The N-formylpeptide receptor (FPR) and a second Gi-coupled receptor mediate fMet–Leu–Phe-stimulated activation of NADPH oxidase in murine neutrophils

doi:10.1016/S0008-8749(02)00564-6

Cellular Immunology

Volume 218, Issues 1–2, July–August 2002, Pages 7-12

https://doi.org/10.1016/S0008-8749(02)00564-6 Get rights and content

Abstract

N-Formylypeptides such as fMet–Leu–Phe (fMLF) potently induce superoxide production through NADPH oxidase activation. The receptors that mediate this response have not been defined. Here, we provide definitive proof using a mouse model that formyl peptide receptor (FPR) is a receptor, but not the only receptor, that mediates fMLF-induced oxidase activation. In wild-type (FPR^+/+) mouse neutrophils, superoxide production is dependent on the concentration of fMLF with an EC₅₀ of ∼5 μM and a peak at ∼50 μM. In contrast, FPR-deficient (FPR^−/−) mouse neutrophils produced markedly less superoxide with an EC₅₀ of ∼50 μM and a peak at ∼200 μM. Yet, FPR^+/+ and FPR^−/− neutrophils showed similar oxidase activation kinetics and G_i protein-dependent pharmacological sensitivities. These results suggested that a second receptor, likely FPR2, mediates superoxide production at high concentrations of fMLF. This less sensitive second pathway may permit continued oxidant generation in response to formyl peptides when FPR is desensitized in high concentrations of the chemotactic gradient.

Introduction

N-Formylpeptides potently stimulate phagocyte migration, degranulation, and production of reactive oxygen species through activation of NADPH oxidase [1], [2], [3], [4]. In vitro functional assays have revealed that both high- [5] and low- [6] affinity human fMet–Leu–Phe (fMLF) receptors, which are both expressed in neutrophils [7] and are termed FPR (formyl peptide receptor) and FPRL1R (formylpeptide receptor-like 1 receptor), respectively, mediate fMLF-stimulated mobilization of intracellular Ca²⁺[7], [8], [9] and chemotaxis [10] in receptor-transfected cells. Studies in FPR-deficient (“knockout”) mice have shown that FPR and FPR2, the murine counterparts of human FPR and FPRL1R, respectively, mediate both of these functions in mouse neutrophils [11]. However, the receptors that activate NADPH oxidase have not been defined.

The receptor-mediated or PMA-stimulated assembly of the essential components of NADPH oxidase, including gp91phox (phagocyte oxidase), p47phox, p67phox, p22phox, and Rac1 or Rac2, catalyzes the oxidation of NADPH and reduction of oxygen to form highly reactive superoxide anions [12]. Superoxide generation by NADPH oxidase in phagocytes such as neutrophils and macrophages is critical for host resistance against life-threatening bacterial and fungal infections, as demonstrated by the oxidase defects observed in individuals afflicted with chronic granulomatous disease (CGD) [12], [13], [14]. Conversely, excess production of superoxide can promote tissue destruction and disease [15]. Therefore, precise delineation of the receptors and signaling pathway(s) involved in activation of this enzyme is an important research goal.

Activation of cells by N-formylpeptides is especially relevant because the prototype N-formylpeptide, fMLF, is a cleavage product of bacterial [16] and mitochondrial [17] proteins, implicating this peptide in bacteria-induced and tissue destruction-induced inflammatory events. Furthermore, fMLF stimulates unusually robust oxidase activity in phagocytes, as compared to other chemoattractants such as the chemokines [18]. Interestingly, two closely related chemokine receptors [19], CXCR1 and CXCR2, both mediate IL-8-stimulated chemotaxis [20] and Ca²⁺ mobilization [19] with similar efficiency, but only CXCR1 is coupled to the respiratory burst [21], illustrating the unpredictability of structure–function correlations for even closely related receptors, such as murine FPR and FPR2. In this study, we provide evidence that FPR and a second G_i-dependent formyl peptide receptor, likely FPR2, mediate NADPH oxidase activation in mouse neutrophils.

