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Department of Medical Physiology, Cardiovascular Research Institute, Texas A&M University System Health Science Center, College Station, TX 77843; and
Department of Chemical Engineering, University of California, Los Angeles, CA 90095
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to iNOS, arginase is another major L-arginine-consuming enzyme in macrophages that converts L-arginine to L-ornithine and urea. There are two isoforms of arginase, arginase I and II. Arginase I is expressed most abundantly, but not exclusively, in the liver (15), while arginase II is expressed in the kidney and many other extrahepatic tissues (16, 17, 18, 19). The main function of the hepatic arginase is to detoxify ammonia (15). However, the biological role of the extrahepatic arginase remains unclear. In macrophages, arginase expression and activity can be induced by LPS (17, 20), TGF-ß (21), and cytokines such as IL-4 and IL-10 (22, 23, 24, 25). However, the biological significance of its induction has not been defined. Because both arginase and iNOS exist in activated macrophages, diversion of their common substrate, L-arginine, from the iNOS pathway to the arginase pathway may afford macrophage arginase a regulatory role in NO production. This contention is supported by our previous studies showing that NO production in activated macrophages is enhanced by blocking arginase activity (26). However, whether arginase is physiologically or pathophysiologically relevant to immune responses where NO production is involved remains unclear. Since IL-13 has been shown to play a counteracting role in the NO-mediated cytotoxic effect of macrophages (11, 13), we tested the hypothesis that IL-13 increases the expression and activity of arginase and thus suppresses NO production. In addition, the signaling mechanisms responsible for the arginase induction by IL-13 were investigated.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All chemicals and drugs, unless otherwise noted, were purchased from Sigma (St. Louis, MO).
Cell culture and treatments
The murine macrophage cell line J774A.1 was obtained from American Type Culture Collection (Manassas, VA) and maintained in 60-mm culture dishes with DMEM supplemented with 10% FBS (Summit Biotechnology, Ft. Collins, CO), 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B, and 50 µg/ml gentamicin at 37°C under a humidified 10% CO2 atmosphere. Medium was changed daily, and cells were passaged after confluence by trypsinization with Dulbecco’s PBS containing 0.25% trypsin (Life Technologies, Rockville, MD) and 0.02% EDTA. Experiments were performed 2 days after the cells reached confluence. To carry out the experiments using a physiological level of L-arginine, the cell culture medium was replaced with modified RPMI 1640 medium (Life Technologies) containing 100 µM L-arginine, 4% FBS, 200 µM glutamine, 10 mM HEPES, and the antibiotics as described above on the day of the experiment.
To study the concentration-dependent effect of IL-13 on arginase activity in macrophages, cells were treated with different concentrations of IL-13 (0, 1, 5, 10, 15, and 20 ng/ml; Intergen, Purchase, NY) for 18 h. In another set of experiments, cells were incubated with 5 ng/ml IL-13 for different periods of time (1, 2, 4, 8, 12, 18, and 24 h) to examine the time course of arginase activation. To investigate the signaling pathway of arginase activation, the effects of various activators or inhibitors of signaling kinases were examined. In this regard, cells were pretreated with the activators or inhibitors for 6 h, followed by an 18-h incubation with IL-13 (5 ng/ml). Parallel control experiments were performed by adding the vehicle solution (i.e., solvent for pharmacologic agents) to the cells with an identical incubation time as that used in the experimental groups, and cell vitality (>95%) was assessed by the trypan blue exclusion method to ensure that the observed effects were not due to cell death. At the indicated times, cells were harvested for assay of arginase activity or for Western blot analysis of arginase and iNOS expression as described below.
To study the effect of IL-13 on NO production, macrophages were treated with LPS (from Escherichia coli 0.111:B4; 1 µg/ml) for iNOS induction in the presence and the absence of IL-13 (5 ng/ml). After 18 h of treatment, nitrite content in the cell culture medium was measured as described below and was used as an index of NO production. To verify the involvement of arginase in the regulation of NO production, a set of parallel experiments was conducted in the presence of an arginase inhibitor, L-norvaline (20 mM).
Arginase assay
To prepare cell lysate for arginase assay, cells were first
rinsed with ice-cold Dulbecco’s PBS twice after each specified
treatment and then scraped into 300 µl of lysis buffer containing 50
mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 0.1 mM EGTA, 1 µg/ml leupeptin, 1
µg/ml aprotinin, and 0.1 mM PMSF. Cells were lysed by sonication at
20 kHz (Sonic & Materials, Newtown, CT) for 30 s (10 s/cycle).
