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(Journal of Leukocyte Biology. 2002;71:359-366.)
© 2002 by Society for Leukocyte Biology

Activation of the RON receptor tyrosine kinase protects murine macrophages from apoptotic death induced by bacterial lipopolysaccharide

Yi-Qing Chen*, Yong-Qing Zhou{dagger} and Ming-Hai Wang*

* Department of Medicine, University of Colorado School of Medicine, Denver Health Medical Center, Denver; and
{dagger} Division of Neurosurgery, The First Affiliated Teaching Hospital, Zhejiang University School of Medicine, Hangzhou, People’s Republic of China

Correspondence: M-H. Wang, Department of Medicine, UCHSC, Denver Health Medical Center, Mail 4000, 777 Bannock Street, Denver, CO 80204. E-mail: ming-hai.wang{at}uchsc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
RON is a receptor tyrosine kinase activated by macrophage-stimulating protein. We demonstrate here that RON activation inhibits LPS-induced apoptosis of mouse peritoneal macrophages and Raw264.7 cells expressing RON or a constitutively active RON mutant. The antiapoptotic effect of RON was accompanied with the inhibition of LPS-induced production of nitric oxide (NO), a molecule responsible for LPS-induced cell apoptosis. This conclusion is supported by experiments using a chemical NO donor GSNO, in which RON activation directly blocked GSNO-induced apoptotic death of Raw264.7 cells and inhibited LPS-induced p53 accumulation. Furthermore, we showed that treatment of cells with wortmannin, which inhibits phosphatidylinositol (PI)-3 kinase, prevents the inhibitory effect of RON on LPS-induced macrophage apoptosis. These results were confirmed further by expression of a dominant inhibitory PI-3 kinase p85 subunit. These data suggest that by activating PI-3 kinase and inhibiting p53 accumulation, RON protects macrophage from apoptosis induced by LPS and NO. The antiapoptotic effect of RON might represent a novel mechanism for the survival of activated macrophages during inflammation.

Key Words: MSP • apoptosis • PI-3 kinase • iNOS


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Macrophage activation is an integral component of inflammatory reactions occurring during bacterial infection, tissue injury, and immune responses [1 , 2 ]. A variety of substances including cytokines, growth factors, and bacterial cell-wall products are capable of activating macrophages and regulating their cellular functions [3 ]. The most notable activator for macrophages is gram-negative bacterial endotoxin, also known as lipopolysaccharide (LPS) [4 ]. LPS has the ability to stimulate multiple signaling pathways in macrophages, which results in production of inflammatory cytokines, regulation of adhesion molecule expression, and induction of inducible nitric oxide synthase (iNOS) expression [5 6 7 ]. Moreover, LPS stimulation causes programmed cell death of monocytes/macrophages under certain conditions [8 , 9 ]. These activities are essential in vivo in controlling and maintaining the balanced inflammatory reactions.

RON (recepteur d’origine nantais) is a receptor tyrosine kinase belonging to the MET protooncogene family [10 ]. The cDNA encoding human RON was cloned from skin keratinocytes [10 ]. The murine homologue of RON was isolated from hematopoietic stem cells and named STK (stem cell-derived tyrosine kinase) [11 12 ]. For simplicity, we will refer to RON/STK as RON in this paper. Mature RON is a 180-kD heterodimeric protein composed of a 40-kD {alpha} chain and a 140-kD ß chain with intrinsic tyrosine kinase activity [10 , 13 ]. {alpha} and ß chains are derived from a single-chain RON precursor by proteolytic conversion [10 , 13 ]. The ligand for RON was identified as a macrophage-stimulating protein (MSP) [13 , 14 ], also known as a hepatocyte growth factor-like protein [15 ]. MSP is a serum protein originally identified by its stimulatory activities in mouse peritoneal resident macrophages [16 ]. RON expression is restricted in certain types of tissue macrophages including those derived from peritoneal cavity, skin, and bone [17 18 19 ]. Monocytes, alveolar macrophages, and spleen macrophages do not express RON [17 ]. Experiments using partial knockout mice have found that the disruption of the RON gene significantly increases the inflammatory reactions in vivo after LPS challenge [20 , 21 ], suggesting that RON might play a critical role in controlling inflammatory activities of macrophages during inflammation and septic shock.

