Absence of calcium-independent phospholipase A2 β impairs platelet-activating factor production and inflammatory cell recruitment in Trypanosoma cruzi-infected endothelial cells

Abstract

Both acute and chronic phases of Trypanosoma cruzi (T. cruzi) infection are characterized by tissue inflammation, mainly in the heart. A key step in the inflammatory process is the transmigration of inflammatory cells across the endothelium to underlying infected tissues. We observed increased arachidonic acid release and platelet-activating factor (PAF) production in human coronary artery endothelial cells (HCAEC) at up to 96 h of T. cruzi infection. Arachidonic acid release is mediated by activation of the calcium-independent phospholipase A2 (iPLA2) isoforms iPLA2 β and iPLA2 γ, whereas PAF production was dependent upon iPLA2 β activation alone. Trypanosoma cruzi infection also resulted in increased cell surface expression of adhesion molecules. Increased adherence of inflammatory cells to T. cruzi-infected endothelium was blocked by inhibition of endothelial cell iPLA2 β or by blocking the PAF receptor on inflammatory cells. This suggests that PAF, in combination with adhesion molecules, might contribute to parasite clearing in the heart by recruiting inflammatory cells to the endothelium.

Keywords: Inflammation; endothelial; phospholipase A2; platelet‐activating factor.

Figure 1.

Changes in calcium‐independent phospholipase A₂ (iPLA₂) activity (A), arachidonic acid release (B), platelet‐activating factor (PAF) production (C), and cell surface expression of ICAM‐1, VCAM‐1, and E‐selectin (D) in human coronary artery endothelial cells infected with Trypanosoma cruzi (MOI 0.2, untreated ● or heat‐killed □ in A–C) for up to 96 h. Values shown are means ± SEM for four separate cell cultures. *P < 0.05, **P < 0.01 when compared to uninfected controls.

Figure 2.

Calcium‐independent phospholipase A₂ (iPLA₂) activity (A), arachidonic acid release (B), platelet‐activating factor (PAF) production (C), and adherence of polymorphonuclear leukocytes (PMN) (D) in human coronary artery endothelial cells infected with Trypanosoma cruzi (MOI 0.2) for up to 96 h. Cells were pretreated with 0.5 μmol/L (R)‐bromoenol lactone (BEL) or (S)‐BEL (10 min) prior to infection with T. cruzi. Values shown are means ± SEM for four separate cell cultures. **P < 0.01 when compared with uninfected controls. ⁺⁺P < 0.01 when comparing values in the presence or absence of BEL.

Figure 3.

PAF production (A) and RAW 264.7 cell adherence (B) in Trypanosoma cruzi infected (MOI 0.2, 48 h) wild‐type or iPLA₂β knockout endothelial cells following (R)‐ or (S)‐BEL pretreatment (0.5 μmol/L, 10 min). *P < 0.05, **P < 0.01 when compared to uninfected cells. ⁺P < 0.05, ⁺⁺P < 0.01 when comparing values in the presence or absence of BEL. Values shown are mean + SEM for six separate cell cultures.