Inhibition of calcium-independent phospholipase A2 prevents inflammatory mediator production in pulmonary microvascular endothelium

Abstract

Inhalation of allergens can result in mast cell degranulation and release of granule contents, including tryptase, in the lung. Injury to human pulmonary microvascular endothelial cells (HMVEC-L) can also result in activation of the coagulation cascade and thrombin generation. We hypothesize that these proteases activate calcium-independent phospholipase A2 (iPLA2), in HMVEC-L, leading to the production of membrane phospholipids-derived inflammatory mediators. Both thrombin and tryptase stimulation of HMVEC-L increased iPLA2 activity that was inhibited by pretreatment with the iPLA2 selective inhibitor bromoenol lactone (BEL). Arachidonic acid and prostaglandin I2 (PGI2) release were also increased in tryptase and thrombin stimulated cells and inhibited by BEL pretreatment. Pretreating the endothelial cells with AACOCF3 a cytosolic PLA2 inhibitor did not inhibit tryptase or thrombin induced arachidonic acid and PGI2 release. In addition thrombin and tryptase also increased HMVEC-L platelet activating factor (PAF) production that significantly contributes to the recruitment and initial adherence of polymorphonuclear neutrophils (PMN) to the endothelium. Tryptase or thrombin stimulated increase in PMN adherence to the endothelium was inhibited by pretreatment of HMVEC-L with BEL or pretreatment of PMN with CV3988, a PAF receptor specific antagonist. Collectively, these data support our hypothesis that iPLA2 activity is responsible for membrane phospholipid hydrolysis in response to tryptase or thrombin stimulation in HMVEC-L. Therefore selective inhibition of iPLA2 may be a pharmacological target to inhibit the early inflammation in pulmonary vasculature that occurs as a consequence of mast cell degranulation or acute lung injury.

Figure 1 a and b. Tryptase and thrombin induced iPLA₂ activity in HMVEC-L

Calcium independent PLA₂ activity was measured in HMVEC-L after stimulation with tryptase (20ng/ml, Figure 1 a) or thrombin (0.1 IU/ml, Figure 1b) with or without pretreatment with BEL (2µM, 10 min.). Data shown represent the mean ±SE for 4 independent experiments.**p< 0.01 when comparing tryptase or thrombin stimulated values vs. corresponding BEL pretreated samples.

Figure 2 a and b. Tryptase and thrombin increased arachidonic acid release

Arachidonic acid release was measured in cells after stimulating with tryptase (20ng/ml, Figure 2a) or thrombin (0.1 IU/ml, Figure 2b) with or without pre-treatment with BEL (2µM,10min.), for increasing time intervals. Cells were also pretreated with AACOCF₃ (5µM, 10min.) prior to tryptase stimulation. Results represent mean ± SE of 4 independent experiments. * and ** P< 0.05 and 0.01 respectively when compared to control cells. + p<0.05 and ++ p<0.01 when compared to tryptase or thrombin treated cells respectively

Figure 3. Dose dependent changes in PGI₂ production in HMVEC-L

HMVEC-L were treated with tryptase (2, 20, 200 ng/ml) or thrombin (0.1, 0.5, 1.0 IU/ml ) for upto 60 min. and PGI₂ production was measured. Values represent mean ± SE for 4 separate experiments. ** P< 0.01 when compared to control cells.

Figure 4. PGI₂ production increased in HMVEC-L

HMVEC-L were treated with tryptase (20 ng/ml, 10 min.) or thrombin (0.1 IU/ml, 10 min.). PGI₂ production was measured with or without pretreatment BEL (2µM, 10 min.), or AACOCF₃. (2µM, 10 min.) for 10 minutes. ** P< 0.01 when compared to control cells, ++ P<0.01 compared to tryptase or thrombin stimulated cells. Results represent mean ± SE of 4 separate experiments.

Figure 5 a and b. PAF production increased in tryptase and thrombin stimulated HMVEC-L

Cells were incubated with tryptase (20ng/ml, Figure 4a) or thrombin (0.1IU/ml, Figure 4b) for 10 and 30 min. or left untreated. PAF production was measured with or without pretreatment with BEL ( 2µM, 10 min). Data represent means ± SE for 4 separate experiments. * P < 0.05 and **P<0.01 compared with control. + P<0.05 and ++ P<0.01 compared to tryptase or thrombin stimulated cells.

Figure 6a. Tryptase increased cell surface expression of selectins

HMVEC-L were stimulated with tryptase (20ng/ml) and cell surface expressiob of P- and E-selectin was measured at 10 and 30 min. Results represent mean ± SE from four separate experiments. * P <0.05 and **P<0.01 when compared with control cells.

Figure 7. Tryptase and thrombin induced neutrophil adherence

Neutrophil adherence was measured in tryptase (20ng/ml, 10 min.) or thrombin (0.1 IU/ml, 10 min.) treated HMVEC-L. Endothelial cells were pretreated with BEL (2µM,10min.). CV 3988 (10µM,10min.) was incubated directly with neutrophils to inhibit PAF receptor. ** P <0.01 compared to control cells, ++ P <0.01 when compared to tryptase or thrombin treated samples. Results represent mean ± SEM of 3 separate experiments.