The present studies were to determine the effects of RON on LPS-induced apoptosis of macrophages and the potential cellular mechanisms. At present, information related to the functions of RON in protecting macrophage survival is not available, although activation of RON resulting in cell growth or apoptotic death of two established erythroleukemia cell lines was described [22 ]. Because RON activation inhibits LPS-induced NO production, and NO is the principle molecule responsible for LPS-induced macrophage apoptosis [23 , 24 ], we reasoned that RON activation might provide survival signals for activated macrophages. The data presented in this study show that RON activation protects macrophages from apoptosis induced by LPS and chemical NO donor S-nitrosoglutathione (GSNO).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells
Peritoneal resident macrophages were collected from C3H/HeN mice (Taconic, Germantown, NY) as described [23 ]. Mouse macrophage-like cell line Raw264.7 was from American Type Culture Collection (ATCC; Manassas, VA). Raw264.7 cells transfected with pcDNA3.1 vector (Invitrogen, Carlsbad, CA) containing a full-length human RON cDNA [10 ] or a dominant inhibitory p85 of phosphatidylinositol (PI)-3 kinase ({Delta}p85) were established as described [25 ]. Cells were cultured in serum-free RPMI 1640 at 37°C in a humidified incubator containing 5% CO2 in air and stimulated with LPS and other reagents as detailed in each experiment.

Reagents
Pure mature human MSP was provided by Dr. E. J. Leonard (National Cancer Institute, Frederick, MD). Mouse monoclonal antibodies (mAb; clone ID2) to the extracellular domain of human RON and rabbit immunoglobulin G (IgG) antibodies to the C-terminal peptide of RON were as described [25 ]. Rabbit IgG antibodies to mouse p53 were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit IgG antibodies to mouse iNOS were from Transduction Laboratories (San Diego, CA). Mouse interferon-{gamma} (IFN-{gamma}) was from Roche (Indianapolis, IN). LPS from Escherichia coli serotype 055:B5 was from Sigma Chemical Co. (St. Louis, MO). Chemical NO donor GSNO, specific PI-3 kinase inhibitor wortmannin, and cycloheximide (CHX) were from Calbiochem (San Diego, CA).

Animal treatment
Induction of macrophage apoptosis in vivo was performed as described previously [26 ]. C3H/HeN mice (four animals in each group) were injected intraperitoneally (i.p.) with recombinant human MSP (10 µg/mouse) or bovine serum albumin (BSA) in 0.2 ml phosphate-buffered saline (PBS). After 30 min, mice were then injected with 75 µg LPS plus 150 µg CHX/mouse in 0.4 ml PBS. Peritoneal macrophages were collected 10 h after LPS injection. The percentages of apoptotic macrophages were determined using Hoechst dye 33258 staining methods [27 ].

Establishment of Raw264.7 cell lines expressing a constitutively active RON mutant
Transfection of Raw264.7 cells with pcDNA3.1 vector containing a mutant RON cDNA with D1232V substitution was performed as described [25 ]. This cDNA encodes a RON mutant that is constitutively active [28 ]. To avoid clonal variation, RON-expressing cells were positively isolated using mAb ID2 and Dynal beads M-450 coated with goat anti-mouse IgG (Dynal Co., Oslo, Norway) Briefly, G418-resistant cells were first mixed with rabbit IgG anti-CD16 (PharMigen, San Diego, CA) to block the Fc receptor on the cell surface. Cells were then incubated with mAb ID2 followed by Dynal beads coated with rabbit anti-mouse IgG. The selected cells were washed and treated immediately with trypsin-ethylenediaminetetraacetate (EDTA) solution to detach magnetic beads. The isolated cells were pooled and cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 8% fetal bovine serum (FBS) in the presence of 800 µg/ml G418. Expression of RON was determined in Western blotting.

