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Down-regulation of microglial cyclo-oxygenase-2 and inducible nitric oxide synthase by lipocortin 1
British Journal of Pharmacology
Abstract
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).
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.
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.
Dexamethasone (DEX, 1–100 nM) and LC1-derived N-terminus peptide (peptide Ac2-26, 1–100 μg 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.
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 μg ml−1).
In conclusion, our findings support the hypothesis that LC1 may foster neuroprotection by limiting microglial activation, through autocrine and paracrine mechanisms.
British Journal of Pharmacology (1999) 126, 1307–1314; doi:10.1038/sj.bjp.0702423
... After comparing these three annexins with sequences from Ribeiro et al. (2014), Vieira et al. (2015) and Ouali et al. (2020) through Basic Local Alignment Search Tool (BLAST) analyses, we noted that these authors already found them confirming previous inferences from the complete genome sequence. Annexins are a family of proteins that are associated with many biological events, including calcium-binding, interaction with membranes, intracellular vesicle trafficking, arachidonic acid release, leukocyte migration, and that also affects several mediators involved in the inflammatory response including cyclo-oxygenase-2 (Cox-2) and inducible nitric oxide synthase (Minghetti et al., 1999;Ferlazzo et al., 2003;Rescher and Gerke, 2004;Gavins and Hickey, 2012). Annexins presents a highly variable amino-terminal domain, possibly resulting in distinct functions specific to the members of a given family (Gerke and Moss, 2002;Moss and Morgan, 2004;Rescher and Gerke, 2004). ...
... Nitric oxide (NO) is released during the acute phase of T. cruzi infection in mice and treatment with inhibitors of NO synthase exacerbates the infection (Vespa et al., 1994;Hölscher et al., 1998). Annexin regulation is associated with nitric oxide synthase (NOS) induction by bacterial lipopolysaccharide in macrophages (Wu et al., 1995;Minghetti et al., 1999;Ferlazzo et al., 2003;Rescher and Gerke, 2004;Gavins and Hickey, 2012). Nitric oxide (NO) is known to have a protective effect on the gastrointestinal tract. ...
... Glucocorticoids inhibit prostaglandins and leukotrienes, the two main products of inflammation, at the level of PLA2 as well as at the level of cyclooxygenase/PGE isomerase (COX-1 and COX-2; Goppelt-Struebe et al., 1989), which potentiate the anti-inflammatory effect (Raynal and Pollard, 1994;Mira et al., 1997;Rao, 2007). Moreover as outlined above, annexin regulation is associated with NOS induction by bacterial lipopolysaccharide in macrophages (Minghetti et al., 1999;Ferlazzo et al., 2003;Rescher and Gerke, 2004;Gavins and Hickey, 2012). However, in triatomine cellular defense is performed by hemocytes, but these cells remains in the hemolymph and were never reported in the digestive tract lumen. ...
Rhodnius prolixus, Panstrongylus megistus, Triatoma infestans, and Dipetalogaster maxima are all triatomines and potential vectors of the protozoan Trypanosoma cruzi responsible for human Chagas' disease. Considering that the T. cruzi's cycle occurs inside the triatomine digestive tract (TDT), the analysis of the TDT protein profile is an essential step to understand TDT physiology during T. cruzi infection. To characterize the protein profile of TDT of D. maxima, P. megistus, R. prolixus, and T. infestans, a shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach was applied in this report. Most proteins were found to be closely related to metabolic pathways such as gluconeogenesis/glycolysis, citrate cycle, fatty acid metabolism, oxidative phosphorylation, but also to the immune system. We annotated this new proteome contribution gathering it with those previously published in accordance with Gene Ontology and KEGG. Enzymes were classified in terms of class, acceptor, and function, while the proteins from the immune system were annotated by reference to the pathways of humoral response, cell cycle regulation, Toll, IMD, JNK, Jak-STAT, and MAPK, as available from the Insect Innate Immunity Database (IIID). These pathways were further subclassified in recognition, signaling, response, coagulation, melanization and none. Finally, phylogenetic affinities and gene expression of annexins were investigated for understanding their role in the protection and homeostasis of intestinal epithelial cells against the inflammation.
... Only dexamethasone has been widely used in clinical practice. In the brain, dexamethasone interferes with microglia activation by inhibiting the expression of MHC class II, and down regulates microglial COX-2 and iNOS production (Minghetti et al., 1999). It also inhibits microglial ramification and proliferation in vitro (Tanaka et al., 1997), and induces the expression of microglial lipocortin that inhibits microglial activation and exerts a neuroprotective effect (Minghetti et al., 1999). ...
... In the brain, dexamethasone interferes with microglia activation by inhibiting the expression of MHC class II, and down regulates microglial COX-2 and iNOS production (Minghetti et al., 1999). It also inhibits microglial ramification and proliferation in vitro (Tanaka et al., 1997), and induces the expression of microglial lipocortin that inhibits microglial activation and exerts a neuroprotective effect (Minghetti et al., 1999). For example, dexamethasone prevents the degenerative effect of a single intranigral injection of LPS on the dopaminergic system (Castaño et al., 2002). ...
... Anti-inflammatory actions of dexamethasone in brain are produced through downregulation of MHC class II, microglial COX-2 and iNOS production (Tanaka et al., 1997;Minghetti et al., 1999), and upregulation of microglial lipocortin (Minghetti et al., 1999), which interferes microglial activation and proliferation. Although dexamethasone was unable to inhibit microglial proliferation, it produced a delay of microglial activation; thus, microglial cells showed phagosome features after thrombin injection, whereas they appeared partially activated after injection of thrombin in rats treated with dexamethasone, featuring a ramified rather than the amoeboid morphology characteristic of phagocytic cells (Frackowiak et al., 1992). ...
... Only dexamethasone has been widely used in clinical practice. In the brain, dexamethasone interferes with microglia activation by inhibiting the expression of MHC class II, and down regulates microglial COX-2 and iNOS production (Minghetti et al., 1999). It also inhibits microglial ramification and proliferation in vitro (Tanaka et al., 1997), and induces the expression of microglial lipocortin that inhibits microglial activation and exerts a neuroprotective effect (Minghetti et al., 1999). ...
... In the brain, dexamethasone interferes with microglia activation by inhibiting the expression of MHC class II, and down regulates microglial COX-2 and iNOS production (Minghetti et al., 1999). It also inhibits microglial ramification and proliferation in vitro (Tanaka et al., 1997), and induces the expression of microglial lipocortin that inhibits microglial activation and exerts a neuroprotective effect (Minghetti et al., 1999). For example, dexamethasone prevents the degenerative effect of a single intranigral injection of LPS on the dopaminergic system (Castaño et al., 2002). ...
... Anti-inflammatory actions of dexamethasone in brain are produced through downregulation of MHC class II, microglial COX-2 and iNOS production (Tanaka et al., 1997;Minghetti et al., 1999), and upregulation of microglial lipocortin (Minghetti et al., 1999), which interferes microglial activation and proliferation. Although dexamethasone was unable to inhibit microglial proliferation, it produced a delay of microglial activation; thus, microglial cells showed phagosome features after thrombin injection, whereas they appeared partially activated after injection of thrombin in rats treated with dexamethasone, featuring a ramified rather than the amoeboid morphology characteristic of phagocytic cells (Frackowiak et al., 1992). ...
Anti-inflammatory strategies receive growing attention for their potential to prevent pathological deterioration in disorders such as Parkinson's disease, which is accompanied by inflammatory reactions that might play a critical role in the degeneration of nigral dopaminergic neurons. We investigated the influence of dexamethasone - a potent synthetic member of the glucocorticoids class of steroid hormones that acts as an anti-inflammatory - on the degeneration of the dopaminergic neurons of rats observed after intranigral injection of thrombin, a serine protease that induces inflammation through microglia proliferation and activation. We evaluated tyrosine hydroxylase (TH)-positive neurons as well as astroglial and microglial populations; dexamethasone prevented the loss of astrocytes but was unable to stop microglial proliferation induced by thrombin. Moreover, dexamethasone produced alterations in the levels of nexin and the thrombin receptor PAR-1, and facilitated accumulation of alpha-synuclein induced by thrombin in dopaminergic neurons. Dexamethasone increased oxidative stress and expression of monoamine oxidase A and B, along with changes on different MAP kinases related to degenerative processes, resulting in a bigger loss of dopaminergic neurons after intranigral injection of thrombin in dexamethasone-treated animals. It is interesting to ascertain that inhibition of monoamine oxidase by tranylcypromine prevented neurodegeneration of dopaminergic neurons, thus suggesting that the deleterious effects of dexamethasone might be mediated by monoamine oxidase.
... ANXA1 is a calcium-dependent phospholipid-binding protein localized in the plasma membrane, the cytoplasm, and the nucleus, and was first identified as a negative regulator of cytosolic phospholipase A2 (PLA2) [44]. ANXA1 exerts anti-inflammatory activity by inhibiting PLA2, cyclooxygenase-2, and inducible nitric oxide synthase [45,46]. ANXA1 inhibits leukocyte transmigration, promotes M2-macrophage differentiation, accelerates apoptotic cell removal, and thus resolves inflammation [44]. ...
... The inhibition mechanism has not been clearly identified yet, but it can be mediated through the binding of extracellular ANXA1 to cell surface formyl peptide receptors, FPR1 and FPR2 [35,56,57]. ANXA1-FRP axis has been known to inhibit cyclooxygenase-2 [45,46,57], a key activator of pro-inflammatory cytokines including CCL2 [58,59]. In this study, anti-ANXA1 neutralizing antibody promoted cancer cell invasion, and ANXA1 overexpression suppressed CCL2 expression in CAFs, which might provide a mechanistic explanation for how miR-196a-ANXA1 axis regulates the inflammatory features of CAFs. ...
Fibroblasts in the tumor microenvironment, known as cancer-associated fibroblasts (CAFs), promote the migration, invasion, and metastasis of cancer cells when they are activated through diverse processes, including post-transcriptional regulation by microRNAs (miRNAs). To identify the miRNAs that regulate CAF activation, we used NanoString to profile miRNA expression within normal mouse lung fibroblasts (LFs) and CAFs. Based on NanoString profiling, miR-196a was selected as a candidate that was up-regulated in CAFs. miR-196a-overexpressed LFs (LF-196a) promoted the migration and invasion of lung cancer cells in co-culture systems (Transwell migration and spheroid invasion assays). ANXA1 was confirmed as a direct target of miR-196a, and adding back ANXA1 to LF-196a restored the cancer cell invasion promoted by miR-196a. miR-196a increased CCL2 secretion in fibroblasts, and that was suppressed by ANXA1. Furthermore, blocking CCL2 impeded cancer spheroid invasion. In lung adenocarcinoma patients, high miR-196a expression was associated with poor prognosis. Collectively, our results suggest that CAF-specific miR-196a promotes lung cancer progression in the tumor microenvironment via ANXA1 and CCL2 and that miR-196a will be a good therapeutic target or biomarker in lung adenocarcinoma.
... La asociación entre la inducción de la COX-2 y la degeneración neuronal sugiere que la COX-2 interviene de manera más selectiva en la pérdida de conexiones neuronales que en su formación. 1 Las células de la microglia son fuente de PG durante procesos inflamatorios; empero, no se detectó inducción de la COX-2 al estimularlas con citocinas, pero sí con lipopolisacárdos (LPS), 18 un hecho que in vivo sólo podría ocurrir por una infección bacteriana directa del cerebro. ...
... The association between COX-2 induction and neural degeneration suggests that COX-2 may play more of a role in the selective loss of neural connections than in their formation. 1 While known as a source of PGs during inflammatory states, the microglial cells did not show induction of COX-2 in response to cytokines, but did with lipopolysaccharides (LPS), 18 an event that would only occur in vivo by direct bacterial infection of the brain. ...
High numbers of proinflammatory cells (PMNLs), which are carried by the blood to ischemic tissue during reperfusion, are considered responsible for inducing the inflammatory response that occurs in ischemia-reperfusion (I/R) injury. Our objective was to determine the controlled reperfusion (CR) interval duration (CRID) that would minimize the injury caused by the PMNLs that infiltrate ischemic tissue. Animal groups were divided into the following groups: Sham group, ovarian I/R group (OIR), and ovarian ischemia controlled-reperfusion groups OICR-1, OICR-2, OICR-3, OICR-4, OICR-5, OICR-6, which had their ovarian artery opened and then closed for 10, 8, 6, 4, 2, or 1 s, respectively. The results show that the COX-2 activity and the gene expression decreased while the COX-1 activity and the gene expression were found to be increased in parallel to the shortening of the period in CRID. From the histopathological examinations, the findings of hemorrhage, edema, congested vascular structures, degenerated cells, and migration and adhesion of PMNLs were scaled as follows: Sham group < OICR-6 < OICR-5 < OICR-4 < OICR-3 < OICR-2 < OICR-1. The results from the histopathological assessments were consistent with the molecular and biochemical findings. In conclusion, our findings suggest that increased COX-2 activity plays a role in I/R injury of the rat ovary, and that controlled reperfusion for 3, 2, or 1 s following 2 h of ischemia may attenuate the effects of I/R injury.
... La asociación entre la inducción de la COX-2 y la degeneración neuronal sugiere que la COX-2 interviene de manera más selectiva en la pérdida de conexiones neuronales que en su formación. 1 Las células de la microglia son fuente de PG durante procesos inflamatorios; empero, no se detectó inducción de la COX-2 al estimularlas con citocinas, pero sí con lipopolisacárdos (LPS), 18 un hecho que in vivo sólo podría ocurrir por una infección bacteriana directa del cerebro. ...
... The association between COX-2 induction and neural degeneration suggests that COX-2 may play more of a role in the selective loss of neural connections than in their formation. 1 While known as a source of PGs during inflammatory states, the microglial cells did not show induction of COX-2 in response to cytokines, but did with lipopolysaccharides (LPS), 18 an event that would only occur in vivo by direct bacterial infection of the brain. ...
Use of non-steroidal anti-inflammatory drugs (NSAIDs) in dogs is limited because of the adverse effects they produce. All NSAIDs inhibit, by different degrees, the cyclooxygenases (COX), enzymes that exist in at least two isoforms: COX-1 and COX-2. Inhibition of COX-1 is believed to be responsible for the adverse effects of NSAIDs, whereas inhibition of COX-2 is thought to be related to the therapeutic effects of this class of drugs. This theory, to some extent, seems to be true for humans suffering from osteoarthritis who receive COX-2 selective inhibitors. If this hypothesis also applies to dogs, COX-2 selective inhibitors should play an important role in canine therapeutics. This review highlights relevant physiopathologic aspects of COX-2 within the organism (e.g., gastrointestinal tract, kidneys, central nervous system), based on this information, the possible uses and adverse effects of selective COX-2 inhibitors (e.g., celecoxib, nimesulide, NS-398) in dogs are discussed. Clinical-pharmacological information on the use of selective COX-2 inhibitors (e.g., carprofen, celecoxib, nimesulide) specifically obtained from dogs is also presented.
... Previous experimental evidence indicates that intrinsic proteins participate in the regulation of prostaglandin synthesis during inflammation. Of particular interest in this regard is annexin A1 (ANXA1), a calcium-dependent phospholipidbinding protein that mediates glucocorticoid action, reduces cytosolic PLA2 by reducing PLA2 gene expression, and limits COX-2 abundance (11,20,31). ANXA1 is secreted in response to glucocorticoids in a cell-specific process involving its phosphorylation and excretion by the ATP-binding cassette transporter ABCA1 (10,38,44). ...
... In cultured microglial cells, the NH 2 -terminal ANXA1 peptide AC2-26 caused a significant downregulation of COX-2 expression and PGE 2 synthesis. In addition, the inhibitory effects of glucocorticoid treatment on COX-2 expression were shown to at least partially depend on ANXA1 (31). Thus our results suggest that the macula densa belongs to the tissues in which ANXA1 exerts an inhibitory effect on COX-2 expression. ...
Annexin A1 (ANXA1) exerts anti-inflammatory effects through multiple mechanisms including inhibition of prostaglandin synthesis. Once secreted, ANXA1 can bind to G protein-coupled formyl peptide receptors (Fpr) and activate diverse cellular signaling pathways. ANXA1 is known to be expressed in cells of the juxtaglomerular apparatus, but its relation to the expression of cyclooxygenase 2 (COX-2) in thick ascending limb and macula densa cells has not been elucidated. We hypothesized that ANXA1 regulates the biosynthesis of COX-2. ANXA1 abundance in rat kidney macula densa was extensively colocalized with COX-2 (95%). Furosemide, an established stimulus for COX-2 induction, caused enhanced expression of both ANXA1 and COX-2 with maintained colocalization (99%). In ANXA1-deficient mice, COX-2-positive cells were more numerous than in control mice (+107%; normalized to glomerular number; P < 0.05) and renin expression was increased (+566%; normalized to glomerular number; P < 0.05). Cultured macula densa cells transfected with full-length rat ANXA1 revealed downregulation of COX-2 mRNA (-59%; P < 0.05). Similarly, treatment with dexamethasone suppressed COX-2 mRNA in the cells (-49%; P < 0.05), while inducing ANXA1 mRNA (+56%; P < 0.05) and ANXA1 protein secretion. Inhibition of the ANXA-1 receptor Fpr1 with cyclosporin H blunted the effect of dexamethasone on COX-2 expression. These data show that ANXA1 exerts an inhibitory effect on COX-2 expression in the macula densa. ANXA1 may be a novel intrinsic modulator of renal juxtaglomerular regulation by inhibition of PGE(2) synthesis.
