Apoptosing cells are actively phagocytoscd in parenchymal tissues, thus preventin~ the inflammatory reaction which could derive from their slow uncontrolled degradation. The molecular mechanisms by which an apoptotic cell is recognized and taken up are largely unknown. We propose that the recognition of apoptotic hepatocytes is mediated by the sugar recognition systems of the liver, particularly the asialoglyeoproteln receptor (ASGP-R), The re~ults presented here demonstrate the participation of ASGP-R in the removal of apoptotie parenchymal cells, and indicate a new perspective for the understanding of its physiological role.

Physiological deletion of cells ensues programmed death which involves formation of apoptotic bodies with fragmented DNA. Here we report that apoptotic hepatocytes are insoluble in detergents, urea, guanidine hydrochloride, reducing agents and thereby can be isolated from rat liver following collagenase treatment. They are wrinkled, spherical structures similar to cornified envelopes of epidermis by phase-contrast microscopy and show irregular, globular morphology by scanning-electron microscopy. Part of their DNA content is cleaved into nucleosomal and oligonucleosomal fragments. Their insolubility, like that of the comified envelope, is evoked by a-(y-glutamyl)lysine and NL,1V8-bis(y-glutamyl)spermidine protein cross-linking bonds formed by transglutaminase.

We recently reported that activation of "tissue" transglutaminase (EC 2.3.2.13; tTG) in liver cells undergoing apoptosis determines extensive cross-linking of cellular proteins resulting in the formation of SDS-insoluble shells in the so-called "apoptotic bodies". In attempt to obtain further insight into the role played by tTG in apoptosis of liver cells, we investigated its expression in primary cultures of neonatal rat liver cells stimulated with epidermal growth factor (EGF). EGF-treatment of neonatal rat liver cells induces first hyperplasia of hepatocytes, followed by involution characterized by a high incidence of apoptosis. The proliferative phase of hepatocytes is paralleled by a 10-fold increase in tTG mRNA level, which is followed, during the phase of involution, by sequential increases in enzyme activity and levels of SDS-insoluble apoptotic bodies. tTG immunostaining at both the light- and electron-microscopic levels shows that the most intensive reaction is present in globular structures showing the typical morphological appearance of mature apoptotic bodies. In early apoptotic stages, tTG protein is localized in the perinuclear region of the cell. Intense immunostaining is also found in the apoptotic bodies present inside phagosomes within the cytoplasm of neighboring cells. This evidence confirms and extends our previous findings, indicating that tTG induction and activation specifically takes place in cells undergoing apoptosis, suggesting a key role for the enzyme in the apoptotic program.

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