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Glucocorticoid-induced annexin I secretion by monocytes and peritoneal leukocytes
British Journal of Pharmacology
Abstract
We have studied the ability of the glucocorticoid, dexamethasone, to induce annexin 1 secretion by either human blood monocytes or rat peritoneal leukocytes.
The in vivo treatment of rats with dexamethasone (1.25 mg kg ⁻¹ ) selectively induced secretion of annexin 1 by peritoneal leukocytes, as assessed by incubating these cells in culture medium. Annexin 1 secretion was also induced in human cultured monocytes, in vitro , by 10 ⁻⁶ m dexamethasone.
Annexin 1 secretion was inhibited in the presence of 20 mM NH 4 C1 or by conducting the experiments at 18°C. In contrast, it was not inhibited by monensin, nocodazole or brefeldin A.
The time necessary for annexin 1 synthesis and secretion was less than 15 min.
These data indicate that glucocorticoids induce annexin 1 secretion by monocytes or peritoneal leukocytes. Because it is not inhibited by monensin, nocodazole or brefeldin A and it is rapid, annexin 1 secretion seems to occur by the secretory pathway similar to that used by several cytosolic proteins such as interleukin‐iβ.
... These might form part of the mechanism by which lipocortin 1 is released from the cells. exteriorisation by the classical secretory pathway ending in exocytosis (Coméra and Russo-Marie, 1995). It is therefore important to discover its location within cells as this may provide clues to the mechanism of its exteriorisation and release. ...
... The cells respond to LC1, which inhibits their growth and blocks EGF-stimulated proliferation (Croxtall and Flower, 1992;Croxtall et al., 1993), and blocks the activation of cytoplasmic phospholipase A2 (Croxtall et al., 1995(Croxtall et al., , 1996. Binding of LC1 to the surface of A549 cells is calciumdependent (Croxtall and Flower, 1992) since, as in other cell types (Coméra and Russo-Marie, 1995;Christian et al., 1996;Goulding et al., 1996), LC1 can be released from cell surfaces into the medium by EDTA. However, to date, the distribution of LC1 within the cells and cell membrane has not been reported. ...
... Christmas et al. (1991) demonstrated secretion of large amounts of annexin I and annexin 5 by the human prostate and detected both annexins in blood plasma. Glucocorticoidstimulated externalisation of annexin I by human blood mononuclear cells and rat peritoneal leukocytes is inhibited by 20 mM NH 4Cl and at 18°C but not by monensin, nocodazol or brefeldin A; furthermore, the interval between annexin I synthesis and secretion is shorter than that required for protein secretion by the classical regulated pathway (Coméra and Russo-Marie, 1995). As with interleukin-1 and some growth factors (Kuchler and Thorner, 1992), which also lack a signal sequence, the mechanism of their secretion remains to be elucidated. ...
Lipocortin 1 (annexin I) is a calcium- and phospholipid-binding annexin protein which can be externalised from cells despite the lack of a signal sequence. To determine its cellular distribution lipocortin 1 in A549 human lung adenocarcinoma cells was localised by light- and electron-microscopic immunocytochemistry and by cell fractionation and western blotting. Lipocortin 1 immunoreactivity is concentrated in prominent patches associated with the plasma membrane. The intensity of these patches varied with the confluence and duration of the culture and was not detectably diminished by an EDTA wash before fixation. Tubulin and cytokeratin 8 were colocalized with lipocortin 1 in the patches. Within the cells lipocortin 1 was distributed throughout the cytoplasm. Electron microscopy revealed prominent immunoreactivity along the plasma membrane with occasional large clusters of gold particles in contact with the membrane surface of the cells; within the cytoplasm the membrane of some vesicle/vacuole structures and some small electron-dense bodies was immunoreactive, but no immunogold particles were associated with the multilamellar bodies. Subcellular fractionation, extraction and western blotting showed that lipocortin 1 in the membrane pellet was present as two distinct fractions; one, intimately associated with the lipid bilayer, which behaved like an integral membrane protein and one loosely attached which behaved like a peripheral membrane protein. The results show that a substantial amounts of lipocortin 1 is concentrated in focal structures associated with and immediately beneath the plasma membrane. These might form part of the mechanism by which lipocortin 1 is released from the cells.
... Neutrophils and monocytes are particularly enriched in ANXA1 and ANXA1 binding sites, and much attention has been focused on the regulation of ANXA1 expression by GCs in these cell types [11][12][13][14]. In contrast, the number of ANXA1 binding sites in lymphocytes is barely detectable, and whether GCs regulate the expression of ANXA1 in these cells is still a matter of debate [15][16][17][18]. ...
... Several in vivo and in vitro studies have shown that exposure to endogenous or exogenous GC hormones increases the cellular turnover of ANXA1, changes its subcellular localisation and stimulates the secretion of this protein [3,11,16,22,24]. However, the mechanisms involved in ANXA1 exportation from the intracellular milieu to the outer surface of the plasma membrane are still not completely understood. ...
... ANXA1 does not contain a hydrophobic signal sequence necessary for the release from the intracellular compartment via the classical endoplasmic reticulum (ER)-Golgi system, unlike most proteins that undergo secretion [1,25]. Some recent studies suggest that its secretion occurs through an unconventional pathway [2,5,11,21], or through a Ca 2+dependent mechanism [6,7], both yet not totally understood. Once outside the cell, ANXA1 gains access to specific highaffinity, saturable and Ca 2+ -dependent binding sites [24,26,27], which have been clearly identified on the plasma membrane of neuroendocrine and phagocytic cells [3,15,16,18]. ...
The mechanisms by which glucocorticoids (GC) regulate annexin I (ANXA1) secretion in different cells are still a matter of debate. The aims of this study were to evaluate the ability of dexamethasone (Dex) to induce ANXA1 secretion and to investigate the roles of the intracellular free Ca2+ concentration ([Ca2+]i), and of the GC receptor, on that process. For this purpose, the human immature lymphoblastic CCRF-CEM cell line was used. Treatment of the cells with Dex, for up to 4 h, significantly reduced the intracellular content of ANXA1 and increased the amount of this protein bound to the outer surface of the plasma membrane, whereas exposure of cells to Dex, for 12 h, induced the synthesis of ANXA1. At the same short time periods, Dex also induced a significant increase in the [Ca2+]i. Incubation of the cells with BAPTA-AM (10 microM), a cell-permeant high affinity Ca2+ chelator, completely inhibited Dex-induced ANXA1 secretion. Furthermore, the Ca2+ ionophore, ionomycin, alone induced ANXA1 cleavage, but not its secretion. Additionally, we used brefeldin A to investigate the involvement of the classical endoplasmic reticulum (ER)-Golgi pathway of protein secretion in the release of ANXA1. The GC receptor antagonist, RU486, neither reverted the Dex-dependent ANXA1 secretion nor inhibited the increase of the [Ca2+]i induced by Dex. Together, our results indicate that Dex induces ANXA1 synthesis and secretion in CCRF-CEM cells. ANXA1 secretion in this cell type show the following characteristics: (i) is unlikely to involve the classical ER-Golgi pathway; (ii) requires a Ca(2+)-dependent cleavage of ANXA1; (iii) involves both Ca(2+)-dependent and independent mechanisms; and (iv) is apparently independent of the GC receptor alpha isoform.
... In experimental models, AnxA1 or AnxA1-derived peptides exert a broad range of anti-inflammatory effects in monocytes, involving transcriptional changes as well as rapid post-translational effects [4,5]. A number of studies have also shown that glucocorticoids induce de novo synthesis as well as translocation of AnxA1 to the cell surface in peripheral blood mononuclear cells (PBMCs) or isolated monocytes/macrophages [6][7][8][9][10][11]. The expression of AnxA1 in circulating PBMCs may thus serve as an indicator of anti-inflammatory actions and glucocorticoid sensitivity. ...
... In order to achieve its biological effects, the protein is translocated from the cytosol to the cell surface where it binds to a G protein-coupled receptor belonging to the formyl peptide receptor family [23]. Glucocorticoids are known to induce both de novo synthesis and translocation of AnxA1 to the cell surface [6,[9][10][11] and there is consistent evidence that AnxA1 is a key mediator of anti-inflammatory actions in monocytes/macrophages [4,5]. Although the effects of AnxA1 has been studied mainly in vitro using exogenous AnxA1, a previous in vivo gene expression profiling study of human PBMCs verified that AnxA1 was a candidate marker of inflammatory modulation [24]. ...
Annexin A1 (AnxA1) is a key player in resolution of inflammation and a mediator of glucocorticoid actions. In atherosclerotic tissue, increased expression of AnxA1 has been associated with protective plaque-stabilizing effects. Here, we investigated the expression of AnxA1 in peripheral blood mononuclear cells (PBMCs) from patients with coronary artery disease (CAD). Blood was collected from 57 patients with stable CAD (SCAD) and 41 healthy controls. We also included a minor group (n = 10) with acute coronary syndrome (ACS). AnxA1 mRNA was measured in PBMCs. Expression of AnxA1 protein (total and surface-bound) and glucocorticoid receptors (GR) were detected in PBMC subsets by flow cytometry. Also, salivary cortisol, interleukin(IL)-6 and IL-10 in plasma, and LPS-induced cytokine secretion from PBMCs, with or without dexamethasone, were assessed. AnxA1 mRNA was found to be slightly increased in PBMCs from SCAD patients compared with controls. However, protein expression of AnxA1 or GRs in PBMC subsets did not differ between SCAD patients and controls, despite SCAD patients showing a more proinflammatory cytokine profile ex vivo. Only surface expression of AnxA1 on monocytes correlated with dexamethasone-mediated suppression of cytokines. In ACS patients, a marked activation of AnxA1 was seen involving both gene expression and translocation of protein to cell surface probably reflecting a rapid glucocorticoid action modulating the acute inflammatory response in ACS. To conclude, surface expression of AnxA1 on monocytes may reflect the degree of glucocorticoid sensitivity. Speculatively, “normal” surface expression of AnxA1 indicates that anti-inflammatory capacity is impaired in SCAD patients.
... The percentage viability was calculated from the total cell counts. The concentration of compounds at which viability was > 95% was used for further studies [10]. ...
... The supernatant was isolated for measurement of membrane bound ANXA1. Subsequent to the removal of membrane associated ANXA1, cells were lysed and the lysate was taken for the calculation of entire intracellular ANXA1 [10]. ...
Purpose: To investigate the modulatory effects of phytoestrogens (coumestrol, daidzein and genistein) and curcumin on the induction and secretion of annexin-1 (ANXA-1) in human peripheral blood mononuclear cells (PBMCs) under inflammatory and non-inflammatory conditions, as well as their effect on the activity of phospholipase A(2)-V (sPLA(2)-V). Methods: The modulatory effects of phytoestrogens and curcumin on the induction ofANXA1 were investigated via sandwich ELISA method, while their effects on the activity of sPLA(2)-V were determined by photometric assays. Besides, the cell viability of these compounds was determined by standard trypan blue exclusion method using PBMCs. Results: The results indicate a significant increase (p < 0.05) in the total content ofANXA1, particularly by coumestrol (p < 0.01), in both inflammatory and non-inflammatory cells. Besides, the compounds also exhibited a dose-dependent inhibition of sPLA(2)-V activity; however, among these compounds, curcumin and genistein were the strongest inhibitors with an IC50 value of 11.1 +/- 0.3 mu M and 13.6 +/- 0.6 mu M respectively. Conclusion: The investigated compounds have a potential to induce synthesis and secretion ofANXA1 as well as inhibitory activity of sPLA(2)-V, suggesting their inhibitory role in phospholipid metabolism and 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). Once externalized, ANXA1 or its NH 2 -terminal peptides may bind to formyl peptide receptors (FPR; Refs. ...
... Effect of Dex on MMDD1 ANXA1 expression and secretion. We next sought to elucidate whether or not release of ANXA1 from macula densa cells was sensitive to stimuli previously shown to be effective in pituitary folliculo-stellate cells (39), monocytes (10), and other leukocytes (11). Treating MMDD1 cells for 8 h with 0.1 M Dex caused a marked increase in the amount of ANXA1 in the medium compared with vehicletreated cells, whereas the intracellular concentration remained stable (Fig. 10A). ...
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.
... Glucocorticoids have in numerous studies been shown to induce both de novo synthesis of AnxA1, and to contribute to its translocation from the cytosol to the cell surface where the protein exerts its biological effects (Comera and Russo-Marie, 1995;Solito et al., 2003). Moreover, there is convincing evidence that AnxA1 is a key mediator of anti-inflammatory effects in monocytes/macrophages, involving both rapid post-translational effects and transcriptional changes, thus promoting a stable anti-inflammatory phenotype (Lange et al., 2007;Strausbaugh and Rosen, 2001). ...
... Annexin I was shown to be secreted from peritoneal leucocytes extracted from rats treated with dexamethasone (Comera and Russo-Marie, 1995). This study also showed secretion of annexin I from human monocytes in response to annexin I. ...
