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Department of Internal Medicine, Division of Pulmonary/Critical Care Medicine, Wayne State University School of Medicine, Detroit, MI 48201
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. We found that
mouse peritoneal resident and Con A-elicited macrophages collected on
day 3 or day 5 express RON. Acute exudate macrophages collected on day
1 did not express RON. Activation of RON inhibited LPS- and
IFN--induced macrophage nitric oxide production and iNOS mRNA
accumulation. Similar inhibition was observed also in Raw264.7
macrophage cell lines transfected with human RON cDNA. In these cells,
MSP induced RON phosphorylation concomitant with reduced iNOS mRNA
expression and protein synthesis. Further, we show that activated RON
inhibited the iNOS gene transcription activity as assessed by
chloramphenicol acetyltransferase activity in Raw264.7 cells expressing
RON. Wortmannin, a specific inhibitor of phosphatidylinositol-3 (PI-3)
kinase, prevented the inhibitory effect of RON on the iNOS gene
promoter activity and on the nitric oxide production induced by LPS and
IFN-. These effects were confirmed further by introducing a
dominant-inhibitory PI-3 kinase p85 subunit in RON-expressing Rwa264.7
cells. Taken together, our results suggest that RON is expressed in
peritoneal macrophages at later stages of inflammation. Activation of
RON by MSP in mature exudate macrophages inhibits LPS- and
IFN--induced iNOS synthesis. PI-3 kinase is an important effector
molecule required for RON-mediated inhibition of iNOS expression in
macrophages. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF-
(3), and controlled by
transcription factors such as NF-
B (4, 5) and IFN-regulatory
factor-1 (6, 7). In inflammation, NO mediates certain aspects of the
immune response and is critical for macrophage-mediated bactericidal
and tumoricidal activities (8, 9, 10). However, under pathologic
conditions, high NO output is also responsible for macrophage apoptosis
(11), autoimmune tissue damage (12, 13), and septic shock (14).
Inhibition of iNOS production significantly reduces the toxic effect of
NO and increases cell survival (11, 15). Cytokines such as TGF-ß,
IL-4, and IL-11 have been found to suppress stimulated iNOS expression
in macrophages (16, 17, 18). Recently, we have demonstrated that
macrophage-stimulating protein (MSP) (19), the ligand for RON
(recepteur d’origine nantais) (20, 21), is a potent endogenous
inhibitor of LPS- and IFN--induced iNOS expression in murine
peritoneal resident macrophages (22). MSP is an 80-kDa heterodimeric protein that was first found to stimulate migration of murine peritoneal resident macrophages (23). MSP is a member of a protein family that includes plasminogen (24, 25) and hepatocyte growth factor/scatter factor (26) and is also known as hepatocyte growth factor-like protein (27). MSP is primarily synthesized by liver cells (28) and circulates in blood as a biologically inactive single-chain pro-MSP (29). Proteolytic conversion of pro-MSP into the two-chain form of mature MSP is required for its binding to the receptor RON (29, 30, 31, 32). MSP stimulates many activities in resident peritoneal macrophages, including cell shape change, chemotactic migration, and phagocytosis (19, 23).
RON is a novel receptor tyrosine kinase (33) belonging to the Met
proto-oncogene family (34). The murine homologue of RON was cloned from
hematopoietic stem cells and named "stem cell-derived tyrosine
kinase" (35, 36). RON is expressed in peritoneal resident macrophages
but not in circulating monocytes or alveolar macrophages (37, 38).
Several studies indicate that RON expression in macrophages is
regulated in monocyte differentiation and inflammation (37).
