Down-regulation of microglial cyclo-oxygenase-2 and inducible nitric oxide synthase expression by lipocortin 1.
1. Activated microglial cells are believed to play an active role in most brain pathologies, during which they can contribute to host defence and repair but also to the establishment of tissue damage. These actions are largely mediated by microglial secretory products, among which are prostaglandins (PGs) and nitric oxide (NO). 2. The anti-inflammatory protein, lipocortin 1 (LC1) was reported to have neuroprotective action and to be induced by glucocorticoids in several brain structures, with a preferential expression in microglia. In this paper we tested whether the neuroprotective effect of LC1 could be explained by an inhibitory effect on microglial activation. 3. We have previously shown that bacterial endotoxin (LPS) strongly stimulates PGE2 and NO production in rat primary microglial cultures, by inducing the expression of the key enzymes cyclo-oxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), respectively. 4. Dexamethasone (DEX, 1-100 nM) and LC1-derived N-terminus peptide (peptide Ac2-26, 1-100 microg ml(-1)) dose-dependently inhibited the production of both PGE2 and NO from LPS-stimulated microglia. The inhibitory effects of DEX on NO and of the peptide on NO and PGE2 synthesis were partially abrogated by a specific antiserum, raised against the N-terminus of human LC1. The peptide Ac2-26 did not affect arachidonic acid release from control and LPS-stimulated microglial cultures. 5. Western blot experiments showed that the LPS-induced expression of COX-2 and iNOS was effectively down-regulated by DEX (100 nM) and peptide Ac2-26 (100 microg ml(-1)). 6. In conclusion, our findings support the hypothesis that LC1 may foster neuroprotection by limiting microglial activation, through autocrine and paracrine mechanisms. (+info)
Modulation of cellular annexin I in human leukocytes infiltrating DTH skin reactions.
Based on our previous studies showing endogenous annexin I being depleted from migrated neutrophils (PMN) in vitro, we have tested whether the levels of this glucocorticoid-regulated protein in PMN and mononuclear cells (PBMC) were modified after adhesion to endothelial monolayers in vitro and extravasation into skin blisters in vivo. In vitro, annexin I levels were depleted more significantly (-70%) in post-adherent PMNs than in monocytes (-25%) and lymphocytes (-50%, only in the positive fraction). In vivo, a significant time-dependent increase (approximately threefold, P < 0.05) in cell-associated annexin I was measured in PBMCs recovered from the blisters, whereas no significant changes were detected in extravasated PMNs. This was associated with annexin I release in the blister fluids (approximately 35 ng/mL), whereas no detectable protein was found in matched-paired plasmas. In conclusion, we report for the first time an activation of the annexin I pathway during an ongoing experimental inflammatory response in humans, which is differently regulated between PMNs and PBMCs. (+info)
Inhibitory effect of annexin I on synovial inflammation in rat adjuvant arthritis.
OBJECTIVE: Annexin I is an endogenous antiinflammatory mediator, expressed in rheumatoid arthritis (RA) synovium, the contribution of which to autoregulation of the synovial inflammatory response has not been examined in models of RA. We investigated the antiinflammatory role of annexin I in rat adjuvant arthritis. METHODS: Rats with adjuvant-induced arthritis (AIA) were treated with a specific anti-annexin I monoclonal antibody (mAb), isotype control IgG, and/or dexamethasone. Clinical outcomes and synovial synthesis of tumor necrosis factor alpha (TNFalpha), prostaglandin E2 (PGE2), and nitric oxide were examined, and annexin I expression was assessed by flow cytometry and reverse transcription-polymerase chain reaction. RESULTS: Anti-annexin I mAb reversed the effects of dexamethasone on the clinical features of AIA and exacerbated AIA in the absence of exogenous glucocorticoid. Clinical exacerbation of AIA by anti-annexin I mAb was accompanied by significantly increased synovial TNFalpha and PGE2, suggesting that annexin I tonically inhibits the production of these mediators. Anti-annexin I mAb treatment was associated with significantly reduced leukocyte intracellular annexin I, despite increased annexin I messenger RNA expression, consistent with a depletion effect of extracellular mAb via the cell surface. CONCLUSION: Annexin I is a key endogenous inhibitory mediator of arthritis via mechanisms that include inhibition of cytokine and effector molecule production. Moreover, a synthesis-independent depletion of intracellular annexin I by extracellular antibody supports the hypothesis that externalization of annexin I is involved in its mode of action. (+info)
Annexin I modulates cell functions by controlling intracellular calcium release.
