Carmen García-Rodríguez

Activation of monocytic cells through Fcγ receptors induces the expression of macrophage-inflammatory protein (MIP)-1α, MIP-1β, and RANTES

Monocytic cells were stimulated with IgG-OVA equivalence immune complexes, mAb reacting with FcγRI, FcγRIIA, and FcγRIII, LPS, TNF-α, 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)-1α, MIP-1β, 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 FcγR with mAb reacting with FcγRI and FcγRIIA, but not with a mAb reacting with FcγRIII, a subtype of receptor not expressed in THP-1 cells, thus indicating that both FcγRI and FcγRIIA 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 FcγR cross-linking, although expression of IFN-γ-inducible protein 10 mRNA was also observed in response to those agonists. The production of MIP-1α, MIP-1β, 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-1α using pharmacological agents and EMSA showed the possible involvement of CCAAT/enhancer-binding protein β sites and ruled out the functional significance of both NF-AT and AP-1 sites.

Anti-inflammatory activity of Cymbopogon citratus leaves infusion via proteasome and nuclear factor-κB pathway inhibition: contribution of chlorogenic acid

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-MS(n). 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-MS(n), 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.

Cymbopogon citratus as source of new and safe anti-inflammatory drugs: Bio-guided assay using lipopolysaccharide-stimulated macrophages

ETHNOPHARMACOLOGICAL RELEVANCE 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. AIM OF THE STUDY 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. MATERIALS AND METHODS 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. RESULTS 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. CONCLUSIONS 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.

Lipopolysaccharide and Sphingosine-1-Phosphate Cooperate To Induce Inflammatory Molecules and Leukocyte Adhesion in Endothelial Cells

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 S1P1/3 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.

A New Pharmacological Effect of Salicylates: Inhibition of NFAT-Dependent Transcription

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-α, TGF-β1, IL-2, lymphotactin, MIP-1α, and MIP-1β was inhibited to different extents. In THP-1 cells, inhibition of the expression of M-CSF, G-CSF, stem cell factor, IFN-γ, TNF-α, TGF-β1, lymphotoxin-β1, MIP-1α, MIP-1β, 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.

Selective attenuation of Toll-like receptor 2 signalling may explain the atheroprotective effect of sphingosine 1-phosphate

AIMS 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. METHODS AND RESULTS 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. CONCLUSION 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.

Differential roles of PI3-Kinase, MAPKs and NF-κB on the manipulation of dendritic cell Th1/Th2 cytokine/chemokine polarizing profile

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.

Viral and bacterial patterns induce TLR-mediated sustained inflammation and calcification in aortic valve interstitial cells.

BACKGROUND Aortic stenosis shares some ethiopathological features with atherosclerosis and increasing evidence links Toll-like receptors (TLRs) to atherogenesis. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Varicose Veins Show Enhanced Chemokine Expression

OBJECTIVES 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. MATERIAL AND METHODS 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). RESULTS 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. CONCLUSIONS 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.

Aging and amyloid β oligomers enhance TLR4 expression, LPS-induced Ca2+ responses, and neuron cell death in cultured rat hippocampal neurons

BackgroundToll-like receptors (TLRs) are transmembrane pattern-recognition receptors of the innate immune system recognizing diverse pathogen-derived and tissue damage-related ligands. It has been suggested that TLR signaling contributes to the pathogenesis of age-related, neurodegenerative diseases, including Alzheimer’s disease (AD). AD is associated to oligomers of the amyloid β peptide (Aβo) that cause intracellular Ca2+ dishomeostasis and neuron cell death in rat hippocampal neurons. Here we assessed the interplay between inflammation and Aβo in long-term cultures of rat hippocampal neurons, an in vitro model of neuron aging and/or senescence.MethodsCa2+ imaging and immunofluorescence against annexin V and TLR4 were applied in short- and long-term cultures of rat hippocampal neurons to test the effects of TLR4-agonist LPS and Aβo on cytosolic [Ca2+] and on apoptosis as well as on expression of TLR4.ResultsLPS increases cytosolic [Ca2+] and promotes apoptosis in rat hippocampal neurons in long-term culture considered aged and/or senescent neurons, but not in short-term cultured neurons considered young neurons. TLR4 antagonist CAY10614 prevents both effects. TLR4 expression in rat hippocampal neurons is significantly larger in aged hippocampal cultures. Treatment of aged hippocampal cultures with Aβo increases TLR4 expression and enhances LPS-induced Ca2+ responses and neuron cell death.ConclusionsAging and amyloid β oligomers, the neurotoxin involved in Alzheimer’s disease, enhance TLR4 expression as well as LPS-induced Ca2+ responses and neuron cell death in rat hippocampal neurons aged in vitro.