Members of the annexin protein family are characterised by their ability to bind to phosphohpids in a calcium-dependent manner. Since their discovery in the late 70s, a number of different family members have been found in mammals, plants and lower eukaryotes, although to date, no function has been unequivocally determined for any member. The restricted tissue-distribution of the annexins suggest that regulation occurs at the gene level. Most controversially, annexin I has been proposed to be steroid-inducible. Both annexins I and VI were found to be unresponsive to steroids at the protein and mRNA level in a range of cell-types. Also, steroids were shown to have no effect on annexin I secretion. Reporter gene analysis confirmed that the promoters for both the annexins I and VI genes are not steroid-inducible. Tmncation mutations were used to characterise the promoter regulatory elements showing that the annexin I promoter requires a functional CAAT and TATA box for activity. In contrast, these elements are not required in the annexin VI promoter. However, it is postulated that this promoter contains an "initiator element". Further, it is shown that both enhancers and repressors operate within the annexin VI promoter, and that a site for the transcription factor SPl is likely to be functional in-vivo. A region within the annexin VI promoter was shown to be homologous to non-coding regions of a number of genes, including interleukin-4. This region was found to be a cell-specific enhancer/repressor of gene activity. In response to activation of the interleukin-4 gene, a cell-specific protein complex was seen to bind to a site within the homologous region. It was shown that this protein is not a member of the STAT, NF-κT or NFκB transcription factor families. Removal of this protein-binding site profoundly influences the enhancer/repressor activity of the homologous region. Lastly, activation of the interleukin-4 gene triggers a marked reduction in the level of annexin VI protein/mRNA specifically in Jurkat cells. This suggests that interleukin-4 and annexin VI expression may be co-ordinately regulated by the action of enhancer/repressor elements within non-coding regions.
... Thirty minutes later, the lumen contents were rinsed away and replaced with the same PBS solution with or without 300 μg/ml of sonicated E171 TiO 2 for a further incubation period of 30 min. We analyzed in parallel the cell lysis by measuring the cytosolic release of lactate dehydrogenase (LDH; as described in [46], using sodium pyruvate, NADH and spectrophotometry) in the lumen recovered after ex vivo experiments performed in control milieu, in the presence of TiO 2 , TAP or both of them. Results were expressed in LDH activity/sec/mg of protein in the corresponding tissue. ...
Background:
Food-grade TiO2 (E171 in the EU) is widely used as a coloring agent in foodstuffs, including sweets. Chronic dietary exposure raises concerns for human health due to proinflammatory properties and the ability to induce and promote preneoplastic lesions in the rodent gut. Characterization of intestinal TiO2 uptake is essential for assessing the health risk in humans. We studied in vivo the gut absorption kinetics of TiO2 in fasted mice orally given a single dose (40 mg/kg) to assess the ability of intestinal apical surfaces to absorb particles when available without entrapment in the bolus. The epithelial translocation pathways were also identified ex vivo using intestinal loops in anesthetized mice.
Results:
The absorption of TiO2 particles was analyzed in gut tissues by laser-reflective confocal microscopy and ICP-MS at 4 and 8 h following oral administration. A bimodal pattern was detected in the small intestine: TiO2 absorption peaked at 4 h in jejunal and ileal villi before returning to basal levels at 8 h, while being undetectable at 4 h but significantly present at 8 h in the jejunal Peyer's patches (PP). Lower absorption occurred in the colon, while TiO2 particles were clearly detectable by confocal microscopy in the blood at 4 and 8 h after treatment. Ex vivo, jejunal loops were exposed to the food additive in the presence and absence of pharmacological inhibitors of paracellular tight junction (TJ) permeability or of transcellular (endocytic) passage. Thirty minutes after E171 addition, TiO2 absorption by the jejunal villi was decreased by 66% (p < 0.001 vs. control) in the presence of the paracellular permeability blocker triaminopyrimidine; the other inhibitors had no significant effect. Substantial absorption through a goblet cell (GC)-associated pathway, insensitive to TJ blockade, was also detected.
Conclusions:
After a single E171 dose in mice, early intestinal uptake of TiO2 particles mainly occurred through the villi of the small intestine, which, in contrast to the PP, represent the main absorption surface in the small intestine. A GC-associated passage and passive diffusion through paracellular TJ spaces between enterocytes appeared to be major absorption routes for transepithelial uptake of dietary TiO2.
... In prednisone-and deflazacort-treated mice, Gzmb (encoding granzyme B) and Ifng (encoding IFN-γ) expression levels were downregulated compared with those of control animals, consistent with the anticipated effect of steroids to promote resolution of the immune response (17). In leukocytes, Anxa1 is induced by GC steroids, suggesting that its upregulation is one mechanism by which GC steroids exert their effect (18). ...
Glucocorticoid steroids such as prednisone are prescribed for chronic muscle conditions such as Duchenne muscular dystrophy, where their use is associated with prolonged ambulation. The positive effects of chronic steroid treatment in muscular dystrophy are paradoxical because these steroids are also known to trigger muscle atrophy. Chronic steroid use usually involves once-daily dosing, although weekly dosing in children has been suggested for its reduced side effects on behavior. In this work, we tested steroid dosing in mice and found that a single pulse of glucocorticoid steroids improved sarcolemmal repair through increased expression of annexins A1 and A6, which mediate myofiber repair. This increased expression was dependent on glucocorticoid response elements upstream of annexins and was reinforced by the expression of forkhead box O1 (FOXO1). We compared weekly versus daily steroid treatment in mouse models of acute muscle injury and in muscular dystrophy and determined that both regimens provided comparable benefits in terms of annexin gene expression and muscle repair. However, daily dosing activated atrophic pathways, including F-box protein 32 (Fbxo32), which encodes atrogin-1. Conversely, weekly steroid treatment in mdx mice improved muscle function and histopathology and concomitantly induced the ergogenic transcription factor Krüppel-like factor 15 (Klf15) while decreasing Fbxo32. These findings suggest that intermittent, rather than daily, glucocorticoid steroid regimen promotes sarcolemmal repair and muscle recovery from injury while limiting atrophic remodeling.
... Annexin A1 is a member of a structurally related family of calcium-and phospholipid-binding proteins and has been implicated as a downstream mediator of anti-inflammatory glucocorticoids88899091. In the context of regeneration, the upregulation of annexin A1 could serve as a mechanism to limit the inflammatory response by restricting pro-inflammatory processes . ...
The inability to functionally repair tissues that are lost as a consequence of disease or injury remains a significant challenge for regenerative medicine. The molecular and cellular processes involved in complete restoration of tissue architecture and function are expected to be complex and remain largely unknown. Unlike humans, certain salamanders can completely regenerate injured tissues and lost appendages without scar formation. A parsimonious hypothesis would predict that all of these regenerative activities are regulated, at least in part, by a common set of genes. To test this hypothesis and identify genes that might control conserved regenerative processes, we performed a comprehensive microarray analysis of the early regenerative response in five regeneration-competent tissues from the newt Notophthalmus viridescens. Consistent with this hypothesis, we established a molecular signature for regeneration that consists of common genes or gene family members that exhibit dynamic differential regulation during regeneration in multiple tissue types. These genes include members of the matrix metalloproteinase family and its regulators, extracellular matrix components, genes involved in controlling cytoskeleton dynamics, and a variety of immune response factors. Gene Ontology term enrichment analysis validated and supported their functional activities in conserved regenerative processes. Surprisingly, dendrogram clustering and RadViz classification also revealed that each regenerative tissue had its own unique temporal expression profile, pointing to an inherent tissue-specific regenerative gene program. These new findings demand a reconsideration of how we conceptualize regenerative processes and how we devise new strategies for regenerative medicine.
... GR homodimerisation favourable to inducing an 'anti-inflammatory profile'? Whilst a great deal of work has focussed on the ability of GR to repress pro-inflammatory gene transcription, it is important to remember that GR also activates genes encoding anti-inflammatory mediators including IjBa, DUSP-1, glucocorticoid-induced Unknown [118,119] Glucocorticoid-induced leucine zipper (GILZ) ...
... Over the past several years, it has become apparent that certain proteins lacking conventional N-terminal signal peptides are secreted via a non-classical pathway (Muesch et al., 1990). For example, annexin-1 (Christmas et al., 1991), IL-1 , galectin 3 (Mehul and Hughes, 1997) and acidic and basic fibroblast growth factors (Abraham et al., 1986) are secreted in the presence of brefeldin A, monensin or nocodazole (Comera and Russo-Marie, 1995;Rubartelli et al., 1990). Although this non-classical secretory pathway(s) is poorly defined, it may involve an active translocation process or plasma membrane evagination (Cooper and Barondes, 1990). ...
An elevation in the circulating level of the squamous-cell carcinoma antigen (SCCA) can be a poor prognostic indicator in certain types of squamous-cell cancers. Total SCCA in the circulation comprises 2 nearly identical, ∼45 kDa proteins, SCCA1 and SCCA2. Both proteins are members of the high-molecular weight serine proteinase inhibitor (serpin) family with SCCA1 paradoxically inhibiting lysosomal cysteine proteinases and SCCA2 inhibiting chymotrypsin-like serine proteinases. Although SCCA1 and SCCA2 are detected in the cytoplasm of normal squamous epithelial cells, neither serpin is detected normally in the serum. Thus, their presence in the circulation at relatively high concentrations suggests that malignant epithelial cells are re-directing serpin activity to the fluid phase via an active secretory process. Because serpins typically inhibit their targets by binding at 1:1 stoichiometry, a change in the distribution pattern of SCCA1 and SCCA2 (i.e., intracellular to extracellular) could indicate the need of tumor cells to neutralize harmful extracellular proteinases. The purpose of our study was to determine experimentally the fate of SCCA1 and SCCA2 in squamous carcinoma cells. Using subcellular fractionation, SCCA-green fluorescent fusion protein expression and confocal microscopy, SCCA1 and SCCA2 were found exclusively in the cytosol and were not associated with nuclei, mitochondria, lysosomes, microtubules, actin or the Golgi. In contrast to previous reports, metabolic labeling and pulse-chase experiments showed that neither non-stimulated nor TNFα/PMA-stimulated squamous carcinoma cells appreciably secreted these ov-serpins into the medium. Collectively, these data suggest that the major site of SCCA1 and SCCA2 inhibitory activity remains within the cytosol and that their presence in the sera of patients with advanced squamous-cell carcinomas may be due to their passive release into the circulation. Int. J. Cancer 89:368–377, 2000. © 2000 Wiley-Liss, Inc.
... Exogenous glucocorticoids have been shown to induce ANXA1 production by peripheral blood mononuclear cells in vivo in man (163). In addition, a recent study demonstrated that ANXA1 expression in neutrophils was strongly correlated with the serum cortisol production, proposing a role for ANXA1 in mediating the anti-inflammatory effects of endogenous glucocorticoids (164). ...
Atherosclerosis is characterized by chronic inflammation involving autoimmune components. The degree of inflammatory activity, as detectable both within the atherosclerotic plaque and in the circulation, is associated with plaque destabilization and atherothrombotic complications. Endogenous glucocorticoids are modulators of innate and acquired immune responses, and as such play a key role in the reciprocal interaction between neuroendocrine and immune systems. Abnormalities in hypothalamic-pituitary-adrenal axis (HPA) function have been described in several chronic inflammatory disorders, and evidence has emerged lately that HPA dysfunction may be implicated also in the pathogenesis of coronary artery disease. This review is an outline of knowledge gained so far by previous studies of glucocorticoids in coronary atherosclerosis and myocardial infarction. The results consistently point towards a dysregulated cortisol secretion that may involve a failure to contain inflammatory activity. A dysfunctional HPA axis and its possible implications for coronary artery disease progress, including the hypothetical link between stress and inflammation, are discussed.
... Stress, immune function and the HPA axis used as a tracer indicate that the exported protein is newly synthesized (Comera & Russo Marie, 1995). In agreement with these findings we have reported that the dexamethasone-induced externalisation of LCl by pituitary cells is abolished by cycloheximide (1.0 ,ig ml-'), an inhibitor of mRNA translation ( Figure 9a, Taylor et al., 1993). ...
... Annexin A1 (lipocortin I) has long been suggested to function as a cellular mediator of anti-inflammatory glucocorticoids since its expression and secretion in several cell types is induced by glucocorticoids (see, for example, Refs. 46,228,308) and since exogenously administered protein exhibits anti-inflammatory activities in several animal models of inflammation (for reviews, see Refs. 85,86,260). ...
Annexins are a family of calcium-dependent phospholipid-binding proteins. They are abundant in the eukaryotic kingdom. Though structurally well investigated in the last twenty years the in vivo function of the annexins is still unclear. The determination of the crystal structure of human annexin V was the first milestone in the structural investigation of this protein family. Succeedingly, a variety of three-dimensional structures of annexin crystals as well as of membrane bound annexins were solved. They should provide tools to understand the in vivo function of the annexin family. Based on the structural knowledge mutagenesis studies and biophysical investigations were started to elucidate possible functions of annexins like membrane binding and ion channel activity.
... Parallel studies confirmed that none of the drugs or nucleotides employed in the experiments interfered with either the running of the gels or the binding of the antibody. The specificity of the anti-LC1 and antiannexin 5 antisera are described elsewhere (19,20); anti-LC1 pAb exhibits weak affinity for annexin 2, but this is unlikely to be a significant factor at the dilution used in the present study (19). ...
