Role of arachidonic acid and its metabolites in the priming of NADPH oxidase in human polymorphonuclear leukocytes by peritoneal dialysis effluent

Abstract

Peritoneal dialysis effluent (PDE) contains a low-molecular-weight solute that will activate and prime the NADPH oxidase of human neutrophils via a phospholipase A2 (PLA2)-dependent mechanism. Since the products of PLA2 are known to activate and prime the oxidase we have investigated their role in the dialysis effluent-mediated activation and priming of human neutrophils. NADPH oxidase activity of PDE-primed and -unprimed neutrophils was measured by lucigenin-enhanced chemiluminescence in the presence of known inhibitors of the arachidonic acid cascade. Incubation of neutrophils with the nonselective PLA2 inhibitor quinacrine (0 to 100 microM) reduced oxidase activity in both primed and unprimed cells. Furthermore, primed cells were more sensitive to the action of quinacrine than were unprimed cells. We were unable to determine the relative roles of secretory PLA2 (sPLA2) and cytosolic PLA2 (cPLA2) since the selective sPLA2 inhibitor scalaradial (0 to 100 microM) inhibited oxidase activity in both groups of cells by similar degrees, while the specific cPLA2 inhibitor AACO-CF3 (0 to 50 microM) failed to affect activity in either group. Inhibition of platelet-activating factor (PAF), cycloxygenase, and 5-lipoxygenase-activating protein by hexanolamino-PAF (0 to 25 microM), flurbiprofen (0 to 25 microM), and MK886 (0 to 5 microM), respectively, had no effect upon oxidase activity. However, the direct inhibition of 5-lipoxygenase by caffeic acid or lipoxin A4 resulted in a similar concentration-dependent attenuation of oxidase activity in both primed and unprimed cells. Leukotriene B4 (LTB4) release from primed neutrophils was comparable to that from unprimed cells with the exception of phorbol myristate acetate-stimulated cells, which released fivefold more LTB4 than control. Taken together, these results suggest that it is arachidonic acid per se, and not its metabolites, that is important in priming of the neutrophil NADPH oxidase by dialysis effluent.

FIG. 1

Effect of quinacrine upon AA release from fMLP (1 μM)-challenged PMN. PMN were incubated in buffer (open squares) or PDE (closed squares). Results are expressed as mean (± SEM) percent inhibitions relative to control (no quinacrine) (n = 6, six dialysis effluents upon PMN from six donors). Absolute control values (100%) were 1.90% ± 0.22% and 4.80% ± 0.55% total radioactivity incorporated for unprimed and primed cells, respectively.

FIG. 2

Effects of quinacrine upon superoxide generation of unstimulated PMN (a) and PMN challenged with 1 μM fMLP (b). PMN were incubated in buffer (open circles) or PDE (closed circles). Results are expressed as mean (± SEM) percent inhibitions relative to control (no quinacrine) (n = 6, six dialysis effluents upon PMN from six donors). Asterisks indicate P values of ≤0.05 (Mann-Whitney U test). Absolute control values (100%) were 28.85 ± 9.40 relative light units (RLU) and 115 ± 23.57 RLU (a) and 1,107 ± 28 RLU and 3,618 ± 153 RLU (b) for unprimed and primed cells, respectively.

FIG. 3

Effect of scalaradial upon AA release from fMLP (1 μM)-challenged PMN. PMN were incubated in buffer (open squares) or PDE (closed squares). Results are expressed as mean (± SEM) percent inhibitions relative to control (no scalaradial) (n = 6, six dialysis effluents upon PMN from six donors). Absolute control values (100%) were 1.80% ± 0.21% and 3.42% ± 0.37% total radioactivity incorporated for unprimed and primed cells, respectively.

FIG. 4

Effect of scalaradial upon superoxide generation by fMLP (1 μM)-challenged PMN. PMN were incubated in buffer (open circles) or PDE (closed circles). Results are expressed as mean (± SEM) percent inhibitions relative to control (no scalaradial) (n = 6, six dialysis effluents upon PMN from six donors). Similar profiles were obtained from unstimulated PMN and PMN challenged with S. epidermidis (2 × 10⁷ ml⁻¹) and PMA (10 ng ml⁻¹). Absolute control values (100%) were 503 ± 42 relative light units (RLU) and 2,241 ± 224 RLU for unprimed and primed cells, respectively.

FIG. 5

Effect of AACO-CF₃ upon AA release from fMLP (1 μM)-challenged PMN. PMN were incubated in buffer (open squares) or PDE (closed squares). Results are expressed as mean (± SEM) percent inhibitions relative to control (no AACO-CF₃) (n = 6, six dialysis effluents upon PMN from six donors). Absolute control values (100%) were 2.20% ± 0.25% and 4.62% ± 0.19% total radioactivity incorporated for unprimed and primed cells, respectively.

FIG. 6

Effect of AACO-CF₃ upon superoxide generation from fMLP (1 μM)-challenged PMN. PMN were incubated in buffer (open circles) or PDE (closed circles). Results are expressed as mean (± SEM) percent inhibitions relative to (no AACO-CF₃) (n = 6, six dialysis effluents upon PMN from six donors). Similar profiles were obtained from unstimulated PMN and PMN challenged with S. epidermidis (2 × 10⁷ ml⁻¹) and PMA (10 ng ml⁻¹). Absolute control values (100%) were 747 ± 83 relative light units (RLU) and 1,842 ± 90 RLU for unprimed and primed cells, respectively.

FIG. 7

Generation of LTB₄ by unstimulated PMN and PMN challenged with fMLP (1 μM), S. epidermidis (2 × 10⁷ ml⁻¹), and PMA (10 ng ml⁻¹). PMN were incubated in buffer (open bars) or PDE (hatched bars). Results are expressed as mean (± SEM) amount of LTB₄ released (ng ml⁻¹) (n = 6, six dialysis effluents upon PMN from six donors). The asterisk indicates a P value of ≤0.05 (Mann-Whitney U test).