Differential activation and regulation of CXCR1 and CXCR2 by CXCL8 monomer and dimer - PubMed (original) (raw)

Comparative Study

. 2009 Sep 1;183(5):3425-32.

doi: 10.4049/jimmunol.0900305. Epub 2009 Aug 10.

Affiliations

Comparative Study

Differential activation and regulation of CXCR1 and CXCR2 by CXCL8 monomer and dimer

Mohd W Nasser et al. J Immunol. 2009.

Abstract

CXCL8 (also known as IL-8) activates CXCR1 and CXCR2 to mediate neutrophil recruitment and trigger cytotoxic effect at sites of infection. Under physiological conditions, CXCL8 could exist as monomers, dimers, or a mixture of monomers and dimers. Therefore, both forms of CXCL8 could interact with CXCR1 and CXCR2 with different affinities and potencies to mediate different cellular responses. In the present study, we have used a "trapped" nonassociating monomer (L25NMe) and a nondissociating dimer (R26C) to investigate their activities for human neutrophils that express both receptors and for RBL-2H3 cells stably expressing either CXCR1(RBL-CXCR1) or CXCR2 (RBL-CXCR2). The monomer was more active than the dimer for activities such as intracellular Ca(2+) mobilization, phosphoinositide hydrolysis, chemotaxis. and exocytosis. Receptor regulation, however, is distinct for each receptor. The rate of monomer-mediated regulation of CXCR1 is greater for activities such as phosphorylation, desensitization, beta-arrestin translocation, and internalization. In contrast, for CXCR2, both monomeric and dimeric CXCL8 mediate these activities to a similar extent. Interestingly, receptor-mediated signal-regulated kinase (ERK) phosphorylation in response to all three CXCL8 variants was more sustained for CXCR2 relative to CXCR1. Taken together, the results indicate that the CXCL8 monomer and dimer differentially activate and regulate CXCR1 and CXCR2 receptors. These distinct properties of the ligand and the receptors play a critical role in orchestrating neutrophil recruitment and eliciting cytotoxic activity during an inflammatory response.

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Figures

Figure 1

Figure 1. Monomer, dimer or native CXCL8 induced intracellular Ca2+ mobilization in human neutrophils

Human neutrophils (5 × 106) were loaded with indo-1AM and stimulated with different concentration of monomer, dimer, and WT CXCL8. The results are representative of one of three experiments performed in triplicate.

Figure 2

Figure 2. Expression and characterization of WT CXCR1 and CXCR2

A) A representative histogram of FACS analysis showing surface expression of CXCR1 and CXCR2 in RBL-2H3 cells after staining with CXCR1 (dotted line) or CXCR2 (broad line) specific antibodies. B) For intracellular Ca2+ mobilization, RBL cells (5 × 106) expressing human CXCR1 (RBL-CXCR1) or CXCR2 (RBL-CXCR2) were loaded with Indo-1AM and stimulated with monomer, dimer, or WT CXCL8. The representative tracings are one of the three experiments.

Figure 3

Figure 3. Phosphoinositide hydrolysis (PI), secretion of β-hexosaminidase release and Chemotaxis

For the generation of inositol phosphates (A and C), cells were cultured overnight in the presence of [3H]inositol (1 μC/ml). Cells were preincubated for 10 min at 37°C with HEPES-buffered saline containing 10 mM LiCl in a total volume of 200 μl and stimulated with different concentrations of monomer, dimer, or WT CXCL8 for 10 min. The supernatant was used to determine the release of inositol phosphates. Data are represented as the fold stimulation over basal. For secretion of β-hexosaminidase (B and D), cells were seeded as indicated above and stimulated with different concentrations of CXCL8 variants for 10 min. The supernatant (15 μl) was removed and β-hexosaminidase release was measured. Data are represented as fold stimulation over basal. Both experiments were repeated three times with similar results. Chemotactic response to CXCL8 variants using RBL-CXCR1 and RBL-CXCR2 (E and F) or HMECs (G and H) were measured as described in Materials and Methods. The results are representative of one of four experiments performed in triplicate. *, P< 0.05 and ** P< 0.01.

