Figure 4

Distinct signal transduction pathways are activated in PMNs by PAR₁ and PAR₂ and are subverted by fungi. WT (a), tlr2^−/−, tlr4^−/−, or myd88^−/− (b) PMNs were stimulated with PAR agonists (PAR-APs) with and without antagonists (PAR-ANTs) for 30 min. Blots of cell lysates were incubated with rabbit anti-ERK or phosphorylated (p) ERK and anti-p38 or pp38 MAPK antibodies. None, cells left unstimulated for 30 min. (c) PMNs stimulated with phorbol 12-myristate β-acetate for 30 min. (b) Zymosan was used to stimulate CD14/TLR2/HEK293 cells transfected with par1siRNA and LPS to stimulate CD14/TLR4/HEK293 cells transfected with par2 siRNA. EMSA was performed 2 h later. The specificity of bands was verified by using antibodies to p65 and p50. (e) WT or myd88^−/− PMNs were sequentially exposed to fungi (30 min) and PAR-AP (30 min) before blotting (none, unstimulated cells). Cells were pre-exposed to the p38 inhibitor SB202190 (10 μM) for 60 min before stimulation with Aspergillus+PAR₂-AP. Initial experiments have shown that MAPK phosphorylation in response to agonists or fungi alone was optimal at 30 min and declined at 60 min (Supplementary Figure S2 online). (f) Antagonistic effects of PAR₁-AP and PAR₂-AP on the respiratory burst (O₂⁻) and matrix metalloproteinase 9 (MMP9) production of PMNs in response to Candida or Aspergillus. PMNs were stimulated with PAR agonists/antagonists and fungi as above. (AI, arbitrary index of scanning densitometry) *P<0.05, treated vs. untreated PMNs. −, unstimulated cells. (g) PMNs from tlr2^−/−, tlr4^−/−, and myd88^−/− mice were exposed to PAR agonists and fungi and assessed for oxidant production as above. Bars are s.e. *P<0.05, treated vs. untreated. EMSA, electrophoretic mobility shift assay; LPS, lipopolysaccharide; MAPK, mitogen-activated kinase; PAR, protease-activated receptor; PMN, polymorphonuclear neutrophil; WT, wild type.

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