Division of Gastroenterology-Hepatology, Department of Internal Medicine, University of Iowa, Iowa City, Iowa 52242
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ABSTRACT |
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Interleukin-10 (IL-10)
and transforming growth factor- (TGF-
) regulate CD4+
T cell interferon-
(IFN-
) secretion in schistosome granulomas. The role of IL-12 was determined using C57BL/6 and CBA mice. C57BL/6 IL-4
/
granuloma cells were stimulated to produce IFN-
when cultured with IL-10 or TGF-
neutralizing monoclonal antibody. In
comparison, C57BL/6 wild-type (WT) control granuloma cells produced
less IFN-
. IL-12, IL-18, and soluble egg antigen stimulated IFN-
release from C57BL/6 IL-4
/
and WT mice. IFN-
production in C57 IL-4
/
and WT granulomas was IL-12 dependent, because IL-12
blockade partly abrogated IFN-
secretion after stimulation. All
granuloma cells released IL-12 (p70 and p40), and IL-12 production remained constant after anti-TGF-
, anti-IL-10, recombinant IL-18, or
antigen stimulation. C57 WT and IL-4
/
mouse granuloma cells expressed IL-12 receptor (IL-12R)
1-subunit mRNA but little
2 mRNA. TGF-
or IL-10 blockade did not influence
1 or
2 mRNA expression. CBA mouse dispersed granuloma cells released no measurable IFN-
, produced IL-12 p70 and little p40, and expressed IL-12R
2
and little
1 mRNA. In T helper 2 (Th2) granulomas of C57BL/6 WT and
IL-4
/
mice, cells produce IL-12 (for IFN-
production) and IL-10
and TGF-
modulate IFN-
secretion via mechanisms independent of
IL-12 and IL-12R mRNA regulation. We found substantial differences in
control of granuloma IFN-
production and IL-12 circuitry in C57BL/6
and CBA mice.
T helper 1 cell; interleukin-12 receptor
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INTRODUCTION |
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IN MURINE
SCHISTOSOMIASIS mansoni, eggs from the parasite settle in the
liver and intestines during the course of natural infection. The eggs
evoke a strong granulomatous response that surrounds the ova. The
granulomas in the liver of mice with schistosomiasis make large amounts
of interleukin-4 (IL-4) and IL-5 and display the phenotypic
characteristics of a T helper 2 (Th2)-type response. However,
granulomas from mice with a C57BL/6 genetic background contain T cells
that produce interferon- (IFN-
) (30). The various
immunoregulatory circuits that control IFN-
production within these
strong Th2-type granulomas are not well defined.
Various cytokines have a role in Th1/Th2 cell differentiation in murine
schistosomiasis. IL-4 is important for phenotypic expression of most
aspects of the Th2 response (8, 26). In contrast to
wild-type controls, the liver granulomas of IL-4 knockout mice produce
no IgE and little IgG1, IL-5, and IL-13. Also, there is a substantial
reduction in eosinophil and mast cell content. Regional lymph node
cells, splenocytes, and granuloma T cells from IL-4 /
mice produce
more IFN-
than cells from their wild-type counterparts. However,
this shift toward Th1 is modest (8, 15, 26, 26). Although
deletion of IL-13 by itself has little effect, the cytokine profile in
schistosomiasis does shift toward Th1 in the IL-4/IL-13 double-mutant
mouse (15). This suggests that IL-4 and IL-13 have a
synergistic inhibitory effect on development of the Th1 phenotype. Some
other factors that influence Th1/Th2 development include IL-10,
transforming growth factor-
(TGF-
), IL-12, and B7 expression
(18, 19).
IFN- production by granuloma cells increases in response to
recombinant IL-12 (rIL-12), suggesting that some granuloma T cells
display the IL-12 receptor (IL-12R). Also, the granulomas make large
amounts of IL-10 and TGF-
that help limit this IFN-
expression.
IL-12, IL-10, and TGF-
all appear to act directly or indirectly on
the granuloma Thy 1.2+ T cells that make IFN-
(30).
