Kenneth S. Kilgore

Osteoarthritis (OA) is a degenerative joint disease characterized by joint pain and a progressive loss of articular cartilage. Studies to elucidate the pathophysiology of OA have been hampered by the lack of a rapid, reproducible animal model that mimics both the histopathology and symptoms associated with the disease. Injection of mono-iodoacetate (MIA), an inhibitor of glycolysis, into the femorotibial joint of rodents promotes loss of articular cartilage similar to that noted in human OA. Here, we describe the histopathology in the subchondral bone and cartilage of rat (Wistar) knee joints treated with a single intra articular injection of MIA (1 mg) and sacrificed at 1, 3, 5, 7, 14, 28, and 56 days postinjection. Histologically, the early time points (days 1-7) were characterized by areas of chondrocyte degeneration/necrosis sometimes involving the entire thickness of the articular cartilage in the tibial plateaus and femoral condyles. Changes to the subchondral bone, as evidenced by increased numbers of osteoclasts and osteoblasts, were noted at by day 7. By 28 days, there was focal fragmentation and collapse of bony trabeculae with fibrosis and increased osteoclastic activity. By 56 days there were large areas of bone remodeling evidenced by osteoclastic bone resorption and newly formed trabeculae with loss of marrow hematopoietic cells. Subchondral cysts and subchondral sclerosis were present in some rats. In conclusion, intra-articular injection of MIA induces loss of articular cartilage with progression of subchondral bone lesions that mimic those of OA. This model offers a rapid and minimally invasive method to reproduce OA-like lesions in a rodent species.

Activation of the complement cascade and subsequent assembly of the membrane attack complex (MAC) occur in a number of pathophysiological settings. When formed on the surface of endothelial cells in sublytic concentrations, the MAC can induce a number of proinflammatory activities, including the secretion of soluble mediators (eg, interleukin (IL)-8 and monocyte chemoattractant protein (MCP)-1) and the up-regulation of cell surface adhesion molecules. Available data indicate that MAC-induced cell activation may occur through several complex signal transduction pathways, but little is known about the intranuclear mechanisms by which complement-derived products promote the up-regulation of inflammatory mediators. Using purified distal complement proteins (C5-9) to assemble functional MAC on early-passage human umbilical vein endothelial cells (HUVECs), we examined mechanisms of MCP-1 and IL-8 induction. Formation of sublytic concentrations of MAC promoted an increase in nuclear factor (NF)-kappa B DNA binding activity within 60 minutes as determined by serial electrophoretic mobility shift assay. Cytosolic to nuclear translocation of NF-kappa B was confirmed by Western immunoblot and immunocytochemical analyses. Formation of the C5b-8 complex also promoted NF-kappa B translocation but to a lesser degree than observed in HUVECs containing complete MAC. No cytosolic to nuclear translocation of the p65 NF-kappa B subunit was observed in unstimulated HUVECs or in cells incubated with the MAC components devoid of C7. Preincubation of HUVECs with pyrrolidine dithiocarbamate prevented MAC-induced increases in IL-8 and MCP-1 mRNA concentrations and protein secretion. A direct cause and effect linkage between MAC assembly and NF-kappa B activation was established through examination of the pharmacological effect of the peptide SN50 on IL-8 and MCP-1 expression. SN50 is a recently engineered 26-amino-acid peptide that contains a lipophilic cell-membrane-permeable motif and a nuclear localization sequence that specifically competes with the nuclear localization sequence of the NF-kappa B p50 subunit. This study provides direct in vitro evidence that the distal complement system (MAC) can promote proinflammatory endothelial cell activation, specifically, increases in IL-8 and MCP-1 mRNA concentrations and protein secretion, and that cytosolic to nuclear translocation of NF-kappa B is necessary for this response.

Although TNF-alpha and several products of the activated complement system (e.g., C3b, iC3b, and C5a) are known to modulate endothelial cell function in vitro, relatively little is known about the potential modulatory role of the membrane attack complex (MAC) in endothelial cell activation. Using an in vitro neutrophil-endothelial adhesion assay and a quantitative whole cell ELISA to measure endothelial E-selectin and intracellular adhesion molecule-1 (ICAM-1) expression, we examined the modulatory role of the MAC in TNF-alpha-induced neutrophil-endothelial cell adhesive interactions. Activation of quiescent human umbilical vein endothelial cells (HUVECs) with TNF-alpha results in a concentration-dependent increase in neutrophil adhesion measured at 4 h. Assembly of sublytic concentrations of the MAC on endothelial cells did not result in changes in neutrophil-HUVEC adhesion measured at 4 h. Activation of HUVECs with TNF-alpha followed by assembly of the MAC resulted in a marked increase in neutrophil binding as compared with that observed in cells treated with TNF-alpha alone. Blocking studies of mAb revealed that in either TNF-alpha-stimulated or TNF-alpha and MAC-activated endothelial cells enhanced neutrophil binding was nearly entirely attributable to E-selectin and ICAM-1. This conclusion was further supported by a whole-cell ELISA, which provided evidence that the MAC augments TNF-alpha-induced up-regulation of both E-selectin and ICAM-1. This study provides data that support the conclusion that the distal complement system (MAC) can enhance TNF-alpha-induced proinflammatory endothelial cell functions.