Amyloid-β Induces Chemotaxis and Oxidant Stress by Acting at Formylpeptide Receptor 2, a G Protein-coupled Receptor Expressed in Phagocytes and Brain
2001; Elsevier BV; Volume: 276; Issue: 26 Linguagem: Inglês
10.1074/jbc.m101031200
ISSN1083-351X
AutoresH. Lee Tiffany, Mark C. Lavigne, You‐Hong Cui, Jiming Wang, Thomas L. Leto, Ji‐Liang Gao, Philip M. Murphy,
Tópico(s)Bone Metabolism and Diseases
ResumoAmyloid-β, the pathologic protein in Alzheimer's disease, induces chemotaxis and production of reactive oxygen species in phagocytic cells, but mechanisms have not been fully defined. Here we provide three lines of evidence that the phagocyte G protein-coupled receptor (N-formylpeptide receptor 2 (FPR2)) mediates these amyloid-β-dependent functions in phagocytic cells. First, transfection of FPR2, but not related receptors, including the other known N-formylpeptide receptor FPR, reconstituted amyloid-β-dependent chemotaxis and calcium flux in HEK 293 cells. Second, amyloid-β induced both calcium flux and chemotaxis in mouse neutrophils (which express endogenous FPR2) with similar potency as in FPR2-transfected HEK 293 cells. This activity could be specifically desensitized in both cell types by preincubation with a specific FPR2 agonist, which desensitizes the receptor, or with pertussis toxin, which uncouples it from Gi-dependent signaling. Third, specific and reciprocal desensitization of superoxide production was observed whenN-formylpeptides and amyloid-β were used to sequentially stimulate neutrophils from FPR −/− mice, which express FPR2 normally. Potential biological relevance of these results to the neuroinflammation associated with Alzheimer's disease was suggested by two additional findings: first, FPR2 mRNA could be detected by PCR in mouse brain; second, induction of FPR2 expression correlated with induction of calcium flux and chemotaxis by amyloid-β in the mouse microglial cell line N9. Further, in sequential stimulation experiments with N9 cells, N-formylpeptides and amyloid-β were able to reciprocally cross-desensitize each other. Amyloid-β was also a specific agonist at the human counterpart of FPR2, the FPR-like 1 receptor. These results suggest a unified signaling mechanism for linking amyloid-β to phagocyte chemotaxis and oxidant stress in the brain. Amyloid-β, the pathologic protein in Alzheimer's disease, induces chemotaxis and production of reactive oxygen species in phagocytic cells, but mechanisms have not been fully defined. Here we provide three lines of evidence that the phagocyte G protein-coupled receptor (N-formylpeptide receptor 2 (FPR2)) mediates these amyloid-β-dependent functions in phagocytic cells. First, transfection of FPR2, but not related receptors, including the other known N-formylpeptide receptor FPR, reconstituted amyloid-β-dependent chemotaxis and calcium flux in HEK 293 cells. Second, amyloid-β induced both calcium flux and chemotaxis in mouse neutrophils (which express endogenous FPR2) with similar potency as in FPR2-transfected HEK 293 cells. This activity could be specifically desensitized in both cell types by preincubation with a specific FPR2 agonist, which desensitizes the receptor, or with pertussis toxin, which uncouples it from Gi-dependent signaling. Third, specific and reciprocal desensitization of superoxide production was observed whenN-formylpeptides and amyloid-β were used to sequentially stimulate neutrophils from FPR −/− mice, which express FPR2 normally. Potential biological relevance of these results to the neuroinflammation associated with Alzheimer's disease was suggested by two additional findings: first, FPR2 mRNA could be detected by PCR in mouse brain; second, induction of FPR2 expression correlated with induction of calcium flux and chemotaxis by amyloid-β in the mouse microglial cell line N9. Further, in sequential stimulation experiments with N9 cells, N-formylpeptides and amyloid-β were able to reciprocally cross-desensitize each other. Amyloid-β was also a specific agonist at the human counterpart of FPR2, the FPR-like 1 receptor. 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Ageing Dev. 1997; 94: 199-211Crossref PubMed Scopus (43) Google Scholar) as well as Aβ induction of calcium flux in HL-60 cells (33Takenouchi T. Munekata E. Peptides. 