by Rong He, Hairong Sang, and Richard D. Ye Serum amyloid A induces IL-8 secretion through a G protein–coupled receptor, FPRL1/LXA4R by Rong He, Hairong Sang, and Richard D. Ye Blood Volume 101(4):1572-1581 February 15, 2003 ©2003 by American Society of Hematology

SAA induces rapid secretion of IL-8 from neutrophils SAA induces rapid secretion of IL-8 from neutrophils.(A) Freshly prepared neutrophils were stimulated with 2 μM SAA at 37°C for 4 hours in serum-free medium. SAA induces rapid secretion of IL-8 from neutrophils.(A) Freshly prepared neutrophils were stimulated with 2 μM SAA at 37°C for 4 hours in serum-free medium. The secreted (from supernatant; closed bars) and cell-associated (from cell lysate; open bars) cytokines were detected by ELISA. The percentage of cell-associated cytokines in SAA-stimulated samples are 22% (IL-1β), 20% (IL-6), 3% (TNF-α), and 36% (IL-8). In unstimulated neutrophils, 77% of IL-8 is present in cell-associated form. (B) Neutrophils were similarly stimulated with LPS from E coli0111:B4 (LPS1) and S minnesota Re595 (LPS2), SAA, and TNF-α for 4 hours, at the indicated concentrations. Cell-associated IL-8 (open bars) and secreted IL-8 (closed bars) were determined by ELISA. All data are presented as mean ± SEM of 3 independent experiments, each performed in duplicate. Rong He et al. Blood 2003;101:1572-1581 ©2003 by American Society of Hematology

SAA-induced IL-8 secretion requires transcription and de novo protein synthesis.(A) Time course of SAA-induced IL-8 production. SAA-induced IL-8 secretion requires transcription and de novo protein synthesis.(A) Time course of SAA-induced IL-8 production. Neutrophils were stimulated with 2 μM SAA, and the secreted and cell-associated IL-8 concentrations were determined by ELISA after 1, 2, 3, and 4 hours. All data are presented as mean ± SEM of 3 independent experiments, each performed in duplicate. (B) Neutrophils, stimulated with 2 μM SAA for 1, 2.5, and 4 hours or incubated in the absence of SAA for 4 hours, were harvested for RNA preparation. RT-PCR was performed with specific primers for human IL-8 and G3PDH. The PCR products were resolved on a 1% agarose gel by electrophoresis and stained with ethidium bromide. Data shown are from one representative experiment of a total of 3, all with similar results. (C) Neutrophils were preincubated for 1 hour with or without actinomycin D (ActD; 10 μg/mL) or cycloheximide (CHX; 100 μM). The cells were then stimulated with 1 μM SAA for 4 hours. Secreted IL-8 was measured by ELISA as described. Maximal production of IL-8 (∼800 pg/106 cells/mL) was observed in the absence of either inhibitor and was set as 100%, against which the effects of ActD and CHX were measured. Data shown are means ± SEM derived from 3 independent experiments, each performed in duplicate. Rong He et al. Blood 2003;101:1572-1581 ©2003 by American Society of Hematology

SAA stimulates NF-κB activation SAA stimulates NF-κB activation.(A) Neutrophils were stimulated with 2 μM SAA for different periods of time. SAA stimulates NF-κB activation.(A) Neutrophils were stimulated with 2 μM SAA for different periods of time. Nuclear proteins were extracted and electrophoretic mobility shift assay was performed as described in “Materials and methods.” The NF-κB/DNA complex was detected by32P-labeled NF-κB oligonucleotide probe. Unlabeled NF-κB probe (100 × in excess) was used as a competitor to determine the specificity of DNA binding (lane 6). (B) Western blotting analysis was used to detect the cytoplasmic level of IκBα in neutrophils. Time-dependent degradation of IκBα was observed in SAA-treated (2 μM) but not the untreated neutrophils. Data shown are from one representative experiment of a total of 3, all with similar results. (C) HL-60 cells were transiently transfected with either a IL-8 luciferase reporter (−272–IL-8) or an NF-κB luciferase reporter (3 × NF-κB) as described in “Materials and methods.” The luciferase assays were performed after stimulation with 10 μM SAA for 4 hours at 37°C. Relative luciferase activities are expressed as fold induction over basal, after normalization of data against coexpressed β-galactosidase activities. (D) HL-60 cells (5 × 105 cells/200 μL) were cultured in serum-free medium with or without SAA for 4 hours. The secreted IL-8 was detected by ELISA. The fold increases of IL-8 production by the stimulated cells over unstimulated cells are shown. All data are presented as mean ± SEM, from 3 independent experiments, each performed in duplicate. Rong He et al. Blood 2003;101:1572-1581 ©2003 by American Society of Hematology

