Proteinase-activated receptor-2 up-regulation by Fcgamma-receptor activation in human neutrophils

Abstract

We shed new light on the expression and function of the proteinase-activated receptor (PAR) family, associated with inflammation and hyperalgesia, in human granulocytes. Resting cells expressed constitutive levels of PAR-2 and PAR-3 mRNA but not PAR-1 or PAR-4. Based on flow cytometry, stimulation with opsonized bacteria (Bop) specifically up-regulated cell surface expression of PAR-2 in a concentration-dependent and time-dependent manner, independent of transcription or de novo protein synthesis. Primary granules were identified as a source of preformed PAR-2 that can readily be mobilized at the surface on fusion with the plasma membrane. Cellular response to PAR-2 activation, measured as changes in intracellular calcium concentration, was enhanced in PAR-2 up-regulated cells. Increase of cell-surface PAR-2 and of cell responsiveness were dependent specifically on the engagement of immunoglobulin (Ig)-binding receptors. Together, our results reveal that mobilization of intracellular granules, in response to Ig-receptor activation, up-regulates PAR-2 surface expression and makes neutrophils more responsive to proteinase activity. This enhanced response to PAR-2 activation indicates that molecular communication between pain and inflammation may be more important than previously believed.

Figure 1

Proteinase-activated receptor mRNA expression in human granulocytes, as determined by real-time PCR. A) PAR mRNA expression in freshly purified, resting human granulocytes. B) Efficiency of primers with genomic DNA from human granulocytes. C) Efficiency of primers with mRNA from mixed PBMCs. Results are expressed as mean ± se threshold cycle ratio (PAR/GAPDH), from n = 5 (A) or n = 3 (B, C) experiments, each performed with cells from a different donor. D) PAR subtype mRNA expression in granulocytes incubated 5 or 60 min at 37°C with a mixture (Mix) of 5 neutrophil agonists (1 nM GM-CSF, 100 ng/ml TNF-α, 0.1 μg/ml LPS, 30 pM IL-1β, and 100 nM fMLP) or with Bop or Bnop Salmonella enterica serovar Typhimurium (25 bacteria/cell). Results are mean ± se threshold cycle ratio relative to resting cells from n = 5 experiments, each performed with a different donor. Primer sequences are provided in Materials and Methods.

Figure 2

Flow cytometry analysis of PAR expression on the granulocyte cell surface. A) Cell surface expression of PAR in resting granulocytes, nonlabeled (n.l.), labeled with matched-isotype IgG, or labeled with anti-(α)-PAR-2 or anti-α-PAR-3. B) Cell surface expression of PAR-2 in granulocytes stimulated for 60 min at 37°C with a mixture (Mix; 1 nM GM-CSF, 100 ng/ml TNF-α, 30 pM IL-1β, 0.1 μg/ml LPS, and 100 nM fMLP) of agonists or with Bop or Bnop (25 bacteria/cell). C) Same, PAR-3. Results for A–C are from one experiment, typical of ≥3 performed under identical conditions. D) Granulocytes from 3 different donors were stimulated for 60 min at 37°C with Mix, Bop, or Bnop bacteria as in C, or with opsonized (Uop) or nonopsonized urate crystals (Unop, 1 mg/ml). Results are means ± se. E) Granulocytes were stimulated with Mix or Bop for 60 min and assessed for PAR-2 surface expression using 3 distinct anti-PAR-2 antibodies. Mouse anti-PAR-2 antibody was raised against the sequence corresponding to aa 37–50; goat anti-PAR-2 antibody reacts with the N-terminal sequence, and rabbit anti-PAR-2 antibody recognizes aa 230–328 of PAR-2. F) Time course for PAR-2 surface expression in granulocytes stimulated with Bop or Bnop, as determined by flow cytometry. G) Concentration response. H) Cells incubated with transcription inhibitor actinomycin D (5 μg/ml) or with protein synthesis inhibitor cycloheximide (20 μg/ml) prior to stimulation with Bop (25 bacteria/cell; 60 min). Results are means ± se for ≥4 experiments. I) Total granulocytes were sorted according to CD16 surface expression; samples were stimulated with indicated Bop concentrations for 60 min at 37°C. Samples were assessed for PAR-2 surface expression by flow cytometry. Results are means ± se for 3 distinct experiments. *P < 0.05.

