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Macrophage stimulating protein (MSP) evokes superoxide anion production by human macrophages of different origin.

ABSTRACT: 1. 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. 2. 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. 3. 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. 4. 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.

SUBMITTER: Brunelleschi S

PROVIDER: S-EPMC1573047 | biostudies-other | 2001 Nov

REPOSITORIES: biostudies-other

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Project description:2-D-Deoxyglucose (2-dGlc) uptake and accumulation into rat peritoneal macrophages was increased by colony-stimulating factor (mCSF) by stimulating the coupling between endofacial hexokinase activity and the sugar transporter. The evidence for this is as follows: (1) mCSF significantly decreased the Km for zero-trans uptake (P less than 0.05), without altering Vmax.; (2) the accumulation of free 2-dGlc was increased by mCSF (P less than 0.05); (3) mCSF retarded the rate of exit of accumulated free 2-dGlc. The mCSF-dependent increase in 2-dGlc uptake by macrophages was enhanced by preincubation of the cells in mCSF-free solution. The activity of the hexose monophosphate shunt (HMPS) measured by the differential uptake of 2-d[1-3H]Glc and 2-d[2,6-3H]Glc was not stimulated by mCSF. Also, in quiescent cells, superoxide production, as determined by cytochrome c reduction, was unaffected by mCSF. Phorbol myristate acetate (PMA; 40 nM) stimulated both the HMPS activity and superoxide production. Both these effects were dependent on the uptake of external sugar (2-dGlc). Incubation of the macrophages with mCSF enhanced the sugar transport and PMA-dependent stimulation of HMPS activity and superoxide production, indicating a role for mCSF in the 'priming' of macrophage functions. Both HMPS activity and superoxide production are entirely dependent on uptake of exogenous sugar, since the potent sugar-transport inhibitor cytochalasin B competitively inhibited 2-dGlc uptake, HMPS activity and superoxide generation in PMA-activated cells (Ki approximately 0.3 microM for all three processes). Over a wide range of 2-dGlc concentrations, 4 mol of superoxide were generated/mol of 2-dGlc metabolized in the HMPS pathway, indicating coupling between these processes. The Km of 2-d[2,6-3H]Glc uptake in PMA-treated cells was 0.45 +/- 0.07 mM, and Vmax. was 1.32 +/- 0.05 mumol.min-1.ml of cell water-1. It is evident that there is a large degree of slippage between HMPS activity and membrane-associated hexokinase activity, since the Km for HMPS activity was 0.06 +/- 0.02 mM and the Vmax. was 0.10 +/- 0.03 mumol.min-1.ml of cell water-1.

Project description:Incubation of human neutrophils with a chemotactic peptide [N-formylmethionyl-leucylphenylalanine (fMLP)] gave rise to an increase in the phosphoinositide 3-kinase (PI3K) activity, phosphorylation of p47phox and superoxide-anion (O2(-)) generation in the same fMLP-concentration-dependent manner. These responses to fMLP were markedly enhanced when the cells had been incubated for 10 min before the addition of fMLP with increasing concentrations of granulocyte-macrophage colony-stimulating factor (GM-CSF) that were only slightly effective themselves. Wortmannin, an inhibitor of PI3K, suppressed all of these fMLP actions in the same concentration-dependent manner in either GM-CSF-primed or non-primed cells. Sustained activation of protein kinase C by the addition of PMA caused marked phosphorylation of p47phox and respiratory burst itself without activation of PI3K. This strong action of PMA was not primed by GM-CSF. The chemotactic peptide was without effect in pertussis-toxin-treated cells, indicating that its actions are mediated by betagamma-subunits liberated from toxin-susceptible heterotrimeric Gi proteins (Gbetagamma). Thus one of the mechanisms of GM-CSF-mediated priming of fMLP-induced respiratory burst is synergistic activation of wortmannin-sensitive PI3K by Gbetagamma in the presence of tyrosine-phosphorylated proteins in GM-CSF-treated cells, as recently indicated in a cell-free system [Kurosu, Maehama, Okada, Yamamoto, Hoshino, Fukui, Ui, Hazeki and Katada (1997) J. Biol. Chem. 272, 24252-24256]. GM-CSF primed fMLP-induced MAP (mitogen-activated protein) kinase activation enormously as well. The MAP kinase activation was primed even in the presence of wortmannin, indicating that PI3K was not the sole site where tyrosine kinase-related and Gbetagamma-mediated intracellular signals converge to elicit the priming. The GM-CSF priming of fMLP-induced PI3K activation and O2(-) generation was much smaller in magnitude in neutrophils in which cAMP accumulated upon incubation with prostaglandin E1 than in the cells without the nucleotide accumulation. Thus the GM-CSF priming site, in addition to PI3K, might be just the target of cAMP-dependent protein kinase A in fMLP-initiated signalling cascades or could be localized immediately downstream thereof.

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Macrophage stimulating protein (MSP) evokes superoxide anion production by human macrophages of different origin.

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