Chitohexaose Activates Macrophages by Alternate Pathway through TLR4 and Blocks Endotoxemia
Figure 2
Binding OF FAg/AgW to human monocytes (in PBMCs) or murine macrophages (in BMDM) is mediated through TLR4.
(A,B) Bone marrow cells of BALB/c mice (A) or purified human PBMCs (B) were incubated with anti-mouse TLR4-PE and anti-human TLR4-PE respectively in the presence or absence of AgW at 4°C for 30 minutes, and CD 14+ cells analyzed by FACS. Representative overlaid histogram shows competitive inhibition of binding of anti-mouse antibodies (A) or anti-human antibodies (B) to TLR4. (C) Soluble TLR4 directly binds to the helminthic antigens. ELISA plates were coated with solubilised extracts of S.digitata, AgW, N.brasiliensis, H.polygyrus, C.elegans and mock-BSA, Phosphorylcholine-BSA, GlcNAc-BSA, Mannose-BSA and LPS. After blocking with skimmed milk-PBS, human PBMC lysates were incubated. The plates were washed and incubated with rabbit anti-human TLR4 antibodies and their binding was detected by using peroxidase conjugated anti-rabbit IgG. The enzyme activity was measured using OPD. % mean ± SEM of two individual experiments are shown. (D) Jurkat cells trasfected with mock or TLR4 plasmid were incubated with anti-human TLR4-PE and analysed by FACS. Representative overlaid histogram shows enhanced expression of TLR4 by transfected cells as compared to mock transfected cells. (E) Enhanced binding to FAg after over expression of TLR 4. Mock or TLR4 transfected jurkat cells were incubated with biotinylated FAg at 4°C for 30 minutes and stained with Streptavidin-PE conjugate. The cells were thoroughly washed to remove unbound Streptavidin-PE and analysed by FACS. Representative overlaid histogram shows enhanced binding of FAg to transfected cells as compared to mock transfected cells.