25 research outputs found
C-terminal fusion of eGFP to the bradykinin B-2 receptor strongly affects down-regulation but not receptor internalization or signaling
A functional comparison was made between the wildtype bradykinin B, receptor (B(2)wt) and the chimera B(2)eGFP (enhanced green-fluorescent protein fused to the C-terminus of B(2)Wt), both stably expressed in HEK 293 cells. There was almost no difference in terms of ligand-inducible receptor phosphorylation and internalization, signal transduction (accumulation of inositol phosphates) or expression and affinity. However, stimulation for up to 8 h with 10 mu M bradykinin (BK) resulted in a strong decrease in surface receptors (by 60% within 5 h) in B(2)Wt, but not in B(2)eGFP. When the expression levels of both constructs where comparably reduced using a weaker promoter, long-term stimulation resulted in a reduction in surface receptors for B(2)wt(low) to less than 20% within 1 h, whereas the chimera B(2)eGFP(low) still displayed 50% binding activity after 2 h. A 1-h incubation in the absence of BK resulted in a recovery of 60% of the binding in B(2)wt(low) after 1-h stimulation with BK, but of only 20% after 7-h stimulation. In contrast, B(2)eGFP(low) levels were restored to more than 70%, even after 7-h stimulation. These data indicate that although the fusion of eGFP to B(2)wt does not affect its ligand-induced internalization, it strongly reduces the down-regulation, most likely by promoting receptor recycling over degradation
Guanosine nucleotides regulate B2 kinin receptor affinity of agonists but not of antagonists: Discussion of a model proposing receptor precoupling to G protein
The effect of nucleotides on binding of the B2 kinin (BK) receptor agonist {[}H-3]BK and the antagonist {[}H-3]NPC17731 to particulate fractions of human foreskin fibroblasts was studied. At 0 degrees C, particulate fractions exhibited a single class of binding sites with a Kd of 2.3 nM for {[}H-3]BK and a K-d Of 3.8 nM for the antagonist {[}H-3]NPC17731. Incubation with radioligands at 37 degrees C for 5 min gave a reduction of agonist, as well as antagonist, binding that was between 0-40% depending on the preparation, even in the absence of guanosine nucleotides. As shown by Scatchard analysis, this reduction in specific binding was due to a shift in the affinity of at least a fraction of the receptors. The presence at 37 degrees C of the guanine nucleotides GTP, GDP and their poorly hydrolyzable analogs left {[}H-3]-NPC17731 binding unaffected, but reduced the receptor affinity for {[}H-3]BK to a K-d Of about 15 nM. The maximal number of receptors, however, was unchanged. This affinity change was strongly dependent on the presence of bivalent cations, in particular Mg2+. It was reversed by incubation at 0 degrees C, The rank order of the guanosine nucleotides for {[}H-3]BK binding reduction was GTP{[}gamma S] = Gpp{[}NH]p > GTP = GDP > GDP{[}beta S]. GMP, ATP, ADP and AMP showed no influence on agonist binding. A model for the interaction of the B2 kinin receptor with G proteins is discussed
Chemokines and galectins form heterodimers to modulate inflammation
Chemokines and galectins are simultaneously upregulated and mediate leukocyte recruitment during inflammation. Until now, these effector molecules have been considered to function independently. Here, we tested the hypothesis that they form molecular hybrids. By systematically screening chemokines for their ability to bind galectin‐1 and galectin‐3, we identified several interacting pairs, such as CXCL12 and galectin‐3. Based on NMR and MD studies of the CXCL12/galectin‐3 heterodimer, we identified contact sites between CXCL12 β‐strand 1 and Gal‐3 F‐face residues. Mutagenesis of galectin‐3 residues involved in heterodimer formation resulted in reduced binding to CXCL12, enabling testing of functional activity comparatively. Galectin‐3, but not its mutants, inhibited CXCL12‐induced chemotaxis of leukocytes and their recruitment into the mouse peritoneum. Moreover, galectin‐3 attenuated CXCL12‐stimulated signaling via its receptor CXCR4 in a ternary complex with the chemokine and receptor, consistent with our structural model. This first report of heterodimerization between chemokines and galectins reveals a new type of interaction between inflammatory mediators that can underlie a novel immunoregulatory mechanism in inflammation. Thus, further exploration of the chemokine/galectin interactome is warranted
Identification of a non-canonical chemokine-receptor pathway suppressing regulatory T cells to drive atherosclerosis
CCL17 is produced by conventional dendritic cells, signals through CCR4 on regulatory T (Treg) cells and drives atherosclerosis by suppressing Treg functions through yet undefined mechanisms. Here we show that conventional dendritic cells from CCL17-deficient mice display a pro-tolerogenic phenotype and transcriptome that is not phenocopied in mice lacking its cognate receptor CCR4. In the plasma of CCL17-deficient mice, CCL3 was the only decreased cytokine/chemokine. We found that CCL17 signaled through CCR8 as an alternate high-affinity receptor, which induced CCL3 expression and suppressed Treg functions in the absence of CCR4. Genetic ablation of CCL3 and CCR8 in CD4+ T cells reduced CCL3 secretion, boosted FoxP3+ Treg numbers and limited atherosclerosis. Conversely, CCL3 administration exacerbated atherosclerosis and restrained Treg differentiation. In symptomatic versus asymptomatic human carotid atheroma, CCL3 expression was increased, whereas FoxP3 expression was reduced. Together, we identified a non-canonical chemokine pathway whereby CCL17 interacts with CCR8 to yield a CCL3-dependent suppression of atheroprotective Treg cells. Doring, van der Vorst, Yan, Neideck et al. present a non-canonical chemokine pathway involving CCL17 signaling through CCR8, which induces CCL3 expression independent of CCR4 and suppresses the functions of atheroprotective Treg cells