Stimulation of RON-expressing cells with MSP rapidly induces tyrosine
phosphorylation of RON (36, 37). Intracellular signaling proteins such
as phosphatidylinositol-3 (PI-3) kinase, phospholipase C-, and
mitogen-activated protein (MAP) kinase (Erk2) were also phosphorylated
and activated upon MSP stimulation (39, 40), suggesting that RON is
capable of stimulating diverse signaling pathways that may be involved
in MSP-induced biologic activities in macrophages (39, 40). Recently,
we have found that activation of RON by MSP in murine peritoneal
resident macrophages results in inhibition of iNOS expression induced
by LPS and proinflammatory cytokines, including IFN- and TNF-
(22). The mechanism for such inhibition is currently unknown. Studies
using RON knockout mice showed that inactivation of the RON gene
increases the susceptibility to LPS-induced septic shock (41). This
effect was accompanied by increased NO production by RON-/-
macrophages (41). Therefore, RON plays a critical role in regulating
macrophage activities in response to inflammatory stimulation (22, 41).
In the present report, we have studied the expression of RON in
peritoneal exudate macrophages and the role of RON in regulation of
LPS-induced NO production. Also, we have established Raw264.7 cell
lines expressing human RON and studied the mechanisms by which RON
activation inhibits LPS-induced iNOS expression. Our results showed
that MSP inhibits iNOS expression in both resident macrophages and
mature exudate macrophages. RON activation by MSP transduces inhibitory
signals that block LPS- and IFN--induced iNOS promoter activity. The
specific PI-3 kinase inhibitor wortmannin and a dominant-inhibitory p85
of PI-3 kinase prevent the inhibitory effect of RON, suggesting that
RON-induced activation of PI-3 kinase is an important intracellular
component that transduces inhibitory signals in MSP-treated
macrophages.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The murine macrophage-like cell line Raw264.7 was obtained from American Type Culture Collection (Manassas, VA) and adapted to macrophage serum-free medium (M-SFM, Life Technologies, Gaithersburg, MD). Mouse peritoneal resident macrophages were obtained from C3H/HeN mice by lavage of the peritoneal cavity with 10 ml of RPMI 1640 containing 1% fetal bovine serum. Peritoneal exudate macrophages were collected from mice at different intervals after i.p. injection of 1 ml of Con A solution (40 mg in PBS, Sigma, St. Louis, MO). Collected cells were washed twice with cold RPMI 1640 at 4°C. All cell cultures were maintained in M-SFM at 37°C in a humidified incubator containing 5% CO2 in air.
Reagents
Purified human plasma MSP (19) was kindly provided by Dr.
E. J. Leonard (National Cancer Institute, Frederick, MD). Rabbit
polyclonal Abs to synthetic C-terminal peptide of human or mouse RON
were generated from rabbits immunized with keyhole limpet
hemocyanin-conjugated peptides. An mAb to human RON (clone ID2) was
used as described (39). Mouse recombinant IFN- (1 x
105 U/ml), IL-4 (5000 U/ml), IL-10 (20,000 U/ml), and
TGF-ß1 were from Boehringer Mannheim (Indianapolis, IN). A mutant
cDNA encoding a dominant-inhibitory p85 of PI-3 kinase (
p85) (42)
was provided by Dr. M. Kasuga (Kobe University, Kobe, Japan). The cDNA
was inserted into the expression vector pcDNA3 (Invitrogen, San Diego,
CA). The mouse iNOS cDNA (43) was provided by Dr. C. Lowenstein (The
Johns Hopkins University, Baltimore, MD). The 5.0-kb DNA fragment
containing the mouse iNOS gene promoter (44) was provided by Dr. H.
Esumi (National Cancer Center, Tokyo, Japan). The cDNA was inserted
into the vector pBLCAT5 to generate the reporter construct piNOSCAT1
(44). LPS prepared from Escherichia coli serotype 055:B5 was
from Life Technologies. Wortmannin was from Calbiochem (San Diego, CA).
Sulfanilamide, naphthylethylenediamine dihydrochloride, sodium nitrite,
and other chemicals were from Sigma.
Establishment of Raw264.7 cell lines expressing human RON
Transfection of Raw264.7 cells with an expression vector pDR2 containing a full length of human RON cDNA was performed using electroporation (44). Briefly, cells (1 x 107) were mixed with 5 µg of the plasmid in 0.5 ml of PBS, pH 7.6, and electroporated at 330 V and 500 µF. Recovered cells were incubated in M-SFM at 37°C for 24 h and then switched to fresh medium containing 400 µg/ml of hygromycin B. The colonies were picked and expanded into cell lines. Expression of RON by individual clones was determined by Western blotting using rabbit IgG to RON after immunoprecipitation. Two cell lines expressing high levels of RON were obtained and designated as RawRON8 and RawRON32.
