm filter, and the protein content was estimated (Pierce,
Rockford, IL). An Ascaris suum–derived antigen was prepared
from Ascaris pseudocoelomic fluid (ABF), as described elsewhere
[17]. Excretory/secretory antigens from A. suum larvae developing
from L2 to L3 (L2/L3 antigen) or from L3 to L4 (L3/L4 antigen)
in culture were prepared, as described elsewhere [18]. Excretory/
secretory antigens from cultured Trichuris suis adult worms were
prepared as described elsewhere [19]. Antigen preparations were
tested for the presence of bacterial endotoxin with the Limulus
lysate assay (BioWhittaker, Walkersville, MD). Endotoxin levels
were undetectable in all samples.
Peripheral blood mononuclear cell (PBMC) proliferation assays. PBMC were isolated by centrifugation on lymphocyte separation medium (Organon Teknika, Durham, NC). The cells were
washed and plated onto 96-well round-bottomed tissue culture
plates (Corning, Cambridge, MA) at 2 3 10 5 cells/mL in a volume
of 200 mL of RPMI 1640 (BioWhittaker) supplemented with 10%
AB serum (BioWhittaker), 0.08 mg/mL gentamicin (Life Technologies, Gibco BRL), and 2 mM L-glutamine (Biofluids, Rockville,
MD). The PBMC were stimulated with purified PPD at 10 mg/mL,
PWM at a dilution of 1:200, and Ascaris and Trichuris antigens,
as described above, at concentrations between 1 and 5 mg/mL, as
indicated by preliminary concentration titration experiments. The
plates were incubated at 377C and at 5% CO2 for 5 days, pulsed
with 1 mCi of tritiated thymidine (DuPont, Boston) for 16 h, and
were harvested onto filter mats for subsequent scintillation counting. Results are expressed as geometric mean stimulation indexes
(SI).
JID 2000;182 (October)
Cytokine ELISPOT. Immulon 4 96-well microtiter plates (Dynex Technologies, Chantilly, VA) were coated overnight at 47C with
the following anti–human monoclonal antibodies in PBS: IL-4
(clone 39D10; Pharmingen, San Diego), IL-5 (clone TRFK5;
DNAX, Palo Alto, CA), IL-10 (clone 9D7; Pharmingen), and IFNg (clone M700A; Endogen, Woburn, MA). After washing with
PBS-Tween 0.05%, the plates were blocked with 5% bovine serum
albumin/PBS/Tween 0.05% for 2 h at room temperature. PBMC
were added to the plates at a concentration of 2.5 3 10 6 cells/mL
(200 mL per well) in culture medium (RPMI 1640; BioWhittaker)
supplemented with 10% fetal calf serum (Atlanta Biologicals, Norcross, GA), 0.08 mg/mL gentamicin (Life Technologies, Gibco
BRL), and 2 mM L-glutamine (Biofluids). Cultures were performed
in the presence or absence of ASC antigen (10 mg/mL). The plates
were incubated at 377C and at 5% CO2 for 18 h (IFN-g and
IL-10) or 48 h (IL-4 and IL-5). After washing, the biotinylated
anti–human cytokine antibodies were added (anti–IL-4 25D2, anti–
IL-5 JES1, and anti–IL-10 JES1 [Pharmingen] and polyclonal rabbit anti–human IFN-g [Endogen]), and the plates were incubated
overnight at 47C. The plates were washed, alkaline phosphatase–conjugated streptavidin (Jackson Immunoresearch, West
Grove, PA) was added, and the plates were incubated for 1 h at
room temperature, followed by the addition of 5-bromo-4-chloro3-indolyl phosphate substrate (Sigma)/2.5% agarose (Gibco BRL).
The spots were counted after 24 h with a dissecting microscope.
ELISPOT data were expressed as the absolute difference between
the number of spots detected in antigen-stimulated cultures and
those detected in cultures with medium alone. To determine the
potential modulatory role of IL-10 in cytokine expression, 10 mg/
mL of an IL-10–neutralizing antibody (clone 19F1; Pharmingen)
or rat IgG2a antibody (Pharmingen) was added to some of the
cultures.
