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Characterization of phospholipase A2 release by elicited-peritoneal macrophage and its relationship to eicosanoid
L A Marshall1, B Bolognese, A Roshak
1SmithKline Beecham Pharmaceuticals, Inflammation & Respiratory Pharmacology, King of Prussia, PA 19406-0939.
Abstract:
Elicited guinea pig peritoneal macrophages released a soluble phospholipase A2 (PLA2) into conditioned media that was biochemically and pharmacologically similar to the recombinant human (rh) type II 14 kDa PLA2. The level of activity found in the 24 h media positively correlated with cell number and was reduced by actinomycin D and cycloheximide suggesting the enzyme was being constitutively synthesized. The enzyme protein accumulated in the media over 10-24 h and remained at maximal levels over 40 h as demonstrated by both activity and ELISA measurements. This was preceded by an increase in total cell-associated sn-2-acylhydrolytic activity which reached maximal levels by 12-17 h of culture and remained elevated to 40 h. Treatment of cell homogenate with dithiothreitol (DTT) revealed a rise in DTT-insensitive sn-2-acylhydrolytic activity which increased between 12 and 24 h. Prostaglandin (PG) E2 but not leukotriene (LT)B4 accumulated in the media and reached maximal levels by 24 h. This paralleled the release of secreted type II 14 kDa-like PLA2 and the rise in DTT-insensitive cell-associated activity, but not upregulation and formation of new cyclooxygenase after aspirin treatment. Overnight exposure to a non-cell permeable selective 14 kDa PLA2 inhibitor or neutralizing mAb interfered with expression of PLA2 activity but not PGE2 accumulation over 24 h. This indicated that the secreted PLA2 was not directly involved in PGE2 biosynthesis. Exposure of the elicited macrophages to the cell permeable 14 kDa PLA2 inhibitor, 12-epi-scalaradial also did not effect PGE2 accumulation. Taken together, elicited guinea-pig macrophages release 14 kDa PLA2 upon culture, but this activity appears not to be related to the concomitant accumulation in PGE2. The role of the cell-associated PLA2 activity(s) in PGE2 formation cannot be ruled out.
Insights
Guinea pig macrophages secrete a phospholipase A2 (PLA2) similar to human type II PLA2. This secreted PLA2 is not directly involved in prostaglandin E2 production, though cell-associated PLA2 activity
Area of Science:
- Immunology
- Biochemistry
- Cell Biology
Background:
- Macrophages play a crucial role in inflammatory responses.
- Phospholipase A2 (PLA2) enzymes are involved in the release of fatty acids for eicosanoid synthesis.
- The specific role of secreted PLA2 in macrophage-mediated inflammation requires further elucidation.
Purpose of the Study:
- To characterize the secreted phospholipase A2 (PLA2) from elicited guinea pig peritoneal macrophages.
- To investigate the relationship between secreted PLA2 activity and prostaglandin E2 (PGE2) accumulation.
- To determine the involvement of secreted PLA2 in PGE2 biosynthesis.
Main Methods:
- Elicited guinea pig peritoneal macrophages were cultured.
- PLA2 activity in conditioned media and cell homogenates was measured.
- Enzyme synthesis was assessed using actinomycin D and cycloheximide.
- ELISA was used to quantify enzyme protein levels.
- Prostaglandin E2 and leukotriene B4 levels were measured.
- Selective 14 kDa PLA2 inhibitors and neutralizing monoclonal antibodies (mAbs) were employed.
Main Results:
- Macrophages released a soluble PLA2 biochemically and pharmacologically similar to human type II 14 kDa PLA2.
- PLA2 secretion was constitutive, with enzyme protein accumulating in the media over time.
- Prostaglandin E2 accumulated in the media, paralleling PLA2 release, but was not affected by PLA2 inhibition.
- Neither secreted PLA2 inhibition nor cell-permeable inhibitor impacted PGE2 accumulation, suggesting no direct role in PGE2 biosynthesis.
Conclusions:
- Elicited guinea pig macrophages constitutively synthesize and release a 14 kDa PLA2-like enzyme.
- The secreted PLA2 is not directly responsible for the observed PGE2 accumulation in cultured macrophages.
- The role of cell-associated PLA2 activities in PGE2 formation remains to be determined.