Cell lyses and Western blotting
These methods were performed as described [12 ]. Briefly, macrophages (3–6x106) were lysed in 0.2 ml lysis buffer [0.1 M Tris, pH 7.6, containing 0.15 M NaCl, 2 mM EDTA, 0.5% Nonidet P-40 (NP-40), 0.5% Triton X-100, 100 µM sodium vanadate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 20 µg/ml soybean trypsin inhibitor]. Cellular proteins were separated in 8% acrylamide gel under reduced conditions. Rabbit IgG antibodies to p53 or iNOS were used as detecting antibodies followed by goat anti-rabbit IgG conjugated with peroxidase. The reaction was developed with enhanced chemiluminescent reagents (Amersham, Arlington Heights, IL) and exposed to film.

Assay for NO-2 production
Macrophages at 2 x 106 cells/ml were incubated in RPMI 1640 in 200 µl/well in a 96-well tissue-culture plate. Cells were stimulated with LPS. Culture fluids were collected 24 h after incubation. Synthesis of NO was determined by measuring NO-2, a stable reaction production of NO with molecular oxygen, using Griess reagents as described previously [23 ]. NO2 concentrations were calculated by comparison with a standard curve prepared with NaNO2.

DNA isolation and fragmentation assay
Macrophages were lysed in 500 µl lysis buffer [10 mM Tris buffer, pH 8.0, containing 1 mM EDTA, 100 mM NaCl, and 1% sodium dodecyl sulfate (SDS)] with 100 U/ml proteinase K and were incubated at 50°C overnight. Lysates were then treated with RNase A for 1 h at room temperature. DNAs were extracted with phenol/chloroform and precipitated at -20°C. DNA (5 µg/sample) was run in a 1% agarose gel, visualized by ultraviolet fluorescence after staining the gel with ethidium bromide, and photographed.

Hoechst dye 33258 fluorochrome nuclear-staining assay for apoptotic cells
The assay was performed as described [27 ]. Briefly, macrophages (2x106) were cultured in a 30-mm-diameter culture dish overnight and then stimulated with 1 µg/ml LPS for 24 h. MSP (5 nM) was added after culture initiation. After stimulation, cells were fixed with 3% paraformaldehyde for 5 min and washed with PBS. Apoptotic cells were stained with Hoechst dye 33258 (1.5 µg/ml) for 10 min followed by extensive wash with distilled water. Stained nuclei were visualized using a fluorescent microscope (Nikon). A minimum of 200 cells was counted in each sample. Apoptotic cells were expressed as a percentage of the total counted nuclei.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of RON activation on apoptosis of primary macrophages induced by LPS
To determine if RON activation provides survival signals for macrophages, peritoneal resident macrophages were stimulated with LPS alone or with IFN-{gamma} in the presence or absence of MSP. Macrophage apoptosis was determined by DNA fragmentation and Hoechst dye 33258 staining assays. The results are shown in Figure 1 . LPS induced DNA fragmentation in peritoneal resident macrophages when cells were cultured under serum-free conditions (Fig. 1A) . DNA fragmentation was also seen when cells were stimulated with LPS plus IFN-{gamma}. In contrast, stimulation of macrophages with MSP alone did not induce DNA ladder formation. However, when MSP was added together with LPS, LPS-induced DNA ladder formation was reduced, indicating that ligand-induced RON activation has the ability to inhibit LPS-induced macrophage apoptosis.



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  		Figure 1. Protective effect of MSP on apoptosis of primary macrophages stimulated with LPS. Mouse peritoneal resident macrophages (2x106/dish) were stimulated with 1 µg/ml LPS alone or with 100 U/ml IFN-{gamma}. MSP (5 nM) was added simultaneously. After incubation of cells for 24 h, apoptotic cells were determined by DNA fragmentation assay (A) or by Hoechst dye 33258 nuclei staining as % of apoptotic cells (mean±SD of triplicate wells; B). CTL, Cytolytic T lymphocytes. Experiments were repeated three times. Results shown here are from one representative experiment.

