K

K., YM-264 and A. the skin and oral mucosa, which serve as a primary barrier between the external environment and the internal tissues. They also provide a barrier to microorganisms, toxins, and various antigens. is among the most important human pathogens; this organism causes various superficial and systemic infections and is often implicated in oral mucositis, including angular cheilitis (24, 33) and denture-induced stomatitis (47). It is also a leading cause of bacterial keratitis (41) and has been implicated Rabbit Polyclonal to Keratin 15 as a causative or exacerbating agent in a broad range of skin diseases, including atopic dermatitis, carbuncles, cellulitis, furuncles, follicles, Kawasaki syndrome, impetigo, psoriasis, and scalded skin YM-264 syndrome (6, 10-12, 29, 34, 43, 50). Additionally, is usually a major cause of wound infection and is thought to delay wound healing (3). is normally an extracellular pathogen; however, it can be internalized by a variety of nonphagocytic host cells in a fibronectin-binding protein (FnBP)-dependent manner. The host cells that have been studied to date include human umbilical vein endothelial cells (25), the human keratinocyte cell line HaCaT (28), corneal epithelial cells (20), osteoblasts (1), and epithelial 293 cells (a human embryonic kidney cell line) (42). In the previous studies the workers examined the conversation of host cells with in vitro, but internalization of by mammary gland epithelial cells has also been exhibited in vivo (8). The uptake of by host cells is usually a receptor-mediated process that has been hypothesized to involve host cell integrins and microbial surface components recognizing adhesive matrix molecules, MSCRAMMs (see reference 35 for a review of MSCRAMMs). Indeed, the internalization of by endothelial cells (25), osteoblasts (1; Nair, unpublished data), and 293 cells (41) has been shown to require the host cell integrin 51 and the fibronectin-binding MSCRAMM FnBPA and/or FnBPB. However, while there is little dispute that this FnBPs are involved in bacterial uptake, studies with a variety of epithelial cells have provided conflicting evidence for an essential role for the YM-264 FnBPs in the internalization process (8, 14, 19, 21). The differences may be due to the various sources of the epithelial cells and their nature (for example, immortalized cells versus normal primary cells). The purpose of this study was to determine the mechanisms of internalization of by primary human keratinocytes and keratinocyte cell lines derived from oral mucosa and skin. We exhibited in this study that internalization of by human keratinocyte cell YM-264 lines, like internalization by other cell types, requires bacterial FnBPs and is mediated by the major fibronectin-binding integrin 51. A second fibronectin-binding integrin found on keratinocytes, v6, does not mediate internalization of by human primary keratinocytes both FnBP-dependent and -impartial pathways are used. clumping factor B (ClfB) has recently been shown to be a major adhesin involved in binding of the bacteria to epithelial cells (32). Using an isogenic mutant defective in ClfB, we found that this molecule is not involved in the internalization of by primary keratinocytes. MATERIALS AND METHODS Chemical and reagents. All chemicals and reagents were obtained from Sigma-Aldrich (Poole, United Kingdom) unless otherwise indicated. Culture media and phosphate-buffered saline without Ca2+ and Mg2+ (PBS) were obtained from Gibco, Invitrogen Ltd. (Paisley, United Kingdom). Function-blocking monoclonal antibody against 51 integrin (JBS5) was obtained from Chemicon Intl. Ltd. (Chandlers YM-264 Ford, Hampshire, United Kingdom). Monoclonal antibody for cytokeratin 10 (DE-K10) was obtained from Dako UK Ltd. (Ely, United Kingdom). A function-blocking monoclonal antibody against av integrin was prepared in our laboratories from hybridoma cells (ATCC HB8449) obtained from the American Type Culture Collection (Rockville, Md.). Human cells and.