Tumor necrosis factor alpha and its receptor: Cytokine regulation of expression in human glioma cell lines

The studies presented in this dissertation investigated one aspect of a central nervous system (CNS) derived immune response, namely, expression and modulation of tumor necrosis factor-$\alpha$ (TNF-$\alpha$) receptor expression and TNF-$\alpha$ gene expression by the cytokines interferon-$\gamma$ (IFN-$\gamma$), interleukin-1$\beta$ (IL-1$\beta$), and TNF-$\alpha$ itself. Two different human malignant astroglioma cell lines, D54-MG and CH235-MG, were utilized.

D54-MG cells were used to investigate the growth regulating effects of TNF-$\alpha$ and the expression of surface receptors for TNF-$\alpha$. Additionally, synthesis and secretion of TNF-$\alpha$ were investigated. D54-MG cells responded mitogenically after incubation with TNF-$\alpha$ for 48 h. Radio-iodinated TNF-$\alpha$ was used to quantify TNF-$\alpha$ receptors on these cells. Scatchard analysis of binding data revealed curvilinear plots consistent with two distinct receptor sites for TNF-$\alpha$ on D54-MG cells. D54-MG cells did not constitutively secrete TNF-$\alpha$; however, after stimulation, TNF-$\alpha$ mRNA and protein were expressed.

The effects of IL-1$\beta$, a CNS derived cytokine, on TNF-$\alpha$ protein production in CH235-MG cells constituted the following studies. Nuclear run off studies demonstrated that IL-1$\beta$ induced TNF-$\alpha$ protein production through transcriptional activation of the TNF-$\alpha$ gene. Treatment of CH235-MG cells with cycloheximide (CHX), a protein synthesis inhibitor, and actinomycin-D, an RNA polymerase inhibitor, revealed that TNF-$\alpha$ expression is regulated at transcriptional and posttranscriptional levels.

Last, second messenger pathway(s) capable of mediating IL-1$\beta$ induced TNF-$\alpha$ gene expression in CH235-MG cells were determined. Studies utilizing activators of protein kinase C (PKC) or PKC specific inhibitors indicated that IL-1$\beta$ mediated its effects through the PKC pathway. Moreover, PKC depletion studies, followed by IL-1$\beta$ treatment, resulted in the inability of IL-1$\beta$ to induce TNF-$\alpha$, confirming that PKC activity is required in this system.

The studies in this dissertation demonstrate that astroglioma cells express TNF-$\alpha$ receptors and express TNF-$\alpha$ protein in response to IL-1$\beta$ and that PKC activation is required for IL-1$\beta$ induced transcriptional activation of the TNF-$\alpha$ gene.