Cytokine ELISA. Supernatant fluids were harvested from the
same cultures used for proliferation assays at 24 h (IL-2, IL-4, and
IL-10) and 5 days (IL-5 and IFN-g). Cytokine ELISA for IL-2,
IFN-g, IL-4, IL-5, and IL-10 were performed, as described elsewhere [5]. Cytokine levels were expressed in picograms per milliliter
as the absolute difference between protein levels in antigen-stimulated cultures and control medium cultures.
Detection of A. lumbricoides–specific antibodies. Levels of A.
lumbricoides–specific total IgG, IgG1–4, IgA, IgM, and IgE were
determined in plasma samples, as described elsewhere [20], with
the following modification: ELISA plates (25805; Corning) were
coated with the PBS-soluble A. lumbricoides adult antigen (ASC)
at 2.5 mg/mL (total IgG and IgG1–3) or 10 mg/mL (IgG4 and IgE)
in carbonate buffer (0.045 M NaHCO3–0.02 M Na2CO3 at pH 9.6).
For analysis of IgA and IgM, plates were coated with A. lumbricoides antigen at 20 and 2.5 mg/mL, respectively. After incubation
with appropriately diluted plasma samples, the plates were incubated with alkaline phosphatase conjugated anti–human IgA
(Sigma) or anti–human IgM (Sigma). Plate development was performed with p-nitrophenyl phosphate in sodium carbonate buffer.
Plates were read on a microtiter plate reader, and unknown values
were interpolated from standard curves derived from pools of positive serum samples, with antibody levels expressed as arbitrary
units. Cutoff levels to determine positive- versus negative-reacting
serum samples for each isotype and IgG subclass were calculated
as the mean 1 3 SD of a pool of serum samples derived from 15
JID 2000;182 (October)
Table 1.
Cytokines and Human Ascaris Infection
1209
Subject characteristics in Ascaris lumbricoides–infected groups and uninfected groups.
A. lumbricoides
Trichuris trichiura
A. lumbricoides
Age, years
Sex,
M/F
Prevalence, %
Intensity, epg
Prevalence, %
Intensity, epg
Uninfected (n p 40)
Infected (n p 73)
32 (14–66)
14 (12–17)
13/27
38/35
0
100
0
6728 (1278–61,200)
0
71.2
0
74 (0–22,093)
NOTE. Data are mean (range), except where noted. No other geohelminth infections were detected. M, male; F,
female; epg, eggs per gram of stool.
healthy uninfected volunteers living in Quito. Appropriate cutoff
values were subtracted from each test serum.
Statistical methods. Data from PBMC proliferation, ELISPOT,
and antibody assays were normalized by loge transformation and
group means compared with Student’s t test. Cytokine supernatant
data were highly skewed and were analyzed with the Mann-Whitney
U test. Comparison of proportions was performed with the x2 test.
Bivariate analysis was done by calculation of Spearman’s rank correlation coefficients. Because of the large number of tests performed,
significance was taken at the 1% level, to reduce the likelihood of
type I errors. Otherwise, statistical significance was inferred by P !
.05. All the described assays were not performed on every subject,
and the analyses were performed on the maximal subject data available for each of the 2 study groups.
Results
Subject characteristics. The demographic and parasitologic
characteristics of the study subjects are shown in table 1. Of the
113 subjects recruited into the study, 73 were infected with A.
lumbricoides and 40 were not. All those recruited from rural
communities had significant infections with A. lumbricoides (geometric mean, 6728 epg). The prevalence of Trichuris trichiura in
the Ascaris–infected group was high (71.2%), but the mean infection intensity was low (74 epg). Hookworm and S. stercoralis
infections were absent in this study group. No helminth infections
were detected among the uninfected subjects. The mean age of
the uninfected group (32 years) was greater than that of the
infected group (14 years), but otherwise there was no difference,
either immunological or anthropometric, between those !20
years old and those 120 years old. No anthropometric evidence
of malnutrition was observed in the group infected with A.
lumbricoides.
