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Macrophage Stimulating Protein (MSP), a serum factor related to Hepatocyte Growth Factor, was originally discovered to stimulate chemotaxis of murine resident peritoneal macrophages. MSP is the ligand for Ron, a member of the Met subfamily of tyrosine kinase receptors. The effects of MSP on human macrophages and the role played in human pathophysiology have long been elusive.
We show here that human recombinant MSP (hrMSP) evokes a dose-dependent superoxide anion production in human alveolar and peritoneal macrophages as well as in monocyte-derived macrophages, but not in circulating human monocytes. Consistently, the mature Ron protein is expressed by the MSP responsive cells but not by the unresponsive monocytes. The respiratory burst evoked by hrMSP is quantitatively higher than the one induced by N-formylmethionyl-leucyl-phenylalanine and similar to phorbol myristate acetate-evoked one.
To investigate the mechanisms involved in NADPH oxidase activation, leading to superoxide anion production, different signal transduction inhibitors were used. By using the non selective tyrosine kinase inhibitor genistein, the selective c-Src inhibitor PP1, the tyrosine phosphatase inhibitor sodium orthovanadate, the phosphatidylinositol 3-kinase inhibitor wortmannin, the p38 inhibitor SB203580, the MEK inhibitor PD098059, we demonstrate that hrMSP-evoked superoxide production is mediated by tyrosine kinase activity, requires the activation of Src but not of PI 3-kinase. We also show that MAP kinase and p38 signalling pathways are involved.
These results clearly indicate that hrMSP induces the respiratory burst in human macrophages but not in monocytes, suggesting for the MSP/Ron complex a role of activator as well as of possible marker for human mature macrophages.
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Macrophage Stimulating Protein (MSP) is a 78 kDa heterodimeric protein composed by a disulfide-linked 53 kDa α chain and a 23 kDa β chain. It belongs to the kringle protein family, that includes plasminogen, prothrombin and Hepatocyte Growth Factor (HGF) (Yoshimura et al., 1993). MSP is synthesized in the liver and circulates in the blood in nanomolar concentration as a biologically inactive single-chain precursor, pro-MSP. Proteolytic cleavage of pro-MSP, yielding the dimeric mature MSP required for the activation of its receptor, occurs on the cell surface of murine peritoneal macrophages and in wound fluids by the pro-MSP convertase activity, a trypsin-like serine protease (Nanney et al., 1998; Wang et al., 1996c). The specific receptor of MSP is the transmembrane tyrosine kinase Ron, which belongs to the Met proto-oncogene family. Ron is a heterodimeric receptor composed of extracellular α (35 kDa) and a transmembrane β (150 kDa) chains (Gaudino et al., 1994; Wang et al., 1994b). MSP ligand stimulation of Ron-expressing cells rapidly induces the tyrosine kinase activity of the receptor, and Ron signalling occurs through association with multiple effectors [e.g., phospholipase C-γ, phosphatidylinositol 3-kinase (PI 3-kinase), focal adhesion kinase, c-Src, mitogen-activated protein kinase (MAP kinase), c-Jun amino terminal kinase (JNK)] as a consequence of the phosphorylation of a unique multifunctional docking site, conserved in all members of the Met family (Danilkovitch & Leonard, 1999; Danilkovitch-Miagkova et al., 2000; Iwama et al., 1996; Wang et al., 1996b). Since its discovery as a serum protein able to induce responsiveness to the chemoattractant C5a, and to enhance spreading, phagocytosis and chemotaxis in mouse resident peritoneal macrophages (Leonard & Skeel, 1979), the MSP spectrum of activity has widened and this protein is now regarded as a pleiotrophic factor. MSP stimulates human and murine bone marrow megakaryocytopoiesis, induces apoptosis in erythroid cell lines, induces proliferation and migration of murine keratinocytes and liver progenitor cells, increases ciliary motility of human nasal cilia, stimulates bone resorption by human osteoclasts, and promotes migration of non-small cell lung tumour cells (Banu et al., 1996; Kurihara et al., 1996; Medico et al., 1996; Sakamoto et al., 1997; Wang et al., 1996a; Willett et al., 1998). An increased expression of mRNA for MSP has been shown in experimental models of inflammation and liver regeneration in the rat (Bezerra et al., 1994), while a decreased hepatic MSP production has been suggested to impair Kupffer cell phagocytosis in patients with fulminant hepatic failure (Harrison et al., 1994).
