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Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and
Biomedical Research Institute, Rockville, MD 20852
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, TNF-
, and inducible NO and the
formation of nonfibrotic granulomas. Elevated serum aspartate
transaminase levels confirmed that mortality was in part attributable
to acute hepatotoxicity. In contrast, the Th2-polarized
IL-10/IL-12-deficient mice developed a progressive wasting disease that
correlated with increased hepatic fibrosis, formation of large
eosinophil-rich granulomas, a 10-fold increase in IL-4 and IL-13, and
significant mortality during the chronic stages of infection.
Surprisingly, IL-10-deficient mice displayed pathological features that
were characteristic of both extremes, while wild-type mice developed
relatively successful long term chronic infections. These data
demonstrate that IL-10 significantly suppresses type 1 and type 2
cytokine development in IL-4- and IL-12-deficient mice, respectively,
thereby impeding the development of severe egg-induced pathology in the
single cytokine-deficient animals. Together, these findings reveal the
central regulatory role of IL-10 in the pathogenesis of schistosomiasis
and illustrate that excessive type 1 and type 2 cytokine responses
trigger distinct, but equally detrimental, forms of pathology following
infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(7), sequestration
of parasite eggs by eosinophil-enriched granulomas, and development of
tissue fibrosis (8). The chronic granulomatous response
and resultant fibrosis can eventually lead to portal hypertension,
establishment of portal-systemic shunts, intestinal bleeding, and
ultimately death. Thus, morbidity and mortality in chronic murine
schistosomiasis were hypothesized to develop as a direct consequence of
the egg-induced Th2-type response (9).
However, recent studies have provided compelling evidence that
challenges the link between Th2-associated immune responses and disease
development during schistosomiasis (10, 11). For example,
Brunet et al. (10) reported that schistosome-infected
IL-4-deficient mice were incapable of developing effective Th2
responses and consequently displayed enhanced morbidity and mortality.
Pathology in these mice was attributed to increased production of
proinflammatory mediators. Fallon and Dunne (11) also
documented enhanced mortality in infected mice that had been previously
tolerized to egg Ags. Similar to the IL-4-deficient mice, the
egg-tolerized animals displayed decreased Th2 and increased
Th1-associated responses following infection. These observations are
thus comparable to those found in infected SCID mice, which also fail
to develop Th2 responses. Here, as observed in the egg-tolerized or
IL-4-deficient mice, morbidity and mortality correlated with the
expression of IFN- and TNF- (12). Therefore, these
reports suggest that Th2 immune responses in schistosomiasis are, in
fact, host protective. Interestingly, this hypothesis is supported by
recent studies of patients exhibiting the hepatosplenic form of
schistosomiasis. Here, high levels of IFN-, TNF-, soluble TNF-
receptors, and ICAM-1 and low levels of IL-5 were detected in infected
individuals exhibiting severe hepatosplenomegaly (13).
Together, mouse and human studies question whether severe morbidity and
mortality in schistosomiasis are attributable to the development of
polarized Th2- or Th1-type immune responses (14).
Recently, we demonstrated that IL-10 is critical for establishing polarized egg-specific Th cell responses in vivo. IL-10-deficient mice challenged i.v. with schistosome eggs (15) or infected with Schistosoma mansoni (16) developed a completely nonpolarized, codominant Th1/Th2-type immune response. Interestingly, the mixed response resulted in a marked increase in the size of egg-induced hepatic granulomas, particularly at the acute stage of infection (16). Nevertheless, it was unclear whether the exacerbated inflammatory response in IL-10-deficient mice was attributable to changes in Th1- or Th2-type cytokine expression. Recently, we crossed IL-10-deficient mice with IL-4- and IL-12-deficient animals to generate double cytokine-deficient mice that develop highly polarized Th1- and Th2-type responses, respectively (17). Thus, the double cytokine-deficient animals provided unique tools to directly compare the contributions of polarized Th1- and Th2-type cytokine responses to the pathogenesis of schistosomiasis in mice that are otherwise genetically matched. Moreover, they provided an excellent system to formally define the role of IL-10, which clearly exhibits significant immunoregulatory activity in both murine and human schistosomiasis (18, 19, 20, 21, 22, 23).
In this study five distinct single or double cytokine-deficient mouse lines were infected with S. mansoni, and the effects on disease development were compared with those in wild-type (WT)2 animals. The results from this study clearly show that the development of extreme Th1, Th2, or mixed Th1/Th2 cytokine-producing phenotypes is highly detrimental during schistosome infection. Nevertheless, the pathological consequences associated with each response are distinct and develop at different times following infection. These findings reveal the central regulatory role of IL-10 to the pathogenesis of schistosomiasis and demonstrate that the maintenance of IL-10 expression during acute and chronic schistosome infection is critical for host survival. Moreover, they clarify the controversial roles of Th1/Th2-type cytokine responses to the pathogenesis of schistosomiasis (9, 24) and illustrate that an imbalance in either Th1- or Th2-type cytokine expression can contribute to morbidity during infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The double cytokine-deficient mice (C57BL/6) used in this study were previously described (17). All mice were obtained from Taconic Farms (Germantown, PA) and were between 6 and 8 wk of age at the start of each study. All mice were housed in a National Institutes of Health American Association for the Accreditation of Laboratory Animal Care-approved animal facility and were fed an enriched diet (NIH-07) fortified with vitamins and minerals as specified by the National Institutes of Health veterinary staff. Cercariae of a Puerto Rican strain of S. mansoni (Naval Medical Research Institute) were obtained from infected Biomphalaria glabrata snails (Biomedical Research Institute, Rockville, MD). Soluble egg Ag preparation (SEA) was derived from homogenized eggs as previously described (25).
