Amyloid-β Induces Chemotaxis and Oxidant Stress by Acting at Formylpeptide Receptor 2, a G Protein-coupled Receptor Expressed in Phagocytes and Brain

Abstract

Amyloid-␤, 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-␤-dependent functions in phagocytic cells. First, transfection of FPR2, but not related receptors, including the other known N-formylpeptide receptor FPR, reconstituted amyloid-␤-dependent chemotaxis and calcium flux in HEK 293 cells. Second, amyloid-␤ 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 G i -dependent signaling. Third, specific and reciprocal desensitization of superoxide production was observed when N-formylpeptides and amyloid-␤ 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-␤ in the mouse microglial cell line N9. Further, in sequential stimulation experiments with N9 cells, N-formylpeptides and amyloid-␤ were able to reciprocally cross-desensitize each other. Amyloid-␤ 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-␤ to phagocyte chemotaxis and oxidant stress in the brain.

Sphingosine 1-Phosphate, a Diffusible Calcium Influx Factor Mediating Store-operated Calcium Entry

“…To further exclude the possibility that PMN responses to S1P were initiated via G␣ icoupled S1PR, we treated PMN with pertussis toxin (PTX, 1 g/ml, for 3 h at 37°C) prior to stimulation with S1P. G␣ icoupled calcium store release in the neutrophil is fully inhibited at this PTX dose (32)(33)(34). We found that, in the neutrophil, PTX had no effect upon S1P-induced calcium entry (Fig.…”

Trp-Lys-Tyr-Met-Val-Met stimulates phagocytosis via phospho-lipase D-dependent signaling in mouse dendritic cells

“…The fMLF also has been reported to elicit superoxide generation in the mouse neutrophils via FPR1 activation (Fillion et al, 2001;Li et al, 2002). Regarding the functional roles of FPR2, the amyloid beta peptide has been reported to induce phagocyte chemotaxis and oxidant stress in phagocytes (Tiffany et al, 2001). Although the functional roles of the activation of the FPR family receptors in the neutrophils were extensively studied, their roles in the DCs have not been fully investigated.…”

“…responses in mice (Gao et al, 1999;Hartt et al, 2000;Liang et al, 2000;Tiffany et al, 2001). Activating FPR1 via its specific agonist, N-formyl-methionyl-leucyl-phenylalanine (fMLF) induced the chemotactic migration of phagocytic cells, such as monocytes and neutrophils.…”

CXCL12 Signaling Is Independent of Jak2 and Jak3

“…Cells were stimulated with CXCL12 in a continuously stirred cuvette at 37°C, and changes in intracellular calcium levels were detected fluorimetrically using a ratio fluorescence spectrometer (model MS-III; Photon Technology Inc., South Brunswick, NJ) (37). Data were recorded every 20 ms as the ratio of fluorescence emitted at 510 nM following sequential excitation at 340 and 380 nM.…”