Arginase activity in the cell lysates was measured as described
previously (26). In brief, cell lysate (50 µl) was added
to 50 µl of Tris-HCl (50 mM; pH 7.5) containing 10 mM
MnCl2. Macrophage arginase was then activated by
heating this mixture at 55–60°C for 10 min. The hydrolysis reaction
of L-arginine by arginase was conducted by incubating the
mixture containing activated arginase with 50 µl of
L-arginine (0.5 M; pH 9.7) at 37°C for 1 h and was
stopped by adding 400 µl of the acid solution mixture
(H2SO4:H3PO4:H2O,
1:3:7). For colorimetric determination of urea,
-isonitrosopropiophenone (25 µl, 9% in absolute ethanol) was
added, and the mixture was heated at 100°C for 45 min. After placing
the sample in the dark for 10 min at room temperature, the urea
concentration was determined spectrophotometrically by the absorbance
at 550 nm measured with a microplate reader (Molecular Devices,
Sunnyvale, CA). The amount of the urea produced was used as an index
for arginase activity.
NO measurement
It has been shown that the primary decomposition product of NO in hemoglobin-free solution is nitrite (27). Our previous studies confirmed this finding and reported that >95% of total NO released from activated macrophages was converted to nitrite in the culture medium (26). Therefore, nitrite accumulation in the supernatant of cell culture was determined by a chemiluminescence NO analyzer (Sievers Instruments, Boulder, CO) and was used as an index of NO production. At the end of each respective experiment, the supernatant of cell culture medium was collected for NO analysis. The collected sample (100 µl) was injected into a reflux chamber containing glacial acetic acid and 1% potassium iodide at room temperature. Under these conditions, nitrite is quantitatively converted to NO. The gaseous NO was then purged into the chemiluminescence NO analyzer and quantified by reference to sodium nitrite standards.
RNA isolation and RT-PCR analysis
Expression of arginase I and arginase II genes was distinguished using RT-PCR. Total RNA was isolated with TRIzol reagent (10 ml/100 mm plate; Life Technologies) following the manufacturer’s procedures. The RNA from each plate was dissolved in 50 µl of RNase-free water and stored at -70°C. Rat liver and kidney tissues were used as positive controls for the arginase I gene and arginase II genes, respectively. First-strand cDNA synthesis was performed in a 20-µl volume using 0.1 µg/µl total RNA, reverse transcriptase (RT), and a gene-specific 3'-oligonucleotide to prime the reaction (Thermoscript RT, Life Technologies). To determine whether the PCR was amplifying genomic DNA, a first-strand cDNA synthesis reaction was performed with and without RT. Two microliters of RT cDNA sample were used to perform a PCR in a 50-µl volume containing 5 µl of 10x cDNA synthesis buffer, 2 µl of 10 mM dNTP, 0.1 µg/µl of each primer, and 2.5 U of Taq DNA polymerase (Roche, Indianapolis, IN). Each primer was designed from mouse sequences and was 24 nucleotides in length (arginase I: sense, 5'-GGG CTG GAC CCA GCA TTC ACC CCG-3'; antisense, 5'-TCA CTT AGG TGG TTT AAG GTA GTC-3'; arginase II: sense, 5'-GAC CCT AAA CTG GCT CCA GCC ACA-3'; antisense, 5'-CTA AAT TCT CAC ACA TTC TTC ATT-3'; GAPDH: sense, 5'-ACA GCC GCA TCT TCT TGT GCA GTG-3'; antisense, 5'-GGC CTT GAC TGT GCC GTT GAA TTT-3'). The PCR was initiated with a denaturation step at 94°C (2 min), followed by 30 cycles of 94°C for 15 s (denaturation), 60°C for 15 s (annealing), and 72°C for 15 s (extension). The PCR was terminated with a final step at 72°C (7 min). The PCR-amplified products were electrophoresed on an 1.8% agarose gel and visualized with ethidium bromide staining.