IL-12 is a powerful inducer of IFN- production and Th1 cell
development (16). Pulmonary and natural models of
granulomatous inflammation show that rIL-12 administration before the
onset of egg deposition can enhance IFN-
production and Th1 cell
development in response to schistosome eggs (19). However,
largely unexplored is the role of intralesional IL-12 and IL-12R in the
regulation of ongoing IFN-
production within granulomas of murine
schistosomiasis, which was the subject of this study.
In C57BL/6 and IL-4-deficient mice, we showed that Th2 granulomas of
murine schistosomiasis produce IL-12 that is required for the ongoing
IFN- response. Although granuloma IFN-
is subject to regulation
by IL-10, TGF-
, and antigen stimulation, none of these factors
affected IL-12 production or IL-12R mRNA expression. Similar studies
using CBA mice revealed substantial stain differences regarding control
of granuloma IFN-
production and IL-12 circuitry.
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MATERIALS AND METHODS |
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Mice and schistosome infection.
This study used normal C57BL/6 and CBA/J mice obtained from the
National Cancer Institute (Bethesda, MD). Also used were C57BL/6 IL-4
/
and C57BL/6 IL-12 p40 knockout mice. Breeding colonies for the
mutant animals were maintained at the University of Iowa. At 7-8
wk of age, mice were infected subcutaneously with 50 cercariae of the
Puerto Rican strain of Schistosoma mansoni.
Granuloma cell and splenocyte dispersal and cell culture. Livers of mice killed during the eighth week of infection were homogenized for 30 s at low speed in a Waring blender. Granulomas were collected by 1 g sedimentation and washed three times in RPMI 1640 medium (RPMI). To prepare a single cell suspension from these granulomas, the intact granulomas were incubated in a shaking water bath at 37°C for 30 min in RPMI containing 0.5% collagenase (type 1 from Clostridium histolyticum, Sigma Chemical, St. Louis, MO). Repeated suction and expulsion through a 1-ml syringe disrupted the softened granulomas further. The dispersed granuloma cell suspensions were passed through sterile gauze to exclude nondispersed fragments. The cells were collected by centrifugation, washed three times in RPMI, and counted. Cell viability was determined by eosin Y exclusion.
Single cell suspensions of splenocytes were prepared from individual spleens from 8-wk infected mice by gentle teasing in RPMI. The cells were briefly resuspended in distilled water to lyse red blood cells. The splenocytes were then washed three times in a large volume of RPMI. For cytokine measurements in supernatants, cells were cultured in microtiter plates at 106 cells/well in medium (200 µl) for 18 h at 37°C in 5% CO2 atmosphere with or without anti-IL-10 (1 µg/ml; SXC1, DNAX, Palo Alto, CA), anti-TGF-T cell line. The D1.1 T cell line was maintained in T25 flasks in complete RPMI medium. For boosting, 6 ml RPMI complete medium containing rabbit IgG (100 µg/ml) was added to T25 flasks containing adherent D1.1 cells. Also added were irradiated (3,000 rad) splenocytes (106/ml). The cultures were split every 2-3 days and maintained in complete medium. Two weeks after boosting, cellular RNA was extracted for mRNA analysis.
RNA extraction and RNase protection assay. Each experiment used RNA from splenocytes or granuloma cells pooled from three to four mice. Total cellular RNA was extracted from cell suspensions by homogenization in guanidinium-acid phenol as previously described (9). Total RNA preparations contained equivalent 18S and 28S RNA bands. RNA extracts were quantified spectrophotometrically.
RPA was used to quantify IL-12RELISAs.
We used ELISAs to measure cytokine concentrations in supernatants. To
measure IFN-, plates were coated with a MAb to IFN-
(HB170,
American Type Culture Collection) and incubated with supernatant. IFN-
was detected with polyclonal rabbit anti-IFN-
(a gift from Dr. Mary Wilson, Department of Medicine, University of Iowa) followed by biotinylated goat anti-rabbit IgG (Accurate Chemical, Westbury, NY),
streptavidin-horseradish peroxidase (HRP), and
2,2'-azino-di(3-ethylbenzthiazoline) sulfonic acid substrate (Zymed,
San Francisco, CA).