Figure 3

Figure 3. Phosphoinositide hydrolysis (PI), secretion of β-hexosaminidase release and Chemotaxis

For the generation of inositol phosphates (A and C), cells were cultured overnight in the presence of [3H]inositol (1 μC/ml). Cells were preincubated for 10 min at 37°C with HEPES-buffered saline containing 10 mM LiCl in a total volume of 200 μl and stimulated with different concentrations of monomer, dimer, or WT CXCL8 for 10 min. The supernatant was used to determine the release of inositol phosphates. Data are represented as the fold stimulation over basal. For secretion of β-hexosaminidase (B and D), cells were seeded as indicated above and stimulated with different concentrations of CXCL8 variants for 10 min. The supernatant (15 μl) was removed and β-hexosaminidase release was measured. Data are represented as fold stimulation over basal. Both experiments were repeated three times with similar results. Chemotactic response to CXCL8 variants using RBL-CXCR1 and RBL-CXCR2 (E and F) or HMECs (G and H) were measured as described in Materials and Methods. The results are representative of one of four experiments performed in triplicate. *, P< 0.05 and ** P< 0.01.

Figure 4

Figure 4. Phosphorylation of CXCR1 and CXCR2

A) 32P-labeled RBL-2H3 cells expressing CXCR1 or CXCR2were incubated for 5 min with or without stimulants as shown. Cells were lysed, immunoprecipitated with specific antibodies against CXCR1 or CXCR2 and then analyzed by SDS-PAGE and autoradiography. The results are from a representative experiment that was repeated three times. B and C, The amount of radioactivity per lane was determined by counting excised phosphorylated bands.

Figure 5

Figure 5. Internalization CXCR1 and CXCR2

RBL-CXCR1 (A) or RBL-CXCR2 (B) cells were treated with 100 nM of monomer, dimer, or WT CXCL8 for different times, washed and assayed for 125I-WT CXCL8 (0.1nM) binding. The value is presented as a percentage of totals, which is defined as the total amount of 125I-WT CXCL8 bound to control (untreated) cells. The experiment was repeated five times with similar results.

Figure 6

Figure 6. CXCR1 and CXCR2-mediated βarrestin 1 translocation to the cell membrane

RBL-CXCR1 and RBL-CXCR2 were transfected with β-arrestin1-GFP. The fluorescence images of GFP were captured upon addition of 100 nM of monomer, dimer, or WT CXCL8 to live cells as described in the method section.

Figure 7

Figure 7. CXCR1 and CXCR2-mediated ERK phosphorylation

RBL-2H3 cells stably expressing CXCR1 or CXCR2 were stimulated with monomer (D), dimmer(G), or WT CXCL8(A) (100 nM) for 0–5 min. ERK1/2 phosphorylation and total ERK were determined by Western blotting using anti-phospho-ERK1/2 (pERK1/2) and anti-total ERK1/2 (ERK1/2) antibodies, respectively. The experiments were repeated three times with similar results(B, C, E, F, H and I). Band density from radiogram was calculated by Image-Pro Plus software (Media Cybernetics) and is represented as relative phospho-ERK1/2 expression (arbitrary units). Data shown are averages of three experiments.

Figure 8

Figure 8. Desensitization of CXCR1 and CXCR2-mediated intracellular calcium mobilization and GTPase activity

RBL-2H3 cells (5×106 cells) expressing (A) CXCR1 or (B) CXCR2 were loaded with Indo-1 AM and exposed to a first dose of either monomer, dimer, or WT CXCL8 (0–100 nM). Cells were rechallenged 3 min later with a second dose of CXCL8 (10 nM) and intracellular Ca2+ mobilization was measured. Data are represented as percentage of maximum which represents intracellular Ca2+ mobilization-induced by CXCL8 (10 nM) in the absence of pretreatment (320±49 and 227±28 for RBL-CXCR1 and RBL-CXCR2, respectively). C) For GTPase activity, the cells were treated with or without ligand (100 nM) for 5 min. Membranes were prepared and assayed for WT CXCL8 (100 nM) stimulated GTP hydrolysis. The data are presented as percentage of maximum which is the net maximal stimulation obtained with untreated cells. Data shown are representative of three experiments performed in triplicate. *, P< 0.05 and ** P< 0.01.

Figure 9

Figure 9. Phosphorylation and internalization of BD199VA

32P-labeled RBL-2H3 cells (5 × 106 cells/60 mm plate) expressing CXCR2 (A, left panel) or BD199VA (A, right panel)were incubated for 5 min with or without stimulants as shown. Cells were lysed, immunoprecipitated with specific antibody against CXCR2 and then analyzed by SDS-PAGE and autoradiography. The results are from a representative experiment that was repeated twice. For internalization (panels B and C), cells were treated with 100 nM of ligands for different times, washed and assayed for 125I-WT CXCL8 (0.1nM) binding. The values are presented as a percentage of totals, which is defined as the total amount of 125I-WT CXCL8 bound to control (untreated) cells. The experiment was repeated three times with similar results.

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