Annexin I is an intracellular protein in search of a function. Ex vivo it has calcium- and phospholipid-binding properties. To evaluate its role in vivo, MCF-7 cells were stably transfected with annexin I in sense or antisense orientations. In cells overexpressing annexin I, calcium release was abrogated on stimulation of purinergic or bradykinin receptors, whereas non-transfected cells or cells with down-regulated annexin I released calcium within seconds. Basal calcium and calcium stores were not affected. The impaired calcium release was paralleled by a down-regulation of the activities of phospholipase C, group II phospholipase A2, and E-cadherin with altered adhesion and enhanced tumor growth on soft agar. Significantly smaller tumors, with the histologically most differentiated cells, were observed in nude mice inoculated with cells transfected with the antisense rather than with the sense plasmid. These observations indicate that annexin I modulates cell functions by controlling intracellular calcium release. Frey, B. M., Reber, B. F. X., Vishwanath, B. S., Escher, G., Frey, F. J. Annexin I modulates cell functions by controlling intracellular calcium release. (+info)
The inhibitory effect of dexamethasone on lymphocyte adhesion molecule expression and intercellular aggregation is not mediated by lipocortin 1.
Glucocorticoids exert their anti-inflammatory activity through multiple pathways which include the inhibition of cell adhesion events. The glucocorticoid-induced protein lipocortin 1 (LC1) has reported anti-inflammatory properties and has been proposed as a putative mediator of the anti-inflammatory effects of glucocorticoids. The role of LC1 in mediating the glucocorticoid inhibition of lymphocyte adhesion and cell adhesion molecule (CAM) expression was investigated in vitro using a microaggregation assay, flow cytometry and confocal microscopy. Lymphocytes stimulated for 96 h with plastic-bound OKT3 antibody showed significant increases in LFA-1 and CD2 expression. Dexamethasone (DEX; 10(-6) M) inhibited this increase but the neutralizing anti-LC1 MoAb 1A (5 microg/ml) failed to reverse the DEX effect; neither was purified human LC1 (50 x 10(-9) M) able to inhibit CAM expression. The biological activity of the LC1 was confirmed by its ability to suppress monocyte phagocytosis and respiratory burst in response to bovine serum albumin (BSA)-anti-BSA complexes. OKT3 stimulation of cultured mononuclear cells resulted in intercellular aggregation, scored microscopically using a visual index. This aggregation was completely reversed by 10-6 M DEX but unaffected by LC1 (50 x 10(-9) M). Significant intracellular expression of lymphocyte LC1 was observed using the anti-LC1 MoAb 1B in saponin-permeabilized cells. Distribution of LC1 had a diffuse, cytoplasmic pattern. LC1 expression was reduced following 3 h treatment with 10(-6) M DEX. These findings indicate that the DEX effects on lymphocyte adhesion and CAM expression are not mediated by LC1. Thus the reported in vivo effects of LC1 on leucocyte adhesion and transmigration probably occur through functional/conformation changes of surface CAM, rather than by alteration in expression. (+info)
The annexin protein lipocortin 1 regulates the MAPK/ERK pathway.