Infections and weight measurements
Mice were percutaneously exposed to S. mansoni
(Puerto Rican strain) by immersion of their tails in water containing
20–30 parasites for 40 min. Weight measurements were taken every
week starting 5 wk postinfection and continued until wk 18
postinfection. The change in weight was expressed as the mean weight at
each time point subtracted from the initial weight at 5 wk
postinfection and expressed as weight gain or weight loss (grams).
Lymphocyte culture, nitrite measurement, and cytokine assays
For in vitro cytokine measurements, mesenteric lymph nodes (MLN)
were removed aseptically 8 wk after infection, and single-cell
suspensions were prepared. Mesenteric nodes were assayed individually,
and cells were plated in 24-well tissue culture plates at a final
concentration of 3 x 106 cells/ml in RPMI
supplemented with 2 mM glutamine, 25 mM HEPES, 10% FCS, 50 µM 2-ME,
penicillin, and streptomycin. Spleens were pooled in each group, and
cells were plated at a final concentration of 4 x
106 cells/ml. Cultures were incubated at 37°C
in an atmosphere of 5% CO2. Cells were
stimulated with Con A (5 µg/ml), SEA (20 µg/ml), or medium alone.
Supernatant fluids were harvested at 72 h and assayed for cytokine
activity. IFN- and IL-5 levels were measured by specific two-site
ELISA as previously described (25). IL-4 levels were
determined using DUOSET reagents supplied by Genzyme (Cambridge, MA),
and IL-13 levels were measured using a capture ELISA kit supplied by
R&D Systems (Minneapolis, MN) following the manufacturer’s
instructions. Cytokine levels were calculated using standard curves
constructed with recombinant murine cytokines. NO production by
cultured spleen cells was assayed by the Greiss reaction from 50 µl
of supernatant collected after 72 h of culture.
Measurement of SEA-specific Ab responses
For assessment of serum Ig levels, sera were collected at 8 wk postinfection. Immulon 4 (Dynatech, Chantilly, VA) microtiter plates were coated overnight at 4°C with SEA (1 µg in 50 µl/well) diluted in PBS. Plates were blocked with 200 µl of 5% nonfat dry milk/PBS for 2 h at 37°C. The blocking solution was aspirated, and the wells were washed six times with PBS/0.05% Tween-20 (Sigma). Individual mouse serum was serially diluted 1/100 to 1/102,500 in 1% BSA/PBS, and 50 µl was added to appropriate wells. Plates were incubated at 37°C for 90 min and then washed six times with PBS/0.05% Tween-20. Fifty microliters of isotype-specific HRP-conjugated rabbit anti-mouse Abs in 1% BSA/PBS diluted at 1/1,000 (measurement of IgG1 and IgG2b; Zymed, San Francisco, CA) were added to the wells and incubated at 37°C for 2 h. Wells were again washed six times with PBS/0.05% Tween 20, 100 µl of 2,2'-azino-di-(3-ethyl-benzthiazoline sulfonate:H2O2 substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added, and the reactions were developed in the dark at room temperature for 20–30 min. Absorbance at 405 nm was determined using a Vmax Kinetic Microplate Reader (Molecular Devices, Palo Alto, CA). Specific SEA-isotype titers were calculated by the product of absorbance and the reciprocal of the serum dilution from an average of two points in the linear portion of the dilution curve.
Total serum IgE Abs were quantitated by ELISA using a protocol provided by PharMingen (San Diego, CA). Briefly, plates were coated with anti-mouse IgE capture mAb from clone R35-72 in 0.1 M NaHCO3 (pH 8.2) overnight at 4°C. The secondary mAb was a biotinylated anti-mouse IgE from clone R35-92, and the streptavidin-peroxidase reagent was diluted 1/1000 in 1% BSA/PBS. A purified mouse IgE from clone IgE-2 (PharMingen) was used as the control standard.
Histopathology and fibrosis measurement
The collagen content of the liver, determined as hydroxyproline, was measured as described previously (26). Approximately half the liver was fixed in Bouin-Hollande solution, and histologic sections were processed and stained with Giemsa (Histo-Path of America, Clinton, MD). The diameters and eosinophil contents of granulomas (30/mouse) surrounding single, mature, and viable eggs were measured using an ocular micrometer, and the volume of each granuloma was calculated assuming a spherical shape. Eggs in the liver and intestines were counted separately after digestion in 4% KOH at 37°C (26).
RT-PCR detection of cytokine mRNAs
Two 25-mg portions of each liver were combined and homogenized
in 1 ml of RNA STAT-60 using a tissue Polytron (Omni, Waterbury, CT),
and total RNA was isolated as recommended by the manufacturer. The RNA
was resuspended in diethylpyrocarbonate-treated water and quantitated
spectrophotometrically. A RT-PCR procedure was performed as previously
described (27) to determine relative quantities of mRNA
for IFN-, TNF-, and hypoxanthine phosphoribosyltransferase
(HPRT). The primers and probes for all genes were previously published
(27, 28). The amplified DNA was analyzed by
electrophoresis, Southern blotting, and hybridization with
cytokine-specific probes. The chemiluminescent signals were quantified
using a 600 ZS scanner (Microtek, Torrance, CA). The amount of PCR
product was determined by comparing the ratio of cytokine specific
signal density to that of HPRT-specific signal density for individual
samples (five mice per group). Arbitrary densitometric units for
individual samples were subsequently multiplied by a factor of 100 and
compared with those for control mice (uninfected mouse liver).
Amplification of HPRT served as an internal control for the amount of
RNA and cDNA from each sample.