MSP also inhibits lipopolysaccharide (LPS)- and/or cytokine-induced nitric oxide (NO) release and iNOS expression in murine peritoneal macrophages (Chen et al., 1998; Wang et al., 1994a), by negative modulation of co-stimulatory signals activating the transcription factor NF-κB (Liu et al., 1999).
The expression of the mouse Ron homologue Stk has been regarded as a marker of terminal differentiation of murine peritoneal macrophages, since this receptor is present in peritoneal macrophages, but not in alveolar macrophages, acute exudate macrophages or circulating monocytes (Iwama et al., 1995).
Knock-out mice lacking the Ron/Stk receptor showed enhanced susceptibility to LPS-induced septic shock, increased NO production as well as increased responses in tests of delayed-type hypersensitivity (Correll et al., 1997). Moreover, it has been demonstrated that mice carrying a targeted loss of MSP have a delayed macrophage activation, although migration of mononuclear phagocytes into the peritoneal cavity is unaltered as compared to wild-type animals (Bezerra et al., 1998).
In mouse peritoneal resident macrophages, as well as in peritoneal exudate macrophages, Ron becomes expressed and tyrosine phosphorylated upon MSP stimulation. Consequently, iNOS expression and NO release from mature peritoneal exudate macrophages are inhibited (Chen et al., 1998). Interestingly, endotoxin-induced NO production down-regulates Ron expression in murine macrophages (Wang et al., 2000).
As far as NO production is concerned, human monocyte/macrophages are at variance from the rodent ones. While murine macrophages rapidly produce large amounts of NO after challenge with classical stimuli such as IFN-γ, TNF-α or LPS, human monocytes and tissue macrophages usually do not. The actual amount of NO metabolites released by human monocyte/macrophages is extremely low when compared to that produced by rodent cells, although human monocytes and macrophages express the iNOS gene (Albina, 1995).
Recently, Ron immunoreactivity has been detected in dermal macrophages of normal human skin as well as in macrophages from burn wound skin (Nanney et al., 1998); however, the functions exerted by MSP on human monocyte/macrophages are still elusive.
This prompted us to investigate the effects of MSP on human mononuclear phagocytes of different origins: circulating monocytes, monocyte-derived macrophages, alveolar macrophages and peritoneal macrophages. In the inflammatory and immune processes, monocytes and macrophages operate phagocytosis, chemotaxis, antigen processing and presentation, secretion of enzymes and cytokines as well as oxy-radical production (Morrissette et al., 1999). Therefore, we used these cells to evaluate the ability of MSP to evoke superoxide anion (O2−) production, as compared to other stimuli such as N-formylmethionyl-leucyl-phenylalanine (FMLP) and phorbol 12-myristate 13-acetate (PMA). Moreover, by using different pharmacological inhibitors, we investigated the signal transduction pathways involved.
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The results presented here indicate that human macrophages of different origin (alveolar, peritoneal and in vitro-differentiated macrophages) express the tyrosine kinase receptor Ron, whereas in human circulating monocytes no Ron protein was detected. These results also confirm and further extend previous observations in murine macrophages (Iwama et al., 1995) as well as in the monocytic THP-1 cell line, which expressed Ron only when differentiated to macrophages by IFN-γ plus TNF-α treatment (Chen et al., 1996). Interestingly, Ron expression has been observed in human dermal macrophages from both normal and burn wound skin (Nanney et al., 1998).
Human Ron mRNA is expressed in the lung (Gaudino et al., 1994) and the protein has been detected at the apical surface of the bronchiolar ciliated epithelium, but not in goblet cells (Sakamoto et al., 1997). Moreover, MSP content ranged from 1.3 to 5.8 ng/ml in BAL fluids obtained from four healthy non-smokers, but the absolute concentration of MSP in the bronchoalveolar space, where AMs are located, was possibly higher as suggested by Sakamoto et al. (1997). It is attractive to speculate that MSP, via circulation (Wang et al., 1996c), can be supplied to the bronchoalveolar spaces, where it binds and activates Ron on AMs to induce the respiratory burst, as suggested by our results. The stimulatory effect of hrMSP on AMs was comparable to the one evoked by the tumour promoter PMA and higher than the chemotactic peptide FMLP-induced one. Similar findings have been reported in human nasal cilia, where MSP ability to increase ciliary motility is similar to that of LTC4 and other inflammatory mediators (Sakamoto et al., 1997). Moreover, by evaluating MSP levels in induced sputum, Takano et al. (2000) found a significant increase in patients with bronchiectasis as compared with normal controls. They also reported that half of the MSP in sputum is present as mature MSP, that is the biologically active alpha/beta chain heterodimer (Takano et al., 2000). Furthermore, the elevated O2− production measured after challenge with hrMSP in AMs isolated from patients with sarcoidosis might suggest a peculiar involvement of MSP in interstitial lung diseases.
