Browsing by Author "Cruz, MT"
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- Contact sensitizer nickel sulfate activates the transcription factors NF-kB and AP-1 and increases the expression of nitric oxide synthase in a skin dendritic cell linePublication . Cruz, MT; Gonçalo, Margarida; Figueiredo, A; Carvalho, AP; Duarte, CBNuclear factor kappa B (NF-kB) and activating protein-1 (AP-1) transcription factors are ubiquitously expressed signaling molecules known to regulate the transcription of a large number of genes involved in immune responses, namely the inducible isoform of nitric oxide synthase (iNOS). In this study, we demonstrate that a fetal skin-derived dendritic cell line (FSDC) produces nitric oxide (NO) in response to the contact sensitizer nickel sulfate (NiSO(4)) and increases the expression of the iNOS protein, as determined by immunofluorescence and Western blot analysis. The sensitizer NiSO(4) increased cytoplasmic iNOS expression by 31.9 +/- 10.3% and nitrite production, as assayed by the Griess reaction, by 27.6 +/- 9.5%. Electrophoretic mobility shift assay (EMSA), showed that 30 min of FSDC exposure to NiSO(4) activates the transcription factor NF-kB by 58.2 +/- 7.0% and 2 h of FSDC exposure to NiSO(4) activates the transcription factor AP-1 by 26.0 +/- 1.4%. Together, these results indicate that NiSO(4) activates the NF-kB and AP-1 pathways and induces iNOS expression in skin dendritic cells.
- Contact sensitizers downregulate the expression of the chemokine receptors CCR6 and CXCR4 in a skin dendritic cell linePublication . Cruz, MT; Gonçalo, Margarida; Paiva, A; Morgado, JM; Figueiredo, A; Duarte, CB; Lopes, MCChemokines are involved in the control of dendritic cell (DC) trafficking, which is critical for the immune response, namely in allergic contact dermatitis (ACD). In this work, we investigated by flow cytometry the effect of the contact sensitizers 2,4-dinitrofluorobenzene (DNFB), 1,4-phenylenediamine (PPD) and nickel sulfate (NiSO(4)), on the surface expression of the chemokine receptors CCR6 and CXCR4 in DC. As an experimental model of a DC we used a fetal skin-derived dendritic cell line (FSDC), which has morphological, phenotypical and functional characteristics of skin DC. Our results show that all the skin sensitizers studied decreased the membrane expression of the chemokine receptors CCR6 and CXCR4. In contrast, 2,4-dichloronitrobenzene (DCNB), the inactive analogue of DNFB without contact sensitizing properties, was without effect on the surface expression of these receptors. Lipopolysaccharide (LPS), which induces the maturation of DC, also reduced surface CCR6 and CXCR4 expression.
- Dexamethasone prevents granulocyte-macrophage colony-stimulating factor-induced nuclear factor-kappaB activation, inducible nitric oxide synthase expression and nitric oxide production in a skin dendritic cell line.Publication . Vital, AL; Gonçalo, Margarida; Cruz, MT; Figueiredo, A; Duarte, CB; Lopes, MCAIMS: Nitric oxide (NO) has been increasingly implicated in inflammatory skin diseases, namely in allergic contact dermatitis. In this work, we investigated the effect of dexamethasone on NO production induced by the epidermal cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) in a mouse fetal skin dendritic cell line. METHODS: NO production was assessed by the method of Griess. Expression of the inducible isoform of nitric oxide synthase (iNOS) protein was evaluated by western blot analysis and immunofluorescence microscopy. Western blot analysis was also performed to evaluate cytosolic IkappaB-alpha (IkappaB-alpha) protein levels. The electrophoretic mobility shift assay was used to evaluate the activation or inhibition of nuclear factor kappa B (NF-kappaB). RESULTS: GM-CSF induced iNOS expression and NO production, and activated the transcription factor NF-kappaB. Dexamethasone inhibited, in a dose-dependent manner, NO production induced by GM-CSF. Addition of dexamethasone to the culture, 30 min before GM-CSF stimulation, significantly inhibited the cellular expression of iNOS. Dexamethasone also inhibited GM-CSF-induced NF-kappaB activation by preventing a significant decrease on the IkappaB-alpha protein levels, thus blocking NF-kappaB migration to the nucleus. CONCLUSIONS: The corticosteroid dexamethasone inhibits GM-CSF-induced NF-kappaB activation, iNOS protein expression and NO production. These results suggest that dexamethasone is a potent inhibitor of intracellular events that are involved on NO synthesis, in skin dendritic cells.
