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*
Schistosomiasis Immunology and Pathology Unit and
Max Planck Institut für Immunbiologie, Freiburg, Germany;
Department of Microbiology/Immunology, Cornell University, Ithaca, NY 14853;
Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892;
¶ Department of Biochemistry, University of Extremadura, Caceres, Spain; and
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Biomedical Research Institute, Rockville, MD 20852
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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suppresses Arg-1 activity,
because type 1 polarized IL-4/IL-10-deficient, IL-4/IL-13-deficient,
and egg/IL-12-sensitized animals fail to up-regulate Arg-1 following
egg exposure. Notably, granuloma size decreases in these
type-1-deviated/Arg-1-unresponsive mice, suggesting an important
regulatory role for Arg-1 in schistosome egg-induced pathology. To test
this hypothesis, we administered difluoromethylornithine to block
ornithine-aminodecarboxylase, which uses the product of arginine
metabolism, L-ornithine, to generate polyamines.
Strikingly, granuloma size and hepatic fibrosis increased in the
ornithine-aminodecarboxylase-inhibited mice. Furthermore, we show that
type 2 cytokine-stimulated macrophages produce proline under strict
arginase control. Together, these data reveal an important regulatory
role for the arginase biosynthetic pathway in the regulation of
inflammation and demonstrate that differential activation of
Arg-1/NOS-2 is a critical determinant in the pathogenesis of granuloma
formation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice infected with the helminth parasite Schistosoma mansoni develop a pathologic reaction that closely mimics the human disease. Some of the eggs laid by adult worm pairs are trapped in the liver, a process that can lead to marked inflammation, tissue eosinophilia, collagen deposition, and ultimately portal hypertension and severe hepatic fibrosis in some patients. In mice the inflammatory response is dominated by the production of type 2-associated cytokines, including IL-4, IL-5, IL-10, and IL-13. Several studies have demonstrated that these type 2 cytokines control many aspects of the egg-induced immunopathologic response (1, 2).
Previously, we showed that much of the granulomatous liver pathology is reduced in infected mice when the egg-specific response is converted from a Th2-dominant to a Th1-dominant reaction. This is accomplished by sensitizing mice to schistosome egg Ag in the presence of the Th1-inducing adjuvant, IL-12, before infection (3). Several type 1-associated cytokines were shown to be involved in the protective response (4), although recent studies suggested that NO synthase-2 (NOS-2)4 expression is particularly important (5). NOS-2-deficient mice sensitized with eggs/IL-12 not only fail to control their egg-induced inflammatory response, but actually display a marked exacerbation in the reaction despite developing a Th1-deviated response. A similar role for NOS-2 was recently described in Mycobacterium avium-infected mice (6). These findings suggest that while Th1 cytokines are critical, the downstream functional activity of NO-producing cells is equally important. Thus, NOS-2 is important not only for its antimicrobial activities (7), but also because it can serve as a potent anti-inflammatory and antifibrotic mediator.
Murine macrophages are a dominant feature of egg-induced granulomas and
represent the main cellular source of inducible NO. However, these
cells possess two inducible enzymes, NOS-2 and arginase, that share
L-arginine as a common substrate. Although it is clear that
NOS-2 plays an important role in schistosomiasis, the specific
contribution of arginine metabolism to the regulation of granulomatous
pathology remains unclear. In previous in vitro studies, we showed that
different combinations of cytokines induce NOS-2 and the hepatic
isoform of arginase (arginase type 1 (Arg-1)). The Th1-associated
cytokines IFN- and TNF-
activate NOS-2, whereas the Th2-type
cytokines IL-4, IL-10, and IL-13 induce Arg-1 (8). Of
interest, the preferential activation of these enzymes is also observed
during Ag presentation by macrophages and dendritic cells to
corresponding CD4+ Th1 or Th2 lymphocytes
(9, 10). Thus, in a manner similar to the Th1/Th2
paradigm, the cytokine-mediated activation of one enzyme is accompanied
by the active suppression of the other.
In mammals, two arginase isoforms are expressed: the cytosolic Arg-1 and the mitochondrial Arg-2 (11). The isoforms catalyze the same reaction, but are encoded by different genes and differ in their tissue distribution. Arg-1 is an essential enzyme of the urea cycle and is expressed at high levels in hepatocytes. This enzyme hydrolyses L-arginine to urea and L-ornithine; therefore, its main function in the liver is the detoxification of ammonia. Although it is expressed at other sites, the exact role of Arg-1 in extrahepatic cells and tissues is not well understood. Because L-ornithine, a product of arginase activity, is a necessary metabolite for the production of polyamines and prolines, which control cell proliferation and collagen production, respectively, arginase activity appears to be critically linked with cell growth and connective tissue production. Notably, both of these activities are key parameters in the pathogenesis of inflammatory responses.
We hypothesized that type 1/type 2 cytokines would regulate the dominance of NOS-2 Arg-1 vs expression in vivo, which, in turn, would regulate the character and magnitude of inflammatory reactions. To test this hypothesis, we examined L-arginine metabolism in two different models of granulomatous disease. Initially, we investigated whether the expression of Arg-1 and NOS-2 was regulated in a manner similar to that described in in vitro studies. Specifically, we examined in several cytokine-deficient mouse strains whether arginase was activated following exposure to schistosome eggs, a potent Th2-inducing stimulus. In separate studies, mice treated with schistosome eggs and IL-12 or Mycobacterium avium-infected animals were used to examine the pattern of Arg-1/NOS-2 expression during type 1-dominated inflammatory responses. Finally, additional in vitro and in vivo studies were designed to determine more directly whether granulomatous pathology is influenced by the activation of arginase.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female 42-day-old C57BL/6, C57BL/10, C57BL/10Ai knockout (KO) IL-10, C57BL/6Ai-[KO] IL-4, and C57BL/6Ai-[KO] IL-10/IL-4 mice were obtained from Taconic Farms (Germantown, NY). Wild-type (WT)-F2, IL-13-KO, and IL-4/IL-13-KO mice (all 129Ola x C57BL/6 (F2)) were provided by Dr. A. McKenzie (Medical Research Council, London, U.K.). All mice were housed under specific pathogen-free conditions in a National Institute of Health American Association for the Accreditation of Laboratory Animal Care-approved animal facility. Mycobacterium avium strain 2-151 SmT was cultured on agar plates as described previously (12). Cercariae of a Puerto Rican (NMRI) strain of S. mansoni (Biomedical Research Institute) were obtained from infected Biomphalaria glabrata snails (Biomedical Research Institute). Soluble egg Ag (SEA) and soluble worm Ag preparations were obtained from homogenized S. mansoni eggs and adult parasites as previously described (3).
Immunizations, infections, and difluoromethylornithine (DFMO) treatment
S. mansoni eggs were extracted from the livers of infected mice (Biomedical Research Institute) and enriched for mature eggs. For the induction of pulmonary granulomas, groups of five mice were immunized with 5000 freshly isolated eggs i.p. and challenged with 5000 eggs i.v. 14 days later. All mice were sacrificed 6 days after the egg challenge. In designated experiments, C57BL/10Ai mice and C57BL/10Ai-[KO] IL-10 mice were also injected i.p. with 0.25 µg rIL-12 on 5 consecutive days beginning on the day of the primary egg immunization.