Expression of a dominant-inhibitory p85 of PI-3 kinase (p85) in
Raw264.7 cells expressing human RON
RawRON8 cells were transfected with 5 µg of pcDNA3 containing
the cDNA encoding p85 using transfection reagent
N-[1-(2,3-dioleoyloxyl)propyl]-N,N,N-trimethylammonium
methyl sulfate (DOTAP; Boehringer Mannheim) as described
previously (20). Transfected cells were selected in 400 µg/ml of
G418. Colonies were picked and expanded into cell lines.
Expression of p85 in each cell line was determined by Western
blotting using rabbit IgG specific to p85 (Transduction Laboratories,
Lexington, KY). Two cell clones, Rp85–1 and Rp85–2, expressing
different levels of p85 were used in functional studies.
Assay for NO2- production
Macrophages at 2 x 106 cells/ml were incubated
in M-SFM in 200 µl/well in a 96-well Microtiter plate. Cells
were stimulated with LPS, IFN-, or their combinations in the
presence or absence of different concentrations of MSP. Cultured fluids
were collected 36 h after incubation. Synthesis of NO was
determined by measuring NO2-, a stable reaction
product of NO with molecular oxygen, using Griess reagent (0.05%
sulfanilamide and 0.05% N-(1-naphthyl) ethylenediamine
dihydrochloride in 2.5% H3PO3) as previously
described (22). The optical densities of each sample were measured by
an ELISA reader at the wavelength of 570 nm. NO2-
concentrations were calculated by comparison with a standard curve
prepared using NaNO2.
Immunoprecipitation and Western blotting
These procedures were performed as described previously (36). Briefly, macrophages were lysed in 0.5 ml of lysis buffer (0.1 M Tris buffer, pH 7.60, containing 0.15 M NaCl2, 2 mM EDTA, 1% Nonidet P-40, 0.5% Triton X-100, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 20 µg/ml soybean trypsin inhibitor, and 100 µM sodium vanadate). Lysates were centrifuged in a microcentrifuge, diluted with TBS-T buffer (100 mM Tris buffer, pH 7.6, containing 150 mM NaCl, 0.5% BSA, and 0.05% Tween-20), and precleared with normal mouse or rabbit IgG bound to protein G-Sepharose beads. Cellular proteins were immunoprecipitated with specific Abs bound to protein G-Sepharose beads. Samples were then solubilized and reduced in 100°C in sample buffer containing 2-ME and separated in 8% acrylamide gel containing SDS. After electrophoresis, proteins were transferred to an Immobilon-P membrane (Millipore, Bedford, MA). Rabbit IgG to human or mouse RON peptide, mAb 4G10 to phosphotyrosine (Upstate Biotechnology, Lake Placid, NY), or rabbit IgG to p85, respectively, was used to detect specific proteins. Goat anti-mouse or rabbit IgG conjugated with peroxidase (Boehringer Mannheim) were used as secondary Ab. The reaction was developed with enhanced chemiluminescent reagent (Amersham, Arlington Heights, IL) and exposed to film. In some instances, membranes were treated with SDS/2-ME erasure buffer (20) and then reprobed with other Abs.
RNA isolation and Northern blot analysis
Total RNA was extracted from cultured macrophages using the RNAzol B (Biotecx Laboratories, Houston, TX) according to the manufacturer’s protocol. All RNA samples had an A260/280 ratio of >1.75. Aliquots containing 15 µg of total RNA were size fractionated in a 1% agarose gel containing 2.2 M formaldehyde. After electrophoresis, RNA was transferred to a nylon membrane (Boehringer Mannheim) and UV cross-linked. The hybridization procedures were conducted as described previously (22). The mouse iNOS cDNA fragment labeled with [32P]dCTP (Amersham) was used as a specific probe. The ß-actin cDNA was used as control. The radioactivity on the membrane was autoradiographed at -80°C on XAR-5 film (Eastman Kodak, Rochester, NY) with the use of intensifying screens (DuPont, Wilmington, DE).