Measurement of serum TNF-, IFN-, and aspartate transaminase
Serum TNF-, IFN-, and aspartate transaminase levels were
measured 8 wk postinfection. Serum TNF- levels were measured by
a capture ELISA kit supplied by R&D Systems, and IFN- levels were
quantitated by capture ELISA as previously described. Cytokine levels
were calculated using standard curves constructed with
recombinant murine cytokines. Measurement of the liver-associated
enzyme aspartate serum transaminase (AST) used a colorimetric assay
modified from a commercial kit (Sigma, St. Louis, MO)
(29).
Statistics
Hepatic fibrosis (adjusted for egg number) decreases with increasing intensity of infection (worm pairs) and was therefore, compared by analysis of covariance, using the log of total liver eggs as the covariate and the log of hydroxyproline per egg. Variables that did not change with infection intensity were compared by one-way ANOVA or Student’s t test. SEA-specific Ab isotype values and in vitro secreted cytokine profiles were graphed according to the StatView program (SAS Institute, Cary, NC). Bars from bottom to top show the 10th, 25th, 50th, 75th, and 90th percentiles, respectively, of the tested samples. Single outliers are indicated as circles. Values for secreted cytokine proteins, semiquantitative RT-PCR, serum Ab data, and serum cytokine levels were compared using Student’s two-tailed t test. p < 0.05 was regarded as significant. A minimum of two separate experiments were performed for all data.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Given the controversial roles of type 1 and type 2 cytokines in
the pathogenesis of schistosomiasis (24, 30, 31), we
examined the regulatory role of IL-10 in mice that manifested extreme
Th1- or Th2-producing phenotypes (17). For these studies,
IL-10/IL-4-deficient and IL-10/IL-12-deficient mice were infected with
20–30 S. mansoni cercariae, and disease progression was
examined in detail during both the acute and chronic stages of
infection and compared with that in WT mice as well as animals
exhibiting single cytokine deficiencies. Evidence that the double
cytokine-deficient mice generated a polarized type 1 (IL-10/IL-4
deficient) or type 2 (IL-10/IL-12 deficient) immune response following
infection was determined by examining the cytokine-producing profiles
of SEA-stimulated lymphocytes ex vivo (Fig. 1
). SEA-specific serum Ab isotype
profiles 8 wk postinfection also indicate the extent of immune
polarization (Fig. 2). As expected
(32), lymphocytes from infected WT mice produced
significant amounts of the Th2-type cytokine IL-5 (Fig. 1B)
but produced relatively little IFN- (Fig. 1A). By
contrast, IL-10-deficient mice developed the most nonpolarized
immunological phenotype of all the mice examined in the study. MLN
(Fig. 1) and spleen cells (data not shown) from IL-10-deficient mice
secreted large amounts of IL-5 and IFN- after in vitro stimulation
with SEA. Additionally, infected IL-10-deficient mice generated high
serum titers of both type 1 (IgG2b; Fig. 2A) and type 2
(IgG1 and IgE; Fig. 2, B and C)-associated Ab
isotypes.
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B), consistent with previous
observations (6, 33, 34). The continued production of
IL-10 in the absence of IL-4 (15) clearly explains the
inability of IL-4-deficient mice to manifest an extreme Th1 phenotype
following S. mansoni infection. Indeed, in this study only
MLN (Fig. 1) and spleen cell cultures (data not shown) prepared from
the double IL-10/IL-4-deficient mice produced abundant IFN- and
little or no IL-5 upon restimulation with SEA. Additionally, high
titers of SEA-specific IgG2b Abs were detected in the sera of
IL-10/IL-4 deficient mice (Fig. 2A). There were also little
SEA-specific IgG1 and no circulating IgE Abs detectable (Fig. 2, B and C), further demonstrating that these mice
were developing a strongly polarized type 1 immune response. In fact,
compared with the other mice in this study, the highest ratios of
SEA-specific IFN-/IL-5 and IgG2b/IgG1 Ab were observed in the mice
simultaneously deficient in IL-4 and IL-10.
Conversely, mice deficient in IL-12 alone showed relatively little
change in their cytokine or Ab responses compared with WT animals
(Figs. 1 and 2). This is probably explained by the lack of a
significant IL-12 response in infected WT animals (35). In
contrast, the double IL-10/IL-12-deficient mice generated a robust
SEA-specific IL-5 response 5–10 times greater than the response in WT
or IL-12-deficient mice. Moreover, unlike the single IL-10-deficient
animals, these mice did not exhibit a marked IFN- response,
demonstrating that a highly polarized and heightened type 2 response
was generated only in the combined absence of IL-10 and IL-12. These
mice also displayed the lowest serum titers of the type 1-associated
IgG2b Ab (Fig. 2A) and the highest total IgE Ab response
(Fig. 2C).
A critical protective role for IL-10 is revealed during chronic S. mansoni-infection
Because the IL-10-deficient (mixed response), IL-10/IL-4-deficient (Th1-dominant) and IL-10/IL-12-deficient (Th2-dominant) mice generated such unique cytokine-producing profiles following infection, the mice provided an excellent opportunity to examine the contribution of IL-10 as well as immune deviation to disease progression during schistosomiasis. Again, as described above, mice were infected with 20–30 S. mansoni cercariae and examined weekly for a total of 18 wk for signs of illness. One set of animals was sacrificed on wk 8 postinfection to determine whether there were significant differences in parasite burdens or fecundity. The remaining animals were monitored for changes in weight and for survival throughout the 18 wk.
