| Elucidation of the cellular signaling pathways that are attributed to the progression of lameness must first begin with the identification of molecules that are involved in the normal homeostasis of hoof horn tissue. Epithelial-mesenchymal cell interactions direct growth and differentiation of hoof horn keratinocytes, but little is recognized about the means by which these interactions contributes to hoof horn disease. A greater understanding of factors orchestrating keratinocyte growth, differentiation and maturation in hoof horn production are pivotal to understanding hoof horn properties in health and disease. The specific objectives were to establish an in vitro culture system of bovine coronette keratinocytes, estimate stem cell numbers in keratinocyte populations, and determine epithelial-mesenchymal cell interactions involving keratinocyte growth and corresponding cytokine and growth factor expression patterns.To establish an in vitro culture system that mimicked in vivo epithelial-mesenchymal cell interactions, we first had to characterize the keratinocyte compartment of the bovine claw. Abaxial coronette from the lateral, hindlimb claw was incubated in 1.5% trypsin (2h, 37C), minced into fine pieces and propagated 14-21d in Dulbecco's Media (10% FBS). Fibroblasts were collected using (0.7muM EDTA, 2min, 25°C) and residual keratinocytes placed in keratinocyte growth medium containing epidermal growth factor (EGF) and bovine pituitary extract (BPE) for 7d. To assess stem cell numbers, keratinocytes were seeded onto bovine dermal fibroblast monolayers at 5 x 103, 7.5 x 103, or 1.0 x 104 viable keratinocytes/cm 2, incubated 10d in DMEM (10% FBS). The expression kinetics of different cytokines, growth factors and cognate receptors were determined at the mRNA level in parallel with functional colony formation assays. Keratinocytes formed 154 +/- 84, 241 +/- 97, 255 +/- 66 colonies at plating densities of 5 x 103, 7.5 x 103, or 1.0 x 104 viable keratinocytes/cm2, respectively. Mesenchymal cell requirements in keratinocyte growth were assessed by plating 5 x 103 and 7.5 x 103 viable keratinocytes directly onto plastic culture dishes lacking dermal fibroblasts or onto confluent monolayers of fibroblasts in DMEM (0.1% FBS). Keratinocytes co-cultured in the presence of dermal fibroblasts formed 144 +/- 31.6, and 183 +/- 53.0 colonies respectively, whereas keratinocytes cultured in the absence of fibroblasts formed zero colonies. Results show, 0.3-2.1% of coronette keratinocytes possessed stem cell qualities that were only expressed in the presence of dermal fibroblasts. The expression profiles established during the progression of colony formation represented a patterns of growth factors (KGF and GM-CSF) and cytokines (IL-1, TNF-alpha, and TGF-beta) expression which was time-dependent, and involved representative receptor (IL-1RTI) and accessory molecule (IL-1RTII and IL-1RA) contributions. These findings demonstrate, (i) the ability of bovine coronette keratinocytes to form colonies in vitro, (ii) a strict dependence of fibroblast for keratinocyte colony formation, and (iii) the potential for cytokines, growth factors and cognate receptors in mediating bovine keratinocyte colony formation in vitro.Finally, we examined the effects bovine mesenchymal-epithelial cell interactions and serum plays on cytokine and growth factor mRNA expression, and demonstrated a double paracrine loop mechanism. In cocultures, established on fibroblast monolayers maintained in 10% FBS, cytokine, growth factor and cognate receptors mRNA expression was serum concentration (1 vs. 10%) and time-dependent. IL-1beta, IL1-receptor type I, KGFR, and KGF were induced by elevated serum (10%) concentration, where GMCSF and TNF-alpha mRNA expression were repressed at both 1% and 10% serum supplementation. Additionally, to mimic the state of resting fibroblast in the dermis, fibroblast were serum starved prior to coculture establishment, and cocultures were maintained in 1% FBS to study mesenchymal-epithelial interactions independent of elevated serum. Interleukin-1-alpha and -1beta, IL1RTI, GM-CSF, and TGF-beta were strongly enhanced, whereas IL1RA, KGFR, and TNF-alpha were down-regulated over time within this keratinocyte-fibroblast coculture model. We postulated that IL-1 was involved in the mutual cross-talk between epidermal keratinocytes and fibroblasts, we substantiated this using IL-1 blocking experiments. Both IL-1-alpha and -1beta neutralizing antibodies and IL-1 receptor antagonist significantly reduced GM-CSF expression in cocultures. Thus IL-1 and GM-CSF act reciprocally as paracrine mediators within the keratinocyte-fibroblast coculture model. (Abstract shortened by UMI.)... |