Chloramphenicol acetyltransferase (CAT) assay for detection of iNOS promoter activity
The method has been described previously (45). Briefly,
RON-expressing Raw264.7 or control cells were cultured in M-SFM. For
transient transfection analysis, cells at 50% of confluence in a 6-mm
culture dish were transfected with 10 µg of the piNOSCAT1 using the
tranfection reagent DOTAP as described (31). The ß-galactosidase
reference plasmid pRSVßgal was used as an internal standard for
transfection efficiency. After 24 h, cells were washed once with
M-SFM and then stimulated with LPS, IFN-, or their combinations in
the presence or absence of MSP. After incubation for an additional
40 h, whole cell extracts were prepared by disrupting cells with
three freeze-thaw cycles. The supernatants were assayed for the amounts
of CAT proteins using a CAT-specific ELISA kit (5-Primer
3-Primer,
Boulder, CO). Experiments were repeated three times. The concentrations
of the CAT protein were normalized for ß-galactosidase activity.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression of RON in murine resident peritoneal macrophages has
been reported previously (37). However, it is unclear whether
inflammatory macrophages express RON. To test this, peritoneal exudate
macrophages were collected at several time intervals after Con A
injection. Western blot analysis was conducted to determine RON
expression. The results shown in Figure 1
A demonstrated that resident
peritoneal macrophages express RON. RON was not detected in acute
inflammatory macrophages collected on day 1 (lane E1)
but was expressed in exudate macrophages harvested on day 3
(lane E3) and day 5 (lane E5). The
level of RON from exudate macrophages collected on day 5 is comparable
with those from resident macrophages. We then tested whether MSP
induces phosphorylation of RON in inflammatory macrophages. The results
presented in Figure 1B show that MSP induced tyrosine
phosphorylation of RON both in mature exudate and resident macrophages.
The time course of RON phosphorylation induced by MSP in mature exudate
macrophages is shown in Figure 1C. The phosphorylated RON
was first detected 5 min after MSP stimulation, peaked at 15 min, and
then gradually reduced to a lower level.
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To determine whether RON activation affects the production of NO
by activated macrophages, Con A-elicited mouse peritoneal macrophages
were collected at different time intervals and analyzed for the effect
of MSP on LPS- and IFN--induced NO production. The results are shown
in Table I. Consistent with our previous
report (22), we showed that MSP inhibited LPS- or LPS plus
IFN--induced NO production by resident macrophages. MSP had no
effect on LPS- and IFN--induced NO formation by acute exudate
macrophages collected on day 1. In contrast, exudate macrophages
collected on day 3 responded to MSP. Accumulation of NO in culture
fluids was significantly reduced in the presence of MSP, with
inhibition ranging from 72% in LPS alone to 76% in LPS- and
IFN--treated cells. These data indicate that RON activation inhibits
LPS- and IFN--stimulated NO production not only in resident cells,
but also in Con A-elicited mature exudate macrophages.
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A. The amounts of
the induced iNOS protein in MSP-treated cells were dramatically
diminished when compared with cells stimulated with LPS. Also, these
results show that MSP inhibited not only LPS-induced iNOS protein
synthesis, but also the synergistic effect of LPS and IFN- on the
iNOS protein expression. In addition, these results show that the
inhibitory effect of MSP is comparable with that of TGF-ß and is much
stronger than that of IL-4. IL-4 only partially inhibited the LPS- and
IFN--induced iNOS protein expression. The Northern blot analysis of
the iNOS mRNA expression is shown in Figure 2B. MSP
inhibited the iNOS mRNA expression induced by LPS and IFN-. This
effect was dose dependent. At 10 nM, MSP inhibited the iNOS mRNA
expression by about 80%. These data suggest that the effect of MSP on
LPS-induced NO production by mature exudate macrophages is targeted at
the level of the iNOS mRNA accumulation.
