[Show abstract][Hide abstract] ABSTRACT: Given that the bioactive lipid sphingosine 1-phosphate is involved in cardiovascular pathophysiology, and since lipid accumulation and inflammation are hallmarks of calcific aortic stenosis, the role of sphingosine 1-phosphate on the pro-inflammatory/pro-osteogenic pathways in human interstitial cells from aortic and pulmonary valves was investigated. Real-time PCR showed sphingosine 1-phosphate receptor expression in aortic valve interstitial cells. Exposure of cells to sphingosine 1-phosphate induced pro-inflammatory responses characterized by interleukin-6, interleukin-8, and cyclooxygenase-2 up-regulations, as observed by ELISA and Western blot. Strikingly, cell treatment with sphingosine 1-phosphate plus lipopolysaccharide resulted in the synergistic induction of cyclooxygenase-2, and intercellular adhesion molecule 1, as well as the secretion of prostaglandin E2, the soluble form of the intercellular adhesion molecule 1, and the pro-angiogenic factor vascular endothelial growth factor-A. Remarkably, the synergistic effect was significantly higher in aortic valve interstitial cells from stenotic than control valves, and was drastically lower in cells from pulmonary valves, which rarely undergo stenosis. siRNA and pharmacological analysis revealed the involvement of sphingosine 1-phosphate receptors 1/3 and Toll-like receptor-4, and downstream signaling through p38/MAPK, protein kinase C, and NF-κB. As regards pro-osteogenic pathways, sphingosine 1-phosphate induced calcium deposition and the expression of the calcification markers bone morphogenetic protein-2 and alkaline phosphatase, and enhanced the effect of lipopolysaccharide, an effect that was partially blocked by inhibition of sphingosine 1-phosphate receptors 3/2 signaling. In conclusion, the interplay between sphingosine 1-phosphate receptors and Toll-like receptor 4 signaling leads to a cooperative up-regulation of inflammatory, angiogenic, and osteogenic pathways in aortic valve interstitial cells that seems relevant to the pathogenesis of aortic stenosis and may allow the inception of new therapeutic approaches.
[Show abstract][Hide abstract] ABSTRACT: Flavonoids from lemongrass – Cymbopogon citratus (DC.) Stapf – leaves infusion, a commonly consumed beverage for the treatment of inflammatory-related conditions, were investigated in this work. Luteolin O-, C- and O,C-glycosides were isolated and identified by nuclear magnetic resonance, being the cassiaoccidentalin B structure fully characterized for the first time in lemongrass. The anti-inflammatory activity of luteolin and its glycosides was evaluated in lipopolysaccharide-stimulated macrophages. Luteolin glycosides demonstrated less cytotoxicity than luteolin itself. Although glycosylation decreases luteolin anti-inflammatory properties, being higher to C-glycosylation, an inhibitory effect on inflammatory mediator production (nitric oxide and IL-1β) was verified for the luteolin 7-O-β-glucopyranoside, without cytotoxic effects. Therefore, luteolin glycosides from lemongrass infusion are evidenced as a less toxic alternative to current anti-inflammatory drugs with promising use in pharmaceutical and food supplement industries. Additionally, this work establishes structure–activity relationships, which constitutes valuable information in the design of anti-inflammatory luteolin glycosides devoid of toxicity.
[Show abstract][Hide abstract] ABSTRACT: Purpose: Given the association of Toll-like receptor (TLR)-4 to calcific aortic stenosis and the relevant function of the lipid mediator sphingosine-1-phosphate (S1P) in the cardiovascular system, the putative role of TLR4 ligands and S1P in the induction of pro-inflammatory and pro-adhesive responses in endothelial cells from human aortic (AVEC) and pulmonary valves (PVEC) was investigated.
Methods: AVEC and PVEC obtained from n =5 heart transplant patients with no valve disease, and AVEC derived from stenotic aortic valves collected from n = 5 patients with degenerative aortic stenosis and indication for valve replacement were studied. Reverse transcriptase PCR assays were used to evaluate the mRNA expression of TLR and S1P receptors. Cytokine secretion and adhesion molecule expression were evaluated by antibody arrays and Western blot, and adhesion of human monocytes to AVEC monolayers was assayed in vitro. Comparison between stenotic and control aortic valve and between aortic and pulmonar valve were performed.
