The lectin-like domain of TNF protects from listeriolysin-induced hyperpermeability in human pulmonary microvascular endothelial cells - a crucial role for protein kinase C-alpha inhibition

Abstract

Listeriosis can lead to potentially lethal pulmonary complications in newborns and immune compromised patients, characterized by extensive permeability edema. Listeriolysin (LLO), the main virulence factor of Listeria monocytogenes, induces a dose-dependent hyperpermeability in monolayers of human lung microvascular endothelial cells in vitro. The permeability increasing activity of LLO, which is accompanied by an increased reactive oxygen species (ROS) generation, RhoA activation and myosin light chain (MLC) phosphorylation, can be completely inhibited by the protein kinase C (PKC) alpha/beta inhibitor GO6976, indicating a crucial role for PKC in the induction of barrier dysfunction. The TNF-derived TIP peptide, which mimics the lectin-like domain of the cytokine, blunts LLO-induced hyperpermeability in vitro, upon inhibiting LLO-induced protein kinase C-alpha activation, ROS generation and MLC phosphorylation and upon restoring the RhoA/Rac 1 balance. These results indicate that the lectin-like domain of TNF has a potential therapeutic value in protecting from LLO-induced pulmonary endothelial hyperpermeability.

Fig. 1

Effect of a 2 h treatment with LLO (250 ng/ml) on A. RhoA activity and B. Rac1 activity in HL-MVEC, both expressed as % of ctrl. Cells were pretreated for 30 min or not with TIP peptide (50 μg/ml). (n=4, *p<0.05 vs. ctrl; #p<0.05 vs. LLO).

Fig. 2

LLO induces a dose-dependent increase in MLC phosphorylation upon a 2 h treatment in HL-MVEC, expressed as relative densitometric units (RDU) of the ppMLC/total MLC ratio (Western Blotting), normalized to the control cell signal. (n=3; *p<0.05 vs. ctrl).

Fig. 3

LLO (250 ng/ml) induces PKC-α activation within 30 min, which is blunted upon 30 min pretreatment with the TIP peptide in HL-MVEC (n=4; *p<0.05 vs. ctrl).

Fig. 4

The PKC-α/βII inhibitor GÖ6976 (1 μM) significantly inhibits A. LLO-mediated MLC phosphorylation (expressed as relative densitometric units (RDU) of the ppMLC/total MLC ratio (Western Blotting), normalized to the control cell signal). n=3, *p<0.05 vs. ctrl; ‡p<0.05 vs. LLO and B. LLO-induced endothelial hyperpermeability (TER in ECIS) in HL-MVEC, upon 30 min preincubation. LLO: 250 ng/ml; (n=3; *p<0.05 vs. ctrl, #p<0.05 vs. LLO).

Fig. 5

A. LLO (250 ng/ml; 6 h incubation) induces ROS generation in ovine PAEC, as measured by means of the EPR technique, and a 30 min pretreatment with the TIP peptide (50 μg/ml) inhibits this effect. The TIP peptide did not induce changes in basal ROS generation (n=6; *p<0.05 vs. ctrl; #p<0.05 vs. LLO). B. LLO treatment increases mRNA concentration of NADPH oxidase 4 in HL-MVEC, as measured by Real-Time RT-PCR and a 30 min pretreatment with the TIP peptide inhibits this activity. (n = 3; *p<0.05 vs. ctrl).

Fig. 6

A. The TIP peptide (50 μg/ml) protects from LLO-induced hyperpermeability in HL-MVEC (TER measurements in ECIS, n=4; t test) analysis demonstrated that TER values in the TIP peptide/LLO groups were significantly different from the corresponding LLO groups between 3 and 10 h post LLO treatment (p<0.05). B. The TIP peptide (50 μg/ml) blunts LLO-induced MLC phosphorylation, measured as relative densitometric units (RDU) of the ppMLC/total MLC ratio, normalized to the control cell signal (2 h incubation, 250 ng/ml LLO) in an amiloride-sensitive (10 μM) manner. (n=3, *p<0.05 vs. ctrl; ‡p<0.05 vs. LLO). C. representative blot assessing total and ppMLC.

Fig. 7

LLO induces a rapid Ca²⁺ influx in BAEC transduced adenovirus encoding the calcium-sensitive photoprotein aequorin (n=6, Church and Fulton, 2006).