Phosphorylation of ERK1/2 and p38 and their potential involvement in SAA-induced IL-8 secretion.(A) Neutrophils (4 × 105 cells) were stimulated with 2 μM SAA for indicated times. Phosphorylation of ERK1/2 and p38 and their potential involvement in SAA-induced IL-8 secretion.(A) Neutrophils (4 × 105 cells) were stimulated with 2 μM SAA for indicated times. Phosphorylation of ERK1/2 and p38 was determined by Western blotting, using specific antibodies against phospho-ERK1/2 and phospho-p38. The unphosphorylated ERK1/2 and p38 were also determined. Three experiments were performed and a representative set of data is shown. (B) Neutrophils were treated with the MEK inhibitor U0126 or the p38 inhibitor SB202190 for 1 hour prior to SAA (2 μM) stimulation. The secreted IL-8 was measured after 4 hours and expressed as percentage changes. Maximal secretion of IL-8 (1090 pg/mL/106 cells) was obtained in the absence of inhibitor and was set as 100%. Data are presented as means ± SEM of 3 independent experiments, each performed in duplicate. Rong He et al. Blood 2003;101:1572-1581 ©2003 by American Society of Hematology

Elevation of intracellular Ca2+ is required for IL-8 secretion Elevation of intracellular Ca2+ is required for IL-8 secretion.(A-B) Indo-1/am–labeled neutrophils were stimulated with SAA (1 μM) in the absence (A) or presence (B) of BAPTA/am (20 μM). Elevation of intracellular Ca2+ is required for IL-8 secretion.(A-B) Indo-1/am–labeled neutrophils were stimulated with SAA (1 μM) in the absence (A) or presence (B) of BAPTA/am (20 μM). Triton X-100 was then added to 0.1% for measurement of total intracellular Ca2+, an indicator of Indo-1/am equal loading. Relative intracellular Ca2+ concentration was expressed as fluorescence ratio (405:485 nm). (C) Secretion of IL-8 and phosphorylation of ERK1/2 by SAA-stimulated neutrophils were determined in the absence or presence of BAPTA/am (20 μM and 40 μM). Maximal secretion (580 pg/mL/106 cells) was set as 100%. Data are presented as means ± SEM of 3 independent experiments, each performed in duplicate. Rong He et al. Blood 2003;101:1572-1581 ©2003 by American Society of Hematology

Inhibition of SAA-induced IL-8 secretion and MAP kinase phosphorylation by PTX.(A) Neutrophils were preincubated with or without either 100 ng/mL or 500 ng/mL pertussis toxin (PTX) at 37°C for 1 hour and then stimulated with 2 μM SAA for 3 hours. Inhibition of SAA-induced IL-8 secretion and MAP kinase phosphorylation by PTX.(A) Neutrophils were preincubated with or without either 100 ng/mL or 500 ng/mL pertussis toxin (PTX) at 37°C for 1 hour and then stimulated with 2 μM SAA for 3 hours. The secreted IL-8 was measured by ELISA and expressed as percent of change, with maximal IL-8 (980 pg/mL/106 cells) set as 100%. Data are presented as means ± SEM of 3 independent experiments, each performed in duplicate. (B) Neutrophils (4 × 105 cells) were incubated with or without PTX (400 ng/mL) for 30 minutes, and then stimulated with 2 μM SAA for indicated times. Induction of both ERK1/2 and p38 phosphorylation was determined by Western blotting. Three experiments were performed and a representative set of results is shown. Rong He et al. Blood 2003;101:1572-1581 ©2003 by American Society of Hematology

SAA induces Ca2+ mobilization through PFRL1/LXA4R in neutrophils SAA induces Ca2+ mobilization through PFRL1/LXA4R in neutrophils.Neutrophils labeled with Indo-1 were stimulated with SAA (1 μM), MMK-1 (10 nM), LXA4 (1.4 μM), and fMLF (10 nM) in different sequence combinations to determine cross-desensitization. SAA induces Ca2+ mobilization through PFRL1/LXA4R in neutrophils.Neutrophils labeled with Indo-1 were stimulated with SAA (1 μM), MMK-1 (10 nM), LXA4 (1.4 μM), and fMLF (10 nM) in different sequence combinations to determine cross-desensitization. Changes of intracellular Ca2+ are expressed as relative fluorescence ratio (405:485 nm). Results shown are representative of 1 of the 2 experiments. Rong He et al. Blood 2003;101:1572-1581 ©2003 by American Society of Hematology

