Organotypic cocultures as skin equivalents: A complex and sophisticated in vitro system

To assess the role of genes required for skin organogenesis, tissue regeneration and homeostasis, we have established in vitro skin equivalents composed of primary cells or cell lines, respectively. In these organotypic cocultures keratinocytes generate a normal epidermis irrespective of the species and tissue origin of fibroblasts. The combination of cells derived from mouse and human tissues facilitates the identification of the origin of compounds involved in epidermal tissue reconstitution and thus the precise analysis of growth regulatory mechanisms

Functional Characterization of the Epidermal Cholinergic System In Vitro

The aim of this study was to analyze the influence of cholinergic and anticholinergic drugs on epidermal physiology using organotypic cocultures (OTCs). Blocking of all acetylcholine receptors (AChRs) by combined treatment with mecamylamine and atropine or treatment with strychnine (blocking α9nAChR) for 7–14 days resulted in a complete inhibition of epidermal differentiation and proliferation. Blockage of nicotinic (n)AChR with mecamylamine led to a less pronounced delay in epidermal differentiation and proliferation than blockage of muscarinic (m)AChR with atropine, evidenced by reduced epithelial thickness and expression of terminal differentiation markers like cytokeratin 2e or filaggrin. In OTCs treated with atropine, mecamylamine, or strychnine, we could demonstrate intracellular lipid accumulation in the lower epidermal layers, indicating a severely disturbed epidermal barrier. In addition, we observed prominent acantholysis in the basal and lower suprabasal layers in mecamylamine-, atropine-, and strychnine-treated cultures, accompanied by a decreased expression of cell adhesion proteins. This globally reduced cell adhesion led to cell death via intrinsic activation of apoptosis. In contrast, stimulation of nAChR and mAChR with cholinergic drugs resulted in a significantly thickened epithelium, accompanied by an improved epithelial maturation. In summary, we show that epidermal AChR are crucially involved in the regulation of epidermal homeostasis

Interleukin-1-Induced Growth Factor Expression in Postmitotic and Resting Fibroblasts.

Tissue homeostasis in skin is regulated by epithelial-mesenchymal interactions, mostly operating via diffusible factors. To study the underlying regulatory mechanisms, in vitro systems have been established to mimic the in vivo situation in skin. In co-cultures, keratinocytes grow either adjacent to irradiated fibroblasts on plastic or on top of collagen gels containing fibroblasts, thus forming 3-dimensional organotypic structures. Keratinocyte growth is supported in part by fibroblast-produced factors induced by keratinocyte mediators such as interleukin-1 (IL-1). To better understand this cellular interaction and its modulation by fibroblast proliferation and extracellular matrix (ECM), we examined the effect of IL-1 on growth factor expression in proliferating and growth-arrested x-irradiated human dermal fibroblasts on plastic and in resting cells embedded in collagen gels. By semiquantitative reverse transcriptase PCR, we demonstrated that IL-1α and IL-1β stimulated the expression of KGF, HGF, IL-1&alpaha;, IL-1β, IL-1RI, and IL-8 in fibroblasts regardless of their physiologic condition, whereas that of TGF-β remained unaffected. The constitutive mRNA levels were usually lower in irradiated postmitotic and ECM-embedded cells than in proliferating fibroblasts. Cells responded to stimulation with IL-1 under all three culture conditions, although to different degrees depending on the growth factor. As demonstrated for HGF, IL-8, and IL-1β, the IL-1α-induced mRNA expression was followed by production and secretion of protein in irradiated fibroblasts. Thus, our findings show that resting and growth-inhibited fibroblasts, reflecting more closely the situation in dermis, exhibit lower constitutive growth factor expression levels but characteristically respond to IL-1 stimulation

Keratinocyte Growth Regulation in Defined Organotypic Cultures Through IL-1-Induced Keratinocyte Growth Factor Expression in Resting Fibroblasts

Balanced keratinocyte proliferation and differentiation resulting in regular tissue organization strictly depend on dermal support. Organotypic cultures represent biologically relevant in vitro models to study the molecular mechanism of the underlying dermal–epidermal interactions. To mimic the state of resting fibroblasts in the dermis, postmitotic (irradiated) fibroblasts were incorporated in the collagen matrix, where they typically support epidermal proliferation and tissue organization. In coculture with keratinocytes, fibroblasts exhibit an enhanced expression of keratinocyte growth factor and the interleukin-1 receptor (type I), which further increase with culture time. In cocultured keratinocytes, keratinocyte growth factor receptor as well as RNA expression and protein release of interleukin-1α and interleukin-1β are upregulated. We hypothesized that the modulated cytokine expression represents a basic mechanism for keratinocyte growth regulation. The functional significance of this double paracrine pathway, i.e., induction of keratinocyte growth factor expression in fibroblasts by keratinocytes via release of interleukin-1, was confirmed by interfering with both signaling elements: (i) interleukin-1-neutralizing antibodies and interleukin-1 receptor antagonist significantly inhibited keratinocyte growth factor release, keratinocyte proliferation, and tissue formation comparable to the effect produced by keratinocyte-growth-factor-blocking antibodies; (ii) addition of keratinocyte growth factor to cocultures with inactivated interleukin-1 pathway completely reverted growth inhibition; (iii) in organotypic cocultures with subthreshold fibroblast numbers both interleukin-1 and keratinocyte growth factor restored the impaired epidermal morphogenesis. Thus, epidermal tissue regeneration in organotypic cocultures is mainly regulated by keratinocyte-derived interleukin-1 signaling, which induces keratinocyte growth factor expression in cocultured fibroblasts. This demonstrates a novel role for interleukin-1 in skin homeostasis substantiating data from wound healing studies in vivo