Pannexin 1 channels link chemoattractant receptor signaling to local excitation and global inhibition responses at the front and back of polarized neutrophils

Abstract

Neutrophil chemotaxis requires excitatory signals at the front and inhibitory signals at the back of cells, which regulate cell migration in a chemotactic gradient field. We have previously shown that ATP release via pannexin 1 (PANX1) channels and autocrine stimulation of P2Y2 receptors contribute to the excitatory signals at the front. Here we show that PANX1 also contributes to the inhibitory signals at the back, namely by providing the ligand for A2A adenosine receptors. In resting neutrophils, we found that A2A receptors are uniformly distributed across the cell surface. In polarized cells, A2A receptors redistributed to the back where their stimulation triggered intracellular cAMP accumulation and protein kinase A (PKA) activation, which blocked chemoattractant receptor signaling. Inhibition of PANX1 blocked A2A receptor stimulation and cAMP accumulation in response to formyl peptide receptor stimulation. Treatments that blocked endogenous A2A receptor signaling impaired the polarization and migration of neutrophils in a chemotactic gradient field and resulted in enhanced ERK and p38 MAPK signaling in response to formyl peptide receptor stimulation. These findings suggest that chemoattractant receptors require PANX1 to trigger excitatory and inhibitory signals that synergize to fine-tune chemotactic responses at the front and back of neutrophils. PANX1 channels thus link local excitatory signals to the global inhibitory signals that orchestrate chemotaxis of neutrophils in gradient fields.

Keywords: Adenosine Receptor; Chemotaxis; Cyclic AMP (cAMP); Local Excitation and Global Inhibition Model; Neutrophil; Pannexin.

FIGURE 1.

PANX1 is essential for neutrophil chemotaxis. A, inhibition of ATP release with the gap junction inhibitor CBX disrupts cell polarization and blocks chemotaxis in response to fMLP. dHL-60 cells stably expressing YFP-actin were used to visualize F-actin polymerization at the leading edge in response to chemotactic cues. Cells were exposed to an fMLP gradient using a micropipette loaded with 100 nm fMLP (location of pipette tip is indicated by asterisks) in the presence or absence of 20 μm CBX. F-actin polarization (YFP channel; yellow) and cell morphology (bright field) were recorded over time, and both image sequences were merged (see also supplemental Movie S1). B, inhibition of PANX1 abolishes gradient sensing. Freshly isolated primary human neutrophils were exposed to a chemotactic gradient using a micropipette as described above, and migration of cells toward the tip of the micropipette was monitored under the microscope. The chemotactic behavior of untreated (control) cells and of cells pretreated for 5 min with a mimetic inhibitory peptide (¹⁰panx1; 200 μm), a scrambled control peptide (^scpanx1 control; 200 μm), or CBX (20 μm) was recorded, and individual cell traces were tracked and plotted (see supplemental Movie S2). C and D, inhibition of PANX1 impairs gradient sensing and migration speed. Cells were considered to migrate in the correct direction if their migration paths did not deviate by more than 60° from a straight path toward the tip of the micropipette. The total length traveled by each cell over the observation time was used to calculate migration speed. Data shown are representative of results obtained with cells isolated from at least three different healthy individuals. Statistical analysis was done with Student's t test, *, p < 0.05.

FIGURE 2.

FPR stimulation induces cAMP signaling via PANX1. A, FPR stimulation induces a transient rise in intracellular cAMP. Freshly isolated human neutrophils were treated with 1 nm fMLP, and intracellular cAMP concentrations were assessed after the indicated times using a cAMP-Screen assay kit. B, exogenously added cAMP impairs neutrophil migration. Human neutrophils migrating in an fMLP gradient field generated with a micropipette were treated with the cell-permeable cAMP analog dibutyryl cAMP-AM (1 μm), and migration speed was recorded as described in the legend for Fig. 1. C, PANX1 is required for fMLP-induced cAMP accumulation. Human neutrophils were pretreated for 10 min with the indicated concentrations of CBX or ¹⁰panx1 or with 200 μm control peptide (^scpanx1). Then cells were stimulated with 1 nm fMLP, and intracellular cAMP concentrations were measured after 5 min. Forskolin (100 μm) was used as a positive control; no stim, no stimulation. Values are expressed as mean ± S.D., and the data shown are representative of three individual experiments performed on different days; statistical analysis was done with Student's t test, *, p < 0.05.

