Dynamics of basement membrane formation by keratinocyte-fibroblast interactions in organotypic skin culture.

The cutaneous basement membrane zone, composed of numerous macromolecules, plays a multifunctional role in tissue regeneration and maintenance. To elucidate the cellular origin and dynamics of basement membrane formation, de novo synthesis, deposition, and ultrastructural assembly of its components were analyzed in organotypic cultures of adult skin keratinocytes on collagen gels with or without collagen-embedded dermal cells. Collagen IV and laminin-1 deposition occurred only in the presence of mesenchymal cells: patchy at day 4 and continuous after 1 week. Chain-specific mRNA expression started at day 2 in both keratinocytes and fibroblasts. It steadily increased up to day 10, however, with a reciprocal induction pattern, mRNA abundance shifting from keratinocytes to fibroblasts. On the other hand, laminin-5 staining was first observed at day 4, but in keratinocyte both mono- and cocultures. This was followed by nidogen, which was detected in cocultures but also in dermal monocultures. Laminin-5 protein persisted throughout day 21, whereas nidogen steadily increased in intensity. Expression kinetics revealed high levels of laminin-5 transcripts early and in keratinocytes only, whereas nidogen was expressed later and predominantly in fibroblasts. Although basement membrane protein deposition was continuous at day 14, the ultrastructural organization was still fragmentary, eventually normalizing at 3 weeks. These data demonstrate a dynamic interaction and cooperation of epithelial and mesenchymal skin cells in basement membrane formation. This interaction is supposedly mediated via diffusible factors. Our findings further extend the scope of epithelial-mesenchymal interactions stressing that both cell compartments are essential to constitute a tissue-specific extracellular matrix structure.

Organotypic and epidermal-dermal co-cultures of normal human keratinocytes and dermal cells: Regulation of transforming growth factor alpha, beta1 and beta2 mRNA levels.

Epidermal-dermal cell-cell interactions are recognized to influence keratinocyte proliferation in vivo as well as in vitro. To study the underlying molecular mechanisms of epithelial-mesenchymal interactions epidermal-dermal cell co-cultures and organotypic cultures were used. Steady-state mRNA levels are described for transforming growth factors (TGF) alpha, beta1 and beta2, factors known to stimulate or inhibit the epidermal proliferation rate. In epidermal-dermal monolayer co-cultures TGF alpha hybridization signals were absent. TGF beta1 mRNA was expressed in all cell types (keratinocytes, fibroblasts and microvascular endothelial cells), yet not regulated. In contrast, TGF beta2 mRNA was significantly induced in mesenchymal cells when they were co-cultured with keratinocytes. In organotypic cultures epidermal proliferation is dependent on the presence of fibroblasts within the gel. TGF beta1 was expressed at low levels in all cell types whereas TGF beta2 transcripts were not detectable at all. TGF alpha mRNA was present in keratinocytes at high levels, independent of epidermal cell proliferation or added epidermal growth factor. These results indicate complex regulative mechanisms for TGF alpha, beta1 and beta2 at the mRNA level. However, post-transcriptional steps are involved in the activation of TGF beta1 and 2 and also have to be considered.

Mutual induction of growth factor gene expression by epidermal-dermal cell interaction.

Epithelial-mesenchymal interactions control epidermal growth and differentiation, but little is known about the mechanisms of this interaction. We have examined the effects of human dermal microvascular endothelial cells (DMEC) and fibroblasts on keratinocytes in conventional (feeder layer) and organotypic cocultures (lifted collagen gels) and demonstrated the induction of paracrine growth factor gene expression. Clonal keratinocyte growth was similarly stimulated in cocultures with irradiated DMEC and fibroblasts as feeder cells. This effect is most probably caused by induction of growth factor expression in cocultured dermal cells. Keratinocytes stimulated mRNA levels for KGF and IL-6 in both mesenchymal cell types and GM-CSF in fibroblasts. The feeder effect could not be replaced by conditioned media or addition of isolated growth factors. In organotypic cocultures with keratinocytes growing on collagen gels (repopulated with dermal cells), a virtually normal epidermis was formed within 7 to 10 d. Keratinocyte proliferation was drastically stimulated by dermal cells (histone 3 mRNA expression and BrdU labeling) which continued to proliferate as well in the gel. Expression of all typical differentiation markers was provoked in the reconstituted epithelium, though with different localization as compared to normal epidermis. Keratins K1 and K10 appeared coexpressed but delayed, reflecting conditions in epidermal hyperplasia. Keratin localization and proliferation were normalized under in vivo conditions, i.e., in surface transplants on nude mice. From these data it is concluded that epidermal homeostasis is in part controlled by complex reciprocally induced paracrine acting factors in concert with cell-cell interactions and extracellular matrix influences.

Localization of neurotensin-immunoreactivity in the spinal cord and peripheral nervous system of the guinea pig.

The mapping of the regional distribution of neurotensin-immunoreactive (NT-IR) fibers and neurons is presented for cervical, thoracic, lumbal and sacral segments of the guinea pig spinal cord. The occurrence of NT-IR fibers in para- and prevertebral ganglia and intramural plexus of viscera is described. It is suggested that NT-IR fibers originating from neurons in the substantia intermedia of the spinal cord are connected with peripheral ganglia.