Immunoblotting
Expression of arginase I and iNOS was detected with the mAbs against mouse liver arginase (arginase I) and mouse macrophage iNOS, respectively (Transduction Laboratories, Lexington, KY). The Western blotting was performed following the manufacturer’s instructions. In brief, cell lysates were prepared as described above, and equal amounts (25 µg) of proteins from each sample were subjected to 10% SDS-PAGE. Following electrophoresis, proteins were electrotransferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). The nitrocellulose membranes were incubated with the primary mAbs against arginase I or iNOS and then with an HRP-conjugated anti-mouse IgG secondary Ab (Transduction Laboratories). The proteins were visualized using the enhanced chemiluminescence detection kits (Amersham Pharmacia Biotech, Piscataway, NJ). The activation of tyrosine kinases and p38 mitogen-activated protein kinase (MAPK) was also investigated by immunoblotting analysis with phosphotyrosine mAb (P-Tyr-100, New England Biolabs, Beverly, MA) and phospho-p38 MAPK polyclonal Ab (New England Biolabs), respectively. The blotting procedures were as described above, except the anti-rabbit IgG secondary Ab (Transduction Laboratories) was used for detecting phosphorylated p38 MAPK. Cell extracts from C-6 glioma cells prepared with anisomycin treatment were provided by New England Biolabs and were used as a positive control for phosphorylated p38 MAPK.
cAMP measurement
Intracellular levels of cAMP were determined by a cAMP enzyme immunoassay kit (Assay Designs, Ann Arbor, MI) following the manufacturer’s instructions.
Protein determination
For all experiments, protein concentrations from each plate of cells were determined by a bicinchoninic acid protein assay (Pierce, Rockford, IL) after lysing the cells. The protein concentrations were used as a basis to normalize the results of each experiment.
Statistical analysis
Results are presented as the mean ± SEM for at least three independent experiments performed in triplicate. Data analysis was performed by one-way ANOVA followed by Fisher’s protected least significant difference test using StatView 4 (Abacus Concepts, Berkeley, CA). Differences were considered statistically significant when p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The concentration- and time-dependent effects of IL-13 on
macrophage arginase activity were examined. Arginase activity
(i.e., urea production) was determined after treating the
cells with various concentrations (0–20 ng/ml) of IL-13 for 18 h.
Fig. 1
A shows that macrophages
exhibited a low resting level of arginase activity in the absence of
IL-13. Arginase activity was significantly increased by IL-13 in a
concentration-dependent manner and reached a maximum at 15 ng/ml. The
time-dependent increase in arginase activity in response to 5 ng/ml of
IL-13 is shown in Fig. 1B. Arginase activity was first
observed to increase at 2 h after the addition of IL-13 and
reached its plateau at 12 h. The arginase activity remained high
after treating the cells for 24 h (Fig. 1B). In the
following experiments, 5 ng/ml IL-13 was used to activate arginase, and
an 18-h incubation time was selected because the maximum arginase
activity was expressed at this time.
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To determine the regulatory level of the arginase activation by
IL-13, actinomycin D (0.5 µg/ml) or cycloheximide (25 µg/ml) was
applied to the cultures 30 min before the addition of IL-13 (5
ng/ml) to block gene transcription and protein translation,
respectively. Following an 18-h incubation with IL-13, cells were
harvested for determination of arginase activity as well as arginase
gene and protein expressions. Fig. 2A shows that the
IL-13-induced arginase activity was completely abolished by actinomycin
D or cycloheximide. To examine which isoform of arginase was
responsible for the increased arginase activity, RT-PCR was performed,
and the liver and kidney tissues from rats were used as positive
controls for arginase I and arginase II, respectively. Fig. 2B shows that arginase I mRNA was expressed in the resting
macrophages and that IL-13 increased the level of arginase I gene
expression. In contrast, arginase II was not detected in either control
or IL-13-treated cells. Western blotting against arginase I protein
shows that arginase expression was below the detectable level in
resting macrophages (liver cell lysate was used as a positive control),
but was induced by IL-13, as demonstrated by the presence of arginase
monomers of 35 and 38 kDa (Fig. 2C). The expression of
arginase I protein was also abolished by either actinomycin D or
cycloheximide.
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The cAMP-responsive element (CRE) and glucocorticoid-responsive
element have been located in the promoter region of the liver arginase
gene and are responsible for the arginase induction (28).