PBMC proliferation. PBMC proliferation to adult and larval
stage Ascaris antigens are shown in figure 1. There were no significant differences in proliferative responses to medium alone,
PWM, or PPD between the 2 groups; however, proliferative responses to adult-derived worm antigens (ASC; uninfected SI, 0.9
[95% confidence interval {CI}, 0.7–1.1] vs. infected SI, 1.9 [95%
CI, 1.5–2.5]) and ABF (uninfected SI, 0.8 [95% CI, 0.6–1.0] vs.
infected SI, 1.3 [95% CI, 1.2–1.6]) were significantly greater in
the A. lumbricoides infection group. Similarly, responses to larval
molt-stage excretory/secretory antigens L2/L3 (uninfected SI, 1.3
[95% CI, 0.9–2.0] vs. infected SI, 3.9 [95% CI, 2.9–5.1]) and L3/
L4 (uninfected SI, 0.8 [95% CI. 0.6–1.1] vs. infected SI, 2.0 [95%
CI, 1.6–2.4]) were significantly greater in the infected group. Re-
sponses to Ascaris antigens were analyzed also by comparing the
frequencies of responders (e.g., SI 1 2 ) in each group: significantly
greater response rates in the infection group were seen to ASC
(0% response rate uninfected vs. 39% infected; P ! .01 ), ABF (8%
vs. 38%; P ! .05), L2/L3 (25% vs. 69%; P ! .01), and L3/L4 (0%
vs. 42%; P ! .01) antigen preparations. Proliferative responses to
the Trichuris-derived antigen were negligible in both groups (data
not shown). Because the uninfected group included older subjects
than the infected group, the former group was stratified into 2
groups (120 years old and !20 years old) in the analysis: there
were no significant age-dependent differences in proliferative responses to adult or larval-stage antigens or in the frequencies of
responders to these antigens (data not shown).
Frequency of cytokine-secreting PBMC.
Previous studies
have demonstrated a direct relationship between the frequency
of cytokine-secreting cells and cytokine protein levels in culture
supernatants [5], and ELISPOT may be a more sensitive method
for the detection of antigen-specific cytokine expression, such as
IL-4, which is not usually detectable in culture supernatants in
Figure 1.
Proliferation of peripheral blood mononuclear cells
(PBMC) in the Ascaris lumbricoides–infected (hatched columns) and
–uninfected (open columns) groups. PBMC were stimulated with pokeweed mitogen (PWM), purified protein derivative (PPD), A. lumbricoides adult antigen (ASC), pseudocoelomic fluid antigen from Ascaris
suum adults (ABF), and L2/L3 and L3/L4 excretory/secretory antigens
from A. suum. Geometric mean stimulation indexes (SI) and 95% confidence intervals are shown. **P ! .01. ***P ! .001.
1210
Cooper et al.
the absence of mitogenic stimulation. The frequencies of cytokine-secreting PBMC stimulated with ASC antigen are shown in
figure 2. Significantly higher frequencies of cells secreting IL-4
and IL-5 were observed in A. lumbricoides–infected subjects, compared with uninfected control subjects. The frequencies of IFNg– and IL-10–secreting cells were similar and very low in both
study groups. When ratios of Th2 to Th1 cytokine frequencies
were calculated, significantly greater Th2/Th1 ratios were observed in the infected group (IL-4/IFN-g uninfected 0.7 [95% CI,
0.5–1.1] vs. infected 7.6 [95% CI, 5.1–11.4], P ! .001; IL-5/IFNg uninfected 0.6 [95% CI, 0.4–0.9] vs. infected 9.6 [95% CI,
6.0–15.4], P ! .001). Addition of a neutralizing antibody to IL10 did not affect the frequencies of PBMC expressing IFN-g,
IL-4, or IL-5 (data not shown). The frequencies of PBMC secreting IL-4 and IL-5 were significantly correlated (r p .893,
P ! .001). The uninfected study group was stratified into 2 age
groups in the analysis (120 years old and !20 years old). There
were no significant age-dependent differences in the frequencies
of cytokine-expressing PBMC for all the cytokines analyzed (data
not shown).
Cytokine protein production. Cytokine protein production
(IFN-g, IL-2, IL-10, and IL-5) to mitogen, PPD, and parasite
antigens is shown in table 2. IL-4 was not detectable in antigenstimulated culture supernatants (data not shown). There were
no significant differences between the 2 groups in production
of IL-2, IL-10, and IFN-g to mitogen, nonparasite antigens,
or larval- or adult-stage A. lumbricoides antigens. In contrast,
production of IL-5 (table 2) was markedly elevated in the infection group to mitogen, the nonparasite antigen PPD, A.
lumbricoides adult-derived antigens (ASC and ABF), and A.
lumbricoides L3/L4 antigen. Similarly, the proportions of subjects with significant net cytokine production of IL-5 (e.g., 110
pg/mL) were significantly greater also in the infected group
(PPD uninfected 4% vs. infected 19%, P ! .05; PWM uninfected
71% vs. infected 98%, P ! .001; ABF uninfected 7% vs. infected
44%, P ! .001; L3/L4 uninfected 21% vs. infected 64%, P !
