PMC

BMS-345541 or wortmannin inhibited LPS-induced nuclear translocation of phospho p65 in chondrocytes as demonstrated by immunofluorescence microscopy. Primary human chondrocyte cultures either served as controls (a-b: without primary antibody; c-d: with primary antibody) or were treated with lipopolysaccharides (LPS) alone (e-f) or were pre-treated with wortmannin (20 nM) (g-h) or with BMS-345541 (5 mM) (i-j) for 12 h before co-treatment with LPS (100 ng/ml) for 24 h before immunolabeling with phospho p65 antibodies and rhodamine-coupled secondary antibodies and counterstained with DAPI to visualize cell nuclei. Images shown are representative of three independent experiments. Magnification x400; bar, 30 nm.

Inhibitory effects of BMS-345541 or/and wortmannin on LPS-induced NF-κB/PI-3K and apoptosis in primary human chondrocytes in vitro. Lipopolysaccharides (LPS)-induced disruption of cartilage may be induced through LPS complex formation with collagen II fibrils and through LPS/Toll-like receptor 4 (TLR4) association. Anti-collagen type II significantly reduced these procollagen-endotoxin complexes. Functional association with TLR4 leads to activation of intracellular downstream signaling pathway NF-κB, nuclear factor-κB (NF-κB)/PI-3K, phosphatidylinositol 3-kinase (PI-3K) inducing upregulation of matrix-degrading enzymes, inflammation and apoptosis. Culturing with specific inhibitors wortmannin (for PI-3K/AKT) and BMS-345541 (for IκB kinases (IKKs)) suppresses LPS-induced inflammatory response indicating the potential for new medical approaches.

Effects of BMS-345541 or/and wortmannin on the LPS-induced proinflammatory and apoptotic signaling in human chondrocytes. Primary human chondrocytes in monolayer cultures were either left untreated or stimulated with 100 ng/ml lipopolysaccharides (LPS), prestimulated with 5 mM BMS-345541, with 20 nM wortmannin or a combination of both inhibitors, BMS-345541 and wortmannin (5 mM and 20 nM) for 12 h and then treated with 100 ng/ml LPS for the indicated times. Whole-cell extracts were fractionated (500 ng protein per lane) on SDS-PAGE and examined by western blot analysis using anti-matrix metalloproteinase (MMP)-9 (A), -MMP-13 (B), -cyclooxygenase-2 (Cox-2) (C) and anti-cleaved caspase-3 (D). The results shown are representative of three independent experiments. Housekeeping protein β-actin served as a loading control.

LPS induced PI-3K/Akt activation and PI-3K inhibitor (wortmannin) suppressed this in a time- and dose-dependent manner. (A-B) Primary human chondrocytes in monolayer culture were either left untreated (controls), treated with lipopolysaccharides (LPS) (0, 0.1, 1, 10, 100 and 1000 ng/ml) for 50 min or stimulated with 100 ng/ml LPS for the indicated times. Nuclear extracts were prepared and assayed for Akt activation by western blotting as described in Materials and methods. (C-D) Primary human chondrocytes in monolayer cultures were either left untreated or were prestimulated with wortmannin for the indicated concentrations and treated with 100 ng/ml LPS for 50 min, or prestimulated with 20 nM wortmannin for 12 h followed by co-treatment with 100 ng/ml LPS for the indicated times. Nuclear extracts were prepared and assayed for Akt activation by western blot analysis as described in Materials and methods. Housekeeping protein β-actin served as a loading control and remained unaffected. PI-3K, phosphatidylinositol 3-kinase.

LPS induced cellular and matrix degradation and apoptosis in cartilage tissue. Primary human chondrocytes were either left untreated (a) or were treated with 100 ng/ml lipopolysaccharides (LPS) (b-d), pretreated with BMS-345541 (5 mM) (e-g), wortmannin (20 nM) (h-j) followed by LPS treatment, or pretreated in combination with BMS-345541 and wortmannin (5 mM and 20 nM) (k-m) for 12 h and then stimulated with LPS for another 24 h. The cells were transferred to high-density culture for 14 days. Ultrastructural morphology was evaluated by electron microscopy. Control cultures of chondrocytes showed well-developed chondrocytes (Ch) embedded in a well-developed extracellular matrix (ECM) (a). Treatment with LPS resulted in matrix breakdown and cell lysis and apoptosis (arrows) (b-d). Pretreatment with BMS-345541 alone (e-g), with wortmannin alone (h-j) or in combination with BMS and wortmannin (k-m) resulted in a marked improvement of chondrocyte phenotype and the formation of cartilage nodules. The formation of a dense extracellular matrix (ECM) surrounding well-developed chondrocytes (Ch) was observed. Magnification x5000; bar, 1 µm.

