Th17 in sensitized human lymphocytes ex vivo ?

Abstract

IntroductionIn recent work we could show that sensitized human T lymphocytes reveal altered STAT6/STAT1 in terms of DNA binding balance compared to normal controls [1,2]. As STAT3 is specific for Th17 signal transduction, and this cell type can play a dominant role in B cell regulation and induction of allergic, inflammatory and autoimmune disorders [3, for review 4], the aim of this paper was to investigate STAT3 activation in the down stream regulation of circulating human lymphocytes ex vivo. Material and methodsHuman PBMC from 5 atopic/non-atopic volunteers (IgE > 500 IU/ml, normal age matched controls: < 50 IU/ml) were stimulated by PHA and anti-CD3/IL-2 (1 ng/ml) in 3-day cultures. Allergic patients were suffering from seasonal hay fever and chronic atopic eczema. IL-23 (10 ng/ml) and histamine (2.5 mM) were added 4 hours post-plated. Cytokines were measured by enzyme linked immune sorbent assay (ELISA) and STAT-DNA interaction by electrophoretic mobility shift assay (EMSA), respectively. For EMSA, the infrared dye labelled double stranded DNA oligonucleotide STAT3 5'GAT CCT TCT GGG AAT TCC TAG ATC 3' (LI-COR Nebraska USA) was applied. PBMC differentiation was identified using cytoflow/FACS analyses. EMSA results were processed using the Gelscan V 5.1 software: P < 0.05 was considered statistically significant. Results and discussionIt could be shown that IL-17E as well as IL-4 was constitutively increased in the atopic samples and that anti-CD3 was a more effective stimulator than PHA compared to the non-atopic matched controls (Tab. 1). For IL-4, PHA was more effective than both anti-CD3 and IFN-g in the non-atopic group (p < 0.05, n = 5, Student t). The most augmented IL-17E-responses could be demonstrated in non-atopics using IL-23 as a stimulator (Tab. 1). The contrary effect was observed in the atopic group, where the initially high levels of IL-17E were down-regulated corresponding to the increase of Th1 (IFN-g) polarization. This is in a good accordance to recent work, where the IL-23/Th17-axis was shown to be the predominant regulator of inflammatory and allergic disorders [5]. In terms of a potential negative feedback of histamine it could be demonstrated that the lymphocyte proliferation and Th1 activity (IFN-g) was increased, thereby inducing down-regulation of the Th2/Th17-IL-17E activity (data not shown). This is in a good agreement to the recent observation indicating that histamine feedback responses were strongly concentration-dependent in human lymphocytes ex vivo [6]. Surprisingly, anti-CD3-stimulated DNA binding of STAT3 was suppressed after Th17-stimulatory IL-23 activation in the presence of IL-1ß with an inverse effect in the atopic group (Fig. 1). After addition of histamine (2.5 mM) the responses turned into the contrary. There was an increase in the atopic group but inhibition in the non-atopic group as it was demonstrated also for STAT1 in recent work [6].From these results it could be concluded that Th17 showed downstream regulatory potency with specific differences in atopic pat...