FIGURE 3.

A_2A receptors regulate FPR signaling via cAMP accumulation. A, FPR-induced cAMP signaling requires A_2A receptor stimulation. Human neutrophils were pretreated for 10 min with the indicated concentrations of the A_2A receptor antagonists CSC or SCH58261 or the A_2A receptor agonist CGS21680. Then the cells were stimulated with 1 nm fMLP, and intracellular cAMP concentrations were assessed after 5 min. B and C, A_2A receptor signaling fine-tunes MAPK activation in response to FPR stimulation. Human neutrophils were treated for 15 min with the indicated concentrations of the PKA inhibitor H89 (B and C) or the A_2A receptor antagonist CSC (C). Then the cells were stimulated with the indicated concentrations of fMLP and p38, and ERK MAPK signaling was determined using Western blotting with antibodies that recognize the phosphorylated and thereby activated forms of these MAPKs. MAPK activation was estimated by comparing data obtained with these antibodies and with corresponding antibodies that recognize the active and inactive forms of these MAPKs. p-p38, phosphorylated p38; p-Erk, phosphorylated ERK. Results are expressed as mean ± S.D., and the data shown are representative of at least three individual experiments performed on different days with cells from different healthy individuals; statistical analysis was done with Student's t test, *, p < 0.05.

FIGURE 4.

A_2A receptors redistribute toward the back of polarized cells. A, A_2A receptors are absent from the leading edge of polarized neutrophils. Cellular distribution patterns of A_2A receptors and F-actin in primary human neutrophils without or with fMLP (1 nm) stimulation for 10 min were assessed with anti-A_2A receptor antibodies (green) and phalloidin (red) staining (see also supplemental Movie S3). Arrows indicate the leading edge. B, A_2A receptors rapidly redistribute from the front toward the back during cell polarization. dHL-60 cells expressing an A_2A receptor-EYFP fusion protein were stimulated with a micropipette loaded with 100 nm fMLP (the location of the micropipette tip is indicated by asterisks), and A_2A receptor redistribution was recorded over time using fluorescence video microscopy (see also supplemental Movie S4). The arrow indicates the leading edge. C, ATP release is required for cell polarization and A_2A receptor translocation. dHL-60 cells expressing A_2A receptor-EYFP were treated for 5 min with or without CBX (50 μm) to block ATP release. Then cells were stimulated with 1 nm fMLP, and the percentages of cells that formed elongated cell shapes (polarization) and clearly showed receptor translocation were enumerated. Data shown are representative of at least three different experiments with similar results. Statistical analysis was done with Student's t test, *, p < 0.05.

FIGURE 5.

A_2A receptors contribute to chemotaxis by regulating migration speed. A, interfering with FPR-induced endogenous A_2A receptor signaling inhibits chemotaxis. Neutrophils were treated with the A_2A receptor antagonist CSC (200 nm), with the A_2A receptor agonist CGS21680 (200 nm), or with the PKA inhibitor H89 (10 μm). Then chemotaxis toward a micropipette containing fMLP (100 nm) was recorded, and individual cell traces were plotted (see also supplemental Movie S5). B and C, interfering with endogenous A_2A receptor signaling blocks migration speed. Correct direction of migration (B) and migration velocity (C) were determined as described above. All values are expressed as mean ± S.D., and the data shown are representative of three or more experiments performed with different cell preparations. Statistical analysis was done with Student's t test, *, p < 0.05.

FIGURE 6.

Proposed model of neutrophil chemotaxis. As previously reported, stimulation of chemoattractant receptors induces local release of ATP through PANX1 channels at the site that first encounters the chemoattractant (10, 11). Autocrine feedback via P2Y₂ receptors amplifies the chemotactic signal and triggers cell polarization, whereby cells assume an elongated shape, and PANX1, CD39 (NTPDase1), and A3 adenosine receptors accumulate at the leading edge. In the current study, we found that A_2A receptors are translocated from the leading edge toward the back of polarized neutrophils and that inhibitory signaling via A_2A receptor-dependent cAMP accumulation inhibits excitatory chemotactic signaling by blocking FPR-dependent ERK and p38 MAPK activation globally with the exception of the leading edge. ALP, alkaline phosphatase; ADO, adenosine; PIP3, phosphatidylinositol (3,4,5)-triphosphate.