It is unclear whether arginase expression in macrophages is regulated
in a similar manner. To address this issue, macrophage arginase
activity was studied after treating the cells with a permeable cAMP
analogue, 8-Br-cAMP (Research Biochemicals International, Natick, MA)
or with dexamethasone. Incubating macrophages with 8-Br-cAMP (10 µM)
for 24 h caused a 5-fold increase in arginase activity, which is
about 60% of that induced by IL-13 (Fig. 3). A higher concentration of 8-Br-cAMP
(100 µM) did not further increase arginase activity (data not shown).
The effect of IL-13 on arginase activity was markedly increased by
pretreating the cells with 8-Br-cAMP (10 µM) for 6 h.
Dexamethasone (3 µM) had no effect on either basal arginase activity
or IL-13-induced arginase activity (Fig. 3). Furthermore, dexamethasone
did not affect the increased arginase activity by IL-13 in
8-Br-cAMP-treated macrophages (Fig. 3). These results suggested that
cAMP, rather than glucocorticoid, was involved in arginase induction in
macrophages associated with IL-13 stimulation.
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. The PKA-specific inhibitor KT5720 (50
nM; Calbiochem, San Diego, CA) had no effect on the resting arginase
activity, but abolished the activity and expression induced by IL-13 or
8-Br-cAMP. Furthermore, the synergistic effect of 8-Br-cAMP and IL-13
on arginase activity and expression was also abolished by KT5720 (Fig. 4). Actinomycin D (0.5 µg/ml) and cycloheximide (25 µg/ml)
completely blocked IL-13-induced arginase activity and expression in
the cells pretreated with 8-Br-cAMP (Fig. 4).
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Because the results presented above indicated that activation of
the PKA pathway was obligatory for induction of arginase by IL-13 (Fig. 4
), the effect of IL-13 on intracellular levels of cAMP was
investigated to gain further insight into the role of cAMP in arginase
activation. As shown in Fig. 5, incubation of macrophages with IL-13 resulted in time-dependent changes
in the intracellular cAMP level. IL-13 produced a transient increase
(
6-fold) in cellular cAMP within 10 min. Although the level of cAMP
started to decline after 10 min of incubation, its content remained
above resting levels for 2 h and then gradually returned toward
the resting value after 5 h of IL-13 treatment (Fig. 5).
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To investigate the possible involvement of other signaling
kinases, e.g., protein kinase C (PKC), MAPK, and tyrosine kinases in
arginase activation, cells were pretreated with respective inhibitors
of these signaling kinases for 6 h before an 18-h incubation with
IL-13. Three different tyrosine kinase inhibitors, tyrphostins (20
µM; Life Technologies), herbimycin A (0.5 µM; Calbiochem), and
genistein (50 µM; Calbiochem), were used to examine the involvement
of tyrosine phosphorylation in IL-13-induced arginase activation. All
three tyrosine kinase inhibitors significantly attenuated IL-13-induced
arginase activation to a similar extent (Fig. 6A). In contrast, the PKC
inhibitor calphostin C (50 nM; Calbiochem) did not cause a significant
change in arginase activity induced by IL-13 (Fig. 6A).
IL-13-induced arginase activity was also not affected by a specific
p42/p44 MAPK kinase inhibitor, PD98059 (5 µM; Calbiochem), but was
attenuated by SB203580 (0.5 µM; Calbiochem), a specific inhibitor of
p38 MAPK (Fig. 6A). The combination of a tyrosine kinase
inhibitor genistein (or herbimycin A; data not shown) and the p38 MAPK
inhibitor SB203580 inhibited IL-13-induced arginase activity to a
similar extent as when tyrosine kinase inhibitors were used alone.
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B shows that proteins at
Mr of 33, 35, 54, 58, 100, 125, and
180 kDa were phosphorylated at 10 min after the IL-13 treatment. The
phosphorylation was significantly inhibited by pretreating the cells
with genistein (50 µM, 6 h). The activation of p38 MAPK was also
detected at 10 min, but not at 20 min or 1 h, after IL-13
treatment (Fig. 6C). Effects of tyrosine kinase inhibitors and p38 MAPK inhibitor on IL-13-increased intracellular cAMP
To determine the involvement of tyrosine kinases and p38 MAPK in
the signaling of cAMP elevation by IL-13, cells were pretreated with
either the tyrosine kinase inhibitors genistein or herbimycin A or with
the p38 MAPK inhibitor SB203580 for 6 h. Intracellular cAMP levels
were then determined 10 min after the addition of IL-13. Fig. 7A shows that IL-13 increased
cellular cAMP by 6- to 7-fold, which is consistent with the results
presented in Fig. 5. Pretreating the cells with genistein or herbimycin
A significantly attenuated the increase in cAMP, while SB203580 had no
effect on the cAMP elevation. Furthermore, the arginase activity
increased by 8-Br-cAMP was not affected by the inhibition of p38 MAPK
(Fig. 7B).
