What Harbours the Cradle of Life? The Progesterone-Dependent Immunomodulation


Halasz M *, Polgar B , Kozma N , Berki T , Szekeres-Bartho J ,

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Objectives: The foeto-maternal relationship during pregnancy is controlled by a complex regulation of immuno-endocrine homeostasis where progesterone-dependent immunmodulation plays a key role. Due to stimulation by foetally derived antigens lymphocytes of healthy pregnant women express progesterone receptors and in the presence of progesterone produce a 34-kDa mediator protein named the progesterone-induced blocking factor (PIBF). PIBF induces a Th2 dominant cytokine production via inhibiting STAT4 and activating STAT6 inhibits maternal NK activity which results in decreased cell-mediated response thus exerts an anti-abortive effect. Though PIBF does not directly bind to IL-4 receptor-alpha our aim was to determine the role of IL-4Ralpha in PIBF signalling. Furthermore we investigated the effects of PIBF on the protein kinase C (PKC)/ Ca++ system which plays a key role in Th1/Th2 differentiation. Materials and Methods: Confocal microscopy was used to detect the localization of IL-4Ralpha and PIBF receptor (PIBFR). To verify the involvement of IL-4Ralpha in PIBF signalling IL-4Ralpha was silenced by oligonucleotides interfering with IL-4Ralpha mRNA. Assuming that the PIBF receptor is a GPI-anchored protein PIBF-induced phosphorylation of STAT6 was tested in phosphatidylinositol-specific phospholipase C (PI-PLC) digested cells by Western blotting. The hypothesis that PIBF receptors float in glycosphyngolipid-cholesterol rafts was tested by depletion of cholesterol using methyl-β-cyclodextrin (MbCD). Proteins from PIBF-treated cells were reacted on Western blots with phospho-specific antibodies recognizing different PKC izoforms. Intracellular free calcium was measured by flow cytometry. Results: Labelling of the IL-4Ralpha and simultaneous activation of the PIBF receptor with a FITC-labelled ligand revealed co-capping of the two binding sites. In IL-4Ralpha deficient cells the STAT6 activating effect of PIBF was markedly reduced. After PI-PLC treatment PIBF did not induce STAT6 phosphorylation while IL-4 retained the same effect. In MbCD treated cells neither PIBF nor IL-4 were able to Tyr-phosphorylate STAT6 suggesting that not only the PIBFR but also the IL-4Ralpha is enriched in lipid rafts. Both IL-4 and PIBF induced PKC phosphorylation which was abrogated by anti- IL-4Ralpha or anti-PIBF IgG pre-treatment. PIBF treatment did not alter intracellular Ca++-levels. Inhibition of PKCzeta or PKCtheta phosphorylation but not that of PKCalpha/beta resulted in the loss of STAT6 and JAK1 phosphorylation by PIBF. Conclusion: Our findings suggest the existence of a novel type of IL-4R composed of the alpha-chain of IL-4R and the PIBFR. The PIBFR is a GPI-anchored protein that lacks cytoplasmic tail thus it uses the intracellular domain of IL-4Ralpha for signalling. Upon ligand-binding the PIBFR enriched in lipid rafts forms a complex with the IL-4Ralpha subunit and activates the JAK1/STAT6 pathway. PIBF phosphorylates PKC via binding to the IL-4R without affecting intracellular Ca++. Phosphorylation of PKCzeta and PKCtheta is required for JAK1 and STAT6 activation whereas PKCalpha/beta is not involved. These findings explain the mechanism by which PIBF supports a Th2 dominant cytokine pattern.