1995; 16 (; Correction (1995) Peptides16, 1557): 1019-1024Crossref PubMed Scopus (15) Google Scholar). This, together with the fact that calcium flux is strongly associated with GPCR activation by chemoattractants, suggested to us that Aβ may act via a GPCR. Since ligand promiscuity is a common property of chemoattractant receptors, we tested this hypothesis by examining the ability of cloned phagocyte chemoattractant receptors to reconstitute Aβ signaling in a transfected cell line. We also investigated receptors mediating Aβ signaling on mouse phagocytes (reported here) and human phagocytes (reported separately). Construction of human embryonic kidney (HEK) 293 cell lines expressing human formylpeptide receptor (FPR), human formylpeptide receptor-like 1 receptor (FPRL1R), mouse FPR, mouse FPR2, mouse lipoxin A4 receptor (encoded by Fpr-rs1), a mouse orphan receptor encoded by Fpr-rs3, and human CCR5 and CX3CR1 has been previously described (34Hartt J.K. Barish G. Murphy P.M. Gao J-L. J. Exp. Med. 1999; 190: 741-747Crossref PubMed Scopus (100) Google Scholar, 35Combadiere C. Ahuja S.K. Tiffany H.L. Murphy P.M. J. Leukocyte Biol. 1996; 60: 147-152Crossref PubMed Scopus (267) Google Scholar, 36Combadiere C. Salzwedel K. Smith E.D. Tiffany H.L. Berger E.A. Murphy P.M. J. Biol. Chem. 1998; 273: 23799-23804Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 37Gao J.-L. Chen H. Filie J.D. Kozak C.A. Murphy P.M. Genomics. 1998; 51: 270-276Crossref PubMed Scopus (116) Google Scholar). Fpr-rs1 andFpr-rs3 were tested because of their high structural similarity to the known formylpeptide receptors and because they are also expressed in phagocytes (37Gao J.-L. Chen H. Filie J.D. Kozak C.A. Murphy P.M. Genomics. 1998; 51: 270-276Crossref PubMed Scopus (116) Google Scholar). Cells were grown in Dulbecco's modified Eagle's medium high glucose medium (Life Technologies, Inc.) containing 10% heat-inactivated fetal calf serum (Hyclone, Logan, UT), 100 units/ml penicillin, 100 μg/ml streptomycin (Hyclone), and 2 mg/ml G418 (Life Technologies) at 37 °C, 5% CO2, and 100% humidity. A human CCR1-expressing HEK 293 cell line has also been previously described (38Combadiere C. Ahuja S.K. Van Damme J. Tiffany H.L. Gao J-L. Murphy P.M. J. Biol. Chem. 1995; 270: 29671-29675Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar); culture conditions were the same except for usage of 200 units/ml hygromycin B (Calbiochem) as the selective antibiotic. A mouse pre-B cell lymphoma cell line (4DE4) expressing human CCR8 has been reported previously (39Tiffany H.L. Lautens L.L. Gao J-L. Pease J. Locati M. Combadiere C. Modi W. Bonner T.I. Murphy P.M. J. Exp. Med. 1997; 186: 165-170Crossref PubMed Scopus (187) Google Scholar). These cells were cultured in RPMI 1640 (Life Technologies) containing 10% heat-inactivated fetal bovine serum, 50 μm β-mercaptoethanol (Sigma), and 2 mg/ml G418. The N9 murine microglial cell line was a kind gift from Dr. P. Ricciardi-Castagnoli (Universita Degli Studi di Milano-Bicocca, Milan, Italy). These cells express typical markers of resting mouse microglia and have been extensively used as representatives of primary mouse microglial cells (40Ferrari D. Villalba M. Chiozzi P. Falzoni S. Ricciardi-Castagnoli P. Di Virgilio F. J. Immunol. 1996; 156: 1531-1539PubMed Google Scholar). The cells were grown in Iscove's modified Dulbecco's medium supplemented with 5% heat-inactivated fetal calf serum, 2 mm glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 50 mm 2-mercaptoethanol. Neutrophils were obtained from the peritoneal cavity of wild type and gene knockout litter mates of F1 and F6 backcrosses of 129/sV FPR−/− mice with C57Bl/6 mice 3–4 h after intraperitoneal injection of a 3% thioglycollate solution, as previously described (41Gao J-L. Lee E.J. Murphy P.M. J. Exp. Med. 1999; 189: 657-662Crossref PubMed Scopus (241) Google Scholar). The cell population was consistently composed of >90% neutrophils, as determined by light microscopy of DiffQuick-stained cytospins. Thus, hereafter we will refer to this cell preparation as