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Vezava antigena na imunoglobulinski receptor BCR izzove aktivacijo in proliferacijo pri zrelih limfocitih B ter apoptozo pri nezrelih limfocitih B. Stimulacija BCR aktivira signalne poti v celici, ki vodijo do transkripcijskega reprogramiranja. Poveča se tudi izraţanje gena Ptger4, ki kodira prostaglandinski receptor EP4, na katerega se veţe naravni ligand prostaglandin E2. Prostaglandini so ţe dlje časa znani kot modulatorji imunskega odziva. Vezava prostaglandina E2 na receptor EP4 povzroči aktivacijo od cikličnega AMP odvisnih in neodvisnih signalnih poti, ki vodijo v modulacijo BCR induciranega fenotipskega odziva limfocitov B. Aktivacija BCR inducira izraţanje gena za interlevkin 6, ki ima v svoji promotorski regiji vezavno mesto za transkripcijski faktor CREB. Namen diplomskega dela je pojasniti vlogo receptorja EP4 pri modulaciji BCR induciranega izraţanja interlevkina 6. Pri eksperimentalnem delu smo se posluţili farmakološkega pristopa. Celično linijo WEHI 231 smo stimulirali z različnimi agonisti, antagonisti in inhibitorji signalnih poti. Vpliv stimulacije receptorja EP4 na BCR inducirano izraţanje gena za IL-6 smo spremljali z metodo veriţne reakcije s polimerazo v realnem času, izločanje IL-6 v celični medij pa s citometričnim citokinskim testom. Z encimsko imunskim testom za cAMP smo ugotavljali z receptorjem EP4 posredovano nastajanje cikličnega AMP v WEHI 231 celicah. Vlogo EP4/cAMP odvisnih signalnih poti pri modulaciji BCR induciranega izraţanja IL-6 smo ugotavljali z uporabo izbranih inhibitorjev encima adenilat ciklaze in protein kinaze A. V zadnjem delu diplomske naloge smo še določili vpliv IL-6 na proliferacijo WEHI 231 celične linije z metodo MTS. Ugotovili smo, da stimulacija WEHI 231 celic s PGE2 poviša z BCR inducirano izraţanje gena za IL-6. Z uporabo specifičnega agonista in antagonista receptorja EP4 smo dokazali, da so učinki PGE2 posredovani preko EP4 podtipa receptorja. Ker stimulacija receptorja EP4 sama po sebi nima vpliva na izraţanje gena za IL-6, sklepamo, da stimulacija receptorja EP4 modulira BCR inducirane signalne poti. Stimulacija receptorja EP4 je povzročila dvig intracelularne koncentracije cAMP. Z inhibicijo encima adenilat ciklaze in encima protein kinaze A smo dosegli popolno inhibicijo z receptorjem EP4 posredovane modulacije BCR induciranega izraţanja in izločanja IL-6. S tem smo dokazali, da poteka modulacija BCR induciranega izraţanja in izločanja IL-6 v nezrelih limfocitih B preko cAMP/PKA odvisne signalne poti, ki najverjetneje vodi v povečano aktivacijo transkripcijskega faktorja CREB. Izločeni IL-6 deluje na celično linijo WEHI 231 stimulatorno, saj se je proliferacija celic v prisotnosti rekombinantnega IL-6 povečala. Kljub povečanemu izraţanju gena za IL-6 ob hkratni stimulaciji receptorja EP4 in BCR, pa stimulacija obeh receptorjev vodi v zniţano izraţanje gena za receptor za IL-6, kar nakazuje obstoj negativne povratne zanke. Ligation of antigen to immunoglobulin receptor BCR induces activation and proliferation of mature B cells and apoptosis of immature B cells. Stimulation of BCR activates signal pathways in cells that induce transcriptional reprogramming. One of the genes, whose expression is highly induced, is also Ptger4, that codes for prostaglandin receptor EP4. Endogenous ligand for this receptor, PGE2, is a well-known immunomodulator. Ligation of PGE2 on EP4 receptor activates cAMP-dependent and independent pathways that promote modulation of BCR induced phenotypic response of B lymphocyte. Activation of BCR induces interleukin 6 gene expression, which has CREB binding site in promotor region of the gene. We investigated the role of receptor EP4 in modulation of BCR induced interleukin 6 gene expression. The experimental work was based on pharmacological treatment of WEHI 231 cell line. We stimulated the cells with agonists, antagonists and inhibitors of signal pathway components. Measuring the interleukin 6 gene expression after stimulation of EP4 receptor and BCR helped us to determine how both receptors modulate the expression of the fore mentioned gene. Besides that we also measured the excretion of interleukin 6 to cell media with cytometric cytokine assay. Using the enzyme-linked immunoassay we measured the concentration of cAMP after ligation of EP4 receptor. The function of EP4/cAMP dependent signal pathways in modulation of BCR induced interleukin 6 gene expression was determined by using relevant inhibitors of adenylate cyclase and protein kinase A. In the last part of our research work we determined the effect of interleukin 6 on proliferation of WEHI 231 cell line using the MTS method. The results clearly show that stimulation of WEHI 231 cells with PGE2 raises BCR induced interleukin 6 gene expression. Using specific EP4 receptor agonist and antagonist we proved, that PGE2 mediates its effects mainly through EP4 receptor. As ligation of EP4 receptor without simultaneous ligation of BCR does not induce interleukin 6 gene expression, we conclude, that ligation of EP4 receptor modulates BCR induced interleukin 6 gene expression. The stimulation of EP4 receptor raised intracellular production of cAMP. By inhibiting the enzyme adenylate cyclase and protein kinase A we managed to completely diminish the effects of EP4 receptor stimulation on BCR induced interleukin 6 gene expression. We summarize, that stimulation of EP4 receptor modulates BCR induced interleukin 6 gene expression via cAMP/PKA dependent signal pathway. Most probably this pathway leads to activation of CREB nuclear factor. Excreted interleukin 6 stimulates proliferation of WEHI 231 cells. Despite the rise in interleukin 6 gene expression after simultaneous stimulation of EP4 receptor and BCR (as we proved in the first part of our research) the expression of interleukin 6 receptor gene remains low. Even more, its expression reduces 4 hours after stimulation of EP4 receptor and BCR which could indicate a negative feedback loop. |