Infection and sensitization of mice with eggs and rIL-12 has been previously described (3). Briefly, groups of 10 C57BL/6Ai mice were injected i.p. with 5000 eggs on three occasions separated by 2-wk intervals. Animals were also injected i.p. with rIL-12 (0.25 µg/dose) on 5 consecutive days beginning on the day of each egg immunization. Naive mice and egg/IL-12-presensitized mice were infected 2 wk after the last egg/IL-12 injection by percutaneous exposure of tail skin for 40 min in water containing 25 cercariae. All mice were sacrificed 8 wk after infection. We noted no mortality in any group up to the point of sacrifice. In designated experiments infected C57BL/6Ai mice were treated with 1% (42.3 mM) or 2% (84.6 mM) (D,L)-2-DFMO (provided by Dr. V. Steele, National Cancer Institute) in the drinking water beginning at wk 5 postinfection. The mice were kept on DFMO-containing water until the end of the experiment.
Groups of five C57BL/6Ai mice were injected i.v. with 108 CFU M. avium strain 2–151 SmT. Mice were sacrificed 2 and 4 wk after infection and compared with noninfected controls. Lung tissue from individual mice was embedded in paraffin and stained with the Fite acid-fast method (American Histolabs, Histo-Path of America, Millersville, MD) to monitor bacterial infection. In addition, bacterial CFU were analyzed in lung tissue samples as previously described (12).
Histopathology, fibrosis measurement, and immunohistochemistry
Approximately half of the liver was fixed in Bouin-Hollande solution, and half of the lung was inflated with fixative. Histologic sections were processed and stained with Giemsa (Histo-Path of America). The diameter and cell composition of granulomas (30/mouse) surrounding single eggs were measured using an ocular micrometer, and the volume of each granuloma was calculated assuming a spherical shape. The number of eosinophils was counted in the same area by an experienced pathologist. Only granulomas around mature, viable eggs were measured in the liver. The collagen content of the liver samples, determined as hydroxyproline, was analyzed as described previously (1).
Formalin-fixed, paraffin-embedded tissue sections were deparaffinized and rehydrated, and the Ab epitopes unmasked by a 5-min incubation in 1% SDS in TBS at room temperature. Endogenous peroxidase activity was quenched by a 5-min incubation in 3% H2O2 in H2O. Arginase expression was detected with mouse anti-Arg-1 IgG1 (BD-Transduction Laboratories, Lexington, KY) or normal mouse IgG1 (Zymed Laboratories, South San Francisco, CA) as a negative control and using the M.O.M. kit (Vector Laboratories, Burlingame, CA) as directed by the manufacturer. The samples were developed with diaminobenzidene (Vector Laboratories) as directed by the manufacturer and counterstained with Vector Green (Vector Laboratories).
Lymphocyte culture and cytokine detection
Spleen and mesenteric lymph node (infection model) or
lung-draining lymph node (pulmonary granuloma model) were removed
aseptically, and single-cell suspensions were prepared. Cells were
plated in 24-well tissue culture plates at a final concentration of
4 x 106 cells/ml (spleen) or 3 x
106 cells/ml (lymph node) in RPMI 1640
supplemented with 10% FCS, 2 mM glutamine, 1 mM sodium pyruvate, 50
µM 2-ME, and antibiotic-antimycotic solution (all from Life
Technologies, Gaithersburg, MD). Cultures were incubated at 37°C in a
humidified atmosphere of 5% CO2. Cells were
stimulated with SEA (20 µg/ml), soluble worm Ag (50 µg/ml), Con A
(1 µg/ml), or medium alone. Supernatant fluids were harvested at
72 h and assayed for cytokine production. IFN- and IL-5 were
measured by two-site ELISA as previously described (3).
Cytokine levels were calculated with standard curves constructed using
recombinant murine cytokines.
RT-PCR detection of mRNA
Relative quantities of mRNA for IFN-, IL-4, IL-5, IL-13,
hypoxanthine-phosphoribosyltransferase (HPRT), NOS-2, Arg-1, Arg-2,
ornithine-aminotransferase (OAT), and ornithine-aminodecarboxylase
(ODC) expressed in inflammatory tissue, were determined by RT-PCR as
previously described (13). The sequences of primers and
probes for HPRT, cytokines (13), and NOS-2
(14) have been published previously. The following primers
and probes were used for arginase isoforms OAT and ODC: Arg-1: sense,
5'-CAGAAGAATGGAAGAGTCAG-3'; antisense, 5'-CAGATATGCAGGGAGTCACC-3';
probe, 5'-GCATCTCTGGCCACGCCAGGGTCC-3'; Arg-2: sense,
5'-TGATTGGCAAAAGGCAGA GG-3'; antisense, 5'-CTAGGAGTAGGAAGGTGGTC-3';
probe, 5'-GGCTCCAGCCACAGGAACCCCTG-3'; ODC: sense,
5'-TGAGCCAGGCAGATACTA-3'; antisense, 5'-TTGGCCGTGACATTACA-3';
probe, 5'-TTCTCAAACAGCATCCAATC-3'; and OAT: sense,
5'-CACATACGGCGGAAACCCACTA-3'; antisense,
5'-TCGCCTCTCGCTCTCACTTTTCT-3'; probe, 5'-TGCCCTCTGACGTTGTGACC-3'. The
amplified DNA was analyzed by electrophoresis, Southern blotting, and
hybridization with nonradioactive specific probes. The chemiluminescent
signals were quantified using a ScanJet IIP (Hewlett-Packard, Palo
Alto, CA). The amount of PCR product was determined by comparing the
ratio of molecule-specific signal density to that of HPRT-specific
signal density for individual samples. Arbitrary densitometric units
for individual samples were subsequently multiplied by 100 and compared
with those for control mice (uninfected mouse tissue).
Determination of arginase activity in tissues and granulomatous liver cell isolation
The granulomatous lung or liver tissue (
100 mg) was crushed
with a pestle in an Eppendorf tube (1.5 ml), resuspended in 0.5 ml 50
mM Tris-HCl buffer, pH 7.5, and homogenized with an Ultra-Turrax (IKA,
Muhlheim, Germany). After addition of 0.5 ml 0.1% Triton X-100, the
tubes were shaken for 10 min at 25°C and centrifuged at 13,000 rpm.
Arginase activity was determined as described previously
(9), and the protein concentration was measured with the
bicinchoninic acid protein assay (Pierce, Rockford, IL). One unit of
arginase activity is the amount of enzyme necessary to hydrolyze 1 µM
arginine/min.