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-induced iNOS synthesis
To study the effect of RON-mediated inhibition of iNOS expression
in more detail, we transfected Raw264.7 cells with human RON cDNA by
electroporation. Raw264.7 cells do not express endogenous RON as
determined by Northern and Western blot analysis (data not shown).
Also, MSP has no effect on LPS or LPS plus IFN--induced NO
production by Raw264.7 cells (our unpublished data). By cDNA
transfection, two cell clones, namely RawRON8 and RawRON32, expressing
high levels of RON were obtained and expanded into cell lines.
Results presented in Figure 3A
show the expression of RON in RawRON8 and RawRON32, as detected by
Western blot after immunoprecipitation of RON with mAb ID2. An
unprocessed, single-chain RON precursor (pro-RON) with a molecular mass
of 180 kDa was observed. About half of the expressed protein is
processed into the mature RON ß-chain heterodimer. This is evident
by the presence of the 145-kDa RON ß-chain. The 40-kDa -chain is
not shown, because our rabbit IgG only recognizes the C-terminal
peptide of the RON ß-chain. In Figure 3B, we show that
stimulation of these cells with MSP induced tyrosine phosphorylation of
RON, indicating that the expressed RON is functional.
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A, LPS
strongly induced expression of the iNOS protein in RawRON8 cells. MSP
totally blocked expression of the iNOS protein induced by LPS, and its
effect was comparable with that of TGF-ß. IL-10 had no effect on
LPS-induced synthesis of the iNOS protein. IL-4 only partially
antagonized the LPS effect. Similar data were obtained with RawRON32
cells (our unpublished data). To determine whether the MSP affects the
iNOS mRNA expression, RawRON8 cells were treated with LPS and IFN-.
Different amounts of MSP were added simultaneously after initiation of
cell culture. The results shown in Figure 4B demonstrate the
dose-dependent inhibition of iNOS mRNA expression by MSP. Again, MSP at
10 nM could totally block the synergistic effect of LPS and IFN- on
iNOS gene expression. These results are similar to those shown in
Figure 2
B, indicating that the inhibitory effect of MSP
could be reproduced in Raw264.7 cells expressing human RON, and that
the action of MSP is targeted at the level of the iNOS mRNA expression.
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To explore the possibility that MSP inhibits iNOS transcription,
we transiently transfected RawRON8 cells with the piNOSCAT1 reporter
gene construct (44). After stimulation of cells with LPS alone or
combined with IFN-, the effect of MSP on the iNOS promoter activity
was determined by the CAT assay. The results presented in Figure 5 show that in cells stimulated with LPS
and IFN-, the amounts of CAT were significantly increased in
comparison with cells without treatment, indicating that LPS and
IFN- activated the iNOS promoter. MSP had no effect by itself on
iNOS promoter activity but significantly inhibited LPS-stimulated CAT
production. MSP also inhibited the synergistic effect of LPS and
IFN- on CAT production. These data suggest that MSP-mediated
inhibition of iNOS transcription is mediated by inhibiting the promoter
activity of the iNOS gene activated by LPS and IFN-. In addition,
MSP did not inhibit the LPS-induced promoter activity of the iNOS gene
in parental (untransfected) Rwa264.7 cells (our unpublished data).
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To determine whether the inhibitory effect of RON on iNOS promoter
could be regulated by specific inhibitors such as wortmannin for PI-3
kinase (46), RON-expressing Raw264.7 cells were pretreated with
wortmannin after transient transfection of piNOSCAT1. Cells were then
treated with LPS to induce CAT reporter gene transcription. The results
in Figure 6 show that preincubation of
RawRON8 cells with 50 nM wortmannin had no effect on LPS-induced CAT
production. However, wortmannin significantly increased the amounts of
LPS-induced CAT proteins in cells treated with MSP, indicating that
wortmannin prevents the inhibitory effect of MSP on the LPS-induced
promoter activity of the iNOS gene.