Our previous studies have demonstrated that lipocortin 1 (LC1, also called annexin 1) is an important mediator of glucocorticoid action in the neuroendocrine system, particularly with regard to the powerful inhibitory actions of the steroids on the secretion of ACTH and its hypothalamic releasing hormones. In the present study, we have used an antisense oligodeoxynucleotide (ODN) unique to LC1 to investigate further the role of this protein in the regulatory effects of dexamethasone on ACTH release in vitro from rat anterior pituitary cells. Pituitary cells dispersed with collagenase retained their functional and morphological integrity in vitro and sequestered ODNs in a time-dependent manner from the incubation medium. LC1 was readily detected in the cells by Western blot analysis or by immunoprecipitation/autoradiography after preloading with 35S-methionine/cysteine; the bulk of the protein was contained within an intracellular pool but a small amount was attached to the outer cell surface (pericellular). Dexamethasone (100 nm, 2.5 h) initiated de novo synthesis of LC1; it also increased the amount of LC1 in the pericellular pool detected by either method and caused a concomitant decrease in intracellular LC1. The responses to the steroid were prevented by the inclusion in the medium of an LC1 antisense ODN (50 nM, 3.5 h) but the corresponding sense and scrambled ODN sequences were inert. None of the ODN sequences tested influence the expression of annexin 5 in the pituitary tissue. CRH-41 (100 pM-1 mM), forskolin (1 nM-1 mM) and an L-Ca2+-channel opener BAY K8644 (100 pM-1 microM) initiated concentration dependent increases in immunoreactive- (ir-) ACTH release from the pituitary cells that were reduced (P < 0.01) by preincubation with dexamethasone (100 nM, 2.5 h). The inhibitory effects of the steroid were reversed by the LC1 antisense ODN (50 nM, P < 0.01), whereas the LC1 sense and scrambled control sequences (50 nM) were both ineffective in this respect (P > 0.05). The results add further support to the view that the acute inhibitory effects of glucocorticoids on the secretion of ACTH by the pituitary gland are dependent on the generation of lipocortin 1.
... The other two forms most likely represent phosphorylated states of the protein. 10 The increase in the low molecular weight forms of the Ax 1 in induced cells is prominent even though protease inhibitors were present suggesting that proteolysis occurs intrinsically. Membrane-associated Ax 1 (Panel B) is not detectable in unstimulated cells (control lane 6). ...
Annexin 1 (Ax 1), a protein whose synthesis and secretion are induced during the inflammatory response, has been proposed as a mediator of the anti-inflammatory action of glucocorticoids. To gain insight into a broader role of Ax 1 during the inflammatory response, the authors have investigated how pro-inflammatory cytokines [interleukin 1 (IL-1), IL-6 and tumour necrosis factor alpha (TNF-alpha)] affect Ax 1 expression and regulation at transcriptional and translational levels. The authors show that induction of the Ax 1 protein and its translocation to the cell membrane are stimulated by interleukin 6. However neither IL-1 nor TNF-alpha display these effects. Analysis of 5'-deletion mutants and the full length Ax 1 promoter fused to a luciferase reporter gene using transient transfections of human lung adenocarcinoma A 549 cells identified a unique 30 bp region of the Ax 1 promoter as critical for the responsiveness of the reporter gene to IL-6 and dexamethasone. Gel retardation and supershift assays showed that IL-6 stimulation is mediated by a C/EBP beta-like transcriptional factor. These data suggest that Ax 1 may participate in host defence as a new acute class II phase protein.
... Our data show that LC1 contributes to the inhibitory actions of glucocorticoids on the release of ACTH, PRL, and TSH from the anterior pituitary gland (11)(12)(13)(14)(15). Subsequently, using fluorescence-activated cell sorting analysis, we demonstrated LC1-binding sites on the surface of the majority of endocrine cells in the rat anterior pituitary gland (16). In addition, we showed that glucocorticoids promote de novo synthesis of LC1 in the adenohypophysis and cause the translocation of intracellular LC1 to pericellular sites (10,13,14,17) as they do in other tissues (18,19). Although the mechanism of externalization of LC1, which lacks a signal sequence, is unknown, externalization appears to be essential for mediating many actions of LC1 in the neuroendocrine system (14). ...
It is now well established that lipocortin 1 (LC1) plays an important role as a mediator of early delayed glucocorticoid feedback action in the hypothalamo-hypophysial system. In both the hypothalamus and anterior pituitary gland, LC1 mimics some of the actions of glucocorticoids; moreover, glucocorticoids stimulate the synthesis of LC1 and cause the translocation of intracellular LC1 to the outer cell surface. The mechanism by which LC1 acts in these tissues is only partially understood, but may involve paracrine and/or autocrine actions. To address these possibilities we have investigated the localization of LC1 in the rat pituitary gland, using double labeling immunohistochemistry to identify the pituitary cell types that express LC1. At the light microscopic level LC1 was not detected in the endocrine cells in cryosections of the pituitary, but it was found in abundance in the surrounding folliculo-stellate (FS) cells. In the anterior and interme diate pituitary lobes, there was a near total colocalization of LC1 and S100, a specific marker of FS cells. By contrast, in the posterior pituitary gland, LC1 immunoreactivity was not colocalized with S100 which labeled most pituicytes, or with OX-42 monoclonal antibody, a marker of the microglial cells. Immunogold electron microscopy confirmed that LC1 is present in the nongranulated FS cells. LC1 im munoreactivity was also present in a mouse pituitary FS-like cell line (TtT/GF), particularly in the periphery of the cytoplasm. The localization of LC1 in the FS cells of the anterior pituitary gland defines LC1 as a new marker of the FS cell population. These results support our hypothesis that LC1 acts as one of the paracrine agents liberated by FS cells that modulate the release of pituitary hormones.
... Although the expression of annexin I was significantly increased in S14 cells, the distribution pattern seems to be similar within the three cell types analyzed. Different groups of investigators have shown that glucocorticoids induce the synthesis of annexin I (31)(32)(33)(34)(35). Thus we tested the following hypothesis: whether glucocorticoids can further increase the expression or alter the distribution of annexin I. ...
Annexin I is an intracellular protein in search of a function. Ex vivo it has calcium- and phospholipid-binding properties. To evaluate its role in vivo, MCF-7 cells were stably transfected with annexin I in sense or antisense orientations. In cells overexpressing annexin I, calcium release was abrogated on stimulation of purinergic or bradykinin receptors, whereas non-transfected cells or cells with down-regulated annexin I released calcium within seconds. Basal calcium and calcium stores were not affected. The impaired calcium release was paralleled by a down-regulation of the activities of phospholipase C, group II phospholipase A2, and E-cadherin with altered adhesion and enhanced tumor growth on soft agar. Significantly smaller tumors, with the histologically most differentiated cells, were observed in nude mice inoculated with cells transfected with the antisense rather than with the sense plasmid. These observations indicate that annexin I modulates cell functions by controlling intracellular calcium release. Frey, B. M., Reber, B. F. X., Vishwanath, B. S., Escher, G., Frey, F. J. Annexin I modulates cell functions by controlling intracellular calcium release.
... The release of annexins I and IV might be attributable to the cytotoxic effect on alveolar cells. Annexin I is induced by glucocorticoids [2,8,17] and inhibits the activity of 100 kDa cytosolic phospholipase A2, which is responsible for the release of arachidonic acid (the prostaglandin precursor) from membrane phosphatidylcholine [14], thereby mediating the antiinflammatory effects of glucocorticoids [6]. In response to the viral inoculation, plasma concentrations of glucocorticoids are assumed to be elevated, and an increased glucocorticoid concentration in turn results in an accumulation annexin I in alveolar cells. ...
Annexins are phospholipid-binding proteins and are abundant in the lung. Annexins I and IV, but not II and VI, have been detected in bronchoalveolar lavage (BAL) fluids from calves inoculated with Pasteurella haemolytica, the pathogen for calf pneumonia. In this study, BAL fluids from calves with experimental pneumonia induced by inoculation to right lung lobes of bovine herpes virus-1 (BHV-1), the major viral pathogen for pneumonia, were examined for detection of annexins I and IV. Of 6 calves inoculated with BHV-1, annexins I and IV were coincidentally detected in BAL fluids from right lung lobes of 4 calves, but not in BAL fluids from left lung lobes of 6 inoculated calves or those from left and right lung lobes of 3 control calves. Annexin II and VI were not found in any BAL fluids examined. These results, together with previous findings on calves inoculated with Pasteurella haemolytica, suggest that the release of annexins I and IV onto the alveolar surface is an essential event occurring in response to pulmonary infections of BHIV-1 and Pasteurella haemolytica.
... Glucocorticoiddependent annexin I externalization has been demonstrated in monocytes, macrophages and PMN. 4,7,8 Consistent with this, treatment of experimental ani-mals with anti-annexin I antibodies prevented the anti-inflammatory effect of dexamathasone in several models of acute inflammation. 9,10 Besides glucocorticoid treatment, we and others have recently shown that annexin I secretion from human PMN occurs during the process of extravasation outside the blood vessel. ...
Specific binding sites for the anti-inflammatory protein annexin I have been detected on the surface of human monocytes and polymorphonuclear leukocytes (PMN). These binding sites are proteinaceous in nature and are sensitive to cleavage by the proteolytic enzymes trypsin, collagenase, elastase and cathepsin G. When monocytes and PMN were isolated independently from peripheral blood, only the monocytes exhibited constitutive annexin I binding. However PMN acquired the capacity to bind annexin I following co-culture with monocytes. PMN incubation with sodium azide, but not protease inhibitors, partially blocked this process. A similar increase in annexin I binding capacity was also detected in PMN following adhesion to endothelial monolayers. We propose that a juxtacrine activation rather than a cleavage-mediated transfer is involved in this process. Removal of annexin I binding sites from monocytes with elastase rendered monocytes functionally insensitive to full length annexin I or to the annexin I-derived pharmacophore, peptide Ac2-26, assessed as suppression of the respiratory burst. These data indicate that the annexin I binding site on phagocytic cells may have an important function in the feedback control of the inflammatory response and their loss through cleavage could potentiate such responses.
... Annexin A1 (lipocortin I) has long been suggested to function as a cellular mediator of anti-inflammatory glucocorticoids since its expression and secretion in several cell types is induced by glucocorticoids (see, for example, Refs. 46,228,308) and since exogenously administered protein exhibits anti-inflammatory activities in several animal models of inflammation (for reviews, see Refs. 85,86,260). ...
Annexins are Ca2+ and phospholipid binding proteins forming an evolutionary conserved multigene family with members of the family being expressed throughout animal and plant kingdoms. Structurally, annexins are characterized by a highly alpha-helical and tightly packed protein core domain considered to represent a Ca2+-regulated membrane binding module. Many of the annexin cores have been crystallized, and their molecular structures reveal interesting features that include the architecture of the annexin-type Ca2+ binding sites and a central hydrophilic pore proposed to function as a Ca2+ channel. In addition to the conserved core, all annexins contain a second principal domain. This domain, which NH2-terminally precedes the core, is unique for a given member of the family and most likely specifies individual annexin properties in vivo. Cellular and animal knock-out models as well as dominant-negative mutants have recently been established for a number of annexins, and the effects of such manipulations are strikingly different for different members of the family. At least for some annexins, it appears that they participate in the regulation of membrane organization and membrane traffic and the regulation of ion (Ca2+) currents across membranes or Ca2+ concentrations within cells. Although annexins lack signal sequences for secretion, some members of the family have also been identified extracellularly where they can act as receptors for serum proteases on the endothelium as well as inhibitors of neutrophil migration and blood coagulation. Finally, deregulations in annexin expression and activity have been correlated with human diseases, e.g., in acute promyelocytic leukemia and the antiphospholipid antibody syndrome, and the term annexinopathies has been coined.
... Consistent with the lack of apparent association of annexin I with intracellular organelles, annexin I lacks the hydrophobic signal sequence that targets proteins to the classical secretory pathway (37). Furthermore, drugs that block various steps in the exocytotic pathway do not alter the cellular export of annexin I that occurs in response to glucocorticoid treatment (38,39). Proteins that are externalized from cells but are not packaged into intracellular vesicles and released by exocytosis must either diffuse through the membrane or be transported across the membrane in some way. ...
Our recent studies on rat pituitary tissue suggest that the annexin I-dependent inhibitory actions of glucocorticoids may not be exerted directly on endocrine cells but indirectly via folliculo-stellate (FS) cells. FS cells contain glucocorticoid receptors and abundant annexin I. We have studied the localization of annexin I in FS cells and the ability of dexamethasone to induce annexin I secretion by an FS (TtT/GF) cell line, using Western blotting and immunofluorescence microscopy. Exposure of TtT/GF cells to dexamethasone (0.1 micro M, 3 h) caused an increase in the amount of annexin I protein in the intracellular compartment and attached to the surface of the cells. In nonpermeabilized cells, immunofluorescence labeling revealed that annexin I immunoreactivity was associated with the cell surface and concentrated in focal patches on the ends of cytoplasmic processes; dexamethasone (0.1 micro M, 3 h) increased both the number and intensity of these foci. Immunogold electron microscopy confirmed in anterior pituitary tissue the presence of immunoreactive-annexin at the surface of FS cell processes contacting endocrine cells. These data support our hypothesis that annexin I is released by FS cells in response to glucocorticoids to mediate glucocorticoid inhibitory actions on pituitary hormone release via a juxtacrine mechanism.
Aims
Previous studies in TB patients showed an immuno-endocrine imbalance characterized by a disease-severity associated increase in plasma levels of proinflammatory cytokines and glucocorticoids (GCs).
To analyze the potential immunomodulatory effect of circulating GCs over peripheral blood mononuclear cells (PBMC) from TB patients, we investigated the expression of positively (anti-inflammatory-related genes ANXA1; FKBP51; GILZ, NFKBIA, and NFKBIB) and negatively (inflammatory genes: IL-6, IL-1β, and IFN-γ) Glucocorticoids Receptors (GR)-regulated genes. Plasma concentrations of cytokines and hormones, together with specific lymphoproliferation were also assessed.
Materials and methods
Gene expression was quantified by RT-qPCR, specific lymphoproliferation by ³H-thymidine incorporation, whereas plasma cytokines and hormones levels by ELISA.