 
To quantitate the antiapoptotic effect of MSP on LPS-stimulated macrophages, the Hoechst dye 33258 staining assay was performed. Results were shown in Figure 1B . In macrophages stimulated with LPS alone or plus IFN-{gamma}, the increased numbers of apoptotic cells were observed. The percentages of LPS- or LPS plus IFN-{gamma}-induced macrophage apoptosis reached about 27% or 23%. However, when MSP was included in cell cultures together with LPS or LPS plus IFN-{gamma}, the percentages of nuclei stained with H33258 were reduced significantly to 8.5% or 6%, respectively. These data suggest that RON activation blocks LPS-induced apoptotic activities in primary peritoneal macrophages.

The protective effect of MSP in vivo against LPS-induced macrophage apoptosis
To confirm the protective effect of RON in vivo, mice were injected i.p. with human MSP for 30 min and then injected with LPS and CHX. Peritoneal macrophages were isolated, and the apoptotic rates were determined 10 h after LPS treatment. The results were shown in Figure 2 . In mice treated with LPS and CHX, the percentages of apoptotic macrophages ranged at about 28%. However, when mice were pretreated with MSP, the numbers of LPS-induced apoptotic macrophages were reduced to only 15%, an almost 50% reduction. These data suggest that activation of RON in vivo exerts a protective effect on peritoneal macrophages against LPS-induced apoptosis. This protective effect could have implication in RON-mediated anti-inflammatory activities in vivo [20 , 21 ].



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  		Figure 2. Protective effect of MSP in vivo on LPS-induced macrophage apoptosis. Four mice from each group were used for injection as described in Materials and Methods. Values are means ± SD from each group.

 
Inhibition by MSP of apoptosis in RON-expressing Raw264.7 cells
To determine whether the protective effect of RON could be reproduced with established macrophage cell lines, murine macrophage-like Raw264.7 cells, molecularly engineered to express human RON, were stimulated with LPS in the presence or absence of MSP. The original Raw264.7 cells do not express murine RON [25 ]. After stimulation, the antiapoptotic effect of MSP was determined by the DNA fragmentation assay. The results are shown in Figure 3 . Again, LPS induced DNA fragmentation in Raw264.7 cells. This effect was prevented by MSP. Also, the synergistic effect of LPS and IFN-{gamma} was blocked significantly after RON activation. By quantitative analysis using the Hoechst dye 33258 nuclear-staining assay, we found that RON activation by MSP significantly prevents LPS-induced Raw264.7 cell apoptosis. The percentages of LPS-induced apoptotic cells (35.4%) were reduced to 12.7% when MSP was included.



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  		Figure 3. Inhibition by MSP of LPS-induced apoptotic death in RON-expressing Raw264.7 macrophages. Experimental conditions were the same as those described in Figure 1 , except RON-expressing Raw264.7 cells were used. Experiments were repeated twice. Results shown here are from one representative experiment.

 
To further demonstrate that RON activation protects macrophages from LPS-induced apoptosis, Raw264.7 cells transfected with a constitutively active RON mutant (D1232V) [26 ] were included in experiments. These cells, together with Raw264.7 cells transfected with the pcDNA3.0 vector alone (Raw-PC), were stimulated with LPS in the presence or absence of MSP. The percentages of apoptotic cells were determined in the Hoechst H33258-staining assay. The results are shown in Figure 4 . LPS induced apoptosis in Raw264.7 cells transfected with pcDNA3.0 vector alone. The addition of MSP had no protective effect, because cells do not express RON. However, the expression of the D1232V mutant rendered cells less sensitive to LPS-induced apoptosis, as evident by the decreased apoptotic cells. The addition of MSP also did not show a significantly protective effect on these cells, because D1232V is a constitutively active RON mutant. These results suggest that RON activation is required for the protection of macrophages from apoptosis.



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  		Figure 4. Inhibition of LPS-induced apoptotic death of Raw264.7 cells by the constitutively active RON mutant. Experimental conditions were the same as described in Figure 1 . Cells were all stimulated with LPS plus IFN-{gamma}. Raw-PC refers to Raw264.7 cells transfected with pcDNA3.0 vector alone. Experiments were repeated three times. Results shown here are from one representative experiment.