Statistical analysis. Data are means ± SE or SD of multiple determinations. Differences between two groups were compared using Student's t-test. ANOVA and Dunnett's t-test were used for multiple comparison data. P < 0.05 was considered significant.
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RESULTS |
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IFN- production within granulomas is subject to regulation.
Dispersed granuloma cells from the livers of C57BL/6 or C57BL/6 IL-4
/
mice secrete IFN-
constitutively when cultured in vitro
(30). Table 1 shows that
granuloma cells cultured with blocking anti-IL-10 or anti-TGF-
MAb
produce substantially more IFN-
, suggesting that IL-10 and TGF-
made by the dispersed cells function to restrain IFN-
synthesis.
Isotype control antibody had no effect. Also, cells incubated with
schistosome SEA, rIL-12, or rIL-18 secreted more IFN-
. As expected,
cells from schistosome granulomas that developed in C57BL/6 IL-4
/
mice made more IFN-
than granuloma cells from IL-4-producing
wild-type control animals (Table 1).
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Inducible IFN- production requires IL-12.
IL-12 induces T lymphocytes to secrete IFN-
and promotes development
of the Th1 CD4+ T cell phenotype. It was determined if
IL-12 was necessary for ongoing IFN-
production within schistosome
granulomas. Dispersed granuloma cells cultured with neutralizing
anti-IL-12 MAb produced less IFN-
than cells grown without blocking
anti-IL-12. In the C57BL/6 IL-4
/
mouse, constitutive production of
IFN-
by the dispersed granuloma cells decreased by ~40%. Also,
IFN-
secreted by these granuloma cells in response to anti-IL-10
MAb, anti-TGF-
MAb, SEA, or rIL-18 decreased by 70-97% (Fig.
1A). Granuloma cells from
C57BL/6 wild-type control mice yielded similar results (Fig. 1B). The amount of blocking anti-IL-12 MAb added to each
culture was sufficient to completely neutralize the biological activity of rIL-12 used at 500 pg/ml in a bioassay.
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Granuloma cells from C57BL/6 IL-12 /
mice make no measurable
IFN-
.
C57BL/6 IL-12
/
mice were infected with S. mansoni to further analyze the role of IL-12 in intragranuloma
IFN-
production. In three separate experiments, the dispersed
granuloma cells from the IL-12
/
animals cultured in vitro failed
to secrete IFN-
(<30 pg/ml) even when stimulated with rIL-12,
rIL-18, SEA, anti-IL-10, or anti-TGF-
. Also, granuloma cells
cultured with rIL-12 (5 ng/ml), anti-IL-10, and/or anti-TGF-
MAb
produced no measurable IFN-
.
Granuloma cells secrete IL-12.
Because culturing granuloma cells with neutralizing anti-IL-12 MAb
substantially decreased inducible IFN- production, we determined
whether the granuloma cells released ample amounts of this cytokine.
Bioactive IL-12 (p70) is a heterodimeric cytokine consisting of p35 and
p40 subunits. The p40 subunit can be produced independently of the p35
subunit. Free p40 and p40 dimers can function as IL-12 inhibitors
(29). Thus we used two separate ELISAs to measure both p70
(active IL-12) and total p40 (p40 + p40/p35 and p40/p40 complexes).
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Manipulation of IFN- production does not change rates of IL-12
p70 or total p40 secretion from granuloma cells cultured in vitro.
As shown above, granuloma IFN-
production is partly dependent on
IL-12, and rIL-12 can strongly stimulate IFN-
secretion in granuloma
cells from C57BL/6 mice. Thus we tested the hypothesis that IL-10,
TGF-
, IL-18, and antigen modulated IFN-
synthesis via regulation
of IL-12 production. Dispersed granuloma cells were cultured in vitro
with or without anti-IL-10 MAb, anti-TGF-
MAb, rIL-18, SEA, or
anti-CD3 MAb. After the incubation, the cell-free supernatants were
assayed for IL-12 p70 and total p40. Table
2 shows that these manipulations had no
affect on IL-12 release.
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IL-12R 1 and
2 mRNA expression in dispersed granuloma cells.
