Age-related cataract: Antibody against β-crystallin killed human lens epithelial cells in vitro

Purpose. In order to examine the hypothesis that autoimmune antibodies are potentially damaging to lens epithelium which may lead to cataract development, the effects of β-crystallin antibody (β-C Ab) on cell death and fiber differentiation in human lens epithelial (HLE) cells in culture were investigated. Methods. Since the cell killing mechanism is known to involve the binding of the antigen-antibody complex, the effects of β-C Ab were studied both in the presence and absence of the complement. Primary cultures of HLE cells were treated with β-C Ab in concentration range of 1:640 to 1:80 for 3 hours and the viability of cells was tested by their ability to exclude trypan blue dye. The effect of Ab treatment on fiber cell differentiation was examined by the ability of the cells to form lentoids. HLE cells were treated with 1:100 dilution of β-C Ab for six days and then passaged on non-protein binding membranes at high cell density. After 4 weeks of culture without the Ab and the complement, lentoids were examined by transmission electron microscopy (TEM). The binding of β-C Ab to the cell surface was observed by TEM using immunocytochemistry and using the conjugate of colloidal gold and β-C Ab. Results. More than 20% of the non-log phase 3rd passage cells treated with β-C Ab at more than 1:160 dilution in the presence of complement were damaged. Cells treated with β-C Ab with complement failed to form typical lentoids and appeared as degenerated epithelial cell aggregates. Unlike typical lentoids, these cell aggregates showed no loss of cytoplasmic organelles and failed to form interdigitations or gap junctions between cells. The gold particles reacted with β-C Ab were observed on the surface of the plasma membrane. Conclusion. β-C Ab bound to the surface of HLE cells, caused the cell death and prevented the cells from undergoing normal fiber differentiation.The results support the hypothesis that autoantibodies against lens antigens are pathogenic agents to the lens epithelial cells involving classical complement mediated pathway.