Sex hormone modulation of cell growth and apoptosis of the human monocytic/macrophage cell line

Sex hormones seem to modulate the immune/inflammatory responses by different mechanisms in female and male rheumatoid arthritis patients. The effects of 17β-oestradiol and of testosterone were tested on the cultured human monocytic/macrophage cell line (THP-1) activated with IFN-γ in order to investigate their role in cell proliferation and apoptosis. Activated human THP-1 cells were cultured in the presence of 17β-oestradiol and testosterone (final concentration, 10 nM). The evaluation of markers of cell proliferation included the NF-κB DNA-binding assay, the NF-κB inhibition complex, the proliferating cell nuclear antigen expression and the methyl-tetrazolium salt test. Apoptosis was detected by the annexin V-propidium assay and by the cleaved poly-ADP ribose polymerase expression. Specific methods included flow analysis cytometry scatter analysis, immunocytochemistry and western blot analysis. Cell growth inhibition and increased apoptosis were observed in testosterone-treated THP-1 cells. Increased poly-ADP ribose polymerase-cleaved expression and decreased proliferating cell nuclear antigen expression, as well as an increase of IκB-α and a decrease of the IκB-α phosphorylated form (ser 32), were found in testosterone-treated THP-1 cells. However, the NF-κB DNA binding was found increased in 17β-oestradiol-treated THP-1 cells. The treatment with staurosporine (enhancer of apoptosis) induced decreased NF-κB DNA binding in all conditions, but particularly in testosterone-treated THP-1 cells. Treatment of THP-1 by sex hormones was found to influence cell proliferation and apoptosis. Androgens were found to increase the apoptosis, and oestrogens showed a protective trend on cell death – both acting as modulators of the NF-κB complex

CTLA4-Ig interacts with cultured synovial macrophages from rheumatoid arthritis patients and downregulates cytokine production

Introduction: Co-stimulatory signal B7(CD80/CD86):CD28 is needed in order to activate T cells in immune response. Cytotoxic T lymphocyte-associated antigen-4-immunoglobulin (CTLA4-Ig) binding to the B7 molecules on antigen-presenting cells downregulates this activation and represents a recent biological treatment in rheumatoid arthritis (RA). Objectives of the study were to investigate the presence of the B7.2 (CD86) molecule and its masking by CTLA4-Ig on cultures of both RA synovial macrophages (RA SM), and of macrophages differentiated from THP-1 cells (M). In addition, the anti-inflammatory effects of CTLA4-Ig on co-cultures of RA SM and M with activated T cells were tested.Methods: All macrophages were co-cultured for 24 hours with activated T cells, without or with CTLA4-Ig (10, 100, 500 \u3bcg/ml for 1 hour, 3 hours and overnight, respectively). Immunofluorescence (IF) staining for B7.2, and an analysis of inflammatory cytokine expression (interleukin (IL) -6, tumor necrosis factor (TNF) \u3b1, IL-1\u3b2, transforming growth factor (TGF) \u3b2) by immunocytochemistry (ICC), western blot (WB) and reverse transcriptase-polymerase chain reaction (RT-PCR) were performed.Results: Macrophages showed intense B7.2 expression. CTLA4-Ig/B7.2 masking was evident for all macrophages, even after only 1 hour of cell culture (range from 10 to 100 \u3bcg/ml). ICC of co-cultures showed a dose-dependent decrease in inflammatory cytokines (P < 0.001 for IL-6, TNF\u3b1, IL-1\u3b2 and TGF\u3b2). Data were confirmed by WB and RT-PCR analysis.Conclusions: Optimal concentrations of CTLA4-Ig for the CTLA4-Ig/B7.2 masking on activated macrophages were identified and were found to induce significant downregulation in the cell production of IL-6, TNF\u3b1, IL1-\u3b2 and TGF\u3b2. In conclusion, macrophages would appear to be a sensitive target for CTLA4-Ig treatment in RA

Uric Acid Promotes Apoptosis in Human Proximal Tubule Cells by Oxidative Stress and the Activation of NADPH Oxidase NOX 4

Mild hyperuricemia has been linked to the development and progression of tubulointerstitial renal damage. However the mechanisms by which uric acid may cause these effects are poorly explored. We investigated the effect of uric acid on apoptosis and the underlying mechanisms in a human proximal tubule cell line (HK-2). Increased uric acid concentration decreased tubule cell viability and increased apoptotic cells in a dose dependent manner (up to a 7-fold increase, p<0.0001). Uric acid up-regulated Bax (+60% with respect to Ctrl; p<0.05) and down regulated X-linked inhibitor of apoptosis protein. Apoptosis was blunted by Caspase-9 but not Caspase-8 inhibition. Uric acid induced changes in the mitochondrial membrane, elevations in reactive oxygen species and a pronounced up-regulation of NOX 4 mRNA and protein (p<0.05). In addition, both reactive oxygen species production and apoptosis was prevented by the NADPH oxidase inhibitor DPI as well as by Nox 4 knockdown. URAT 1 transport inhibition by probenecid and losartan and its knock down by specific siRNA, blunted apoptosis, suggesting a URAT 1 dependent cell death. In summary, our data show that uric acid increases the permissiveness of proximal tubule kidney cells to apoptosis by triggering a pathway involving NADPH oxidase signalling and URAT 1 transport. These results might explain the chronic tubulointerstitial damage observed in hyperuricaemic states and suggest that uric acid transport in tubular cells is necessary for urate-induced effects