- Differential activation of nuclear factor kappa B subunits in a skin dendritic cell line in response to the strong sensitizer 2,4-dinitrofluorobenzenePublication . Cruz, MT; Duarte, CB; Gonçalo, Margarida; Figueiredo, A; Carvalho, AP; Lopes, MCDendritic cell (DC) maturation is essential for the initiation of T-dependent immune responses. Nuclear factor kappa B (NF-kappaB) transcription factors are ubiquitously expressed signalling molecules, known to regulate the transcription of a large number of genes involved in immune responses, including cytokines and cell surface molecules. In this work, we studied the time-dependent activation of five members of the NF-kappaB family, p50, p52, p65, RelB and cRel, in a mouse skin DC line in response to stimulation with the strong sensitizer, 2,4-dinitrofluorobenzene (DNFB). Western blot assay revealed that exposure of fetal skin DC (FSDC) to DNFB induced the degradation of the inhibitor of NF-kappaB (IkappaB). Three out of its five members, i.e. p50, p52, and RelB, were similarly activated upon DNFB stimulation, with subsequent translocation of these subunits from the cytosol to the nucleus, but with different kinetics. In contrast, p65 expression was diminished in both the nucleus and the cytosol. The electrophoretic mobility shift assay (EMSA) showed that exposure of FSDC to DNFB induced DNA binding to NF-kappaB. Together, these results show that DNFB differentially activates the various members of the NF-kappaB family in skin DC.
- Differential modulation of CXCR4 and CD40 protein levels by skin sensitizers and irritants in the FSDC cell linePublication . Neves, BM; Cruz, MT; Francisco, V; Gonçalo, Margarida; Figueiredo, A; Duarte, CB; Lopes, MCThe development of non-animal methods for skin sensitization testing is an urgent challenge. Some of the most promising in vitro approaches are based on the analysis of phenotypical and functional modifications induced by sensitizers in dendritic cell models. In this work, we evaluated, for the first time, a fetal skin-derived dendritic cell line (FSDC) as a model to discriminate between sensitizers and irritants, through analysis of their effects on CD40 and CXCR4 protein expression. The chemicals concentrations were chosen based on a slight cytotoxicity effect (up to 15%). Protein levels were evaluated by Western blot and immunocytochemistry, after stimulation with the skin sensitizers 2,4-dinitrofluorobenzene (DNFB), 1,4-phenylenediamine (PPD) and nickel sulphate (NiSO(4)), the non-sensitizer 2,4-dichloronitrobenzene (DCNB), and the irritants sodium dodecyl sulphate (SDS) and benzalkonium chloride (BC). All sensitizers tested increased CD40 and CXCR4 levels. In contrast, irritants decreased both proteins levels, with a more pronounced effect on CXCR4. In agreement with these results, dendritic cells derived from human peripheral blood monocytes-derived dendritic cells (MoDC) showed a similar response pattern to the skin sensitizer and irritant tested, PPD and SDS, respectively. In conclusion, evaluation of CD40 and CXCR4 proteins in chemical-treated FSDC may represent a useful tool in a future in vitro test for sensitizing assessment
- Effect of lipopolysaccharide, skin sensitizers and irritants on thioredoxin-1 expression in dendritic cells: relevance of different signalling pathwaysPublication . Francisco, V; Neves, BM; Cruz, MT; Gonçalo, Margarida; Figueiredo, A; Duarte, CB; Lopes, MCThioredoxin-1 is a ubiquitous protein involved in phenotypical and functional changes in dendritic cells (DC). We investigated the effect of lipopolysaccharide (LPS), skin sensitizers, and irritants on thioredoxin-1 by Western blot and immunofluorescence and on mRNA by real-time PCR. As DC models, we used a skin DC line and DC derived from human blood monocytes. We observed that all tested chemicals increased thioredoxin-1 expression, which is only transient for irritants, being the strongest effect observed for LPS (63 +/- 15%). To address the involvement of thioredoxin-1 in DC maturation, we analysed the effect of an activator of thioredoxin-1 expression, hydrogen peroxide, on CD86 expression, a marker of DC maturation. We found that hydrogen peroxide increases thioredoxin-1 and CD86 expression reinforcing thioredoxin-1 involvement in DC maturation. Because mitogen-activated protein kinases and PI3K are activated upon DC maturation, we also analysed their involvement in thioredoxin-1 modulation. We verified that LPS-induced upregulation of thioredoxin-1 expression was dependent on PI3K pathway.
- Effect of skin sensitizers on inducible nitric oxide synthase expression and nitric oxide production in skin dendritic cells: role of different immunosuppressive drugsPublication . Cruz, MT; Neves, BM; Gonçalo, Margarida; Figueiredo, A; Duarte, CB; Lopes, MCNitric oxide (NO) is involved in the pathogenesis of acute and chronic inflammatory conditions, namely in allergic contact dermatitis (ACD). However, the mechanism by which NO acts in ACD remains elusive. The present study focuses on the effects of different contact sensitizers (2,4-dinitrofluorbenzene, 1,4-phenylenediamine, nickel sulfate), the inactive analogue of DNFB, 2,4-dichloronitrobenzene, and two irritants (sodium dodecyl sulphate and benzalkonium chloride) on the expression of the inducible isoform of nitric oxide synthase (iNOS) and NO production in skin dendritic cells. It was also studied the role of different immunosuppressive drugs on iNOS expression and NO production. Only nickel sulfate increased the expression of iNOS and NO production being these effects inhibited by dexamathasone. In contrast, cyclosporin A and sirolimus, two other immunosuppressive drugs tested, did not affect iNOS expression triggered by nickel.