Key findings
Transcripts of ANXA1, GILZ, NFKBIB, and NFKBIA appeared significantly increased in patients, whereas FKBP51, IL-6, IL-1β, and NF-κB remained unchanged. Upon analyzing according to disease severity, mRNA levels for ANXA1 and NFKBIB were even higher in moderate and severe patients. GILZ was increased in moderate cases, with NFKBIA and IL-1 β being higher in severe ones, who also displayed increased GRβ transcripts. TB patients had reduced plasma DHEA concentrations together with increased pro and anti-inflammatory cytokines (IFN-γ, IL-6, and IL-10) cortisol and cortisol/DHEA ratio, more evident in progressive cases, in whom their PBMC also showed a decreased mycobacterial-driven proliferation. The cortisol/DHEA ratio and GRα expression were positively correlated with GR-regulated genes mainly in moderate patients.
Significance
The increased expression of cortisol-regulated anti-inflammatory genes in TB patients-PBMC, predominantly in progressive disease, seems compatible with a relatively insufficient attempt to downregulate the accompanying inflammation.
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.
Annexin A2 (ANX2) is a calcium (Ca(2+))-binding protein that binds to acidic phospholipids and is known to play a crucial role in many cellular regulatory processes. In particular, ANX2 has been described as a crucial receptor for thrombolysis by the tissue-type plasminogen activator (tPA) and plasmin system. In the nervous system, tPA is involved in processes of neuronal plasticity such as hippocampal long-term potentiation and in the dorsal horn pain in several pain models. We investigated detailed changes in expression of ANX2 after nerve injury and evaluated the interaction between tPA using the rat spared nerve injury (SNI) model. SNI-induced the expression of ANX2 in L4/5 DRG neurons. In the spinal cord, constitutive ANX2-immunoreactivity was expressed in laminae I-II. Peripheral nerve injury increased the ANX2 immunoreactive terminals mainly in laminae I-V of the dorsal horn on the side ipsilateral to the nerve injury. Double labeling analysis revealed the co-localization of ANX2 with tPA in the axons of primary afferents in the dorsal horn. Experimental inhibition of ANX2 and tPA interaction by intrathecal administration of homocysteine significantly prevented and reversed SNI-induced mechanical allodynia. Thus, alterations of ANX2 may be involved in tPA-dependent plasticity after peripheral nerve injury and have an important role in neuropathic pain.
Im Rahmen der Embryonalentwicklung oder der Gewebshomöostase entstehen kontinuierlich apoptotische Zellen, die effizient von professionellen Phagozyten, aber auch von nicht-professionellen Phagozyten aufgenommen werden. Im Gegensatz zur Aufnahme von Pathogenen ist die Phagozytose apoptotischer Zellen (Efferozytose) ein anti-inflammatorischer Prozess. Somit trägt die Efferozytose essentiell zur Erhaltung der peripheren Toleranz bei, indem sie Immunreaktionen gegen Autoantigene verhindert. Die molekularen Mechanismen des immunsuppressiven Effekts der Efferozytose sind bis heute nur unzureichend aufgeklärt. Annexin A1 (AnxA1), ein intrazelluläres, Phospholipid-bindendes Protein, gehört zu den beschriebenen eat me-Signalen und transloziert an die Membran früh-apoptotischer Zellen. Darüber hinaus ist AnxA1 in der Lage, die Toll like Rezeptor (TLR)-vermittelte, pro-inflammatorische Antwort in dendritischen Zellen (DC) zu supprimieren. Die Schlüsselfrage nach dem AnxA1-Rezeptor, der diese Immunsuppression auf DC vermittelt, blieb bislang jedoch unbeantwortet. Im Rahmen der vorliegenden Arbeit wurde deswegen eine neue UV-Kreuzvernetzungsmethode etabliert, um die Bindung von AnxA1 an seinen putativen Rezeptor mittels einer kovalenten Bindung zu stabilisieren. Dadurch konnte das Low density lipoprotein receptor-related protein 1 (LRP1) als potentieller AnxA1-Rezeptor identifiziert werden. Die Interaktion von LRP1 mit AnxA1, sowie mit weiteren Annexinen, wurde anhand verschiedenster Bindungsstudien in vitro validiert. Des Weiteren ermittelten quartz crystal mircobalance-Studien eine hohe, Calcium-abhängige Bindungsaffinität von LRP1 an verschiedene Annexine. Anhand DC- und Makrophagen-spezifischer LRP1 knock out-Mäuse wurde die Rolle von LRP1 auf die AnxA1-vermittelte Immunsuppression untersucht. In dieser Studie konnte erstmalig gezeigt werden, dass AnxA1, neben der Suppression in DC, auch die TLR-induzierte Antwort in Makrophagen supprimiert. Diese Immunsuppression erfolgte sowohl in Makrophagen als auch in DC unabhängig von LRP1. Da LRP1 und AnxA1 im Kontext der Efferozytose beschrieben sind, wurde anschließend die Relevanz der Interaktion für die Phagozytose apoptotischer Zellen untersucht. Durch die putative Redundanz von eat me-Signalen und deren Rezeptoren konnte kein Einfluss des AnxA1-LRP1-Komplexes auf die Efferozytose nachgewiesen werden. Jedoch konnte in einem System, in dem einzelne eat me-Signale isoliert betrachtet wurden, gezeigt werden, dass LRP1 die Phagozytose von AnxA1-gekoppelten Beads in DC und nicht-professionellen Phagozyten beeinflusst. Zusammengefasst konnte in der vorliegenden Arbeit erstmalig LRP1 als neuer Rezeptor für Annexine identifiziert und charakterisiert werden. Die AnxA1-LRP1-Interaktion vermittelt dabei nicht die Toleranzinduktion apoptotischer Zellen, sondern spielt eine wichtige Rolle bei Phagozytose-Prozessen. Durch die gezielte Manipulation der Interaktion von LRP1 mit verschiedenen Annexinen in Patienten mit Autoimmunerkrankungen, die auf einer defekten Efferozytose beruhen, könnte der AnxA1-LRP1-Komplex im therapeutischen Kontext von großem Interesse sein.
Unregulated inflammation underlies many diseases, including sepsis. Much interest lies in targeting anti-inflammatory mechanisms to develop new treatments. One such target is the anti-inflammatory protein annexin A1 (AnxA1) and its receptor, FPR2/ALX. Using intravital videomicroscopy, we investigated the role of AnxA1 and FPR2/ALX in a murine model of endotoxin-induced cerebral inflammation [intraperitoneal injection of lipopolysaccharide (LPS)]. An inflammatory response was confirmed by elevations in proinflammatory serum cytokines, increased cerebrovascular permeability, elevation in brain myeloperoxidase, and increased leukocyte rolling and adhesion in cerebral venules of wild-type (WT) mice, which were further exacerbated in AnxA1-null mice. mRNA expression of TLR2, TLR4, MyD-88, and Ly96 was also assessed. The AnxA1-mimetic peptide, AnxA1(Ac2-26) (100 μg/mouse, ∼33 μmol) mitigated LPS-induced leukocyte adhesion in WT and AnxA1-null animals without affecting leukocyte rolling, in comparison to saline control. AnxA1(Ac2-26) effects were attenuated by Boc2 (pan-FPR antagonist, 10 μg/mouse, ∼12 nmol), and by minocycline (2.25 mg/mouse, ∼6.3 nmol). The nonselective Fpr agonists, fMLP (6 μg/mouse, ∼17 nmol) and AnxA1(Ac2-26), and the Fpr2-selective agonist ATLa (5 μg/mouse, ∼11 nmol) were without effect in Fpr2/3(-/-) mice. In summary, our novel results demonstrate that the AnxA1/FPR2 system has an important role in effecting the resolution of cerebral inflammation in sepsis and may, therefore, provide a novel therapeutic target.-Gavins, F. N. E., Hughes, E. L., Buss, N. A. P. S., Holloway, P. M., Getting, S. J., Buckingham, J. C. Leukocyte recruitment in the brain in sepsis: involvement of the annexin 1-FPR2/ALX anti-inflammatory system.
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
Annexin I and II were studied immunohistochemically in arteries involved by Takayasu arteritis. Results suggest that they are important in the pathophysiologic function of macrophages and endothelial cells.
The Hong Kong Chinese population has an unusually high incidence of colorectal cancer in the young, suggestive of hereditary susceptibility. To search for a genetic basis for this predisposition, we studied the incidence of microsatellite instability (MSI) in paraffin-embedded colectomy specimens of 124 young (<50 years old) Chinese colorectal cancer patients referred to the Hong Kong Hereditary Gastrointestinal Cancer Registry from 1995 to 1998. By medical record review and personal interview, we searched for distinct clinical features associated with the manifestation of MSI in this group of patients. For patients with MSI tumours, blood was taken for detection of germline mutation in 2 mismatch repair (MMR) genes. MSI was present in 33 tumours from 23 males and 10 females (26.6%). Ongoing mutation analysis has so far identified MMR gene mutations in 8 patients with MSI tumours. The incidence of MSI increased significantly with decreasing age at cancer diagnosis. For patients aged 30 to 49, MSI tumours were located mainly at the proximal colon. However, for exceptionally young patients (<30 years), MSI tumours tended to be at the distal large bowel. This observation suggested a differential activity of the MMR pathway in colorectal carcinogenesis in different age groups. On multivariate analysis, young age at cancer diagnosis, proximal tumour location, a strong family history of colorectal cancer, and a personal history of metachronous cancer were independent predictors for MSI status. This knowledge may have an impact on the management of young colorectal cancer patients and their families. Int. J. Cancer 89:356–360, 2000. © 2000 Wiley-Liss, Inc.
The mechanism of short-term glucocorticoid (GC) inhibition of the hypothalamic-pituitary-adrenal axis is not well understood. The direct anti-inflammatory activities of lipocortins (LCs) have suggested a role for them as extra- and intracellular mediators of the biological effects of GCs. It has been reported that recombinant human (rh) LC1 inhibits corticotropin (ACTH) release from pituitary tissue in vitro but not from AtT-20 D16:16 corticotrophs. Using the same cell line we have tested whether other exogenous rhLCs or native LC extracted from polymorphonucleate neutrophils (neLC), likely LC1, have an effect on ACTH secretion. It is shown that: (1) basal release was not affected by a short-term incubation with neLC; (2) secretion induced by corticotropin-releasing factor (CRF) and other secretagogues (phorbol ester, potassium ion or calcium ionophore) was inhibited by neLC; (3) GC inhibition of CRF-stimulated release was reverted by a monoclonal anti-neLC antibody; (4) rhLC2, rhLC5 and the fragment 212-234 of rhLC5 were without effect. Thus, only neLC is effective on AtT-20 D16:16 cells, suggesting for this annexin a role in the early phase GC inhibition of ACTH secretion.
The lipocortins are a family of structurally related proteins that have been shown to be implicated in multiple aspects of cell biology. Subsequent research has shown that lipocortin 1 (LC1) participates in the physiological and pathological functioning of the CNS and neuroendocrine system. In the present study, the effects of 12-O-tetradecanoylphorbol 13-acetate (TPA), dibutyryl cyclic AMP (Bt2cAMP) or dexamethasone (DEX) on expression of LC1 were investigated by a sandwich enzyme immunoassay and reverse transcription polymerase chain reaction (RT-PCR) in rat astrocytoma (C6) cells. Time-dependent experiments revealed that the intracellular protein content and the mRNA of rat LC1 increased significantly 4 h after TPA (10 nM) or DEX (1 μM) addition. TPA and DEX elicited a prominent induction of LC1 at 10−8 M and 10−6 M, respectively. Bt2cAMP (0.5 mM) also appeared to induce, but the induction was not statistically significant. In addition, DEX increased the extracellular secretion of LC1 without cytotoxicity. These results suggest that LC1 synthesis is chemically induced and selectively released from C6 cells by dexamethasone.
Ischaemia–reperfusion injury underlies many of the most important cardiovascular diseases such as myocardial infarction, thrombotic stroke, embolic vascular occlusions and peripheral vascular insufficiency. Neutrophils feature prominently in this inflammatory component of post-ischaemic injury. Experimental therapies, shown to reduce neutrophil-mediated ischaemia–reperfusion injury include neutrophil depletion, direct inhibitors of neutrophil activators, antibodies against neutrophil adhesion molecules and the endothelial adhesion molecules. However, aside from these approaches, it is increasingly recognised that glucocorticoids are potent inhibitors of neutrophil-mediated injury. The anti-inflammatory actions of glucocorticoid include the activation of classical cytoplasmic receptors leading to changes in gene transcription as well as the induction of regulatory proteins, such as annexin 1. Annexin 1 is a potent inhibitor of neutrophil extravasation in vivo. Administration of the annexin 1 or peptides derived from its N-terminal domain, reduce neutrophil extravasation in models of acute inflammation. In addition, as reviewed by this article, annexin 1 protects against ischaemia–reperfusion in the heart and mesenteric microcirculation, as well as in multiple organ failure associated with splanchnic ischaemia–reperfusion. Such findings would suggest annexin 1 is a novel anti-inflammatory agent with a potential for the treatment of cardiovascular pathologies associated with neutrophil activation and recruitment.
The mechanism by which lipocortin 1 (LC1) is extruded from cells in the brain and periphery in response to a glucocorticoid challenge is unknown. This study examined the influence of three inhibitors of the classical endoplasmic reticulum-Golgi pathway of protein secretion on the dexamethasone-induced (0.1 microM, 2-3 h) cellular exportation of LC1 in vitro in brain (cortex, hippocampus, hypothalamus), anterior pituitary tissue and peritoneal macrophages. In all instances, the steroid-induced exportation of LC1 was unaffected by brefeldin A (1.4 microM), monensin (10 microM) and nocodazole (3.3 microM); however, these drugs readily blocked the release of corticotrophin from pituitary tissue. These data suggest that LC1 is exported by a mechanism distinct from the classical pathway of protein secretion.