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To determine whether IL-13-induced arginase activation contributes
to the inhibition of NO production from activated macrophages, the
effects of IL-13 on LPS-induced NO production were examined in the
presence and the absence of a specific arginase inhibitor
L-norvaline. In this regard, macrophages were coincubated
with LPS (1 µg/ml) and IL-13 (5 ng/ml) in the presence and the
absence of L-norvaline (20 mM) for 18 h. As shown in
Fig. 8, NO production (i.e., nitrite
formation) from resting macrophages was negligible, but increased
significantly in cells treated with LPS. In addition, the increase in
NO production was associated with induction of iNOS in macrophages.
IL-13 caused an induction of arginase and a 65% reduction in NO
production from LPS-activated macrophages (Fig. 8). It should be noted
that iNOS expression was also down-regulated by IL-13.
L-Norvaline slightly, but significantly, increased
LPS-induced NO production without affecting iNOS expression.
Furthermore, IL-13-suppressed NO production was partially (70%)
restored by L-norvaline (Fig. 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study despite the basal level of arginase I gene
expression being detected in resting macrophages, arginase protein
expression and enzyme activity were relatively low in these cells.
Nevertheless, macrophage arginase can be robustly induced in response
to an appropriate stimulation. For example, macrophage arginase
activity was reported to increase 4- to 100-fold in response to IL-4,
IL-10, and TGF-ß, with a 50% effective concentration of 1–5 ng/ml
(21, 22, 23). In our study the 50% effective concentrationfor
IL-13 was about 2.5 ng/ml, and this is comparable to that reported for
other cytokines in arginase activation. Both actinomycin D and
cycloheximide completely abolished the effect of IL-13, indicating that
de novo synthesis of both mRNA and protein was necessary for the
increased arginase activity. Although both isoforms of arginase have
been detected in LPS-activated macrophages (17, 20),
results from the RT-PCR study indicated that only type I arginase was
up-regulated by IL-13 (Fig. 2). It appears that different isoforms of
the arginase gene can be induced independently in response to different
stimuli.
Earlier studies on the promoter region of liver arginase had revealed
sequences matching CRE and glucocorticoid-responsive element
(28), and the expression of liver arginase was
up-regulated by cAMP and glucocorticoid (29, 30). In
contrast to the liver cells, we found that macrophage arginase is
up-regulated by a cAMP analogue but not by dexamethasone (Fig. 3),
suggesting that the arginase activation is independent of
glucocorticoid responsiveness. It should be noted that the
concentration of dexamethasone (3 µM) used in the present study was
effective because the LPS-induced NO production by these cells was
reduced by >40% with the same level of dexamethasone in our
preliminary studies (data not shown). Although the promoter region of
arginase in macrophages has not been investigated, recent studies have
shown that dexamethasone can enhance arginase II induction in the
macrophage cell line RAW264.7 (31). This finding might
explain the ineffectiveness of dexamethasone in our study, because
arginase I was the only isoform induced by IL-13 (Fig. 2B).
Because the up-regulation of arginase by IL-13 and by a cAMP analogue
was abolished by a specific PKA inhibitor (Fig. 4), it is believed that
activation of cAMP/PKA is the primary signaling pathway responsible for
arginase induction and the subsequent increase in arginase activity by
IL-13.
