Adrenomedullin, a neuropeptide with immunoregulatory properties induces semi-mature tolerogenic dendritic cells.

Dendritic cells (DC) play a pivotal role in tolerance. Adrenomedullin (AM), a neuropeptide with anti-apoptotic and anti-inflammatory effects, may decrease T helper type 1 effector cells and induce regulatory T (Treg) cells. The aim of this study was to evaluate AM effects on murine dendritic cell (DC) maturation and functions. Bone marrow-derived DC were produced and stimulated with CpG motifs, lipopolysaccharide or AM for 24 hr. Then, DC maturation and expression of AM and AM receptors were evaluated. Compared with lipopolysaccharide-stimulated or CpG-stimulated DC, AM-stimulated DC had lower levels of co-stimulatory molecule expression and pro-inflammatory cytokine release. The AM induced high levels of interferon-γ but not of interleukin-10. Importantly, AM inhibited lipopolysaccharide-induced maturation of DC. However, allogeneic T-cell stimulation and endocytic capacity of AM-stimulated DC were comparable to those of semi-mature and mature DC. Moreover, DC expressed AM and its receptors at a basal level, and AM receptor expression increased with DC maturation. The AM stimulation induced indoleamine 2,3-dioxygenase (IDO) expression, promoting Treg cell expansion. For the first time, we describe the DC maturation phenotype by a neuropeptide (AM). We have demonstrated that AM and its receptors are expressed in DC and that exogenous AM can modify the DC phenotype and functions and can induce a semi-mature DC phenotype with IDO expression. These results indicate close interactions among immune system regulation mechanisms and calcitonin-like peptides.

Adrenomedullin(22-52) combats inflammation and prevents systemic bone loss in murine collagen-induced arthritis.

OBJECTIVE: Adrenomedullin(22-52) is a truncated peptide derived from adrenomedullin, a growth factor with antiapoptotic and immunoregulatory properties. It can act as an agonist or an antagonist depending on cell type. Its in vivo effects are unknown, but adrenomedullin(22-52) could possess immunomodulatory properties. This study was undertaken to evaluate the effect of adrenomedullin(22-52) in a mouse model of arthritis. METHODS: DBA/1 mice with collagen-induced arthritis (CIA) were treated with 1.2 µg/gm adrenomedullin(22-52) , adrenomedullin, or saline at arthritis onset. Bone mineral density was measured at the beginning of the experiment and when mice were killed. Mouse joints were processed for histologic analysis and protein studies, and spleens were examined for Treg cell expression. Cytokine expression was studied in mouse joint tissue and serum. RESULTS: In mice with CIA, adrenomedullin and adrenomedullin(22-52) reduced clinical and histologic arthritis scores and shifted the pattern of articular and systemic cytokine expression from Th1 to Th2, as compared to untreated mice with CIA (controls). Tumor necrosis factor α, interleukin-6 (IL-6), and IL-17A levels were significantly decreased in the joints of mice with CIA treated with adrenomedullin or adrenomedullin(22-52) as compared to controls, whereas IL-4 and IL-10 levels were increased. Adrenomedullin(22-52) was more effective than adrenomedullin in modulating cytokine content and enhanced Treg cell function without changing Treg cell expression compared to controls. Adrenomedullin receptor binding and transcriptional adrenomedullin receptor expression were markedly increased in joints from controls, whereas adrenomedullin receptor binding was considerably decreased in treated animals. Mice with CIA treated with adrenomedullin or adrenomedullin(22-52) had considerably fewer apoptotic chondrocytes and diminished cartilage degradation. Adrenomedullin(22-52) completely prevented systemic bone loss by preserving osteoblastic activity, but without changes in osteoclastic activity. CONCLUSION: Our findings indicate that adrenomedullin(22-52) , which has no vasoactive or tumor-inducing effects, is a potent antiinflammatory and bone-protective agent in this arthritis model.

Adrenomedullin increases fibroblast-like synoviocyte adhesion to extracellular matrix proteins by upregulating integrin activation.

