HUMAN KERATINOCYTES MAINTAIN REVERSIBLE ANTI-APOPTOTIC DEFENSES IN-VIVO AND IN-VITRO

Human keratinocytes proliferate and differentiate in an epidermal envi ronment where induction of apoptosis can be triggered by ultraviolet r adiation (UVR), activated lymphocytes and cytokines. The purpose of th is study was to determine whether keratinocytes were susceptible to ap optosis induced by ionophore, ultraviolet radiation, cytokines or cros slinking of CD95 (Fas/APO-1). In normal human skin exposed to two mini mal erythema doses of ultraviolet radiation, suprabasal cells were the first keratinocytes to demonstrate apoptotic nuclei, and by 48 h apop totic cells were identified throughout the mid to upper epidermis. How ever, most keratinocytes resisted apoptosis and UVR-induced apoptosis was not observed in basal cells, or in the most differentiated epiderm is. Human keratinocytes and keratinocyte cell lines cultured in vitro developed maximal apoptosis 48 h after radiation. Human keratinocytes cultured in full growth factor supplements were resistant to UVR-induc ed apoptosis compared to keratinocyte cell lines or to a lymphoid cell line (HL60) susceptible to apoptosis. Keratinocyte cell lines were co mpletely resistant to apoptosis induced by interferon-gamma, interfero n-alpha, IL-2, IL-6, TNF-alpha, IL-1Ra, and GM-CSF. A subset of the ce lls in cultures of keratinocytes and transformed keratinocyte cell lin es died by apoptosis in response to anti-Fas, IL-1 alpha and TNF-alpha plus IFN-gamma and ionophore. Second passage freshly isolated human k eratinocytes were much more resistant to apoptosis induced by ionophor e, anti-fas and cytokines than were transformed keratinocyte cell line s. Calcium shift to induce differentiation in second-passage keratinoc yte cultures made keratinocytes even more resistant to UVR-induced apo ptosis. This parallels the lack of UVR-induced apoptosis observed in t he most differentiated keratinocytes in irradiated human skin. Both ke ratinocytes and keratinocytecell lines express rather low levels of th e anti-apoptotic proteins bcl-2 and bcl-x compared to other apoptosis- resistant cell types. The differences between keratinocytes and kerati nocyte cell lines in susceptibility to apoptosis are not explained by difference in expression of bcl-2 or bcl-x. Finally, withdrawal of gro wth factors from keratinocytes decreased cell survival following UVR a nd increased the induction of apoptosis. Inhibition of protein synthes is with cycloheximide also made keratinocytes more susceptible to UVR- induced apoptosis, indicating that anti-apoptotic defences in cultured keratinocytes are dependent on active protein synthesis. These experi ments show that the strong keratinocyte defences against apoptosis are stratified within the epidermis, and can be altered by differentiatio n and growth factor withdrawal.