neutrophils. To monitor intracellular Ca2+ concentration, adherent cells were harvested by incubation in phosphate-buffered saline at 37 °C for 15 min and then incubated in phosphate-buffered saline containing 2.5 μmFura-2/AM at 37 °C for 45 min. Cells were washed twice with HBSS (Life Technologies) and suspended in HBSS at 1–2 × 106/ml. One ml of cells was added to 1 ml of HBSS and stimulated with ligand in a continuously stirred cuvette at 37 °C in a fluorimeter (model MS-III; Photon Technology Inc., South Brunswick, NJ). Data were recorded every 200 ms as the relative ratio of fluorescence emitted at 510 nm following sequential excitation at 340 and 380 nm. The following ligands were evaluated: Aβ (nonfibrillated, human residues 1–42; California Peptide Research; Napa, CA), fMet-Leu-Phe (fMLF; Sigma), ATP (Life Technologies), and the chemokines RANTES, SDF-1, I-309, fractalkine, MIP-1α, and KC (Peprotech, Rocky Hill, NJ). The particular chemokines tested were chosen because of their specificity for phagocyte targets. All chemokines were human with the exception of KC, which is mouse. The receptor targets for these chemokines are as follows: RANTES, CCR1, CCR3 and CCR5; SDF-1, CXCR4; I-309, CCR8; fractalkine, CX3CR1; MIP-1α, CCR1, and CCR5; CXCR2. Aβ, chemokines and ATP were dissolved in water and stored at −20 °C; fMLF was dissolved in Me2SO and stored at −20 °C. In some experiments, the cells were incubated in 250 ng/ml pertussis toxin (PTX; Calbiochem) for 4 h at 37 °C in medium, harvested, and loaded with Fura-2/AM as described above. Immediately after harvesting, murine neutrophils were incubated in 1–2 × 106/ml of phosphate-buffered saline containing 2.5 μm Fura-2/AM for 45 min at 37 °C. Neutrophils were washed twice in HBSS and suspended to 1–2 × 106/ml for analysis. Calcium flux was performed with N9 cells preincubated in the presence or absence of 300 ng/ml lipopolysaccharide (LPS) (37 °C, 24 h) using similar procedures. HEK 293 cells were harvested from tissue culture flasks by incubation in trypsin (0.05%)/EDTA (0.1%) (Quality Biologicals, Inc., Gaithersburg, MD) for 5 min at 37 °C. Cells were suspended evenly by vigorous pipetting, and excess Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum was then added to block trypsin. Cells were washed twice in Dulbecco's modified Eagle's medium and suspended to a concentration of 4 × 106 cells/ml in chemotaxis medium (RPMI 1640; 20 mm HEPES (Life Technologies) and 1% bovine serum albumin (ICN Biomedicals Inc., Aurora, OH)). Chemoattractants, diluted in chemotaxis medium, were added to the bottom wells of a 96-well chemotaxis plate (Neuro Probe, Inc., Gaithersburg, MD). A 12-μm pore size membrane was placed on top, and 25 μl of cell suspension containing ∼100,000 cells was placed in the upper chamber. Cells were incubated for 5 h at 37 °C, 100% humidity, 5% CO2. The membrane was carefully removed, and cells in the bottom well were counted using a hemacytometer. Methods for murine neutrophils were the same except that ∼200,000 cells were added to the top of a 5-μm pore size membrane. Chemotaxis assays for N9 cells incubated with or without LPS (300 ng/ml, at 37 °C for 24 h) were performed with 48-well chemotaxis chambers (Neuro Probe). Polycarbonate filters with 8-μm pore size and 90-min incubation at 37 °C were used for measurement of microglial cell migration. Mouse neutrophils were suspended in HBSS containing Ca2+ and Mg2+ at 106 cells/ml. 50 μl (5 × 104 cells) were distributed into wells of a 96-well microtiter chemiluminescence plate and incubated at 37 °C for 5 min. Then a mixture of the superoxide-specific chemiluminescence indicator reagent Diogenes (National Diagnostics, Atlanta, GA) was added to the cells (50% of total reaction volume) with appropriate stimuli or vehicle control, and superoxide dismutase-inhibitable chemiluminescence was measured in a luminometer (Labsystems Luminoskan; Helsinki, Finland). Data are expressed as integrated luminescence (relative light units) observed during 0.5-s readings obtained at 12-s intervals over a time course of 10 min. For sequential stimulation experiments, 5 × 104 FPR −/− neutrophils were distributed into