Norris D A, Middleton M H, Whang K, Schleicher M, McGovern T, Bennion S D, David-Bajar K, Davis D, Duke R C
Department of Dermatology, University of Colorado School of Medicine, Denver 80262, USA.
Apoptosis. 1997;2(2):136-48. doi: 10.1023/a:1026456229688.
Human keratinocytes proliferate and differentiate in an epidermal environment where induction of apoptosis can be triggered by ultraviolet radiation (UVR), activated lymphocytes and cytokines. The purpose of this study was to determine whether keratinocytes were susceptible to apoptosis induced by ionophore, ultra-violet radiation, cytokines or crosslinking of CD95 (Fas/APO-1). In normal human skin exposed to two minimal erythema doses of ultraviolet radiation, suprabasal cells were the first keratinocytes to demonstrate apoptotic nuclei, and by 48 h apoptotic cells were identified throughout the mid to upper epidermis. However, most keratinocytes resisted apoptosis and UVR-induced apoptosis was not observed in basal cells, or in the most differentiated epidermis. 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-induced apoptosis compared to keratinocyte cell lines or to a lymphoid cell line (HL60) susceptible to apoptosis. Keratinocyte cell lines were completely resistant to apoptosis induced by interferon-gamma, interferon-alpha, IL-2, IL-6, TNF-alpha, IL-1Ra, and GM-CSF. A subset of the cells in cultures of keratinocytes and transformed keratinocyte cell lines died by apoptosis in response to anti-Fas, IL-1alpha and TNF-alpha plus IFN-gamma and ionophore. Second passage freshly isolated human keratinocytes were much more resistant to apoptosis induced by ionophore, anti-Fas and cytokines than were transformed keratinocyte cell lines. Calcium shift to induce differentiation in second-passage keratinocyte cultures made keratinocytes even more resistant to UVR-induced apoptosis. This parallels the lack of UVR-induced apoptosis observed in the most differentiated keratinocytes in irradiated human skin. Both keratinocytes and keratinocyte cell lines express rather low levels of the anti-apoptotic proteins bcl-2 and bcl-x compared to other apoptosis-resistant cell types. The differences between keratinocytes and keratinocyte cell lines in susceptibility to apoptosis are not explained by difference in expression of bcl-2 or bcl-x. Finally, withdrawal of growth factors from keratinocytes decreased cell survival following UVR and increased the induction of apoptosis. Inhibition of protein synthesis with cyclo-heximide also made keratinocytes more susceptible to UVR-induced apoptosis, indicating that anti-apoptotic defences in cultured keratinocytes are dependent on active protein synthesis. These experiments show that the strong keratinocyte defences against apoptosis are stratified within the epidermis, and can be altered by differentiation and growth factor withdrawal.
人类角质形成细胞在表皮环境中增殖和分化，在该环境中，紫外线辐射（UVR）、活化淋巴细胞和细胞因子可触发细胞凋亡。本研究的目的是确定角质形成细胞是否易受离子载体、紫外线辐射、细胞因子或CD95（Fas/APO-1）交联诱导的细胞凋亡影响。在暴露于两个最小红斑量紫外线辐射的正常人皮肤中，基底层以上的细胞是最早显示凋亡细胞核的角质形成细胞，到48小时时，整个表皮中、上部均能识别出凋亡细胞。然而，大多数角质形成细胞抵抗凋亡，在基底细胞或最分化的表皮中未观察到UVR诱导的凋亡。体外培养的人角质形成细胞和角质形成细胞系在辐射后48小时出现最大程度的凋亡。与角质形成细胞系或易发生凋亡的淋巴细胞系（HL60）相比，在完全生长因子补充剂中培养的人角质形成细胞对UVR诱导的凋亡具有抗性。角质形成细胞系对干扰素-γ、干扰素-α、IL-2、IL-6、TNF-α、IL-1Ra和GM-CSF诱导的凋亡完全具有抗性。角质形成细胞和转化角质形成细胞系培养物中的一部分细胞因抗Fas、IL-1α、TNF-α加干扰素-γ和离子载体而发生凋亡。第二代新鲜分离的人角质形成细胞比转化角质形成细胞系对离子载体、抗Fas和细胞因子诱导的凋亡更具抗性。在第二代角质形成细胞培养物中通过钙转移诱导分化，使角质形成细胞对UVR诱导的凋亡更具抗性。这与在受照射人皮肤中最分化的角质形成细胞中未观察到UVR诱导的凋亡情况相似。与其他抗凋亡细胞类型相比，角质形成细胞和角质形成细胞系均表达相当低水平的抗凋亡蛋白bcl-2和bcl-x。角质形成细胞和角质形成细胞系在凋亡易感性方面的差异不能用bcl-2或bcl-x表达的差异来解释。最后，从角质形成细胞中撤除生长因子会降低UVR后的细胞存活率，并增加凋亡诱导。用环己酰亚胺抑制蛋白质合成也使角质形成细胞更容易受到UVR诱导的凋亡影响，表明培养的角质形成细胞中的抗凋亡防御依赖于活跃的蛋白质合成。这些实验表明，角质形成细胞对凋亡的强大防御在表皮内是分层的，并且可以通过分化和生长因子撤除而改变。