Section snippets

Cell preparation

The FPR-deficient (−/−; “knockout”) mouse, which was derived from FPR^+/−×FPR^+/− matings of an F1 backcross of FPR^+/− 129/Sv with wild-type C57Bl/6 mice, has been described previously [22]. Leukocytes were harvested from the peritoneal cavities of wild-type (FPR^+/+) or FPR-deficient (FPR^−/−) mice approximately 4 h following intraperitoneal injection of each mouse with 1 ml of 3% thioglycollate. Greater than 90% of the cells recovered were neutrophils as indicated by the morphologic appearance of

fMLF stimulates superoxide production in mouse neutrophils through FPR-dependent and FPR-independent pathways

To confirm that fMLF activates NADPH oxidase in primary cells through FPR, we compared fMLF-stimulated superoxide production in FPR^+/+ and FPR^−/− mouse neutrophils. The neutrophils were isolated from mouse peritoneal cavities approximately 4 h following intraperitoneal injection of thioglycollate. As reported previously [22], there was no significant difference in the number of neutrophils harvested from peritoneal cavities of FPR^+/+ and FPR^−/− mice injected with thioglycollate. fMLF-stimulated

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The contradictory data were obtained for high- and low affinity receptors signaling to NADPH oxidase. Lavigne et al. [19] deduced that common Gi-coupled signal transduction pathways function downstream of both receptors in mouse neutrophils [19]. Whereas Fu et al. [20] supposed a difference in signaling between two receptors by virtue of sensitivity to phosphoinositide binding peptide PBP10.
Polymorphonuclear neutrophils (PMNs) express the high and low affinity receptors to formylated peptides (mFPR1 and mFPR2 in mice, accordingly). RhoA/ROCK (Rho activated kinase) pathway is crucial for cell motility and oxidase activity regulated via FPRs. There are contradictory data on RhoA-mediated regulation of NADPH oxidase activity in phagocytes. We have shown divergent Rho GTPases signaling via mFPR1 and mFPR2 to NADPH oxidase in PMNs from inflammatory site. The present study was aimed to find out the role of RhoA/ROCK in the respiratory burst activated via mFPR1 and mFPR2 in the bone marrow PMNs. Different kinetics of RhoA activation were detected with 0.1 μM fMLF and 1 μM WKYMVM operating via mFPR1 and mFPR2, accordingly. RhoA was translocated in fMLF-activated cells towards the cell center and juxtamembrane space versus uniform allocation in the resting cells. Specific inhibition of RhoA by CT04, Rho inhibitor I, weakly depressed the respiratory burst induced via mFPR1, but significantly increased the one induced via mFPR2. Inhibition of ROCK, the main effector of RhoA, by Y27632 led to the same effect on the respiratory burst. Regulation of mFPR2-induced respiratory response by ROCK was impossible under the cytoskeleton disruption by cytochalasin D, whereas it persisted in the case of mFPR1 activation. Thus we suggest RhoA to be one of the regulatory and signal transduction components in the respiratory burst through FPRs in the mouse bone marrow PMNs. Both mFPR1 and mFPR2 binding with a ligand trigger the activation of RhoA. FPR1 signaling through RhoA/ROCK increases NADPH-oxidase activity. But in FPR2 action RhoA/ROCK together with cytoskeleton-linked systems down-regulates NADPH-oxidase. This mechanism could restrain the reactive oxygen species dependent damage of own tissues during the chemotaxis of PMNs and in the resting cells.
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Several recent studies have shown that activation of FPR2 is potentially proatherogenic (Fig. 1). The first evidence is that FPR2 activation may contribute to superoxide production when neutrophils are stimulated with N-formyl-Met–Leu–Phe (fMLF), a potent chemoattractant for phagocytes [5]. This effect is mediated by NADPH oxidase (NOX) activation and it was found that reactive oxygen species (ROS) production result from NOX activation might accelerate atherogenesis [6].
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The need for novel anti-inflammatory drugs justifies the search for innovative targets that could satisfy this goal. For quite some time now, we have proposed the study of endogenous anti-inflammation as a distinctive approach to the discovery of new drugs. This approach requires development of new compounds that activate specific receptor targets to downregulate the cellular and tissue pathways operative in the host during inflammation. Here we dwell on a family of G-protein coupled receptors (GPCR) termed FPRs, acronym for formyl-peptide receptors. With three and seven members in man and mouse, respectively, these receptors harness many biological functions, spanning odour perception and hair growth, to the control of multiple