As shown in Table I, there were no
differences in the establishment of infection in any group of mice. All
groups harbored a similar number of worm pairs, and total worm and
tissue egg burdens were not significantly different. Egg viability was
not assessed. Nevertheless, there were marked differences in how the
animals dealt with their infections. There were no signs of cachexia in
any mice up to 5 wk postinfection. Interestingly, however, after the
onset of egg laying (5 wk postinfection), several groups of
cytokine-deficient mice began losing weight (Fig. 3A). The single
IL-10-deficient mice did not show significant weight change up to wk 12
postinfection, at which time they began a gradual decline. Shortly
following the drop in weight, a high rate of mortality was observed
(Fig. 3B), with <50% of the mice surviving through wk 18.
Surprisingly, those mice that survived past wk 16 showed a significant
weight gain during the last weeks of the study. Mortality increased,
however, when the IL-10 deficiency was coupled with a deficiency in
either IL-12 or IL-4. Most dramatic was the rapid weight loss seen
in the infected IL-10/IL-4-deficient mice (Fig. 3A). These
mice lost an average of >2 g of body weight between 6 and 8 wk
postinfection and were all dead by 9 wk (Fig. 3B). Mice
deficient for both IL-10 and IL-12 (Th2-polarized) also displayed a
higher rate of mortality than the single IL-10-deficient animals,
although it was much more gradual than in the IL-10/IL-4-deficient
(Th1-polarized) mice. Indeed, the IL-10/12-deficient mice gained weight
up to wk 9 postinfection, at which point they then began a very gradual
decline. The weight loss again correlated closely with some mortality.
Although the IL-4-deficient mice also showed excessive weight loss
between wk 7 and 13 postinfection (Fig. 3A), there was no
significant decrease in survival compared with infected WT controls
(Fig. 3B). Moreover, similar to the single IL-10-deficient
mice, the IL-4-deficient animals gradually gained weight as they
entered the chronic stage of infection. In marked contrast to the other
mice in the study, the IL-12-deficient and WT mice gained weight
throughout the study. This correlated well with the near complete
absence of mortality among these animals.
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and TNF-
In an attempt to explain the increased mortality of mice deficient
in IL-10, we examined in detail the production of IFN- and TNF-,
two candidate markers of cytokine-induced shock (36).
Lymphocytes obtained from both IL-10- and IL-10/IL-4-deficient mice
produced the most IFN- in vitro when restimulated with SEA (Fig. 1A). Therefore, we also examined the cytokine response in
vivo by examining the serum levels of IFN- and TNF- (by ELISA)
and by analyzing the cytokine mRNA response within the granulomatous
tissues (by RT-PCR) at 7.5–8 wk postinfection. Consistent with their
lymphocyte responses, mice harboring deficiencies in IL-10 or
IL-10/IL-4 had the highest levels of IFN- in the serum (Fig. 4B) and IFN- mRNA
expression in the liver (Fig. 4A). The pattern of TNF-
mRNA expression was similar in these mice, although WT and
IL-4-deficient mice also exhibited a marked response (Fig. 4C). In contrast to their tissue mRNA responses, the pattern
of TNF- expression in the serum was more variable among the groups,
although it was interesting to note that all the mice that displayed
some weight loss during infection (Fig. 3A) also exhibited
detectable serum levels of TNF- (Fig. 4D). Thus, a
combined increased local and systemic IFN- and TNF- response
correlated most closely with the high degree of mortality observed in
the IL-10- and IL-10/IL-4-deficient mice. Nevertheless, these findings
failed to explain the significant mortality observed in the
IL-10/IL-12-deficient mice, as these animals clearly exhibited a
markedly reduced IFN- response, both locally and systemically (Figs. 1A and 4, A and B).
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Overproduction of NO can induce tissue damage (37)
and may contribute to morbidity during schistosome infection
(38). Because IFN- and TNF- are both potent
activators of NO (39, 40), and both cytokines were
markedly up-regulated in IL-10- and IL-10/IL-4-deficient mice, we
examined NO production following infection. Splenocytes were obtained
from the various cytokine-deficient mice 8 wk postinfection and were
placed in culture in the presence (Fig. 5B) or absence (Fig. 5A) of SEA. Interestingly, NO levels (as assessed by nitrite
production) closely mirrored the IFN- response of SEA-stimulated MLN
cells (Fig. 1A) as well as the tissue (Fig. 4A)
and serum (Fig. 4B) IFN- expression profiles. A slight
increase in nitrite levels was detected even in unstimulated cultures
from the IL-10-, IL-10/IL-4-, and IL-4-deficient mice (Fig. 5A). A similar, but more marked, expression followed SEA
stimulation, although the IL-10- and IL-10/IL-4-deficient mice
displayed the strongest response. Little or no induction of NO was
detected in the cultures obtained from WT, IL-12-deficient, or
IL-10/IL-12-deficient mice.
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, AST levels were significantly elevated
in all infected animals. However, the most dramatic increase was
observed in IL-10- and IL-10/IL-4-deficient mice. These mice also
displayed a robust type 1 response (high IFN-, TNF-, and iNO),
demonstrating a strong correlation among type 1 cytokines, AST
expression, and morbidity following infection. Importantly, the double
IL-10/IL-12-deficient animals confirmed the association with a robust
type 1 response for this activity, because these animals
(Th2-polarized) failed to exhibit a similarly elevated AST response.
Nevertheless, the latter animals also exhibited significant mortality,
suggesting that other mechanisms contribute to morbidity during
infection.
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Differences in the egg-induced granulomatous response could also
contribute to the morbidity and mortality observed in the IL-10 and
double cytokine-deficient mice. Consistent with many previous
observations, WT mice generated hepatic granulomas (Fig. 7A) rich in eosinophils (Fig. 7B) and displayed increased levels of hydroxyproline (Fig. 7C), a quantitative measure of tissue fibrosis, 8 wk
postinfection. Given the lack of a significant change in the cytokine
response of infected IL-12-deficient mice (Fig. 1), it was not too
surprising that hepatic pathology was not significantly affected by the
single IL-12 deficiency. In marked contrast to these animals, however,
IL-10-deficent mice developed large granulomas (Fig. 7A).