Liver tissue samples from infected mice were passed through a sterile stainless sieve and washed twice with cold PBS. The pellet, containing sedimented granulomas, single cells, and tissue debris, was resuspended in RPMI 1640 containing 100 U/ml collagenase D (Roche, Indianapolis, IN) and 4 U/ml DNase I (Sigma) and digested for 20 min at 37°C. The digested material was passed through a cell strainer (100 µm) to remove debris and obtain a single-cell suspension. The cells were washed twice with RPMI 1640. Erythrocytes were lysed with ACK buffer (BioWhittaker, Walkersville, MD), and cells were plated at 106 cells/well in 48-well culture plates. Cells were washed twice with cold PBS and lysed in 250 µl 0.1% Triton X-100, 1x protease inhibitors. Arginase activity was determined as described previously (9).
Detection of Arg-1 and NOS-2 protein expression
Equal amounts of lung tissue lysate from individual mice were pooled in each experimental group. SDS-PAGE was performed as described by Laemmli (15) in a 10% polyacrylamide gel and electrotransferred onto nitrocellulose membranes (16). Immunodetection was performed with rabbit polyclonal Ab raised against rat Arg-1 (17, 18), which cross-reacts with murine Arg-1. NOS-2 was detected by Ab from BD-Transduction Laboratories. After further incubation with HRP-anti-IgG, immunoreactive proteins were revealed using the Supersignal kit (Pierce).
Analysis of macrophage proline production
Murine bone marrow-derived macrophages (BMM) were obtained by flushing the femurs of CBF1 mice. Cells were cultured for 8 days in hydrophobic Teflon bags (Biofolie 25, Heraeus, Hanau, Germany) in DMEM containing 10% inactivated FCS, 5% horse serum, 1 mM sodium pyruvate, 2 mM glutamine, 60 µM 2-ME, penicillin (100 U/ml), and streptomycin (100 µg/ml; Life Technologies, Paisley, Scotland) and the supernatant of L929 fibroblasts at a final concentration of 15% (v/v) as a source of M-CSF, which drives cell proliferation toward a >95% pure population of BMM.
To induce arginase activity, BMM (105 cells in
triplicate) were pretreated with IL-4, IL-13 (both from R&D Systems,
Wiesbaden, Germany), IL-10 (PeproTech, Rocky Hill, NJ; all 20 U/ml), or
IFN- (gift from Dr. Adolf, Ernst-Bender Institute, Vienna, Austria;
500 U/ml) and in designated wells with 1 mM
L-hydroxyarginine (Clinalfa, Laufelfingen, Switzerland) for
24 h at 37°C and with 10% CO2 in 0.2 ml
DMEM supplemented with 10% inactivated FCS, 1 mM sodium pyruvate, 2 mM
glutamine, and 60 µM 2-ME. The cells were washed and placed in DMEM
without arginine containing 2% FCS and pulsed with 0.2 µCi
L-[U-14C]arginine (NEN, Boston, MA;
sp. act., 348 mCi/mM) or 0.2, 0.4, or 0.6 µCi
L-[1-14C]ornithine (NEN; sp. act.,
52 mCi/mM). Cytokines and inhibitor were added at the original
concentration, and after 24 h the cultures were frozen at
-80°C. The synthesized radioactive proline was isolated in a 20-µl
aliquot of the culture medium by TLC in silica-coated plates (Merck,
Darmstadt, Germany) using ethanol/water (30/70, v/v) as solvent. The
separated compounds were detected with ninhydrine in a reference band
with nonradioactive standards, and the radioactive spots with the same
Rf value were placed in scintillation vials and counted.
Statistical analysis
Hepatic fibrosis per single egg decreases with increasing intensity of infection (total number hepatic eggs per tissue) in the infection experiments (19). These variables were, therefore, compared by analysis of covariance, using the log of total liver eggs as the covariate and the log of hydroxyproline per egg. All other variables were compared by Student’s t test. In all cases, results were considered significant at p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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WT mice and mice deficient for IL-10, IL-4, and IL-10/IL-4 were
sensitized i.p. with freshly isolated schistosome eggs and 2 wk later
were challenged i.v. with a second bolus of eggs. On day 6
postchallenge, all animals were sacrificed, and pulmonary granuloma
size and eosinophilia were assessed by microscopy. Lung tissues were
also assayed for arginase activity. Large granulomas formed in WT and
IL-10-deficient mice, while lesion size moderately decreased in the
absence of IL-4 (Fig. 1
A),
which was consistent with previous observations (20, 21).
In contrast to the findings in the WT and single KO animals, granuloma
size (Fig. 1A) and granuloma eosinophils (Fig. 1B) were both significantly decreased in mice deficient in
IL-4 and IL-10. Strikingly, lung arginase activity, examined by three
different assays, closely correlated with these pathologic observations
(Fig. 1, C–E). Arginase enzyme activity was induced in the
WT and IL-10 KO mice after egg injection, and these mice displayed no
impairment in lesion development. In marked contrast, enzyme activity
was almost absent in the double IL-10/IL-4 KO mice that were highly
defective for granuloma formation. Most of the arginase activity
derived from the cytosolic Arg-1 isoform, as suggested by both RT-PCR
(Fig. 1D) and Western blot (Fig. 1E). Consistent
with the enzyme activity assays (Fig. 1C), Arg-1 mRNA (Fig. 1D) and protein expression (Fig. 1E) were almost
undetectable in the double IL-10/IL-4 KO mice, while high levels were
readily observed in the egg-injected WT and IL-10-deficient mice.
IL-4-deficient mice displayed an intermediate phenotype in all three
assays, which was again consistent with their pattern of lesion
formation and tissue eosinophilia. Although it has been reported that
the mitochondrial arginase (Arg-2) can also be differentially regulated
in cytokine-activated macrophages (22), there was no
evidence that Arg-2 mRNA expression was induced following egg injection
(data not shown).
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F). The
dominance of type 1 vs type 2 cytokine expression in the granulomatous
tissues was also confirmed (20) by RT-PCR (Fig. 1, G and H). The results from these experiments
showed that WT and IL-10/IL-4-deficient mice develop completely
divergent type 1/type 2 cytokine and NOS-2/Arg-1 responses, while
IL-10- and IL-4-deficient mice display intermediate phenotypes. IL-5
and IL-13 mRNA expression was maximally induced (Fig. 1H),
while little IFN- mRNA was detectable in the lungs of egg-injected
WT mice (Fig. 1G), which was consistent with previous
findings (20, 21). There was also little NOS-2 mRNA
expression in these animals (Fig. 1F). In contrast, the
IL-10/IL-4-deficient mice showed high levels of IFN-, but almost
undetectable IL-5 and IL-13 mRNA (Fig. 1H). These mice,
unlike the WT group, displayed a highly significant NOS-2 mRNA response
and correspondingly low levels of Arg-1 (Fig. 1, D and
F). Of interest, although IFN- mRNA expression was
markedly increased in the IL-10-deficient animals, little induction of
NOS-2 was evident, and Arg-1 remained the dominant enzymatic response.