Kinin release from human kininogen by 10 aspartic proteases produced by pathogenic yeast Candida albicans
Chronic Intake of the Selective Serotonin Reuptake Inhibitor Fluoxetine Enhances Atherosclerosis
Objective Cardiovascular diseases and depression are the leading causes of disability in Western countries. Clinical data on potential cardiovascular effects of serotonin reuptake inhibitors (SSRIs), the most commonly used antidepressant drugs, are controversial. In addition to blocking serotonin reuptake transporter in the brain, SSRIs deplete the major peripheral serotonin (5-hydroxytryptamine [5-HT]) storage by inhibiting serotonin reuptake transporter-mediated uptake in platelets. In this study, we aimed to investigate the effect of chronic SSRI intake on the development of atherosclerosis. Approach and Results Treatment of apolipoprotein E-deficient mice with the SSRI fluoxetine for 2, 4, or 16 weeks increased atherosclerotic lesion formation, with most pronounced effect during early plaque development. Intravital microscopy of carotid arteries revealed enhanced myeloid cell adhesion on fluoxetine treatment. Mechanistically, we found that fluoxetine augmented vascular permeability and increased chemokine-induced integrin-binding activity of circulating leukocytes. In vitro stimulation of murine blood demonstrated that fluoxetine, but not 5-HT, could directly promote 1 and 2 integrin activation provided C-C motif chemokine ligand 5 was also present. Similar effects were observed with the SSRI escitalopram. Enhanced C-C motif chemokine ligand 5-induced integrin activation by fluoxetine was also confirmed in a human neutrophil-like cell line. In contrast to the proatherogenic properties of fluoxetine, pharmacological inhibition of the peripheral 5-HT synthesizing enzyme tryptophan hydroxylase 1 did not promote atherosclerosis, suggesting that the proatherogenic effect of fluoxetine occurs independent of peripheral 5-HT depletion. Conclusions SSRI intake may promote atherosclerosis and therefore potentially increase the risk for acute cardiovascular events by a mechanism that is independent of 5-HT depletion
Deficiency of Monoacylglycerol Lipase Enhances IgM Plasma Levels and Limits Atherogenesis in a CB2-Dependent Manner.
Cross-Linking GPVI-Fc by Anti-Fc Antibodies Potentiates Its Inhibition of Atherosclerotic Plaque- and Collagen-Induced Platelet Activation
SummaryTo enhance the antithrombotic properties of recombinant glycoprotein VI fragment crystallizable (GPVI-Fc), the authors incubated GPVI-Fc with anti-human Fc antibodies to cross-link the Fc tails of GPVI-Fc. Cross-linking potentiated the inhibition of human plaque- and collagen-induced platelet aggregation by GPVI-Fc under static and flow conditions without increasing bleeding time in vitro. Cross-linking with anti-human-Fc Fab2 was even superior to anti-human-Fc immunoglobulin G (IgG). Advanced optical imaging revealed a continuous sheath-like coverage of collagen fibers by cross-linked GPVI-Fc complexes. Cross-linking of GPVI into oligomeric complexes provides a new, highly effective, and probably safe antithrombotic treatment as it suppresses platelet GPVI-plaque interaction selectively at the site of acute atherothrombosis
CD27 co-stimulation increases the abundance of regulatory T cells and reduces atherosclerosis in hyperlipidaemic mice
Aims The co-stimulatory receptor CD27 modulates responses of T cells, B cells, and NK cells. Various T cell subsets participate in atherogenesis. However, the role of CD27 in atherosclerosis remains unexplored. Methods and results Here we investigated the effect of bone marrow-derived and systemic CD27 deficiency in Apolipoprotein E-deficient (Apoe(-/-)) mice in early and advanced stages of atherosclerosis. Lethally-irradiated Apoe(-/-) mice reconstituted with Cd27(-/-)Apoe(-/-) bone marrow and consuming an atherogenic diet displayed a markedly increased plaque size and lesional inflammation compared to mice receiving Cd27(+/+)Apoe(-/-) bone marrow. Accordingly, chow diet-fed Cd27(-/-)Apoe(-/-) mice showed exacerbated lesion development and increased inflammation at the age of 18 weeks. At a more advanced stage of atherosclerosis (28 weeks), lesion size and phenotype did not differ between the two groups. Systemic and bone marrow-derived CD27 deficiency reduced the abundance of regulatory T cells (Treg) in blood, lymphoid organs, and the aorta. Numbers of other immune cells were not affected while expression of inflammatory cytokine genes (e.g. IL-1 beta and IL-6) was increased in the aorta when haematopoietic CD27 was lacking. In vitro, Tregs of CD27-deficient mice showed similar suppressive capacity compared with their wild-type controls and migrated equally towards CCL19 and CCL21. However, thymic Cd27(-/-) Tregs underwent increased apoptosis and expressed fewer markers of proliferation in vivo. Reconstitution of Cd27(-/-)Apoe(-/-) mice with Cd27(+/+)Apoe(-/-) Tregs reversed the increase in atherosclerosis. Conclusion We demonstrate that CD27 co-stimulation increases the number of Tregs and limits lesion development and inflammation in experimental atherosclerosis, particularly during early stages of disease. Thus, our study suggests that promotion of CD27 function may mitigate atherosclerosi