Nevertheless, while their granulomas were larger, there was no
significant increase in hepatic fibrosis (Fig. 7C), compared
with that in WT mice. There was also no change in the cellular
composition of the granulomas, which was consistent with previous
observations (16). Granuloma size was increased in
IL-4-deficient mice, although the magnitude of the increase was less
marked than that in IL-10-deficient animals (Fig. 7A).
Moreover, unlike the IL-10-deficient mice, tissue fibrosis decreased
(Fig. 7C), and significantly fewer eosinophils were observed
in the granulomas of IL-4-deficient vs WT mice (Fig. 7B).
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A). Again, it is important to note that these mice, unlike
their single cytokine-deficient counterparts, developed the most
extreme Th1-polarized response following infection (Fig. 1). The near
complete ablation of the type 2 response in the IL-10/IL-4-deficient
mice also resulted in the most dramatic change in the phenotype of the
developing granulomas. In fact, their lesions were almost completely
devoid of eosinophils (Fig. 7B), and the fibrotic response
was significantly reduced compared with that in WT mice (Fig. 7C). In contrast to these observations, the double
IL-10/IL-12-deficient animals developed large (Fig. 7A)
eosinophil-rich granulomas (Fig. 7B) similar in size to
those in IL-10-deficient mice. More importantly, however, in contrast
to the WT or single cytokine-deficient animals, the double
IL-10/IL-12-deficient mice displayed a significant increase in hepatic
fibrosis (Fig. 7C). Indeed, these mice were the only animals
in the study that displayed a marked and highly significant increase in
this important parameter of disease compared with WT mice. Production of the profibrogenic cytokines, IL-4 and IL-13, is increased in infected IL-10- and IL-10/IL-12-deficient mice
Previous studies demonstrated that IL-4 directly stimulates
collagen production in fibroblasts (4). More recently,
IL-13 was shown to exhibit a similar functional activity in vitro, and
in vivo studies demonstrated that IL-13 plays the dominant role in the
development of hepatic fibrosis during infection with S.
mansoni (6). The pattern of IL-4 and IL-13 production
was therefore investigated in the various cytokine-deficient mice 8 wk
postinfection. Consistent with previous observations, WT mice showed
significant SEA-specific IL-13 (Fig. 8A) and IL-4 (Fig. 8B) responses following infection (6), and
there was no dramatic change in this response in IL-12-deficient mice.
In marked contrast, however, almost a 10-fold increase in both IL-4 and
IL-13 was observed in the MLN cell cultures prepared from IL-10- and
IL-10/IL-12-deficient mice. Thus, there was a strong correlation
between maximal hepatic fibrosis (Fig. 7C) and IL-4 and
IL-13 levels, particularly in the double IL-10/IL-12-deficient mice, in
which the levels of anti-fibrotic IFN- (7) were
clearly diminished (Fig. 1A). Perhaps not surprisingly,
IL-13 production was significantly decreased in IL-4-deficient mice and
was almost undetectable in the majority of IL-10/IL-4-deficient mice,
which, again, correlated well with the modest fibrotic response
observed in these animals. IL-4 and IL-13 mRNA expression in the
granulomatous livers showed a similar pattern (data not shown).
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, A and
D, both groups exhibited a significant egg-induced
inflammatory response 8 wk postinfection. Nevertheless, collagen, which
forms an impressive ring around lesions in IL-10/IL-12-deficient mice
(Fig. 9B), was almost absent from the granulomas in the
IL-10/IL-4-deficient group (Fig. 9E). The polarized light
image shown in Fig. 9C illustrates the dense pattern of
collagen deposition that formed around the granulomas in
IL-10/IL-12-deficient mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although pathological changes were noted in all knockout animals, the
most dramatic outcome of infection was observed in the double
IL-10/IL-4-deficient mice. These mice uniformly lost weight beginning
at the onset of egg-laying, and all had succumbed to infection within 9
wk postinfection. Given the rapid onset of cachexia in these mice,
overproduction of type 1 cytokines was suspected as a possible
explanation (1, 2, 10). Indeed, cells from these mice,
unlike the other animals in the study, produced abundant IFN- and
little of the Th2 cytokines IL-4, IL-5, and IL-13. There was also
detectable systemic IFN- and TNF-, and splenocytes restimulated
with SEA produced abundant iNO, suggesting a heightened state of
macrophage activation in IL-10/IL-4-deficient mice. The marked
elevation in AST levels suggested that significant hepatocyte damage
probably contributed to the acute mortality. Moreover, although the
mice developed egg-induced granulomas, the phenotype of the lesions was
completely different from that in the other mice in the study. In fact,
their granulomas were almost devoid of eosinophils and were much less
fibrotic. Thus, this atypical granulomatous response could also
contribute to morbidity by failing to efficiently contain or neutralize
hepatotoxins produced by the developing miracidia within the egg
(41).
Brunet et al. reported similar infection-related mortality in mice
deficient for IL-4 (10). In their studies, IL-4-deficient
mice began to lose weight at the onset of egg deposition, and almost
100% mortality was reported by 8–10 wk postinfection. Similar to our
observations in infected IL-10/IL-4-deficient mice, they observed
elevated expression of type 1 cytokines. They suggested that IL-4 and
possibly other type 2 cytokines were required to inhibit the
deleterious effects of the proinflammatory mediators TNF- and iNO.
