NUCLEAR DEGENERATION IN THE DEVELOPING LENS AND ITS REGULATION BY TNF-ALPHA

DNA fragmentation in lens fibre cell nuclei undergoing programmed dege neration was identified by terminal deoxynucleotidyl transferase (TdT) -mediated biotin-dUTP nick end labelling (TUNEL). Lens epithelial cell s in culture were induced to differentiate into lens fibre-like clumps of cells (lentoids) by insulin and it was shown that the TUNEL method was also an effective means of labelling degenerating nuclei in lento id cells in lens epithelial cell cultures. Using immuno-fluorescence a nd confocal microscopy, it was shown that TNF alpha and TNF receptor ( TNFR1, and TNFR2) immunoreactivity was present in sections of chick em bryo lenses. TNF alpha immunoreactivity was associated with the lens e pithelium and lens fibres. TNFR1 immunoreactivity was present in lens epithelial cells, cortical lens fibres, and lens fibre cell nuclei, wh ile TNFR2 immunoreactivity had a similar distribution to that of TNFR1 , but was not associated with nuclei. Similar patterns of TNF alpha, T NFR1, and TNFR2 immunoreactivity were observed in lens epithelial cell cultures. When added to lens epithelial cell cultures, TNF alpha, at concentrations of 50 to 100 ng ml(-1), and agonistic antibodies to bot h TNFR1 and TNFR2 significantly (P < 0.05) enhanced the number of dege nerating (TUNEL-positive) nuclei. On the other hand, a neutralising an tibody to TNF alpha significantly (P < 0.05) reduced the number of TUN EL-positive nuclei. These results demonstrate that TUNEL is an effecti ve means of labelling degenerating lens fibre nuclei during lens fibre and lentoid differentiation, and suggest a potential role for TNF alp ha-like factors and their receptors in the degeneration of lens fibre cell nuclei during lens differentiation. We further suggest that the n uclear degeneration of lens fibre cells is analogous to the nuclear ev ents that occur during apoptosis, and that in lens cells the nuclear d egeneration is uncoupled from the plasma membrane events of apoptosis that normally lead to cell death. (C) 1998 Academic Press Limited.