Harlequin Ichthyosis Keratinocytes in Lifted Culture Differentiate Poorly by Morphologic and Biochemical Criteria

Philip Fleckman, Barbara Hager, Beverly A. Dale
1997 Journal of Investigative Dermatology  
Harlequin ichthyosis (HI) is a severe congenital ichthyosis in which massively thickened stratum corneum with abnormal barrier function often results in death of affected newborns. Survivors evolve into a severe nonbullous ichthyosiform erythroderma. Previously we have ascertained three biochemical phenotypes of HI, based on abnormal profilaggrin and K6 and K16 expression in epidermis. Submerged cultures of HI keratinocytes differentiated abnormally, but the three phenotypes were
more » ... le in vitro. We hypothesized that differentiation in submerged culture was insufficient to reflect iu vivo biochemical abnormalities or that dermal components might be necessary for expression. To test these hypotheses HI keratinocytes and fibroblasts (n = 3) were grown on collagen gels at the air-medium interface in a cross-over design with normal keratino-H arlequin ichthyosis (HI) is a severe disorder in which infants are born encased in a shroud of thickened fissured epidermis that li1. 11its n1ovement and breathing and produces ectropion, eclabium, and ear dysmorphology. Newborns usu ally die at birth from barrie r dysfunction and breathing difficulties. The us e of oral retinoids has facilitated survival. Long-term survivors evolve into a phenotype of severe nonbullous congenital ichthyosiform erythroderma (Lawlor, 1988; Roberts, 1989). We have studied epidermis and keratinocytes from a number of affected individuals and describe d three biochemical phenotypes, based on the expression ofprofilaggrin and the K6/K16 keratin pair (Dale et nl, 1990). The bioc hemical phenotype ofinfants born within the same farnily remains the same. Abnorn1al lamellar granules are observed in all three phenotypes (Dale et al, 1990; Mili1er et nl, 1992; Akiyama el nl, 1994). Submerged cultures of HI kerati.nocytes are morphologically abnormal but do not exhibit the bioch e mical changes observed in extracts of epidermis from which the keratinocytes are cultured (Dale et nl, 1990). We hypothesized that either differentiation of the submerged HI cultures was not sufficient to express the abnormalities observed in vi11o or that dermal factors might be n ecessa ry for exp ression of the HI phenotype. In an attempt to address both possibilities, HI keratinocytes were grown u1 lifte d c ulture, where Manuscript Abbreviation : Hl, Harl equin ichthyosis. cytes and fibroblasts. Epithelia derived from lifted cultures were studied by light microscopy and immunocytochemistry and extracted for western blot analysis. In contrast to our prediction, lifted cultures of HI keratinocytes formed a poorly differentiated epithelium, and normal keratinocytes formed an epidermal-like tissue with expression of K1 and expression and processing of profilaggrin to filaggrin. In addition, the presence of HI fibroblasts consistently altered differentiation of both HI and normal keratinocytes, resulting in less complete morphologic differentiation. The findings suggest that both epithelial and mesenchymal elements of the skin from HI are affected but that the primary abnormality lies in the keratinocytes. Key words: epidermal dijfeJ'eutiationlpro-.filaggriu/keratius. J Invest Dermatol 109:36-38, 1997 more complete morphologic and bioch e mical diffe re ntiation of normal epidermal cells is observed (Asselineau el nl, 1990 ). An additional advantage of the lifted culture system used was the abi li ty to seed the collagen gel with co ntrol or HI fibroblasts. Thus, a cross-over study was undertaken with control and HI keratinocytes growing on gels seeded with control or HI fibroblasts . ln this study, we show that keratinocytes from HI become less differe ntiated when cultured at the air-medium inte rfa ce, under conditions where in. vitro differentiation normally improves. MATERIALS AND METHODS Culture Keratinocytes and fibrob lasts were cultured from biopsy specimens as described (Dale et al, 1990) . Lifted cu ltures were grown by the me thod of Asselineau and Bell (Asselineau and Prunicras, 1984), with either H arleq uin or nonnal newborn foreskin keratinocytcs seeded on a collagen gel implanted with Harlequin or normal adult or foreskin fibroblasts. Lifted cultures were maintained in Dulbecco's modified Eagle's medjum with 20')1 0 fetal bovine serw11 , 0.4 JLg hydrocortisone per ml, 10 ng epidermal growth factor per ml , and 10 -10 M cholera toxin. Keratinocytes were studi ed through the third passage. ConBu ent submerged cultures and cultures that had been grown at the air-medium interface for 21 d were studied. Immunocytochemistry Lifted cultures were fixed in methyl Carney's, embedded in paraftin, and sectioned for hematoxylin and eosin staining and immunocytochemistry as previously described (Kautsky et a/ , 1995) . Primary antibody was followed by secondary biotinylated anti-mouse lgG, amplification with immunoperoxidase (Vectastain ABC kit, Vector Laboratories, Burlingame, CA), and visualization with diaminobcn zidinc as chromagen. Monoclonal antibodies AE3, SClO (generous gifts of T.-T. Sun, Department of Dermatology, New York University Medi cal School), and AKI-11 were used to identifY neutral-basic keratins, Kl, and profilaggrin /fi lagg.:in , respectively.
doi:10.1111/1523-1747.ep12276450 pmid:9204952 fatcat:g3xlr3dwb5c3pdzdt4gcbev464