Immunolocalization of keratin polypeptides in human epidermis using monoclonal antibodies

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Abstract
Three monoclonal antibodies (AE1, AE2 and AE3) prepared against human epidermal keratins were used to study keratin expression during normal epidermal differentiation. Immunofluorescence staining data suggested that the antibodies were specific for keratin-type 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. AE1 reacted with 56 and 50 kdalton keratins, AE2 and 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 AE1 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; as was shown by direct analysis of keratins extracted from serial, horizontal skin sections using the immunoblot technique. Results suggested that the 65- to 67-kdalton keratins were present only in cells above the basal layer; the 58-kdalton keratin was detected throughout the entire epidermis including the basal layer; the 56-kdalton keratin was absent in the basal layer and first appeared probably in the upper spinous layer; and the 50-kdalton keratin, the only other major keratin detected in the basal layer was normally eliminated during stratum corneum formation. The 56 and 65-67-kdalton keratins characteristic of epidermal cells undergoing terminal differentiation, may be regarded as molecular markers for keratinization.