N-terminal peptides derived from the anti-inflammatory peptide, annexin-1, inhibit neutrophil function but can also induce pro-inflammatory effects. Although equine annexin-1 has been sequenced, its cellular expression and properties have not been reported. This study has examined whether annexin-1 is present in equine leucocytes and how the N-terminal peptide, Ac2-26, affects equine neutrophil superoxide production. Annexin-1 expression in equine neutrophils and mononuclear cells and the ability of Ac2-26 to activate neutrophil p42/44 MAPK were determined by immunoblotting. Equine neutrophil superoxide production was measured by the reduction of cytochrome (cyt) C following stimulation with Ac2-26 and the formyl peptide receptor (FPR) agonists, FMLP, WKYMVm and WKYMVM. Responses were examined in the presence of the pan-FPR antagonist, BOC-2, and the role of p42/44 MAPK in agonist-induced effects was determined using PD98059. The effect of Ac2-26 on superoxide production in response to serum-treated zymosan (STZ) was also investigated, and the roles of FPR and p42/44 MAPK ascertained. Annexin-1 was detected in both equine neutrophils and mononuclear cells using a polyclonal rabbit anti-human annexin-1 antibody. Ac2-26 (5x10(-5)M) induced superoxide production in cytochalasin B-primed (48+/-8 versus 21+/-9 (unstimulated cells) nmol cyt C/10(6) neutrophils) and un-primed cells (37+/-10 versus 11+/-5 nmol cyt C/10(6) neutrophils). FMLP and WKYMVm, but not WKYMVM, also caused superoxide production in primed neutrophils, suggesting the response was mediated by FPR receptor binding. This was supported by the marked inhibitory effect of BOC-2 on the responses to Ac2-26 and FMLP although, interestingly, the effects of WKYMVm were not significantly reduced (50+/-5 (WKYMVm) versus 45+/-5 (WKYMVm+BOC-2) nmol reduced cyt C/10(6) neutrophils). Inhibition of p42/44 MAPK activation with PD98059 significantly attenuated superoxide production in response to Ac2-26, FMLP and WKYMVm and Western blotting showed that Ac2-26 induced p42/44 MAPK activation. At a concentration which did not cause superoxide production, Ac2-26 (10(-5)M) significantly reduced the response to STZ (84+/-17% inhibition). This inhibitory effect was attenuated by both BOC-2 and PD98059. These results suggest that if activation of equine leucocytes in vivo leads to the release and subsequent cleavage of annexin-1, the N-terminal peptides formed could bind to neutrophil FPR and decrease free radical production in response to particulate stimuli. This could help to reduce local tissue damage but, as Ac2-26 can also stimulate superoxide production at higher concentrations in an FPR-dependent manner, the amount of free radical production may depend on the concentration of peptide present.
Apoptotische Zellen entstehen kontinuierlich im Rahmen der Entwicklung und Gewebehomöostase multizellulärer Organismen. Ihre Beseitigung erfolgt durch Phagozyten wie Dendritische Zellen (DC). Während die Aufnahme von Pathogenen DC aktiviert und zur Auslösung einer Immunantwort führt, wirkt die Phagozytose apoptotischer Zellen antiinflammatorisch. Dies dient dem Schutz des Organismus vor Immunreaktionen gegen Selbst-Antigene aus apoptotischen Zellen und damit vor Autoimmunerkrankungen. Durch welche Mechanismen apoptotische Zellen DC beeinflussen ist jedoch weitgehend ungeklärt. In dieser Arbeit wurde gezeigt, dass das Protein Annexin 1, das spezifisch auf der Oberfläche frühapoptotischer Zellen präsentiert wird, zu deren antiinflammatorischem Effekt beiträgt, indem es die Signaltransduktion von Toll-like-Rezeptoren (TLR) und somit die Aktivierung von DC hemmt. Die Modulation der Immunantwort durch apoptotische Zellen und insbesondere durch das Protein Annexin 1 wurde in in vitro-Experimenten mit DC oder DC-ähnlichen Zelllinien aus Maus und Mensch untersucht. Vorinkubation mit apoptotischen Zellen oder rekombinantem Annexin 1 führt zu einer verminderten Aktivierbarkeit der DC durch TLR-Liganden. Dies zeigt sich in einer reduzierten Sekretion proinflammatorischer Zytokine wie TNF, bei unveränderter Produktion des antiinflammatorischen Zytokins IL-10. Der Einfluss auf die Zytokinsekretion spiegelt sich in einer entsprechenden Regulation der Zytokin-mRNAs wider. Genexpressionsanalysen zeigen einen globalen negativ regulatorischen Einfluss von Annexin 1 auf die durch Lipopolysaccharid (LPS) induzierten proinflammatorischen Gene. Diese Effekte beruhen auf einer Inhibition der TLR-induzierten Signaltransduktion. Vorinkubation mit Annexin 1 bewirkt eine Reduktion der TLR-induzierten Phosphorylierung und Aktivierung von MAP-Kinasen sowie eine verminderte Aktivierung des Transkriptionsfaktors NF-kB. Die Regulation betrifft beide TLR-induzierte Signalwege, die von den Adaptormolekülen MyD88 bzw. TRIF abhängen: MyD88- und TRIF-abhängige Zytokine sind gleichermaßen inhibiert, und ein Effekt von Annexin 1 ist auch in MyD88-defizienten Mäusen zu beobachten. Dementsprechend beeinflusst Annexin 1 sowohl die Aktivierung über den ausschließlich TRIF-abhängigen TLR3 als auch die MyD88-abhängige Signaltransduktion von TLR2 und dem IL-1-Rezeptor. Die Stimulierbarkeit der DC über den nicht mit den TLR verwandten TNF-Rezeptor wird durch Annexin 1 ebenfalls vermindert, was auf eine umfassendere Inhibition proinflammatorischer Signaltransduktion hinweist. Experimente mit dem Translationsinhibitor Cycloheximid weisen darauf hin, dass der antiinflammatorische Effekt von einer durch Annexin 1 induzierten Proteinsynthese abhängt. Die Analyse der Annexin 1-vermittelten Effekte auf DC trägt zum Verständnis des Mechanismus bei, wie apoptotische Zellen das Immunsystem beeinflussen und möglicherweise zu peripherer Toleranz führen. Apoptotic cells are generated continuously during development and tissue homeostasis of multicellular organisms. They are removed by phagocytes such as dendritic cells (DC). In contrast to pathogens, which activate DC upon uptake and lead to initiation of an immune response, apoptotic cells show an anti-inflammatory effect on DC. This protects the organism from immune reactions against self-antigens derived from apoptotic cells and, thus, from autoimmune diseases. Mechanistically, however, not much is known about the tolerogenic effect of apoptotic cells. This work shows that the protein annexin 1, which is presented specifically on the surface of early apoptotic cells, contributes to the anti-inflammatory effect by inhibiting the signal transduction of Toll-like receptors (TLR) and thus the activation of DC. The modulation of the immune response by apoptotic cells and the protein annexin 1 in particular was investigated in in vitro experiments, using DC or DC-like cell lines derived from mouse or humans. Pre-incubation with apoptotic cells or recombinant annexin 1 leads to a reduced reactivity of DC towards TLR ligands. This results in a reduced secretion of pro-inflammatory cytokines such as TNF, whereas the production of the anti-inflammatory cytokine IL-10 is unaffected. The influence on the secretion is mirrored by a corresponding regulation of the cytokine mRNAs. Gene expression analyses show a global negative impact of annexin 1 on LPS-induced proinflammatory genes. These effects are due to an inhibition of TLR-induced signal transduction. Pre-incubation with annexin 1 leads to a reduction of TLR-induced activation of MAP-kinases and NF-kB. Both pathways induced by TLR, depending on the adaptors MyD88 and TRIF, respectively, are affected: MyD88- and TRIF-dependent cytokines are inhibited to a similar extent, and the effect of annexin 1 can still be observed in MyD88-deficient mice. Correspondingly, annexin 1 inhibits activation mediated by the TRIF-dependent TLR3 as well as the MyD88-dependent signal transduction of TLR2 and the IL-1 receptor. The reactivity of the DC towards TNF, which acts via a non-TLR-related receptor, is reduced by annexin 1 as well, indicating a broad inhibition of pro-inflammatory signal transduction pathways. Experiments using the translational inhibitor cycloheximide indicated that the anti-inflammatory effect of annexin 1 depends on protein synthesis. The analysis of the effects of annexin 1 on DC contributes to a better understanding of the mechanism how apoptotic cells influence the immune system and possibly lead to peripheral tolerance.
Leukocyte extravasation occurs in many pathophysiological conditions, including inflammation, neoplasia and asthma. In recent years many studies have elucidated the steps that promote the initial interaction between extravasating cells and endothelium of the post-capillary venule; the sequential role of several classes of adhesion molecules (cell-specific chemokines) and activators (multipotent cytokines) is well established. In this review, Mauro Perretti focuses on a less well investigated mechanism by which the host downregulates extravasation at the leukocyte-endothelium interface. The neutrophilic polymorphonuclear leukocyte is used as the prime example of a leukocyte that interacts with the endothelium, and particular emphasis is given to the possibility that novel anti-inflammatory therapies might be developed from a better understanding of the inhibitory mechanisms activated by endogenous mediators such as adenosine, lipocortin 1, NO, prostacyclin and cathepsin G.
The glucocorticoid-induced antiinflammatory protein lipocortin 1 is present in arthritic synovium but its ability to regulate joint inflammation has not previously been studied. We investigated the role of lipocortin 1 in the antiinflammatory activity of glucocorticoids in an acute arthritis model induced by intraarticular injection of carrageenan. Compared to control joints (0.09 +/- 0.08 x 10(6) synovial fluid cell count), carrageenan injected joints exhibited marked infiltration of PMN (10.2 +/- 0.7 x 10(6), p < 0.001). Both intraperitoneal (1.0 mg/kg) and intraarticular administration (5 micrograms) of dexamethasone (DEX) significantly suppressed arthritis severity (p < 0.001 and 0.005, respectively), and the effects of DEX were significantly prevented by intra-articular injection of antilipocortin 1 mAb (p < 0.05). Carrageenan arthritis was also significantly inhibited by intraarticular administration of the N-terminal lipocortin 1 peptide Ac2-26 at doses of 1 or 2 mg/kg (p < 0.01). Intraarticular injection antilipocortin 1 mAb in the absence of DEX also significantly exacerbated arthritis severity (p < 0.005). In vitro treatment of PMN with DEX was associated with significant inhibition of phagocytosis (p < 0.005) and reactive oxygen species (ROS) generation (p < 0.001). Antilipocortin 1 mAb significantly reduced the inhibitory effects of DEX (p < 0.01 and 0.005, respectively). These results demonstrate that lipocortin 1 mediates the effects of exogenous glucocorticoids on neutrophil migration in carrageenan-induced acute arthritis, exerts an endogenous antiinflammatory influence, and mediates glucocorticoid inhibition of neutrophil activation.
To gain insight into the molecular basis of annexin gene expression we have analysed the annexin I and VI gene promoters. A previously described 881 bp sequence immediately upstream of the annexin I transcription start site and a similar size fragment proximal to the annexin VI transcription start site both drove expression of the luciferase reporter gene in fibroblasts and epithelial cells. Neither promoter displayed any sensitivity to dexamethasone, suggesting that the putative glucocorticoid response element in the annexin I promoter is non-functional. Consistent with this, endogenous annexin I gene expression was unaffected by dexamethasone at the mRNA and protein levels in A431 cells. A series of 5' deletions of the two promoters were examined to define the minimal active sequences. For annexin I this corresponded to a sequence approx. 150 bp upstream of the transcription start site that included CAAT and TATA boxes. Unexpectedly, the annexin VI promoter, which also contains CAAT and TATA boxes, was fully active in the absence of these elements, a 53 bp sequence between these boxes and the transcription start site having maximal activity. Electrophoretic mobility-shift assays with nuclear extracts from A431 and HeLa cells with probes corresponding to this region revealed an SP1-binding site. These results show that the annexin I and VI genes have individual modes of transcriptional regulation and that if either annexin I or annexin VI has an anti-inflammatory role, then this is in the absence of steroid-induced gene expression.
The effects of endogenous production of NO., catalysed by the mitochondrial nitric oxide synthase (NOS), on mitochondrial metabolism were studied. The respiratory rates of intact mitochondria in State 4 were decreased by 40% and 28% with succinate and malate-glutamate, respectively, in the presence of L-arginine (L-Arg); conversely, the O2 uptake with NG-methyl-L-arginine (NMMA), a competitive inhibitor of NOS, was increased. The production of NO. and the inhibition of the respiratory rates were dependent on the metabolic state in which mitochondria were maintained: NO. production was probably supported by mitochondrial NADPH, the latter maintained by the energy-dependent transhydrogenase. In addition to the decline in the respiratory rate, an inhibition of ATP synthesis was also observed (40-50%) following supplementation with L-Arg. The dependence of the respiratory rates of mitochondria in State 3 and cytochrome oxidase activities on O2 concentrations with either L-Arg or NMMA indicated that both processes were competitively inhibited by NO. at the cytochrome oxidase level. This inhibition can be explained by the interaction of NO. with cytochrome oxidase at the binuclear centre. The role of NO. as a physiological modulator of cytochrome oxidase is discussed in terms of cellular metabolism.