The maintenance of a significant type 2 cytokine response in these
animals may explain this unexpected pattern (Fig. 1H)
(20). The findings from IL-4-deficient mice support this
hypothesis, showing a much more impaired type 2 response and
correspondingly increased expression of both IFN- and NOS-2 mRNA
(Fig. 1, F–H).
We also examined the contribution of IL-13 to the regulation of Arg-1,
because recent studies showed that IL-13 cooperates with IL-4 to
generate egg-induced granulomas (21). Here, experiments
conducted with IL-13-deficient and double IL-4/IL-13 mutant mice to a
great extent duplicated the findings generated with IL-4- and
IL-4/IL-10-deficient mice (Fig. 2).
Arginase activity and egg-induced pathology were reduced, but not
completely abrogated, in the IL-13-deficient mice. Strikingly, however,
the double IL-4/IL-13-deficient mice displayed a much more impaired
granulomatous response (Fig. 2, A and B), which
correlated perfectly with an almost complete absence of arginase
activity in the tissue (Fig. 2C). Thus, in the schistosome
egg-granuloma model, the expression and activation of Arg-1 in vivo
directly correlates with a type 2 cytokine response and maximum
egg-induced pathology, whereas peak NOS-2 expression correlates with
polarized type 1 cytokine expression and reduced inflammation.
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To determine whether IL-12 and type 2 to type 1 immune deviation
would affect arginase activity in the pulmonary granuloma model, we
immunized WT and IL-10-deficient mice with schistosome eggs and rIL-12,
challenged them with eggs i.v. 2 wk later and then sacrificed the mice
on day 6. WT and IL-10-deficient mice were selected for these studies
because they showed the highest arginase activity and formed large
eosinophil-rich granulomas when not sensitized with eggs and IL-12
(Fig. 1). As shown in Fig. 3, granuloma
size (Fig. 3A) and tissue eosinophilia (Fig. 3B)
were markedly decreased in both strains when sensitized in the presence
of IL-12. Arginase activity was also significantly decreased in these
mice (Fig. 3C), and this correlated with a marked alteration
in the type 1/type 2 cytokine response. The normal egg-specific type 2
response in WT mice was converted to an IFN--dominant response by
the egg/IL-12 sensitization procedure (Fig. 3D). Notably,
however, although IFN- increased in the non-IL-12-treated IL-10 KO
mice, arginase activity and granuloma formation were completely
preserved in these animals. Granuloma size and arginase activity were
only decreased in these mice when the animals were sensitized with eggs
and IL-12. In contrast to the nonsensitized KO animals, type 2 cytokine
production decreased dramatically in the sensitized mice, suggesting
that type 2 cytokine expression was the most critical factor for
maintaining arginase activity and granuloma formation (Fig. 3D).
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C57BL/6 mice were infected with M. avium
(108 CFU/mouse), and lung tissue was analyzed for
expression and activation of Arg-1 and NOS-2 on wk 2 and 4 following
infection. The local cytokine response was also examined by
semiquantitative RT-PCR to show that a dominant type 1 response was
induced. In addition, Fite-stained lung tissue sections were analyzed
to confirm that mice were infected (data not shown). Finally, lung
samples were homogenized to determine bacterial loads. The mean
infection intensity on day 14 postinfection was 2.24 x
105 ± 1.04 x 105
CFU/mg tissue; by day 28 this number rose to 3.29 x
106 ± 8.32 x 105
CFU/mg. In contrast to the findings with schistosome eggs, RT-PCR
analysis of M. avium-infected lungs showed a dominant type 1
cytokine mRNA response (Fig. 4A). IFN- was up-regulated
by day 14 postinfection and remained at high levels through day 28. In
contrast, IL-4 was barely detectable on day 14, although a slight
increase was observed by day 28 (Fig. 4A). Consistent with
these observations, Arg-1 mRNA levels (Fig. 4B) and Arg-1
enzymatic activity (Fig. 4C) showed little change over
background on day 14, and only a slight, but nevertheless significant,
increase by day 28. Indeed, compared with the Arg-1 response observed
in the lungs of schistosome egg-challenged mice, the small induction
detected following M. avium infection appeared minor (Fig. 4, B and C). Western blot analysis confirmed this
conclusion, because we were not able to detect Arg-1 protein expression
at any time point (data not shown). In contrast to these observations,
NOS-2 mRNA expression was clearly induced in the lungs of M.
avium-infected mice (Fig. 4D). NOS-2 protein expression
was also easily detectable by day 28 postinfection (Fig. 4E). By comparison, NOS-2 mRNA was only slightly induced in
the lungs of schistosome egg-challenged mice (Fig. 4D), and
no protein was detected by Western blot analysis (data not shown).
Thus, Arg-1 activity does not simply result from an ongoing
inflammatory reaction, but rather appears to be strictly dependent on a
type 2-driven response.
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Because one of the main functions of the liver is the
detoxification of ammonia, Arg-1 is highly expressed in hepatocytes,
where the enzyme is essential for the urea cycle. Therefore, the liver
displays basal levels of arginase activity much higher than any other
tissue. Because of its high background expression, the liver did not
provide a convenient setting to investigate the differential regulation
of NOS-2/Arg-1 expression in vivo. Therefore, we initially focused on
the lung, where NOS-2 and Arg-1 are both almost undetectable in naive
mice. Nevertheless, in schistosomiasis, much of the morbidity stems
from the fibrotic pathology that develops in response to eggs deposited
in the liver. As such, we were interested in extending our findings
from the pulmonary granuloma model to the livers of infected animals.
Tissue sections prepared from the liver and intestine of mice infected
with cercariae from S. mansoni and killed 8 wk after
infection were stained with anti-Arg-1 Ab. Immunohistochemical
staining shows a significant accumulation of arginase-positive cells in
areas surrounding the deposited eggs in both tissues (Fig. 5, A and D). Not
surprisingly, high background levels were detected in the liver
parenchyma of both infected and uninfected mice. Nevertheless, more
intense staining was clearly visible in the granulomas. Interesting
differences in the cellular localization of arginase were also noted in
these sections. Specifically, dense staining for arginase was observed
in cells found in the liver parenchyma, which was similar to findings
in a previous report (23), while arginase-positive cells
in the granuloma showed a more diffuse pattern (Fig. 5, A
and B).
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), this sensitization procedure effectively deviated the
normal type 2 cytokine response to a dominant type 1 phenotype
(3). Unlike the findings from the pulmonary granuloma
model, however, little regulation of Arg-1 activity was detected in
whole liver tissue by wk 8 postinfection (Fig. 6A) compared with that in
uninfected animals. As indicated by arginase staining of normal liver
tissue (Fig. 5, A and B), liver arginase activity
in uninfected mice was extremely high, which probably masked any
up-regulation that may have occurred following infection. Nevertheless,
when the liver inflammatory cell infiltrate was separated from
hepatocytes and analyzed, a clear reduction in arginase activity was
observed in the infected egg/IL-12-sensitized mice (Fig. 6B). These data thus support the pulmonary granuloma studies
and suggest that regulation of Arg-1 in the liver is largely restricted
to the areas of granulomatous inflammation. It is important to note,
however, that our experiments did not allow us to identify the exact
cell types that were expressing arginase. In contrast to arginase
findings, however, NOS-2 was readily detected in the livers of the type
1-deviated mice (Fig. 6A), which was consistent with
previous observations (5).
