Cholesterol is believed to serve as the common receptor for the cholesterol-dependent cytolysins (CDCs). One member of this toxin family, Streptococcus intermedius intermedilysin (ILY), exhibits a narrow spectrum of cellular specificity that is seemingly inconsistent with this premise. We show here that ILY, via its domain 4 structure, binds to the glycosyl-phosphatidylinositol-linked membrane protein human CD59 (huCD59). CD59 is an inhibitor of the membrane attack complex of human complement. ILY specifically binds to huCD59 via residues that are the binding site for the C8alpha and C9 complement proteins. These studies provide a new model for the mechanism of cellular recognition by a CDC.

Arthrofibrosis is a fibrotic joint disorder that begins with an inflammatory reaction to insults such as injury, surgery and infection. Excessive extracellular matrix and adhesions contract pouches, bursae and tendons, cause pain and prevent a normal range of joint motion, with devastating consequences for patient quality of life. Arthrofibrosis affects people of all ages, with published rates varying. The risk factors and best management strategies are largely unknown due to a poor understanding of the pathology and lack of diagnostic biomarkers. However, current research into the pathogenesis of fibrosis in organs now informs the understanding of arthrofibrosis. The process begins when stress signals stimulate immune cells. The resulting cascade of cytokines and mediators drives fibroblasts to differentiate into myofibroblasts, which secrete fibrillar collagens and transforming growth factor-β (TGF-β). Positive feedback networks then dysregulate processes that normally terminate healing processes. We propose two subtypes of arthrofibrosis occur: active arthrofibrosis and residual arthrofibrosis. In the latter the fibrogenic processes have resolved but the joint remains stiff. The best therapeutic approach for each subtype may differ significantly. Treatment typically involves surgery, however, a pharmacological approach to correct dysregulated cell signalling could be more effective. Recent research shows that myofibroblasts are capable of reversing differentiation, and understanding the mechanisms of pathogenesis and resolution will be essential for the development of cell-based treatments. Therapies with significant promise are currently available, with more in development, including those that inhibit TGF-β signalling and epigenetic modifications. This review focuses on pathogenesis of sterile arthrofibrosis and therapeutic treatments.

The rapid movement of phospholipids (PL) between plasma membrane leaflets in response to increased intracellular Ca 2؉ is thought to play a key role in expression of platelet procoagulant activity and in clearance of injured or apoptotic cells. We recently reported isolation of a ϳ37-kDa protein in erythrocyte membrane that mediates Ca 2؉ -dependent movement of PL between membrane leaflets, similar to that observed upon elevation of Ca 2؉ in the cytosol (Bassé , F., Stout, J. G., Sims, P. J., and Wiedmer, T. (1996) J. Biol. Chem. 271, 17205-17210). Based on internal peptide sequence obtained from this protein, a 1,445-base pair cDNA was cloned from a K-562 cDNA library. The deduced ''PL scramblase'' protein is a proline-rich, type II plasma membrane protein with a single transmembrane segment near the C terminus. Antibody against the deduced Cterminal peptide was found to precipitate the ϳ37-kDa red blood cell protein and absorb PL scramblase activity, confirming the identity of the cloned cDNA to erythrocyte PL scramblase. Ca 2؉ -dependent PL scramblase activity was also demonstrated in recombinant protein expressed from plasmid containing the cDNA. Quantitative immunoblotting revealed an approximately 10-fold higher abundance of PL scramblase in platelet (ϳ10 4 molecules/cell) than in erythrocyte (ϳ10 3 molecules/ cell), consistent with apparent increased PL scramblase activity of the platelet plasma membrane. PL scramblase mRNA was found in a variety of hematologic and nonhematologic cells and tissues, suggesting that this protein functions in all cells.

Phospholipid scramblase 1 (PLSCR1) is an endofacial plasma membrane protein proposed to participate in transbilayer movement of phosphatidylserine and other phospholipids. In addition to its putative role in the reorganization of plasma membrane phospholipids, PLSCR1 is a substrate of intracellular kinases that imply its possible participation in diverse signaling pathways underlying proliferation, differentiation, or apoptosis. Because PLSCR1 is prominently expressed in a variety of blood cells, we evaluated PLSCR activity in platelets and erythrocytes, and cytokine-dependent growth of hematopoietic precursor cells, of PLSCR1 knock-out mice. Adult PLSCR1 ؊/؊ mice showed no obvious hematologic or hemostatic abnormality, and blood cells from these animals normally mobilized phosphatidylserine to the cell surface upon stimulation. Whereas blood cell counts in adult PLSCR1 ؊/؊ mice were normal, in both fetus and newborn animals neutrophil counts were significantly depressed relative to age-matched wild type (WT). Furthermore, when compared with WT, hematopoietic precursor cells from PLSCR1 ؊/؊ mice showed defective colony formation and impaired differentiation to mature granulocytes as stimulated by stem cell factor and granulocyte colony-stimulating factor (G-CSF). By contrast, PLSCR1 ؊/؊ cells showed normal colony formation stimulated by interleukin-3 or granulocyte-macrophage CSF, and expansion of megakaryocytic and erythroid progenitors by thrombopoietin or erythropoietin was unaffected. Stem cell factor and G-CSF were also found to induce marked increases in PLSCR1 levels in WT cells. Consistent with in vitro assays, PLSCR1 ؊/؊ mice treated with G-CSF showed less than 50% of the granulocytosis observed in identically treated WT mice. These data provide direct evidence that PLSCR1 functionally contributes to cytokine-regulated cell proliferation and differentiation and suggest it is required for normal myelopoiesis. (Blood. 2002;99: 4030-4038)