An Immunochemical Analysis of Alcoholic Hyaline and its Relationship to Keratin Intermediate Filaments

Alcoholic Hyaline (AH) and keratin intermediate filaments possess ultrastructural, biochemical and immunochemical similarities. This study compares human AH, human stratum corneum keratin (HSCK) and normal human liver cytokeratin (HCK) by quantitative immunochemical means, using polyclonal as well as monoclonal antibodies. Enzyme-linked immunosorbent assays (ELISAs) were employed, as well as immunoblotting and immunocytochemical techniques. HCK was isolated using Triton X-100 and high-salt” buffers and exhibited seven major polypeptide bands on SDS-PAGE (41-58 kd). Antisera were made toward HCK and the two major HCK bands. Other immunochemicals included antisera toward AH, the three major AH bands, and HSCK, as well as 3 monoclonal antibodies which were produced toward AH. Immunoblot analysis of AN demonstrated the presence of HCK-like determinants in each of the major bands, but no HSCK crossreactivity was noted. Quantitative ELISA demonstrated crossreactive components to be particularly abundant in the major AH bands. Studies using antisera toward HCK and intact AH demonstrated a significant, though moderate quantity of shared immunoreactive material, while the major and most consistent AH bands appeared to be composed principally of HCK-like constituents. Comparison of AH and HCK with epidermal keratin (HSCK), on the other hand, demonstrated common immunoreactive components to be but minor constituents, indicating a greater degree of tissue specificity than previously recognized. This specificity was underscored by monoclonal antibody IIB3, which reacted strongly with liver cytokeratins (AH and HCK) but did not bind HSCK. Two monoclonal antibodies with a more restricted immunocrossreactivity were also produced. Monoclonal antibody IC1 was directed toward a unique AH determinant not normally present in the liver, the nature of which is not completely understood at this time, and antibody IIID4 reacted with a determinant in the outer layers of the epidermis in a pattern previously associated with keratinization-specific determinants. Such determinants may be involved in AH pathogenesis. In summary, while intact AH appears to possess non-cytokeratin, and possibly unique constituents in addition to HCK-like components, the major and most consistent AH polypeptides appear to be composed chiefly of immunoreactive-HCK These results would support a mechanism of AH formation which involves the condensation and collapse of normal hepatocellular intermediate filaments as one of the steps