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Because wortmannin reduces the inhibitory effect of MSP on the
LPS-induced transcription activity of the iNOS gene promoter, we wanted
to determine whether it could prevent the MSP-induced inhibition of NO
production. To test this hypothesis, RawRON8 cells were pretreated with
wortmannin and then stimulated with LPS and IFN-. The amount of
nitrite in culture fluid was determined by Griess reaction. The results
presented in Figure 7A show
that pretreatment of Rwa264.7 cells with wortmannin had no effect on
LPS- and IFN--induced NO production. However, in the presence of
MSP, wortmannin prevented the inhibitory effect of MSP on NO production
induced by LPS and IFN-. The results demonstrating the
dose-dependent restoration of LPS- and IFN--induced NO production
are shown in Figure 7B. Wortmannin at 50 nM concentration
prevented completely the MSP-mediated inhibition of NO.
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To investigate further whether activation of PI-3 kinase is
required for the RON-mediated inhibition of iNOS expression, RawRON8
cells were introduced with an expression vector containing the
dominant-inhibitory p85 of PI-3 kinase. The p85 has been used
effectively to block endogenous PI-3 kinase activity (42). Transfected
cells expressing p85 were obtained. The results in Figure 8A show the levels of p85
expressed by three individual clones. Using these cells, together with
RawRON8 cells, the effect of p85 on MSP-induced inhibition of NO
production was tested. The results in Figure 8B show that in
LPS- and IFN--stimulated RON-expressing Raw264.7 cells, MSP-mediated
inhibition of NO production was prevented significantly by wortmannin.
Similarly, in RawRON8 cells expressing p85, the inhibitory effect of
MSP was significantly reduced. The levels of MSP-mediated NO inhibition
were reduced to about 35% (clone 1) and 18% (clone 2) in these two
cell lines, suggesting that inhibition of the endogenous PI-3 kinase by
p85 prevented the MSP-mediated inhibition of LPS- and
IFN--induced NO production. Taken together, these data indicate that
MSP-induced activation of PI-3 kinase mediated RON-induced inhibition
of iNOS synthesis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MSP inhibition of iNOS synthesis was first described in LPS- and
cytokine-stimulated murine resident peritoneal macrophages (22).
Physiologic concentrations of MSP induced a long lasting inhibitory
effect on stimulated iNOS expression (22). MSP also blocked the iNOS
mRNA expression induced by different iNOS inducers, including LPS,
IFN-, TNF-, IL-2, or their different combinations (22). We found
that the inhibitory effect of MSP was specific for iNOS, because
LPS-induced cytokine production such as monocyte chemoattractant
protein 1 was not affected by MSP (22). The data presented here show
that the action of MSP is not limited to resident macrophages. Exudate
peritoneal macrophages 3 days after Con A injection express RON and
respond to MSP. In contrast, acute exudate macrophages do not express
RON. This effect can be reproduced by expression of RON in the
established mouse macrophage-like cell line Raw264.7.
Several studies, including ours, have indicated that expression of RON
is restricted to certain types of tissue macrophages (37, 38) and is
regulated during inflammation (37). Mouse resident peritoneal
macrophages express RON and respond to MSP with increased chemotactic
migration, cell shape change, and phagocytosis (19). Osteoclasts, a
member of the mononuclear phagocyte system, also express RON
(47), and MSP stimulation facilitates the bone resorption
activity of osteoclasts in vitro (47). In contrast, macrophages derived
from alveolus, spleen, and bone marrow do not express RON and respond
to MSP (47). These studies imply that RON expression is regulated such
that not all populations of tissue-derived macrophages express RON. The
restricted expression pattern also suggests that the transcription of
the RON gene is controlled by the environment in which macrophages
reside (37, 47). Iwama et al. (37) have presented evidence showing that
expression of RON by peritoneal resident macrophages is related to
macrophage differentiation in peritoneal cavity. Using a specific RON
Ab and a marker Ab for macrophage, they demonstrated that blood
monocytes do not express RON but gradually become predominantly RON
positive several days after entrance into the peritoneal cavity (37).
