Expression of phospholipases A2 in primary human lung macrophages: role of cytosolic phospholipase A2-alpha in arachidonic acid release and platelet activating factor synthesis

Abstract

Macrophages are a major source of lipid mediators in the human lung. Expression and contribution of cytosolic (cPLA(2)) and secreted phospholipases A(2) (sPLA(2)) to the generation of lipid mediators in human macrophages are unclear. We investigated the expression and role of different PLA(2)s in the production of lipid mediators in primary human lung macrophages. Macrophages express the alpha, but not the zeta isoform of group IV and group VIA cPLA(2) (iPLA(2)). Two structurally-divergent inhibitors of group IV cPLA(2) completely block arachidonic acid release by macrophages in response to non-physiological (Ca(2+) ionophores and phorbol esters) and physiological agonists (lipopolysaccharide and Mycobacterium protein derivative). These inhibitors also reduce by 70% the synthesis of platelet-activating factor by activated macrophages. Among the full set of human sPLA(2)s, macrophages express group IIA, IID, IIE, IIF, V, X and XIIA, but not group IB and III enzymes. Me-Indoxam, a potent and cell impermeable inhibitor of several sPLA(2)s, has no effect on arachidonate release or platelet-activating factor production. Agonist-induced exocytosis is not influenced by cPLA(2) inhibitors at concentrations that block arachidonic acid release. Our results indicate that human macrophages express cPLA(2)-alpha, iPLA(2) and several sPLA(2)s. Cytosolic PLA(2)-alpha is the major enzyme responsible for lipid mediator production in human macrophages.

Figure 1. Western blot detection of cPLA₂-α in human lung macrophages

Lane 1 is macrophage lysate, and lane 2 is recombinant cPLA₂-α protein. Additional details are given in Experimental Procedures.

Figure 2. Presence of sPLA₂s in human lung macrophages

Macrophage lysates were analyzed for sPLA₂ protein content by time-resolved fluorescence immunoassay (See “Methods”). Approximate detection limits (pg sPLA₂ per assay well) were: GIB, 10; GIIA, 20; GIID, 500; GIIE, 40; GIIF, 300; GIII, 300; GV, 60; GX, 4; GXIIA, 600. Data are expressed as ng of sPLA₂/3×10⁶ cells and are the mean ± SE of five different donors. * p < 0.05 vs. respective unstimulated.

Figure 3

Structures of the cPLA₂-α and sPLA₂ inhibitors used in this study.

Figure 4. Effect of cPLA₂ and sPLA₂ inhibitors on AA release from PMA-(upper panel) and A23187-stimulated (lower panel) human lung macrophages

[³H]AA-labeled human lung macrophages were preincubated (30 min, 37°C) with increasing concentrations (0.01–10 μM) of AZ-1 (○), pyrrolidine-1 (●) or Me-Indoxam (■) and then stimulated (30 min, 37°C) with 1 μM PMA (upper panel) or A23187 (lower panel). At the end of the incubation, supernatants were collected and centrifuged twice (1000 g, 4°C, 5 min) for subsequent determination of AA release. Values are the mean ± SE of four different experiments. * p < 0.05 vs. respective stimulus alone ** p < 0.01 vs. respective stimulus alone

Figure 5. Effect of cPLA₂ and sPLA₂ inhibitors on AA release from PMA + A23187-stimulated human lung macrophages

[³H]AA-labeled human lung macrophages were preincubated (30 min, 37°C) with increasing concentrations (0.01–10 μM) of AZ-1 (○), pyrrolidine-1 (●) or Me-Indoxam (■) and then stimulated with 1 μM PMA (10 min, 37°C) and subsequently with 1 μM A23187 (30 min, 37°C). At the of the incubation, supernatants were collected and centrifuged twice (1000 g, 4°C, 5 min) for subsequent determination of AA release. Values are the mean ± SE of three different experiments. * p < 0.05 vs. PMA + A23187 ** p < 0.01 vs. PMA + A23187

Figure 6. Effects of PPD, LAM and PGN-SA on AA release from human lung macrophages

[³H]AA-labeled human lung macrophages were stimulated (1 h, 37°C) with increasing concentrations (0.3–50 μg/ml) of PPD, LAM, or PGN-SA. At the end of the incubation, supernatants were collected and centrifuged twice (1000 g, 4°C, 5 min) for subsequent determination of AA release. Values are the mean ± SE of three different experiments. * p < 0.05 vs. unstimulated ** p < 0.01 vs. unstimulated

Figure 7. Effect of cPLA₂ inhibitors on AA release from PPD-stimulated human lung macrophages

[³H]AA-labeled human lung macrophages were preincubated (30 min, 37°C) with increasing concentrations (0.01–10 μM) of AZ-1 or pyrrolidine-1 and then stimulated (1 h, 37°C) with 30 μg/ml PPD. At the end of the incubation, supernatants were collected and centrifuged twice (1000 g, 4°C, 5 min) for subsequent determination of AA release. Values are the mean ± SE of three different experiments. § p < 0.01 vs. unstimulated * p < 0.05 vs. PPD alone ** p < 0.01 vs. PPD alone

Figure 8. Effect of cPLA₂ inhibitors on AA release from LPS-stimulated human lung macrophages

[³H]AA-labeled human lung macrophages were preincubated (30 min, 37°C) with increasing concentrations (0.01–10 μM) of AZ-1 or pyrrolidine-1 and then stimulated (2 h, 37°C) with 1 μg/ml LPS. At the end of the incubation, supernatants were collected and centrifuged twice (1000 g, 4°C, 5 min) for subsequent determination of AA release. Values are the mean ± SE of three different experiments. § p < 0.01 vs. unstimulated ** p < 0.01 vs. LPS alone

Figure 9. Effect of cPLA₂ inhibitors on PAF synthesis from A23187-stimulated human lung macrophages

Human lung macrophages were preincubated (30 min, 37°C) with increasing concentrations (0.01–10 μM) of AZ–1 (○) or pyrrolidine–1 (●) and then stimulated with 1 μM A23187 (10 min, 37°C) in the presence of [³H]acetic acid. At the end of the incubation, lipid were extracted from both cells and supernatants by the Bligh and Dyer technique. PAF was separated by TLC and quantitated by lipid scintillation counting. Values are the mean ± SE of three different experiments. ** p < 0.01 vs. A23187 alone

Figure 10. Effect of cPLA₂ and sPLA₂ inhibitors on β-glucuronidase release from A23187-stimulated human lung macrophages

Human lung macrophages were preincubated (30 min, 37°C) with increasing concentrations (0.01–10 μM) of pyrrolidine-1, AZ-1 or Me-Indoxam and then stimulated with 1 μM A23187 (90 min, 37°C). At the of the incubation, supernatants were collected and centrifuged twice (1000 g, 4°C, 5 min). β-Glucuronidase release was determined by a colorimetric technique. The values are expressed as the percentage of the total cellular content determined in cell aliquots lysed with 0.1% Triton X-100. The data are the mean ± SE of three different experiments. § p < 0.01 vs. unstimulated.