We also demonstrate that the tyrosine kinase receptor Ron is expressed in MDMs and PMs isolated from ascitic fluid of cirrhotic patients, and that hrMSP induces a dose-dependent respiratory burst. In this case, too, hrMSP induced a higher amount of O2−, as compared to FMLP, strongly implying this factor as a potent regulator of macrophage activity.
The Ron receptor is functional in the macrophage lineage cells but not in circulating monocytes, since hrMSP activates only human macrophages of different origin to produce superoxide anion. This is a novel activity proposed for MSP, not described so far, that suggests a role of MSP in host defense and control of infection.
The source of reactive oxygen intermediates is the NADPH oxidase, a multi-component enzyme complex which transfers a single electron from NADPH to oxygen, resulting in the production of O2−. The O2− so generated is in turn the key element for the production of hydrogen peroxide, hydroxyl radical, singlet oxygen and long-living oxidants.
NADPH oxidase, which comprises both cytosolic and membrane components, is inactive until the cell is stimulated by phagocytosis or various inflammatory mediators. Activation is a multi-step process, not completely elucidated, which comprises partial activation of p47phox, translocation of p47phox, p67phox and p40phox to the membrane, final phosphorylation of p47phox, coupled to the acquisition of the catalytic activity of the enzyme (Babior, 1999).
NO has been demonstrated to inhibit the oxidase by preventing its assembly during activation (Fujii et al., 1997) and it is tempting to speculate that hrMSP- evoked O2− production in our cells might result, as least in part, from its ability to inhibit NO release, as demonstrated in murine macrophages (Chen et al., 1998; Liu et al., 1999).
The tyrosine kinase activity of Src-like protein kinases, as well as PI 3-kinase and MAP kinases, have also been associated to activation of the NADPH oxidase (Downey et al., 1998; Erdreich-Epstein et al., 1999; Lal et al., 1999; Rane et al., 1997). The majority of these studies have been performed using human neutrophils, while few of them were conducted on monocyte/macrophages. In rat AMs, different MAP kinases have been shown to be responsible for NADPH oxidase activation in response to zymosan-activated serum (Torres & Forman, 1999); in another macrophage population, the human bone marrow-derived macrophages, tyrosine kinases of the Src family have been demonstrated to play a key role in O2− production evoked by immune complexes (Erdreich-Epstein et al., 1999).
The mechanism of hrMSP-induced O2− production in human macrophages is still to be defined. However, PI 3-kinase does not seem to play a key role in the process, although this enzyme has been implicated in MSP/Ron signal transduction in other cell types. Our data show that wortmannin had no significant inhibitory effect on hrMSP-evoked O2− production, while markedly inhibited FMLP-evoked (but not PMA-evoked) respiratory burst. These observations are consistent with the implication of PI 3-kinase in chemotaxis and in NADPH oxidase activation of human eosinophils elicited by agonists acting through G protein-coupled receptors (Elsner et al., 1996). Moreover, also in the respiratory burst of human neutrophils, wortmannin has been repetitively reported to inhibit FMLP- but not PMA- induced responses (Kodama et al., 1999; Tudan et al., 1999).
The involvement of tyrosine kinases in NADPH oxidase activation in PMs was evaluated by using selective and non-selective inhibitors. The non-selective tyrosine kinase inhibitor genistein inhibited hrMSP- and FMLP-evoked O2− production in human PMs, while inactive against PMA, so confirming previous data in human neutrophils (Tan et al., 1998). The increased O2− production that we observed in the presence of the tyrosine phosphatase inhibitor sodium vanadate further confirmed the role of genistein in the respiratory burst. Sodium vanadate enhanced by 40% hrMSP- and FMLP-evoked respiratory burst, but did not affect PMA-evoked one. It has to be taken into account that the role of this phosphatase inhibitor in PMA-stimulated macrophages is controversial, being either inhibitory (Conde et al., 1995) or stimulatory (Green & Phillips, 1994).
