Presence of LPS in the extracellular matrix in cartilage tissue. (A) Primary human chondrocytes (1 × 106 cells) were cultured in high-density culture with lipopolysaccharides (LPS) (100 ng/ml) or without LPS (Co) for different times. Whole-cell extracts were prepared, and cell lysates were resolved by SDS-PAGE, electrotransferred to nitrocellulose membrane, and then probed for LPS expression by western blot analysis using antibodies to this protein. β-Actin served as an internal control. LPS control peptide (Co Pep) was used as a control. M = marker for molecular weights. (B) Primary human chondrocytes (Ch) (1 × 106 cells) were cultured for 7 days in high-density culture and then incubated with LPS (100 ng/ml) for 12 h before evaluation by immunoelectron microscopy. Secondary gold particle-labeled antibody was observed directly on the collagen bundles or clustered (arrows) together indicating the existence of LPS between the mesh of the collagen fibers in the extracellular matrix (ECM). (C-D) Primary human chondrocytes (Ch) (1 × 106 cells) were cultured for 7 days in high-density culture, and then first preincubated with anti-collagen type II (100 ng/ml) (C) or control rabbit IgG (100 µl/ml) (D) for 24 h and followed by incubation with LPS (100 ng/ml) for 12 h before evaluation with immunoelectron microscopy. Opposite to preincubated cultures with control rabbit IgG, only a very small amount of secondary gold particle-labeled antibodies was observed on the collagen bundles or clustered (arrows) in the extracellular matrix (ECM) in preincubated cultures with anti-collagen type II. Magnification x35,000; bar, 0.25 µm.
Lorenz et al. Arthritis Research & Therapy 2013 15:R111 doi:10.1186/ar4291