... After its first discovery, ANXA1 was described as an endogenous modulator of anti-inflammatory actions. As such, the protein was shown to up-regulate the production of anti-inflammatory cytokines and to control pro-inflammatory mediator release (Minghetti et al., 1999;Ferlazzo et al., 2003). ...
Oriented cell divisions (OCDs) represent a fundamental mechanism for tissue morphogenesis, repair and differentiation where the mitotic spindle is oriented along a specific polarity axis. Early research identified the evolutionarily conserved Gαi/LGN/NuMA ternary complex that mediates orientation of the mitotic spindle by being restricted to specific cortical regions. The mechanisms that control the recruitment of these proteins to the cortex remain unfolding, particularly in epithelial systems such as the mammary gland. The mammary gland represents a unique organ that develops predominantly after birth where postnatal morphogenesis of the mammary gland drives dramatic tissue turnover and remodelling. Thus, differentiation and proliferation are constantly balanced to allow normal mammary gland development and homeostasis. How mammary epithelial cells regulate mitotic spindle orientation, hence OCDs, to accompany the rapid and constant tissue turnover is not well understood. This study aimed to identify novel factors that regulate the LGN-mediated spindle orientation machinery and determine how their dysregulation affects OCDs in mammary epithelial cells. By combining co-immunoprecipitation with mass spectrometry, the LGN interactome at the cell cortex of mitotic mammary epithelial cells was characterised and the membrane-associated protein Annexin A1 (ANXA1) was identified as a novel partner of LGN. Confocal and time-lapse microscopy demonstrated a critical role of ANXA1 in regulating the position and planar orientation of the mitotic spindle by instructing the accumulation and restriction of the LGN complex at the lateral cortex. Moreover, loss of ANXA1 leads to mitotic spindle misassembly and chromosome segregation defects, affecting the dynamics and progression of mitosis. Collectively the present study identified ANXA1 as a novel intrinsic cue of OCDs in mammary epithelial cells. Given increasing evidence of a link between OCD and tumorigenesis, this work is not only important for advancing our understanding of normal epithelial biology but also elucidating how imbalance of OCDs can contribute to the abnormal cell behaviour observed in cancer.
... Annexin A1 also binds and activates cell messengers that activate apoptosis machinery Gavins and Hickey, 2012), which prevents necrosis and release of inflammatory factors. It has a role in controlling release of nitric oxide and inhibits cyclooxygenase 2 expression, thereby upregulating potent anti-inflammatory cytokines such as interleukin (IL)-10 (Minghetti et al., 1999). A recent study shows a role for annexin A1 in the regulation of nucleotide-binding oligomerization (NOD-), LRR-and pyrin domain-containing protein (NLRP3) inflammasome activation (Sanches et al., 2020a). ...
Sepsis is a continuing problem in modern healthcare, with a relatively high prevalence, and a significant mortality rate worldwide. Currently, no specific anti-sepsis treatment exists despite decades of research on developing potential therapies. Annexins are molecules that show efficacy in preclinical models of sepsis but have not been investigated as a potential therapy in patients with sepsis. Human annexins play important roles in cell membrane dynamics, as well as mediation of systemic effects. Most notably, annexins are highly involved in anti-inflammatory processes, adaptive immunity, modulation of coagulation and fibrinolysis, as well as protective shielding of cells from phagocytosis. These discoveries led to the development of analogous peptides which mimic their physiological function, and investigation into the potential of using the annexins and their analogous peptides as therapeutic agents in conditions where inflammation and coagulation play a large role in the pathophysiology. In numerous studies, treatment with recombinant human annexins and annexin analogue peptides have consistently found positive outcomes in animal models of sepsis, myocardial infarction, and ischemia reperfusion injury. Annexins A1 and A5 improve organ function and reduce mortality in animal sepsis models, inhibit inflammatory processes, reduce inflammatory mediator release, and protect against ischemic injury. The mechanisms of action and demonstrated efficacy of annexins in animal models support development of annexins and their analogues for the treatment of sepsis. The effects of annexin A5 on inflammation and platelet activation may be particularly beneficial in disease caused by SARS-CoV-2 infection. Safety and efficacy of recombinant human annexin A5 are currently being studied in clinical trials in sepsis and severe COVID-19 patients.
... Although Nos2-encoded iNOS is representative of proinflammatory reactive oxygen species signaling and is highly expressed by proinflammatory M1 macrophages, it was described to be downregulated by Ac2-26 treatment in a recent study. 41 MARCO is known as a coactivator of inflammatory responses on macrophages, leading to the activation of NF-κB signaling and the release of proinflammatory cytokines. 42 Finally, we could find higher levels of FPR2/ALX and FPR1 in the colitis group at POD 3 compared with the control and Ac2-26-NP groups. ...
Background
Although in most patients with inflammatory bowel diseases, conservative therapy is successful, a significant proportion of patients still require surgery once in their lifetime. Development of a safe perioperative treatment to dampen colitis activity without disturbance of anastomotic healing is an urgent and unmet medical need. Annexin A1 (ANXA1) has been shown to be effective in reducing colitis activity. Herein, a nanoparticle-based perioperative treatment approach was used for analysis of the effects of ANXA1 on the resolution of inflammation after surgery for colitis.
Methods
Anxa1-knockout mice were used to delineate the effects of ANXA1 on anastomotic healing. A murine model of preoperative dextran sodium sulfate colitis was performed. Collagen-IV-targeted polymeric nanoparticles, loaded with the ANXA1 biomimetic peptide Ac2-26 (Ac2-26-NPs), were synthesized and administered perioperatively during colitis induction. The effects of the Ac2-26-NPs on postoperative recovery and anastomotic healing were evaluated using the disease activity index, histological healing scores, and weight monitoring. Ultimately, whole-genome RNA sequencing of the anastomotic tissue was performed to unravel underlying molecular mechanisms.
Results
Anxa1-knockout exacerbated the inflammatory response in the healing anastomosis. Treatment with Ac2-26-NPs improved preoperative colitis activity (P < 0.045), postoperative healing scores (P < 0.018), and weight recovery (P < 0.015). Whole-genome RNA sequencing revealed that the suppression of proinflammatory cytokine and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling was associated with the treatment effects and a phenotypic switch toward anti-inflammatory M2 macrophages.
Conclusions
Proresolving therapy with Ac2-26-NPs promises to be a potent perioperative therapy because it improves colitis activity and even intestinal anastomotic healing by the suppression of proinflammatory signaling.
... Indeed, dexamethasone (DEX) treatment could reduce the production of pro-inflammatory cytokines including IL1 and TNFα [19], and inhibit microglial ramification and proliferation in vitro [20]. A previous study reported that DEX plays a neuroprotective role through inhibition on microgliosis via expression of microglial lipocortin [21]. Overall, the above evidence suggests that DEX may be a potential agent to inhibit inflammatory responses and to protect neurons. ...
Prolonged neuroinflammation is a driving force for neurodegenerative disease, and agents against inflammatory responses are regarded as potential treatment strategies. Here we aimed to evaluate the prevention effects on gliosis by dexamethasone (DEX), an anti-inflammation drug. We used DEX to treat the nicastrin conditional knockout (cKO) mouse, a neurodegenerative mouse model. DEX (10 mg/kg) was given to 2.5-month-old nicastrin cKO mice, which have not started to display neurodegeneration and gliosis, for 2 months. Immunohistochemistry (IHC) and Western blotting techniques were used to detect changes in neuroinflammatory responses. We found that activation of glial fibrillary acidic protein (GFAP) positive or ionized calcium binding adapter molecule1 (Iba1) positive cells was not inhibited in nicastrin cKO mice treated with DEX as compared to those treated with saline. These data suggest that DEX does not prevent or ameliorate gliosis in a neurodegenerative mouse model when given prior to neuronal or synaptic loss.
... It inhibits the formation of lipid mediator by blockade of enzyme phospholipase A 2 , thereby regulating cellular growth and differentiation, neuroendocrine secretion, central nervous system response to cytokines and accumulation of neutrophil to different tissues [70][71][72]. It blocks the release of inflammatory mediators by inhibition of cytosolic phospholipase A 2 enzyme, thus prevents the formation of pro-inflammatory eicosanoid, inducible cyclooxygenase and nitric oxide synthase enzymes [73]. It may also affects on inducible nitric oxide synthase (iNOS) expression in rats with septic shock. ...
Inflammation is a physiological intrinsic host response to injury meant for removal of noxious stimuli and maintenance of homeostasis. It is a defensive body mechanism that involves immune cells, blood vessels and molecular mediators of inflammation. Glucocorticoids (GCs) are steroidal hormones responsible for regulation of homeostatic and metabolic functions of body. Synthetic GCs are the most useful anti-inflammatory drugs used for the treatment of chronic inflammatory diseases such as asthma, chronic obstructive pulmonary disease (COPD), allergies, multiple sclerosis, tendinitis, lupus, atopic dermatitis, ulcerative colitis, rheumatoid arthritis and osteoarthritis whereas, the long term use of GCs are associated with many side effects. The anti-inflammatory and immunosuppressive (desired) effects of GCs are usually mediated by transrepression mechanism whereas; the metabolic and toxic (undesired) effects are usually manifested by transactivation mechanism. Though GCs are most potent anti-inflammatory and immunosuppressive drugs, the common problem associated with their use is GC resistance. Several research studies are rising to comprehend these mechanisms, which would be helpful in improving the GC resistance in asthma and COPD patients. This review aims to focus on identification of new drug targets in inflammation which will be helpful in the resolution of inflammation. The ample understanding of GC mechanisms of action helps in the development of novel anti-inflammatory drugs for the treatment of inflammatory and autoimmune disease with reduced side effects and minimal toxicity.
... Indeed, in ischemia-reperfusion animal models, where the oxidative injury is mostly mediated by impaired blood supply-induced hypoxia or anoxia, LXA 4 reduced tissue damage in the spinal cord and myocardium [31][32][33], where its therapeutic effect has been linked to the induction of the Keap/Nrf2-dependent antioxidant response [32,34]. Interestingly, annexin A1, a distinct nonlipid FPR2/ALX ligand, is involved in the induction of iNOS [35][36][37], which further strengthens the role of the FPR2 transduction axis in regulating the cellular redox state. ...
Specialized proresolving mediators (SPMs) are a novel class of endogenous lipids, derived by ω -6 and ω -3 essential polyunsaturated fatty acids such as arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) that trigger and orchestrate the resolution of inflammation, which is the series of cellular and molecular events that leads to spontaneous regression of inflammatory processes and restoring of tissue homeostasis. These lipids are emerging as highly effective therapeutic agents that exert their immunoregulatory activity by activating the proresolving pathway, as reported by a consistent bulk of evidences gathered in the last two decades since their discovery. The production of reactive oxygen (ROS) and nitrogen (RNS) species by immune cells plays indeed an important role in the inflammatory mechanisms of host defence, and it is now clear that oxidative stress, viewed as an imbalance between such species and their elimination, can lead to many chronic inflammatory diseases. This review, the first of its kind, is aimed at exploring the manifold effects of SPMs on modulation of reactive species production, along with the mechanisms through which they either inhibit molecular signalling pathways that are activated by oxidative stress or induce the expression of endogenous antioxidant systems. Furthermore, the possible role of SPMs in oxidative stress-mediated chronic disorders is also summarized, suggesting not only that their anti-inflammatory and proresolving properties are strictly associated with their antioxidant role but also that these endogenous lipids might be exploited in the treatment of several pathologies in which uncontrolled production of ROS and RNS or impairment of the antioxidant machinery represents a main pathogenetic mechanism.
... Depending on dose, glucocorticoids can induce macrophage apoptosis [481] or microglial cell death [253]. They induce expression of microglial lipocortin, which inhibits microglial activation and is neuroprotective [362]. ...
... This protein is also reported to inhibit the cyclooxygenase-2 expression and, thus, blocks the production of prostaglandins and other proinflammatory mediators. AnxA1 is considered as an effector molecule in the mechanism of action of the anti-inflammation effect of steroid drugs [22][23][24]. In addition, AnxA1 is also believed to be responsible for the regulation of the immune system. ...
Objective: The aims of this study were to investigate the comparative pharmacodynamics effect of methylprednisolone (MP) innovator, MP branded generic, and MP generic products to the serum concentration of annexin A1 (AnxA1).Methods: It was conducted by two-way crossover design in male rabbits. AnxA1 was measured at 0, 0.5, 1, 2, 3, 5, 7, and 9 h after the administration of the drugs. The peak concentration (Cmax), the time at which the peak concentration was achieved (Tmax), and the area under the plasma concentration-time curve (AUC) were also determined.Results: The highest concentration and widest AUC of AnxA1 were obtained in MP innovator drug. MP innovator and branded generic reaches the peak time (Tmax) at the third 3rd h, while the MP generic reaches the peak time at the 5th h. The results showed that there was no significant difference in the serum concentration of AnxA1 between MP tablets after analyzed with a one-way analysis of variance.Conclusion: It could be concluded that the innovator drug of MP tablet gave the same effect on the serum concentration of AnxA1 than its generic counterparts, but an onset of action MP innovator and branded generic is faster than the generic product.
... This protein is also reported to inhibit the cyclooxygenase-2 expression and, thus, blocks the production of prostaglandins and other proinflammatory mediators. AnxA1 is considered as an effector molecule in the mechanism of action of the anti-inflammation effect of steroid drugs [22][23][24]. In addition, AnxA1 is also believed to be responsible for the regulation of the immune system. ...
Objective: The aims of this study were to investigate the comparative pharmacodynamics effect of methylprednisolone (MP) innovator, MP branded generic, and MP generic products to the serum concentration of annexin A1 (AnxA1).Methods: It was conducted by two-way crossover design in male rabbits. AnxA1 was measured at 0, 0.5, 1, 2, 3, 5, 7, and 9 h after the administration of the drugs. The peak concentration (Cmax), the time at which the peak concentration was achieved (Tmax), and the area under the plasma concentration-time curve (AUC) were also determined.Results: The highest concentration and widest AUC of AnxA1 were obtained in MP innovator drug. MP innovator and branded generic reaches the peak time (Tmax) at the third 3rd h, while the MP generic reaches the peak time at the 5th h. The results showed that there was no significant difference in the serum concentration of AnxA1 between MP tablets after analyzed with a one-way analysis of variance.Conclusion: It could be concluded that the innovator drug of MP tablet gave the same effect on the serum concentration of AnxA1 than its generic counterparts, but an onset of action MP innovator and branded generic is faster than the generic product.
... In addition, ANXA1 induces apoptosis, leading the pro-survival signals that cause prolonged lifespan of neutrophils at the inflammatory site, promoting the recruitment and clearance of apoptotic neutrophils by resolving macrophages [24]. ANXA1 regulates several mediators that are involved in the inflammatory response, such cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and the production and secretion of pro-inflammatory cytokines by macrophages [25,26]. ANXA1 is important in the regulation of dendritic cell maturation and activation [27] and plays important roles in the regulation of toll like receptor and type-1 interferon responses in macrophages [28], enhancing the activation of transcription factors such as nuclear factor-kappa-B (NF-kB) [27] and interferon response factor-3 associating with upstream signal proteins TANK-binding kinase [28]. ...
Targeting inflammation in cancer has shown promise to improve and complement current therapies. The tumor microenvironment plays an important role in cancer growth and metastasis and -tumor associated macrophages possess pro-tumoral and pro-metastatic properties. Annexin A1 (ANXA1) is an immune-modulating protein with diverse functions in the immune system and in cancer. In breast cancer, high ANXA1 expression leads to poor prognosis and increased metastasis. Here, we will review ANXA1 as a modulator of inflammation, and discuss its importance in breast cancer and highlight its new role in alternative macrophage activation in the tumor microenvironment. This review may provide an updated understanding into the various roles of ANXA1 which may enable future therapeutic developments for the treatment of breast cancer.
... It is widely established that IL-10 production can be increased by the ERK signalling pathway that is activated upon ANXA1 binding to its FPR thereby suggesting the presence of a looping mechanism to control both IL-10 and nitric oxide release [24]. Interestingly, ANXA1 also inhibits cyclo-oxygenase-2 (COX-2) expression, hence, controlling pro-inflammatory mediator release; a phenomenon unique to microglial cells [25]. ...
Annexin A1 (ANXA1) has long been classed as an anti-inflammatory protein due to its control over leukocyte-mediated immune responses. However, it is now recognized that ANXA1 has widespread effects beyond the immune system with implications in maintaining the homeostatic environment within the entire body due to its ability to affect cellular signalling, hormonal secretion, foetal development, the aging process and development of disease. In this review, we aim to provide a global overview of the role of ANXA1 covering aspects of peripheral and central inflammation, immune repair and endocrine control with focus on the prognostic, diagnostic and therapeutic potential of the molecule in cancer, neurodegeneration and inflammatory-based disorders.