.001). Of interest are the elevated levels of IL-5 detected in
response to L2/L3 antigen in both groups. Production of IFNg, IL-5, and IL-10 by PBMC stimulated with Trichiura antigen
was negligible in both groups (data not shown). Among the
infected group, the frequency of PBMC stimulated with A.
lumbricoides antigen (ASC) that secreted IL-5 was significantly
associated with the levels of IL-5 protein production in supernatants from the same subjects (r p .433; P ! .001). Similar
associations were not observed for IFN-g (r p 2.041) and IL10 (r p .204). The uninfected study group was stratified into 2
groups in the analysis of cytokine data (120 years old or !20
years old): no significant differences between the 2 stratifications were observed.
A. lumbricoides–specific antibody levels. There were significant differences in parasite-specific antibody levels between
the A. lumbricoides–infected groups and the uninfected groups
(figure 3) in levels of total IgG, IgG1, IgG2, IgG3, IgM, and
JID 2000;182 (October)
Figure 2.
Frequencies of peripheral blood mononuclear cells
(PBMC) secreting interleukin (IL)–10, interferon (IFN)–g, IL-4, or IL5 in the Ascaris lumbricoides–infected (hatched columns) and –uninfected (open columns) groups. Cultures were performed in the presence
of absence of A. lumbricoides adult antigen, and cellular frequencies
were assessed by ELISPOT. Findings are expressed as the frequency
of cytokine-positive cells per million PBMC. Geometric mean ratios
and 95% confidence intervals are shown for each group. ***P ! .001.
IgE. Levels of parasite-specific IgE were negligible in the uninfected group, and levels of IgG4 were negligible in both
groups. It is of interest that levels of parasite-specific IgA were
greater in the uninfected group. The uninfected study group
was stratified into 2 age groups, as noted above: there were no
significant intergroup differences in antibody isotype levels. The
associations between levels of parasite-specific immunoglobulin
isotypes and IgG subclasses and infection intensity with A.
lumbricoides were also examined. A number of significant correlations were seen. Levels of total IgG (r p .336; P ! .001),
IgG1 (r p .399; P ! .001), and IgG2 (r p .254; P ! .01) were
positively correlated with A. lumbricoides egg counts; total IgG
levels were correlated with levels of IgG1 (r p .801; P ! .001),
IgG2 (r p .687; P ! .001), and IgG3 (r p .380; P ! .001); and
IgE levels were correlated with total IgG (r p .373; P ! .001)
and IgG1 (r p .391; P ! .001).
Discussion
Human helminth infections induce strong and often polarized
cytokine responses (IL-4 and IL-5). This holds true for tissue
helminth parasites such as filariae [5, 6] and schistosomes [21].
Although geohelminth infections including A. lumbricoides are
associated with high levels of circulating IgE [22] and a peripheral
eosinophilia [23] that are known to be induced by IL-4 and IL5, there are no published reports of the cytokine phenotypes
associated with these infections in endemic communities. In this
study, we demonstrated that young adults infected with A. lumbricoides both produce and express greater levels of the type 2
JID 2000;182 (October)
Cytokines and Human Ascaris Infection
1211
Table 2. Production of interleukin (IL)–10, interferon (IFN)–g, and IL-5 in peripheral blood mononuclear cell culture supernatants from the
Ascaris lumbricoides–infected and –uninfected groups.