 
Effect of RON activation on chemical NO donor-induced macrophage apoptosis
To determine whether RON activation has an inhibitory effect on NO-induced apoptosis, RON-expressing Raw264.7 cells were stimulated with a toxic dose of NO donor GSNO in the presence or absence of MSP. LPS was used as the control. The results are shown in Figure 5 . GSNO at a concentration of 100 µM induced DNA fragmentation in RON-expressing Raw264.7 cells. The effect of GSNO is comparable with that induced by LPS. The addition of MSP not only inhibited LPS-induced cell death but also blocked GSNO-induced DNA fragmentation (Fig. 5A) . These results are in line with those derived from Hoechst dye 33258 staining (Fig. 5B) . Activation of RON by MSP significantly reduced the percentages of H33258-stained nuclei in GSNO-treated cells.



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  		Figure 5. Effect of RON activation on GSNO-induced apoptosis of Raw264.7 cells. RON-expressing Raw264.7 cells were stimulated with GSNO (100 µM) for 24 h in the presence or absence of 5 nM MSP. LPS was used as the positive control. Apoptotic cells were determined by DNA fragmentation assay (A) or by Hoechst dye 33258 nuclei staining (B). The data shown in B are presented as mean ± SD of triplicate wells and derived from one of three experiments with similar results.

 
Correlation of MSP-induced iNOS inhibition and cell survival in LPS-stimulated Raw264.7 cells
Because LPS-induced NO is the principle molecule responsible for macrophage apoptosis, we sought to determine the effect of RON activation on LPS-induced iNOS expression to correlate the apoptotic effect of LPS. The results are shown in Figure 6 . LPS induced the iNOS protein expression in a time-dependent manner. RON activation inhibited LPS-induced iNOS expression. The levels of iNOS 24 h after LPS and MSP treatment are only about 8% of those induced by LPS alone (Fig. 6A) . Similarly, LPS-induced NO accumulated in culture fluids (23.7 µM/24 h incubation) was also reduced to 5.3 µM when MSP was included. The percentages of apoptotic cells after treatment with LPS or LPS plus MSP are shown in Figure 6B . A progressive increase of apoptotic cells was observed after LPS treatment. However, in the presence of MSP, the numbers of cells stained with Hoechst dye 33258 were reduced significantly. These data suggest that the effect of RON on macrophage apoptosis is accompanied with the inhibition of LPS-induced iNOS expression



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  		Figure 6. Correlation of MSP-induced inhibition of iNOS expression and cell apoptosis in Raw264.7 cells expressing RON. Macrophages (2x106/ml) were stimulated with 1 µg/ml LPS in the presence or absence of 5 nM MSP. At time 0, 8, or 24 h, cells were lysed or stained with Hoechst dye 33258. (A) Cellular proteins (50 µg/sample) were separated in 12% SDS-polyacrylamide gel electrophoresis (PAGE) under reduced conditions. Rabbit IgG against iNOS was used in Western blotting. Lanes 1, 3, and 5, stimulated with LPS alone; lanes 2, 4, and 6, stimulated with LPS and MSP. (B) Cells were stained with H33258 and counted under microscope. Open bars, stimulated with LPS alone; shaded bars, stimulated with LPS and MSP. Results are shown as mean ± SD of triplicate wells. Experiments were repeated twice with similar results.

 
Inhibitory effect of RON on LPS-induced p53 expression
To study the protective effect of RON in more detail, we measured the levels of the p53 protein induced by LPS or GSNO in the presence or absence of MSP. Peritoneal resident macrophages were stimulated with LPS alone or with IFN-{gamma}. The effect of MSP on p53 expression was determined in Western blotting with specific anti-p53 antibodies. The results are shown in Figure 7A . The p53 protein was not expressed in unstimulated macrophages. Also, MSP alone did not induce p53 expression. As expected, LPS induced p53 expression. However, in the presence of MSP, LPS-induced p53 expression was inhibited. The levels of p53 in cells treated with LPS and MSP were only about 27% of those induced by LPS. RON-expressing Raw264.7 cells were also used to see the inhibitory effect of RON on p53 expression. As shown in Figure 7B , MSP significantly inhibited LPS-induced p53 expression. Moreover, GSNO-induced p53 expression was also inhibited by MSP in a dose-dependent manner. The inhibition ranges at 44% or 71% in 2 or 10 MSP-treated cells (Fig. 7C) . These results suggest that RON activation blocks LPS or GSNO-induced p53 expression not only in peritoneal resident macrophages but also in established RON-expressing Raw264.7 cells.