Figure 3

Test for subcellular localization of a PAR-2 reservoir and effect of PAR-2 activation on the degranulation process. PAR-2, CD63, and CD66b surface expression in granulocytes stimulated with 1 μM fMLP or 100 nM PMA for 5 min at 37°C in the absence or presence of CB (5 μM), as determined by flow cytometry. A) Raw data for PAR-2 from a typical experiment among ≥3 performed under identical conditions. B) Results for PAR-2, CD63, and CD66b; means ± se; n = 3. *P < 0.05. C) PAR-2, CD63, and CD66b surface expression in granulocytes stimulated with Bop (25 bacteria/cell) for 60 min at 37°C, as determined by flow cytometry. D) CD63 and CD66b surface expression in granulocytes incubated as in C, then stimulated with 100 μM SLIGKV, trypsin (1:10 dilution) or thrombin (1U/ml), for an additional 30 min at 37°C. Results are means ± se for 3 experiments with cells from different donors.

Figure 4

Effect of PAR-2 activation on intracellular Ca²⁺ responses. A–H) Granulocytes were incubated with or without Bop (25 bacteria/cell) for 5 min (A–D) or 60 min (E–H) at 37°C and then stimulated with the PAR-2 agonist peptide SLIGKV (100 μM; A, E), trypsin (1:10; B, F), IL-8 (100 nM; C, G), or thrombin (1U/ml; D, H) at t = 10 s. Samples were monitored for intracellular Ca²⁺ levels, as described in Materials and Methods. Results from one experiment are shown, typical of ≥3 distinct experiments performed in identical conditions with cells from different donors. Arrowheads indicate injection time. I) Intracellular Ca²⁺ responses were compiled by calculating the surface below the curves between t = 10 and 60 s. Results are mean ± se percentage change between cells treated with and without agonists; n = 3 distinct experiments. *P < 0.05 vs. no stimulation; **P < 0.05 vs. saline before stimulation; ***P < 0.05 vs. 5 min Bop before stimulation. J) Granulocytes were incubated with Bop for 60 min and stimulated with indicated concentrations of SLIGKV. Results show mean ± se intracellular Ca²⁺ increase vs. control (incubated 60 min with saline); n = 3 distinct experiments with cells from different donors. *P < 0.05 vs. no stimulation; **P < 0.05 vs. 100 μM SLIGKV stimulation.

Figure 5

Involvement of FcγRs on PAR-2 expression. A) Granulocytes were preincubated 5 min at 37°C with blocking antibodies for IgG-binding receptors or CRs (4 μg/ml), then stimulated for 60 min with either Bop (25 bacteria/cell) or aggregated IgGs (1 mg/ml). Samples were assessed for PAR-2 surface expression by flow cytometry. Results are mean ± se percentages of PAR-2 positive cells; n = 3 distinct experiments with cells from different donors. B) Granulocytes were preincubated 5 min at 37°C with 1 μg/ml blocking antibodies for IgG-binding receptors before incubation with Bop (25 bacteria/cell) for 60 min, and stimulation with indicated agonists. Samples were monitored for intracellular Ca²⁺ levels. Results from one experiment are shown, typical of ≥3 distinct experiments performed in identical conditions with cells from different donors. Arrowheads indicate injection time. C) Intracellular Ca²⁺ changes were compiled by calculating surface below curves between t = 10 and 60 s, above resting levels. Results are mean ± se percentage change between cells treated with and without Bop; n = 3 distinct experiments. *P < 0.05 vs. absence of blocking antibodies.