Results: Reverse transcriptase PCR assays revealed the expression of TLRs at the mRNA level in AVEC, being TLR4 the most abundant, as well as the expression of S1P receptors, the S1P1 being the most abundant subtype. As regards pro-inflammatory molecules, the exposure of control AVECs to the bacterial lipopolysaccharide (LPS), a TLR4 ligand, induced the secretion of several cytokines such as interleukin (IL)-6, granulocyte macrophage colony-stimulating factor (GM-CSF), and monocyte chemotactic protein-1. Moreover, LPS cooperated with S1P to further up-regulate IL-6 and GM-CSF secretion. In addition, AVEC exposure to LPS induced the expression of the adhesion molecule ICAM-1, which was further up-regulated in the presence of S1P. Interestingly, the effect was more prominent in endothelial cells from the aortic valve than from the pulmonary valve, which rarely undergoes stenosis, and more potent in cells from stenotic valves. The cooperative effect on ICAM-1 up-regulation was inhibited by S1P1 receptor antagonists. In adhesion assays, LPS and S1P cooperated to increase the number of monocytes that adhered to the activated AVEC monolayers, and the effect was more marked in stenotic than in control AVECs.
Conclusions: TLR4 and S1P1 receptor cooperate to up-regulate pro-inflammatory/pro-adhesive molecules in AVECs, what might be relevant for the pathogenesis of aortic stenosis given the stronger effect on stenotic aortic valves and the lower effect on pulmonary valves, and could open the way for new therapeutic approaches for the disease.
Preview · Article · Aug 2013 · European Heart Journal
[Show abstract][Hide abstract] ABSTRACT: Ethnopharmacological relevance Cymbopogon citratus (DC.) Stapf leaves infusion is used in traditional medicine for the treatment of inflammatory conditions, however little is known about their bioactive compounds. Aim of the study Investigate the compounds responsible for anti-inflammatory potential of Cymbopogon citratus (Cy) on cytokines production induced by lipopolysaccharide (LPS) in human and mouse macrophages, and the action mechanisms involved. Materials and methods An essential oil-free infusion of Cy was prepared and polyphenol-rich fractions (PFs) were obtained from it by column chromatography. Chlorogenic acid (CGA) was identified, by HPLC/PDA/ESI-MSn. The expression of cytokines, namely TNF-α and CCL5, was analyzed by real-time RT-PCR, on LPS-stimulated human macrophages. Activation of nuclear factor (NF)-κB, a master regulator of inflammation, was investigated by western blot and gene reporter assay. Proteasome activity was assessed using a fluorogenic peptide. Results Cymbopogon citratus extract and its polyphenols inhibited the cytokine production on human macrophages. This supports the anti-inflammatory activity of Cy polyphenols in physiologically relevant cells. Concerning the effect on the activation of NF-κB pathway, the results pointed to an inhibition of LPS-induced NF-κB activation by Cy and PFs. CGA was identified, by HPLC/PDA/ESI-MSn, as the main phenolic acid of the Cy infusion, and it demonstrated to be, at least in part, responsible by that effect. Additionally, it was verified for the first time that Cy and PFs inhibited the proteasome activity, a complex that controls NF-κB activation, having CGA a strong contribution. Conclusions The results evidenced, for the first time, the anti-inflammatory properties of Cymbopogon citratus through proteasome inhibition and, consequently NF-κB pathway and cytokine expression. Additionally, Cy polyphenols, in particular chlorogenic acid, were highlighted as bioactive compounds.
Full-text · Article · Apr 2013 · Journal of ethnopharmacology
[Show abstract][Hide abstract] ABSTRACT: Given that TLRs and sphingosine-1-phosphate (S1P) are key players in inflammation, we explored the potential interplay between TLRs and S1P in the adhesion/inflammatory pathways in primary human endothelial cells. As determined by Western blot and flow cytometry, cells treated with LPS (a TLR4 ligand) and S1P showed significantly enhanced expression of adhesion molecules such as ICAM-1 and E-selectin compared with the effect of either ligand alone. Cell-type differences on E-selectin upregulation were observed. In contrast, no cooperation effect on ICAM-1 or E-selectin was observed with a TLR2/TLR1 ligand. Consistent with an increase in adhesion molecule expression, endothelial cell treatment with LPS plus S1P significantly enhanced adhesion of PBMCs under shear stress conditions compared with the effect of either ligand alone and exhibited comparable levels of cell adhesion strength as those after TNF-α treatment. Moreover, LPS and S1P cooperated to increase the expression of proinflammatory molecules such as IL-6, cyclooxygenase-2, and prostacyclin, as determined by ELISA and Western blot. The analysis of signaling pathways revealed the synergistic phosphorylation of ERK upon LPS plus S1P treatment of HUVEC and human aortic endothelial cells and cell-type differences on p38 and NF-κB activation. Moreover, pharmacological and small interfering RNA experiments disclosed the involvement of S1P(1/3) and NF-κB in the cooperation effect and that cell origin determines the S1P receptors and signaling routes involved. Sphingosine kinase activity induction upon LPS plus S1P treatment suggests S1P- Sphingosine kinase axis involvement. In summary, LPS and S1P cooperate to increase proinflammatory molecules in endothelial cells and, in turn, to augment leukocyte adhesion, thus exacerbating S1P-mediated proadhesive/proinflammatory properties.