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  		Figure 7. Inhibitory effect of MSP on LPS or GSNO-induced p53 expression. Mouse peritoneal resident macrophages (A) or RON-expressing Raw264.7 cells (B) at 2 x 106 cells/ml were stimulated with LPS in the presence or absence of 2 nM MSP for 8 h at 37°C. RON-expressing Raw264.7 cells were also stimulated with 100 µM GSNO in the presence of 2 or 10 nM MSP for 8 h (C). Western blotting was performed as described in Materials and Methods. Experiments were repeated three times. Results shown here are from one representative experiment.

 
Effect of MSP-induced activation of PI-3 kinase on LPS-induced apoptosis
To determine if PI-3 kinase is involved in the RON-mediated inhibitory effect, RON-expressing Raw264.7 cells were pretreated with wortmannin and then stimulated with LPS in the presence of MSP. The results are shown in Figure 8 . Wortmannin alone had no effect on cell survival even added together with LPS. However, when wortmannin was used with MSP, the percentages of LPS-induced macrophage apoptosis were increased significantly, indicating that wortmannin prevents the inhibitory effect of RON on LPS-induced macrophage apoptosis. These data suggest that by inhibiting RON-mediated activation of PI-3 kinase, wortmannin prevents the RON-mediated inhibition of macrophage apoptosis induced by LPS.



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  		Figure 8. Effect of wortmannin on RON-mediated inhibition of macrophage apoptosis. RON-expressing Raw264.7 cells were pretreated with 50 or 200 nM wortmannin (WT) for 30 min at room temperature and then stimulated with 1 µg/ml LPS for 24 h. MSP (5 nM) was added with LPS simultaneously. Nuclei staining with H33258 was performed as described in Materials and Methods. The results are expressed as the percentages of stained nuclei (mean±SD of triplicate wells). Experiments were repeated three times with similar results.

 
To further confirm the role of PI-3 kinase, RON-expressing Raw264.7 cells transfected with a dominant-negative p85 of PI-3 kinase ({Delta}p85; clones R{Delta}p85-1 and R{Delta}p85-2) were used. After stimulation of these cells with LPS in the presence or absence of MSP, the percentages of apoptotic macrophages were determined. The results are shown in Figure 9 . As usual, MSP inhibited apoptosis of control Raw264.7 cells (expressing RON only) induced by LPS. However, in cells expressing RON and {Delta}p85, the inhibitory effect of MSP on LPS-induced apoptosis was diminished significantly. The numbers of H33258-stained nuclei were increased to about 18% (clone R{Delta}p85-1) and 21% (clone R{Delta}p85-2) in these two cell lines, suggesting that inhibition of endogenous PI-3 kinase by {Delta}p85 prevented the RON-mediated inhibition of LPS-induced macrophage apoptosis.