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Hydrolysis of L-arginine by the enzyme arginase
produces L-ornithine, which is a necessary metabolite for
the synthesis of both polyamines and proline (Fig. 7). Generation of these products is
controlled by two enzymes, ODC and OAT, respectively, both of which are
constitutively expressed in normal liver and lung tissues (data not
shown). ODC metabolizes L-ornithine to putrescine and is
therefore the rate-limiting enzyme for polyamine synthesis. Because
arginase is an essential enzyme of the urea cycle, arginase inhibitors
proved toxic (M. Hesse and T. A. Wynn, unpublished
observations), which made it impossible to directly examine its role in
the pathogenesis of schistosomiasis. Therefore, we turned our attention
to ODC, because it operates directly downstream from arginase, and its
enzymatic activity is easily blocked with the inhibitor DFMO. DFMO is a
selective inhibitor of ODC and is well tolerated in rodents and humans
(24). The compound is commonly used as a chemotherapeutic
agent for neoplasms, but, notably, has also demonstrated efficacy as
curative treatment for some protozoal infections (25).
Because of its documented antiparasitic activity, we initially
investigated whether DFMO was toxic for schistosomes. It is important
that DFMO did not affect either the viability or fecundity of adult
worms cultured in vitro, even when the parasites were exposed to
relatively high (31 mM) concentrations of the compound (data not
shown).
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A) as well as granuloma size
(Fig. 8B). Although the granulomatous response worsened in
the treated mice, no differences in the cellular make-up of their
lesions were detected. Indeed, the percentages of granuloma-associated
eosinophils were nearly identical in all three groups (Fig. 8C). Strikingly, the expression of IL-4 and IL-5 was totally
unaffected (Fig. 8D). These findings suggest that the
additional increase in fibrosis in DFMO-treated animals compared with
that in infected, nontreated mice is unlikely to be explained by the
enhanced production of type 2 cytokines (21, 26, 27).
While induction of a type 2 cytokine response is clearly necessary to
regulate pathology in infected mice (2) and to control
arginase activity (Figs. 1 and 2), we hypothesize the increased
bioavailability of L-ornithine for its competing
enzyme, OAT, may be a more likely explanation for the increased
fibrotic response observed in the DFMO-treated mice (Fig. 7).
|
Because we were unable to test the downstream function of the
opposing enzyme, OAT, due to the lack of a specific inhibitor, we took
a more direct in vitro approach and examined whether proline production
was enhanced in macrophages that were stimulated with IL-4, IL-13, or
IL-10. In these studies BMM were stimulated with the cytokines in the
presence or the absence L-hydroxyarginine, a potent
physiologic inhibitor of arginase produced by NOS-2 (see Fig. 7), to
provide additional proof that proline production in macrophages is
regulated by the activity of arginase.
As shown in Fig. 9A, IL-4 and
IL-13 markedly up-regulated proline production, while addition of
L-hydroxyarginine to the cultures completely
blocked the response. IL-10, in contrast, stimulated only very weak
proline production. As described previously (8, 9),
arginase activity was also significantly up-regulated in the
cytokine-stimulated cultures (Fig. 9A), confirming that the
BMM were developing an alternatively activated phenotype. The extent of
arginase induction correlates with the amount of detected proline.
|
B). Here, proline levels were only slightly affected by the
addition of IL-4 and IL-13. Thus, while IL-4 stimulated a small, but
significant, increase in proline in the presence of excess ornithine,
the increase was only 2-fold, not nearly as dramatic as the 13-fold
increase observed in arginine-enriched cultures (Fig. 9A).
Moreover, even unstimulated macrophages increased proline production
10-fold (Fig. 9B) compared with control macrophages when
provided the arginase substrate, arginine (Fig. 9A).
Finally, incubation of macrophages with the anti-fibrotic cytokine
IFN- (4) failed to inhibit proline production,
providing additional proof that cytokines have little or no influence
on OAT activity. As such, the data from these experiments demonstrate
that proline production is both significant in macrophages and tightly
regulated by the type 2 cytokine-dependent activation of arginase. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, IL-1, and TNF- stimulate NO
production by macrophages and therefore serve as the key signals that
promote the development of classically activated macrophages
(7). In contrast, recent in vitro experiments demonstrate
that several Th2-associated cytokines, including IL-4, IL-13, and
IL-10, are involved in the generation of alternatively activated cells,
characterized by up-regulation of Arg-1, the hepatic isoform of
arginase (8, 9, 10). These in vitro data suggest that the
reciprocal pattern of L-arginine metabolism exhibited by
activated macrophages might play an important role in regulating their
effector functions during immune responses. Nevertheless, previous to
this study, there was no direct evidence that these mechanisms were
operating or important in vivo.
Our studies conducted in the schistosome lung granuloma model in
cytokine-deficient mice demonstrate a strict requirement for type 2
cytokines in the inducible expression and activation of Arg-1 in vivo,
and in agreement with in vitro data (9), IL-4 and IL-13
are the major arginase inducers (Figs. 1 and 2). The results in
IL-10-deficient mice show that IL-10, while important for polarizing
the egg-specific Th2 response, is not essential to generate
alternatively activated, Arg-1-expressing cells, although synergistic
effects between IL-10 and IL-4/IL-13 remain possible. This conclusion
is also supported by our finding that IL-10 induces only very marginal
production of proline by macrophages in vitro (Fig. 9A).
Consistent with these observations, little induction of Arg-1
expression was observed in M. avium-infected mice even after
4 wk of infection (Fig. 4). Instead, NOS-2 remained the dominant,
although not exclusive, response in infected mice. Modest arginase
activity was reported in IFN-/TNF--stimulated macrophages, a
finding that was attributed to autocrine production of IL-10 by the
activated cells (10). Of interest, although IFN- is the
dominant response during M. avium infection, significant
production of IL-10 has also been reported (34), which may
explain the low level arginase activity in the lungs of these mice.
Thus, IL-10 could induce arginase to counterbalance NOS-2 activity
during Th1 responses. In general, the incidence of pulmonary fibrosis
during mycobacteria infection is low, although there might be strain
differences. Interestingly, data from murine models suggest that the
fibrotic response in mycobacteria infections may be attributable to an
underlying type 2 cytokine response (35, 36). Regardless,
these data complement the findings with schistosome eggs and
demonstrate that NOS-2/Arg-1 expression is tightly regulated by type
1/type 2 cytokines. Together, these results demonstrate for the first
time the differential regulation of NOS-2 and Arg-1 by type 1/type 2
cytokines at sites of pathogen-induced inflammation.