LPS induced TLR4 expression and associated with TLR4 in human chondrocytes. (A) Primary human chondrocytes were either left untreated (b, basal Co) or were treated with 10 ng/ml (c) or 100 ng/ml lipopolysaccharides (LPS) (d) for 12 h, then were immunostained with anti-Toll-like receptor 4 (TLR4) (red) and counterstained with DAPI (blue). Control, in which primary antibodies were replaced with control rabbit IgG (a). Results are representative of three experiments using human chondrocytes. Magnification x400; bar, 30 nm. (B) Lysates prepared from human chondrocytes exposed to LPS (100 ng/ml) or untreated (c) (negative control) were incubated with anti-LPS. Immunoprecipitates were analyzed by western blotting with anti-TLR4. Presence of band of the correct size (approximately 100 kDa) corresponding to chondrocytes treated with LPS confirms the binding of LPS to TLR4. The original samples were probed with an antibody to anti-TLR4. Data shown are representative of three independent experiments. IgH: immunglobulin heavy chain.

LPS induced the phosphorylation and translocation of p65 and the IKK inhibitor BMS-345541 or PI-3K inhibitor wortmannin suppressed this in a time- and dose-dependent manner. (A-B) Primary human chondrocytes in monolayer culture were either left untreated (controls), treated with lipopolysaccharides (LPS) (0, 0.1, 1, 10, 100 and 1000 ng/ml) for 30 min (A) or stimulated with 100 ng/ml LPS for the indicated times (B). Nuclear extracts were prepared and assayed for nuclear factor-κB (NF-κB) (p65) activation by western blot analysis as described in Materials and methods. (C-F) Primary human chondrocytes in monolayer cultures were either left alone or pretreated with BMS-345541 or with wortmannin for the indicated concentrations followed by treatment with 100 ng/ml LPS for 30 min (C and E), or prestimulated with 5 mM BMS-345541 or with 20 nM wortmannin for 12 h and co-treated with 100 ng/ml LPS for the indicated times (D and F). Nuclear extracts were prepared and assayed for NF-κB (p65) activation by western blot analysis as described in Materials and methods. Synthesis of poly(ADP-ribose) polymerase (PARP) remained unaffected in nuclear extracts. IKK, IκB kinase; PI-3K, phosphatidylinositol 3-kinase.

Effects of BMS-345541 and wortmannin on LPS signalling in chondrocytes. (A) Effects of BMS-345541 or wortmannin on LPS-induced IκB-α phosphorylation and degradation in chondrocytes in monolayer cultures. Primary human chondrocytes in monolayer culture were either stimulated with 100 ng/ml lipopolysaccharides (LPS) or prestimulated with 5 mM BMS-345541 or with 20 nM wortmannin for 12 h followed by LPS treatment (100 ng/ml) for the indicated times. Cytoplasmic extracts were prepared, fractionated (500 ng protein per lane) on 10% SDS-PAGE and electrotransferred onto nitrocellulose membranes. Western blot analysis was performed with anti-phospho-specific-IκB-α (II), anti-IκB-α (I) and anti-β-actin (II, control). The results shown are representative of three independent experiments. (B) Effects of BMS-345541 or wortmannin on LPS-induced IKK activation in chondrocytes in monolayer cultures. Primary human chondrocytes in monolayer culture were either stimulated with 100 ng/ml LPS or prestimulated with 5 mM BMS-345541 or with 20 nM wortmannin for 12 h followed by treatment with 100 ng/ml LPS for the indicated times. Whole-cell extracts were immunoprecipitated with an antibody against IkB kinase (IKK) and then analyzed by an immune complex kinase assay as described in Materials and methods. To examine the effect of LPS, BMS-345541 or wortmannin on the level of activation of IKK proteins, whole-cell extracts were fractionated (500 ng protein per lane) on SDS-PAGE and examined by western blot analysis using anti-phospho-specific-IκB-α and anti-IκB-β (I). LPS, BMS-345541 or wortmannin had no direct effect on the expression of IKK-α or IKK-β proteins (II, III). The results shown are representative of three independent experiments.

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.