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  		Figure 9. Effect of dominant-negative p85 of PI-3 kinase in RON-mediated inhibition of macrophage apoptosis. R{Delta}p85-1 (top panel) or R{Delta}p85-2 (middle panel) cells were stimulated with LPS in the presence or absence of MSP as described in Figure 7 . Raw264.7 cells expressing RON only (bottom panel) were used as the positive control. Wortmannin was used at the concentration of 200 nM. H33258 staining was conducted as described. The top and middle panels represent R{Delta}p85-1 and R{Delta}p85-2 cells, respectively. The bottom panel represents RON-expressing Raw264.7 cells. Experiments were repeated twice. Results shown here are from one representative experiment and are expressed as the percentages of stained nuclei (mean±SD of triplicate wells).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The involvement of receptor tyrosine kinases in protecting macrophages from apoptosis has been described previously [29 , 30 ]. The typical example is the colony-stimulating factor-1 receptor encoded by the c-fms protooncogene [30 ]. We have now demonstrated that RON is also a receptor that transduces survival signals in macrophages. Activation of RON by MSP protects primary resident macrophages or established Raw264.7 cells from apoptosis induced by LPS and chemical NO donor GSNO. As demonstrated in our experiments, activated RON has the ability to antagonize LPS-induced apoptotic action by inhibiting DNA fragmentation, reducing the numbers of cell nucleus stained with Hoechst dye 33258, and suppressing LPS-induced p53 expression. Our results also showed that PI-3 kinase is one of the critical signaling transducers involved in RON-mediated inhibition of macrophage apoptosis. The significance of these findings is important for two reasons. First, RON is a receptor tyrosine kinase displaying dual functions in macrophages [16 , 23 ]. The stimulatory activities of RON include macrophage cell-shape change, chemotactic migration, and phagocytosis [16 ]. These functions are essential for macrophages to move to inflammatory sites and to eliminate invading pathogens and damaged cells. Conversely, RON activation inhibits LPS or inflammatory cytokine-induced NO production [23 , 25 ]. NO is a toxic molecule responsible for numerous pathophysiological consequences of LPS, including macrophage apoptosis [31 ]. In this sense, the inhibition of apoptosis by RON may provide a protective mechanism for activated macrophages and allow them to perform their functional activities during bacterial infection and tissue injury. Second, RON has in vivo anti-inflammatory and septic activities. Mice with the disrupted RON gene have shown increased inflammatory reactions and were more susceptible to death in LPS-induced septic shock [20 , 21 ]. These abnormal responses are in part a result of the unbalanced production of NO by RON-/- macrophages [20 , 21 ]. Considering these activities of RON, it is reasonable to think that by inhibiting NO production and protecting macrophage from LPS-induced apoptosis, RON activation may facilitate macrophage survival, migration, and phagocytosis at the sites of inflammation. The results shown Figure 2 indicated that this might be the case.

Macrophage apoptosis occurs under various pathological conditions [32 ]. Many stimuli including LPS, bacterial lipoprotein, and Fas ligand are capable of inducing macrophage apoptosis [9 , 33 , 34 ]. We show here that peritoneal resident macrophages were susceptible to LPS-induced apoptosis, as evident by the formation of DNA fragmentation and the increased percentages of the nuclei stained with the DNA-specific fluorochrome Hoechst dye 33258. These cellular damaging activities of LPS were inhibited following RON activation. Moreover, the protective effect of RON could be reproduced by using Raw264.7 macrophage-like cells molecularly engineered to express human RON. These results indicate that ligand-dependent activation of RON is required to block the apoptotic signals induced by LPS. This notion is supported further by the results derived from experiments using Raw264.7 cells expressing a constitutively active RON mutant. In these cells, LPS-induced DNA fragmentation was reduced significantly, even in the absence of MSP.

One of the effector molecules that mediate LPS-induced macrophage apoptosis is NO [24 ]. In murine macrophages, NO has emerged as a principle molecule responsible for LPS-induced apoptosis occurring in macrophages derived from different tissues [9 , 34 , 36 ]. NO-induced apoptosis also occurs in established human or murine monocyte/macrophage cell lines including Raw264.7 cells [28 ]. Our data showed that RON activation not only inhibits LPS but also NO donor GSNO-induced apoptotic death of Raw264.7 cells expressing human RON. These results suggest that the effects of RON are probably mediated by blocking LPS-induced production of NO and GSNO-induced p53 expression. We have demonstrated previously that activation of RON results in inhibition of LPS-induced NO formation but not cytokine production in primary macrophages and Raw264.7 cells [23 ]. Our results presented in Figure 6 further confirmed that MSP inhibits LPS-induced iNOS expression in a time-dependent manner, which is correlated with the appearance of cellular DNA fragmentation. As we showed previously, activated RON transduces signals that block the promoter activities of the iNOS gene and the iNOS mRNA expression induced by LPS and IFN-{gamma} [25 ]. Moreover, these effects were mediated by blocking LPS-induced nuclear factor-{kappa}B (NF-{kappa}B) activation [37 ]. Because LPS-induced macrophage apoptosis is mediated mainly by NO, and RON is an endogenous NO inhibitor, we reasoned that the inhibitory effect of RON on LPS-induced apoptosis is mediated by inhibition of NO production. However, because the toxic effect of NO is only a part of LPS activities, which causes macrophage apoptosis, more studies are needed to determine if other mechanisms are also involved. As shown in Figure 5 , RON activation inhibits macrophage apoptosis induced by chemically generated NO. This result suggests a different mechanism is involved in RON-mediated inhibition of NO-induced macrophage apoptosis. Inhibition of p53 expression as shown in Figure 7 might be one of the mechanisms. Thus, the antiapoptotic effects of RON are not limited in inhibiting iNOS expression. Other mechanisms such as blocking p53 expression may also apply.