Plasminogen activator inhibitor 2 (PAI-2) is a serine protease inhibitor (serpin) that is secreted and accumulated intracellularly by monocytes. We investigated PAI-2 synthesis by isolated human peripheral blood monocytes and found that a 47-kDa nonglycosylated form of PAI-2 was abundant in conditioned medium from monocytes. Secretion of PAI-2 by monocytes was not inhibited by agents that inhibit either ER-Golgi pathway-dependent secretion, brefeldin A, or N-linked glycosylation, tunicamycin. IL-1beta served as a control for a protein that is secreted by an ER-Golgi-independent pathway, and secretion of IL-1beta was not inhibited by brefeldin A. This was in contrast to secretion of TNFalpha, which was dependent on the ER-Golgi pathway. None of the treatments was cytotoxic toward monocytes, as measured by release of the intracellular enzyme lactate dehydrogenase (LDH) into the conditioned medium. Subcellular fractionation revealed that PAI-2 and IL-1beta were colocalized. The mechanism for secretion of PAI-2 was not dependent on calcium or intracellular trafficking via the classical vesicular mechanism(s), distinguishing it from IL-1beta secretion. These studies show that PAI-2 is secreted by primary human monocytes via an ER-Golgi-independent pathway.
Annexin I is a local mediator in neural-endocrine feedback control of inflammation. J. Neurophysiol. 80: 3120-3126, 1998. Activation of primary afferent nociceptors induces a neural endocrine-mediated inhibition of the inflammatory response via a circuit that includes ascending spinal pathways and activation of the hypothalamic-pituitary adrenal (HPA) axis. This circuit inhibits sympathetic neuron-dependent plasma extravasation (PE) in the rat knee joint produced by bradykinin (BK), but not sympathetic neuron-independent PE produced by platelet activating factor (PAF). Noxious (25 mA) but not non-noxious (2.5 mA) electrical stimulation significantly increased plasma corticosterone concentrations, and intravenous infusion of corticosterone (5 micrograms/min) mimicked inhibition of BK-induced PE produced by noxious stimulation. However, perfusion of corticosterone locally through the knee joint, at doses that do not have a systemic action (i.e., </=1 microM), did not inhibit BK-induced PE. Annexin I (lipocortin-1), a 37-kDa member of a family of phospholipid and calcium binding proteins, can mediate local anti-inflammatory effects of glucocorticoids via a mechanism that is partially dependent on inhibition of phospholipase A2 activity and adhesion and transmigration of polymorphonuclear leukocytes. Because BK-induced PE is dependent on both polymorphonuclear leukocytes and phospholipase A2 activity, we tested the hypothesis that the action of corticosterone to inhibit BK-induced PE is mediated by stimulating the production and release of annexin I. Perfusion of BK (150 nM) through the rat knee joint induces a rapid and sustained increase in PE. Co-perfusion of BK with annexin I (100 ng/ml) through the knee joint mimics the inhibition of BK-induced PE produced by noxious electrical stimulation or by intravenous corticosterone. Co-perfusion of BK with annexin I antibody (LCPS1, 1:60 dilution) prevented the inhibition of BK-induced PE produced by noxious electrical stimulation or intravenous corticosterone adminstration. PAF-induced PE, which is not dependent on polymorphonuclear leukocytes, was not inhibited by local perfusion of annexin I. These data suggest that the inhibitory effect of C-fiber activity on BK-induced PE, acting via an HPA circuit, is mediated by annexin I in the knee joint.
Lipocortin 1 (LC1, also called annexin 1), a Ca2(+)- and phospholipid-binding protein, is an important mediator of glucocorticoid action in the anterior pituitary gland. Previous studies based on immunoprecipitation and Western blot analysis suggest that LC1 is found intracellularly both in the cytoplasm and in association with membranes and also on the cell surface where it attaches to the membrane by a Ca2(+)-dependent mechanism. However, as yet it is unclear which anterior pituitary cell types express the protein. Accordingly, we have developed a method based on a combination of fluorescence activated cell (FAC) analysis/sorting and electron microscopy to detect and quantify intracellular LC1 in rat anterior pituitary cells and to identify the cell types in which it is expressed. In addition, we have measured cell surface LC1 and examined the influence of glucocorticoids on the cellular disposition of the protein. Anterior pituitary cells were dispersed with collagenase. For experiments measuring intracellular LC1, three cell fixation/permeabilisation methods were examined initially, i.e. (1) Zamboni's fluid (30 min) and Triton-X-100 (0.12%, 1 or 12 h); (2) paraformaldehyde (2%, 1 h) and Triton-X-100 (0.2%, 10 min); and (3) paraformaldehyde (0.2%, 15 min) and saponin (0.1%, 5 min). The protocol using paraformaldehyde/Triton-X-100 provided optimal preservation of cell ultrastructure and of LC1 immunoreactivity (ir-LC1) while also effectively permeabilising the cells; it was therefore used in subsequent studies. Using an anti-LC1 monoclonal antibody as a probe, 82+/-5% of the secretory cells in the heterogeneous anterior pituitary cell preparation were shown by FAC analysis to display specific fluorescence for intracellular ir-LC1. Morphological analysis and immunogold-histochemistry of cells separated by FAC sorting identified corticotrophs, lactotrophs, somatotrophs and gonadotrophs in the population displaying LC1 immunofluorescence. LC1 was also detected on the surface of anterior pituitary cells by FACS analysis. Incubation of anterior pituitary cells with dexamethasone or corticosterone (0.1 and 1.0 microM) prior to fixation and analysis produced a significant, concentration-dependent decrease in intracellular ir-LC1 and a concomitant increase in the amount of ir-LC1 detected on the surface of the cells; the effects of the two steroids were indistinguishable quantitatively. In conclusion, we report a novel method which permits (1) the detection and semi-quantitative measurement of intracellular and surface LC1 in anterior pituitary cells; and (2) the identification of the cell types in which the protein is found.
Immunoblotting and immunohistochemical analysis were performed to identify the cells expressing and secreting annexin 1 during experimental rat colitis induced by trinitrobenzene sulfonic acid. Annexin 1 expression was increased during the inflammation. Likewise, annexin 1 secretion was induced in inflamed colons at one, three, six, and nine days after trinitrobenzene sulfonic acid treatment but was not detected in colons from controls and rats at 12 days. Immunohistochemistry showed that the rise in annexin 1 expression resulted from the infiltration of two types of leukocytes highly positive for annexin 1: neutrophils (the most abundant) and macrophages. At day 1 after treatment, neutrophils of the inflammatory site, in mucosa and submucosa, are the only cells expressing annexin 1. Immunoblotting showed that they secreted annexin 1 whereas neutrophils from blood or tunica muscularis did not. This indicates that, during this colitis, annexin 1 can be secreted by neutrophils located in the inflammatory site.
Glucocorticoids exert their anti-inflammatory activity through multiple pathways which include the inhibition of cell adhesion events. The glucocorticoid-induced protein lipocortin 1 (LC1) has reported anti-inflammatory properties and has been proposed as a putative mediator of the anti-inflammatory effects of glucocorticoids. The role of LC1 in mediating the glucocorticoid inhibition of lymphocyte adhesion and cell adhesion molecule (CAM) expression was investigated in vitro using a microaggregation assay, flow cytometry and confocal microscopy. Lymphocytes stimulated for 96 h with plastic-bound OKT3 antibody showed significant increases in LFA-1 and CD2 expression. Dexamethasone (DEX; 10(-6) M) inhibited this increase but the neutralizing anti-LC1 MoAb 1A (5 microg/ml) failed to reverse the DEX effect; neither was purified human LC1 (50 x 10(-9) M) able to inhibit CAM expression. The biological activity of the LC1 was confirmed by its ability to suppress monocyte phagocytosis and respiratory burst in response to bovine serum albumin (BSA)-anti-BSA complexes. OKT3 stimulation of cultured mononuclear cells resulted in intercellular aggregation, scored microscopically using a visual index. This aggregation was completely reversed by 10-6 M DEX but unaffected by LC1 (50 x 10(-9) M). Significant intracellular expression of lymphocyte LC1 was observed using the anti-LC1 MoAb 1B in saponin-permeabilized cells. Distribution of LC1 had a diffuse, cytoplasmic pattern. LC1 expression was reduced following 3 h treatment with 10(-6) M DEX. These findings indicate that the DEX effects on lymphocyte adhesion and CAM expression are not mediated by LC1. Thus the reported in vivo effects of LC1 on leucocyte adhesion and transmigration probably occur through functional/conformation changes of surface CAM, rather than by alteration in expression.
Annexin-1 (ANXA1, lipocortin 1) is a pleiotrophic protein produced by many cell types including peripheral blood leucocytes. Although it has been shown to inhibit "macroscopic" inflammatory processes in animal models, its direct effects on antigen-activated human T cells have not been studied.
To test the hypothesis that ANXA1-derived peptides inhibit antigen-driven prototype Th1 and Th2-type human T cell responses of clinical relevance and lectin-driven responses in vitro.
Peripheral blood mononuclear cells (PBMC) were isolated from 14 atopic subjects sensitized to house dust mite allergen (Dermatophagoides pteronyssinus, Der p) and purified protein derivative (PPD) of Mycobacterium tuberculosis. PBMC (1 x 106/mL) were cultured with phytohaemagglutinin (PHA; 5 microg/mL; 4 days), Der p (25 microg/mL; 6 days), PPD (10 microg/mL, 6 days) or medium control. Two ANXA1-derived peptides, Ac2-26 and AF-2 (5-500 microM), were assessed for possible inhibition of PHA-and antigen-induced T cell proliferation (measured by 3H-thymidine uptake), while Ac2-26 was assessed for inhibition of Der p-induced interleukin (IL)-5 release and PPD-induced interferon-gamma (IFN-gamma) release (measured by ELISA). Comparison was made with dexamethasone as an established inhibitory control. Endogenous production by PBMC of cell surface-associated and intracellular ANXA1 in response to PHA, Der p and PPD in the presence and absence of dexamethasone was measured by specific ELISA.
Both PHA- and antigen-induced T cellular proliferation were inhibited by dexamethasone. Although neither ANXA1-derived peptide significantly altered PHA-induced proliferation, both effected concentration-dependent reductions in antigen-induced proliferation, Ac2-26 being the more potent. Peptides of identical amino acid composition to Ac2-26 and AF-2, but of random sequence, were ineffective at equivalent concentrations. In addition, Ac2-26 and dexamethasone inhibited Der p-induced IL-5 release and PPD-induced IFN-gamma release in a concentration-dependent fashion. Endogenous ANXA1 was detectable in PBMC, but at concentrations approximately 104-fold lower, in molar terms, than the effective concentrations of the exogenously added, ANXA1-derived inhibitory peptides. Endogenous production was not significantly altered by any of the T cell stimuli employed in this study, in the presence or absence of dexamethasone.
In prototype Th1 and Th2-type human T cell responses, ANXA1-derived peptides can inhibit antigen-driven cellular proliferation and cytokine production.
Annexin I and II are classical types of the annexin family and known for their physiological functions in inflammatory processes and thrombus formation. Takayasu arteritis, on the other hand, is a vasculitis of unknown etiology characterized by the inflammatory involvement of the internal vessel wall and thrombus formation. It causes the stenotic and/or obstructive changes of the arterial wall leading to the characteristic clinical feature of pulselessness. Expression of annexin I and II in vessel walls obtained from nine patients with Takayasu arteritis was studied immunohistochemically to investigate their roles in this morbid condition. Expression of annexin I was recognized in foamy cells in the thickened intima and/or medial layer and in inflammatory cells in vessel walls, but neither in endothelial cells nor in normal or proliferated smooth muscle cells. Strong expression of annexin II was present in foamy cells as well as in endothelial cells both in thickened intima and in proliferated smooth muscle cells in the intima, but not in smooth muscle cells in the media. The stronger expression of annexin II was seen in the endothelial cells in the vasa vasorum of both thickened and normal adventitia. These results suggest the participation of annexin I and II in the process of this vasculitis, and an essential role of vasa vasorum in the pathogenesis of Takayasu arteritis.
Annexins are primarily intracellular proteins as would be predicted from their lack of hydrophobic signal sequences. However, we now report that the human prostate gland selectively secretes high concentrations of annexin 1 (also called lipocortin 1 and p35) and a proteolytic cleavage product, des1-29-annexin 1, into seminal plasma. Secreted annexin 1 had a blocked amino terminus and was structurally indistinguishable from intracellular annexin 1. Although annexin 1 and the structurally related protein, annexin 4, co-localized to many of the same cells of the ductal epithelium of the prostate, annexin 4 was not secreted. Thus, the secretion of annexin 1 appears to involve a highly selective mechanism that does not involve targeting to the endoplasmic reticulum by a hydrophobic signal sequence.
In eukaryotes, most proteins which are transported to the extracellular space, into mitochondria or into chloroplasts are synthesized as precursor polypeptides containing cleavable N-terminal signal or targeting sequences. We have searched the literature for proteins that are exported from the cytosol without being proteolytically processed. Some of these proteins contain uncleaved signal or targeting sequences. However, among secretory proteins there is a class that does not possess hydrophobic signal sequences and appears to leave the cell by a secretory pathway clearly distinct from the classical route through the endoplasmic reticulum and Golgi apparatus.