PP1, a selective inhibitor of the Src-family tyrosine kinase activity, dose-dependently inhibited hrMSP- and PMA-evoked O2− production, while less active versus FMLP. This finding is partially at variance with previous observations in bone marrow-derived human macrophages (Erdreich-Epstein et al., 1999), showing a large inhibitory effect (about 80% with PP1 10 μM) in immune complexes-induced respiratory burst but no effect on PMA-evoked one. We have no definitive explanation for this fact, but differences in the macrophage cell type might be envisaged, since, as reported by the authors (Erdreich-Epstein et al., 1999), 99.5% of bone marrow-derived human macrophages (recovered from the screens used to filter bone marrow after harvest, grown and differentiated in M-CSF) were CD14 positive. Recent investigations in epithelial cells have shown that Ron activation is integrin-dependent and that adhesion of RON-expressing epithelial cells to extracellular matrix requires the tyrosine kinase activity of c-Src and Ron itself as well. Actually, collagen-induced Ron phosphorylation becomes reduced in cells transfected with a dominant-negative kinase-inactive Src construct, suggesting that Ron is phosphorylated by activated Src (Danilkovitch-Miagkova et al., 2000). The fact that Src can phosphorylate Ron in epithelial cells when stimulated by collagen, but not by MSP, indicates that the pattern of Ron tyrosine phosphorylation induced by extracellular matrix is distinct from that induced by MSP. Moreover, MSP addition to collagen-adherent cells resulted in an increased Ron phosphorylation and kinase activity as compared to MSP or collagen alone (Danilkovitch-Miagkova et al., 2000). Whether the known Src inhibitor PP1 affects MSP-evoked respiratory burst either through the direct inhibition of Src or indirectly, requires further investigations. However, in our opinion, our observations on macrophages further strengthen the role of Src in MSP/Ron signalling.
The dual specificity kinase MEK (MAP kinase kinase) activates ERKs (Extracellular-regulated protein kinases) and p38, which are members of the MAP kinase family. MEK inhibition by PD098059 prevents O2− production in zymosan-stimulated neutrophils (Downey et al., 1998), while conflicting results have been reported for FMLP (Downey et al., 1998; Kuroki & O'flaherty, 1997). On the other hand, inhibition of p38 by SB203580 prevents NADPH oxidase activation by FMLP and by PMA (Lal et al., 1999).
Our results indicate that PD 098059 inhibited hrMSP- and FMLP- evoked respiratory burst, while a slight increase was observed at 10 μMversus PMA. On its turn, the p38 inhibitor SB 203580 displayed a clear-cut inhibitory effect on all evaluated stimuli, i.e. hrMSP, FMLP and PMA, in accordance with results obtained in human neutrophils (Lal et al., 1999).
These results are in agreement with the observation that in Ron-transfected MDCK cells, MSP activates Ras by activation and translocation of the nucleotide exchange factor SOS (Li et al., 1995) and confirm that ERKs and p38 are primary targets of the pathway induced by Ron, via the Ras/MAP kinase pathway. It is well known that the activity of NADPH oxidase can be regulated also by Rac, a small GTP binding protein of the Ras family (Babior, 1999; Irani & Goldschmidt-Clermont, 1998), that has been shown activated by several tyrosine kinase receptors (Babior, 1999; Boehm et al., 1999). A possibility therefore exists that the pattern of activation of NADPH oxidase by MSP could also rely on Rac.
However, the occasional discrepancies observed in some experiments on signal transduction inhibitors are due to the pathways elicited by the different stimuli, given that FMLP acts through a G-protein-coupled receptor, unlike MSP and PMA. Another explanation relates to possible structural differences in the mechanism of NADPH oxidase activation between macrophages and other phagocytes (e.g., neutrophils, where most experiments have been conducted).
The role of reactive oxygen species has recently been re-evaluated and growing evidence points to their physiological function as signalling molecules in the regulation of intracellular pathways; protein tyrosine kinases, protein tyrosine phosphatases, phospholipase A2 and the transcription factor NF-κB were identified as potential targets of hydrogen peroxide (Finkel, 1998). Reactive oxygen species produced by the NADPH oxidase could regulate the dynamic equilibrium between the opposing effects of the tyrosine kinases and tyrosine phosphatases (Finkel, 1998). Therefore, one can speculate that the O2− produced by MSP in human macrophages of different origin might also act as a second messenger for signal transduction.