PPD
Cytokine
IL-2
Uninfected
Infected
IL-10
Uninfected
Infected
IFN-g
Uninfected
Infected
IL-5
Uninfected
Infected
PWM
ASC
ABF
L2/L3
L3/L4
Mean (range)
%
Mean (range)
%
Mean (range)
%
Mean (range)
%
Mean (range)
%
Mean (range)
%
1.9 (0–212.5)
2.1 (0–315.4)
23
21
222.6 (50.4–661)
253.6 (43.6–2653.1)
100
100
1.3 (0–538.2)
1.1 (0–117.6)
17
15
1.2 (0–217.5)
2.1 (0–323.5)
20
29
4.2 (0–358.8)
4.5 (0–134.3)
40
42
1.2 (0–56.5)
3.2 (0–737.6)
17
32
2.5 (0–89.8)
1.4 (0–57.7)
33
16
35.6 (0–220.7)
46.7 (0–291.1)
93
95
2.3 (0–65.4)
3.5 (0–346.1)
30
33
1.3 (0–48.2)
1.0 (0–79.1)
20
16
1.3 (0–50.2)
1.1 (0–124.2)
20
19
11.6 (0–100.1)
11.1 (0–1331.4)
63
60
46.9 (0–1226.7)
20.8 (0–3463.7)
71
60
1825.4 (3.0–9961.0)
1151.8 (0–9961.0)
96
84
2.4 (0–171.4)
1.9 (0–107.5)
36
23
1.8 (0–66.1)
0.9 (0–44.4)
29
11
4.0 (0–53.4)
5.1 (0–238.3)
40
40
3.3 (0–68.8)
5.1 (0–238.3)
39
42
1.2 (0–30.1)
a
2.5 (0–596.3)
4
a
19
21.2 (0–192.5)
c
168.5 (0–897.9)
71
c
98
1.8 (0–32.9)
a
4.6 (0–224.8)
18
37
1.5 (0–39.3)
b
4.8 (0–100.2)
7
c
44
6.7 (0–179.1)
14.3 (0–507.3)
50
60
2.5 (0–87.1)
b
9.1 (0–176.4)
21
c
64
NOTE. Cultures were stimulated with purified protein derivative (PPD), pokeweed mitogen (PWM), PBS-soluble A. lumbricoides adult antigen (ASC), Ascaris
pseudocoelomic fluid (ABF), and L2/L3 and L3/L4 excretory/secretory antigens from Ascaris suum. Shown are geometric mean (range) and the percentage of subjects
producing 110 pg/mL of cytokine.
a
P ! .05.
b
P ! .01.
c
P ! .001.
cytokines IL-4 and IL-5 than do uninfected endemic control
subjects. Although IL-4 and IL-5 cytokine expression was predominant, there was no evidence of impaired IFN-g expression
in the infected group, compared with the uninfected control
group, which may indicate a mixed Th1/Th2 response, a response
that is analogous to that seen early in infection in experimental
animals after tissue helminth infections.
Concurrent helminth infections may alter the immune response to nonparasite antigens, as is suggested by the findings
of experimental animal models of helminth infections [10–12]
and human studies of the impact of concurrent tissue helminth
infections on postvaccination immune responses [13, 14, 20].
Helminth infections may alter the immune response to nonparasite antigens through bystander effects: an IL-4–rich environment at the site of immune interactions between helminth
antigens and immune cells would create a Th2-polarizing environment for nonparasite antigens presented to the immune
system in the immune compartment. Geohelminth infections
might affect the response to nonparasite antigens presented at
the mucosal surface, and, in the case of geohelminth infections
with a pulmonary phase of larval migration, could affect responses in both the pulmonary and intestinal mucosa. For example, the pulmonary migrations of Ascaris larvae would create
a highly polarized Th2 immune environment in the lung mucosa, which might enhance Th2 cytokine production to nonparasite antigens (e.g., aeroallergens or pathogens such as Mycobacterium tuberculosis) at the same site, enhancing allergic
reactivity to environmental allergens and thus contributing to
the enhanced allergic pulmonary reactivity that has been associated with geohelminth infections [15, 24]. Two observations
in this study would support such a hypothesis: significantly
greater levels of IL-5 were secreted by the infection group in
response to L3/L4 Ascaris antigens (e.g., lung-stage parasites);
and infected subjects secreted significantly greater amounts of
IL-5 after stimulation with PPD. An inappropriate immune
response (i.e., Th2 cytokine secretion) among individuals with
significant exposure to A. lumbricoides might have an impact
on susceptibility to pulmonary infections with M. tuberculosis
and merits further investigation.
