Immunomodulatory Effects Of Rye Grass Pollen Allergen Lol p 5 On The Prostaglandin E2 Pathway and Kallikrein-Kinin System Of Respiratory Epithelial Cells

Abstract

Several important aeroallergens are known to modulate respiratory epithelial (RE) function. The rye grass pollen Group 5 allergen is a significant contributor to pollen allergy but its immunomodulatory effects on RE function are unknown. Rye grass pollen allergen rLol p 5 and its N- and C-terminal domains were expressed in E. coli. Physicochemical studies were conducted using SDS-PAGE, native-PAGE, HPLC, circular dichroism and Phyre2 modelling. Allergenicity was determined by ELISA and immunoblot, and ribonuclease activity by enzyme assay. RE IL-8 release was examined using ELISA and A549, 16HBE14sigma- and Detroit562 cell lines. mRNA level expression of enzymes/proteins associated with the prostaglandin E2 (PGE2) pathway and kallikrein-kinin system (KKS) by RE cells were determined using RT-PCR. 3D-modelling showed rLol p 5 to be structurally similar to Timothy grass pollen Group 5 allergen; circular dichroism analysis showed heat stable proteins with a predominance of α-helices and coils, consistent with the 3D structures. HPLC and native PAGE indicated trimerization (mature and N-terminal domain) and dimerization (C-terminal domain). Each protein was enzymatically active, and reacted with IgE from seven rye grass pollen sensitive patients. All three cell lines released IL-8, and A549 cells showed PGE2 release. RT-PCR with A549 showed up-regulation of COX-2, mPGES-1, mPGES-2 and cPGES in the PGE2 pathway, and gC1qR, UPAR, UPA, HSP90α and PAI associated with the KKS. The ribonuclease Lol p 5 and its component domains were allergenic, existed as oligomers, induced IL-8 and PGE2 and up-regulated proinflammatory PGE2 and KKS pathways by, as yet, unknown mechanisms.