No preview · Article · Oct 2012 · The Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: The pathogenesis of chronic venous disease, a complex and common pathology affecting the lower extremities, is still poorly understood. Accumulating evidence supports the role of inflammation as a mechanism underlying the physiopathology of chronic venous disease. In this review, we will carry out an overview of the role of inflammation in the varicose vein pathology, focusing on recent data that supports the use of pharmacological tools to decrease the production of inflammatory mediators or to block their effects, as a complementary therapeutic approach. The most accepted mechanism linking venous hypertension to the changes in the macro and microcirculation is the leukocyte "trapping" model, according to which leukocytes infiltrate the venous wall and valves and migrate across the postcapillarvenules endothelium, thus leading to wall remodeling and valvular destruction. An incipient inflammatory focus in the vascular intima or media, the activation of endothelial cells by hypoxia, or altered hemodynamics, might cause the leukocytes to leave the circulation. In addition, fluid shear stress can contribute to leukocyte activation, and nitric oxide donors and inflammatory mediators can modulate the response. In the last decade, several reports have shown the involvement of molecules implicated in the inflammatory response such as adhesion molecules and cytokines, especially vascular cellular adhesion molecule-1, transforming growth factor-β, interleukin (IL)-6, IL-8 and matrix metalloproteinases, as well as the transcription factor hypoxia inducible factor-1 α in varicose vein disease. Additionally, in support of the leukocyte "trapping" model, recent data from our group has disclosed a correlation between elevated levels of chemotactic cytokines and varicose veins, specifically monocyte-chemoattractant protein-1, IL-8, interferon-inducible protein-10, RANTES, macrophage-inflammatory protein (MIP)-1α and MIP-1β. Furthermore, recent findings show that acetylsalicylic acid (ASA) treatment of patients with varicose syndrome accelerated healing and delayed recurrence of venous ulceration, suggesting that drugs that diminish leukocyte activation seemed to benefit ulcer healing and could be used as a complementary treatment. In addition to studies with human tissues, several animal model studies have suggested that inhibition of the early stages of inflammation offers potential targets that could be effective for the treatment of venous disease. In summary, recent reports highlight the impact of inflammation and hypoxia on varicose vein pathogenesis, and open the avenue to future preventive and therapeutic designs.
[Show abstract][Hide abstract] ABSTRACT: Aortic stenosis shares some ethiopathological features with atherosclerosis and increasing evidence links Toll-like receptors (TLRs) to atherogenesis.
TLR-mediated inflammation and osteogenesis were investigated in human interstitial cells isolated from stenotic and non-stenotic aortic valves. TLR expression and signalling were evaluated by quantitative RT-PCR, flow cytometry, Western blot analysis, ELISA, and cytokine arrays. Osteogenesis was evaluated by measuring alkaline phosphatase activity.
Interstitial cells from control valves express most TLRs, being TLR4 the most abundant, whereas cells from stenotic valves express higher TLR4 and TLR2 and lower TLR5 and TLR9 transcript levels. When pro-inflammatory pathways were analyzed, we observed that TLR4, TLR2 and TLR3 ligands induced an early activation of NF-κB and p38 MAPK activation in cells from control and stenotic valves. Strikingly, when TLRs sensing viral patterns were studied, a sustained TLR3-mediated activation of NF-κB, a κB-independent induction of catalytically active cyclooxigenase (COX)-2 and ICAM-1 expression, and induction of expression of several chemokines were observed. TLR4, but not TLR2, engagement produced a similar but NF-κB-dependent effect. Moreover, TLR3 and TLR4 agonists induced alkaline phosphatase expression and activity.