facets (pain; cell migration; oxidative burst; xenobiotic engulfment) of the inflammatory reaction. We focus on FPR biology with particular attention to molecules able to produce pharmacological effects by interacting with these GPCRs, describing endogenous agonists of FPRs and, more relevantly, the current development of synthetic agonists. Besides being potential leads for the development of the anti-inflammatory therapeutics of the future, these compounds could also help clarify the properties and roles that each FPR might play in the complex network of pathways that is inflammation. We conclude that FPR2 agonists could be valid warhorses for defining a novel philosophy for anti-inflammatory drug discovery programmes: mimicking – with new compounds – the way our body disposes of inflammation could be a viable approach to regulate aberrant inflammatory responses as in the case of several chronic rheumatic and cardiovascular pathologies.
ClC-3 and ICl<inf>swell</inf> are required for normal neutrophil chemotaxis and shape change
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Chemotaxis Assays—For the EZ-TAXIScan™ and DIAS chemotaxis assays, we chose to use fMLF as the CA, because it has been studied extensively in both human and murine PMN chemotaxis. Although murine PMNs lack receptors for human PMN CAs, such as CXCR1, CXCL8 (interleukin-8 receptor), and neutrophil-activating peptide-2 receptors, both species have receptors responsible for sensing fMLF spatial gradients (20, 21). EZ-TAXIScan™ Assays—EZ-TAXIScan™ assays (Effector Cell Institute, Tokyo) were performed at 37 °C using the assembled chemotaxis apparatus, composed of the chip holder and silicone chip perfused in EZT buffer, as described (22).
Polymorphonuclear leukocytes undergo directed movement to sites of infection, a complex process known as chemotaxis. Extension of the polymorphonuclear leukocyte (PMN) leading edge toward a chemoattractant in association with uropod retraction must involve a coordinated increase/decrease in membrane, redistribution of cell volume, or both. Deficits in PMN phagocytosis and trans-endothelial migration, both highly motile PMN functions, suggested that the anion transporters, ClC-3 and ICl_swell, are involved in cell motility and shape change (Moreland, J. G., Davis, A. P., Bailey, G., Nauseef, W. M., and Lamb, F. S. (2006) J. Biol. Chem. 281, 12277-12288). We hypothesized that ClC-3 and ICl_swell are required for normal PMN chemotaxis through regulation of cell volume and shape change. Using complementary chemotaxis assays, EZ-TAXIScan™ and dynamic imaging analysis software, we analyzed the directed cell movement and morphology of PMNs lacking normal anion transporter function. Murine Clcn3^-/- PMNs and human PMNs treated with anion transporter inhibitors demonstrated impaired chemotaxis in response to formyl peptide. This included decreased cell velocity and failure to undergo normal cycles of elongation and retraction. Impaired chemotaxis was not due to a diminished number of formyl peptide receptors in either murine or human PMNs, as measured by flow cytometry. Murine Clcn3^-/- and Clcn3^+/+ PMNs demonstrated a similar regulatory volume decrease, indicating that the ICl_swell response to hypotonic challenge was intact in these cells. We further demonstrated that ICl_swell is essential for shape change during human PMN chemotaxis. We speculate that ClC-3 and ICl_swell have unique roles in regulation of PMN chemotaxis; ICl_swell through direct effects on PMN volume and ClC-3 through regulation of ICl_swell.
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The N-formylpeptide receptor (FPR) and a second Gi-coupled receptor mediate fMet–Leu–Phe-stimulated activation of NADPH oxidase in murine neutrophils

Abstract

Introduction

Section snippets

Cell preparation

fMLF stimulates superoxide production in mouse neutrophils through FPR-dependent and FPR-independent pathways

Pharmacol. Ther.

Blood

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

FEBS Lett.

J. Biol. Chem.

J. Biol. Chem.

Blood

J. Biol. Chem.

Neutrophil signal transduction and activation of the respiratory burst

Physiol. Rev.

Chemoattractant stimulus-response coupling

The human N-formylpeptide receptor. Characterization of two cDNA isolates and evidence for a new subfamily of G-protein-coupled receptors

Biochemistry

Signal transducing properties of the N-formyl peptide receptor expressed in undifferentiated HL60 cells

J. Immunol.

A seven-transmembrane, G protein-coupled receptor, FPRL1, mediates the chemotactic activity of serum amyloid A for human phagocytic cells

J. Exp. Med.

The N-formylpeptide receptor (FPR) and a second G_i-coupled receptor mediate fMet–Leu–Phe-stimulated activation of NADPH oxidase in murine neutrophils