Surprisingly, although the production of IFN- was markedly
up-regulated in schistosome egg-injected IL-10 KO mice, Arg-1 was
induced to the same extent as in WT animals (Figs. 1 and 3). This was
somewhat unexpected, given the established inhibitory role played by
IFN- on Arg-1 activity (8). Nevertheless, these animals
displayed no impairment of their type 2 cytokine response (IL-4/IL-13),
which could explain the maintenance of both Arg-1 expression and
granuloma formation (Fig. 1). To test this hypothesis, we sensitized WT
and IL-10-deficient mice with eggs and IL-12 before the i.v. egg
challenge. In these experiments IL-10 KO mice showed a marked reduction
in Arg-1 activity and a corresponding decrease in Th2-type cytokine
expression and granuloma formation (Fig. 3). These studies suggest that
both the timing and the degree of exposure to Th1 and Th2 cytokines
play important roles in determining the effector cell phenotype. Thus,
the initial phase of an immune response is especially critical for
determining the effector cell phenotype as well as the magnitude of the
ensuing inflammatory response. Recent studies with rat macrophages also
support this conclusion (37, 38). These findings also
demonstrate that the degree of NOS-2 vs Arg-1 expression in vivo is a
much better predictor of the overall magnitude of schistosome
egg-induced pathology than is the relative dominance of type 1/type 2
cytokines in vivo.
Although we do not yet know the exact mechanism by which arginase
regulates the granulomatous response in murine schistosomiasis or which
cells besides macrophages express the enzyme, peak granuloma formation
correlated with high arginase activity in every case. In contrast,
NOS-2 was induced in mice sensitized with eggs and IL-12 (Fig. 6A), and the animals developed smaller and much less
fibrotic granulomas when subsequently infected (5).
Suppressing the type 2 response in NOS-2-deficient animals, in contrast
to WT animals, completely failed to reduce egg-induced liver pathology
and actually worsened the reaction (5). The inability to
down-regulate pathology in the type 1-deviated NOS-2-deficient mice may
be explained in part by the lack of
L-hydroxyarginine, the most potent
physiologic inhibitor of arginase (39, 40). It is
synthesized by NOS-2 during the oxidation of arginine to NO (Fig. 7),
but would be absent in NOS-2-deficient mice. Therefore, direct
competition for arginine is eliminated in NOS-2-deficient mice, as is
the arginase inhibitory activity of
L-hydroxyarginine, which could increase the
arginase-dependent production of L-ornithine and,
in turn, polyamine and proline levels (Fig. 7). Indeed, this hypothesis
is strongly supported by the fact that proline production by
alternatively activated macrophages is blocked by
L-hydroxyarginine (Fig. 9A) and is
regulated exclusively by the availability of
L-ornithine (Fig. 9B).
To directly test the involvement of the arginase biosynthetic pathway
in the pathogenesis of schistosomiasis, we blocked the
L-ornithine-dependent synthesis of polyamines with a
specific inhibitor. Here, for the first time, we demonstrate that
arginase activity is functionally related to the development of
granulomatous pathology. When ODC was inhibited in infected mice,
fibrosis and granuloma size increased significantly (Fig. 8). We
hypothesize that by blocking ODC, competition between ODC and OAT for
L-ornithine is eliminated. This would favor the utilization
of L-ornithine by OAT, thereby increasing the synthesis of
prolines, which could explain the increase in collagen deposition (Fig. 7). Early studies of schistosomiasis pathology showed that liver
fibrosis is directly influenced by the availability of proline
(41, 42). We now provide a mechanistic explanation for
these findings by showing a direct connection between the immune
response and the arginine metabolic pathways. As our data clearly
demonstrate, IL-4/IL-13-activated macrophages are an important source
of proline (Fig. 9), and by regulating the availability of this amino
acid, macrophages may act as key regulators of the fibrotic reaction.
Nevertheless, at this time we cannot exclude the possible contributions
of other proline-expressing cells.
Type 2 cytokine-controlled arginase activity may also serve to
counter-regulate the tissue-destructive potential of excessive NO
production (8, 43, 44, 45). In some situations it may also
promote the survival of pathogens by limiting the ability of activated
macrophages to kill invading organisms (46). Macrophages
pretreated with IL-4 or IL-13 become refractory to IFN-/TNF-
stimulation and are incapable of producing NO due to substrate
depletion mediated by arginase (8, 45). In these
macrophages, NOS-2 expression is up-regulated normally in response to
IFN-, which suggests that substrate competition is the primary
explanation for the inability of type 2 cytokine-primed macrophages to
produce NO. Strikingly, excess arginine can restore NO production and
killing of Toxoplasma gondii by IFN--activated
IL-4-pretreated cells (45). Thus, cytokine-mediated
regulation of Arg-1 expression might be an important mechanism to
control the effector functions of classically activated macrophages and
prevent NO-mediated immunopathology (47, 48, 49).
Extrahepatic arginase activity has also been observed during wound
healing (50). Alternatively activated macrophages can
support the healing process by providing proline (Fig. 9), which is
required for collagen synthesis, and/or by increasing polyamine-induced
cell proliferation (51, 52). Our findings, when viewed
together, demonstrate that macrophages as well as other
NOS-2/Arg-1-expressing cells play key roles in the pathogenesis of
chronic inflammatory diseases. The data also suggest that once the
functional phenotype of downstream effector cells is determined, the Th
response may have only a limited effect on the development of
granulomatous pathology. In mice several studies now indicate that the
balance between arginase/NOS-2 activity is an important mechanism
controlling the functionality of macrophages (5, 8, 9, 45, 46, 53). Our results add to these findings and demonstrate that,
depending on the specific inflammatory response in question, excess
activation of Arg-1 or NOS-2 can be either tissue protective or
destructive. Because therapies for chronic inflammatory diseases
typically begin long after the initiation of an immune response, novel
strategies that target important effector cell functions, such as those
described here, may prove more effective than traditional approaches
targeting Th activity.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 M.H. and M.M. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Thomas A. Wynn, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 7 Center Drive, Room 7/318, Bethesda, MD 20892. E-mail address: twynn{at}niaid.nih.gov
4 Abbreviations used in this paper: NOS-2, NO synthase-2; Arg-1, arginase-1; BMM, bone marrow-derived macrophages; DFMO, difluoromethylornithine; HPRT, hypoxanthine-phosphoribosyltransferase; KO, knockout; OAT, ornithine-aminotransferase; ODC, ornithine-aminodecarboxylase; SEA, soluble egg Ag; WT, wild type.
Received for publication July 19, 2001. Accepted for publication October 1, 2001.