Lipocortins are structurally related, glucocorticoid-inducible proteins that inhibit phospholipase A2 (PLA2), thereby reducing the liberation of arachidonic acid from phospholipids and so limiting the synthesis of eicosanoid inflammatory mediators. This study is the first demonstration of one lipocortin, lipocortin 1 (Lc 1; 37 kDa), in human lung lavage supernatants. In lavage fluid from healthy volunteers, a higher percentage (greater than 70%) of the detected Lc 1 was in its native form, compared to that from patients with abnormal lungs. In patients' lavage fluids, Lc 1 was more likely to be partially degraded (34 kDa). In abnormal bronchoalveolar lavage fluid (BALF), the more polymorphonuclear neutrophils (PMN)/lavage, the lower the proportion of Lc 1 in the native (37 kDa) form (n = 7 pairs, rs = -0.8214, p less than 0.05). Furthermore, when BALF cells were cultured and the harvested conditioned media incubated with pure human recombinant Lc 1, degradation of the 37 kDa form increased with the percentage of PMN (n = 10 pairs, s = -0.7200 after 1 hr; n = 6 pairs, rs = -0.9241 after 6 hr). These results suggest that factors released from the PMN are responsible for Lc 1 degradation in man. When recombinant human Lc 1 was incubated with human neutrophil elastase, the enzyme degraded Lc 1 in a dose-dependent way, suggesting that neutrophil elastase may be one such factor. Since PMNs are ubiquitous at sites of inflammation, it is possible that Lc 1 degradation is a permissive mechanism, which ensures that sufficient inflammation occurs to destroy the provocative stimulus. However, it is equally possible that, in some circumstances, the mechanism may be pathological and that the inactivation of Lc 1 leads to chronic, uncontrolled inflammation.
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Interleukin 1 (IL-1) is a major soluble mediator of inflammation. Two human IL-1 genes, alpha and beta, have been isolated, which encode polypeptides with only 20-30% amino acid sequence homology. Unlike most secreted proteins, the two cytokines do not have a signal sequence, an unexpected finding in view of their biological role. Here we show that IL-1 beta is actively secreted by activated human monocytes via a pathway of secretion different from the classical endoplasmic reticulum--Golgi route. Drugs which block the intracellular transport of IL-6, of tumour necrosis factor alpha and of other secretory proteins do not inhibit secretion of IL-1 beta. Secretion of IL-1 beta is blocked by methylamine, low temperature or serum free medium, and is increased by raising the culture temperature to 42 degrees C or by the presence of calcium ionophores, brefeldin A, monensin, dinitrophenol or carbonyl cyanide chlorophenylhydrazone. IL-1 beta is contained in part within intracellular vesicles which protect it from protease digestion. In U937 cells large amounts of IL-1 beta are made but none is secreted. In these cells IL-1 beta is not found in the vesicular fraction, and all the protein is accessible to protease digestion. This suggests that intracellular vesicles that contain IL-1 beta are part of the protein secretory pathway. We conclude that IL-1 beta is released by activated monocytes via a novel mechanism of secretion which may involve translocation of intracellular membranes and is increased by stress conditions.
Glucocorticoids inhibit superoxide (O2-) generation by phagocytes through a mechanism that remains unclear. We investigated this effect by using dexamethasone on guinea pig alveolar macrophages. O2- generation was induced either by the calcium ionophore A23187, a potent stimulus of phospholipase A2, or by the protein kinase C activator, phorbol myristate acetate (PMA). Dexamethasone inhibited O2- generation initiated by A23187 by 50-55%. This inhibition required: (a) more than 45 min incubation and was maximal after 2 h; (b) glucocorticoid receptor occupancy; and (c) protein synthesis. The inhibitory effect of dexamethasone could not be explained by an interaction with the respiratory burst enzyme NADPH oxidase since O2- generation was only weakly affected upon PMA stimulation. Lipocortin I, a glucocorticoid inducible and phospholipase A2 inhibitory protein, inhibited O2- generation initiated by A23187 but failed to modulate the respiratory burst activated by PMA. These results were obtained with lipocortin I purified from mouse lungs, human blood mononuclear cells, and with human recombinant lipocortin I. We propose that lipocortin I is capable of inhibiting the activation of NADPH oxidase only when membrane signal transduction involves phospholipase A2. By mimicking the effect of dexamethasone, lipocortin I may extend its potential anti-inflammatory action to the partial control of the formation of oxygen reactive species by phagocytes.
The amino acid sequence of anchorin CII, a collagen-binding protein isolated originally from chondrocyte membranes, was previously determined by sequencing of cDNA and proteolytic fragments of the protein. Computer analysis of the protein sequence revealed four internal repeats of approximately 70-80 residues, each containing a highly conserved consensus sequence of 17 residues. These repeats show considerable homology with sequences in human and bovine calpactin, lipocortin, endonexin and protein II, which are members of a family of Ca2+- and phospholipid-binding proteins, as well as major substrates of tyrosine kinases. While these proteins have been located at the inner side of the plasma membrane of fibroblasts and epithelial cells, here we present experimental evidence that anchorin CII is at least partially released from cells and binds to the outer cell surface. Biosynthesis studies in cell-free systems and in cell culture indicate that anchorin CII is not processed, which is consistent with the absence of signal sequences from the protein. Yet, pulse-chase experiments show that anchorin is released into the culture medium of fibroblasts after 30 min, and in chondrocyte cultures after 20 h. Anchorin CII was located to the outer cell surface of chondrocytes by lactoperoxidase-catalyzed cell surface iodination as well as by antibody labeling both at light- and electron-microscopical level. The pericellular localization of anchorin CII is consistent with the notion that this protein is involved in the interaction of chondrocytes and fibroblasts with extracellular collagen.
We have examined the ability of a highly purified 38-kD phospholipase-inhibitory protein (p38) isolated from human placental membranes that is also a preferred substrate for the epidermal growth factor-urogastrone (EGF-URO) receptor/kinase, to block the release of arachidonate from zymosan-stimulated murine peritoneal macrophages in vitro and to exhibit antiinflammatory activity in a carrageenin rat paw edema test in vivo. The ability of glucocorticoids to increase the amounts of this protein in macrophage cultures was also examined. p38 represents the naturally occurring, intact, NH2-terminally blocked human placental form of the protein termed calpactin II (or lipocortin I), for which partial amino acid sequence data and a complete amino acid sequence deduced from cDNA analysis have been reported. Our data demonstrated that, whereas p38 was an effective inhibitor of pancreatic phospholipase A2 in vitro, it was unable to inhibit either the release of arachidonate from cultured zymosan-stimulated mouse peritoneal macrophages or inflammation in a rat paw edema test. At comparatively high protein concentrations, p38 enhanced either arachidonate release from intact macrophages in vitro (0.5-10 micrograms/ml) or carrageenin-induced paw swelling in vivo (2.5 or 25 micrograms per injection). Furthermore, we were unable to detect induced amounts of p38 in cultures of glucocorticoid-treated peritoneal macrophages obtained from either mice or rats. Our data indicate that the antiphospholipase activity of p38 in vitro and the ability of p38 to serve as a receptor/kinase substrate may in no way relate to the putative ability of the protein to modify eicosanoid release from macrophages in vivo, so as to modulate the inflammatory process. Our data also raise the possibility that p38 (calpactin II) may not be a true representative of the lipocortin family of glucocorticoid-inducible antiinflammatory proteins, despite its ability to inhibit phospholipase A2 in vitro.
Monocytes lack lactoferrin and have much less myeloperoxidase than neutrophils. They also acquire a potential catalyst for .OH production (tartrate-resistant acid phosphatase) as they differentiate into macrophages. Consequently, the nature of free radicals produced by these cells was examined using the previously developed spin-trapping system. When stimulated with either PMA or OZ neither monocytes nor monocyte-derived macrophages (MDM) exhibited spin trap evidence of .OH formation. Pretreatment with IFN-gamma failed to induce MDM .OH production. When provided with an exogenous Fe+3 catalyst, both stimulated monocytes and MDM, but not PMN, exhibited sustained .OH production, presumably due to the absence of lactoferrin in mononuclear phagocytes. Sustained production of .OH could contribute to the microbicidal activity of mononuclear phagocytes as well as inflammatory tissue damage under in vivo conditions where catalytic Fe+3 may be present.
Monocytes lack lactoferrin and have much less myeloperoxidase than neutrophils. They also acquire a potential catalyst for .OH production (tartrate-resistant acid phosphatase) as they differentiate into macrophages. Consequently, the nature of free radicals produced by these cells was examined using the previously developed spin-trapping system. When stimulated with either PMA or OZ neither monocytes nor monocyte-derived macrophages (MDM) exhibited spin trap evidence of .OH formation. Pretreatment with IFN-gamma failed to induce MDM .OH production. When provided with an exogenous Fe+3 catalyst, both stimulated monocytes and MDM, but not PMN, exhibited sustained .OH production, presumably due to the absence of lactoferrin in mononuclear phagocytes. Sustained production of .OH could contribute to the microbicidal activity of mononuclear phagocytes as well as inflammatory tissue damage under in vivo conditions where catalytic Fe+3 may be present.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Lipocortin I, in some cells, may be a potent inhibitor of phospholipase A2 activity. These studies evaluated the relative amounts of lipocortin I in human alveolar macrophages compared with blood monocytes, using a specific polyclonal antibody and the technique of Western analysis. Lipocortin I was detected in all isolates of human alveolar macrophages and had molecular masses of 37,000 and 33,000 D. Corticosteroids increased amounts of lipocortin I in these cells in a dose-dependent manner. This effect was specific for corticosteroids as related steroids had no effect. Blood monocytes, when compared with alveolar macrophages, contained relatively small amounts of lipocortin I. We conclude that lipocortin I is present in relatively large amounts in human alveolar macrophages and that amounts of the protein can be induced by corticosteroids. We further speculate that the relative amounts of lipocortin I within monocytes/macrophages may be a marker of differentiation.
Basic fibroblast growth factor (bFGF) modulates functions of a variety of cell types. Whereas bFGF is known to act extracellularly, the protein lacks a transient signal peptide. No defined mechanism for bFGF secretion has been characterized besides release from dead or injured cells. To study this problem we devised an experimental system to examine bFGF-mediated migration of isolated single cells. Under these conditions individual cells are not affected by bFGF derived from other cells. By this method we have previously shown that bFGF released by NIH 3T3 cells transfected with bFGF cDNA modulates migration in an autocrine manner. We have now examined the effects on cell motility of drugs or treatments known to affect various pathways of protein secretion. Drugs that block secretion via the endoplasmic reticulum (ER)-Golgi complex or via multidrug resistance proteins did not inhibit cell motility. Migration was enhanced by the calcium ionophore A23187, which stimulates exocytosis, and was inhibited by methylamine, serum-free, and low temperature (18 degrees C) conditions, which block endo- and exocytosis. The reversal of these effects by the concomitant addition of affinity-purified anti-bFGF IgG or recombinant bFGF showed that the alterations in cell migration were mediated by changes in bFGF externalization. Thus bFGF can be released via a mechanism of exocytosis independent of the ER-Golgi pathway.
The effect of dexamethasone on mRNA and protein synthesis of lipocortins (LCT) 1, 2 and 5 has been investigated in U-937 cells. A constitutive expression of both mRNAs and proteins was detected in undifferentiated U-937 cells. This constitutive level was increased time- and dose-dependently by incubation with phorbol myristate acetate (PMA). In U-937 cells differentiated by 24 h incubation with 6 ng/ml PMA, dexamethasone (DEX) (1 microM for 16 h) caused an increased synthesis of the mRNA level of LCT-1 and 2, but not of LCT-5, over the level induced by PMA. DEX had no effect in undifferentiated cells. Moreover, DEX stimulated the extracellular release of LCT-1 and 5, but not of LCT-2, and inhibited the release of PGE2 and TXB2 only in the differentiated U-937 cells. These results suggest that the responsiveness of these cells to glucocorticoids is dependent on the phase of cell differentiation. The selective release of lipocortins by differentiated U-937 cells may explain, at least in part, the inhibition by DEX of the prostanoid release.
The presence and amount of the anti-inflammatory protein lipocortin 1 was determined in plasma and peripheral blood leucocytes by a highly specific, enzyme-linked immunosorbent assay. Within 120 min of a single intravenous dose of 100 mg hydrocortisone, the intracellular concentrations of lipocortin 1 in peripheral monocytes in 7 of 8 healthy men increased by a median of 225% (range 129-507%) compared with pretreatment levels, and mononuclear cell-surface lipocortin increased by a median of 224% (range 76-483%). Placebo injections had no effect. There was no increase at any time in free plasma or polymorph-associated lipocortin. In 3 of 4 subjects, induction of lipocortin was also observed when whole unseparated blood was incubated in vitro after steroid administration, but cells which had first been isolated and purified were refractory to such induction. Thus rapid changes in the concentration of an active anti-inflammatory protein can occur in man after normal therapeutic doses of hydrocortisone.
Calcium-dependent phospholipid binding and phospholipase A2 inhibitory proteins were isolated from human mononuclear cells. Lipocortins I and II were present whereas lipocortin IV (endonexin I) was not. The other proteins were purified to homogeneity and shown to have molecular masses of 35, 36, 32 and 73 kDa. The 36-kDa and 73-kDa proteins are related, the smaller appears to be part of the larger. The 73-kDa protein is related to the 67-kDa calelectrin and to lipocortin VI; the 32-kDa protein is different from endonexin I but related to chromobindin 7 and to lipocortin V. The 35-kDa protein has been identified by tryptic peptide sequencing as lipocortin III. All these proteins inhibit phospholipase A2 activity in vitro and the three smaller ones inhibit the [3H]arachidonic acid release from prelabelled monocytes induced by the calcium ionophore A23187 in a dose-dependent manner.
Lipocortin I is a corticosteroid-inducible protein that has potent anti-inflammatory activity. To determine whether lipocortin I is present on the epithelial surface of the human lung, we used a specific polyclonal antibody by the technique of Western blotting to evaluate bronchoalveolar lavage (BAL) fluid of normal individuals and patients with idiopathic pulmonary fibrosis. Lipocortin I was a normal constituent of the epithelial surface of the normal lung and comprised 0.23 +/- 0.03% of BAL fluid proteins. Four separate immunoreactive species were detected, at 37, 36, 34, and 33 kDa, consistent with previously published results. Corticosteroids increased the amounts of lipocortin present in normal volunteers and in patients with idiopathic pulmonary fibrosis. These results demonstrate that lipocortin I is normally present in the human lung and further suggest that lipocortin I may be an important modulator of the anti-inflammatory effects of corticosteroids in the lung.