... Anxa1 has shown antipyretic and neuroprotective activities in a variety of animal models. One study showed that exogenous Anxa1 inhibited lipopolysaccharideinduced upregulation of pro-inflammatory mediators in microglial cultures (Minghetti et al. 1999). Another study demonstrated that excitotoxic injury induced by an NMDA receptor agonist rats were inhibited with active Anxa1 fragment and administration of Anxa1 markedly reduced infarct size (Black et al. 1992) and cerebral edema induced by mid-cerebral artery occlusion (MCAO) in rats (Relton et al. 1991). ...
A growing body of evidence has shown bisphenol A (BPA), an estrogen-like industrial chemical, has adverse effects on the nervous system. In this study, we investigated the transcriptional behavior of long non-coding RNAs (lncRNAs) and mRNAs to provide the information to explore neurotoxic effects induced by BPA. By microarray expression profiling, we discovered 151 differentially expressed lncRNAs and 794 differentially expressed mRNAs in the BPA intervention group compared with the control group. Gene ontology analysis indicated the differentially expressed mRNAs were mainly involved in fundamental metabolic processes and physiological and pathological conditions, such as development, synaptic transmission, homeostasis, injury, and neuroinflammation responses. In the expression network of the BPA-induced group, a great number of nodes and connections were found in comparison to the control-derived network. We identified lncRNAs that were aberrantly expressed in the BPA group, among which, growth arrest specific 5 (GAS5) might participate in the BPA-induced neurotoxicity by regulating Jun, RAS, and other pathways indirectly through these differentially expressed genes. This study provides the first investigation of genome-wide lncRNA expression and correlation between lncRNA and mRNA expression in the BPA-induced neurotoxicity. Our results suggest that the elevated expression of lncRNAs is a major biomarker in the neurotoxicity induced by BPA.
... ANXA1 specifically targets cytosolic phospholipase A2 via inhibition and suppression of cytokine-induced enzyme activation 26,27 . ANXA1 inhibits the expression of other inflammatory mediators such as iNOS or NOS2 in macrophages and inducible cyclooxygenase (COX-2) in activated microglia 28 . Moreover, ANXA1 could inhibit the chemotaxis of neutrophils and monocytes during inflammation 27,29 . ...
Periodontal (gum) disease is a highly prevalent infection and inflammation accounting for the majority of tooth loss in adult population worldwide. Porphyromonas gingivalis is a keystone periodontal pathogen and its lipopolysaccharide (PgLPS) acts as a major virulence attribute to the disease. Herein, we deciphered the overall host response of human gingival fibroblasts (HGFs) to two featured isoforms of tetra-acylated PgLPS1435/1449 and penta-acylated PgLPS1690 with reference to E. coli LPS through quantitative proteomics. This study unraveled differentially expressed novel biomarkers of immuno-inflammatory response, antioxidant defense and cytoskeletal dynamics in HGFs. PgLPS1690 greatly upregulated inflammatory proteins (e.g. cyclophilin, inducible nitric oxide synthase, annexins, galectin, cathepsins and heat shock proteins), whereas the anti-inflammatory proteins (e.g. Annexin A2 and Annexin A6) were significantly upregulated by PgLPS1435/1449. Interestingly, the antioxidants proteins such as mitochondrial manganese-containing superoxide dismutase and peroxiredoxin 5 were only upregulated by PgLPS1690. The cytoskeletal rearrangement-related proteins like myosin were differentially regulated by these PgLPS isoforms. The present study gives new insight into the biological properties of P. gingivalis LPS lipid A moiety that could critically modulate immuno-inflammatory response, antioxidant defense and cytoskeletal dynamics in HGFs, and thereby enhances our understanding of periodontal pathogenesis.
... Using rodent microglia cultures, it was shown that the N-terminal fragment of ANXA1, Ac2-26, prevents lipopolysaccharide (LPS) mediated stimulation of cyclo-oxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS), as well as the release of nitric oxide (NO) 39,40 . In addition, ANXA1 is known to inhibit phospholipase A2 activity 41 , thereby preventing the release of arachidonic acid (AA), an essential fatty acid for prostanoid synthesis. ...
Annexin A1 (ANXA1) is a protein known to have multiple roles in the regulation of inflammatory responses. In this study, we find that after oxygen glucose deprivation/reoxygenation (ODG/R) injury, activated PKC phosphorylated ANXA1 at the serine 27 residue (p27S-ANXA1), and promoted the translocation of p27S-ANXA1 to the nucleus of BV-2 microglial cells. This in turn induced BV-2 microglial cells to produce large amounts of pro-inflammatory cytokines. The phenomenon could be mimicked by either transfecting a mutant form of ANXA1 with its serine 27 residue converted to aspartic acid, S27D, or by using the PKC agonist, phorbol 12-myristate 13-acetate (PMA) in these microglial cells. In contrast, transfecting cells with an ANXA1 S27A mutant (serine 27 converted to alanine) or treating the cells with the PKC antagonist, GF103209X (GF) reversed this effet. Our study demonstrates that ANXA1 can be phosphorylated by PKC and is subsequently translocated to the nucleus of BV-2 microglial cells after OGD/R, resulting in the induction of pro-inflammatory cytokines.
... ANXA1 is a calcium/phospholipid binding protein associated with plasma membrane phospholipids and vesicles [36] and is involved in intracellular trafficking [37], plasma membrane repair [38], apoptosis [39] and leukocyte migration [40]. ANXA1 has been also suggested to exert a protective role in central nervous system after ischemia through the prevention of microglia activation [40,41]. ...
... Anti-inflammatory protein annexin A1 is the founding member of the annexin family of calcium-and phospholipid-binding proteins. It is widely expressed throughout the body and has been shown to mediate anti-inflammatory glucocorticoid effects in the innate and the adaptive immune system (Minghetti et al. 1999, Kim et al. 2001, D'Acquisto et al. 2008. It is also involved in the regulation of intracellular calcium signalling (Monastyrskaya et al. 2009, Swa et al. 2012) and may exert cytoprotective effects during phases of energy depletion, acidosis or membrane damage . ...
AimThe anti-inflammatory protein annexin A1 (AnxA1) and its formyl peptide receptor 2 (FPR2) have protective effects in organ fibrosis. Their role in chronic kidney disease (CKD) has not yet been elucidated. Our aim was to characterize the AnxA1/FPR2 system in models of renal fibrosis.Methods
Rats were treated with angiotensin receptor antagonist during the nephrogenic period (ARAnp) to induce late-onset hypertensive nephropathy and fibrosis. Localization and regulation of AnxA1 and FPR2 were studied by quantitative real time PCR and double labeling immunofluorescence. Biological effects of AnxA1 were studied in cultured renal fibroblasts from AnxA1-/- and wild type mice.ResultsARAnp kidneys displayed matrix foci containing CD73+ fibroblasts, alpha smooth muscle actin (a-SMA)+ myofibroblasts, and CD68+ macrophages. TGF-β and AnxA1 mRNAs were ~3-fold higher than in controls. AnxA1 was localized to macrophages and fibroblasts; myofibroblasts were negative. FPR2 was localized to fibroblasts, myofibroblasts, macrophages and endothelial cells. AnxA1 and FPR2 immunoreactive signals were increased in the foci, with fibroblasts and macrophages expressing both proteins. AnxA1-/- fibroblasts revealed higher α-SMA (7-fold) and collagen 1a1 (Col1A1; 144-fold) mRNA levels than controls. Treatment of murine WT fibroblasts with TGF-β (22.5 ng/ml/24h) increased mRNA levels of α-SMA (9.3 fold) and Col1A1 (4 fold). These increases were greatly attenuated upon overexpression of AnxA1 (1.5 and 1.7-fold, respectively; p<.05). Human fibroblasts reacted similarly when receiving the FPR2 inhibitor WRW4.Conclusion
Our results demonstrate that AnxA1 and FPR2 are abundantly expressed in the renal interstitium and modulate fibroblast phenotype and extracellular matrix synthesis activity.This article is protected by copyright. All rights reserved.
... The central antipyretic effect of GCs could be mediated by lipocortin-1, a GC-inducible protein which is a potent inhibitor of phospholipase A2. This idea is supported by the observations that central injections of lipocortin-1 reduce fever (98) and that lipocortin-1 downregulates the LPS-induced expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in microglial cells (99). As it has been discussed above for neuropeptides, the antipyretic effects of GCs seem to be mediated by multiple mechanisms. ...
... Administration of dexamethasone over a one-month period at 6 months of age restored some functions as assessed using the wheel activity test. Dexamethasone blocks microglial activation by inhibiting MHC class II expression through down-regulating cyclooxygenase 2 and inducible nitric oxide synthase production Page 20 of 39 The International Journal of Neuropsychopharmacology (Minghetti et al., 1999). This glucocorticoid has also been shown to prevent neuronal death triggered by the injection of the TLR4 agonist lipopolysaccharide (Castano et al., 2002). ...
Accumulating evidence supports a role for the immune system in the pathogenesis of Parkinson's disease (PD). Importantly, recent preclinical studies are now suggesting a specific contribution of inflammation to the α-synuclein (αSyn) induced pathology seen in this condition.
We used flow cytometry and western blots to detect toll-like receptor (TLR) 2 and 4 expression in blood and brain samples of PD patients and mice overexpressing human αSyn. To further assess the effects of αSyn overexpression on the innate immune system, we performed a longitudinal study using Thy1.2-αSyn mice that expressed a bicistronic DNA construct (reporter genes luciferase) and green fluorescent protein) under the transcriptional control of the murine TLR2 promoter.
Here, we report increases in TLR2 and TLR4 expression in circulating monocytes and of TLR4 in B cells and in the caudate/putamen of PD patients. Monthly bioluminescence imaging of Thy1.2-αSyn mice showed increasing TLR2 expression from 10 months of age, although no change in TLR2 and TLR4 expression was observed in the blood and brain of these mice at 12 months of age. Dexamethasone treatment starting at 5 months of age for one month significantly decreased the microglial response in the brain of these mice and promoted functional recovery as observed using a wheel-running activity test.
Our results show that TLR2 and TLR4 are modulated in the blood and in the brain of PD patients and that overexpression of αSyn leads to a progressive microglial response, the inhibition of which as a beneficial impact on some motor phenotypes of an animal model of α-synucleinopathy.
© The Author 2014. Published by Oxford University Press on behalf of CINP.
... Les stéroïdes comme la dexaméthasone ont un effet désactivateur sur les cellules microgliales. Cette molécule diminue la production de NO (Minghetti et al., 1999). Les médiateurs neuronaux désactivent les cellules microgliales. ...
... In the same study, an AnxA1 fragment blocked iNOS in macrophages stimulated with LPS, indicating that the extracellular release of AnxA1 mediates the effect of GCs on the expression of iNOS (Wu et al., 1995). A study using microglial cells demonstrated that the N-terminus peptide of AnxA1 Ac2-26 inhibits the LPS-induced expression of both iNOS and COX-2 (Minghetti et al., 1999). ...
... This glucocorticoid-regulated protein annexin A1, mainly expressing in subcellular granules of neutrophils and monocytes [29], has been implicated in a number of biological events, including both acute [30] and chronic [31] inflammations, leukocyte trafficking [32], monocyte migration [33], and apoptotic leukocytes clearance [34]. annexin A1 may also affect a number of mediators that are involved in the inflammatory response, including IL-10, cyclooxygenase-2 (Cox-2), and inducible nitric oxide synthase (iNOS) [35,36]. Nevertheless, the response of annexin A1 in the process of A. baumannii infection and whether this novel mediator is able to be used as a new biomarker in the diagnosis of sepsis remain to be elucidated. ...
Xuebijing (XBJ) injection is a herbal medicine that has been widely used in the treatment of sepsis in China; however, its role in the development and progression of Acinetobacter baumannii sepsis and the underlying mechanisms remain uninvestigated. In the present study, fifty-four male Wistar rats were randomly assigned to normal-control group, sepsis-control group, and sepsis + XBJ group, each containing three subgroups of different treatment time periods (6, 12, and 24 hrs following injection, resp.). The sepsis model was established by intraperitoneal injection of A. baumannii ATCC 19606. For XBJ treatment, 4 mL/kg XBJ was administrated simultaneously by intravenous injection through caudal vein every 12 hrs. All animals demonstrated ill state, obvious intestinal dysfunction, histopathological lung damages, and overactive inflammatory responses after A. baumannii infection, and these events could be partially reversed by XBJ treatment from the beginning of infection. XBJ induced an increase in the expression of anti-inflammatory mediator annexin A1; however, two proinflammatory cytokines, interleukin-8 (IL-8) and tumor necrosis factor- α (TNF- α ), were decreased at the each monitored time point. These findings suggested that XBJ via its cytokine-mediated anti-inflammatory effects might have a potential role in preventing the progression of A. baumannii infection to sepsis by early administration.
... AnxA1 may also play a role in the regeneration of skeletal muscle tissue by stimulating the migration of satellite cells via the modulation of myoblast cell differentiation, which in turn causes skeletal muscle differentiation (Hawke and Garry, 2001;Bizzarro et al., 2012). In addition, AnxA1 has a valuable role in inhibiting the negative feedback effects of glucocorticoids on the release of corticotrophin (ACTH) and hypothalamic-releasing hormones (Buckingham et al., 2006), and also affects a number of mediators that are involved in the inflammatory response, including cyclo-oxygenase-2 (Cox-2) and inducible nitric oxide synthase (iNOS; Minghetti et al., 1999;Ferlazzo et al., 2003;Perretti and Dalli, 2009). ...
Inflammation is the body’s way of defending itself against noxious stimuli and pathogens. Under normal circumstances, the body is able to eliminate the insult and subsequently promote the resolution of inflammation and the repair of damaged tissues. The concept of homeostasis is one that not only requires a fine balance between both pro-inflammatory mediators and pro-resolving/anti-inflammatory mediators, but also that this balance occurs in a time and space-specific manner. This review examines annexin A1, an anti-inflammatory protein that, when used as an exogenous therapeutic, has been shown to be very effective in limiting inflammation in a diverse range of experimental models, including myocardial ischemia/reperfusion injury, arthritis, stroke, multiple sclerosis, and sepsis. Notably, this glucocorticoid-inducible protein, along with another anti-inflammatory mediator, lipoxin A4, is starting to help explain and shape our understanding of the resolution phase of inflammation. In so doing, these molecules are carving the way for innovative drug discovery, based on the stimulation of endogenous pro-resolving pathways.
... Additionally, activation of microglia by TLR can be modulated by further PGE2 synthesis. Although factors such as TGF-β [79], TNF-α [80], norepinephrine [81], adenosine, and PGE2 [82], can act as COX-2 positive regulators, other factors, such as IFN-γ [83], IL-10 [79], NO [83], and lipocortin [84] are negative regulators of COX-2 expression and activation. Interestingly, PGE2 synthesis is rapidly augmented when microglia are treated with phosphatidylserine (PS) liposomes in a manner that is dependent on the COX-1/mPGES-2 axis [85]. ...
The local and systemic production of prostaglandin E(2) (PGE(2)) and its actions in phagocytes lead to immunosuppressive conditions. PGE(2) is produced at high levels during inflammation, and its suppressive effects are caused by the ligation of the E prostanoid receptors EP(2) and EP(4), which results in the production of cyclic AMP. However, PGE(2) also exhibits immunostimulatory properties due to binding to EP(3), which results in decreased cAMP levels. The various guanine nucleotide-binding proteins (G proteins) that are coupled to the different EP receptors account for the pleiotropic roles of PGE(2) in different disease states. Here, we discuss the production of PGE(2) and the actions of this prostanoid in phagocytes from different tissues, the relative contribution of PGE(2) to the modulation of innate immune responses, and the novel therapeutic opportunities that can be used to control inflammatory responses.
... ANXA1 is induced by glucocorticoids in inflammatory cells and mediates many of the anti-inflammatory actions of these drugs (reviewed in [51,52]). In LPS-stimulated microglia, ANXA1 potently inhibits induction of inducible nitric oxide synthase and COX-2, which are major players in neurodegenerative disorders with an inflammatory component [53]. Of note, ANXA1 displays pronounced effects on migratory properties of immunocompetent cells. ...
Microglia, the immunocompetent cells of the CNS, are rapidly activated in response to injury and microglia migration towards and homing at damaged tissue plays a key role in CNS regeneration. Lysophosphatidic acid (LPA) is involved in signaling events evoking microglia responses through cognate G protein-coupled receptors. Here we show that human immortalized C13NJ microglia express LPA receptor subtypes LPA1, LPA2, and LPA3 on mRNA and protein level. LPA activation of C13NJ cells induced Rho and extracellular signal-regulated kinase activation and enhanced cellular ATP production. In addition, LPA induced process retraction, cell spreading, led to pronounced changes of the actin cytoskeleton and reduced cell motility, which could be reversed by inhibition of Rho activity. To get an indication about LPA-induced global alterations in protein expression patterns a 2-D DIGE/LC-ESI-MS proteomic approach was applied. On the proteome level the most prominent changes in response to LPA were observed for glycolytic enzymes and proteins regulating cell motility and/or cytoskeletal dynamics. The present findings suggest that naturally occurring LPA is a potent regulator of microglia biology. This might be of particular relevance in the pathophysiological context of neurodegenerative disorders where LPA concentrations can be significantly elevated in the CNS.
