Immunolocalization of keratin polypeptides in human epidermis using monoclonal antibodies

Abstract

Three monoclonal antibodies (AE1, AE2, and AE3) were prepared against human epidermal keratins and used to study keratin expression during normal epidermal differentiation.Immunofluorescence staining data suggested that the antibodies were specific for keratintype intermediate filaments.The reactivity of these antibodies to individual human epidermal keratin polypeptides (65-67, 58, 56, and 50 kdaltons) was determined by the immunoblot technique.AEI reacted with 56 and 50 kdalton keratins, AE2 with 65-67 and 56-kdalton keratins, and AE3 with 65-67 and 58 kdalton keratins.Thus all major epidermal keratins were recognized by at least one of the monoclonal antibodies.Moreover, common antigenic determinants were present in subsets of epidermal keratins.To correlate the expression of specific keratins with different stages of in vivo epidermal differentiation, the antibodies were used for immunohistochemical staining of frozen skin sections.AE1 reacted with epidermal basal cells, AE2 with cells above the basal layer, and AE3 with the entire epidermis.The observation that AEI and AE2 antibodies (which recognized a common 56 kdalton keratin) stained mutually exclusive parts of the epidermis suggested that certain keratin antigens must be masked in situ.This was shown to be the case by direct analysis of keratins extracted from serial, horizontal skin sections using the immunoblot technique.The results from these immunohistochemical and biochemical approaches suggested that: (a) the 65-to 67-kdalton keratins were present only in cells above the basal layer, (b) the 58kdalton keratin was detected throughout the entire epidermis including the basal layer, (c) the 56-kdalton keratin was absent in the basal layer and first appeared probably in the upper spinous layer, and (d) the 50-kdalton keratin was the only other major keratin detected in the basal layer and was normally eliminated during s.corneum formation.The 56 and 65-67kdalton keratins, which are characteristic of epidermal cells undergoing terminal differentiation, may be regarded as molecular markers for keratinization.Keratins are a family of water-insoluble proteins of 40,000 to 70,000 daltons (40 to 70 kdaltons [kd]).These proteins form tonofilaments (a class of intermediate fdament) in epidermis (2,4,6,9,13,19,20,21, 27,37,39, 41,46,49,54) as well as in almost all other epithelia (10--12, 30, 36, 45, 47, 48).The process of normal epidermal differentiation (keratinization) is characterized by a series of morphological and biochemical changes as cells progress from the germinative basal layer through the spinous and granular layers to the outer cornified layer (55).Analysis of keratins extracted from horizontal sections of the epidermis suggests that cells of inner 580 layers contain primarily small keratins, whereas cells of outer layers contain large keratins in addition to small ones (14, 40).Furthermore, antisera specific for the high molecular weight (65-67 kd) keratins demonstrate preferential binding to cells above the basal layer (44; cf.52, 53).These data suggest that keratin composition changes during epidermal differentiation and, more specifically, that the 65-to 67-kd keratins first appear in the spinous layer.Other keratins of the epidermis, however, have not.beenlocalized precisely and thus the functional significance of these keratins remains unclear.To facilitate the immunolocalization of specific keratins in