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, IL-12, and TNF- are required to maintain reduced liver pathology in mice vaccinated with Schistosoma mansoni eggs and IL-12. J. Immunol. 161:4201. Adrenoceptor regulation of macrophage arginase activity. Surgery 127:412.[Medline]
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 T. Nakano, H. Inoue, S. Fukuyama, K. Matsumoto, M. Matsumura, M. Tsuda, T. Matsumoto, H. Aizawa, and Y. Nakanishi Niflumic Acid Suppresses Interleukin-13-induced Asthma Phenotypes Am. J. Respir. Crit. Care Med., June 1, 2006; 173(11): 1216 - 1221. [Abstract] [Full Text] [PDF]
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A. Yeramian, L. Martin, N. Serrat, L. Arpa, C. Soler, J. Bertran, C. McLeod, M. Palacin, M. Modolell, J. Lloberas, et al. Arginine Transport via Cationic Amino Acid Transporter 2 Plays a Critical Regulatory Role in Classical or Alternative Activation of Macrophages J. Immunol., May 15, 2006; 176(10): 5918 - 5924. [Abstract] [Full Text] [PDF]
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M. Baumgart, F. Tompkins, J. Leng, and M. Hesse Naturally Occurring CD4+Foxp3+ Regulatory T Cells Are an Essential, IL-10-Independent Part of the Immunoregulatory Network in Schistosoma mansoni Egg-Induced Inflammation J. Immunol., May 1, 2006; 176(9): 5374 - 5387. [Abstract] [Full Text] [PDF]
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L. Martin, M. Comalada, L. Marti, E. I. Closs, C. L. MacLeod, R. Martin del Rio, A. Zorzano, M. Modolell, A. Celada, M. Palacin, et al. Granulocyte-macrophage colony-stimulating factor increases L-arginine transport through the induction of CAT2 in bone marrow-derived macrophages Am J Physiol Cell Physiol, May 1, 2006; 290(5): C1364 - C1372. [Abstract] [Full Text] [PDF]
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A. Prasse, D. V. Pechkovsky, G. B. Toews, W. Jungraithmayr, F. Kollert, T. Goldmann, E. Vollmer, J. Muller-Quernheim, and G. Zissel A Vicious Circle of Alveolar Macrophages and Fibroblasts Perpetuates Pulmonary Fibrosis via CCL18 Am. J. Respir. Crit. Care Med., April 1, 2006; 173(7): 781 - 792. [Abstract] [Full Text] [PDF]
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R. M. Reiman, R. W. Thompson, C. G. Feng, D. Hari, R. Knight, A. W. Cheever, H. F. Rosenberg, and T. A. Wynn Interleukin-5 (IL-5) Augments the Progression of Liver Fibrosis by Regulating IL-13 Activity Infect. Immun., March 1, 2006; 74(3): 1471 - 1479. [Abstract] [Full Text] [PDF]
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P. G. Woodruff, L. L. Koth, Y. H. Yang, M. W. Rodriguez, S. Favoreto, G. M. Dolganov, A. C. Paquet, and D. J. Erle A Distinctive Alveolar Macrophage Activation State Induced by Cigarette Smoking Am. J. Respir. Crit. Care Med., December 1, 2005; 172(11): 1383 - 1392. [Abstract] [Full Text] [PDF]
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C. Rozenfeld, R. Martinez, S. Seabra, C. Sant'Anna, J. G. R. Goncalves, M. Bozza, V. Moura-Neto, and W. De Souza Toxoplasma gondii Prevents Neuron Degeneration by Interferon-{gamma}-Activated Microglia in a Mechanism Involving Inhibition of Inducible Nitric Oxide Synthase and Transforming Growth Factor-{beta}1 Production by Infected Microglia Am. J. Pathol., October 1, 2005; 167(4): 1021 - 1031. [Abstract] [Full Text] [PDF]
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R. D. Stout, C. Jiang, B. Matta, I. Tietzel, S. K. Watkins, and J. Suttles Macrophages Sequentially Change Their Functional Phenotype in Response to Changes in Microenvironmental Influences J. Immunol., July 1, 2005; 175(1): 342 - 349. [Abstract] [Full Text] [PDF]
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L. Brys, A. Beschin, G. Raes, G. H. Ghassabeh, W. Noel, J. Brandt, F. Brombacher, and P. D. Baetselier Reactive Oxygen Species and 12/15-Lipoxygenase Contribute to the Antiproliferative Capacity of Alternatively Activated Myeloid Cells Elicited during Helminth Infection J. Immunol., May 15, 2005; 174(10): 6095 - 6104. [Abstract] [Full Text] [PDF]
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M. Munder, F. Mollinedo, J. Calafat, J. Canchado, C. Gil-Lamaignere, J. M. Fuentes, C. Luckner, G. Doschko, G. Soler, K. Eichmann, et al. Arginase I is constitutively expressed in human granulocytes and participates in fungicidal activity Blood, March 15, 2005; 105(6): 2549 - 2556. [Abstract] [Full Text] [PDF]
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G. Raes, L. Brys, B. K. Dahal, J. Brandt, J. Grooten, F. Brombacher, G. Vanham, W. Noel, P. Bogaert, T. Boonefaes, et al. Macrophage galactose-type C-type lectins as novel markers for alternatively activated macrophages elicited by parasitic infections and allergic airway inflammation J. Leukoc. Biol., March 1, 2005; 77(3): 321 - 327. [Abstract] [Full Text] [PDF]
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B. Arendse, J. Van Snick, and F. Brombacher IL-9 Is a Susceptibility Factor in Leishmania major Infection by Promoting Detrimental Th2/Type 2 Responses J. Immunol., February 15, 2005; 174(4): 2205 - 2211. [Abstract] [Full Text] [PDF]
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S. Donnelly, S. M. O'Neill, M. Sekiya, G. Mulcahy, and J. P. Dalton Thioredoxin Peroxidase Secreted by Fasciola hepatica Induces the Alternative Activation of Macrophages Infect. Immun., January 1, 2005; 73(1): 166 - 173. [Abstract] [Full Text] [PDF]
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M. G. Nair, I. J. Gallagher, M. D. Taylor, P. Loke, P. S. Coulson, R. A. Wilson, R. M. Maizels, and J. E. Allen Chitinase and Fizz Family Members Are a Generalized Feature of Nematode Infection with Selective Upregulation of Ym1 and Fizz1 by Antigen-Presenting Cells Infect. Immun., January 1, 2005; 73(1): 385 - 394. [Abstract] [Full Text] [PDF]
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K. Kwong, R. A. Vaishnav, Y. Liu, N. Subhedar, A. J. Stromberg, M. L. Getchell, and T. V. Getchell Target ablation-induced regulation of macrophage recruitment into the olfactory epithelium of Mip-1{alpha}-/- mice and restoration of function by exogenous MIP-1{alpha} Physiol Genomics, December 15, 2004; 20(1): 73 - 86. [Abstract] [Full Text] [PDF]
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 V. Bansal, P. Rodriguez, G. Wu, D. C. Eichler, J. Zabaleta, F. Taheri, and J. B. Ochoa Citrulline Can Preserve Proliferation and Prevent the Loss of CD3 {zeta} Chain Under Conditions of Low Arginine JPEN J Parenter Enteral Nutr, November 1, 2004; 28(6): 423 - 430. [Abstract] [Full Text] [PDF]