- Granulocyte-macrophage colony-stimulating factor activates the transcription of nuclear factor kappa B and induces the expression of nitric oxide synthase in a skin dendritic cell line.Publication . Cruz, MT; Duarte, CB; Gonçalo, Margarida; Figueiredo, A; Carvalho, AP; Lopes, MCNitric oxide (NO) produced by skin dendritic cells and keratinocytes plays an important role in skin physiology, growth and remodelling. Nitric oxide is also involved in skin inflammatory processes and in modulating antigen presentation (either enhancing or suppressing it). In this study, we found that GM-CSF stimulates the expression of the inducible isoform of nitric oxide synthase (iNOS) in a fetal-skin-derived dendritic cell line (FSDC) and, consequently, increases the nitrite production from 11.9 +/- 3.2 micromol/L (basal level) to 26.9 +/- 4.2 micromol/L. Pyrrolidinedithiocarbamate (PDTC) inhibits nitrite production, with a half maximal inhibitory concentration (IC50) of 19.3 micromol/L and the iNOS protein expression in FSDC. In addition, western blot assays revealed that exposure of FSDC to GM-CSF induces the phosphorylation and degradation of the inhibitor of NF-kappaB (IkB), with subsequent translocation of the p50, p52 and RelB subunits of the transcription nuclear factor kappa B (NF-kappaB) from the cytosol to the nucleus. Electrophoretic mobility shift assays (EMSA) showed that FSDC exposure to GM-CSF activates the transcription factor NF-kappaB. Together, these results show that GM-CSF induces iNOS expression in skin dendritic cells by a mechanism involving activation of the NF-kappaB pathway.
- Involvement of JAK2 and MAPK on type II nitric oxide synthase expression in skin-derived dendritic cellsPublication . Cruz, MT; Duarte, CB; Gonçalo, Margarida; Carvalho, AP; Lopes, MCIn this report, we demonstrate that a fetal mouse skin-derived dendritic cell line produces nitric oxide (NO) in response to the endotoxin [lipopolysaccharide (LPS)] and to cytokines [tumor necrosis factor-alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF)]. Expression of the inducible isoform of NO synthase (iNOS) was confirmed by immunofluorescence with an antibody against iNOS. The tyrosine kinase inhibitor genistein decreased LPS- and GM-CSF-induced nitrite (NO(-2)) production. The effect of LPS and cytokines on NO(-2) production was inhibited by the Janus kinase 2 (JAK2) inhibitor tyrphostin B42. The p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB-203580 also reduced the NO(-2) production evoked by LPS, TNF-alpha, or GM-CSF, but it was not as effective as tyrphostin B42. Inhibition of MAPK kinase with PD-098059 also slightly reduced the effect of TNF-alpha or GM-CSF on NO(-2) production. Immunocytochemistry studies revealed that the transcription factor nuclear factor-kappaB was translocated from the cytoplasm into the nuclei of fetal skin-derived dendritic cells (FSDC) stimulated with LPS, and this translocation was inhibited by tyrphostin B42. Our results show that JAK2 plays a major role in the induction of iNOS in FSDC.
- LPS induction of I kappa B-alpha degradation and iNOS expression in a skin dendritic cell line is prevented by the janus kinase 2 inhibitor, Tyrphostin b42Publication . Cruz, MT; Duarte, CB; Gonçalo, Margarida; Carvalho, AP; Lopes, MCThe Janus kinase (JAK) family of protein tyrosine kinases are activated in response to a wide variety of external stimuli. Here we have investigated whether the janus kinase 2 (JAK2) is involved in the induction of nitric oxide synthase type II (iNOS) expression in a mouse fetal skin dendritic cell line (FSDC). In FSDC the expression of iNOS protein and nitric oxide production, in response to the lipopolysaccharide (LPS) stimulus (5 microg/ml), is inhibited by the specific inhibitor of the JAK2, tyrphostin B42 with an half maximal inhibitory concentration (IC(50)) of 9.65 microM. The antioxidant compound pyrrolidinedithiocarbamate (PDTC) inhibits both the nitrite production with an IC(50) of 16.6 microM and the iNOS protein expression in FSDC. In addition, LPS induces the activation of NF-kappa B, and tyrphostin B42 prevents the degradation of the cytosolic factor I kappa B-alpha and blocks the translocation of the NF-kappa B p65 protein subunit into the nucleus. These results indicate that the JAK family of protein kinases and the transcription factor NF-kappa B are involved in the induction of iNOS protein expression in FSDC stimulated with LPS.