Exposure of aortic valve interstitial cells to viral and Gram-negative bacteria molecular patterns induces distinct and long-term TLR-mediated pro-inflammatory and pro-osteogenic responses that might be relevant to the pathogenesis of degenerative aortic stenosis.
No preview · Article · Jan 2011 · International journal of cardiology
[Show abstract][Hide abstract] ABSTRACT: Aqueous extracts of Cymbopogon citratus (Cy) leaves are used in traditional medicine for the treatment of inflammatory conditions, however, little is known about their mechanism of action.
The aim of this study is to explore the anti-inflammatory properties of Cymbopogon citratus leaves and their polyphenol-rich fractions (PFs), as well its mechanism of action in murine macrophages.
A lipid- and essential oil-free infusion of Cy leaves was prepared (Cy extract) and fractionated by column chromatography. Anti-inflammatory properties of Cy extract (1.115 mg/ml) and its PFs, namely phenolic acids (530 μg/ml), flavonoids (97.5 μg/ml) and tannins (78 μg/ml), were investigated using lipopolysaccharide (LPS)-stimulated Raw 264.7 macrophages as in vitro model. As inflammatory parameters, nitric oxide (NO) production was evaluated by Griess reaction, as well as effects on cyclooxygenase-2 (COX-2), inducible NO synthase (iNOS) expression and on intracellular signaling pathways activation, which were analyzed by Western blot using specific antibodies.
Cy extract inhibited iNOS expression, NO production and various LPS-induced pathways like p38 mitogen-activated protein kinase (MAPK), c-jun NH(2)-terminal kinase (JNK) 1/2 and the transcription nuclear factor (NF)-κB. The extracellular signal-regulated kinase (ERK) 1/2 and the phosphatidylinositol-3-kinase (PI3K)/Akt activation were not affected by Cy extract. Both phenolic acid- and tannin-rich fractions significantly inhibited NF-κB activation, iNOS expression and NO production but none of the PFs modulated MAPKs or PI3K/Akt activation. Neither Cy extract nor PFs affected LPS-induced COX-2 expression but LPS-induced PGE(2) production is inhibited by Cy extract and by phenolic acid-rich fraction.
Our data provide evidence that support the usage of Cymbopogon citratus leaves extract in traditional medicine, and suggest that Cy, in particular its polyphenolic compounds, could constitute a natural source of a new and safe anti-inflammatory drug.
Full-text · Article · Nov 2010 · Journal of ethnopharmacology
[Show abstract][Hide abstract] ABSTRACT: Leucocyte infiltration in the wall of varicose veins has been reported previously. This study was designed to investigate the expression of pro-inflammatory cytokines and chemokines in control and in patients with varicose veins and to test the effect of treating varicose vein patients with acetylsalicylic acid (ASA) on cytokine expression prior to removal of varices.
Sections of vein were removed during operation from both patient groups, and ribonuclease protection assays (RPAs) were performed to assess the expression of chemokines. Group I included non-varicose saphenous veins from healthy patients undergoing amputation for trauma. Varicose veins were obtained from patients with primary varicose undergoing surgical treatment who received no drug (group II) or treatment with 300 mg day(-1) of ASA for 15 days before surgery (group III).
Non-varicose veins constitutively expressed low levels of monocyte-chemoattractant protein (MCP-1) and interleukin (IL)-8 mRNA. Varicose veins had a distinct chemokine expression pattern, since significant up-regulation of MCP-1 and IL-8 and a marked expression of IP-10, RANTES, MIP-1alpha and MIP-1beta mRNA were detected. Removal of the endothelium did not alter this pattern. Varicose veins obtained from patients treated with ASA showed a consistent decrease in chemokine expression, although it did not reach statistical significance.
Varicose veins showed increased expression of several chemokines compared to control veins. A non-significant reduction of activation was observed following treatment with ASA for 15 days.