IL-12 functions through a receptor with a binding
1-subunit and a
signaling
2-subunit. Using an RNase protection assay, we examined
the expression and regulation of these subunits within the granulomas
of murine schistosomiasis. Mice on the C57BL/6 genetic background
strongly expressed the
1-subunit. Compared with the GAPDH
housekeeping gene, granuloma cells from C57BL/6 IL-4
/
mice had
amounts of IL-12
1 mRNA similar to those of cells from C57BL/6
wild-type control animals. In both mouse strains,
2 mRNA was
detected only at exceedingly low levels (Table
3).
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IL-10 and/or TGF- blockade enhance granuloma cell IFN-
production even with rIL-12 added to cultures.
rIL-12 stimulates dispersed granuloma cells from C57 BL/6 wild-type and
IL-4
/
mice to produce IFN-
. We also determined whether
anti-IL-10 and anti-TGF-
MAbs could enhance IFN-
secretion even
with rIL-12 added to the cultures. IL-10 and TGF-
neutralization enhanced IFN-
production beyond that induced by rIL-12 used at 0.01, 0.1, 0.5, or 5 µg/ml. rIL-12 at 0.5 and 5 µg/ml were both at the
top of the IL-12 dose-response curve (see Table 1 for results using
rIL-12 at 0.5 µg/ml with anti-IL-10/TGF-
).
IFN- production in CBA mice is regulated differently.
Experiments evaluated IFN-
production from dispersed granuloma cells
of 8-wk infected CBA mice. These cells made no detectable IFN-
constitutively or after stimulation with rIL-12, rIL18, anti-TGF-
,
anti-IL-10, or SEA (Table 1). Table 2 shows that dispersed granuloma
cells from CBA mice constitutively secreted the bioactive form of IL-12
(p70) at similar rates to that of C57BL/6 granuloma cells. However, in
contrast, granuloma cells from CBA mice released little free p40. Also,
analysis of IL-12R mRNA expression yielded an unexpected result. These
granuloma cells expressed IL-12R
2 mRNA in excess to that of IL-12R
2 (Table 3). Similar to the C57BL/6 granuloma cells, neither IL-10 nor TGF-
blockade affected IL-12 secretion (Table 2) or IL-12R mRNA
expression (Table 3) during short-term cultures.
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DISCUSSION |
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Granulomas in the liver of mice with schistosomiasis make large
amounts of IL-4 and IL-5 and display the phenotypic characteristics of
a Th2-type response. However, the granulomas from mice on a C57BL/6
genetic background produce some IFN- that appears to derive from
granuloma T cells (30).
IL-12 is a component of the innate immune response important for
induction of Th1 cells. Its early release at the onset of inflammation
promotes IFN- production and Th1 cell development and inhibits Th2
cell differentiation (36). Macrophages and dendritic cells
probably are the major sources of IL-12 (21). There are T
cell-dependent and -independent mechanisms for induction of IL-12
(13). The T cell-dependent pathway involves CD40-CD40L interaction (13, 31). Lipopolysaccharide, endotoxin, and
various microorganisms can induce IL-12 expression independently of T cells (3). IL-12 production can be upregulated by IFN-
(24) and PGE2 and downregulated by IL-10
(2, 11), TGF-
, and other factors (1, 10,
37). Also, IL-4 and IL-13 can suppress IL-12 production, helping
to prevent emergence of the Th1 response (12, 23).
Thus it was surprising to discover that schistosome Th2-type granulomas
from mice on the C57BL/6 genetic background produced IL-12 as well as
IL-10, TGF-, IL-4, and IL-13. This constitutive IL-12 secretion
appeared to be independent of IL-10 and TGF-
regulation, because
IL-10 and TGF-
blockade had no effect on IL-12 release (p40 or p70).
Granulomas in IL-4
/
mice make no IL-4 and little IL-13
(8). IL-12 secretion from granulomas of IL-4-deficient
mice was only modestly higher, suggesting that IL-4 and IL-13 were not
major determinants of IL-12 production within schistosome granulomas.