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P. Misson, S. van den Brule, V. Barbarin, D. Lison, and F. Huaux Markers of macrophage differentiation in experimental silicosis J. Leukoc. Biol., November 1, 2004; 76(5): 926 - 932. [Abstract] [Full Text] [PDF]
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N. E. King, M. E. Rothenberg, and N. Zimmermann Arginine in Asthma and Lung Inflammation J. Nutr., October 1, 2004; 134(10): 2830S - 2836S. [Abstract] [Full Text] [PDF]
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M. Kaviratne, M. Hesse, M. Leusink, A. W. Cheever, S. J. Davies, J. H. McKerrow, L. M. Wakefield, J. J. Letterio, and T. A. Wynn IL-13 Activates a Mechanism of Tissue Fibrosis That Is Completely TGF-{beta} Independent J. Immunol., September 15, 2004; 173(6): 4020 - 4029. [Abstract] [Full Text] [PDF]
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A.-L. Pauleau, R. Rutschman, R. Lang, A. Pernis, S. S. Watowich, and P. J. Murray Enhancer-Mediated Control of Macrophage-Specific Arginase I Expression J. Immunol., June 15, 2004; 172(12): 7565 - 7573. [Abstract] [Full Text] [PDF]
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D. P. Beiting, S. K. Bliss, D. H. Schlafer, V. L. Roberts, and J. A. Appleton Interleukin-10 Limits Local and Body Cavity Inflammation during Infection with Muscle-Stage Trichinella spiralis Infect. Immun., June 1, 2004; 72(6): 3129 - 3137. [Abstract] [Full Text] [PDF]
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S. Duleu, P. Vincendeau, P. Courtois, S. Semballa, I. Lagroye, S. Daulouede, J.-L. Boucher, K. T. Wilson, B. Veyret, and A. P. Gobert Mouse Strain Susceptibility to Trypanosome Infection: An Arginase-Dependent Effect J. Immunol., May 15, 2004; 172(10): 6298 - 6303. [Abstract] [Full Text] [PDF]
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P. Kropf, M. A. Freudenberg, M. Modolell, H. P. Price, S. Herath, S. Antoniazi, C. Galanos, D. F. Smith, and I. Muller Toll-Like Receptor 4 Contributes to Efficient Control of Infection with the Protozoan Parasite Leishmania major Infect. Immun., April 1, 2004; 72(4): 1920 - 1928. [Abstract] [Full Text] [PDF]
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L. C. Gavrilescu, B. A. Butcher, L. Del Rio, G. A. Taylor, and E. Y. Denkers STAT1 Is Essential for Antimicrobial Effector Function but Dispensable for Gamma Interferon Production during Toxoplasma gondii Infection Infect. Immun., March 1, 2004; 72(3): 1257 - 1264. [Abstract] [Full Text] [PDF]
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M. Hesse, C. A. Piccirillo, Y. Belkaid, J. Prufer, M. Mentink-Kane, M. Leusink, A. W. Cheever, E. M. Shevach, and T. A. Wynn The Pathogenesis of Schistosomiasis Is Controlled by Cooperating IL-10-Producing Innate Effector and Regulatory T Cells J. Immunol., March 1, 2004; 172(5): 3157 - 3166. [Abstract] [Full Text] [PDF]
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S. El-Gayar, H. Thuring-Nahler, J. Pfeilschifter, M. Rollinghoff, and C. Bogdan Translational Control of Inducible Nitric Oxide Synthase by IL-13 and Arginine Availability in Inflammatory Macrophages J. Immunol., November 1, 2003; 171(9): 4561 - 4568. [Abstract] [Full Text] [PDF]
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N. G. Sandler, M. M. Mentink-Kane, A. W. Cheever, and T. A. Wynn Global Gene Expression Profiles During Acute Pathogen-Induced Pulmonary Inflammation Reveal Divergent Roles for Th1 and Th2 Responses in Tissue Repair J. Immunol., October 1, 2003; 171(7): 3655 - 3667. [Abstract] [Full Text] [PDF]
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A. C. La Flamme, K. Ruddenklau, and B. T. Backstrom Schistosomiasis Decreases Central Nervous System Inflammation and Alters the Progression of Experimental Autoimmune Encephalomyelitis Infect. Immun., September 1, 2003; 71(9): 4996 - 5004. [Abstract] [Full Text] [PDF]
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P. C. Rodriguez, A. H. Zea, J. DeSalvo, K. S. Culotta, J. Zabaleta, D. G. Quiceno, J. B. Ochoa, and A. C. Ochoa L-Arginine Consumption by Macrophages Modulates the Expression of CD3{zeta} Chain in T Lymphocytes J. Immunol., August 1, 2003; 171(3): 1232 - 1239. [Abstract] [Full Text] [PDF]
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Y. Liu, J. A. Van Ginderachter, L. Brys, P. De Baetselier, G. Raes, and A. B. Geldhof Nitric Oxide-Independent CTL Suppression during Tumor Progression: Association with Arginase-Producing (M2) Myeloid Cells J. Immunol., May 15, 2003; 170(10): 5064 - 5074. [Abstract] [Full Text] [PDF]
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L. Martinez-Pomares, D. M. Reid, G. D. Brown, P. R. Taylor, R. J. Stillion, S. A. Linehan, S. Zamze, S. Gordon, and S. Y. C. Wong Analysis of mannose receptor regulation by IL-4, IL-10, and proteolytic processing using novel monoclonal antibodies J. Leukoc. Biol., May 1, 2003; 73(5): 604 - 613. [Abstract] [Full Text] [PDF]
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M. G. Chiaramonte, M. Mentink-Kane, B. A. Jacobson, A. W. Cheever, M. J. Whitters, M. E.P. Goad, A. Wong, M. Collins, D. D. Donaldson, M. J. Grusby, et al. Regulation and Function of the Interleukin 13 Receptor {alpha} 2 During a T Helper Cell Type 2-dominant Immune Response J. Exp. Med., March 17, 2003; 197(6): 687 - 701. [Abstract] [Full Text] [PDF]
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D. M. Mosser The many faces of macrophage activation J. Leukoc. Biol., February 1, 2003; 73(2): 209 - 212. [Full Text] [PDF]
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C. Stempin, L. Giordanengo, S. Gea, and F. Cerban Alternative activation and increase of Trypanosoma cruzi survival in murine macrophages stimulated by cruzipain, a parasite antigen J. Leukoc. Biol., October 1, 2002; 72(4): 727 - 734. [Abstract] [Full Text] [PDF]
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A. Mencacci, C. Montagnoli, A. Bacci, E. Cenci, L. Pitzurra, A. Spreca, M. Kopf, A. H. Sharpe, and L. Romani CD80+Gr-1+ Myeloid Cells Inhibit Development of Antifungal Th1 Immunity in Mice with Candidiasis J. Immunol., September 15, 2002; 169(6): 3180 - 3190. [Abstract] [Full Text] [PDF]
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). *, Group is significantly
different from uninfected controls. Pulmonary expression of NOS-2
protein was also evaluated by Western blot (E).