Preview · Article · Oct 2009 · European journal of vascular and endovascular surgery: the official journal of the European Society for Vascular Surgery
[Show abstract][Hide abstract] ABSTRACT: Dendritic cells (DC) are professional antigen-presenting cells with a unique capacity to initiate and modulate immune responses by their ability to prime naïve T-cells. Upon stimuli, DC experience several morphologic, phenotypic and functional changes in a process referred to as maturation. This process is crucial to the biological functions of DC since their maturation status confer them the ability to polarize distinct T-cell subsets. In this work we explored the relevance of PI3-Kinase, Mitogen-Activated Protein Kinases (MAPKs) and NF-kappaB on cytokines/chemokines and co-stimulatory molecules expression. As experimental model, we used a fetal skin-derived dendritic cell line (FSDC) induced to mature by treatment with lipopolysaccharide (LPS). Morphology and ultrastructure were analyzed by confocal and electron microscopies, respectively. Levels of phosphorylated proteins were evaluated by Western blot, production of cytokines/chemokines was analyzed by protein arrays and the expression of surface molecules was evaluated by flow cytometry. The effect of specific inhibitors of the studied signaling pathways on the transcription of cytokines/chemokines and co-stimulatory molecules was accessed by Quantitative Real-Time RT-PCR. The results showed that LPS induces significant morphological and ultrastructural changes in FSDC. Western blot analysis revealed that LPS challenge promotes an early and transient activation of NF-kappaB, ERK1/2, p38 MAPK, along with a more sustained PI3 kinase/AKT activation. The co-stimulatory CD40, CD80, CD86 and antigen-presenting MHC class I and II molecules were increased and among secreted molecules, interleukin IL-6, CCL5, G-CSF, CCL2, CXCL2 were strongly up-regulated. Using a pharmacological approach we observed that LPS-induced increase of these molecules was differentially regulated by the distinct signaling pathways. Moreover, the polarizing T(h)2 cytokines/chemokines induced by LPS in FSDC were found to be positively regulated by NF-kappaB and ERK and negatively modulated by p38 MAPK. Altogether these results suggest that the use of pharmacological inhibitors to manipulate DC maturation, namely the polarizing T(h)1/T(h)2 cytokine/chemokine profile, may be useful in the development of more specific immunotherapeutic protocols.
[Show abstract][Hide abstract] ABSTRACT: Vascular inflammation is a major atherogenic factor and Toll-like receptor (TLR) 2 ligands, including bacterial and serum lipoproteins, seem to be involved in atherogenesis. On this basis, we analysed the effect of lipoproteins and different lipid components on TLR2-dependent signalling.
In TLR2-transfected human embryonic kidney 293 cells and human monocytes, oxidized low-density lipoproteins inhibited nuclear factor (NF)-kappaB-driven transcriptional activity and chemokine gene expression in response to TLR2 ligands. Sphingosine 1-phosphate (S1P) and oxidized palmitoyl-arachidonoyl-phosphatidylcholine, but not lipoprotein-carried lysophospholipids, inhibited TLR2 activation. Silencing experiments in TLR2-transfected 293 cells showed that the S1P-mediated attenuation effect is mediated by S1P receptors type 1 and type 2. To address the physiological significance of these findings, additional experiments were performed in human peripheral blood monocytes and monocyte-derived macrophages. In both cell types, S1P selectively attenuated TLR2 signalling, as NF-kappaB and extracellular signal-regulated kinase activation, but not c-Jun amino terminal kinase phosphorylation, were inhibited by physiologically relevant concentrations of S1P. Moreover, the attenuation of TLR2 signalling was partially reverted by pharmacological inhibition of phosphoinositide 3-kinase (PI3K) and Ras pathways. In addition, S1P inhibited the chemokine gene expression elicited by TLR2, but not by TLR4 ligands.
These findings disclose a cross-talk mechanism between lipoprotein components and TLR in which engagement of S1P receptors exert selective attenuation of TLR2-dependent activation via PI3K and Ras signalling. A corollary to these data is that the negative cross-talk of S1P receptors and TLR2 signalling might be involved in the atheroprotective effects of S1P.