IL-12 is a heterodimeric molecule composed of a light (p35) and heavy
(p40) chain. Together, they constitute the active form of the molecule
(p70). Cells that make IL-12 can produce the p40 chain in great excess
over that of the biologically active p70 heterodimer. The free p40
chain can bind to IL-12R 1 and may act as a physiological antagonist
of IL-12 (29). We therefore determined if changes in
IFN-
secretion by granuloma cells after antigen stimulation or
cytokine manipulation also changed the relative production of p40 to p70.
C57BL/6 mice produced twice as much free p40 as p70. However, SEA,
anti-IL-10 MAb, anti-TGF- MAb, rIL18, or anti-CD3 MAb stimulation
did not affect the secretory rate of either the free p40 or p70
component. Thus it is unlikely that an alteration in the relative
production of free p40 to p70 is the mechanism underlying IL-10,
TGF-
, SEA, or anti-CD3 modulation of IFN-
secretion.
Although TGF- IL-10 and SEA did not appear to govern the ongoing
granuloma IL-12 secretion, IL-12 blocking experiments did suggest that
IFN-
produced in response to these agents required the presence of
IL-12. Also notable was the capacity of anti-IL-12 MAb to block
rIL18-induced IFN-
secretion. IFN-
released constitutively from
the granuloma cells was only moderately decreased by IL-12 blockade.
Thus IL-12 produced locally within the granuloma is necessary for
complete expression of IFN-
by granuloma cells in murine schistosomiasis.
To further evaluate the relevance of IL-12 in granuloma IFN-
synthesis, we also infected C57BL/6 IL-12 p40
/
mice with schistosomes. These IL-12
/
animals formed granulomas that made no
detectable IFN-
either constitutively or after stimulation with
rIL-12, rIL18, or other biological agents. This confirms the importance
of IL-12 for the development of IFN-
-producing T cells within these
granulomas. Experiments using lethally schistosome-infected IL-4/IL-12
double knockout mice suggest that at least splenocytes can overcome the
IL-12 deficiency to produce IFN-
(28).
The IL-12R consists of two covalently linked components: IL-12R 1
and
2, each encoded by separate genes. Either unit alone binds IL-12
at low affinity, whereas coexpression of both units can result in
high-affinity binding sites. The IL-12R
2-subunit likely is most
involved in intracellular signaling (16).
The components of the IL-12R are subject to regulation. Activation of T
and natural killer cells results in upregulation of the IL-12R.
Some of the factors that induce IL-12R display include T cell receptor
activation, B7 costimulation (20), IFN-, and IL-12
(7, 34). IL-4, TGF-
, and IL-10 are reported to
downregulate this expression (16). Under various
circumstances, either
1- or
2-subunits are susceptible to
immunoregulation in mice and humans (5, 38). Naive T cells
stimulated with IL-4 selectively lose the
2-subunit, suggesting that
this subunit is a critical regulator of Th1/Th2 differentiation
(32). It is proposed that IL-12 responsiveness is
controlled predominantly through regulation of the
2-subunit
(16).
TGF- has been shown to attenuate IL-12R expression in mice
(17). Thus it was possible that TGF-
and IL-10 blockade
stimulated IFN-
secretion through modulation of IL-12R
1- or
2-transcription. Therefore, we examined the expression of IL-12R
1 and
2 mRNA in the Th2 granulomas of murine schistosomiasis.
Granulomas from mice on the C57BL/6 genetic background strongly
expressed IL-12R 1 mRNA, but little
2. The level of expression was similar in granuloma cells from C57BL/6 IL-4
/
and wild-type controls. This suggested that neither IL-4 nor IL-13 produced within
the granuloma were critically important for regulation of the IL-12R,
at least at the mRNA level. Similarly, anti-IL-10 MAb, anti-TGF-
MAb, and SEA did not alter either IL-12R
1 or
2 mRNA expression.
Thus these factors do not regulate granuloma IFN-
production by
altering transcription of the IL-12R. It remains possible that IL-10
and TGF-
affect IL-12R protein expression posttranscriptionally or
modulate IL-12 intracellular signaling (6). The
stimulation of IFN-
production in the presence of blocking IL-12 MAb
(Fig. 1) provides some evidence that IL-10 and TGF-
may work at
least in part through mechanisms that do not affect IL-12 signaling.