INTRODUCTION: Rheumatoid arthritis (RA) is characterized by bone and cartilage invasion by fibroblast-like synoviocytes (FLSs). Adrenomedullin, a peptide with anabolic and antiapoptotic properties, is secreted by rheumatoid FLSs. Adrenomedullin also increases the expression of adhesion molecules in endothelial cells and keratinocytes. Here, we investigated whether adrenomedullin mediated FLS adhesion to extracellular matrix (ECM) proteins. METHODS: FLSs were isolated from synovial tissues from RA and osteoarthritis (OA) patients. Plates were coated overnight with the ECM proteins vitronectin, fibronectin, and type I collagen (Coll.I). Adrenomedullin was used as a soluble FLS ligand before plating. We tested interactions with the adrenomedullin receptor antagonist (22-52)adrenomedullin and with the protein kinase A (PKA) inhibitor H-89, and inhibition of co-receptor RAMP-2 by siRNA. Cell adhesion was measured by using color densitometry. Activation of α2 and ß1 integrins was evaluated by fluorescent microscopy; integrin inhibition, by RGD peptides; and the talin-integrin interaction, by immunoprecipitation (IP). RESULTS: Adrenomedullin specifically increased RA-FLS adhesion to vitronectin, fibronectin, and Coll.I; no such effect was found for OA-FLS adhesion. Basal or adrenomedullin-stimulated RA-FLS adhesion was inhibited by (22-52)adrenomedullin, H-89, and RAMP-2 siRNA. Adrenomedullin-stimulated adhesion was inhibited by RGD peptides, and associated with α2 and ß1 integrin activation. This activation was shown with IP to be related to an integrin-talin interaction and was significantly decreased by (22-52)adrenomedullin. CONCLUSIONS: Adrenomedullin-stimulated RA-FLS adhesion was specific for ECM proteins and mediated by α2 and ß1 integrins. This effect of adrenomedullin was dependent on adrenomedullin receptors. These results support a new role for adrenomedullin in rheumatoid synovial fibroblast pathobiology.

Interaction of the electrophilic ketoprofenyl-glucuronide and ketoprofenyl-coenzyme A conjugates with cytosolic glutathione S-transferases.

Carboxylic acid-containing drugs are metabolized mainly through the formation of glucuronide and coenzyme A esters. These conjugates have been suspected to be responsible for the toxicity of several nonsteroidal anti-inflammatory drugs because of the reactivity of the electrophilic ester bond. In the present study we investigated the reactivity of ketoprofenyl-acylglucuronide (KPF-OG) and ketoprofenyl-acyl-coenzyme A (KPF-SCoA) toward cytosolic rat liver glutathione S-transferases (GST). We observed that KPF-SCoA, but not KPF-OG inhibited the conjugation of 1-chloro-2,4-dinitrobenzene and 4-nitroquinoline N-oxide catalyzed by both purified cytosolic rat liver GST and GST from FAO and H5-6 rat hepatoma cell lines. Photoaffinity labeling with KPF-SCoA suggested that the binding of this metabolite may overlap the binding site of 4-methylumbelliferone sulfate. Furthermore, high-performance liquid chromatography and mass spectrometry analysis showed that both hydrolysis and transacylation reactions were observed in the presence of GST and glutathione. The formation of ketoprofenyl-S-acyl-glutathione could be kinetically characterized (apparent K(m) = 196.0 +/- 70.6 microM). It is concluded that KPF-SCoA is both a GST inhibitor and a substrate of a GST-dependent transacylation reaction. The reactivity and inhibitory potency of thioester CoA derivatives toward GST may have potential implications on the reported in vivo toxicity of some carboxylic acid-containing drugs.

Irreversible inhibition of glucose-6-phosphate dehydrogenase by the coenzyme A conjugate of ketoprofen: a key to oxidative stress induced by non-steroidal anti-inflammatory drugs?

Oxidative damage by non-steroidal anti-inflammatory drugs (NSAIDs) has been considered relevant to the occurrence of gastro-intestinal side-effects. In the case of chiral arylpropionate derivatives like ketoprofen (KPF), this mechanism has been evidenced for the R-enantiomer, especially when chiral inversion was observed, and lets us suppose the involvement of CoA conjugates. Glucose-6-phosphate dehydrogenase (G6PD) is the crucial enzyme to regenerate the GSH pool and maintain the intracellular redox potential. This enzyme is known to be down-regulated by palmitoyl-CoA thioester. We hypothesised then that G6PD is the target of carboxylic NSAIDs, via their CoA metabolites. We used molecular docking to localise a putative site in the human G6PD then we chose the Yeast orthologue, as the most suitable species to study experimentally the precise molecular interaction. KPF-CoA was effectively shown to bind covalently to the unique cysteine residue of the yeast enzyme. Binding was found to occur in the same site as palmitoyl-CoA. It was decreased in the presence of an allosteric inhibitor of G6PD, phospho(enol)pyruvate, and was not detected with G6PD of Leuconostoc mesenteroides, which does not possess the allosteric site. This site is distinct from the catalytic site, and probably allosteric, explaining the observed non-competitive inhibition of its activity by KPF-CoA. KPF-CoA was shown to induce the production of reactive oxygen species in Caco-2 cells, where its inhibition of G6PD activity was observed.