Lipocortin 1 (annexin 1) is a calcium- and phospholipid-binding protein that modulates anti-inflammatory responses including those induced by lipopolysaccharide. To investigate the precise role of lipocortin 1 in regulating the lipopolysaccharide-induced signal transduction pathways, we generated stable RAW 264.7 macrophage cell lines expressing decreased and increased lipocortin 1 protein. Several RAW 264.7 clones with increased lipocortin 1 protein levels showed constitutive activation of the mitogen-activated protein kinase extracellular signal-regulated kinase, which was down-regulated following lipopolysaccharide treatment. Conversely, clones with decreased lipocortin 1 protein expression showed prolonged extracellular signal-regulated kinase activity, following lipopolysaccharide activation. Lipocortin 1 specifically regulates the components of the extracellular signal-regulated kinase pathway, since changes in lipocortin 1 protein expression had no affect on the related mitogen-activated protein kinases p38 and c-Jun N-terminal kinase. Lipocortin 1 modulated upstream components of the extracellular signal-regulated kinase pathway and associated with the adaptor protein growth factor binding protein. The downstream consequences of altered extracellular signal-regulated kinase activity were independent of the proinflammatory transcription factor nuclear factor kappa B. These data indicate that lipocortin 1 specifically regulates proximal signaling components of the extracellular signal-regulated kinase signal transduction pathway, resulting in the modulation of biochemical functions in RAW macrophages. (+info)
Structural basis of the Ca(2+)-dependent association between S100C (S100A11) and its target, the N-terminal part of annexin I.
BACKGROUND: S100C (S100A11) is a member of the S100 calcium-binding protein family, the function of which is not yet entirely clear, but may include cytoskeleton assembly and dynamics. S100 proteins consist of two EF-hand calcium-binding motifs, connected by a flexible loop. Like several other members of the family, S100C forms a homodimer. A number of S100 proteins form complexes with annexins, another family of calcium-binding proteins that also bind to phospholipids. Structural studies have been undertaken to understand the basis of these interactions. RESULTS: We have solved the crystal structure of a complex of calcium-loaded S100C with a synthetic peptide that corresponds to the first 14 residues of the annexin I N terminus at 2.3 A resolution. We find a stoichiometry of one peptide per S100C monomer, the entire complex structure consisting of two peptides per S100C dimer. Each peptide, however, interacts with both monomers of the S100C dimer. The two S100C molecules of the dimer are linked by a disulphide bridge. The structure is surprisingly close to that of the p11-annexin II N-terminal peptide complex solved previously. We have performed competition experiments to try to understand the specificity of the S100-annexin interaction. CONCLUSIONS: By solving the structure of a second annexin N terminus-S100 protein complex, we confirmed a novel mode of interaction of S100 proteins with their target peptides; there is a one-to-one stoichiometry, where the dimeric structure of the S100 protein is, nevertheless, essential for complex formation. Our structure can provide a model for a Ca(2+)-regulated annexin I-S100C heterotetramer, possibly involved in crosslinking membrane surfaces or organising membranes during certain fusion events. (+info)
Detection of annexins I and IV in bronchoalveolar lavage fluids from calves inoculated with bovine herpes virus-1.
Annexins are phospholipid-binding proteins and are abundant in the lung. Annexins I and IV, but not II and VI, have been detected in bronchoalveolar lavage (BAL) fluids from calves inoculated with Pasteurella haemolytica, the pathogen for calf pneumonia. In this study, BAL fluids from calves with experimental pneumonia induced by inoculation to right lung lobes of bovine herpes virus-1 (BHV-1), the major viral pathogen for pneumonia, were examined for detection of annexins I and IV. Of 6 calves inoculated with BHV-1, annexins I and IV were coincidentally detected in BAL fluids from right lung lobes of 4 calves, but not in BAL fluids from left lung lobes of 6 inoculated calves or those from left and right lung lobes of 3 control calves. Annexin II and VI were not found in any BAL fluids examined. These results, together with previous findings on calves inoculated with Pasteurella haemolytica, suggest that the release of annexins I and IV onto the alveolar surface is an essential event occurring in response to pulmonary infections of BHIV-1 and Pasteurella haemolytica. (+info)