Differential effects of cyclo-oxygenase pathway metabolites on cytokine production by T lymphocytes

Cyclo-oxygenase pathway metabolites released in the microenvironment by activated platelets and endothelial cells are potential local modulators of the immune response. In the present study, we have investigated the modulatory role of PGE2, Iloprost (prostacyclin analogue) U-46619 (thromboxane analogue) on the release of IL-2, IFN-gamma, TNF-alpha and IL-6 by T lymphocytes. Our results show that PGE2, and prostacyclin differ in the regulation of cytokine production. PGE2, inhibited the release of IL-2 and IFN-gamma, while Iloprost did not affect their production. The addition of PGE2, or Iloprost greatly decreased the amount of TNF-alpha measured in the supernatants, although the rates of inhibition differed according-to the kind of stimulation. Unlike that of PGE2, inhibition by Iloprost was stronger in alloactivated cultures than in PHA-stimulated ones. In vitro IL-6 production was stimulated by PGE2, in alloactivated cultures and by Iloprost, whatever the stimulus. These results are probably due to other cellular subsets contaminating the T-lymphocyte preparations. After complete removal of monocytes from cell cultures, there were inhibitory effects of Iloprost and PGE2, on IL-6 released in the supernatants. We did not observe any significant effect of thromboxane analogue on the production of either cytokine

Cytokine production in scleroderma patients: effects of therapy with either iloprost or nifedipine

Objective. To compare the long-term effects of intermittent infusion of iloprost with those of oral nifedipine on the in vitro production of cytokines in patients with systemic sclerosis (SSc), and to evaluate their relationship with the effects of the two treatments on clinical parameters. Methods. The production of cytokines by alloactivated circulating mononucleated cells was assessed before and after one year of treatment in a subset of 31 patients enrolled in a 12-month randomized clinical trial. Nineteen patients were treated with a 5-day (8 hr per day), 20 ng/kg per minute infusion followed by a 1-day infusion every 6 weeks: 12 patients were treated with an oral slow-release formulation of nifedipine, 20 mg twice daily. Quantitative determinations of interleukin-1 \u3b2(IL1-\u3b2) and interleukin-6 (IL6) in the culture supernatants were performed with a commercial ELISA: the levels of tumor necrosis factor-\u3b1 (TNF-\u3b1), and interferon-\u3b3 (IFN-\u3b3) were measured by specific radioimmunometric assays. Results. The production of IL1-\u3b2 was significantly lower in the iloprost group than in the nifedipine group. Both the cutaneous fibrosis and the capillaroscopic patterns were better in patients treated with iloprost than in patients treated with nifedipine. There was a significant positive covariance between IL-1\u3b2 changes and the changes in both the skin score and the capillaroscopic score. Conclusion. There are several mechanisms by which iloprost could exert its clinical efficacy. Vasodilatation and inhibition of platelet aggregation are certainly important, but they are transient. We, suggest that the long-lasting modulation of the cytokine network observed in the present study could be another potential mechanism responsible for the persistent efficacy of iloprost despite its intermittent administration

Deregulated FGFR3 mutants in multiple myeloma cell lines with t(4;14): comparative analysis of Y373C, K650E and the novel G384D mutations.

The t(4;14)(p16.3;q32) chromosomal translocation occurs in approximately 20\% of multiple myelomas (MM) and leads to the apparent deregulation of two genes located on 4p16.3: the fibroblast growth factor receptor 3 (FGFR3) and the putative transcription factor WHSC1/MMSET. Interestingly, FGFR3 mutations known to be associated with autosomal dominant human skeletal disorders have also been found in some MM cell lines with t(4;14) but their pathogenetic role in MM is still controversial. Since cell lines may represent useful models for investigating the effects of deregulated FGFR3 mutants in MM, we analysed the expression, activation, signaling pathways and oncogenic potential of three mutants identified so far: the Y373C and K650E in the KMS-11 and OPM-2 cell lines respectively, and the novel G384D mutation here identified in the KMS-18 cell line. All of the cell lines present a heterozygous FGFR3 gene mutation and transcribe the mutated allele; unlike KMS-11 and OPM-2 (which express the IIIc isoform), the KMS-18 cell line expresses prevalently the isoform IIIb. We demonstrated that, under serum-starved conditions, KMS-11 and OPM-2 cells express appreciable levels of phosphorylated FGFR3 mutants indicating a constitutive activation of the Y373C and K650E receptors; the addition of the aFGF ligand further increased the level of receptor phosphorylation. Conversely, the FGFR3 mutant in KMS-18 does not seem to be constitutively activated since it was phosphorylated only in the presence of the ligand. In all three MM cell lines, ligand-stimulated FGFR3 mutants activated the MAP kinase signaling pathway but did not apparently involve either the STAT1 or STAT3 cascades. However, when transfected in 293T cells, G384D, like Y373C and K650E, was capable of activating MAPK, STAT1 and STAT3 under serum-starved condition. Finally, a focus formation assay of NIH3T3 cells transfected with FGFR3-expressing plasmid vectors showed that Y373C and K650E (albeit at different levels) but not G384D or the wild-type receptor, can induce transformed foci. Overall, our results support the idea that FGFR3 mutations are graded in terms of their activation capability, thus suggesting that they may play a critical role in the tumor progression of MM patients with t(4;14)

Increased interferon-gamma (IFN-γ) levels produced in vitro by alloactivated T lymphocytes in systemic sclerosis and Raynaud's phenomenon

The aim of the present study was to analyse the in vitro proliferation and cytokine production by alloantigen-stimulated peripheral blood mononuclear cells (PBMC) obtained from patients affected by systemic sclerosis (SSc) and patients with Raynaud's phenomenon (RP). In SSc patients the proliferation of PBMC stimulated in vitro with alloantigens was significantly increased compared with healthy subjects, while no differences were observed for RP patients. Lymphocytes from SSc patients also produced larger amounts of IFN-γ compared with healthy controls. However, patients with clinically active disease had lower IFN-γ levels than those found in clinically stable patients. Patients affected by RP showed significantly higher levels of IFN-γ than healthy subjects. Analysis at the clonal level of the lymphocyte subsets involved in alloantigen stimulation in one patient affected by active SSc, and one subject with RP confirmed the results obtained using PBMC. In particular, in the RP patient but not in the SSc patient, we observed a population of CD4+ T cells which proliferated to alloantigens in vitro and produced high levels of IFN-γ. We suggest that T lymphocytes producing high levels of IFN-γ might play a protective role in RP patients and in established scleroderma