NF-KB involvement for CTLA4-IG intracellular signaling in human macrophages

Background Transcription factor NF-kB (nuclear factor kB) play an important role in the regulation of immune and inflammatory responses [1]. Previous studies showed that a significant downregulation of pro-inflammatory cytokine expression (TNF\u3b1, IL-1\u3b2 and IL-6) was evident for cultured human macrophages treated with CTLA4-Ig fusion protein [2]. Objectives The aim of the study was to investigate the NF-kB pathway as possible intracellular signaling target involved in the CTLA4-Ig modulation of cytokine production (TNF\u3b1, IL-1\u3b2 and IL-6) in cultured human macrophages. Methods THP1 cell line, differentiated by PMA (0.5 \u3bcg/ml for 3 hours) into adherent macrophages, were seeded in tissue culture dishes in culture medium with CTLA4-Ig [100 and 500 \u3bcg/ml for 3 and 12 hours] or without CTLA4-Ig, for mRNA extraction and qRT-PCR analysis. At the end of incubation adherent cells were harvested and mRNA was extracted and processed by qRT-PCR analysis for NF-kB1 (p50 subunit) and for TNF\u3b1, IL-1\u3b2, IL-6. Experiments were done in triplicate. Results The qRT-PCR analysis of NFKB1 mRNA, after 3 and 12 hours from CTLA4-Ig treatment, showed a significant downregulation (p<0.001) of the gene expression vs. cnt at both concentrations [100 and 500 \u3bcg/ml]. At the same time, the qRT-PCR analysis of inflammatory cytokine mRNA, already after 3 hours from treatment, showed that CTLA4-Ig [100 \u3bcg/ml] induced a significant decrease for TNF\u3b1 and IL-6 gene expression (p<0.05), compared to untreated macrophages (cnt). CTLA4-Ig [500 \u3bcg/ml] was able to reduce TNF\u3b1 gene expression vs. cnt in a larger extent (p<0.001). After 12 hours from treatment with CTLA4-Ig [100, 500 \u3bcg/ml], it was still evident a significant downregulation in cytokine gene expression for IL-1\u3b2 and IL-6 vs. cnt (p<0.001). Interestingly, TNF\u3b1 downregulation was not still significant after 12 hours from treatment. Conclusions The action of CTLA4-Ig may be exerted at level of the intracellular signaling (trascription factors) by downregulating NF-kB1 gene expression, together with a significant reduction in gene expression for tested inflammatory cytokines

Lysine triggers apoptosis through a NADPH oxidase-dependent mechanism in human renal tubular cells

Progressive chronic kidney disease (CKD) is common in lysinuric protein intolerance (LPI), a primary inherited aminoaciduria characterized by massive Lysine excretion in urine. However, by which mechanisms Lysine may cause kidney damage to tubule cells is still not understood. This study determined whether Lysine overloading of human proximal tubular cells (HK-2) in culture enhances apoptotic cell loss and its associated mechanisms. Overloading HK-2 with Lysine levels reproducing those observed in urine of patients affected by LPI (10 mM) increased apoptosis (+30%; p < 0.01 vs.C), as well as Bax and Apaf-1 expressions (+30-50% p < 0.05), while downregulated Bcl-2 (-40% p < 0.05). Apoptosis induced by high Lysine was no longer observed after addition of caspase-9 and caspase-3 inhibitors while caspase-8 inhibitor had no protective effect. High Lysine induced elevations in ROS generation and NADPH oxidase subunits mRNAs (p22 (phox) +106 +/- 23%, p67 (phox) +108 +/- 22% and gp91 (phox) +75 +/- 4% p < 0.05-0.01). In addition, the NADPH oxidase inhibitor DPI prevented both ROS production and apoptosis. Treating HK-2 with antioxidants, such as Cysteine and its analog, N-acetyl-l-cysteine (NAC), rescued the HK-2 from apoptosis induced by Lysine. In summary, our data show that high Lysine in vitro increases the permissiveness of proximal tubule kidney cells to apoptosis by triggering a pathway involving NADPH oxidase signaling. This event may represent a key cellular effect in the increasing the susceptibility of human tubular cells to apoptosis when the tubules cope with a high Lysine load. This effect is instrumental to renal damage and disease progression in patients with LPI