Author: Ilse Sofia Daehn
Daehn, Ilse Sofia, 2007 Effect of growth factors on T-lymphocyte induced keratinocyte apoptosis, Flinders University, School of Medicine
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Atopic eczema is a T-lymphocyte mediated chronic inflammatory skin disorder. The interaction of CD4+ T-lymphocytes with epidermal keratinocytes results in dysregulated, chronic inflammation and altered barrier function. T-lymphocyte induced keratinocyte apoptosis has been proposed as a mechanism by which epidermal integrity is impaired in eczema. Apoptosis of keratinocytes is thought to result from Tlymphocyte associated Fas ligand (FasL) binding to the death receptor Fas on keratinocytes. The primary aim of this project was to characterize the induction of keratinocyte apoptosis by T-lymphocytes and address the hypothesis that insulin-like growth factor-I (IGF-1), transforming growth factor [beta]1 (TGF[beta]1) and a milk derived growth factor extract containing TGF[beta] and IGF-I (whey growth factor extract; WGFE) protect keratinocytes from T-lymphocyte mediated apoptosis. To address the aims of this project, an in vitro co-culture model was developed combining T-lymphocytes with keratinocytes. Co-cultures were initially established using human Jurkat T-lymphocytes and human HaCaT keratinocytes with more extensive characterisation undertaken using primary CD4+ T-lymphocytes together with HaCaTs or normal human epidermal keratinocytes (NHEK). Annexin V and propidium iodide staining was established as the primary method for measuring keratinocyte apoptosis with this validated using sodium butyrate a known inducer of apoptosis. Changes in nuclear fragmentation and cell morphology were also examined as a key feature of apoptosis. The involvement of the Fas pathway was investigated by assessing T-lymphocyte FasL expression, keratinocyte Fas expression and downstream caspase activation. Inflammatory cytokines IFN[gamma] and TNF[alpha] were also examined due to their ability to induce Fas expression. Studies performed with T-lymphocytes demonstrated that keratinocyte apoptosis was induced, with this due primarily to direct T-lymphocytes and keratinocytes interactions, rather than soluble mediators in the co-culture milieu. Activated T-lymphocytes were found to have high levels of FasL and to upregulate keratinocyte Fas expression. The increased keratinocyte Fas was associated with increased IFN[gamma] levels in the co-culture media and activation of the caspase cascade. A Fas blocking antibody prevented T-lymphocyte induced keratinocyte apoptosis demonstrating that this was a Fas dependent event. As the primary function of keratinocytes is to terminally differentiate, the differentiation status of the cells induced to undergo apoptosis was examined. It was demonstrated that T-lymphocytes decrease the intensity of ?6 integrin expression by the keratinocytes. This marker identifies undifferentiated basal cells as high expressors of [alpha]6, with cells in the early stages of differentiation pathway found to be low expressors of [alpha]6. Co-staining with Annexin V demonstrated that the apoptotic keratinocytes were low expressors of [alpha]6 and thus cells committed to the early stages of differentiation. This suggested that the T-lymphocytes initiated the onset of keratinocyte terminal differentiation with this linked to the cells being more susceptible to death induced by T-lymphocyte by activation of the Fas pathway. The ability of TGF[beta]1, IGF-I and WGFE to inhibit T-lymphocyte induced keratinocyte apoptosis was examined. A combination of recombinant TGF[beta] (10ng) & IGF-I (100ng) was able to significantly inhibit keratinocyte apoptosis. A similar result was obtained with WGFE, and although these growth factor treatments were able to reduce the elevated IFN[gamma] levels in the co-culture media, they did not reduce T-lymphocyte induced Fas upregulation. The TGF?1 and IGF-I combination as well as WGFE did however prevent the T-lymphocyte induced shift from [alpha]6 bright to dim expressing keratinocytes. As such, the growth factor combinations appeared to protect the keratinocytes from T-lymphocyte mediated apoptosis by preventing them from committing to terminal differentiation. The studies in this thesis have characterised the Fas associated mechanisms by which T-lymphocytes induce keratinocyte apoptosis and suggest specific growth factor combinations may have the potential to ameliorate the reduced barrier function associated with inflammatory skin conditions such as atopic eczema.
Keywords: keratinocyte,apoptosis,eczema,growth factors
Subject: Medicine thesis, Biotechnology thesis
Thesis type: Doctor of Philosophy
School: School of Medicine
Supervisor: Dr Timothy Rayner