Expression of the Type-II Phospholipase A2 in Alveolar Macrophages

Abstract

We have shown previously that guinea pig alveolar macrophages (AM) synthesize a secretory phospholipase A2 (PLA2) during in vitro incubation. Here, we report the molecular cloning of this enzyme and show that it has structural features closely related to all known mammalian type-II PLA2. The mRNA and PLA2 activity were undetectable in freshly collected AM, but their levels increased dramatically to reach maximal values after 16 h of culture. Thereafter, the PLA2 activity remained constant with a parallel secretion in the medium, in contrast to mRNA level which returned to near basal values after 32 h. Incubation of AM for 16 h with the inflammatory secretagogue peptide f-Met-Leu-Phe (fMLP) markedly reduced the PLA2 activity and mRNA levels. This inhibition was prevented by preexposure of AM to pertussis toxin, an inhibitor of G-protein. In contrast, when AM were first cultured for 16 h and then incubated with fMLP, no significant change was observed in their PLA2 activity. In conditions where the type-II PLA2 was completely abrogated by fMLP, the latter did not alter the lipopolysaccharide-induced accumulation of tumor necrosis factor alpha mRNA or the release of arachidonic acid induced by the subsequent addition of the calcium ionophore A23187. These studies show that the inflammatory peptide fMLP down-regulates the expression of the type-II PLA2 by AM through a process mediated by G-protein. A possible negative control of the type-II PLA2 expression during AM activation is suggested.

Arachidonic Acid Differentially Affects Basal and Lipopolysaccharide-Induced sPLA2-IIA Expression in Alveolar Macrophages through NF-κB and PPAR-γ–Dependent Pathways

“…We have shown previously that cultured AMs spontaneously synthesized an enzyme with characteristics similar to those of the low-molecular-weight sPLA 2 (Hidi et al, 1993). The molecular cloning of this enzyme allowed us to identify it as an sPLA 2 -IIA (Vial et al, 1995), the expression of which was enhanced after LPS stimulation (Arbibe et al, 1997). In addition, this enzyme was found in the cell homogenates and supernatants, and its catalytic activity was completely abolished by LY311727, a specific inhibitor of the low-molecularweight sPLA 2 activity (Arbibe et al, 1997).…”

“…Total RNAs were extracted by an RNeasy kit, electrophoresed (10 g/lane), and transferred to a nylon membrane. The blots were hybridized at 65°C using ␣-32 P-labeled (random priming) full-length guinea pig sPLA 2 -IIA cDNA (Vial et al, 1995) as a probe. Finally, blots were washed off and rehybridized with murine ␤-actin cDNA at 62°C.…”

“…Among sPLA 2 s, the type IIA sPLA 2 (sPLA 2 -IIA), also referred to as synovial PLA 2 , is a proinflammatory enzyme found to be highly elevated both in the circulation and locally in the tissue, in association with a number of pathological conditions such as atherosclerosis, asthma, and acute lung injury (ALI) (Chilton et al, 1996;Arbibe et al, 1997;Hurt-Camejo et al, 1997). The expression of sPLA 2 -IIA is found to increase in many inflammatory cells, including guinea pig alveolar macrophages (AM) (Vial et al, 1995), upon stimulation with a variety of proinflammatory stimuli such as cytokines (IL-1␤, TNF-␣, IL-6) and LPS.…”

Inhibitory Effects of Surfactant Protein A on Surfactant Phospholipid Hydrolysis by Secreted Phospholipases A2

“…Recombinant guinea pig sPLA 2 -IIA (r-GP sPLA 2 -IIA) was produced in our laboratory (22). The recombinant mouse sPLA 2 s were expressed and purified as described previously (9,10,23).…”

Generation of lyso-phospholipids from surfactant in acute lung injury is mediated by type-II phospholipase A2 and inhibited by a direct surfactant protein A-phospholipase A2 protein interaction.

“…Antiserum was raised in rabbits immunized against the peptide "YDRLMKRGCGTKFL" conjugated to sunflower globulin using the bis-diazoted benzidine (30). This peptide, corresponding to 51-64 amino acids of the mature guinea pig sPLA2-II protein (31), is not present in other known secreted PLA2s.…”