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

Abstract

We have previously reported (Badolato, R., J.M. Wang, W.J. Murphy, A. R. Lloyd, D.F. Michiel, L.L. Bausserman, D.J. Kelvin, and J.J. Oppenheim. 1994. J. Exp. Med. 180:203; Xu, L., R. Badolato, W.J. Murphy, D.L. Longo, M. Anver, S. Hale, J.J. Oppenheim, and J.M. Wang. 1995. J. Immunol. 155:1184.) that the acute phase protein serum amyloid A (SAA) is a potent chemoattractant for human leukocytes in vitro and mouse phagocytes in vivo. To identify the signaling mechanisms, we evaluated patterns of cross-desensitization between SAA and other leukocyte chemoattractants. We found that the chemotactic bacterial peptide, N-formyl- methionyl-leucyl-phenylalanine (fMLP), was able to specifically attenuate Ca2+ mobilization in human phagocytes induced by SAA, but only at very high concentrations, suggesting that SAA uses a low affinity fMLP receptor. Here we demonstrate that SAA selectively induced Ca2+ mobilization and migration of HEK cells expressing FPRL1, a human seven-transmembrane domain phagocyte receptor with low affinity for fMLP, and high affinity for lipoxin A4. Furthermore, radiolabeled SAA specifically bound to human phagocytes and FPRL1-transfected 293 cells. In contrast, SAA was not a ligand or agonist for FPR, the high affinity fMLP receptor. Thus, SAA is the first chemotactic ligand identified for FPRL1. Our results suggest that FPRL1 mediates phagocyte migration in response to SAA.

Figure 1

Cross-desensitization of Ca²⁺ mobilization in human monocytes between SAA and fMLP. Fura-2–loaded monocytes were sequentially stimulated with SAA and fMLP (A) or vice versa (B and C), and the ratio of fluorescence at 340 and 380 nm wavelengths was recorded and calculated with the FLWinLab program.

Figure 2

Calcium mobilization in FPRL1-transfected HEK 293 cells. FPRL1/293 cells were loaded with Fura-2 and were stimulated with various concentrations of fMLP (A) or SAA (B). SAA does not induce Ca²⁺ mobilization in FPR-expressing 293 cells (C) or mock-transfected 293 cells (D). E shows the sequential stimulation of FPRL1/293 cells with SAA and fMLP or vice versa.

Figure 3

Chemotactic activity of SAA for human monocytes and cells transfected to express chemoattractant receptors. Different concentrations of SAA were placed in the lower wells of the chemotaxis chamber. Monocytes, FPRL1/293 cells, or FPR-expressing ETFR cells were placed in the upper wells. After incubation, the cells migrated across the polycarbonate filter were counted and photographed (A: SAA 0.8 μM, fMLP 100 nM). The cell migration was expressed as CI representing the fold increase of the cells migrating in response to stimulants over control medium. (B) Migration of FPRL1/293 cells in response to SAA and fMLP. (C) Migration of FPR-expressing ETFR cells in response to SAA and fMLP. (D) Effect of HDL on SAA induced FPRL1/293 cell migration. HDL at 1,000 μg/ml mixed with 0.8 μM SAA was preincubated at 37°C for 4 h. The mixture was then tested for chemotactic activity on FPRL1/293 cells. The HDL/SAA mixture without preincubation was also tested for chemotactic activity and yielded similar results.

Figure 3

Chemotactic activity of SAA for human monocytes and cells transfected to express chemoattractant receptors. Different concentrations of SAA were placed in the lower wells of the chemotaxis chamber. Monocytes, FPRL1/293 cells, or FPR-expressing ETFR cells were placed in the upper wells. After incubation, the cells migrated across the polycarbonate filter were counted and photographed (A: SAA 0.8 μM, fMLP 100 nM). The cell migration was expressed as CI representing the fold increase of the cells migrating in response to stimulants over control medium. (B) Migration of FPRL1/293 cells in response to SAA and fMLP. (C) Migration of FPR-expressing ETFR cells in response to SAA and fMLP. (D) Effect of HDL on SAA induced FPRL1/293 cell migration. HDL at 1,000 μg/ml mixed with 0.8 μM SAA was preincubated at 37°C for 4 h. The mixture was then tested for chemotactic activity on FPRL1/293 cells. The HDL/SAA mixture without preincubation was also tested for chemotactic activity and yielded similar results.

Figure 4

Binding of ¹²⁵I-labeled SAA to human monocytes and FPRL1/293 cells. rhSAA was radio-iodinated with chloramine T method and the binding of ¹²⁵I-labeled SAA to FPRL1/293 cells (A) or monocytes (B) was measured by adding a constant concentration of ¹²⁵I-labeled SAA to the cells in the presence of increasing concentrations of unlabeled SAA. The data was analyzed and plotted with the Macintosh computer aided program LIGAND. C shows the displacement of ¹²⁵I-labeled SAA binding on monocytes by unlabeled SAA and fMLP. The same results were obtained with neutrophils (data not shown) and FPLR1/293 cells (D).