Abscisic acid is an endogenous cytokine in human granulocytes with cyclic ADP-ribose as second messenger

Abstract

Abscisic acid (ABA) is a phytohormone involved in fundamental physiological processes of higher plants, such as response to abiotic stress (temperature, light, drought), regulation of seed dormancy and germination, and control of stomatal closure. Here, we provide evidence that ABA stimulates several functional activities [phagocytosis, reactive oxygen species and nitric oxide (NO) production, and chemotaxis] of human granulocytes through a signaling pathway sequentially involving a pertussis toxin (PTX)-sensitive G protein/receptor complex, protein kinase A activation, ADP-ribosyl cyclase phosphorylation, and consequent cyclic-ADP-ribose overproduction, leading to an increase of the intracellular Ca(2+) concentration. The increase of free intracellular ABA and its release by activated human granulocytes indicate that ABA should be considered as a new pro-inflammatory cytokine in humans. This discovery is an intriguing example of conservation of a hormone and its signaling pathway from plants to humans and provides insight into the molecular mechanisms of granulocyte activation, possibly leading to the development of new antiinflammatory drugs.

Fig. 1.

ABA increases the [Ca²⁺]_i in FURA-2-AM loaded human granulocytes. (A) ABA (20 μM) was added to cells in HBSS (trace 1) or in Ca²⁺-free HBSS containing 0.1 mM EGTA (trace 2). (B) ABA (20 μM) was added to untreated cells in HBSS (trace 1), to cells pretreated with xestospongin (10 μM for 10 min) (trace 2), with U73122 (5 μM for 10 min) (trace 3), with 8-Br-cADPR (100 μM for 90 min) (trace 4), with both U73122 and 8-Br-cADPR (trace 5), or with PTX (2 μg/ml for 1 h) (trace 6). Traces from one of three different experiments, yielding comparable results, are shown. Arrows indicate the addition of ABA.

Fig. 2.

Intracellular cADPR, IP₃ and cAMP levels, ADPRC activity, and CD38 phosphorylation in ABA-stimulated granulocytes. (A) After addition of 20 μM ABA, [cADPR]_i levels were determined on untreated cells (squares, n = 9), or on cells pretreated with a specific PKA inhibitor (10 μM for 15 min) (triangles, n = 3) or with PTX (2 μg/ml for 1 h) (rhombus, n = 3). The basal [cADPR]_i recorded in unstimulated cells was 31.48 ± 14.15 pmol/10⁹ cells, n = 9. (B) [IP₃]_i levels were determined on untreated cells (squares, n = 4) or on cells pretreated with PTX (2 μg/ml for 1 h) (rhombus, n = 4). The basal [IP₃]_i measured in unstimulated cells was 1.32 ± 0.44 pmol/10⁶ cells, n = 4. (C) ADP- (rhombus) and GDP- (square) ribosyl cyclase activities. (D) [cAMP]_i levels were determined on untreated cells (filled squares, n = 20), on cells pretreated with PTX (2 μg/ml for 1 h) (filled rhombus, n = 3), with U73122 (5 μM for 10 min) (opened squares, n = 3), or with a specific PKC inhibitor (50 nM for 15 min) (white rhombus, n = 4). Higher concentrations of U73122 and of the PKC inhibitor did not further increase the percentage of inhibition. Results are expressed as percentage of basal values, recorded on untreated cells. (E and F) CD38 was immunopurified (see SI Materials and Methods) as follows: from ABA-treated (for 0, 5, 15, and 60 min with 20 μM ABA) granulocytes (E) and from control, 8-Br-cAMP- (500 μM for 15 min) and IL-8- (100 nM for 15 min) treated granulocytes (F). Samples were run in duplicate; Western blots were stained with the anti-CD38 antibody (Left) or with an anti-phosphoserine mAB (Right). Results from one of three different experiments, yielding comparable results, are shown.

Fig. 3.

Mechanism of the ABA-induced [Ca²⁺]_i increase. The interaction of ABA with a G protein-coupled plasmamembrane receptor triggers is shown as follows: (i) activation of PLC, overproduction of IP₃, and stimulation of a PKC-dependent AC; (ii) activation of AC, overproduction of cAMP, PKA-mediated stimulation of ADPRC, and increase of [cADPR]_i. Downstream of cADPR, two mechanisms (dotted lines) might cooperate to induce the observed increase of the [Ca²⁺]_i: extracellular Ca²⁺ influx through store-operated Ca²⁺ entry (a) or direct gating of a plasmamembrane Ca²⁺ channel by cADPR (b). Site-specific inhibitors of the ABA-signaling pathway are indicated in red. PTX, pertussis toxin; U73122, PLC inhibitor; xestospongin, IP₃-specific Ca²⁺-channel blocker; I-PKA and I-PKC, PKA- and PKC-specific myristoylated (peptide inhibitors); 8-Br-cADPR, specific cADPR antagonist; Ry, Ryanodine (cADPR-specific Ca²⁺-channel blocker). The increased [Ca²⁺]_i levels stimulate functional responses: phagocytosis, release of ROS and NO, chemokinesis, and chemotaxis to ABA.

Fig. 4.

Effect of PMA and temperature on granulocyte ABA content. Cells (5 × 10⁷/determination) were incubated in 2.0 ml of HBSS for 30 min at 39°C or at 20°C without (control) or with PMA. The ABA content in cells and supernatants was determined by HPLC-MS. Results are expressed as picomoles of ABA detected in the cells (white bars) or in the supernatants (gray bars). Results shown are mean values ± SD (n = 3 for PMA, n = 9 for temperature) of granulocytes from different subjects. Black bars, sum of mean values of intracellular and released ABA.