Modification and expulsion of keratins by human epidermal keratinocytes upon hapten exposure in vitro.

Allergic contact dermatitis is the most prevalent form of human immunotoxicity. It is caused by reactive low molecular weight chemicals, that is, haptens, coming in contact with the skin where hapten-peptide complexes are formed, activating the immune system. By using sensitizing fluorescent thiol-reactive haptens, that is, bromobimanes, we show how keratinocytes respond to hapten exposure in vitro and reveal, for the first time in a living system, an exact site of haptenation. Rapid internalization and reaction of haptens with keratin filaments were visualized. Subsequently, keratinocytes respond in vitro to hapten exposure by release of membrane blebs, which contain haptenated keratins 5 and 14. Particularly, cysteine 54 of K5 was found to be a specific target. A mechanism is proposed where neoepitopes, otherwise hidden from the immune system, are released after hapten exposure via keratinocyte blebbing. The observed expulsion of modified keratins by keratinocytes in vitro might play a role during hapten sensitization in vivo and should be subject to further investigations

Modification and Expulsion of Keratins by Human Epidermal Keratinocytes upon Hapten Exposure in Vitro

Allergic contact dermatitis is the most prevalent form of human immunotoxicity. It is caused by reactive low molecular weight chemicals, that is, haptens, coming in contact with the skin where hapten−peptide complexes are formed, activating the immune system. By using sensitizing fluorescent thiol-reactive haptens, that is, bromobimanes, we show how keratinocytes respond to hapten exposure in vitro and reveal, for the first time in a living system, an exact site of haptenation. Rapid internalization and reaction of haptens with keratin filaments were visualized. Subsequently, keratinocytes respond in vitro to hapten exposure by release of membrane blebs, which contain haptenated keratins 5 and 14. Particularly, cysteine 54 of K5 was found to be a specific target. A mechanism is proposed where neoepitopes, otherwise hidden from the immune system, are released after hapten exposure via keratinocyte blebbing. The observed expulsion of modified keratins by keratinocytes in vitro might play a role during hapten sensitization in vivo and should be subject to further investigations

Contact Sensitizers Induce Keratinocytes to Release Epitopes - Tools for In Vitro Tests and Implications for Autoimmunity

Allergic contact dermatitis (ACD) is the most prevalent form of human immunotoxicity. It is caused by skin exposure to haptens, i.e., protein-reactive, low-molecular-weight chemical compounds, which form hapten–protein complexes (HPCs) in the skin, triggering the immune system. These immunogenic HPCs are elusive. In this study a series of thiol-reactive caged fluorescent haptens, i.e., bromobimanes, were deployed in combination with two-photon fluorescence microscopy, immunohistochemistry, and proteomics to identify possible hapten targets in proteins in human skin. Key targets found were the basal keratinocytes and the keratins K5 and K14. Particularly, cysteine 54 of K5 was found to be haptenated by the bromobimanes. In addition, elevated levels of anti-keratin antibodies were found in the sera of mice exposed to bromobimanes in vivo. The results indicate a general mechanism in which thiol-reactive haptens generate cryptic epitopes normally concealed from the immune system. In addition, keratinocytes and keratin seem to have an important role in the mechanism behind ACD, which is a subject for further investigations