Preview · Article · Jun 2008 · Cardiovascular Research
[Show abstract][Hide abstract] ABSTRACT: Toll-like receptors (TLRs) are a family of proteins involved in the recognition of pathogen-associated molecular motifs that activate both innate and adaptive immune responses. TLR4 is the member of the family that binds to bacterially produced lipopolysaccharide (LPS). Although still on debate, increasing evidences show that TLRs are also able to recognize endogenous molecules. Two recent reports have highlighted the role of TLR4 signaling pathways as sensors of endogenous ‘danger’ signals and their association to pathogenesis. Vogl and colleagues describe a connection between TLR4 and septic shock through the calciumbinding S100 family of myeloid-related proteins (Mrp) 8 and 14, two proinflammatory molecules secreted by phagocytes upon activation. Apetoh and colleagues describe a new pathway whereby tumor cells dying after chemotherapy release the high-mobility group box 1 protein (HMGB1), which is recognized by TLR4 and induces an immune response that is required for the success of therapy. These interesting studies not only increase our understanding of the biology of TLR4, but also support the ‘danger’ model since both reports identify ‘self’ molecules, produced after tissue damage or chemotherapy-induced cell death, that act as ‘alarm’ signals activating the immune response through TLR4- dependent pathways. Moreover, these articles identify new targets for developing novel therapeutic treatments for septic shock and for the improvement of the efficacy of anticancer drugs.
[Show abstract][Hide abstract] ABSTRACT: Francisella tularensis is a virulent Gram-negative intracellular pathogen. To address the signaling routes involved in the response of host cells
to LPS from F. tularensis live vaccine strain (LVS), experiments were performed in transiently transfected 293 cells. Induction of κB-driven transcriptional
activity by 2.5 μg ml−1 F. tularensis LPS isolated by phenol–water and ether–water extraction, was observed in cells transfected with Toll-like receptor (TLR)
4 and MD-2, although CD14 was required for optimal induction. Conversely, TLR2, TLR2/TLR1 or TLR2/TLR6 transfected cells did
not show κB-driven transcriptional activity in the presence of F. tularensis LPS. In human monocytic cells, F. tularensis LPS activated extracellular signal-regulated kinases and the production of pro-inflammatory proteins. Concentrations of 5–10
μg ml−1 F. tularensis LPS elicited a similar pattern of mRNA and protein induction than 0.1 μg ml−1 E. coli LPS, including the expression of CXC chemokines (IL-8, Gro and IFN-γ-inducible protein-10); CC chemokines (monocyte chemoattractant
protein-1 and -2, macrophage-derived chemoattractant, macrophage inflammatory protein-1α and -1β and RANTES (regulated upon
activation, normal T cell expressed and secreted) and pro-inflammatory cytokines (IL-6 and tumor necrosis factor α). Altogether,
these data indicate that LPS from F. tularensis LVS signals via TLR4 at higher concentrations than those required for E. coli LPS, which may explain the inflammatory reaction and the low endotoxic response associated to vaccination with LVS in humans.
Preview · Article · Jun 2006 · International Immunology
[Show abstract][Hide abstract] ABSTRACT: The anti-inflammatory effects of salicylates, originally attributed to inhibition of cyclooxygenase activity, are currently known to involve additional mechanisms. In this study we investigated the possible modulation by salicylates of NFAT-mediated transcription in lymphocytic and monocytic cell lines. RNase protection assays showed that 2-acetoxy-4-trifluoromethylbenzoic acid (triflusal) inhibited, in a dose-dependent manner, mRNA expression of several cytokine genes, most of which are NFAT-regulated and cyclosporin A (CsA)-sensitive. In Jurkat cells, the expression of IL-3, GM-CSF, TNF-alpha, TGF-beta1, IL-2, lymphotactin, MIP-1alpha, and MIP-1beta was inhibited to different extents. In THP-1 cells, inhibition of the expression of M-CSF, G-CSF, stem cell factor, IFN-gamma, TNF-alpha, TGF-beta1, lymphotoxin-beta1, MIP-1alpha, MIP-1beta, and IL-8 was observed. Sodium salicylate and aspirin only showed significant effects at 5 mM. The transcriptional activity of two genes that contain NFAT sites, a GM-CSF full promoter and a T cell-specific enhancer from the IL-3 locus, was also inhibited by salicylates. Transactivation experiments performed with several NFAT-dependent and AP-1-dependent reporter genes showed that triflusal strongly inhibited NFAT-dependent transcription at concentrations as low as 0.25 mM. Sodium salicylate and aspirin were less potent. The triflusal inhibitory effect was reversible and synergized with suboptimal doses of CsA. Experiments to address the mechanism of action of salicylates in the NFAT activation cascade disclosed a mechanism different from that of CsA, because salicylates inhibited DNA-binding and NFAT-mediated transactivation without affecting phosphorylation or subcellular localization of NFAT. In summary, these data describe a new pharmacological effect of salicylates as inhibitors of NFAT-dependent transcription.