... Prostaglandins inhibit the production of TNFα and IL-1 after activation of the cells with LPS [8]. Steroids such as dexamethasone also have a deactivating effect on activated microglia by decreasing the production of NO [9]. Neuronal mediators, such as β-adrenergic agonists and glutamate, decrease the activation of microglia. ...
Microglial cells are the resident phagocytic cells of the central nervous system (CNS). They possess a wide range of receptors
allowing them to identify and internalize numerous pathogens. We will discuss here the role of the most important receptors
of microglia involved in non-opsonin-dependent phagocytosis (mannose receptor, β-glucan receptor, scavenger receptor) and that of receptors involved in the opsonin-dependent phagocytosis, namely the complement
3 (CR3) and the Fcγ receptors (FcγR). First, the molecular and cellular mechanisms induced when these receptors are conducting a phagocytic event are presented.
In the second part, we will discuss the role these receptors may play in multiple sclerosis and Alzheimer’s disease, in the
elimination by phagocytosis of myelin and beta amyloid peptide respectively.
... enriched monolayers (Fig. 3A) was derived from NOS-2 protein expressed by astrocytes. COX-2 expression, another gene induced in both astrocytes and microglia (Hewett, 1999;Minghetti et al., 1999), was similarly reduced, but not eliminated, by pretreatment with 75 mM LME, providing further support for the notion that protein expression induced in enriched monolayers was astrocyte-derived (Fig. 5B). ...
Microglia, resident phagocytic cells of the central nervous system, are frequent contaminants of astrocyte cultures. Unfortunately and not always fully appreciated, contamination by microglia can confound results of studies designed to elucidate the molecular mechanisms underlying astrocyte-specific responses. The paradigm described herein employs the mitotic inhibitor, cytosine β-D: -arabinofuranoside, followed by the lysosomotropic agent, leucine methylester, to maximally deplete microglia, thereby generating highly enriched astrocyte monolayers that remain viable and functional. Successful removal of microglia from confluent monolayers of primary astrocyte cultures is achieved without the need for cell passage and successful reduction is confirmed by depletion of microglial-specific markers.
... Many subsequent studies have revealed additional and potentially more significant roles in the inhibition of adhesion and transmigration of leukocytes, thereby limiting the intensity and duration of the inflammatory response (7). Inflammatory mediator production is dampened by ANXA1 repression of inducible nitric oxide synthase 2 and cyclooxygenase-2 (COX-2) in macrophages (8). ANXA1 Ϫ/Ϫ mice have increased expression of COX-2 and PLA 2 (9), an exaggerated response to carrageenin-or zymosan-induced inflammation, and are partially resistant to the antiinflammatory effects of glucocorticoids (10). ...
The role of the calcium- and phospholipid-binding protein annexin I (ANXA1) in cell cycle regulation has been investigated in estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 breast tumor cell lines. In MCF-7 cells, ANXA1-targeting small interfering RNA (siRNA) reduced ANXA1 mRNA and protein levels and attenuated cell proliferation induced by FCS, estradiol, or epidermal growth factor. Well-characterized agonists for the known ANXA1 receptor, FPR2, including the ANXA1 N-terminal proteolytic product ANXA1(2-26), lipoxin A(4) (LXA(4)), and the synthetic peptide, Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm), stimulated proliferation of MCF-7 and MDA-MB-231 cells that was attenuated by incubation with FPR2 antagonists WRW(4) (1 μM) or Boc2 (100 nM) or by siRNA against FPR2. FCS-induced mitogenic responses were attenuated by each of the FPR antagonists and by siRNA against FPR2 and, to a lesser extent, FPR1. LXA(4) increased phosphorylation of Akt, p70(S6K) but not ERK1/2. Increases in cyclin D1 protein induced by FCS or LXA(4) were blocked by the PI3 kinase inhibitor, LY294002, and attenuated by FPR2 antagonism using Boc2. In invasive breast cancer, immunohistochemistry revealed the presence of ANXA1 and its receptor, FPR2, in both tumor epithelium and stromal cells. These observations suggest a novel signaling role for ANXA1 in mitogen-activated proliferation of breast tumor epithelial cells that is mediated via activation of FPR1 and FPR2.
... Dahingegen zeigten Cop-1, die Phosphodiesterase-Inhibitoren Rolipram und Pentoxifyllin, die Zytokine IFNβ und IL-10 und die Steroide Estradiol, Testosteron und DHEA keinen modulierenden Einfluss.Einfluss von Steroiden auf die iNOS/NO-ProduktionAufgrund der Untersuchung mehrerer verschiedener Glucocorticosteroide, war es möglich die Substanzen untereinander zu vergleichen und Rückschlüsse auf die Art der beobachteten Hemmung zu ziehen. In früheren Studien war lediglich ein inhibierender Effekt von Dexamethason auf die LPS-induzierte iNOS-Synthese in primärer Mikroglia gezeigt worden(MINGHETTI et al., 1999). Die Effiziens der Inhibition nahm in der Reihenfolge Methylprednisolon =Dexamethason > Hydrocortison > Progesteron ab. ...
Die Multiple Sklerose (MS) ist eine chronisch-entzündliche, demyelinisierende Erkrankung des Gehirns. Bei der Pathogenese der MS spielt unter anderem das freie Radikal Stickoxid (NO) eine Rolle. Dieses wird bei Entzündungsprozessen durch die induzierbare NO-Synthase (iNOS) gebildet. Die iNOS wurde bei MS-Patienten in Astrozyten und in Mikroglia nachgewiesen.
In der Modellerkrankung der MS, der Experimentellen Allergischen Enzephalomyelitis (EAE), wurde durch den Einsatz von iNOS-Inhibitoren eine Beeinflussung der klinischen Symptomatik beobachtet. Davon ausgehend wurde die Hypothese entwickelt, dass eine Inhibition der iNOS mit für die Wirkung der MS-Therapeutika verantwortlich sein könnte. Um darüber Aufschluss zu erhalten, wurden in dieser Studie verschiedene Glucocorticosteroide, bereits angewendete sowie experimentell eingesetzte Therapeutika daraufhin untersucht ob sie die iNOS/NO-Synthese in Mikroglia modulieren können. Dafür wurden primäre Mikroglia-Kulturen neugeborener Ratten verwendet, die nach der Induktion mit LPS (Lipopolysaccharide) das iNOS-Protein und NO synthetisieren.
Von den Glucocorticosteroiden erwiesen sich Methylprednisolon, Dexamethason, Hydrocortison und Progesteron als kompetente Inhibitoren der LPS-induzierten iNOS-Synthese. Estradiol, Testosteron, Cholesterol und DHEA hatten keinen Effekt. Die bereits eingesetzten Pharmaka Interferon-beta (IFNß) und Copolymer-1 (Cop-1) übten in unseren Untersuchungen keinen hemmenden Effekt auf die iNOS/NO-Synthese aus. Von den bei der EAE mit Erfolg angewendeten Therapeutika zeigte der Transforming Growth Factor-beta (TGFß) eine inhibierende Wirkung. Die Phosphodiesterasehemmer Rolipram und Pentoxifyllin, der Matrix-Metalloproteinasen-Inhibitor BB 3103 und das Zytokin Interleukin-10 zeigten in den Untersuchungen keinen Effekt.
Diese Untersuchungen zeigen, dass die Hypothese, dass die Hemmung der iNOS-Synthese ein allgemeines Wirkprinzip von MS-Therapeutika darstellt, nicht bestätigt werden konnte.
Hepatocellular carcinoma (HCC) is the most common liver cancer and is featured with prominent disparity in incidence and mortality rate between males and females. Phospholipases are enzymes that hydrolyze phospholipids (PLs) either with acylhydrolase or phosphodiesterase activity. Phospholipases are involved in lipid metabolism, membrane dynamics, cellular signaling, migration, growth, and cell death. Alterations of phospholipases in hepatic tissues play important roles in pathogenesis, progression, and gender disparity of HCC. Thus, inhibition of phospholipases may be novel targets for HCC chemotherapeutics. This book chapter summarizes the different types of phospholipases and their expressions in cancers, focuses on the potential mechanisms by which these esterases mediate carcinogenesis, and explains how the inhibition of phospholipases may be targets for HCC treatment.
In the context of the electroacupuncture (EA) neurobiological mechanisms, we have previously demonstrated the involvement of formyl peptide receptor 2 (FPR2/ALX) in the antihyperalgesic effect of EA. The present study investigated the involvement of peripheral FPR2/ALX in the antihyperalgesic effect of EA on inflammatory cytokines levels, oxidative stress markers and antioxidant enzymes in an animal model of persistent inflammatory pain. Male Swiss mice underwent intraplantar (i.pl.) injection with complete Freund's adjuvant (CFA). Mechanical hyperalgesia was assessed with von Frey monofilaments. Animals were treated with EA (2/10 Hz, ST36-SP6, 20 minutes) for 4 consecutive days. From the first to the fourth day after CFA injection, animals received i.pl. WRW4 (FPR2/ALX antagonist) or saline before EA. Levels of inflammatory cytokines (TNF, IL-6, IL-4 and IL-10), antioxidant enzymes (catalase and superoxide dismutase), oxidative stress markers (TBARS, protein carbonyl, nitrite/nitrate ratio), and myeloperoxidase activity were measured in paw tissue samples. As previously demonstrated, i.pl. injection of the FPR2/ALX antagonist prevented the antihyperalgesic effect induced by EA. Furthermore, animals treated with EA showed higher levels of IL-10 and catalase activity in the inflamed paw, and these effects were prevented by the antagonist WRW4. EA did not change levels of TNF and IL-6, SOD and MPO activity, and oxidative stress markers. Our work demonstrates that the antihyperalgesic effect of EA on CFA-induced inflammatory pain could be partially associated with higher IL-10 levels and catalase activity, and that these effects may be dependent, at least in part, on the activation of peripheral FPR2/ALX.
Long noncoding RNAs (lncRNAs) are defined as transcripts of more than 200 nucleotides that have little or no coding potential. LncRNAs function as key regulators in diverse physiological and pathological processes. However, the roles of lncRNAs in lipopolysaccharide (LPS)-induced acute liver injury (ALI) are still elusive. In this study, we report the roles of lncRNA Gm26917 induced by LPS in modulating liver inflammation. As key components of the innate immune system, macrophages play critical roles in the initiation, progression and resolution of ALI. Our studies demonstrated that Gm26917 localized in the cytoplasm of hepatic macrophages and globally regulated the expression of inflammatory genes and the differentiation of macrophages. In vivo study showed that lentivirus-mediated gene silencing of Gm26917 attenuated liver inflammation and protected mice from LPS-induced ALI. Furthermore, mechanistic study showed that the 3′-truncation of Gm26917 interacted with the N-terminus of Annexin A1, a negative regulator of the NF-κB signaling pathway. We also found that Gm26917 knockdown suppressed NF-κB activity by decreasing the ubiquitination of Annexin A1 and its interaction with NEMO. In addition, expression of Gm26917 in inflammatory macrophages was regulated by the transcription factor forkhead box M1 (FOXM1). LPS treatment dramatically increased the binding of FOXM1 to the promoter region of Gm26917 in macrophages. In summary, our findings suggest that lncRNA Gm26917 silencing protects against LPS-induced liver injury by regulating the TLR4/NF-κB signaling pathway in macrophages.
Glucocorticoid steroids are widely used as immunomodulatory agents in acute and chronic conditions. Glucocorticoid steroids such as prednisone and deflazacort are recommended for treating Duchenne Muscular Dystrophy where their use prolongs ambulation and life expectancy. Despite this benefit, glucocorticoid use in Duchenne Muscular Dystrophy is also associated with significant adverse consequences ranging from adrenal suppression, growth impairment, poor bone health and metabolic syndrome. For other forms of muscular dystrophy like the limb girdle dystrophies, glucocorticoids are not typically used. Here we review the experimental evidence supporting multiple mechanisms of glucocorticoid action in dystrophic muscle including their role in dampening inflammation and myofiber injury. We also discuss alternative dosing strategies as well as novel steroid agents that are in development and testing, with the goal to reduce adverse consequences of prolonged glucocorticoid exposure while maximizing beneficial outcomes.
The protein annexin A1 (ANXA1) belongs to the danger-associated molecular patterns (DAMPs) that alert the innate immune system about tissue perturbations. In the context of immunogenic cell death (ICD), ANXA1 is released from the cytoplasm of dying cells and, once extracellular, acts on formyl peptide receptor 1 (FPR1) expressed on dendritic cells to favor long-term interactions between dying and dendritic cells. As a result, the accumulation of extracellular ANXA1 constitutes one of the hallmarks of ICD. In the past, the detection of ANXA1 was based on semiquantitative immunoblots. More recently, a commercial enzyme-linked immunosorbent assay (ELISA) has been developed to measure ANXA1 in an accurate fashion. Here, we detail the protocol to measure the concentration of ANXA1 in the supernatants of cancer cells treated with chemotherapy.
Acute inflammation is a self-limiting process of the immune system, which resolves through the initiation of a program referred to as the resolution of inflammation. It has been argued that uncontrolled inflammation may be the basis of a variety of chronic inflammatory and autoimmune diseases. The resolution of inflammation is an active process coordinated by the production of proresolving mediators. The release of proresolving mediators prevents further migration of granulocytes, and increases leukocyte apoptosis. Moreover, some proresolving molecules are able to promote the infiltration of nonphlogistic macrophages, which are fundamental cells to efferocyte of apoptotic granulocytes. This event, in turn, triggers macrophage reprogramming towards more restorative and resolutive roles, thereby promoting resolution and reestablishment of tissue homeostasis. Here, we summarize the most prominent pro-resolving mediators relevant to the resolution of inflammation.
Annexin A1 (AnxA1) is a glucocorticoidregulated protein known for its antiinflammatory and proresolving effects. We have previously shown that cAMP enhancing compounds rolipram (ROL - a PDE4 inhibitor) and db-cAMP (cAMP mimetic) drive caspasedependent resolution of neutrophilic inflammation. In this follow up study, we investigated whether AnxA1 could be involved in the proresolving properties of these compounds using a model of LPS-induced inflammation in BALB/c mice. The treatment with ROL or db-cAMP at the peak of inflammation shortened resolution intervals, improved resolution indices and increased AnxA1 expression. In vitro studies showed that ROL and db-cAMP induced AnxA1 expression and phosphorylation and this effect was prevented by PKA inhibitors, suggesting the involvement of PKA on ROL-induced AnxA1 expression. Akin to these in vitro findings, H89 prevented ROL and db-cAMP-induced resolution of inflammation, and it was associated with decreased levels of intact AnxA1. Moreover, two different strategies to block the AnxA1 pathway - by using BOC-1 a nonselective AnxA1 receptor antagonist or by using an anti-AnxA1 neutralizing antiserum -prevented ROL and db-cAMP-induced resolution and neutrophil apoptosis. Likewise, the ability of ROL or db-cAMP to induce neutrophil apoptosis was impaired in AnxA knockout mice. Finally, in in vitro settings ROL and db-cAMP overrode the survival-inducing effect of LPS in human neutrophils in an AnxA1dependent manner. Our results show that AnxA1 is at least one of the endogenous determinants mediating the proresolving properties of cAMP elevating agents and cAMPmimetic drugs.
These anti-inflammatory drugs that are used to treat acute gout are discussed in detail. Their administration, pharmacology, and toxicity are considered. Then, urate-lowering therapy is thoroughly described, again considering the administration, pharmacology, and toxicity of these agents. The widespread mismanagement of gout in general and even specialty medical practice makes this information important for patients and their physicians.
The elevation of body temperature during fever reaction and endogenous antipyretic effect reflects the existing balance between 2 components directed to the maximal positive use of the fever effects and simultaneous prevention of their side effects. The mechanisms involved in the control of balance between factors known as the fever triggers and endogenous antipyretic factors. The possible mechanisms of action of various endogenous antipyretic system components (glucocorticoids, neuropeptides, cytokines etc.) capable to regulate the duration and quantity of fever reaction are discussed in this review.
Annxine-1 (Anx-1), a member of the annexin superfamily of calcium- and phospholipid- binding proteins, is induced by glucocorticoids (GC) and functions as a mediator of their anti-inflammatory effects. The wide range of effects of Anx-1 includes inhibition of leukocyte recruitment, suppression of the production of inflammatory mediators and cytokines, and induction of apoptosis in inflammatory cells. This profile of activity suggests that the inhibitory effects of Anx-1 would be beneficial in the pathological context of rheumatoid arthritis (RA). Anx-1 is expressed in human RA synovial tissue and cells, and has recently been identified as an important endogenous anti-inflammatory mediator in multiple animal models of RA. Emerging data on the mechanisms of action of Anx-1 suggest it acts to inhibit mitogen activated protein (MAP) kinases, through as yet unidentified mechanisms, thereby inhibiting pro-inflammatory pathways known to be important in RA. Anx-1 treatment strategies, especially as an alternative to GC therapy, may be valuable in RA and other inflammatory diseases.