Surprisingly, in our studies we observed little mortality in the
infected IL-4-deficient group, despite excessive weight loss during the
acute stage of infection. In fact, similar survival was noted for
IL-4-deficient and WT mice through wk 18 postinfection. The
IL-4-deficient mice used in both studies were on the same genetic
background (C57BL/6), and the parasites used (Puerto Rican strain) were
from the same source, so we have no specific explanation for the
apparent discrepancy. However, differences in the numbers of parasites
used to infect our mice, diet (42), and housing conditions
could affect disease progression during infection. Regardless, the data
collected from the infected IL-10/IL-4-deficient mice support the
deleterious role for highly polarized type 1 cytokine responses during
infection with S. mansoni.
Interestingly, the maintenance of a significant IL-10 response in the
absence of IL-4 (6) is a likely explanation for the
minimal morbidity and mortality observed in our infected IL-4-deficient
mice. Metwali et al. reported little default toward a more dominant
Th1-type response in infected IL-4-deficient animals, which is
consistent with our observations (34). Although a modest
increase in IFN- was observed in IL-4-deficient mice, a much more
dramatic increase in IFN- was found in mice deficient for both IL-4
and IL-10. In fact, the major immunological difference measured between
the IL-4-deficient and the IL-10/IL-4-deficient mice is in IFN-
levels (Ag specific (Fig. 1A), hepatic message (Fig. 4A), and systemic (Fig. 4B)). Elevated IFN-
levels probably contribute to the high mortality observed in the
IL-10/4- vs IL-4 deficient animals. These data formally demonstrate
that IL-10 reduces the Th1 default and provides a significant degree of
protection to IL-4-deficient mice. Results from a previous study
suggested that this IL-10 is probably derived from a source other than
Th2 lymphocytes, because little IL-10 is produced by Ag-stimulated
cells from IL-4-deficient mice (6). In this previous study
IL-10 mRNA expression was increased in the granulomatous tissues,
whereas expression of the prototypical Th2 cytokines IL-5 and IL-13 was
diminished (6), further suggesting that the majority of
IL-10 is derived from cells other than Th2 lymphocytes.
Surprisingly, the single IL-10-deficient mice showed higher mortality
than animals exhibiting a deficiency in IL-4 (Fig. 3B). This
was surprising because, unlike the IL-4-deficient mice, expression of
Th2-type cytokines was clearly unimpaired in the IL-10-deficient group.
In fact, production of IL-13 and IL-4 was elevated compared with that
in WT mice (Fig. 8). It was previously hypothesized that Th2-type
cytokines might protect mice from the lethal effects of proinflammatory
mediators (10, 24). Interestingly, however,
IL-10-deficient mice also displayed a marked type 1 response, which was
of a similar magnitude as the double IL-10/IL-4-deficient animals.
Elevated serum levels of IFN-, TNF-, and the liver enzyme AST
suggested that these mice were also suffering from the hepatotoxic
effects of sustained type 1 cytokine production despite the presence of
a significant type 2 response. Thus, while some protection was
apparently conferred by the type 2 response (IL-10-deficient animals
lived longer than the Th1-polarized IL-10/IL-4-deficient mice), there
was no evidence that proinflammatory mediator production was impaired.
This suggests that the maintenance of IL-10, rather than a classical
Th2-type response, is the more critical factor contributing to host
survival during infection.
In previous studies we showed that development of egg-induced hepatic
fibrosis in schistosomiasis is significantly ameliorated by sensitizing
mice to egg Ags in the presence of IL-12 before infection with S.
mansoni (25). These animals developed smaller and
less fibrotic granulomas than the control infected mice and showed
markedly increased Th1 and decreased Th2-type cytokine expression.
Importantly, however, there was no mortality in these mice, even
through the chronic stages of infection, despite the development of a
sustained and polarized type 1 cytokine response. Nevertheless, unlike
the IL-10- and IL-10/IL-4-deficient mice that were incapable of
producing IL-10, the egg/IL-12-sensitized Th1-polarized mice developed
a significantly elevated IL-10 response in the granulomatous tissues,
which was sustained at least through wk 12 postinfection
(25). In fact, the production of IL-10 in IL-12-treated
mice is commonly observed in vivo (43, 44). Thus, it
appears that IL-12-vaccinated animals benefit from an increased IFN-
(antifibrotic) and reduced IL-4/IL-13 (profibrogenic) response, but,
perhaps even more importantly, an unimpaired IL-10 response spares them
from the potentially harmful and toxic effects of the polarized type 1
response. Similar IL-12-based vaccination studies are being performed
in IL-10-deficient mice to confirm this hypothesis. Regardless, these
findings suggest that a highly polarized type 1 response, while
beneficial in terms of preventing tissue fibrosis, is potentially
harmful, particularly when established in an IL-10-deficient
setting.
Given the pathogenic role of the type 1 response in infected IL-10- and
IL-10/IL-4-deficient mice, an improved course of infection was probable
in the double IL-10/IL-12-deficient animals, because these mice were
relatively deficient in type 1 cytokines due to the absence of an
endogenous IL-12 response. Surprisingly, however, these mice exhibited
a high rate of mortality following infection, which exceeded even the
rate observed in the single IL-10-deficient group. In fact, <50% of
the IL-10/IL-12-deficient mice survived through wk 13 postinfection.
