title: Modulation of Th1 and Th2 type immune responses
Creators: name: Schulz, Kerstin Ingrid
Publisher: name: University of Glasgow
Year: 2001
Subjects: subject: Immunology.

description: The Th1/Th2 cell balance is crucial for the outcome of many infections and autoimmune diseases. The identification of novel Th1 and Th2 cell specific markers can expand our knowledge about Th1 and Th2 cell function, and these markers can be used to manipulate Th1/Th2 cell responses. Recently, ST2L (an orphan receptor of the IL-1R family) was identified to be selectively expressed on Th2, but not on Th1 cells. Factors that determine this differential expression pattern are still unknown. Antibodies against ST2L as well as a ST2-Fc fusion protein, mimicking the soluble form (ST2), were able to decrease Th2 effector functions. ST2 is thought to compete with ST2L for the ligand and therefore prevent ST2L signalling. These studies demonstrated that ST2L functions are closely linked to Th2 responses in vitro and in vivo. However, with the ligand of ST2L still unidentified, the function of ST2L and especially of ST2 are controversial. The aim of my thesis was to expand the knowledge about the role and regulation of ST2 and ST2L in murine T helper cells. In my initial experiments (Chapter 3) I established a method to express and purify ST2 from baculovirus-infected insect cells and from stably transfected mammalian CHO cells. The protein was tested for its biological activity on LPS-stimulated macrophages, where it was able to decrease the production of inflammatory cytokines (IL-6 and IL-12). Next, I examined the effects of ST2 on CD4(+) T cell activation (Chapter 4). ST2 could bind to naTve CD4(+) T cells and suppressed Th1 and Th2 type cytokine production. It worked directly on Th cells without the need for APC. CD4(+) T cells, whose TCR had been engaged before, did not bind ST2 and therefore did not show any decrease in cytokine expression. I then assessed the regulatory effect of Th1 and Th2 cytokines on the gene expression of ST2 and ST2L at protein, mRNA and promoter level to clarify whether these cytokines are responsible for the differential expression of ST2 and ST2L (Chapter 5). IL-4, a major cytokine for Th2 development and proliferation, increased ST2L protein expression, while IFN-gamma, the main Th1 cytokine, decreased ST2L protein expression. Similar results were obtained at the mRNA level, where IL-4 upregulated and IFN-gamma downregulated the ST2L mRNA expression. Interestingly, IL-4 induced an early increase in ST2 mRNA expression and a delayed increase in ST2L mRNA, suggesting differential regulation of the two splice variants. The upstream promoter showed increased activity when treated with IL-4, but decreased activity when IFN-gamma was added. Hence, IL-4 was able to upregulate ST2L and drive Th2 cell development simultaneously. IFN-gamma had opposite effects to IL-4. In order to stably transfect Th cells with a ST2L antisense construct, toxicity studies for G418, the selection marker, were carried out on Th1 and Th2 cells (Chapter 6). G418 and its structural analogue gentamicin were found to modulate the Th1/Th2 balance in vitro by selectively suppressing Th2 cells to a greater extent than Th1 cells. To investigate the ability of gentamicin to regulate the Th1/Th2 cell balance in vivo, I treated Leishmania major-infected BALB/c mice with high doses of gentamicin for 12 days. Complete suppression of the disease was achieved, providing gentamicin treatment was started early after infection. Analysis of antigen-specific proliferation, cytokine release and antibody production revealed a complete suppression of antigen-responding Th lymphocyte activity. However, a shorter period of gentamicin treatment (7 days), started immediately after infection, selectively suppressed Th2 but not Th1 cytokine expression, confirming the ability of gentamicin to modulate Th1/Th2 type responses. Gentamicin also caused a partial suppression of macrophage viability. The appearance and activity of parasites changed upon short term gentamicin treatment, but this did not affect their infectivity in vivo. In vitro experiments suggested an increased uptake of the aminoglycoside by macrophages in the presence of parasites, promoting increased clearance of the organism. In this thesis I undertook a series of defined studies which together further advance our knowledge on the regulation of Th1 and Th2 cells. This is based on the function of ST2L/ST2 and gentamicin, both of which have significant effects on the balance between Th1 and Th2 cells. These results can contribute to our ability to modulate some of the most important infectious and inflammatory diseases.