Lipocortin 1 (LC1) has been shown to increase in neuronal damage and act as a neuroprotectant and a neurotrophic factor. IL-1β acts as a mediator of inflammation and has been reported as a potent inducer of various neurotrophic factors including nerve growth factor and fibroblast growth factor. In this study, we investigated the relationship between LC1 and IL-1β in cultured rat astrocytes. Time–and dose–dependent experiments of IL-1β on rat cortical astrocytes in culture revealed that the expression of LC1 mRNA was significantly augmented by IL-1β at 8 h, 10 ng/ml. In addition, IL-1β evoked an extracellular secretion of LC1 without its cytotoxic effects. The effect of IL-1β was completely abolished when we treated cells with inhibitor of mitogen-activated protein kinases (MAPKs) (PD98059) (25 μM), phospholipase A2 inhibitor mepacrine (30 μM) and protein synthesis inhibitor cycloheximide (CHX) (10 μg/ml). This suggests that induction of LC1 by IL-1β is through a MAPKs and phospholipaseA2 pathway and requires protein synthesis. These results indicate that IL-1β released in the central nervous system (CNS) injury can stimulate the transcription of the LC1 gene. Subsequent synthesis and release of LC1 may provide trophic support to neurons and modulate the action of IL-1β in brain damage.
Increasing experimental, clinical, and epidemiological studies point to the pivotal role of inflammation in the pathogenesis of acute and chronic neurodegenerative diseases and to the protective effects of nonsteroidal antiinflammatory drug (NSAID) therapies. Nonetheless, NSAID long-term therapies are limited by their significant adverse effects on gastrointestinal tract and kidneys. Nitroflurbiprofen (NO-flurbiprofen) belongs to a novel class of antiinflammatory agents obtained by derivatization of conventional NSAIDs with a nitric oxide (NO)-releasing moiety, which strongly reduces their untoward side effects without altering the antiinflammatory effectiveness. The recent evidence of neuroprotective effects of NO-NSAIDs in animal models of chronic brain inflammation prompted us to investigate the activities of NO-flurbiprofen and its parent molecule flurbiprofen on activated rat microglia, the brain resident macrophages. We found that NO-flurbiprofen was as potent as flurbiprofen in preventing prostaglandin E2 synthesis in lipopolysaccharide-activated microglial cultures. At variance with previous observations on peripheral macrophages is that NO-flurbiprofen did not show any additional capacity to inhibit interleukin-1β synthesis compared with flurbiprofen. Moreover, NO enhanced the expression of the inducible NO synthase; this effect was most likely attributable to the NO released from the drug, as suggested by experiments performed in the presence of the NO donor Deta-NONOate, which similarly to NO-flurbiprofen is characterised by a slow and long-lasting release. Our findings indicate that NO-NSAIDs may differently affect peripheral and brain macrophages. Given their potential therapeutic role in brain inflammation, further in vivo and in vitro studies are required to understand fully their mechanism of action in the CNS. J. Neurosci. Res. 66:715–722, 2001. © 2001 Wiley-Liss, Inc.
In a previous study, we reported a new bioactive sesquiterpenoid, named dysidotronic acid, to be a potent, selective human synovial phospholipase A2 inhibitor. Dysidotronic acid is a novel, non-complex manoalide analogue lacking the pyranofuranone ring. We now investigate the effect of this compound on cytokine, nitric oxide and prostanoid generation on the mouse macrophage cell line RAW 264.7, where it showed a dose-dependent inhibition with inhibitory concentration 50% values in the micromolar range. This effect was also confirmed in the mouse air pouch injected with zymosan. Dysidotronic acid inhibited the production of tumor necrosis factor alpha and interleukin-1 beta as well as the production of nitric oxide, prostaglandin E2 and leukotriene B4. Decreased nitric oxide generation was the consequence of inhibition of the expression of nitric oxide synthase, whereas PGE2 and LTB4 reduction was due to inhibition of arachidonic acid bioavailability through a direct inhibitory effect of dysodotronic acid on secretory phospholipase A2.
The antiinflammatory glucocorticoids are potent inhibitors of cyclooxygenase, a key regulator of prostaglandin synthesis; yet, the mechanism(s) by which this occurs is not fully understood. We have cloned a 4.1-kilobase (kb) cDNA, distinct from the previously cloned cyclooxygenase (2.8 kb), that confers cyclooxygenase activity to transfected cells. The mRNA for this newly discovered cyclooxygenase is unique for its long 3' untranslated region containing many AUUUA repeats. Levels of the 4.1-kb cyclooxygenase mRNA are rapidly increased by serum or interleukin 1 beta in mouse fibroblasts and human monocytes, respectively, and decreased by glucocorticoids, whereas levels of the 2.8-kb cyclooxygenase mRNA do not change. Similar effects are seen in the presence of cycloheximide where the 4.1-kb, but not the 2.8-kb, mRNA is greatly superinduced. Thus, there are both constitutive (2.8 kb) and regulated (4.1 kb) cyclooxygenase species, the latter most likely being a major mediator of inflammation.
Nitric oxide (NO) mediates several biological actions, including relaxation of blood vessels, cytotoxicity of activated macrophages, and formation of cGMP by activation of glutamate receptors in cerebellar slices. Nitric oxide synthase (EC 1.14.23.-) immunoreactivity is colocalized with nicotinamide adenine di-nucleotide phosphate diaphorase in neurons that are uniquely resistant to toxic insults. We show that the nitric oxide synthase inhibitors, N omega-nitro-L-arginine (EC50 = 20 microM) and N omega-monomethyl-L-arginine (EC50 = 170 microM), prevent neurotoxicity elicited by N-methyl-D-aspartate and related excitatory amino acids. This effect is competitively reversed by L-arginine. Depletion of the culture medium of arginine by arginase or arginine-free growth medium completely attenuates N-methyl-D-aspartate toxicity. Sodium nitroprusside, which spontaneously releases NO, produces dose-dependent cell death that parallels cGMP formation. Hemoglobin, which complexes NO, prevents neurotoxic effects of both N-methyl-D-aspartate and sodium nitroprusside. These data establish that NO mediates the neurotoxicity of glutamate.
Lipocortin-1 (annexin-1) is an endogenous peptide with antiinflammatory properties. We have previously demonstrated lipocortin immunoreactivity in certain glial cells and neurons in the rat brain (Strijbos, P.J.L.M., F.J.H. Tilders, F. Carey, R. Forder, and N.J. Rothwell. 1990. Brain Res. In press.), and have shown that an NH2-terminal fragment (1-188) of lipocortin-1 inhibits the central and peripheral actions of cytokines on fever and thermogenesis in the rat in vivo (Carey, F., R. Forder, M.D. Edge, A.R. Greene, M.A. Horan, P.J.L.M. Strijbos, and N.J. Rothwell. 1990. Am. J. Physiol. 259:R266; and Strijbos, P.J.L.M., J.L. Browning, M. Ward, R. Forder, F. Carey, M.A. Horan, and N.J. Rothwell. 1991. Br. J. Pharmacol. In press.). We now report that intracerebroventricular administration of lipocortin-1 fragment causes marked inhibition of infarct size (60%) and cerebral edema (46%) measured 2 h after cerebral ischemia (middle cerebral artery occlusion) in the rat in vivo. The lipocortin-1 fragment was effective when administered 10 min after induction of ischemia. Ischemia caused increased expression of lipocortin-1 around the area of infarction as demonstrated by immunocytochemistry. Intracerebroventricular injection of neutralizing antilipocortin-1 fragment antiserum increased the size of infarct (53%) and the development of edema (29%). These findings indicate that lipocortin-1 is an endogenous inhibitor of cerebral ischemia with considerable therapeutic potential.
We report here that the bacterial lipopolysaccharide endotoxin induces human blood monocytes in a time- and dose-dependent manner to release prodigious amounts of prostaglandins with thromboxane A2, the major metabolite formed. Cells responded to as little as 1 ng/ml lipopolysaccharide to release prostaglandin E2 and thromboxane A2 with maximal stimulation at 10 micrograms/ml. Lipopolysaccharide was found to induce increased activity of cyclooxygenase enzyme without affecting the activities of phospholipase and thromboxane synthase or the formation of 5-lipoxygenase products (e.g. leukotriene B4). The glucocorticoid dexamethasone completely blocked the lipopolysaccharide-induced prostanoid release by inhibiting the activity of monocyte cyclooxygenase. Dexamethasone did not affect phospholipase and thromboxane synthase activities or leukotriene formation. Immunoprecipitation of [35S]methionine-labeled cyclooxygenase confirmed that the effect of lipopolysaccharide and dexamethasone on the monocyte prostanoid production could be attributed to an increase or decrease, respectively, in cellular cyclooxygenase de novo synthesis.
Administration of Escherichia coli lipopolysaccharide (LPS; 10 mg/kg i.v.) to male Wistar rats caused within 240 min (i) a sustained fall (approximately 30 mmHg) in mean arterial blood pressure, (ii) a reduction (> 75%) in the pressor responses to norepinephrine (1 microgram/kg i.v.), and (iii) an induction of nitric oxide synthase (iNOS) as measured in the lung. Dexamethasone (1 mg/kg i.p. at 2 h prior to LPS) attenuated the hypotension and the vascular hyporeactivity to norepinephrine and reduced (by approximately 77%) the expression of iNOS in the lung. These effects of dexamethasone were prevented by pretreatment of LPS-treated rats with a neutralizing antiserum to lipocortin 1 (anti-LC1; 60 mg/kg s.c. at 24 h prior to LPS) but not by a control nonimmune sheep serum. Stimulation of J774.2 macrophages with LPS (1 microgram/ml for 24 h) caused the expression of iNOS and cyclooxygenase 2 (COX-2) protein and significantly increased nitrite generation; this was prevented by dexamethasone (0.1 microM at 1 h prior to LPS), which also increased cell surface lipocortin 1. Pretreatment of J774.2 cells with anti-LC1 (1:60 dilution at 4 h prior to LPS) also abolished the inhibitory effect of dexamethasone on iNOS expression and nitrite accumulation but not that on COX-2 expression. A lipocortin 1 fragment (residues 1-188 of human lipocortin 1; 20 micrograms/ml at 1 h prior to LPS) also blocked iNOS in J774.2 macrophages activated by LPS (approximately 78% inhibition), and this too was prevented by anti-LC1. We conclude that the extracellular release of endogenous lipocortin 1 (i) mediates the inhibition by dexamethasone of the expression of iNOS, but not of COX-2, and (ii) contributes substantially to the beneficial actions of dexamethasone in rats with endotoxic shock.
We have recently put forward the hypothesis that the dual inhibition of proinflammatory nitric oxide (NO) and prostaglandins (PG) may contribute to the antiinflammatory properties of nitric oxide synthase (NOS) inhibitors. This hypothesis was tested in the present study. A rapid inflammatory response characterized by edema, high levels of nitrites (NO2-, a breakdown product of NO), PG, and cellular infiltration into a fluid exudate was induced by the administration of carrageenan into the subcutaneous rat air pouch. The time course of the induction of inducible nitric oxide synthase (iNOS) protein in the pouch tissue was found to coincide with the production of NO2-. Dexamethasone inhibited both iNOS protein expression and NO2- synthesis in the fluid exudate (IC50 = 0.16 mg/kg). Oral administration of N-iminoethyl-L-lysine (L-NIL) or NG-nitro-L-arginine methyl ester (NO2Arg) not only blocked nitrite accumulation in the pouch fluid in a dose-dependent fashion but also attenuated the elevated release of PG. Finally, carrageenan administration produced a time-dependent increase in cellular infiltration into the pouch exudate that was inhibited by dexamethasone and NOS inhibitors. At early times, i.e., 6 h, the cellular infiltrate is composed primarily of neutrophils (98%). Pretreatment with colchicine reduced both neutrophil infiltration and leukotriene B4 accumulation in the air pouch by 98% but did not affect either NO2- or PG levels. In conclusion, the major findings of this paper are that (a) selective inhibitors of iNOS are clearly antiinflammatory agents by inhibiting not only NO but also PG and cellular infiltration and (b) that neutrophils are not responsible for high levels of NO and PG produced.
The reactive nitrogen species, nitric oxide (NO), plays an important role in the pathogenesis of neurodegenerative diseases. The suppression of NO production may be fundamental for survival of neurons. Here, we report that pretreatment of human ramified microglial cells with nearly physiological levels of exogenous NO prevents lipopolysaccharide (LPS)/tumor necrosis factor alpha (TNF alpha)-inducible NO synthesis, because by affecting NF-kappa B activation it inhibits inducible Ca(2+)-independent NO synthase isoform (iNOS) mRNA expression. Using reverse transcriptase polymerase chain reaction, we have found that both NO donor sodium nitroprusside (SNP) and authentic NO solution are able to inhibit LPS/TNF alpha-inducible iNOS gene expression; this effect was reversed by reduced hemoglobin, a trapping agent for NO. The early presence of SNP during LPS/TNF alpha induction is essential for inhibition of iNOS mRNA expression. Furthermore, SNP is capable of inhibiting LPS/TNF alpha-inducible nitrite release, as determined by Griess reaction. Finally, using electrophoretic mobility shift assay, we have shown that SNP inhibits LPS/TNF alpha-elicited NF-kappa B activation. This suggests that inhibition of iNOS gene expression by exogenous NO may be ascribed to a decreased NF-kappa B availability.
The cellular localization of inducible (iNOS) and constitutive (cNOS) nitric oxide synthase was studied in rats by immunocytochemical techniques involving specific iNOS and cNOS directed antibodies and by NADPH-diaphorase histochemistry. Paraformaldehyde-fixed vibratome sections of brains and cryostat sections of peripheral lymph nodes were studied of rats treated with endotoxin (2.5 micrograms/kg or 2.5 mg/kg i.v.), rats infected with rabies virus, and rats exposed to experimental allergic encephalomyelitis (EAE). Endotoxin-treated animals showed no appearance of immunoreactive iNOS (ir-iNOS) cells in the brain with the exception of a few microglial cells near the median eminence and some meningeal macrophages. In the same animals however, iNOS-immunoreactive cells were found in peripheral lymph nodes. Neurons that stain positive for cNOS and for NADPH-diaphorase could be observed in brains of control as well as of endotoxin-treated animals with a similar distribution and staining intensity. In contrast, animals that had been infected with rabies virus or subjected to EAE, showed the appearance of ir-iNOS-positive cells in several brain areas. These cells are located near blood vessels and lesion sites. The majority of these cells are GSA-I-B4 isolectin-positive and therefore are likely to represent macrophages. Our data suggest that increased production of nitric oxide may play a role in the altered brain functions in rabies-infected and EAE rats. On the contrary, increased nitric oxide production is probably not involved in the non-specific symptoms of sickness induced by endotoxin.
Rat ameboid microglia are able to lyse rat oligodendrocytes in vitro. The lysis is inhibited by transforming growth factor-beta, antagonists of nitric oxide (NO) production, as well as antibodies to TNF-alpha, intercellular adhesion molecule-1 (ICAM-1), and leukocyte functional Ag-1. Ameboid microglial cells spontaneously produce detectable levels of the NO metabolite nitrite (NO2-). Stimuli such as PMA, LPS, and/or IFN-gamma induce micromolar concentrations of NO2- within 24 h. TNF-alpha increases IFN gamma but not LPS-induced NO2- production. Incubation with target oligodendrocytes also increases NO2- production in a contact-dependent manner. NO2- production is inhibited by NO synthase antagonists, transforming growth factor-beta, and anti TNF-alpha. Neither antileukocyte functional Ag-1 nor anti-ICAM-1 inhibit NO2- production by microglia in the presence or absence of oligodendrocytes. Indeed, anti-ICAM-1 treatment increases NO2- production. There is a correlation between ameboid microglial cell killing of oligodendrocytes and NO2- production suggesting NO may be a mechanism of death of the oligodendrocyte and possibly play a role in lesion formation in multiple sclerosis.