Based on these data, it was suggested that terminal differentiation of
monocytes/macrophages in the peritoneal environment results in the
expression of RON (37). We show that acute exudate peritoneal
macrophages collected 1 day after induction of peritonitis by Con A do
not express RON. RON is expressed in mature exudate macrophages 3 or 5
days after Con A stimulation, suggesting that RON is expressed by
peritoneal macrophages at the later stage of inflammation. The
mechanisms that regulate the RON expression in different types of
tissue macrophages are unknown. Inflammatory mediators, such as
cytokines, have been suggested to be involved in regulation of RON
expression (48). Recent in vitro studies have demonstrated that human
monocyte cell line THP1 could be induced to express RON mRNA during
cell differentiation into macrophages (48). Inflammatory cytokines such
as IFN- and TNF- facilitate the RON expression (48). Considering
the promoter structures of RON that contain several cytokine-responsive
elements (49), it is possible that inflammatory cytokines released
during inflammation stimulate the RON gene transcription by
inflammatory macrophages. Thus, it will be of interest to determine
which cytokines or growth factors are capable of regulating RON
expression in primary macrophages.
The ability of RON to inhibit LPS- and IFN--induced iNOS expression
in both resident and mature exudate macrophages suggests that RON
activation may play a role in regulating macrophage activities during
inflammation. Because RON is restricted to certain types of tissue
macrophages, the differences in RON-positive and -negative macrophages
may account for differences in susceptibility of specific tissues to
injuries during inflammation. Our early studies have shown that
activation of RON by MSP induces stimulatory activities in resident
macrophages, including cell shape change, chemotactic migration to C5a,
and phagocytosis of C3bi-coated erythrocytes (19). These observations
imply that MSP may be involved in inflammation by activating tissue
macrophages at the site of injury and stimulating their phagocytic
activities. We have also observed increased ingestion of C3bi-coated
Listeria monocytogenes by macrophages treated with MSP (31)
and increased expression of RON in macrophages recruited into the
peritoneal cavity in Con A-induced peritonitis. However, MSP inhibits
LPS- and inflammatory cytokine-induced iNOS gene expression (Ref. 22
and present data). This activity is of particular interest, because it
indicates that RON has dual functions in regulating macrophage
activities during inflammation. Since excessive formation of NO is a
major cause for tissue damage and septic shock (11, 12, 13, 14), it is
reasonable to speculate that blocking NO production by MSP might
attenuate toxic effects of NO and regulate inflammatory responses.
Recent in vivo studies using mice with the disrupted RON gene
(knockout) have demonstrated that inactivation of RON significantly
increased LPS- or IFN--induced NO production by peritoneal
macrophages and caused severe inflammatory reactions in delayed-type
hypersensitivity (41). RON-deficient mice were also more susceptible to
death induced by LPS at a dose that is sublethal to wild-type mice
(41). These data suggest that RON expression can mediate outcome in
LPS-induced septic shock. Although the causes of the increased
inflammation in RON knockout mice are not known, it was suggested that
lack of RON-mediated inhibition of macrophage iNOS synthesis is at
least partially responsible for these pathophysiologic effects (41).
Considering our present data showing that RON activation inhibits iNOS
expression by inflammatory macrophages, the lack of such inhibition
could be one of the mechanisms that lead to increased inflammation in
vivo.
Many cytokines or growth factors have been found to inhibit macrophage
iNOS expression through different mechanisms (16, 17, 18, 50). TGF-ß
inhibits iNOS expression by decreasing iNOS mRNA stability without
affecting the transcription of the iNOS gene (16). IL-11 blocks iNOS
expression by inhibition of NF-B-dependent transcriptional
activation (50). Evidence from this study indicates that the inhibition
of NO production by MSP occurs at the transcriptional level. Activation
of RON down-regulates LPS- and IFN--induced expression of the iNOS
mRNA in resident and mature exudate macrophage in a dose-dependent
manner. Expression of iNOS mRNA by Raw264.7 cells expressing human RON
was also inhibited. The inhibition of mRNA accumulation correlates with
reduction of the iNOS protein expression and NO production by these
cells. Moreover, these inhibitory effects can be reproduced in Raw264.7
cells expressing human RON. This notion is further supported by
evidence from the studies of the iNOS gene promoter activity. RON
activation inhibited LPS- and IFN--induced iNOS gene promoter
activity, indicating that the action of RON is targeted at the
transcription level. However, since we have not studied the rate of the
iNOS gene transcription and the mRNA stability, other inhibitory
mechanisms cannot be excluded.