Initially, the animals appeared to improve when compared with the
IL-10/IL-4-deficient mice, because they clearly gained weight through
wk 9 postinfection and demonstrated no mortality. Nevertheless, they
slowly began to lose weight as they entered the more chronic stages of
the infection, which was quite dramatic compared with that in the WT
and IL-12-deficient groups (Fig. 3A). The modest iNO
response of SEA-stimulated splenocytes confirmed the absence of a
significant type 1 response (Fig. 5), and the relatively low serum AST
level indicated that there was little hepatotoxicity following
infection (Fig. 6). These observations suggested that
IL-10/IL-12-deficient animals were probably dying from a mechanism
distinct from that operating in the IL-10/IL-4-deficient mice.
Unlike the IL-10/IL-4-deficient mice, which developed smaller lesions,
the IL-10/IL-12-deficient mice formed granulomas that were as large as
those in the IL-10-deficient group (Fig. 7A). This suggests
that the increase in granulomatous inflammation observed in
IL-10-deficient mice is probably controlled by changes in type 2 rather
than type 1 cytokine expression. Perhaps more importantly, however, the
IL-10/IL-12-deficient mice, in contrast to the IL-10- or
IL-10/IL-4-deficient animals, also showed a marked and highly
significant increase in hepatic fibrosis (Fig. 7C). Although
the IL-10-deficient mice also displayed a similarly elevated type 2
response (Fig. 8), we hypothesize that the simultaneous expression of
IFN- (Fig. 1A) prevents the development of an
overexuberant fibrotic response in these animals. Thus, the severe
hepatic fibrosis observed in the IL-10/IL-12-deficient mice is probably
attributable to both the increased expression of the
profibrogenic cytokines IL-4 and IL-13 (6, 45, 46) and the
decreased production of the collagen-suppressing cytokine, IFN-
(7). Thus, morbidity in the double IL-10/IL-12-deficient
mice may be attributable to the marked exacerbation in both egg-induced
inflammation and hepatic fibrosis. Such a hypothesis is consistent with
the delayed morbidity and mortality in the IL-10/IL-12-deficient
compared with the IL-10/IL-4-deficient mice. Moreover, collagen
deposition slowly accumulates throughout infection and would probably
contribute to morbidity only during the more chronic stages of the
infection.
TNF- could also be contributing to the morbidity of the infected
IL-10/IL-12-deficient mice. Serum levels of TNF- were markedly
increased in all mice exhibiting a deficiency in IL-10 (Fig. 4D). Indeed, this attribute appeared to be somewhat
unrelated to the dominance of type 1/type 2 cytokines, although
IL-4-deficient mice also displayed a response. It was particularly
interesting that all the animals that exhibited some degree of weight
loss during infection also developed detectable serum levels of
TNF-. Only WT and IL-12-deficient mice continued to gain weight
throughout the 18 wk of the study (Fig. 3A), which
correlated perfectly with the absence of a detectable serum TNF-
response in these mice (Fig. 4D). Given the well-known
antagonistic nature of type 2-associated cytokines to the production of
TNF-, it was somewhat unexpected to see such a response in the
IL-10/IL-12-deficient group. Nevertheless, previous studies have
suggested that IL-10 may be the most critical type 2 cytokine
responsible for suppressing TNF- production (47). A
role for TNF- in the development of acute morbidity in infected type
1-polarized mice was recently described in schistosomiasis
(10). Interestingly, TNF- was also characterized as a
critical cofactor for a Th2-dependent response (48).
Therefore, as proposed previously in an experimental tuberculosis
model, TNF- may exhibit unique functional activities in type 1
vs type 2 cytokine-dominant responses (49). We are
currently investigating the possible dichotomous role of TNF- in
schistosomiasis by neutralizing the cytokine in our Th1-polarizing
(IL-10/IL-4) and Th2-polarizing (IL-10/IL-12) mice.
The question was recently raised whether schistosome pathology in humans results from type 1 vs type 2 dominant responses. Fallon (24) and others have suggested from both murine and human studies that Th2 cytokine responses do not produce serious pathology and that their primary function in schistosomiasis is protective, because they serve as anti-inflammatory mediators. However, the results from the current study clearly show that highly polarized type 2 as well as type 1 responses exhibit proinflammatory activity. Therefore, in the setting of schistosomiasis it seems inappropriate to strictly label type 1 responses as proinflammatory and type 2 responses as anti-inflammatory. In fact, although egg-induced inflammation can be either type 1- or type 2-mediated, unique pathological features are associated with each response. Although we would agree that one function of the type 2 response is to regulate potentially pathogenic type 1 responses (24, 30), we believe that the results from this study show that type 2 cytokines are not just regulatory factors (50), but also induce important pathological changes and contribute to morbidity in murine schistosomiasis.
Recently, we described the distinct and nonredundant contributions of
the type 2 cytokines, IL-4 and IL-13, to the development of egg-induced
hepatic pathology (6). Although IL-4 played the dominant
role in the generation of the egg-specific type 2 response, both IL-4
and IL-13 were required for the development of eosinophil-rich
granulomas. More importantly, however, IL-13 was shown to be the
dominant mediator of hepatic fibrosis, and this activity was believed
to be a direct effect of the cytokine because it stimulated collagen
deposition by fibroblasts. Previous studies documented the
collagen-suppressing activity of the type 1 cytokines IFN- and IL-12
(7, 25). Together, these findings suggest that type 2
rather than type 1 cytokines are the key mediators of murine hepatic
fibrosis. The results of the current study are consistent with these
previous observations and show that IL-10 significantly regulates IL-13
production during infection (Fig. 8A). Nevertheless, IL-13
production has not been systematically investigated in human
hepatosplenic schistosomiasis, so its link to the development of severe
disease in humans cannot be evaluated at present.