Although the signal transduction pathways induced by IL-13 have not
been explored in detail, tyrosine kinases were recently suggested to
play a role in the signaling pathway following the binding of IL-13 to
its receptor (32, 33). More specifically, IL-13 was shown
to induce rapid tyrosine phosphorylation of several proteins, including
its receptor subunit IL-4R (140 kDa) (33), insulin
receptor substrate-1 (170 kDa) (33, 34), and the Janus
kinase family members JAK1 (120 kDa) (32, 33, 34), JAK2
(130 kDa) (33), and TyK-2 (135 kDa) (33, 34) in different cell types. In the present study,
immunoblotting with anti-phosphotyrosine also detected
tyrosine-phosphorylated proteins migrated at 33, 35, 54, 58, 100,
125, and 180 kDa after 10 min of IL-13 stimulation (Fig. 6B). Based on the Mr, the
proteins at 180 and 125 kDa may be insulin receptor substrate-1 and
JAK, respectively. However, further studies are required to determine
the identities of these tyrosine-phosphorylated proteins as well as
their functional correlation with the activation of arginase by IL-13.
Because adenylyl cyclase has been shown to be activated through a
tyrosine kinase-dependent pathway (35), it is possible
that activation of tyrosine kinases by IL-13 leads to arginase
transcription through cAMP/PKA-mediated activation of CRE-binding
proteins (CREB). This contention is supported by the surge of
intracellular cAMP in the early phase of IL-13 stimulation (i.e., 10
min) and the corresponding inhibition of cAMP elevation and arginase
activity by tyrosine kinase inhibitors. It seems that a sustained
increase in cAMP is not required for the arginase activation because
the increase in arginase activity significantly lagged behind (i.e.,
>2 h) the increased cAMP signal. It is worth noting that tyrosine
kinase inhibitors failed to abolish the IL-13-induced increase in cAMP
and arginase activity, but a PKA inhibitor completely eliminated the
IL-13 effect. Therefore, it is likely that the remaining elevated cAMP
mediates the arginase activation in a tyrosine kinase-independent
manner.
Interestingly, cAMP is not a potent arginase activator per se because
8-Br-cAMP (10 and 100 µM) induced only 60% of the IL-13-induced
arginase activity (Fig. 4). These results suggest that IL-13 may
activate another signal transduction system to facilitate the cAMP/PKA
pathway during IL-13 activation. For example, we found that p38 MAPK is
also partially responsible for the arginase activation by IL-13.
Furthermore, the immunoblotting analysis revealed that the activation
of p38 MAPK by IL-13 was rapid (i.e., 10 min; Fig. 6C) and
was coincident with the tyrosine kinase activation (Fig. 6B)
and cAMP elevation (Fig. 5). The ability of p38 MAPK to phosphorylate
activating transcription factor (ATF), a putative substrate of p38 MAPK
and also a member of the CREB family, suggests that p38 MAPK may play a
role in modulating the expression of genes containing CRE (36, 37). Although CREB can be activated by PKA and bind to CRE as a
homodimer, it has been shown to bind ATF to form a heterodimer, which
then stimulates gene expression (37, 38). It is possible
that the formation of this heterodimer, as a result of ATF
phosphorylation by p38 MAPK, facilitates arginase transcription through
its action on CRE. Therefore, arginase activity induced by IL-13 (i.e.,
with both the cAMP/PKA pathway and the p38 MAPK pathway activated) is
higher than that induced by 8-Br-cAMP alone (i.e., only the cAMP/PKA
pathway is activated). Under these conditions, a sustained increase in
intracellular cAMP (i.e., incubation of the cells with 8-Br-cAMP) may
further facilitate the IL-13 effect through the up-regulated p38
MAPK/CRE signaling. This may explain why priming the macrophages with
exogenous cAMP synergistically enhanced arginase induction by IL-13
(Fig. 4). On the other hand, a brief increase in cAMP by IL-13 alone
might limit the overall capacity for arginase activation. The
inhibition of arginase induction by tyrosine kinase inhibitors was not
further enhanced by blockage of the p38 MAPK pathway (Fig. 6),
suggesting that tyrosine kinases and p38 MAPK kinase are in series of
the same signaling pathway for arginase activation. Furthermore,
inhibition of p38 MAPK had no effect on either the IL-13-induced
increase in intracellular cAMP (Fig. 7A) or the
8-Br-cAMP-induced arginase activation (Fig. 7B), suggesting
that p38 MAPK signaling is in parallel to cAMP/PKA signaling. These
results further support the idea that the role of p38 MAPK is to
modulate cAMP/PKA-mediated arginase induction. On the other hand,
arginase activation was not affected by PKC and p42/p44 MAPK inhibitors
(Fig. 6), which is in agreement with the finding of Welham et al. that
IL-13 fails to activate p44/p42 MAPK in lymphohemopoietic cells
(34). Therefore, we conclude that cAMP/PKA signaling and
p38 MAPK signaling are two parallel pathways that lead to arginase
activation following the stimulation of IL-13. In addition, both
pathways are dependent on tyrosine kinase activation. While the
activation of cAMP/PKA is obligatory for IL-13 to induce arginase, the
p38 MAPK may modulate cAMP/PKA signaling for optimal activation of
arginase.