The goal of our study was to assess whether the human immunodeficiency virus (HIV) coat protein gp120 induces functional alterations in astrocytes and microglia, known for their reactivity and involvement in most types of brain pathology. We hypothesized that gp120-induced anomalies in glial functions, if present, might be mediated by changes in the levels of intracellular messengers important for signal transduction, such as cAMP. Acute (10 min) exposure of cultured rat cortical astrocytes or microglia to 100 pM gp120 caused only a modest (50-60%), though statistically significant, elevation in cAMP levels, which was antagonized by the beta-adrenergic receptor antagonist propranolol. More importantly, the protein substantially depressed [by 30% (astrocytes) and 50% (microglia)] the large increase in cAMP induced by the beta-adrenergic agonist isoproterenol (10 nM), without affecting that induced by direct adenylate cyclase stimulation by forskolin. Qualitatively similar results were obtained using a glial fibrillary acidic protein (GFAP)-positive human glioma cell line. The depression of the beta-adrenergic response had functional consequences in both astrocytes and microglia. In astrocytes we studied the phosphorylation of the two major cytoskeletal proteins, vimentin and GFAP, which is normally stimulated by isoproterenol, and found that gp120 partially (40-50%) prevented such stimulation. In microglial cells, which are the major producers of inflammatory cytokines within the brain, gp120 partially antagonized the negative beta-adrenergic modulation of lipopolysaccharide (10 ng/ml)-induced production of tumor necrosis factor alpha. Our results suggest that, by interfering with the beta-adrenergic regulation of astrocytes and microglia, gp120 may alter astroglial "reactivity" and upset the delicate cytokine network responsible for the defense against viral and opportunistic infections.
Lipocortin-1 (annexin-1) is an endogenous peptide with antiinflammatory properties. We have previously demonstrated lipocortin immunoreactivity in certain glial cells and neurons in the rat brain (Strijbos, P.J.L.M., F.J.H. Tilders, F. Carey, R. Forder, and N.J. Rothwell. 1990. Brain Res. In press.), and have shown that an NH2-terminal fragment (1-188) of lipocortin-1 inhibits the central and peripheral actions of cytokines on fever and thermogenesis in the rat in vivo (Carey, F., R. Forder, M.D. Edge, A.R. Greene, M.A. Horan, P.J.L.M. Strijbos, and N.J. Rothwell. 1990. Am. J. Physiol. 259:R266; and Strijbos, P.J.L.M., J.L. Browning, M. Ward, R. Forder, F. Carey, M.A. Horan, and N.J. Rothwell. 1991. Br. J. Pharmacol. In press.). We now report that intracerebroventricular administration of lipocortin-1 fragment causes marked inhibition of infarct size (60%) and cerebral edema (46%) measured 2 h after cerebral ischemia (middle cerebral artery occlusion) in the rat in vivo. The lipocortin-1 fragment was effective when administered 10 min after induction of ischemia. Ischemia caused increased expression of lipocortin-1 around the area of infarction as demonstrated by immunocytochemistry. Intracerebroventricular injection of neutralizing antilipocortin-1 fragment antiserum increased the size of infarct (53%) and the development of edema (29%). These findings indicate that lipocortin-1 is an endogenous inhibitor of cerebral ischemia with considerable therapeutic potential.
Nitric oxide (NO) mediates several biological actions, including relaxation of blood vessels, cytotoxicity of activated macrophages, and formation of cGMP by activation of glutamate receptors in cerebellar slices. Nitric oxide synthase (EC 1.14.23.-) immunoreactivity is colocalized with nicotinamide adenine di-nucleotide phosphate diaphorase in neurons that are uniquely resistant to toxic insults. We show that the nitric oxide synthase inhibitors, N omega-nitro-L-arginine (EC50 = 20 microM) and N omega-monomethyl-L-arginine (EC50 = 170 microM), prevent neurotoxicity elicited by N-methyl-D-aspartate and related excitatory amino acids. This effect is competitively reversed by L-arginine. Depletion of the culture medium of arginine by arginase or arginine-free growth medium completely attenuates N-methyl-D-aspartate toxicity. Sodium nitroprusside, which spontaneously releases NO, produces dose-dependent cell death that parallels cGMP formation. Hemoglobin, which complexes NO, prevents neurotoxic effects of both N-methyl-D-aspartate and sodium nitroprusside. These data establish that NO mediates the neurotoxicity of glutamate.
The NF-κB/Rel family of transcription factors participates in the activation of a diverse range of genes involved in inflammation, immune response, lymphoid differentiation, growth control and development. The present review provides a brief overview of NF-κB/Rel activation and a detailed analysis of important biological and biochemical inhibitors of the NF-κB/Rel pathway. Given the pleiotropic role of NF-κB in controlling cytokines and other immunoregulatory genes, the inhibition of NF-κB activation by steroid hormones, antioxidants, protease inhibitors and other compounds may provide a pharmacological basis for interfering with pathological inflammatory conditions, cancer and AIDS.
Increased levels of prostanoids have been implicated in various neuropathological diseases, although little is known about their cellular sources inside the brain. In this study, we analyzed the expression of cyclooxygenase-2 (COX-2), a key enzyme in arachidonic acid metabolism, in rat microglia. COX-2 mRNA and protein as well as prostaglandin E2 formation were almost undetectable in unstimulated microglial cultures but were found to be strongly upregulated in response to lipopolysaccharide. However, in contrast to most peripheral cells, proinflammatory cytokines such as tumor necrosis factor α, interleukin-1 β or interleukin-6 failed to markedly induce COX-2 expression. Similar effects were observed by analyzing transcription nuclear factor-κ B (NF-κB) which was strongly activated in microglia by lipopolysaccharide but not by incubation with cytokines. Moreover, known inhibitors of NF-κB activation, such as dexamethasone and the antioxidant pyrrolidine dithiocarbamate, as well as the protein kinase C (PKC) inhibitor Gö6976, strongly reduced lipopolysaccharide-induced COX-2 transcription, indicating the involvement of NF-κB and PKC in COX-2 expression. Our results suggest that microglia may represent an important source of prostanoids in the brain, thus reinforcing their prominent role in cerebral inflammatory processes.
The role played by endogenous lipocortin 1 in the anti-migratory action exerted by dexamethasone (Dex) on monocyte recruitment in an in vivo model of acute inflammation was investigated by use of several neutralizing polyclonal antibodies raised against lipocortin 1 or a lipocortin 1-derived N-terminus peptide (peptide Ac2-26). The efficacy of peptide Ac2-26 in inhibiting monocyte and polymorphonuclear leucocyte (PMN) recruitment was also tested.
Intraperitoneal (i.p.) injection of zymosan A (1 mg) produced a time-dependent cell accumulation into mouse peritoneal cavities which followed a typical profile of acute inflammation: PMN influx was maximal at 4 h post-zymosan (between 15 and 20×106 cells per mouse), and this was followed by an accumulation of monocytes which peaked at the 24 h time-point (between 10 and 15×106 cells per mouse).
Dex administration to mice reduced zymosan-induced 4 h PMN infiltration and 24 h monocyte accumulation with similar efficacy: approximately 50% of inhibition of recruitment of both cell types was achieved at the dose of 30 μg per mouse (∼1 mg kg−1, subcutaneously (s.c.)). Maximal inhibitions of 64% and 67% on PMN and monocyte recruitment, respectively, were measured after a dose of 100 μg per mouse (∼3 mg kg−1, s.c.).
Dex (30 μg s.c.) inhibited monocyte (53%) and PMN (69%) accumulation in response to zymosan application in mice which had been treated with a non-immune sheep serum (50 μl s.c.). In contrast, the steroid was no longer active in reducing cell accumulation in mice which had been passively immunized against full length human recombinant lipocortin 1 (serum LCS3), or against lipocortin 1 N-terminus peptide.
Treatment of mice with vinblastine (1 mg kg−1, intravenously (i.v.)) produced a remarkable leucopenia as assessed 24 h after administration. This was accompanied by a 60% reduction in 4 h-PMN influx, and by a 27% reduction in 24 h-monocyte accumulation, measured after zymosan administration. The inhibitory effect of Dex on monocyte recruitment was not significantly modified in vinblastine-treated mice, with 36% and 57% of inhibition calculated at the dose of 30 μg Dex, and 70% and 60% of inhibition at 100 μg Dex, in vehicle- and vinblastine-treated mice, respectively.
Treatment of mice with peptide Ac2-26 dose-dependently attenuated PMN influx at 4 h post-zymosan with a significant effect at 100 μg per mouse (45% of inhibition, n=9, P<0.05) and a maximal effect of 61% inhibition at the highest dose tested of 200 μg s.c. (n=14, P<0.05). No effect of peptide Ac2-26 (200 μg s.c.) was seen on zymosan-induced 24 h monocyte recruitment. In contrast, administration of 200 μg peptide Ac2-26 every 6 h was effective in reducing the number of monocytes harvested from the inflamed peritoneal cavities at 24 h post-zymosan: 9.40±0.58×106 monocytes per mouse (n=13) and 5.74±0.34 monocytes per mouse (n=14) in vehicle- and peptide Ac2-26-treated mice, respectively (P<0.05).
Finally, peptide Ac2-26 produced a concentration-dependent inhibition of the rate of phagocytosis of mouse resident peritoneal macrophages as measured by flow cytometry, with a maximal reduction of 34% at the highest concentration tested of 100 μg ml−1 (n=8 experiments performed in duplicate; P<0.05).
In conclusion, this study suggests that in vivo monocyte recruitment during acute inflammation is, at least in part, under the negative modulatory control of endogenous lipocortin 1 (as seen after administration of Dex by using the specific antisera) and exogenous lipocortin 1 mimetics (as observed with peptide Ac2-26). In addition to the neutrophil, we can now propose that the monocyte also can be a target for the in vivo anti-inflammatory action of lipocortin 1.
British Journal of Pharmacology (1997) 120, 1075–1082; doi:10.1038/sj.bjp.0701029
A multi-faceted approach was used to investigate the effect of an anti-inflammatory peptide derived from human lipocortin 1 N-terminus region (amino acid 2–26; termed human Ac2–26) on human neutrophil activation in vitro. When incubated with purified human neutrophils. human Ac2–26 produced a concentration-dependent inhibition of elastase release stimulated by formyl-Met-Leu-Phe (fMLP), platelet-activating factor, or leukotriene B4, with an approximate EC50 of 33 μM (100 μg/ml). At this concentration, human Ac2–26 also inhibited (77%) the release of [3H]-arachidonic acid from neutrophils stimulated with fMLP. The peptide, however, did not inhibit the up-regulation of the β2-integrin CD11b and the concomitant shedding of L-selectin from neutrophil plasma membrane induced by fMLP. In adhesion experiments, human Ac2–26 inhibited neutrophil adhesion to endothelial monolayers when this was stimulated with fMLP, but not when this followed endothelial cell activation with histamine or platelet-activating factor. Again, the effect of the peptide was concentration-dependent, and an approximate EC50 of 33 μM was calculated. When a preparation of 125I-labeled human Ac2–26 was incubated with the neutrophils, the peptide was internalised in an energy-dependent fashion. All together, these observations lead us to propose a model in which this peptide derived from the N-terminus of human lipocortin 1 alters a common cellular mechanism producing a selective inhibition of neutrophil activation.
The ability of the glucocorticoid‐induced protein lipocortin 1 (LC1) to inhibit arachidonic acid release and cell proliferation in A549 cells may be mimicked by a sequence taken from the N‐terminal, LC1 13–25 (FIENEEQEYVQTV). We have now synthesized and tested for biological activity a library of 25 smaller peptides derived from this sequence.
Peptides were tested in two assays: A549 cells were prelabelled with tritiated arachidonic acid and thapsigargin (50 n M ) and EGF (10 n M ) used to stimulate the release of this fatty acid. Cell proliferation was determined by counting cell numbers following 3 day incubation with these peptides, or controls.
Many of the peptides were highly insoluble but could be more readily dissolved in aqueous solution in the presence of commercial liposomes or phosphatidyl serine (5 μ M ). Since neither of these agents alone had any effect on arachidonic acid release or cell proliferation, all peptides were tested in the presence of 5 μ M phosphatidyl serine. Under these conditions LC1 13–25 was active in both assay systems with an IC 40 of 40.7 and 57.0 μ M respectively.
Deletion of amino acids from the C‐terminus of the peptide progressively diminished (2–3 fold) the molar potency of LC1 13–25 in both assays: after the removal of Val ²² biological activity was virtually undetectable or very weak (<30% of LC1 13–25 ).
Removal of amino acids from the N‐terminus also lead to a progressive reduction (3–5 fold) in the molar potency of the peptides and biological activity became undetectable, or very weak, after the removal of Glu ¹⁸ .
All active peptides contained the core sequence EQEYV(Glu‐Gln‐Glu‐Tyr‐Val) which seems to represent a crucial component of the pharmacophore, although this sequence on its own was inactive and the shortest peptide with significant activity was LC1 18–25 (EQEYVQTV).
Methoxylation of Tyr ²¹ abolished the ability of LC1 18–25 to inhibit cell proliferation and arachidonic acid release. A cyclized version of LC1 18–25 was also tested and found to be inactive.
LC1 18–25 (178 μ M ) inhibits cPLA 2 activation in A549 cells as judged by a band‐shift assay, whereas equimolar concentrations of an inactive peptide LC1 19–25 were without effect in this assay system.
Several possible mechanisms whereby these peptides act are discussed in the light of LC1 biology and of the effect of glucocorticoids on cell function.
British Journal of Pharmacology (1998) 123 , 975–983; doi: 10.1038/sj.bjp.0701679
The role of inflammatory cytokines in the pathogenesis of neurological diseases is not well understood. The neurotoxic effects of cytokines could be mediated by immunostimulation of glial cells to produce toxic concentrations of nitric oxide (NO) and reactive nitrogen oxides. Cultured microglia and meningeal fibroblasts, but not Type 1 astrocytes, were induced by lipopolysaccharides and cytokines to synthesize NO and reactive nitrogen oxides from L-arginine. In co-cultures of immunostimulated microglia and cerebellar granule neurons, neurotoxicity was blocked by an inhibitor of NO synthase, NG-nitroarginine, and by oxyhemoglobin, which inactivates NO. Microglial-induced neurotoxicity was also partially attenuated by the N-methyl-D-aspartate (NMDA) receptor antagonists, MK-801 and 2-amino-5-phosphovalerate (APV). Superoxide dismutase, which stabilizes NO through inactivation of superoxide anion, augmented microglial-mediated neurotoxicity either alone or in combination with MK-801 or APV. Hence, immunostimulated microglia mediate neurotoxicity by NO, reactive nitrogen oxides, superoxide anion and NMDA-like substances. These findings suggest a novel role for microglial-produced NO and reactive nitrogen oxides as a neurotoxic agent in neurodegenerative disease states.
Activated microglial have been proposed to play a pathogenetic role in immune-mediated neurodegenerative diseases. To test this hypothesis, purified murine neonatal microglial were cocultured with neuronal cells derived from fetal brain. Activation with IFN-gamma and LPS of these cocultures brought about a sharp decrease in uptake of gamma-amino butyric acid and a marked reduction in neuronal cell survival. These effects varied with the density of microglia, the concentrations of the activation signals (IFN-gamma and LPS), and the duration of coculture. Inasmuch as addition of NG-monomethyl-L-arginine blocked these effects, a L-arginine-dependent neurocytotoxic mechanism was implicated. Abundant nitrite, a metabolite of the free radical nitric oxide (NO) derived from L-arginine, was detected in activated microglial/neuronal cell cocultures and in purified microglial cell cultures but not in purified astrocyte or neuronal cell cultures, suggesting that microglial were the principal source of the NO. These findings support the hypothesis that microglia are the source of a neurocytotoxic-free radical, and shed light on an additional mechanism of immune-mediated brain injury.
Lipocortin-1 (annexin-1), an endogenous phospholipid and calcium binding protein, has been shown to significantly attenuate the damage produced by focal cerebral ischaemia in the rat. In the present study we have therefore investigated its effect on N-methyl-D-aspartate (NMDA) induced neuronal damage. Unilateral intrastriatal infusion of a potent and selective NMDA agonist, cis-2,4-methanoglutamate (MGlu), induced an extensive lesion of the striatum in the rat, which was inhibited (greater than 80%) by prior injection of MK801 (4 mg/kg, i.p.). Infusion of 1.2 micrograms of an active fragment of lipocortin-1 (N-terminal 1-188 aa) immediately after MGlu significantly reduced the extent of damage by 44.2 +/- 8.0%. In contrast, infusion of 3 microliters of neutralizing anti-lipocortin-1 antibody with MGlu increased lesion size by 158.9 +/- 22.0%. These findings indicate that the damage produced by intrastriatal infusion of MGlu is mediated by the NMDA receptor. Lipocortin-1 fragment markedly attenuated, and the neutralizing antibody increased, this NMDA mediated neuronal damage. These observations may explain the neuroprotective action of lipocortin following cerebral ischaemia.
Western blotting and densitometry have been used to investigate the lipocortin content of post-mortem central nervous system (CNS) tissue samples from multiple sclerosis (MS) patients and normal controls. Lipocortins 1, 2, 4 and 5 were all detected in normal control grey and white matter. In white matter samples from MS patients these lipocortins were found to be significantly increased, a further elevation in lipocortin content was observed in MS plaque tissue. The implications of these findings with respect to the role of these proteins in inflammatory CNS disease and a possible mechanism of steroid action in the therapy of MS are discussed.