microcentrifuge tubes, and test substances were added. The mixture was then immediately transferred to a chemiluminescence plate. After incubation at 37 °C for 8 min (when fMLF was the first stimulus) or 9 min (when Aβ was the first stimulus), Diogenes reagent plus the final stimulus was added, and the activity was monitored for 10 min. To control for desensitization of NADPH oxidase by the first stimulus, cells were stimulated with PMA (100 ng/ml) after the second stimulation, and superoxide was measured for 10 min. To control for scavenging of superoxide by fMLF or Aβ, neutrophils were stimulated simultaneously with (i) PMA (100 ng/ml) and Me2SO (vehicle for fMLF; 0.2% of the volume in which cells were stimulated); (ii) PMA (100 ng/ml) and fMLF (50 μm); (iii) PMA (100 ng/ml); or (iv) PMA (100 ng/ml) plus Aβ (10 μm). Each condition was tested in triplicate, and the mean of the mean number (grand mean) of superoxide dismutase-inhibitable relative light units throughout the duration of the assay and the corresponding standard errors of grand means were calculated. Differences between conditions were tested for significance by two-tailed paired t tests or unequal variance tests (Mann-Whitney rank sum) where appropriate. A value ofp < 0.05 indicated significant differences. Wild type littermates of an F7 backcross of 129/sV FPR −/− mice with C57Bl/6 mice were euthanized by cervical dislocation, and brains from three mice were removed, pooled, and washed in phosphate-buffered saline for 15 min at room temperature. Brain tissue was sliced in a Petri dish on ice using a clean razor blade and homogenized in an ice-cold Teflon homogenizer. RNA was extracted using the RNA STAT-60 kit (Tel-Test, Inc., Friendswood, TX) according to the manufacturer's instructions. RNA was reverse-transcribed using the cDNA Cycle Kit (Invitrogen, San Diego, CA) following the manufacturer's instructions. Gene-specific primers were used for PCR amplification of the cDNA using the GeneAmp PCR System 9700 (PerkinElmer Life Sciences). For mouse FPR2, the 5′ primer 5′-TCTACCATCTCCAGAGTTCTGTGG and 3′ primer 5′-TTACATCTACCACAATGTGAACTA were used to generate a 268-base pair product. The PCR conditions for amplification were 3 min at 95 °C for the initial melting followed by 30 cycles of 1 min of melting at 95 °C, 1 min of annealing at 55 °C, 2 min of synthesis at 72 °C, with a final extension of 10 min at 72 °C and cooling to 4 °C. PCR products were analyzed by gel electrophoresis using a 1% agarose gel in TBE containing 10 μg of ethidium bromide/100 ml. Data were recorded on a UVP Gel Imaging System (Appropriate Technical Resource, Laurel, MD). For analysis of the N9 microglial cell line, RT-PCR was performed with 0.5 μg of total RNA extracted from cells treated with 300 ng/ml LPS for different time periods (High Fidelity ProSTARTM HF System, Stratagene, Kingsport, TN). The procedure consisted of a 15-min reverse transcription at 37 °C, 1-min inactivation of Moloney murine leukemia virus reverse transcriptase at 95 °C, and 40 cycles of denaturing at 95 °C (30 s), annealing at 55 °C (30 s), and extension at 72 °C (1 min), with a final extension for 10 min at 72 °C. Primers for murine β-actin gene were used as controls (Stratagene). The RT-PCR products at different dilutions were electrophoresed on 1% agarose gel and visualized with ethidium bromide staining. Using induction of calcium flux as a highly sensitive and specific real time assay of receptor activation, we screened a panel of stable cell lines transfected with plasmids encoding the known phagocyte formylpeptide receptors (human and mouse FPR, human FPRL1R, and mouse FPR2), four chemokine receptors (human CCR1, CCR5, CCR8, and CX3CR1), the mouse lipoxin A4 receptor (encoded by mouseFpr-rs1), and an orphan receptor highly related in sequence to formylpeptide receptors (Fpr-rs3), as well as untransfected control cells, for responsiveness to 10 μmAβ (Fig. 1). This concentration was chosen based on Aβ dose-response studies published previously for human neutrophils and monocytes and rat microglial cells (13Bianca V.D. Dusi S. Bianchini E. Pra I.D. Rossi F. J. Biol. Chem. 1999; 274: 15493-15499Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). The lipoxin A4 receptor and Fpr-rs3 were included because of their high sequence similarity to the formylpeptide receptors (37Gao J.