A potential confounding factor between the 2 study groups
was the difference in mean age, with a mean age in the infected
group of 14 years, compared with 32 years in the uninfected
control group. Age, which may be a surrogate for exposure in
endemic areas, may influence the immune response to helminth
antigens, although this is only likely to occur in areas with
significant transmission. In fact, within the uninfected group,
Figure 3.
Levels of Ascaris lumbricoides–specific antibodies in
plasma from the A. lumbricoides–infected (hatched columns) and –uninfected (open columns) groups. Geometric mean antibody levels (arbitrary units) and 95% confidence intervals are shown for IgG (TIgG)
and each of the other isotypes. *P ! .05. **P ! .001.
1212
Cooper et al.
there were no age-dependent differences in the various parameters of the antibody and cellular responses measured in this
study. Low-level exposure in the control group was indicated
by the presence of A. lumbricoides–specific IgG, IgM, and IgA
antibodies in almost all subjects and by the measurable parasitespecific cellular response, particularly to L2/L3 antigens.
The objective of this study was to define the specific cellular
responses to A. lumbricoides in a group of young adults with
significant parasite burdens, living in endemic communities. An
uninfected control group was chosen from a nearby town, where
transmission of A. lumbricoides was likely to be of low intensity.
Uninfected individuals from endemic communities with highintensity transmission cannot be guaranteed to be free of infection for two reasons. First, such individuals might have lowlevel infection below the limits of detection of the Kato-Katz
and stool concentration tests. Second, anthelminthic treatments
(both conventional and traditional) are widely available and
widely used even in rural communities, and the absence of
infection may merely indicate recent treatment. Truly infectionfree individuals in endemic communities may have either more
effective protective responses (putatively immune) [17] or low
exposure. Therefore, the selection of a control uninfected group
from highly endemic communities might result in a group with
highly heterogeneous immune responses and would not have
provided the most appropriate control group for this study.
Most of the Ascaris-derived antigenic preparations used in
this study were derived from A. suum. A. lumbricoides and A.
suum are very closely related and are capable of cross-infecting
both humans and pigs [25]. In fact, it is difficult to distinguish
the 2 species in epidemiologic surveys because the eggs are
morphologically indistinguishable; for many years, it was controversial whether the 2 species were distinct. Although the 2
species can be differentiated by use of rDNA probes [26], and
recent evidence suggests that the 2 species represent reproductively isolated populations [26], it is very likely that the 2 species
possess sufficient antigenic homology [27] to be useful in investigating stage-specific cellular immune responses in humans.
We were not able to detect a measurable systemic cellular
response to Trichuris antigen. The reasons for a lack of responsiveness to this excretory/secretory antigen of T. suis may
include the lack of immunologic cross-reactivity between T. suis
and T. trichiura; the absence of significant systemic stimulation
by Trichuris infection, which does not have an invasive migratory stage; and the low infection intensity in the A. lumbricoides
infection group (mean, 74 epg) may have not been sufficient to
stimulate a measurable systemic response detectable in circulating parasite antigen-specific T cells. Trichuris antigen-specific
T cells are present in significant numbers in experimental animal
models by assay of spleen and mesenteric lymph node lymphocyte populations, but such animals generally receive a high
infective dose [9].
In conclusion, PBMC from young adults living in an A.
lumbricoides–endemic region in Ecuador produced a highly po-
JID 2000;182 (October)
larized Th2 cytokine response to Ascaris antigens. For comparison, we chose an uninfected control group from an endemic
community in which there is less intense parasite transmission.
The uninfected group, however, did demonstrate detectable antiparasite immunity, with elevated levels of specific antibodies
and cellular responses directed against larval-stage antigens.
These data provide the first detailed description of the phenotype of cellular immunity to a human geohelminth parasite in
an area where such parasites are endemic.
Acknowledgments
We thank the community representatives and schoolteachers in the
study communities in Canton Pedernales (Ecuador) for their cooperation. We thank also the following for their assistance in the completion
of this study: Moises Botta (Chief Medical Officer, Canton Pedernales);
Marcelo Aguilar (National Director of Health, Ministry of Public
Health, Quito); and Ronald Guderian (Hospital Vozandes, Quito). The
assistance of Kenneth Farr (US Agency for International Development,
Quito) is gratefully acknowledged, as is the editorial assistance of
Brenda Rae Marshall (National Institutes of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, MD).
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