While it is clear that type 1 cytokines can also trigger significant
morbidity during infection (10), their participation in
the establishment of chronic pathology remains ill defined. It is
intriguing to speculate that type 1 responses might be elevated in the
late stages of the disease in a futile attempt to reverse the damage
done by years of exposure to the reparatory collagen-inducing type 2
cytokines. This scenario might explain the high serum levels of IFN-
and TNF- reported in hepatosplenic schistosomiasis patients
(13). Detailed longitudinal studies would be needed to
investigate this hypothesis. Regardless, the data presented herein
highlight the central regulatory role of IL-10 in schistosomiasis and
demonstrate that type 1 and type 2 cytokines are both potentially
pathogenic. Moreover, this study reveals a mechanism by which parasite
egg-induced IL-10 is not only important for suppressing type 1
cytokines, but is also critical for dampening pathogenic type 2 immune
responses during infection.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: WT, wild type; SEA, soluble egg Ag; MLN, mesenteric lymph nodes; AST, aspartate serum transaminase; iNO, inducible NO; HPRT, hypoxanthine phosphoribosyltransferase.
Received for publication January 24, 2000. Accepted for publication March 29, 2000.
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A. S. MacDonald, E. A. Patton, A. C. La Flamme, M. I. Araujo, C. R. Huxtable, B. Bauman, and E. J. Pearce Impaired Th2 Development and Increased Mortality During Schistosoma mansoni Infection in the Absence of CD40/CD154 Interaction J. Immunol., May 1, 2002; 168(9): 4643 - 4649. [Abstract] [Full Text] [PDF]
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D. E. Jones, M. R. Ackermann, U. Wille, C. A. Hunter, and P. Scott Early Enhanced Th1 Response after Leishmania amazonensis Infection of C57BL/6 Interleukin-10-Deficient Mice Does Not Lead to Resolution of Infection Infect. Immun., April 1, 2002; 70(4): 2151 - 2158. [Abstract] [Full Text] [PDF]
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L. R. Schopf, K. F. Hoffmann, A. W. Cheever, J. F. Urban Jr., and T. A. Wynn IL-10 Is Critical for Host Resistance and Survival During Gastrointestinal Helminth Infection J. Immunol., March 1, 2002; 168(5): 2383 - 2392. [Abstract] [Full Text] [PDF]
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A. S. MacDonald, M. I. Araujo, and E. J. Pearce Immunology of Parasitic Helminth Infections Infect. Immun., February 1, 2002; 70(2): 427 - 433. [Full Text] [PDF]
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S. K. Lundy and D. L. Boros Fas Ligand-Expressing B-1a Lymphocytes Mediate CD4+-T-Cell Apoptosis during Schistosomal Infection: Induction by Interleukin 4 (IL-4) and IL-10 Infect. Immun., February 1, 2002; 70(2): 812 - 819. [Abstract] [Full Text] [PDF]
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B. Vaillant, M. G. Chiaramonte, A. W. Cheever, P. D. Soloway, and T. A. Wynn Regulation of Hepatic Fibrosis and Extracellular Matrix Genes by the Th Response: New Insight into the Role of Tissue Inhibitors of Matrix Metalloproteinases J. Immunol., December 15, 2001; 167(12): 7017 - 7026. [Abstract] [Full Text] [PDF]
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N. Tsuji, K. Suzuki, H. Kasuga-Aoki, Y. Matsumoto, T. Arakawa, K. Ishiwata, and T. Isobe Intranasal Immunization with Recombinant Ascaris suum 14-Kilodalton Antigen Coupled with Cholera Toxin B Subunit Induces Protective Immunity to A. suum Infection in Mice Infect. Immun., December 1, 2001; 69(12): 7285 - 7292. [Abstract] [Full Text] [PDF]
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A. C. La Flamme, E. A. Patton, and E. J. Pearce Role of Gamma Interferon in the Pathogenesis of Severe Schistosomiasis in Interleukin-4-Deficient Mice Infect. Immun., December 1, 2001; 69(12): 7445 - 7452. [Abstract] [Full Text] [PDF]
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M. K. Park, K. F. Hoffmann, A. W. Cheever, D. Amichay, T. A. Wynn, and J. M. Farber Patterns of Chemokine Expression in Models of Schistosoma mansoni Inflammation and Infection Reveal Relationships between Type 1 and Type 2 Responses and Chemokines In Vivo Infect. Immun., November 1, 2001; 69(11): 6755 - 6768. [Abstract] [Full Text] [PDF]
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J. A. Pedras-Vasconcelos, L. R. Brunet, and E. J. Pearce Profound effect of the absence of IL-4 on T cell responses during infection with Schistosoma mansoni J. Leukoc. Biol., November 1, 2001; 70(5): 737 - 744. [Abstract] [Full Text] [PDF]
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C. G. Lee, R. J. Homer, Z. Zhu, S. Lanone, X. Wang, V. Koteliansky, J. M. Shipley, P. Gotwals, P. Noble, Q. Chen, et al. Interleukin-13 Induces Tissue Fibrosis by Selectively Stimulating and Activating Transforming Growth Factor {beta}1 J. Exp. Med., September 17, 2001; 194(6): 809 - 822. [Abstract] [Full Text] [PDF]
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 R. N. Germain The Art of the Probable: System Control in the Adaptive Immune System Science, July 13, 2001; 293(5528): 240 - 245. [Abstract] [Full Text]
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P. G. Fallon, P. Smith, E. J. Richardson, F. J. Jones, H. C. Faulkner, J. Van Snick, J.-C. Renauld, R. K. Grencis, and D. W. Dunne Expression of Interleukin-9 Leads to Th2 Cytokine-Dominated Responses and Fatal Enteropathy in Mice with Chronic Schistosoma mansoni Infections Infect. Immun., October 1, 2000; 68(10): 6005 - 6011. [Abstract] [Full Text] [PDF]
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