-L. Chen H. Filie J.D. Kozak C.A. Murphy P.M. Genomics. 1998; 51: 270-276Crossref PubMed Scopus (116) Google Scholar). Aβ induced a response in HEK 293 cells expressing FPRL1R and FPR2, which are human and mouse low affinity formylpeptide receptors, respectively. Activation of each receptor produced a robust transient that was similar in magnitude and duration to the response induced by the prototypical N-formylpeptide fMLF in the same cells (Fig. 2, A and B) and was similar kinetically to the transients induced by other classic chemoattractants and chemokines (Fig. 1). Aβ was specific for these receptors, since none of the other cell lines tested responded. The CCR1, CCR5, CCR8, and CX3CR1 and the human and mouse FPR (high affinity formylpeptide receptor) cell lines did respond to appropriate known agonists as previously described (34Hartt J.K. Barish G. Murphy P.M. Gao J-L. J. Exp. Med. 1999; 190: 741-747Crossref PubMed Scopus (100) Google Scholar, 35Combadiere C. Ahuja S.K. Tiffany H.L. Murphy P.M. J. Leukocyte Biol. 1996; 60: 147-152Crossref PubMed Scopus (267) Google Scholar, 36Combadiere C. Salzwedel K. Smith E.D. Tiffany H.L. Berger E.A. Murphy P.M. J. Biol. Chem. 1998; 273: 23799-23804Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). The Fpr-rs1 andFpr-rs3 cell lines were unresponsive to fMLF but did respond to ATP through an endogenous signaling pathway. Although RNA forFpr-rs1 and Fpr-rs3 is present in these two cell lines, we have not yet obtained direct evidence of receptor protein expression. Aβ signaling could be completely blocked by pretreatment of the cells with pertussis toxin (Fig. 1, column 1,tracing labeled FPR2 + PTX), which inactivates Gi type G proteins. Pertussis toxin also blocks signaling by other FPR2 agonists (34Hartt J.K. Barish G. Murphy P.M. Gao J-L. J. Exp. Med. 1999; 190: 741-747Crossref PubMed Scopus (100) Google Scholar, 42Liang T.S. Wang J.-M. Murphy P.M. Gao J.-L. Biochem. Biophys. Res. Commun. 2000; 270: 331-335Crossref PubMed Scopus (81) Google Scholar,43Hartt J.K. Liang T. Sahagun-Ruiz A. Wang J.-M. Gao J.-L. Murphy P.M. Biochem. Biophys. Res. Commun. 2000; 272: 699-704Crossref PubMed Scopus (38) Google Scholar). When FPR2- and FPRL1R-expressing cells were sequentially stimulated with 10 μm Aβ, they responded to the first but not the second stimulus (Fig. 1, column 1,tracing labeled FPR2, and data not shown) indicating homologous desensitization of the signal transduction pathway, which is characteristic of G protein-coupled receptors (44Ali H. Richardson R.M. Haribabu B. Snyderman R. J. Biol. Chem. 1999; 274: 6027-6030Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). Moreover, Aβ and fMLF reciprocally interfered with each other's signaling at FPR2 (Fig. 2, A and B) in a concentration-dependent manner, providing further evidence that both agonists act at the same receptor. This was specific, since Aβ did not affect signaling by agonists acting at any of the other receptors considered (Fig. 1 and data not shown). Aβ induced calcium flux in both FPR2- and FPRL1R-transfected HEK 293 cells in a graded concentration-dependent manner, with an EC50 of 5 μm (Fig.3 A). In contrast, HEK 293 cells expressing either mouse or human FPR did not respond to Aβ from 0.5 to 20 μm (Fig. 3 A). However, all four cell lines responded to fMLF in a concentration-dependent manner, with EC50 consistent with those previously reported (data not shown; Ref. 34Hartt J.K. Barish G. Murphy P.M. Gao J-L. J. Exp. Med. 1999; 190: 741-747Crossref PubMed Scopus (100) Google Scholar). To test whether native FPR2 also functions as an Aβ receptor, we first focused on primary mouse neutrophils, which, as we have previously shown, express FPR2 endogenously (34Hartt J.K. Barish G. Murphy P.M. Gao J-L. J. Exp. Med. 1999; 190: 741-747Crossref PubMed Scopus (100) Google Scholar) and which can be analyzed in an FPR-deficient background due to the availability of FPR knockout mice (41Gao J-L. Lee E.J
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