Our IL-10 and TGF- blockade experiments suggest that IL-10 and
TGF-
are factors that help limit IFN-
production within the
ongoing schistosome granulomas. Although we showed no effect on IL-12
secretion, IL-10 can inhibit macrophage secretion of various cytokines
and macrophage expression of major histocompatibility complex class II
and B7 (33). B7 interactions can influence IFN-
responses in murine schistosomiasis (18). The multiple direct effects of IL-10 on antigen-presenting cells and perhaps T cells
could account partly for IL-10 suppression of IFN-
synthesis in
granulomas. Similarly, TGF-
has multiple suppressive actions on T and B cells, macrophages, and other cell types (22,
25). TGF-
can disrupt early IL-12 signaling events in T cells
(6, 17, 27), which could be one point of regulation.
The granulomas in the liver of CBA and C57BL/6 mice have a similar
morphological appearance. A surprising observation was that IFN- and
IL-12 circuitry was substantially different in the granulomas of CBA
and C57BL/6 mice. As opposed to the granuloma cells of C57BL/6 mice,
CBA granuloma cells expressed IL-12R
2 mRNA and little IL-12R
1
mRNA. They also secreted much less IL-12 p40, but similar amounts of
the bioactive form of IL-12 (p70). The dispersed granuloma cells from
CBA mice also produced no detectable IFN-
even after stimulation.
rIL-12, rIL18, anti-IL-10, anti-TGF-
, and SEA used alone or in
combination had no effect on IFN-
secretion. However, CBA granuloma
T cells separated from the other granuloma cell types do make IFN-
when cultured in vitro (4). In fact, granuloma T cells
highly enriched using magnetic beads can produce IFN-
(in ng/ml
quantities) on stimulation with adherent anti-CD3 MAb (unpublished
observation). This suggests that these T cells retain the ability to
make IFN-
and that perhaps there are unique regulatory circuits
within the CBA granuloma environment limiting T cell IFN-
expression.
Our findings are in agreement with Todt et al. (35), who
showed with CBA mice that the granuloma-bearing livers contained mRNA
for IFN-, IL-12, and IL-12R. Also, similar to our results, their
data (35) revealed that dispersed granuloma cells cultured even with rIL-12 failed to secrete IFN-
. However, Todt et al. (35) failed to detect IL-12 secretion from the dispersed
granuloma cells. This latter apparent contradiction to our observations may have resulted from their (35) use of an IFN-
bioassay to indirectly measure IL-12 production rather than a direct
measurement of IL-12 protein using sensitive and highly specific ELISA
assays. Todt et al. (35) also reported IL-12R
1 and no
2 in intact granuloma-bearing livers of 8-wk-infected CBA mice,
which is contrary to our findings using isolated granulomas. This could
be a consequence of liver tissue contamination of their
(35) RNA preparations. While we showed that blocking IL-10
had no immediate effect on IFN-
production, IL-12 secretion, or
IL-12R mRNA expression in dispersed CBA granuloma cells cultured in
vitro, the findings of Todt et al. (35) suggest that IL-10
neutralization in vivo during early granuloma development can lead to
liver granulomas expressing stronger Th1 features.
In summary, this study showed that the Th2 granulomas of murine
schistosomiasis in the C57BL/6 mouse make IL-12, which is a necessary
cofactor for maintenance of the ongoing IFN- response within the
granuloma of natural infection. Granuloma IFN-
is subject to
regulation by IL-10, TGF-
, and antigen stimulation. However, none of
these factors affect IL-12 production or IL-12R mRNA expression. We
also found substantial differences between CBA and C57BL/6 mice in
granuloma IFN-
production, IL-12 p40 secretion, and IL-12R
1 and
2 mRNA expression. This study clearly reveals that there are many
complex interactions involved in the regulation of IFN-
production
in schistosome Th2-type granulomas.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-38327, DK-02428, and DK-25295, the Crohn's and Colitis Foundation of America, and the Veterans Administration.
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FOOTNOTES |
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Address for reprint requests and other correspondence: K. Qadir, Dept. of Internal Medicine, 4607 JCP, Univ. of Iowa, Iowa City, Iowa 52242.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 12 January 2001; accepted in final form 21 June 2001.
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