Preview · Article · Dec 2004 · The Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: Endotoxins displaying differences in the chemical structure of their lipid A were used to induce the expression of chemokines in the human monocytic THP-1 cell line. LPS from two enterobacterial species such as Escherichia coli and Yersinia enterocolitica induced mRNA expression of IFN-gamma-inducible protein (IP)-10, macrophage-inflammatory protein (MIP)-1alpha, MIP-1beta, monocyte chemoattractant protein (MCP)-1 and IL-8. LPS from the non-enterobacterial genera Brucella and Ochrobactrum induced the expression of these chemokines to a lower extent. Attempts to address the signaling routes involved in these responses were carried out in transiently transfected HEK293 cells. Induction of kappaB-driven transcriptional activity by enterobacterial LPS was observed in cells transfected with TLR-4 alone, although co-transfection of TLR-4, MD-2 and CD14 provided optimal induction. The response to Brucella spp. and Ochrobactrum anthropi LPS was only significant at the concentration of 10 microg/ml. These data indicate that LPS from Brucella spp. and O. anthropi, which contain lipid A moieties with structural features different from those of Enterobacteriaceae elicit biochemical signaling via TLR-4 only at high concentrations. Neither TLR-1, TLR-2 and TLR-6 nor heterodimeric combinations of these receptor molecules are involved. Conversely, the ability of LPS to activate the TLR-4 route is a reliable molecular biomarker for endotoxicity.
Preview · Article · Nov 2004 · International Immunology
[Show abstract][Hide abstract] ABSTRACT: In resting cells, the NFAT1 transcription factor is kept inactive in the cytoplasm by phosphorylation on multiple serine residues.
These phosphorylated residues are primarily contained within two types of serine-rich motifs, the SRR-1 and SP motifs, which
are conserved within the NFAT family. Several different kinases have been proposed to regulate NFAT, but no single candidate
displays the specificity required to fully phosphorylate both types of motifs; thus, the identity of the kinase that regulates
NFAT activity remains unclear. Here we show that the NFAT1 serine motifs are regulated by distinct kinases that must coordinate
to control NFAT1 activation. CK1 phosphorylates only the SRR-1 motif, the primary region required for NFAT1 nuclear import.
CK1 exists with NFAT1 in a high-molecular-weight complex in resting T cells but dissociates upon activation. GSK3 does not
phosphorylate the SRR-1 region but can target the NFAT1 SP-2 motif, and it synergizes with CK1 to regulate NFAT1 nuclear export.
We identify a conserved docking site for CK1 in NFAT proteins and show that mutation of this site disrupts NFAT1-CK1 interaction
and causes constitutive nuclear localization of NFAT1. The CK1 docking motif is present in proteins of the Wnt, Hedgehog,
and circadian-rhythm pathways, which also integrate the activities of CK1 and GSK3.
Full-text · Article · Jun 2004 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: Monocytic cells were stimulated with IgG-OVA equivalence immune complexes, mAb reacting with FcgammaRI, FcgammaRIIA, and FcgammaRIII, LPS, TNF-alpha, and the combination of ionomycin and phorbol ester, to address their effects on the expression of the mRNAs encoding for chemokines. Stimulation of monocytes with immune complexes induced a rapid expression of macrophage-inflammatory protein (MIP)-1alpha, MIP-1beta, and IL-8 mRNAs. In contrast, RANTES mRNA was already detectable in resting cells and only increased after 16 h of stimulation. A similar pattern was observed following homotypic stimulation of FcgammaR with mAb reacting with FcgammaRI and FcgammaRIIA, but not with a mAb reacting with FcgammaRIII, a subtype of receptor not expressed in THP-1 cells, thus indicating that both FcgammaRI and FcgammaRIIA are involved in the response. The pattern of chemokine induction elicited by LPS and the combination of ionomycin and PMA showed some similarities to those produced by FcgammaR cross-linking, although expression of IFN-gamma-inducible protein 10 mRNA was also observed in response to those agonists. The production of MIP-1alpha, MIP-1beta, and RANTES proteins encompassing the induction of their mRNAs was confirmed by specific ELISA. Experiments to address the transcription factors involved in the regulation of MIP-1alpha using pharmacological agents and EMSA showed the possible involvement of CCAAT/enhancer-binding protein beta sites and ruled out the functional significance of both NF-AT and AP-1 sites.
Preview · Article · Oct 2002 · The Journal of Immunology