PI-3 kinase is a critical effector molecule involved in the
RON-mediated signaling pathway that leads to inhibition of iNOS
expression. Various molecular targets for PI-3 kinase have been
identified. Among them are cytoskeletal proteins, protein kinases, and
transcription factors (51, 52). Activated PI-3 kinase regulates cell
growth, migration, and morphologic changes (51, 52). Thus, PI-3 kinase
is regarded as an important regulator involved in many physiologic
processes (51, 52). MSP-induced activation of PI-3 kinase has been
reported in epithelial cells as well as in murine peritoneal resident
macrophages (39). In human epithelial cells, MSP stimulates tyrosine
phosphorylation of PI-3 kinase p85 subunit and induces association of
RON with PI-3 kinase (39). These effects are believed to account for
the ability of MSP to induce cell migration and morphologic change
(39). Recently, the role of PI-3 kinase in regulation of iNOS
expression has been documented (53, 54). Wright et al. (53) reported
that IL-13-induced inhibition of iNOS expression in cytokine-stimulated
HT-29 colonic epithelial cells was reversed by LY294002, a specific
inhibitor for PI-3 kinase (53). Their results demonstrated that
the activation of PI-3 kinase by IL-13 is a key signal that is
responsible for the inhibition of iNOS transcription in activated
epithelial cells (53). The effect of wortmannin on LPS-induced NO
production by murine peritoneal macrophages was also reported (54). It
was shown that wortmannin directly enhances LPS-induced NO production,
suggesting that PI-3 kinase plays an important role in transducing the
signals that are involved in LPS-stimulated macrophage activation (54).
Consistent with these studies, our data show that activation of PI-3
kinase is required for RON-mediated iNOS inhibition. Three lines of
evidence support this conclusion. First, wortmannin at a nanomolar
concentration prevented the MSP-induced inhibition of iNOS gene
promoter activity in LPS-stimulated RawRON8 macrophages. Second,
wortmannin reversed the MSP-induced inhibitory effect on NO production
stimulated by LPS and IFN-. Third, the expression of the
dominant-inhibitory p85 of PI-3 kinase, which inhibits the endogenous
PI-3 kinase (42), significantly blocked the RON-mediated inhibition of
NO production. These data strongly suggest that the inhibition of
MSP-mediated activation of PI-3 kinase blocks the RON-transduced iNOS
inhibitory signals. We speculate that lipid products of activated PI-3
kinase may inhibit LPS or IFN--stimulated signal molecules or
transcription factors that are essential for iNOS synthesis. However,
because RON is capable of activating other signaling pathways,
including the MAP kinase cascade (40), other signaling proteins, such
as MAP kinase 1 (MEK1), may be involved in RON-mediated inhibition of
LPS-induced iNOS expression as well. Indeed, our recent studies have
found that the inhibitory effect of MSP on iNOS expression can be
increased further by the presence of PD98059, a specific inhibitor of
MEK1 (55). Studies are currently underway to determine the role of MEK1
in RON-mediated inhibition of iNOS expression in LPS- and
IFN--stimulated macrophages.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to M.-H. Wang, Department of Internal Medicine, Wayne State University School of Medicine, Elliman Research Building, Room 2221, 421 East Canfield, Detroit, MI 48201. E-mail address:
3 Abbreviations used in this paper: NO, nitric oxide; NOS, nitric oxide synthase; iNOS, inducible form of nitric oxide synthase; MSP, macrophage-stimulating protein; RON, recepteur d’origine nantais; MAP, mitogen-activated protein; MEK1, mitogen-activated protein kinase 1; CAT, chloramphenicol acetyltransferase; PI-3, phosphatidylinositol-3; M-SFM, macrophage serum-free medium; DOTAP, N-[1-(2,3-dioleoyloxyl)propyl]-N,N,N-trimethylammoniummethyl sulfate.
Received for publication March 25, 1998. Accepted for publication June 25, 1998.
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