The effect of endogenous glucocorticoids on the expression of the cyclooxygenase enzyme was studied by contrasting cyclooxygenase expression and prostanoid synthesis in adrenalectomized and sham-adrenalectomized mice with or without the concurrent administration of endotoxin. Peritoneal macrophages obtained from adrenalectomized mice showed a 2- to 3-fold induction in cyclooxygenase synthesis and activity when compared to sham controls. Intravenous injection of a sublethal dose of endotoxin (5 micrograms/kg) further stimulated cyclooxygenase synthesis, resulting in a 4-fold increase in prostaglandin production. Similar cyclooxygenase induction can be achieved in macrophages obtained from normal mice but only after high doses of endotoxin (2.5 mg/kg) that are 100% lethal to adrenalectomized mice. Restoration of glucocorticoids in adrenalectomized animals with dexamethasone completely inhibited the elevated cyclooxygenase and protected these animals from endotoxin-induced death. In contrast, no signs of cyclooxygenase induction were observed in the kidneys of the adrenalectomized mice, even when treated with endotoxin. Dexamethasone did not affect the constitutive cyclooxygenase activity and prostaglandin production present in normal and adrenalectomized kidneys. These data indicate the existence of a constitutive cyclooxygenase that is normally present in most cells and tissues and is unaffected by steroids and of an inducible cyclooxygenase that is expressed only in the context of inflammation by proinflammatory cells, like macrophages, and that is under glucocorticoid regulation. Under normal physiological conditions glucocorticoids maintain tonic inhibition of inducible cyclooxygenase expression. Depletion of glucocorticoids or the presence of an inflammatory stimulus such as endotoxin causes rapid induction of this enzyme, resulting in an exacerbated inflammatory response that is often lethal.
The annexins are a widespread family of calcium-dependent membrane-binding proteins. No common function has been identified for the family and, until recently, no crystallographic data existed for an annexin. In this paper we draw together 22 available annexin sequences consisting of 88 similar repeat units, and apply the techniques of multiple sequence alignment, pattern matching, secondary structure prediction and conservation analysis to the characterisation of the molecules. The analysis clearly shows that the repeats cluster into four distinct families and that greatest variation occurs within the repeat 3 units. Multiple alignment of the 88 repeats shows amino acids with conserved physicochemical properties at 22 positions, with only Gly at position 23 being absolutely conserved in all repeats. Secondary structure prediction techniques identify five conserved helices in each repeat unit and patterns of conserved hydrophobic amino acids are consistent with one face of a helix packing against the protein core in predicted helices a, c, d, e. Helix b is generally hydrophobic in all repeats, but contains a striking pattern of repeat-specific residue conservation at position 31, with Arg in repeats 4 and Glu in repeats 2, but unconserved amino acids in repeats 1 and 3. This suggests repeats 2 and 4 may interact via a buried saltbridge. The loop between predicted helices a and b of repeat 3 shows features distinct from the equivalent loop in repeats 1, 2 and 4, suggesting an important structural and/or functional role for this region. No compelling evidence emerges from this study for uteroglobin and the annexins sharing similar tertiary structures, or for uteroglobin representing a derivative of a primordial one-repeat structure that underwent duplication to give the present day annexins. The analyses performed in this paper are re-evaluated in the Appendix, in the light of the recently published X-ray structure for human annexin V. The structure confirms most of the predictions and shows the power of techniques for the determination of tertiary structural information from the amino acid sequences of an aligned protein family.
The effect of glucocorticoids on the production of NO2- and NO by the macrophage cell line J774 was investigated. Stimulation of the cells with lipopolysaccharide (LPS) resulted in a time-dependent accumulation of NO2- in the medium, reaching a plateau after 48h. Concomitant incubation of the cells for 24h with dexamethasone (0.001-1.0 microM) or hydrocortisone (0.01-10.0 microM) caused a concentration-dependent inhibition of NO2- formation. The cytosol of J774 cells stimulated with LPS and IFN-gamma produced a time-dependent increase in the release of NO. This was blocked in a concentration-dependent manner by dexamethasone and hydrocortisone, but not progesterone, administered concomitantly with the immunological stimulus. None of these compounds had any effect on the release of NO once the enzyme had been induced. The inhibitory effect of hydrocortisone on NO formation was blocked by cortexolone. These data suggest that part of the anti-inflammatory and immunosuppressive actions of glucocorticoids is due to their inhibition of the induction of the NO synthase.
The effect of human recombinant lipocortin-1 (hrLC-1) on the pyrogenic actions of the synthetic polyribonucleotide polyinosinic:polycytidylic acid (poly I:C) has been studied in conscious rabbits. Poly I:C (2.5 micrograms kg-1) given i.v. produced a biphasic fever with a first peak after 90-105 min and a second peak between 225-240 min. hrLC-1 (50 micrograms kg-1) given i.v. simultaneously with the poly I:C produced a significant reduction in the febrile response but without complete suppression. The thermal response index over 5 h (TRI5) was 4.69 +/- 0.51 for poly I:C given with saline and the TRI5 for poly I:C given with hrLC-1 was 2.66 +/- 0.45 (values are for n = 5 +/- s.e. mean, P less than 0.05). hrLC-1 administered alone had no effect on body temperature and its antipyretic activity was lost on heating. In a separate series of experiments 1 h pretreatment with dexamethasone (1 mg kg-1) given i.v. reduced the pyrogenic response (TRI5) to poly I:C (2.5 micrograms kg-1) from 4.87 +/- 0.54 without dexamethasone to 2.00 +/- 0.25 (n = 5, P less than 0.05) and dexamethasone given alone had no effect on body temperature. These data demonstrate that LC-1 possesses antipyretic actions and raises the possibility that the antipyretic actions of dexamethasone are mediated through the induction of LC-1.
Stimulation of prostaglandin (PG) release in rat astroglial cultures by various substances, including phorbol esters, melittin, or extracellular ATP, has been reported recently. It is shown here that glucocorticoids (GCs) reduced both basal and stimulated PGD2 release. Hydrocortisone, however, did not inhibit ATP-, calcium ionophore A23187-, or tetradecanoyl phorbol acetate (TPA)-stimulated arachidonic acid release, and only TPA stimulations were affected by dexamethasone. GC-mediated inhibition of PGD2 release thus appeared to exclude regulation at the phospholipase A2 (PLA2) level. Therefore, the effects of GCs on the synthesis of lipocortin I (LC I), a potent, physiological inhibitor of PLA2, were studied in more detail. Dexamethasone was not able to enhance de novo synthesis of LC I in freshly seeded cultures and failed to increase LC I synthesis in 2-3-week-old cultures. It is surprising that LC I was the major LC synthesized in those cultures, and marked amounts accumulated with culture time, reaching plateau levels at approximately day 10. In contrast, LC I was barely detectable in vivo. This tonic inhibition of PLA2 is the most likely explanation for unsuccessful attempts to evoke PG release in astrocyte cultures by various physiological stimuli. GC receptor antagonists (progesterone and RU 38486) given throughout culture time reduced LC I accumulation and simultaneously increased PGD2 release. Nonetheless, a substantial production of LC I persisted in the presence of antagonists. Therefore, LC I induction did not seem to involve GC receptor activation. This was confirmed in serum- and GC-free brain cell aggregate cultures. Here also a marked accumulation of LC I was observed.(ABSTRACT TRUNCATED AT 250 WORDS)
Since a review on this topic in this Journal appeared (Wolfe, 1982), the CNS has proved to be a major focus in eicosanoid research. Although our knowledge is limited at the moment, the research in this field is rapidly growing. In this short review, we summarize recent progress of research (1982-1989) in this field with special attention directed to eicosanoid metabolism, functions of eicosanoids in the neuroendocrine system and synaptic transmission, current information on eicosanoid receptors, and the link between eicosanoids and cerebral circulation. Knowledge of the eicosanoids has paved the way to a better understanding of intercellular signal transduction systems, including neuronal functions.
A tetrazolium salt has been used to develop a quantitative colorimetric assay for mammalian cell survival and proliferation. The assay detects living, but not dead cells and the signal generated is dependent on the degree of activation of the cells. This method can therefore be used to measure cytotoxicity, proliferation or activation. The results can be read on a multiwell scanning spectrophotometer (ELISA reader) and show a high degree of precision. No washing steps are used in the assay. The main advantages of the colorimetric assay are its rapidity and precision, and the lack of any radioisotope. We have used the assay to measure proliferative lymphokines, mitogen stimulations and complement-mediated lysis.
The amount of messenger RNA encoding human inducible nitric oxide synthase and the presence and distribution of NADPH diaphorase were determined in tissue sections from multiple sclerosis (MS) and control brains. Levels of human nitric oxide synthase messenger RNA were markedly elevated in MS brains when compared to normal control brains. NADPH diaphorase activity, a histochemical stain reflecting nitric oxide synthase catalytic activity, was detected in reactive astrocytes in active demyelinating MS lesions and at the edge of chronic active demyelinating lesions. Control brains did not contain NADPH diaphorase-positive astrocytes. These results implicate the free radical nitric oxide in the pathogenesis of demyelinating MS lesions.
We have used purified microglial cultures obtained from neonatal rat cerebral cortex to investigate the ability of microglia to release prostanoids after exposure to bacterial lipopolysaccharide, a classic macrophage activator. Release of prostaglandin E2, prostaglandin D2, and thromboxane A2 was low in basal conditions and increased in a dose- and time-dependent way upon lipopolysaccharide treatment (1-100 ng/ml), by a mechanism requiring de novo protein synthesis. When compared with astrocytes, microglial cells appeared to respond more effectively to lipopolysaccharide, being able to release prostanoids after exposure to a 100-fold lower concentration of lipopolysaccharide. In addition to prostanoids, we also measured the release of leukotriene B4; although lipopolysaccharide failed to stimulate leukotriene B4 release by microglial cells, it doubled the basal production in astrocytes. Lipopolysaccharide enhanced the release of preloaded [3H]arachidonic acid from microglial membrane phospholipids by a mechanism inhibited by the protein synthesis inhibitor cycloheximide, which suggests that the increased availability of arachidonic acid contributed to the enhanced prostanoid production. Lipopolysaccharide, however, also stimulated prostanoid synthesis by inducing cyclooxygenase activity, as shown by determining the activity of newly synthesized enzyme after inactivating the endogenous enzyme with aspirin and by assessing the level of the inducible form of cyclooxygenase by western blot analysis. Among the mechanisms potentially involved in the regulation of microglial prostanoid production, we studied the effect of beta-adrenergic receptor activation. The beta-agonist isoproterenol was inactive by itself but doubled the effect of lipopolysaccharide.(ABSTRACT TRUNCATED AT 250 WORDS)
Alzheimer's disease is the most common cause of progressive intellectual failure. The lesions that develop, called senile plaques, are extracellular deposits principally composed of insoluble aggregates of beta-amyloid protein (A beta), infiltrated by reactive microglia and astrocytes. Although A beta, and a portion of it, the fragment 25-35 (A beta (25-35)), have been shown to exert a direct toxic effect on neurons, the role of microglia in such neuronal injury remains unclear. Here we report a synergistic effect between A beta and interferon-gamma (IFN-gamma) in triggering the production of reactive nitrogen intermediates and tumour-necrosis factor-alpha (TNF-alpha) from microglia. Furthermore, using co-culture experiments, we show that activation of microglia with IFN-gamma and A beta leads to neuronal cell injury in vitro. These findings suggest that A beta and IFN-gamma activate microglia to produce reactive nitrogen intermediates and TNF-alpha, and this may have a role in the pathogenesis of neuronal degeneration observed in ageing and Alzheimer's disease.
We have investigated the mechanism of nitric oxide-induced damage in glial cells. Genomic DNA isolated from astrocytes and microglia, treated for 18 h with varying concentrations of a nitric oxide donor, was analysed by electrophoresis. No DNA damage was evident. Oligodendrocytes, treated with 2 mM nitric oxide for 3-48 h, showed single stranded breaks at 48 h but no laddering of nucleosomic fragments of DNA. When analysed by electron microscopy, ultrastructural changes in oligodendrocytes treated with 1 mM nitric oxide for 24 h showed intact nuclei but alterations in membranes and organelles characteristic of necrosis, including disrupted mitochondria with dissolution of their christae. Astrocytes, a glial cell type that we have previously shown to be much less sensitive to nitric oxide-induced damage, did not show ultrastructural changes. DNA analysis by flow cytometry of glial cells treated with nitric oxide supported the apparent necrotic-type death in oligodendrocytes. Double staining of oligodendrocytes, using Hoechst 33342 and propidium iodide for the simultaneous assessment of both apoptotic and necrotic cells, demonstrated that, while the proportion of dead cells increased with time and increasing concentrations of nitric oxide, the death was due to necrosis and not apoptosis. In this study, we demonstrate that direct exposure to soluble nitric oxide, produced in vitro from a nitric oxide donor chemical, ultimately kills oligodendrocytes by necrosis. Microglia and astrocytes maintain DNA and organelle integrity when exposed to exogenous nitric oxide.
Cyclo‐oxygenase metabolizes arachidonic acid to prostaglandin H 2 (PGH 2 ) and exists in at least two isoforms. Cyclo‐oxygenase‐1 (COX‐1) is expressed constitutively whereas COX‐2 is induced by lipopolysaccharide (LPS) and some cytokines in vitro and at the site of inflammation in vivo . Epithelial cells may be an important source of prostaglandins in the airways and we have, therefore, investigated the expression of COX‐1 or COX‐2 isoforms in primary cultures of human airway epithelial cells or in a human pulmonary epithelial cell line (A549).
COX‐1 or COX‐2 protein was measured by western blot analysis using specific antibodies to COX‐2 and selective antibodies to COX‐1. The activity of COX was assessed by the conversion of either endogenous or exogenous arachidonic acid to four metabolites, PGE 2 , PGF 2α , thromboxane B 2 or 6‐oxo PGF 1α measured by radioimmunoassay. Thus, COX‐1 or COX‐2 activity was measured under two conditions; initially the accumulation of the COX metabolites formed from endogenous arachidonic acid was measured after 24 h. In other experiments designed to measure COX activity directly, cells were treated with cytokines for 12 h before fresh culture medium was added containing exogenous arachidonic acid (30 μ m ) for 15min after which COX metabolites were measured.
Untreated primary cells or A549 cells contained low amounts of COX‐1 or COX‐2 protein. Bacterial LPS (1 μg ml ⁻¹ for 24 h) induced COX‐2 protein in the primary cells, a process which was enhanced by interferon‐γ, with no further increase in the presence of a mixture of cytokines (interleukin‐1β, tumour necrosis factor‐α and interferon‐γ, 10 ng ml ⁻¹ for all). In contrast, A549 cells contained only low levels of COX‐2 protein after exposure to LPS or LPS plus interferon‐γ, but contained large amounts of COX‐2 protein after exposure to the mixture of cytokines.
Untreated human pulmonary primary cells or A549 cells released low levels of all COX metabolites measured over a 24 h incubation period. This release was enhanced by treatment of either cell type with the mixture of cytokines (interleukin‐10, tumour necrosis factor‐α and interferon‐γ, 10ng ml ⁻¹ for all). PGE2 was the principal COX metabolite released by cytokine‐activated epithelial cells. The release of PGE 2 induced by cytokines occurred after a lag period of more than 6 h.
The glucocorticosteroid, dexamethasone (1 μ m ; 30 min prior to cytokines) completely suppressed the cytokine‐induced expression of COX‐2 protein and activity in both primary cells and A549 cells.
In experiments where COX‐2 activity was supported by endogenous stores of arachidonic acid, treatment of A549 cells with interleukin‐10 but not tumour necrosis factor‐α or interferon‐γ alone caused a similar release of PGE 2 to that seen when the cytokines were given in combination. However, both interleukin‐10 and necrosis factor‐α alone produced similar increases in COX‐2 activity (measured in the presence of exogenous arachidonic acid) as seen when the mixture of interleukin‐1β, tumour necrosis factor‐α and interferon‐γ were used to stimulate the cells.
These findings show that COX‐2 expression correlates with the exaggerated release of prostaglandins from cytokine‐activated human pulmonary epithelial cells and that the induction of the enzyme is suppressed by a glucocorticosteroid. These findings may be relevant to inflammatory diseases of the lung, such as asthma.
The effects of prostaglandin (PG) E2 on glutamate-induced cytotoxicity were examined using primary cultures of rat cortical neurons. The cell viability was significantly reduced when cultures were briefly exposed to either glutamate or N-methyl-D-aspartate (NMDA) then incubated with normal medium for 1 h. Similar cytotoxicity was observed with the brief application of ionomycin, a calcium ionophore, and S-nitrosocysteine, a nitric oxide (NO)-generating agent. PGE2 at concentrations of 0.01-1 microM dose-dependently ameliorated the glutamate-induced cytotoxicity. PGE1, butaprost, an EP2 receptor agonist, and 8-bromo-cAMP were also effective in protecting cultures against glutamate cytotoxicity. By contrast, neither 17-phenyl-omega-trinor-PGE2, an EP1 receptor agonist, nor M&B 28767, an EP3 receptor agonist, affected glutamate-induced cytotoxicity. NMDA-induced cytotoxicity was ameliorated by PGE2, butaprost, MK-801, N-omega-nitro-L-arginine, a NO synthase inhibitor, and hemoglobin, which binds NO. These agents excluding MK-801 ameliorated the ionomycin-induced cytotoxicity. The cytotoxicity induced by S-nitrosocysteine was prevented only by hemoglobin but not by the other agents including PGE2. These findings indicate that PGE2 protects cultured cortical neurons against NMDA receptor-mediated glutamate neurotoxicity via EP2 receptors. EP2 receptor stimulation may suppress a step in NO formation triggered by Ca(2+)-influx through NMDA receptors.
