Der p2 Internalization by Epithelium Synergistically Augments Toll-like Receptor-Mediated Proinflammatory Signaling (1)
Sensitization to Der p2, one of the major allergens of Der p, was implicated in 87.8% of these patients. Myeloid differentiation-2 (MD2), is an LPS-binding member of the Toll-like receptor 4 (TLR4) signaling complex. Structures of MD2 and Der p2 exhibit homology. Der p2 can facilitate TLR4 signaling through direct interactions with the TLR4 complex, reconstituting LPS-promoted TLR4 signaling in the absence of MD2 and facilitating such signaling in the presence of MD2.
BEAS-2B monolayers were cultured with rDer p2-EGFP or rEGFP alone. Fluorescent images of living cells were obtained by confocal microscopy. Der p2-EGFP was observed in the cellular cytoplasm following 4 hours and 24 hours of incubation. Internalized Der p2 in the cytoplasm co-localized with ER following 24 hours of incubation.
Expression of mRNA-encoding MD2 was identified in the lysed cells by RT-PCR after 4 hours of culture. Both Der p2 and MD2 proteins were identified in the cellular lysates by immunoblotting with anti-Der p2 and anti-MD2 antibodies following 24 hours of incubation.
Secretion of IL-6/IL-8 by BEAS-2B cells was concentration-dependently augmented by LPS from 2.0- to 19.1/78.8-fold for IL-6 and from 3.3- to 31.7/104.9-fold for IL-8, respectively. IL-6/IL-8 secretion was reduced in the presence of anti-MD2 antibody from 25.7- to 11.5-fold for IL-6 and from 44.6- to 28.5-fold for IL-8, respectively.
Cells were transiently transfected with MD2 to induce overexpression. cells overexpressing MD2 were more sensitive to rDer p2 stimulation for IL-6 secretion.
Der p2 concentration-dependently increased the expression of mRNA encoding IL-6 and IL-8 in these cells over the 16-hours time period. The mRNA expression of TLR2 and IL-1β was somewhat increased, especially in conjunction with LPS.
The most effective inhibitors on IL-6 mRNA expression were dexamethasone, SP600125 (JNK inhibitor), SB203580 (p38 inhibitor), calcitriol, and BAY 11-7082 (IκB inhibitor). The most effective inhibitors on IL-8 mRNA expression were dexamethasone, SP600125, calcitriol, and BAY 11-7082.
All the transcription factor-signaling inhibitors reduced secretion of both IL-6 and IL-8. The most effective inhibitors on IL-6/IL-8 protein secretion were dexamethasone, SP600125 (JNK inhibitor), SB203580 (p38 inhibitor), anti-TLR2 neutralizing antibody, and calcitriol.
The spectrum of responsiveness to TLR2 is wider than that to TLR4 because both receptors respond to LPS, but TLR2 additionally responds to lipoproteins, lipopeptides, and peptidoglycans. In mouse airway smooth muscle (ASM) cells, Der p2 was shown to activate ASM cells in a TLR2/MyD88-dependent manner. Native, but not recombinant, Der p2 which lacks glycosylated lectin ligands can stimulate monocyte-derived dendritic cells through its interaction with DC-SIGN, a lectin receptor.