SAA induces Ca2+ mobilization and ERK1/2 phosphorylation in FPRL1/LXA4R-transfected cells.(A) Stable transfectants, or the untransfected RBL-2H3 cells, were loaded with Indo-1/am and stimulated with SAA (2 μM) or MMK-1 (100 nM). SAA induces Ca2+ mobilization and ERK1/2 phosphorylation in FPRL1/LXA4R-transfected cells.(A) Stable transfectants, or the untransfected RBL-2H3 cells, were loaded with Indo-1/am and stimulated with SAA (2 μM) or MMK-1 (100 nM). Elevation of intracellular Ca2+ was observed in FPRL1/LXA4R-transfected but not the untransfected RBL or FPR-transfected RBL cells. (B) SAA and MMK-1 stimulate activation of ERK1/2 in FPRL1/LXA4R-transfected cells but not in the untransfected cells, as measured by Western blotting using an antiphospho-ERK1/2 antibody (upper blot). Equal loading of samples was determined by an antibody against unphosphorylated ERK1/2 (lower blot). Rong He et al. Blood 2003;101:1572-1581 ©2003 by American Society of Hematology

Characterization of rabbit antiserum against FPRL1/LXA4R Characterization of rabbit antiserum against FPRL1/LXA4R.(A) Histogram of flow cytometry demonstrating binding of the rabbit antiserum (1:50; solid line) as compared with preimmune serum (same dilution; shaded curve). Characterization of rabbit antiserum against FPRL1/LXA4R.(A) Histogram of flow cytometry demonstrating binding of the rabbit antiserum (1:50; solid line) as compared with preimmune serum (same dilution; shaded curve). The original data of side scattering versus FITC (from the secondary antibody) are shown in dot plots below. Mean fluorescence intensities are 14 ± 3.31 with preimmune serum and 27 ± 2.41 with anti-FPRL1. Data presented are from 1 representative experiment of a total of 3. (B) RBL-2H3 cells, the stable transfectants RBL/FPR and RBL/FPRL1, and the HEK293-transient transfectants 293/FPRL1 were incubated with preimmune serum (1:50 dilution; shaded curve) or anti-FPRL1 (same dilution; solid line), followed by FITC-conjugated secondary antibody. The histograms of flow cytometry were shown. Data are representative of 1 of the 2 experiments. (C) Neutrophils were preincubated with either preimmune serum or anti-FPRL1 (1:50 dilution) for 30 minutes at 37°C, and loaded with Indo-1/am. Elevation of intracellular Ca2+ by 1 μM SAA stimulation was blocked by anti-FPRL1. The results are representative of 1 experiment of a total of 3. (D) Inhibition of IL-8 secretion. Neutrophils were preincubated with or without either preimmune serum or anti-FPRL1 (1:50 dilution each) for 30 minutes at 37°C, and then stimulated with 1 μM SAA for 4 hours. IL-8 secretion was measured by ELISA. The secreted IL-8 (1060 pg/106/mL) by 1 μM SAA without serum pretreatment was set as 100%. Data are presented as means ± SEM of 2 independent experiments each performed in duplicate. Rong He et al. Blood 2003;101:1572-1581 ©2003 by American Society of Hematology

Induction of IL-8 expression in FPRL1/LXA4R-transfected cells Induction of IL-8 expression in FPRL1/LXA4R-transfected cells.(A) HeLa cells were transiently transfected with vector (mock) or a FPRL1/LXA4R expression construct, together with one of the luciferase reporters as indicated. Induction of IL-8 expression in FPRL1/LXA4R-transfected cells.(A) HeLa cells were transiently transfected with vector (mock) or a FPRL1/LXA4R expression construct, together with one of the luciferase reporters as indicated. Two days after transfection, cells were stimulated with SAA and the expressed luciferase activities were determined. Data shown are mean ± SEM from 3 separate experiments, each performed in duplicate. (B) The cell surface expression of the receptor was confirmed in FPRL-transfected HeLa cells (open curve), but not in vector (pRK5)–transfected HeLa cells (shaded curve), by flow cytometry using anti-FPRL1 and FITC-conjugated secondary antibody. Rong He et al. Blood 2003;101:1572-1581 ©2003 by American Society of Hematology

Inhibition of MAP kinase phosphorylation and IL-8 secretion by LXA4 Inhibition of MAP kinase phosphorylation and IL-8 secretion by LXA4.(A) Neutrophils were stimulated with 1 μM SAA or 5 μM LXA4, or with SAA and LXA4. Inhibition of MAP kinase phosphorylation and IL-8 secretion by LXA4.(A) Neutrophils were stimulated with 1 μM SAA or 5 μM LXA4, or with SAA and LXA4. After 15 minutes, samples were collected for detection of ERK1/2 and p38 phosphorylation by antiphospho antibodies against these kinases. The results are from a representative experiment of 3 separate experiments. (B) Neutrophils were stimulated with SAA, in the presence or absence of LXA4 as indicated. After 4 hours, the secreted IL-8 was measured by ELISA. The induction of IL-8 in the absence and presence of LXA4 was compared. Data are shown as mean ± SEM of 3 independent experiments, each performed in duplicate. Rong He et al. Blood 2003;101:1572-1581 ©2003 by American Society of Hematology