Arachidonic acid and its metabolites are released in brain extracellular fluids as a result of ischemia and may participate in either damaging or protecting neural tissues. This study investigates the neuroprotective effect of prostacyclin (PGI2) on hypoxia (5 h)/reoxygenation (3 h) and on the excitotoxic neurotransmitter, glutamate (10 microM), in rat cortical neuron cultures. At microM concentrations, PGI2 inhibits lactate dehydrogenase release, a cell-injury marker. These results, showing a direct cytoprotective effect of PGI2 on brain cells, reinforce its beneficial properties on vessels and circulating cells in cerebral ischemia.
The growing evidence that glutamate may be an important agent mediating ischemic damage to neurons, led us to investigate the possible protective effects of pharmacological agents against glutamate in a model system of cortical neurons. In this study we examined, in particular, the cytoprotective effect of prostaglandins. Experiments were carried out in vitro by using rat cortical neurons in culture for 10 days. They were incubated for 3h with glutamate (10 microM) in the presence or absence of various pharmacological agents including prostaglandins (PGD2, PGE1, PGE2, PGF2 alpha, PGI2, 6-Keto-PGF1 alpha, carba-TXA2, carba-PGI2 and PGF2 alpha-methylester). Increase in lacticodehydrogenase (LDH) release into the culture medium has been measured as an index of cell injury. When neurons were incubated with glutamate they released LDH due to NMDA-receptor activation since D-L-2-amino-5-phosphonovaleric acid, a specific receptor antagonist, protected the cells. The protective activity of oxypurinol, amflutizole, superoxide dismutase, NG nitro-L-arginine and quinacrine, also suggests that xanthine oxidase activation, the generation of superoxide radical, and nitrix oxide, as well as phospholipase A2 stimulation are responsible for neuron injury (i.e. LDH release). All the tested prostaglandins, except PGF2 alpha-methylester, afforded significant protection at concentrations between 0.1 and 10 microM. The order of potency of the prostanoids was: PGF2 alpha = PGE2 > Carba-TXA2 > PGE1 > PGD2 > PGI2 = Carba-PGI2 > 6-Keto-PGF1 alpha. Additional experiments showed that prostaglandins did not compete for the NMDA binding site and that they did not inhibit free radical-related membrane damage.(ABSTRACT TRUNCATED AT 250 WORDS)
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Brain macrophages (BM), a subpopulation of microglia, have the ability to kill neurons by producing reactive oxygen intermediates. Cocultures of neurons and macrophages derived from the cerebral cortex of rat embryos were used to look for regulation of BM neurotoxicity. Isoproterenol (10(-7) M), a beta-adrenergic agonist, induced a significant inhibition of BM neurotoxicity and this effect was abolished in the presence of propranolol, a beta-adrenergic antagonist. BM neurotoxicity was also reduced in the presence of prostaglandin E2 (10(-8), 10(-6) M), a metabolite derived from arachidonic acid. These results suggest endogenous mechanisms of neuroprotection operating either during development or following lesions.
A549 cells grown under serum free conditions do not express cyclooxygenase 2 (cox 2). However, addition of IL-1 beta results in the induction of cox 2 in a time and dose dependent manner; actinomycin D completely blocks this induction. Dexamethasone completely suppressed the induction of cox 2 only if present during the first 3.5h of IL-1 beta treatment but not if added after 3.5h. Treatment with a neutralising monoclonal antibody (1A, Biogen) specific to lipocortin-1 failed to reverse this inhibition by dexamethasone. However, using this same antibody we were able to reverse completely the effects of dexamethasone on the suppression of PGE2 release. These observations support the idea that glucocorticoids directly regulate cox 2 expression at the transcriptional level and this phenomenon is not mediated by lipocortin-1.
Lipocortin (annexin) 1 is a putative mediator of the inflammatory effects of glucocorticoids. By flow cytometric analysis (FACS) we have studied the effect of dexamethasone on the cellular localization of lipocortin 1. U-937 cells were incubated with or without 10 nM phorbol 12-myristate 13-acetate (PMA) to induce cell differentiation. Then 1 microM dexamethasone was added and incubation carried out for increasing times (1-24 h). Dexamethasone caused a time-dependent biphasic translocation of lipocortin 1 from the intracellular compartment to the cell membrane with maximal membrane expression at 4 and 24 h. In differentiated U-937 cells the steroid-induced membrane accumulation of lipocortin 1 was significantly higher than that of undifferentiated cells. The accumulation of the protein in the cell membrane may precede its release which is stimulated by dexamethasone in differentiated U-937 cells. Since extracellular lipocortin 1 has anti-inflammatory properties the modulation of the translocation/secretion process of the protein by glucocorticoids may be part of their mechanism of action.
Lipocortin-1, a 37 kDa member of the annexin superfamily of proteins, originally evoked interest as one of the 'second messengers' of the anti-inflammatory actions of the glucocorticoids. Subsequent research has shown that the protein plays a major regulatory role in systems as diverse as cell-growth regulation and differentiation, neutrophil migration, CNS responses to cytokines, neuroendocrine secretion and neurodegeneration. The role of lipocortin-1 in mediating glucocorticoid-induced effects in these systems has been demonstrated using immunoneutralization strategies and by mimicking steroid actions with highly purified or recombinant lipocortin-1 or its biologically active peptide fragments. Originally the mode of action of lipocortin-1 seemed to be largely through inhibition of prostaglandin formation, but it is now clear that it can modify other aspects of cell function, perhaps pointing to a more fundamental mechanism than was originally envisaged. In this article Rod Flower and Nancy Rothwell review the nature, possible mechanisms and clinical relevance of these diverse actions of lipocortin-1.
The cytokine interleukin-1 (IL-1) is synthesised within the brain and acts as a mediator of host defence responses to disease and injury. Several of these central actions of IL-1 are inhibited by an endogenous calcium and phospholipid binding protein, lipocortin-1. Synthesis of IL-1 and lipocortin-1 in the brain is markedly increased by neuronal damage, and inhibition of the actions of endogenous IL-1 by central injection of IL-1 receptor antagonist in the rat significantly inhibits ischaemic and excitotoxic brain damage. Lipocortin-1 appears to act as an endogenous neuroprotective agent that markedly attenuates ischaemic and excitotoxic damage. In contrast, inhibition of the actions of lipocortin-1 by injection of neutralising antiserum exacerbates both forms of neurodegeneration. The mechanisms underlying these effects of IL-1 and lipocortin-1 are largely unknown, but are probably independent of changes in body temperature. Actions of these molecules on corticotrophin releasing factor, arachidonic acid, excitatory amino acids, and nitric oxide, and the possible involvement of these factors in brain damage are discussed.
Immunohistochemical localization of two Ca(++)-binding proteins, Lipocortin 1 (LC1) and S100-beta, demonstrates two distinct classes of primitive glia in the floor plate of rat embryos. With proper fixation (formalin-lysine-periodate-acetic acid), dendritic glia in the CNS of adult rats also apparently stain for either LC1 or S100-beta in the ratio of 1:3. In order to further distinguish and identify these two glial classes, we have examined their population density, topography, and responses to localized neuron death. Neurons of the ipsilateral thalamus undergo apoptosis following cortical ablation; the contralateral thalamus serves as control. By eight days post-lesion, the number of LC1 cells in the ipsilateral thalamus has increased > 4-fold, the increase comprising primarily activated phagocytes adjacent to degenerating neurons. The S100-beta glia in the same region are virtual- ly indistinguishable from control; but background staining (apparently representing extra-cellular S100-beta) is increased. Thus, the responses of dendritic LC1 glia resemble these previously described for microglia and are quite different from the astrocytes identified by S100-beta immunoreactivity. Both dendritic and activated forms of LC1 glia stain with the microglial marker, Griffonia simplicifolia iso-lectin B4. However, before the correspondence of LC1 glia and microglia can be confirmed, two anomalies require resolution: (1) the LC1 glia are greater in number and more evenly distributed than microglia marked with other methods; (2) the dendritic LC1 glia apparently are progeny of primitive glia that form the midline raphe of the embryonic floor plate. The participation of LC1 glia in the removal of CNS debris supports the hypothesis that LC1 plays anti-inflammatory and/or immunosuppressive roles in phagocytes.
Spontaneous recovery from acute experimental allergic encephalomyelitis (EAE) by the Lewis rat is probably mediated by endogenous corticosteroids. It has been proposed that the anti-inflammatory actions of the glucocorticoids may be effected via the induction of mediator proteins termed lipocortins and recently we have demonstrated increased levels of lipocortin 1 in the central nervous system (CNS) of EAE-diseased rats (Bolton C., A-J. Elderfield and R.J. Flower (1990), J. Neuroimmunol. 29: 173-181). In this study, utilizing antisera raised against recombinant human lipocortin 1, immunohistochemistry and light microscopy have been used to determine the distribution of the protein in the cervical spinal cord of Lewis rats during EAE. In normal animals lipocortin 1 immunoreactivity was localized predominantly in the walls of larger blood vessels and to a lesser extent capillaries. The same staining pattern was found in adjuvant-inoculated controls. In sections from EAE-inoculated animals there was no change during the induction phase, but with the onset of clinical symptoms and the appearance of inflammatory infiltrates in the CNS, a marked increase in lipocortin 1 immunostaining was observed. This additional staining was due to widespread immunoreactivity of the lesions, was maximal at the height of disease and decreased following recovery and lesion regression. Within the lesions the vast majority of infiltrating lymphocytes and macrophages were positive for lipocortin 1, including some very heavily stained macrophage-like cells. Measurement of corticosterone in the sera of these animals showed that changes in lipocortin 1 immunostaining in the CNS during EAE closely parallel serum corticosterone levels.
Post-ischemic metabolism of arachidonic acid by cyclooxygenase results in the elaboration of numerous eicosanoids and in the generation of free radicals. Accordingly, the effect of cyclooxygenase inhibition by ibuprofen on post-ischemic eicosanoid production and delayed neuronal death was evaluated in Wistar-Kyoto rats subjected to incomplete forebrain ischemia. In control (C) and ibuprofen-treated groups (n = 5 each), pre- and post-ischemic eicosanoid production in the caudate nucleus (CN) and dorsal hippocampus (HPC) were evaluated by microdialysis. The ibuprofen-treated animals were given ibuprofen, 15 mg/kg i.v., prior to insertion of microdialysis probes. Forebrain ischemia was induced by bilateral carotid artery occlusion (BCAO) for 10 min with simultaneous hypotension to 35 Torr. The concentrations of thromboxane B2 (TxB2), 6-keto-PGF1 alpha and PGF2 alpha in the microdialysate were measured by radioimmunoassay. In two additional concurrent groups of rats (n = 10 each), neuronal injury in the HPC, CN and cortex (parietal, temporal and entorhinal regions) was evaluated histologically three days after 10 min of forebrain ischemia with and without pre-ischemic ibuprofen administration. In the control microdialysis group, levels of TxB2, 6-keto-PGF1 alpha and PGF2 alpha increased in both CN and HPC after probe insertion. These probe related increases were substantially reduced in the ibuprofen group. After ischemia and reperfusion in the control group, the levels of TxB2 and PGF2 alpha increased in both CN and HPC. Levels of 6-keto-PGF1 alpha increased in the CN but not in the HPC. The administration of ibuprofen substantially reduced post-ischemic TxB2 and PGF2 alpha levels in both CN and HPC and decreased 6-keto-PGF1 alpha levels in the CN. The results of these initial microdialysis studies left the possibility that, in the ibuprofen group, the reduction in eicosanoid levels after probe penetration might have influenced the subsequent post-ischemic eicosanoid production. Therefore, in an additional group of animals (n = 5), ibuprofen was administered after probe insertion. Only PGF2 alpha levels were measured in this group. Increased levels of PGF2 alpha comparable to the original control group were detected after probe penetration. Nonetheless, after ibuprofen administration, the pre- and post-ischemic levels of PGF2 alpha were again significantly reduced. In the histologic evaluation groups, overall neuronal injury was significantly less in the ibuprofen treated animals. This protective effect of ibuprofen was most clearly evident in the CA3 sector of the HPC.(ABSTRACT TRUNCATED AT 400 WORDS)
The activity of the steroid-inducible protein lipocortin-1 (LC1; with a primary sequence of 346 amino acids; also called annexin 1), a fragment corresponding to amino acids 1-188 and a short peptide from the N-terminus (amino acid 2-26) were tested for anti-inflammatory actions in three models of acute inflammation in the mouse in comparison with a mAb anti-CD11b (αCD11b). In the mouse air-pouch model LC1, fragment 1-188 and peptide Ac2- 26 exhibited powerful inhibitory effects (ED50 ≃ 5.2, 38 and 88 μg/mouse, respectively) on leukocyte migration elicited by IL-1. LC1 was approximately 200 times more potent than Ac2-26 on a molar basis although both gave maximal inhibitions, in contrast fragment 1-188 only produced a partial dose-response curve. LC1 was approximately 20 times more potent on a molar basis in this assay than the αCD11b mAb. Peptide Ac2-26 and the mAb αCD11b also blocked cell migration into the air-pouch induced by IL-8 with approximately the same potency. In the mouse skin edema and zymosan peritonitis assays Ac2-26 was inhibitory (ED50 of 200 μg/mouse) but less so than the αCD11b antibody (ED50 ≃ 0.5 mg/mouse). Both LC1 (10 μg) and Ac2-26 (200 μg) completely blocked FMLP-induced neutropenia in the mouse. Studies using an inactivated LC1 preparation, which binds to the same high affinity binding sites as the biologically active material, indicated that the short peptide acts on the same sites as the native LC1. This study confirms the activity of LC1 in another model of experimental inflammation and suggests that it acts partly through inhibition of leukocyte activation with an overall effect qualitatively comparable to the blocking of CD11b portion of a β2-integrin complex. It also shows that peptides derived from the N-terminal domain of LC1 may mimic the activity of the full length molecule and points the way for a new family of anti-inflammatory substances that inhibit leukocyte trafficking.
A local pre‐injection of 1 μg dexamethasone sodium phosphate strongly inhibited (> 60% inhibition at 3 h; P < 0.001 at all time points) the development of carrageenin‐induced paw oedema in the rat induced by a subplantar injection of 0.1 ml, 2% carrageenin.
Coinjection of a polyclonal rabbit antiserum raised against human 1–188 recombinant lipocortin 1, which also recognised the rat protein, reversed the inhibitory action of dexamethasone ( P < 0.05 at 4 h and 5 h). At the highest volume used (40 μl) control antisera were without any effect.
These data further support the concept that lipocortin 1 is involved in the anti‐inflammatory mechanism of action of the glucocorticoids.
Incubation of human A549/8 cells with human interleukin-1 beta (50 units/ml), interferon-gamma (100 units/ml), and tumor necrosis factor-alpha (10 ng/ml) (cytomix) resulted in a marked expression of the mRNA of the inducible nitric oxide synthase (NOS II). This induction was prevented by cycloheximide. Dexamethasone markedly reduced cytokine-induced NOS II mRNA concentrations; this reduction was prevented by RU 38486 (mifepristone). Pyrrolidine dithiocarbamate, an inhibitor of nuclear factor-kappa B (NF-kappa B) activation, also significantly decreased cytomix-induced NOS II mRNA levels. When A549/8 cells were transfected with a construct containing 1570-bp 5'-flanking sequence of the murine NOS II gene cloned before a reporter gene, the murine NOS II promoter was induced up to 20-fold with cytomix but not with bacterial lipopolysaccharide. Dexamethasone as well as pyrrolidine dithiocarbamate inhibited this induction. In electrophoretic mobility shift assays, nuclear protein extracts from cytomix-induced, but not from unstimulated cells, significantly slowed the migration of an oligonucleotide containing the NF-kappa B-binding site. This band shift was markedly reduced by dexamethasone. On the other hand, cytomix-induced nuclear protein content of NF-kappa B p65 and NF-kappa B p50 was not reduced by dexamethasone (as analyzed by Western blot). Dexamethasone also did not reduce cytomix-induced expression of NF-kappa B p65 mRNA or enhance the expression of NF-kappa B inhibitor mRNA. The human and murine NOS II promoters also contain consensus sequences for activating protein-1 (AP-1) binding. However, AP-1 binding activity of nuclear extracts of A549/8 cells was not enhanced by cytomix or inhibited by dexamethasone. These data suggest that the activated glucocorticoid receptor prevents (by a protein/protein interaction) the binding of transcription factor NF-kappa B, but not AP-1, to the NOS II promoter, thereby inhibiting the induction of NOS II transcription.