Lipocortin-1 protein synthesis in resting monocytes is under the control of glucocorticoid steroids. This induction occurs at reasonable dexamethasone concentrations, may require concomitant synthesis of transcriptional factors, appears to be cell type specific, and has been observed only in primary tissues in our hands. Variability in the magnitude of the induction suggests that the regulation is complex, involving either additional factors or particular differentiation states. In addition to the induction of intracellular lipocortin-1, steroids cause the appearance of labelled lipocortin-1 on the outer surface of the cells. Whether cell breakage can account for this effect is unclear. Considerable microheterogeneity was found in preparations of recombinant-lipocortin-1. Aspects of N-terminal post-translational processing, N-terminal proteolysis, conformational states and the existence of an air-denatured form lacking alpha-helical structure contributed to this heterogeneity. We believe that these aspects are responsible for the variable biological potency of different preparations. It remains unclear whether this protein actually plays a physiological role in the regulation of the inflammatory response or achieves its effects through membrane binding and subsequent non-physiological perturbation of the cells.
Human peripheral blood monocytes can produce interleukin-1 (IL-1) beta following the addition of picogram amounts of bacterial lipopolysaccharides (LPS). The activation of IL-1 production by these cells can be mimicked by manipulation of specific biochemical pathways which appear to be independent of, and synergistic with, the pathways activated by LPS. Such pathways may be used by other physiological systems to modulate the production of IL-1. Both negative and positive modulation of IL-1 production can be described. Selected chemical antagonists of the arachidonic acid cascade have been shown to inhibit IL-1 production. The activity of such compounds does not appear to be related to their activity as inhibitors of cyclooxygenase or lipooxygenase enzymes or to activity as antioxidants. The intracellular form of IL-1 beta is limited to the precursor, which is found cytoplasmically. The release of IL-1 by activated cells appears to be regulated, in part, by the integrity of the microtubule system of the cells.
Human recombinant lipocortin 1 has been tested for anti-inflammatory activity in a conventional model of acute inflammation. Microgram amounts of the protein, locally administered, inhibited edema of the rat paw when induced by subplantar injections of carrageenin: the ED50 was 10-20 micrograms per paw, and inhibition (maximum of 60-70%) was not dependent upon an intact adrenal cortex. Doses of lipocortin that produced approximately 50% inhibition in the carrageenin test were inactive against edema elicited by bradykinin, serotonin, platelet-activating factor-acether, or dextran, whereas edema caused by Naja mocambique venom phospholipase A2 was strongly inhibited by lipocortin. The protein inhibited edema when rats were pretreated with agents that depleted mast-cell amines, kininogen, or polymorphonuclear leukocytes prior to initiation of the carrageenin edema but had no inhibitory action when rats were pretreated with the dual cyclooxygenase/lipoxygenase inhibitor BW 755C. These results demonstrate that human recombinant lipocortin has potent local anti-inflammatory activity, probably through selectively interfering with eicosanoid generation. Lipocortin is relatively ineffective against edema caused by mast-cell degranulation or kinins, except when degranulation is caused by phospholipase A2.
In polymorphonuclear neutrophils, phospholipase A2 activity is the rate-limiting step for platelet-activating factor (PAF) formation and for the biosynthesis of arachidonic acid derivatives, leukotrienes and prostaglandins. Glucocorticosteroids inhibit phospholipase A2 activity by inducing in target cells the synthesis and release of phospholipase A2 inhibitory proteins named 'lipocortins'. Here, we report that rat pleural inflammatory polymorphonuclear neutrophils, treated with dexamethasone, decrease their production of eicosanoids and PAF. Evidence is presented which may implicate lipocortin 's' in these inhibitions since (i) phospholipase A2 inhibitory proteins are found in the supernatant of dexamethasone-treated cells, (ii) this supernatant inhibits the formation of lipid mediators in untreated cells, inhibition being reversed either by incubating the supernatant with a monoclonal antibody against rat lipocortin or by boiling it and (iii) a 36 kDa lipocortin from mice lungs mimics the effects of dexamethasone when added exogenously on untreated cells. Our results favour the hypothesis that the newly formed lipocortin 's' could be responsible for the antiphospholipase A2 activity of glucocorticosteroids.
The anti-inflammatory action of glucocorticoids has been attributed to the induction of a group of phospholipase A2 inhibitory proteins, collectively called lipocortin. These proteins are thought to control the biosynthesis of the potent mediators of inflammation, prostaglandins and leukotrienes, by inhibiting the release of their common precursor, arachidonic acid, a process that requires phospholipase A2 hydrolysis of phospholipids. Lipocortin-like proteins have been isolated from various cell types, including monocytes, neutrophils and renal medullary cell preparations. The predominant active form is a protein with an apparent relative molecular mass (Mr) of 40,000 (40K). These partially purified preparations of lipocortin mimic the effect of steroids, and mediate anti-inflammatory activity in various in vivo model systems. Using amino-acid sequence information obtained from purified rat lipocortin, we have now cloned human lipocortin complementary DNA and expressed the gene in Escherichia coli. Our studies confirm that lipocortin is a potent inhibitor of phospholipase A2 activity.
The guinea-pig perfused isolated lung, used in conjunction with the cascade superfusion system to measure the release of thromboxane A2(TXA2), is a simple and convenient model for assessing the inhibition by glucocorticoids of eicosanoid formation. Dexamethasone inhibits the release of TXA2 from the lung when it is stimulated by agents such as RCS-RF2 of leukotrienes, but not when bradykinin or arachidonic acid are used. Using this model we have shown that the glucocorticoids suppress eicosanoid generation by cells through the induction of a family of phospholipase A2-inhibitory proteins now termed the 'lipocortins'. Recently the primary structure of one form of lipocortin has been elucidated and the human gene cloned. Lipocortin 1 is a polar monomeric protein with anti-phospholipase properties in vitro and we now report that when infused into guinea-pig lung preparations this protein has the same inhibitory profile as the glucocorticoids but with a more rapid onset of action. This is the first demonstration that eicosanoid formation can be inhibited by a recombinant phospholipase inhibitory protein applied extracellularly.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
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We have reviewed the evidence that amines accumulate in intracellular vesicles of low pH, such as lysosomes and endosomes. There is consequent elevation of intravesicular pH, and inhibition of receptor-ligand dissociation often results from this pH change. We have argued that the capacity for fusion of such vesicles is also reduced by the high pH. We suggest that the variety of effects of amines on membrane flow and macromolecular transport we describe are at least partly due to such reduced fusion (Figs. 1 and 2). We propose that an internal low pH may facilitate heterologous vesicle-vesicle and vesicle-plasma membrane fusion. There is some evidence that clathrin can accelerate phospholipid vesicle fusion in vitro at low pH (Blumenthal et al., 1983) but no direct evidence on the role of intravesicular pH. This idea is consistent not only with the preceding discussion, but also with the fact that the intracellular membrane-bound compartments least involved in fusion events (e.g. mitochondria) are of neutral or alkaline internal pH. Membrane fusion is certainly required for the formation of vesicles at the periphery of the Golgi apparatus, and possibly earlier in the transport and processing of biosynthetic products in the Golgi (Bergeron et al., 1982). Thus the accumulation of amines in the Golgi may be responsible for several effects on the flow of macromolecules along their translocation pathways. The status of the plasma membrane in this view is complex. It might be argued that the pH dictating the fusion step in endocytosis is that of the extracellular fluid, in which case the inhibitory effects of amines on this process are not explained. However, the rapidity of acidification of the newly formed endocytic vesicles allows the possibility that plasma membrane invaginations might temporarily sequester areas which are of lower pH than that of the bulk extracellular fluid even before fusion, since the proton pumping enzyme(s) are probably present on the plasma membrane. Were this the case, then an acid pH could again be a factor determining membrane fusion at the plasma membrane. The inhibition of endocytosis by weak bases thus may again reflect elevation of pH in a sequestered compartment. From the data on the dependence of response on the concentration of amines, we anticipate that most responses involving membrane flow will be biphasic, with inhibitory effects at low amine concentration, giving way to stimulatory ones at higher concentrations. We suggest that the reported dichotomy between different amines in intracellular membrane fusion systems (D'Arcy Hart, 1982) may result from this concentration dependence.(ABSTRACT TRUNCATED AT 400 WORDS)
Human unfractionated mononuclear leukocyte (UFMNL) preparations and T-cell-depleted (TCD) mononuclear leukocyte preparations, obtained from 400 ml of whole peripheral blood (PB) or from continuous-flow centrifugation leukapheresis (CFCL) concentrates, were cleanly separated by counterflow centrifugation-elutriation (CCE) into highly purified lymphocyte and monocyte populations, each with excellent recovery and function. Lymphocytes isolated from UFMNL preparations ranged in number from 1.88 × 108 to 2.22 × 109 with mean purities in excess of 93%. Lymphocytes isolated from TCD preparations yielded up to 1.90 × 109 lymphocytes devoid of all detectable sheep RBC receptor-bearing cells with mean purity and recovery in excess of 93 and 90%, respectively. Monocytes negatively selected from UFMNL preparations obtained from whole PB resulted in yields of 1.08 × 108 monocytes with 92% purity and 94% recovery; UFMNL obtained from CFCL concentrates resulted in yields of 4.22 × 108 monocytes with 92% purity. TCD preparations obtained from PB yielded 7.19 × 107 monocytes in 95% purity and 94% recovery, and from CFCL concentrates, 6.21 × 108 monocytes in 94% purity and 79% recovery. Recovered lymphocytes and monocytes have excellent overall viability, and the purified monocytes have normal phagocytic and chemotactic functional capabilities. Counterflow centrifugation-elutriation (CCE) is a valuable technique for obtaining monocyte and monocyte-depleted (i.e., lymphocyte) cell preparations that are negatively selected in high yield, with excellent purity and viability.
Annexin 1 (named p35, lipocortin I or calpactin II), initially described as a glucocorticoid induced protein, belongs to a new characterized family of intracellular proteins. In the skin, the role of annexins has still not been elucidated. In a previous study, we reported the localization of annexin 1 in both freshly isolated human epidermal cells and in cultured keratinocytes using immunofluorescence, FACS analysis and immunoblotting techniques. The protein was characterized by Western blot and immunoprecipitation as a 35 kDa protein. Results from in vivo studies confirmed the presence of annexin 1 in basal and suprabasal layers of normal human skin with modified reactivity patterns in hyperproliferative lesions. In the present study, the role of glucocorticoids in annexin 1 regulation was investigated in epidermal cells by Western blot and immunoprecipation assays. In contrast to other studies, we found that glucocorticoid treatment of epidermal cells led to a decrease in annexin 1 content in the cytoplasm and the membranes of cells. As annexin 1 was not detected in the nucleus of cells, we conclude that there was a down regulation of annexin 1 after glucocorticoid treatments rather than a translocation of the protein to the nucleus. Despite the absence of the signal peptide sequence necessary for protein secretion, annexin 1 was released in the keratinocyte culture medium. We found that the protein was secreted only in low Ca2+ medium (0.15 mM), this process required an active metabolism.
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.
We have studied the occurrence, distribution and disposition of lipocortins (annexins) 1, 2 and 5 in mixed peritoneal leucocytes obtained from rats in which glucocorticoid levels were altered by adrenalectomy, administration of the glucocorticoid antagonist, RU486, or by injection of dexamethasone or hydrocortisone, as well as from rats in which the peritoneal cells were elicited by inflammatory stimuli.
In cells obtained from untreated rats with an intact adrenal cortex, lipocortins 1, 2 and 5 were readily detectable: the majority of each of the proteins was apparently located intracellularly with much smaller amounts in the membrane. Lipocortin 1 and to a lesser extent lipocortin 5 were also seen in a Ca ²⁺ ‐dependent association with the external plasma membrane. Following administration of RU486 (2 × 20 mg kg ⁻¹ ) the amounts of lipocortin 1 and 2 in cells were greatly reduced. Conversely, injection of hydrocortisone (1 mg kg ⁻¹ ) or dexamethasone (0.08 mg kg ⁻¹ ) caused an increase in the amount of lipocortin 1 and 2 in peritoneal cells within 30 min. Lipocortin 5 was unchanged by any manipulation of glucocorticoid levels.
Lipocortins 1 and 2 were elevated in both intracellular and membrane‐associated fractions of macrophages elicited by intraperitoneal injection in inflammogens. This phenomenon also occurred in adrenalectomized animals.
Our data indicate that glucocorticoids control the synthesis of some members of the lipocortin family in rat mixed peritoneal cells but also suggest the existence of a separate system for controlling the generation of this protein. The significance of these observations is considered in relation to the mechanism of glucocorticoid hormone action on eicosanoid production.
Anti-inflammatory lipocortin 1 production by peripheral blood leukocytes in response to hydrocortisone Brefeldin A: Insight into the control of membrane traffic and organelle structure Cleavage of structural proteins during the assembly to the head of bacteriophage
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Review article: effects of exogenous amines on mammalian cells with particular reference to membrane flow
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DEAN, R.T., JESSUP, W. & ROBERTS, C.R. (1984). Review article:
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Cloning and expression of human lipocortin, a phospholipase A2 inhibitor with potential anti-inflammatory activity
- B P Mattaliano
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- R L Tizard
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- K L Pepinsky
Review article: effects of exogenous amines on mammalian cells with particular reference to membrane flow
- DEAN
A novel pathway for secretory proteins?
- MUESH