A majority of studies of the regulation of macrophage NO release have
focused on the expression of iNOS (6, 39, 40). However,
the substrate L-arginine is used not only by iNOS but also
by arginase. Therefore, the regulation of L-arginine
availability to iNOS by arginase might play a role in modulating NO
production from macrophages. Our previous study supports this
contention, because inhibition of arginase activity enhances NO
production from LPS-activated macrophages (26). Recently,
arginase activity has been shown to be up-regulated by endogenous
factors such as TGF-ß (21) and Th-2 cell-derived
cytokines IL-4 and IL-10 (22, 23, 24, 25). Interestingly, these
factors were also reported to suppress NO production (21, 22, 41), implying that arginase might be an intrinsic modulator of
NO production. However, direct evidence for the functional
interrelationship between the up-regulated arginase and the
down-regulated NO production is lacking. In the present study we
demonstrated that the NO production from LPS-activated macrophages was
attenuated by IL-13 (Fig. 8), indicating that IL-13 is a suppressor of
NO production. It should be noted that IL-13 also reduced iNOS protein
expression, a result that agrees with previous findings of iNOS gene
expression by Bogdan et al. in macrophages (11) and by
Saura et al. in mesangial cells (10). This down-regulation
of iNOS protein might also account for the observed reduction in NO
production. Nevertheless, inhibition of arginase activity by
L-norvaline significantly restored the NO production (by
70%) regardless of the suppressed iNOS expression, indicating that
the activation of arginase contributes significantly to the inhibition
of NO production by IL-13. However, L-norvaline was not
able to completely restore the reduced NO production, presumably due to
the down-regulation of iNOS expression. Therefore, we conclude that the
IL-13-reduced NO production is regulated by at least two mechanisms,
the up-regulated arginase activity and the down-regulated iNOS
expression. These results also suggest that the induced arginase, by
inhibiting NO production from activated macrophages, may contribute at
least in part to the observed anti-inflammatory effect of IL-13 in
vivo (42). It is worth noting that L-norvaline
specifically inhibits arginase activity at the enzyme kinetic level
(15, 26), and it does not affect iNOS expression, as shown
in the present study.
Recent studies have shown that IL-13 expression is elevated at sites of inflammation (43, 44). The inhibitory effect of IL-13 on NO production implies that IL-13 can be a potentially important therapeutic anti-inflammatory cytokine. Gene transfer of IL-13 has been demonstrated to improve survival in lethal endotoxemia in mice (42), presumably through its inhibitory effect on NO production. As mentioned earlier, in addition to IL-13, other anti-inflammatory cytokines, such as IL-4, IL-10, and TGF-ß, have been shown to activate arginase in macrophages. It is thus possible that activation of macrophage arginase, in response to anti-inflammatory cytokines, is used as a general mechanism in the immune system to counteract the possible overproduction of NO under pathophysiologic conditions.
We conclude that IL-13 is a potent activator of arginase in macrophages. The signalings of tyrosine kinases, cAMP/PKA, and p38 MAPK appear to be involved in the activation of arginase by IL-13. In particular, the cAMP/PKA pathway is imperative in mediating the arginase activation. It appears that the induced arginase in combination with the down-regulated iNOS might contribute to the immunosuppressive activity of IL-13 in macrophages through the inhibition of NO production.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprints requests to Dr. Lih Kuo, Department of Medical Physiology, Cardiovascular Research Institute, Texas A&M University System Health Science Center, College Station, TX 77843-1114.
3 Abbreviations used in this paper: NOS, NO synthase; iNOS, inducible NOS; CRE, cAMP-responsive element; 8-Br-cAMP, 8-bromo-cAMP; PKA, protein kinase A; PKC, protein kinase C; MAPK, mitogen-activated protein kinase; CREB, CRE-binding protein; ATF, activating transcription factor.
Received for publication December 22, 1999. Accepted for publication May 25, 2000.
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