Statins enhance the effects of corticosteroids on the balance between regulatory T cells and Th17 cells.

BACKGROUND: Plasticity of CD4(+) lymphocyte Th17/regulatory T cell (Treg) subsets is involved in the pathogenesis of chronic airway inflammatory diseases, such as asthma. Reversal of Th17/Treg cell balance towards Treg cells may be beneficial for the suppression of chronic Th2 cell-mediated inflammatory diseases, such as asthma. However, the effect of the combination of corticosteroids and a statin on the ratio of Treg/Th17 cells is unknown. OBJECTIVE: We investigated the in vitro effects of the combination of simvastatin and fluticasone propionate (FP) on the numbers of Treg and Th17 cells in asthmatic patients after co-incubation with monocyte-derived DCs (mDCs), and explored the underlying signalling pathways involved. METHODS: Using flow cytometry, we determined the effects of FP and simvastatin on Treg/Th17 balance after co-incubation of asthmatic CD4(+) T cells with mDCs. We also measured the relevant Treg and Th17-polarizing cytokines released from mDCs and also investigated the role of indoleamine 2, 3-dioxygenase (IDO) in this response. RESULTS: The combination of simvastatin and FP significantly increased Treg and concomitantly reduced Th17 cell numbers to a greater extent than FP or statin treatment alone. The enhancing effects of simvastatin on FP effects were mediated through the up-regulation of indoleamine 2, 3-dioxygenase and interleukin (IL)-10, together with down-regulation of IL-6 and IL-23 expression in mDCs. CONCLUSION: On the basis of this in vitro model of asthma, we suggest that the combination of a statin and a corticosteroid could augment the Treg/Th17 cell ratio and thus more effectively suppress airway inflammation in asthma patients. This may be particularly relevant in the treatment of severe asthma where Th17 cells are activated and linked to neutrophilic inflammation.

Metabolism of exogenous sn-1-alkyl-sn-2-lyso-glucosaminyl-phosphatidylinositol in HeLa D cells: accumulation of glucosaminyl(acyl)phosphatidylinositol in a metabolically inert compartment.

The somatic genetic defect in paroxysmal nocturnal haemoglobinuria (PNH) involves a block in the transfer of GlcNAc from UDP-GlcNAc to phosphatidylinositol (PI), the first step in the biosynthetic pathway for glycosylphosphatidylinositols (GPIs). We asked whether an exogenous lipid corresponding to an early intermediate in this pathway can be taken up by cells in culture and proceed through the GPI pathway. This approach could offer a strategy to bypass the block in PNH. To address this question we incubated HeLa D cells with sn-1-alkyl-sn-2-lyso-GlcN-[(3)H]PI (lyso-alkyl-GlcN-[(3)H]PI) for 24 h and analysed the cellular lipids. We found three lipid products: unaltered lyso-alkyl-GlcN-[(3)H]PI, GlcN-[(3)H]PI and GlcN(acyl)[(3)H]PI (GlcN-PI with a fatty acid acyl group on inositol). Since the latter two lipids are intermediates in the GPI biosynthetic pathway, this observation demonstrates that an exogenous lipid can enter and proceed partially through this pathway. However, the conversion of GlcN(acyl)PI to downstream mannosylated GPI intermediates in the GPI pathway was inefficient, both for GlcN(acyl)PI produced from the exogenous lipid as well as from that obtained by metabolic labelling with [(3)H]inositol. We investigated this poor conversion by examining whether GlcN(acyl)PI, radioactively labelled sequentially by [(14)C]inositol and [(3)H]inositol, resided in one compartment and could be readily metabolized to downstream intermediates. Isotope ratios indicated that the turnover of GlcN(acyl)PI was slower than those of either downstream mannosylated GPIs or even GPI anchors on proteins, the final products of GPI pathway. This result is incompatible with the one-compartment model and indicates that GlcN(acyl)PI in HeLa D cells accumulates largely in a compartment that is inert to subsequent mannosylation.

The observation of immunosuppressor(s) derived from cultured tumor cells and its partial neutralization with OK-432.