Cellular mechanisms by which LPS and NO induce macrophage apoptosis have been studied extensively [30 , 38 , 39 ]. Induction of p53 accumulation, regulation of Bcl-2 expression, and activation of members of the caspase family are the most commonly observed changes in LPS or NO-stimulated macrophages, which are in the process of apoptosis [30 , 38 , 39 ]. Although defining the detailed mechanisms of how RON inhibits macrophage apoptosis is a subject for future investigation, our current results do provide some clues, which may explain the actions of RON in protecting macrophages from apoptotic death. We found that LPS-induced p53 accumulation was inhibited following MSP treatment. RON activation also partially blocks GSNO-induced p53 expression. The p53 protein is well-known for its role in the cellular response to DNA damage caused by exogenous stimuli including NO [40 ]. It has been demonstrated that p53 activation results in cell-cycle arrest or apoptosis of the affected cells [41 ]. In NO-stimulated Raw264.7 cells, the correlation between accumulation of p53 and induction of apoptosis has been established [20 ]. Biochemical analysis has shown that LPS-induced p53 accumulation is mainly a result of the NO-mediated inhibition of cellular proteasome activities [42 ]. With regard to the effect of RON on LPS-induced p53 expression, we do not know what kind of mechanisms are involved in the action of RON. However, it will be of interest in the future to determine whether RON activation transduces signals that affect p53 expression at the gene level or controls p53 degradation through posttranslational mechanisms.

One of the interesting findings in this study is that PI-3 kinase is involved in RON-mediated inhibition of macrophage apoptosis. PI-3 kinase is well-known for its role in regulating cell growth, migration, differentiation, and survival [43 ]. In PC-12 cells, inhibition of PI-3 kinase by wortmannin or a dominant-negative p85 of PI-3 kinase blocks the cell-surviving effect of the nerve-growth factor [44 ]. Activation of PI-3 kinase is also required for macrophage survival when cultured in serum-free condition [45 ]. We have shown previously that RON activation stimulates PI-3 kinase and causes association of RON with p85 of PI-3 kinase [46 ]. Functional studies also demonstrated that blocking PI-3 kinase activities results in inhibition of RON-mediated macrophage cell-shape change and migration [46 ]. More importantly, suppressing PI-3 kinase prevents RON-mediated inhibition of NO production induced by LPS and inflammatory cytokines [25 ]. We now show that inhibition of RON-mediated activation of PI-3 kinase by specific inhibitor wortmannin or the dominant-negative p85 of PI-3 kinase restores the apoptotic effect of LPS on macrophages. These accumulated evidences provide a functional connection between PI-3 kinase-mediated inhibition of NO production and macrophage apoptosis. We postulate that in macrophages, LPS-induced NO acts on cellular DNA and causes apoptotic death of macrophages. However, in the presence of MSP, activated PI-3 kinase transduces signals that block LPS-induced NO synthesis and p53 accumulation. By antagonizing these LPS-induced toxic effects, activated RON provides survival signals for macrophages. The protective activities of RON could play an important role in regulating macrophage functions during bacterial infection.


   ACKNOWLEDGEMENTS
 
This work is supported by Public Health Service grant RO1 AI43516 from the National Institutes of Health to M-H. W. We thank Dr. E. J. Leonard (National Cancer Institute, Frederick, MD) for providing MSP, Dr. G. Gaudino (University of Torino, Novara, Italy) for the cDNA encoding the RON mutant, and Dr. H. Esumi (National Cancer Institute, Tokyo, Japan) for the cDNA of {Delta}p85 of PI-3 kinase.

Received February 22, 2001; revised October 6, 2001; accepted October 9, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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