Establishing regulatory mechanisms of the human epidermal keratinocyte stem cell-containing proliferative subset in systems which model the complex in vivo multifactorial microenvironment is critical to understanding both healthy homeostasis and altered homeostasis of the epidermis in inflammatory disease. Our hypothesis is that a critical mechanism by which keratinocyte stem cells are overly recruited into cell cycle in lesional psoriasis may be due to the direct cooperative action of lymphokines released by skin-infiltrating T lymphocytes in lesional psoriatic skin. Anticipated results will provide fundamental information on how the immune system can regulate proliferative homeostasis of tissues, with direct relevance to epidermal stem cell biology and to psoriasis. The first major aim of the proposal is to model the in vivo T cell activation milieu that is capable of stimulating hyperproliferation of beta1 integrin +K1/K10- G-O stem cells. Sources of proliferation- inducing signals for psoriatic keratinocytes will be determined by testing cloned lesional psoriatic T cells in isolation, fresh T cells activated in the presence of lesional psoriatic antigen presenting cells, and freshly obtained lesional T cells selected for those undergoing in vivo activation. The focus of the second aim is to identify the stimulatory lymphokines themselves by physicochemical methods, neutralizing antibodies and recombinant lymphokine studies. In addition, cooperative effects of growth factors and extracellular matrix on lymphokine-stimulated keratinocyte stem cell growth will be dissected. The third major aim is to determine the basis by which the epidermal keratinocyte beta1 integrin+ subset of psoriatics responds preferentially to proliferation-promoting T cell lymphokines. This will involve determining whether T cell activity on the hyper-responsive psoriatic keratinocyte is localized primarily to a true stem cell (G-O-G-1 recruitment into cell cycle) or to a transitional cell in early G-1 or to a transient amplifying cell in G-1 (induction of progression to S of cells already committed to G-1).
This aim will also determine whether commitment to differentiation is accelerated or delayed during such stimulation. The methodology to accomplish these aims is as follows: The effect of activated intralesional T cell supernatants or their critical components upon keratinocytes will be investigated using a primary fresh ex vivo epidermal cell proliferation assay, in which increased cell cycle entry of specific keratinocyte subpopulations is determined by flow cytometric analysis. Distinction of recruitment of true stem cells (G-O) will be determined by the use of BrdU quenching of Hoechst DNA binding, in concert with the use of sequential G- 1 markers.