Malignant tumors such as brain tumors have been reported to be associated with immunosuppression caused by certain tumor-secreted cytokines. The reversion of tumor-derived immunosuppression has not been described. The use of OK-432, an immunomodulatory agent prepared from Su-strain of Streptococcus pyogenes A3, to activate peripheral blood mononuclear cells from a patient with glioblastoma multiforme has demonstrated a sharp rise in proliferative response. This proliferative response was compromised in the presence of living and irradiated autogeneic cancer cells. The conditioned media from cultured cells of glioblastoma multiforme, astrocytoma, and cholangiocarcinoma were tested for immunosuppressive ability. We found that conditioned media from 3 of 4 cases of glioblastoma, all 3 cases of astrocytoma, and 1 case of cholangiocarcinoma exhibited immunosuppressive activity toward the proliferative response of allogeneic peripheral blood mononuclear cells to phytohemagglutinin. This is the first report that cholangiocarcinoma produces soluble immunosuppressor(s). Our finding suggested that soluble substance(s) as well as direct cell-cell contact between tumor cells and mononuclear cells play roles in the observed tumor-derived immunosuppression.

The analysis of peri-tumor necrosis following the subcutaneous implantation of autologous tumor cells transfected with an episome transcribing an antisense insulin-like growth factor 1 RNA in a glioblastoma multiforme subject.

A subject inflicted with glioblastoma multiforme who received partial tumor resection and radiotherapy was recruited for an ex vivo gene therapy protocol using irradiated autologous tumor cells that had been engineered to suppress the expression of insulin-like growth factor I as the tumor vaccine. After subcutaneous injection for 8 weeks, the subject developed peri-tumor necrosis with mass effect. The authors wondered whether this event could have resulted from the tumor vaccine. The tissue section bordering the necrotic tumor tissue to the viable normal tissue was examined for nature of any infiltrated cells and their activities. Lymphocytes, macrophages, and a small number of neutrophils diffused into the necrotic tumor tissue were found. The infiltrated lymphocytes consisted of both CD4+ and CD8+ T cells. The functional activity of these lymphocytes was demonstrated by the active production of interferon y and tumor necrosis factor alpha based on the respective immunofluorescent staining localized to these cells. This finding is compatible with the proposed mechanism underlying the tumor vaccination. However, the contribution of radiation treatment to this event cannot be clearly ruled out.

Uptake of exogenous sn-1-acyl-2-lyso-phosphatidylinositol into HeLa S3 cells. Reacylation on the cell surface and metabolism to glucosaminyl(acyl)phosphatidylinositol.

A HeLa S3 subline is unusual in accumulating relatively large amounts of glucosaminyl(acyl)phosphatidylinositol (GlcN(acyl)PI), a derivative of phosphatidylinositol (PI) in which both GlcN and a fatty acid are linked to inositol hydroxyl groups (D. Sevlever, D. Humphrey, and T.L. Rosenberry, submitted for publication). This lipid is a proposed intermediate on the biosynthetic pathway for glycosyl-PI (GPI) anchors of membrane proteins. In this study we demonstrate for the first time that exogenous inositol phospholipids can enter this biosynthetic pathway and be metabolized to GlcN(acyl)PI. When HeLa S3 cells were incubated for 24 h with exogenous PI or sn-1-acyl-2-lyso-phosphatidyl-inositol (lyso-PI) labeled with 3H in the inositol group, 25-30% of the label was recovered in cell-associated lipids and most of the remaining 70-75% in hydrophilic metabolites in the medium. The predominant labeled lipid was PI, with smaller amounts of lyso-PI, phosphatidylinositol 4-phosphate (PIP), and GlcN(acyl)PI. Both exogenous lipid precursors gave the same distribution of labeled lipids, and a similar distribution was observed for endogenous inositol phospholipids metabolically labeled with [3H]inositol. Addition of excess inositol had no effect on the conversion of [3H]lyso-PI to [3H]GlcN(acyl)PI, indicating that the conversion did not result from breakdown to [3H]inositol followed by resynthesis. The cellular orientation of incorporated PI and lyso-PI was determined by incubating cells at 4 degrees C with PI-specific phospholipase C (PI-PLC). This enzyme cleaves only PI and lyso-PI on the outer leaflet of the cell membrane. After 24-h incubation with either precursor, only about 15% of cell-associated [3H]PI or [3H]lyso-PI was on the outer leaflet. However, more than 60% of the [3H]PI was on the outer leaflet after 1-h incubation with either precursor, suggesting that substantial sn-2 acylation of exogenous [3H]lyso-PI occurred in the outer leaflet. This suggestion was confirmed by examining labeled lipids in cells after uptake of [3H]lyso-PI at 4 degrees C. No transmembrane translocation of lyso-PI, PI phosphorylation, or PI glycosylation occurred at this temperature, but some sn-2 acylation was apparent and more than 90% of the [3H]PI formed was on the outer leaflet. These data indicate that sn-2 acylation can occur in the outer leaflet of the cell membrane, perhaps by transacylation from other cell surface phospholipids.